Revealing Reef Fish Eggs at the Species Level: Insights from DNA Barcoding Aligned with Egg Morphology and Spawning Times

Revealing Reef Fish Eggs at the Species Level: Insights from DNA Barcoding Aligned with Egg Morphology and Spawning Times | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Revealing Reef Fish Eggs at the Species Level: Insights from DNA Barcoding Aligned with Egg Morphology and Spawning Times Timothy Patrick van Wagensveld, Sander van Lopik, Jeroen Paul Ham This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6314167/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study utilized DNA-barcoding to identify pelagic reef fish eggs on a species-specific level around Bonaire. Eggs were collected at three different sites (Bachelor Beach: 12.12577° N, 68.28827° W; Angel City: 12.10302° N, 68.28824° W; Pink Beach: 12.06414° N, 68.28336° W; WGS84). Furthermore, egg morphology was described using established criteria, revealing common features such as a spherical shape, transparent and smooth chorion, notable differences such as oil globule colour, and the presence of melanophores. This study provides valuable insights into the reproduction and the morphology of eggs of five distinct fish species and a single genus. In duplicates, we identified eggs belonging to 5 different taxa; Haemulon genus, Rypticus saponaceus , Paraconger caudilimbatus , Heteroconger longissimus , and scarus taeniopterus. A single identification was made for Katsuwonus pelamis. All eggs were collected during October and November in 2022 and 2023. The long-term goal of this study is to establish a fish egg database for the Caribbean including photographic and genetic information, and a fish spawning calendar. Further research is needed and ongoing to uncover other species and to study whether morphology within species and regions vary. Bonaire DNA-barcoding fish eggs fish spawning fish egg morphology Figures Figure 1 Figure 2 Figure 3 Introduction Coral reef ecosystems are one of the most biodiverse and productive ecosystems on the planet, however, they are threatened by a variety of anthropogenic disturbances, including overfishing [ 1 ], pollution [ 2 ], and climate change [ 3 ]. Hence, the global coral cover has declined by 50% since the 1950s [ 4 ]. The loss of coral reefs negatively affects marine biodiversity [ 5 ], food security [ 6 ] and tourism [ 7 ]. Therefore, it is important to ensure the well-being of marine species by identifying critical habitats and critical phases in their life cycle. Reef fish are vital for the health of coral reefs due to their unique roles in the ecosystem. The grazing of algae overgrowth is such an example whereby the survival of juvenile corals increases, and overgrowth of existing corals is mitigated, thus improving the resilience of coral ecosystems [ 8 – 13 ]. Due to overexploitation, reef fish biomass has been negatively affected in the Caribbean, and it may take decades to recover [14–15]. In order to restore affected fish populations, it is necessary to support vulnerable species in their natural habitat, particularly during critical points in their life cycle e.g. spawning events. Yet, knowledge of many reef fish species and their spawning events remain critically absent. Fish have a variety of reproductive strategies including benthic egg layers, vivipary, mouthbrooding or pelagic reproduction. Pelagic reproduction, the simultaneous release of gametes directly into the water column, is the most common method of propagation in bony fish [ 16 ]. After fertilization, buoyant or semi-buoyant eggs are taken by ocean currents to develop into larvae. The ability to capture and identify these pelagic fish eggs on a species-specific level provides important information on the reproductive biology of reef fishes, thus improving our knowledge on the critical reproductive life stage. However, morphological identification of fish eggs at a species level remains a challenging task. Therefore, researchers typically raise fish eggs into larvae to identify species [ 17 ]. Notably, larval rearing is complicated and often associated with high mortality during the first feeding stages due to insufficient knowledge on the nutritional requirements [18]. Recently, DNA-barcoding of pelagic fish eggs has become an emerging research method to identify species composition, critical habitats and seasonal spawning [ 19 – 25 ]. It has been proven to be an effective method to identify species by sequencing Cytochrome-C oxidase subunit 3 (COI-3) of mitochondrial DNA. COI-3 is a commonly used gene for DNA-barcoding due to the relatively high mutation rate of this gene between closely related species. COI-3 codes of marine fish are available in The Barcode of Life Data System (BOLD). These sequences are provided by The Fish Barcode of Life Initiative (FISH-BOL). FISH-BOL is a collaborative international research effort that aims to efficiently study evolution and improve taxonomy by establishing a reference library. DNA-barcoding pelagic fish eggs is an effective method to study reproduction of broadcast spawning reef fish as the eggs do not actively move through the water column as opposed to fish larvae. Using fish larvae as a spawning indicator is less accurate as larvae can be up to weeks or months old when captured [ 26 ], meaning they could already have travelled long distances both in space and time. In addition, fish eggs in tropical waters have a relatively short period until hatching [ 27 ], Therefore, the origin of sampled eggs must be relatively close in proximity to the spawning time and location. In the past, an elaborate study by Munro et al. [28] demonstrated the seasonal spawning for 35 species and provided spawning observations on 83 fish species in the Caribbean Sea. According to a large-scale survey, the number of fish species is estimated to be around 362 in Bonaire National Marine Park [ 29 ]. This illustrates the significant knowledge gap on the spawning patterns of many fish species, with minimal new research released on spawning patterns since then [ 30 – 31 ]. In addition, studying the timing of reproduction and comparing it to past studies may elucidate the effects of global warming. During this study, DNA-barcoding was used on pelagic fish eggs to determine the corresponding species that spawned on the reefs of Bonaire. Moreover, the fish eggs were photographed before DNA-barcoding in order to document and provide morphological characteristics. Offering detailed illustrations of fish eggs can enhance their future identification without the need for DNA-barcoding. The establishment of a comprehensive fish egg database could prove cost-effective, and adopting this illustration-based method could serve as a potential protocol for developing such a database. Therefore, the objectives of this pilot study were to elucidate if (1) fish eggs in this study area can be identified on a species level using DNA barcoding (2) whether there are morphological differences within a species, so that sampled eggs in the future can be identified based on photographs only, 3) whether our method is sufficient in creating a fish egg database and (4) to study the feasibility of making a year-round spawning calendar for reef fish using our method. Method Study area Bonaire is an island which is part of the Caribbean Netherlands in the Caribbean Sea located North of the coast of Venezuela. The average seawater temperature ranges from 26–29ᵒC annually [ 32 ]. This study was conducted on the fringing reefs of Bonaire on the leeward side (west coast) of the island. Bonaire’s reefs have been protected since 1979 when the marine park was established, whereby ongoing conservation, research and management efforts are undertaken by the marine national park ‘’Stichting Nationale Parken’’ (STINAPA). In addition, several no-dive sites and protected areas have been created. Sample collection The fish eggs were collected passively approximately 50 meters off the coast near the reef slopes at three different locations: Bachelor Beach: 12.12577° N, 68.28827° W; Angel City: 12.10302° N, 68.28824° W; Pink Beach: 12.06414° N, 68.28336° W (WGS84), (Fig. 1 ). Samples were collected in October 2022 and subsequently in October and November 2023. In order to collect eggs, floating plankton nets of 1,5 meters long with a circular opening with a diameter of 32 cm and a mesh size of 200 µm were used (Fig. 2 ). The net opening was weighted down to ensure a continuous opening. The nets were attached to the marine park moorings at approximately 1700 hr. The next day the plankton nets were collected in the morning around 0800 hr. At the rear of these plankton nets, plastic bottles were attached to collect the fish eggs. A constant flow of fresh seawater was present by having a 200 µm mesh at the bottom of the bottle. In the morning the bottles with eggs were transported to the laboratory in a cooling box filled with seawater taken from the sampling locations to maintain a constant seawater temperature. The bottles were then emptied into a transparent jar in order to study the eggs. Microscopic photography After settlement of debris, the eggs were sucked up from the surface layer in the jar using a pipette and analysed with an optic microscope (Euromex BioBlue BB.4260). Next, two photographs using different focal points were taken with a microscope camera (Amscope AF205). One photo is focussed on the oil globule, and the other on the larvae. Smaller eggs were photographed using 40x magnification and larger eggs were photographed using 20x magnification. Photograph settings were set to default modes on the Amscope software, except for ‘white balance’. Finally, after morphological study, each egg was individually placed in a 1,5-milliliter plastic Eppendorf tube with 96% denatured ethanol using a sterile pipette. The eggs were sampled for DNA-analysis in duplicates based on their morphological characteristics (oil droplet, diameter, chorion, perivitelline space, yolk segmentation) for DNA-barcoding [33]. DNA-barcoding 76 fish eggs were fixated in 96% ethanol and sent to Macrogen Europe for DNA-barcoding. The fish eggs were sorted in groups based on morphology. Only 41 samples yielded a successful amplification (≥ 97% similarity score in BOLD) of which thirteen are presented in this paper (full list of sequences available in Appendix I). Only morphological duplicates that have at least two successfully barcoded replicates are presented. Whole genomic DNA was extracted from single fish eggs by using a DNA-isolation kit (Macrogen). Next, PCR-amplification was executed using the following primers; VF2_t1: 5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3‘ and FishF2_t1: 5‘- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’. With reverse FishR2_t1: 5’-TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’ and FR1d_t1: 5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’ [ 34 ] and two M13 entailed primers for sequencing [ 35 ]. Sequence products ranged from 700 to 1700 base pairs of the COI-3 gene. Sequence products were then analysed using the Bold systems Identification V4 using animal identification setting on species level barcode records. Only samples with a > 97% similarity score in BOLD were taken into account. Software Images of the selected fish eggs were analysed using ImageJ software. The diameters of the chorion, oil globules, and the widest part of the perivitelline space were measured to provide morphological distinctions [33]. Results A total of 3838 fish eggs were collected, of which 76 were subjected to DNA-barcoding. 13 eggs with morphological duplicates are presented in this paper (Fig. 3 ). In general, fish eggs exhibit a spherical shape and possess a transparent, smooth chorion. However, the eggs of Scarus taeniopterus deviate by being ellipsoid in shape (Fig. 3 g & 3 h). DNA-barcoding Six different taxa were identified (Table 1 ); five on a species-level: Heteroconger longissimus , Katsuwonus pelamis , Paraconger caudilimbatus , Rypticus saponaceus and Scarus taeniopterus . Samples from the Haemulidae family were too closely related to distinguish on a species level based on COI-3 gene sequencing. In addition, Scarus taeniopterus reversed sequences had no matching result for both samples. Morpholgy The two fish eggs from the Haemulon genus exhibit morphological similarities. The eggs have a light-yellow oil globule and scattered melanophores on the embryo (Fig. 3 a & 3 b). The fish eggs were approximately 0,86 mm with oil globules 0,18–0,19 mm in size. The perivitelline space has a width of 0,07–0,12 mm (Table 1 ). This paper presents two duplicates of R . saponaceaus confirming that morphology can differ within a species (Fig. 3 c – 3 f). The fish eggs of R . saponaceus varied in size from 0,8–0.9 mm in size with perivitelline spaces of 0,08–0,12 mm. The oil globules with a round shape were 0,19–0,21 mm in diameter. The odd shaped oil globules were 0,14–0,15 mm in width and 0,27–0,29 mm in length. The fish eggs are characterized by a distinct bright yellow oil globule and a white embryo. The fish eggs were collected in October and November 2022 and 2023. This study presents new spawning information on this species as there is no known information available of R . saponaceaus spawning during these months. The fish eggs of S. taeniopterus have a characteristic ellipsoid shape with smaller yellow oil globules (Fig. 3 g & 3 h). The duplicates are 1,2–1,3 mm in length with small oil globules of 0,09 and 0,11 mm. The perivitelline space has a width of 0,22–0,25 mm. The eggs were captured in November 2023 at two different locations (Pink Beach and Angel City). The egg of K . pelamis (Fig. 3 i) features a white, heavily pigmented embryonic body and a black oil globule located caudally to the embryo. The morphology resembled the eggs of the Haemulon genus, but our results show that eggs with morphological similarities are not necessarily from the same taxa. The morphology matches the description by Kawakami et al. [ 36 ]. Eggs of H . longissimus (Fig. 3 j & 3 k) and P . caudilimbatus (Fig. 3 l & 3 m) are notably larger than the other collected eggs, necessitating multiple oil globules for buoyancy. H . longissimus has less oil globules (13) than P . caudilimbatus (40–45). The duplicates of H . longissimus have the same amount of oil globules whereas P . caudilimbatus have a different amount (Table 1 ). Both species are similar in diameter 2,3–2,5 mm. The perivitelline spaces are wide. H . longissimus has a width of 0,54–0,62 mm. Whereas, P. caudilimbatus has a width of 0,46–0,58 mm. The oil globules for both species are small compared to the other eggs, ranging from 0,047–0,12 mm. The egg shown in Fig. 3 j has brighter yellow oil globules compared to its counterpart in Fig. 3 k. Both species were captured in October 2023. Table 1 Fish eggs, corresponding capture date, location and DNA-barcoding results. FW = Results forward primer, RV = Results reversed primer. Morphological characteristics are further presented in the second part of the table Sample Date Location Genus Species FW (% ID) Species RV (% ID) a 29-10-2023 Bachelor Beach Haemulon Flavolineatum (99.5) & aurolineatum (99.39) Flavolineatum & aurolineatum (100) b 29-10-2023 Bachelor Beach Haemulon Flavolineatum (99) Flavolineatum (100) & aurolineatum (99.85) c 9-11-2023 Pink Beach Rypticus Saponaceus (99.85) Saponaceus (99.46) d 15-10-2022 Bachelor Beach Rypticus Saponaceus (99.85) Saponaceus (99.46) e 9-11-2023 Pink Beach Rypticus Saponaceus (99.48) Saponaceus (99.46) f 9-11-2023 Pink Beach Rypticus Saponaceus (99.85) Saponaceus (99.54) g 14-11-2023 Angel City Scarus Taeniopterus (99.67) No match h 14-11-2023 Pink Beach Scarus Taeniopterus (98.14) No match i 17-10-2022 Bachelor Beach Katsuwonus Pelamis (99.83) Pelamis (98.48) j 29-10-2023 Pink Beach Heteroconger Longissimus (98.73) Longissimus (98.42) k 29-10-2023 Pink Beach Heteroconger Longissimus (78.99) Longissimus (98.46) l 29-10-2023 Pink Beach Paraconger Caudilimbatus (100) Caudilimbatus (100) m 29-10-2023 Pink Beach Paraconger Caudilimbatus (99.69) Caudilimbatus (99.51) Sample Egg diameter (mm) Oil globule diameter (mm) Perivetelline space (mm) Colour of chorion Surface of chorion Yolk Oil globule (n =) Colour of oil globule Melanophores a 0.869 0.193 0.12 Transparent Smooth Homogenous 1 Light yellow/brown + b 0.857 0.18 0.07 Transparent Smooth Homogenous 1 Light yellow/brown + c 0.813 0.193 0.08 Transparent Smooth Homogenous 1 Yellow/green - d 0.897 0.21 0.12 Transparent Smooth Homogenous 1 Yellow/green - e 0.81 0.296 L − 0.152 W 0.09 Transparent Smooth Homogenous 1 Yellow/green - f 0.82 0.269 L − 0.14 W 0.08 Transparent Smooth Homogenous 2 Yellow/green - g 1.3 L − 0,48 W 0.11 0.25 Transparent Smooth Homogenous 1 Ligit yellow - h 1.2 L − 0.49 W 0.09 0.22 Transparent Smooth Homogenous 1 Light yellow - i 0.88 0.19 0.19 Transparent Smooth Homogenous 1 Yellow/black + j 2.5 0.047–0.101 0.54 Transparent Smooth Homogenous 13 Yellow - k 2.5 0.047–0.121 0.62 Transparent Smooth Homogenous 13 Light yellow - l 2.5 0.040–0.113 0.58 Transparent Smooth Homogenous 40 Light yellow - m 2.3 0.029–0.094 0.46 Transparent Smooth Homogenous 45 Light yellow - Temporal patterns – first results of spawning calendar The eggs belonging to the Haemulon family were caught in October, 1 day after full moon (Table 2 ). Rypticus saponaceus eggs were caught in October and November, 6 days after full moon and 4 days before new moon. The two S. taeniopterus eggs were both caught in November, 1 day after full moon. The K. pelamis egg was caught in October at last quarter. Heterconger longissimus and P. caudilimbatus eggs were all caught in October, 1 day after full moon. Table 2 Spawning calendar of collected and DNA-barcoded fish eggs. Eggs indicated in green were collected during waning moon phases, blue are eggs that were collected during waxing moon phases. Numbers indicate how many days after or before full moon or new moon the eggs were collected. The indicated number of eggs collected per species are presented per day relative to the moon phase. For example, 2 Haemulon eggs were collected during the waning moon phase of October, 1 day after full moon. FM = full moon, HM = half moon, NM = New moon FM 1 2 3 4 5 6 7 HM 7 6 5 4 3 2 1 Taxa Common name Month NM 1 2 3 4 5 6 7 HM 7 6 5 4 3 2 1 Haemulon Grunt Oct 2 Rypticus saponaceus Greater Soapfish Oct/Nov 1 3 Scarus taeniopterus Princess parrotfish Nov 2 Katsuwonus pelamis Skipjack tuna Oct 1 Heteroconger longissimus Brown garden eel Oct 2 Paraconger caudilimbatus Margintail conger Oct 2 Discussion Spawning and Egg Morphology This study provides valuable insights into the reproductive spawning of five distinct fish families (Congridae, Haemulidae, Scaridae, Scombridae, and Serranidae) and the morphological characteristics of their eggs within the studied region. Eleven samples were identified at the species level, representing five distinct species: H. longissimus , K. pelamis , P. caudilimbatus , R. saponaceus , and S. taeniopterus . Additionally, two samples were identified at the genus level ( Haemulon ). The collection of fish eggs followed by DNA barcoding provides a comprehensive approach to studying fish reproduction. Eggs were collected using plankton nets, a cost-effective and efficient method. Prior to DNA barcoding, eggs were photographed to facilitate morphological characterization. Standardized criteria were applied to describe egg morphology, allowing for species differentiation and enabling comparisons with similar studies [ 36 ]. Most eggs exhibited common features, including a spherical shape, a transparent and smooth chorion, except for S. taeniopterus , which displayed an ellipsoid shape. Distinct morphological features such as oil globule colouration and the presence of melanophores on the chorion or embryonic bodies enabled species differentiation. Egg size and perivitelline space were also valuable indicators, with unusually shaped oil globules observed in R. saponaceus . This study presents the first documentation of spawning for R. saponaceus , P. caudilimbatus , and H. longissimus in the Caribbean Sea during October, as well as the first visual documentation of their eggs. Previously, only K. pelamis and Haemulon spp . had documented egg images. When comparing K. pelamis images from Kawakami et al. [ 36 ] with our findings, no morphological differences were observed, suggesting consistency across regions. Eggs of the Haemulon genus closely resembled images from Barden et al. [ 37 ], particularly in melanophore distribution. However, embryonic bodies in our samples appeared more pigmented, potentially due to variations in microscope and camera equipment. Documented spawning times for Haemulon species include peak reproductive activity for H. sciurus in Bermuda during May–June, with lunar periodicity, while H. flavolineatum exhibits year-round spawning, peaking in March [ 38 ]. No S. taeniopterus eggs with advanced embryonic development were captured, suggesting that S. taeniopterus spawn early in the morning, as previously described by Colin and Clavijo [ 39 ]. Eggs of R. saponaceus exhibited morphological variation within the species, emphasizing the need for additional sampling. The unusual egg shapes may be attributed to elevated sea temperatures during the pilot study, as thermal stress has been associated with developmental deformities [ 40 ]. The K. pelamis egg highlights that morphologically similar eggs may originate from different species. Consequently, when establishing a fish egg database, it is essential to document morphological features, as visual identification alone may be insufficient. Challenges in DNA-barcoding DNA barcoding success was limited due to transportation and storage constraints. However, strong sequence overlap with the Barcode of Life Database (BOLD) supports the accuracy of species identification for most samples. Only sequences with ≥ 97% similarity to BOLD entries were included, except for S. taeniopterus , which exhibited ≥ 97% overlap with the forward primer but lacked alignment with reverse primer database entries, introducing uncertainty regarding sample identity. On-site DNA barcoding is recommended to enhance sequencing success rates. Additionally, COI-3 sequences were insufficient to distinguish Haemulon species at the species level, suggesting that COI-3 may not be suitable for fine-scale identification within this genus. Photography Photographing fish eggs from multiple angles presented technical challenges. Eggs were placed on a microscope slide with a droplet of seawater, however, the oil globules always caused them to float upright with the oil globules on top, complicating multi-angle imaging. Removal of seawater using a pipette sometimes repositioned the eggs, enabling additional perspectives. However, standardizing this process remains challenging and requires further methodological refinement. Fish Egg Dispersal and Spawning Ground Identification The precise dispersal patterns of fish eggs remain unclear due to the absence of surface current data for hindcasting, making it difficult to pinpoint spawning locations. Despite this limitation, our findings contribute valuable insights by mapping egg distribution patterns and defining a reproductive timeframe for the Haemulon genus, K. pelamis , H. longissimus , S. taeniopterus , P. caudilimbatus , and R. saponaceus . The feasibility of establishing a fish egg database using the methods presented in this study is promising. However, additional research is required to expand species representation and include samples from diverse geographic regions to assess intraspecific morphological variation. A comprehensive fish egg database and improved spawning documentation will support conservation efforts and the development of sustainable fishery policies. More broadly, this approach can be applied to the identification of other marine organisms that begin their life cycle in the planktonic stage, such as corals, echinoderms, and jellyfish. Conclusion Our findings confirm that DNA barcoding is an effective tool for species-level identification of fish eggs. However, we anticipate that the ability to sequence samples locally on Bonaire would significantly enhance our current success rate of approximately 54%. Additionally, local sequencing capabilities would allow for an increase in sample size, improving the robustness of our dataset. Regarding our second research objective, our results indicate that morphological variation in fish eggs can occur within a single species. Further research is required to determine in which species these variations exist and to what extent they manifest. The feasibility of establishing a fish egg database (third research objective) appears promising. However, the presence of slight morphological differences among duplicates underscores the need for additional investigation. Moreover, eggs exhibiting morphological similarities do not always belong to the same species. Therefore, while it is feasible to develop a fish egg database based on photographic records, we emphasize the importance of including detailed morphological characteristics. Solely relying on visual examination for species identification is insufficient. Nevertheless, this study demonstrates the effectiveness of egg collection, photography, and the documentation of morphological traits. To ensure consistency and comparability in the database, we recommend that future researchers utilize the same microscope and camera equipment. Finally, our results indicate that this approach can be successfully employed to develop a spawning calendar for reef fish (research objective 4), much to the like of coral reproduction calendars [ 41 ]. Despite a limited number of sampling days, we documented reproductive activity in six different taxa, three of which had no previously recorded spawning information. Continued year-round sampling and an increased number of DNA-barcoded fish eggs will further refine and expand upon the preliminary spawning calendar presented in this study. Declarations Ethics approval All fish egg collections have been conducted with consent as per the guidelines of the National Parks Foundations, STINAPA, Bonaire, Caribbean Netherlands. Only appointed mooring lines were used for passive fish egg collection. The Basel declaration was signed online by the authors and was referred to as ethical guidance in our use of collected fish eggs. Consent to Participate declaration not applicable Funding This work was financially supported by The World Wide Fund for Nature - Dutch Caribbean (project number 202379) and Stichting Koninklijke Rotterdamse Diergaarde – Rotterdam Zoo. Competing interests The authors have no relevant financial or non-financial interests to disclose. Data availability Statement All data supporting the findings of this study are available within the paper and its supplementary Information. All raw data, regarding forward and reverse primer sequences are presented in this paper and have been included in Appendix I, along with original reference describing the microsatellites used in this study Author Contribution S.V.L drafted the collection methodology. All authors were involved in collecting fish eggs in the field and field equipment maintenance. T.V.W. drafted the manuscript and analysed the data together with J.H. Both J.H and S.V.L. contributed to the manuscript with valuable feedback and input. Acknowledgement We gratefully acknowledge grants from The World Wide Fund for Nature – NL (WWF-NL) and Stichting Koninklijke Rotterdamse Diergaarde (Rotterdam Zoo). The Dutch Caribbean Nature Alliance (DCNA) generously supported the project by providing workspace and project guidance. Permission to work in the national marine park of Bonaire was granted by STINAPA. In addition, much appreciation goes out to their dedicated marine park rangers for their assistance and guidance at sea. Lastly, we are very grateful to our passionate students and volunteers. References Shantz AA, Ladd MC, Burkepile DE (2020) Overfishing and the ecological impacts of extirpating large parrotfish from Caribbean coral reefs. Ecological Monographs, 90 (2), e01403. https://doi.org/10.1002/ecm.1403 . Wear SL, Acuña V, McDonald R, Font C (2021) Sewage pollution, declining ecosystem health, and cross-sector collaboration. Biological Conservation, 255 , 109010. https://doi.org/10.1016/j.biocon.2021.109010 . 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DNA sequencing of fish eggs and larvae reveals high species diversity and seasonal changes in spawning activity in the southeastern Gulf of California. Marine Ecology Progress Series , 592 , 159–179. https://doi.org/10.3354/meps12446 . Azmir IA, Esa Y, Amin SMN, Md Yasin IS, Md Yusof FZ (2017) Identification of larval fish in mangrove areas of Peninsular Malaysia using morphology and DNA barcoding methods. Journal of Applied Ichthyology , 33(5), 998–1006. https://doi.org/10.1111/jai.13425. Burrows M, Browning JS, Breitbart M, Murawski SA, Peebles EB (2019) DNA barcoding reveals clear delineation between spawning sites for neritic versus oceanic fishes in the Gulf of Mexico. Fisheries Oceanography , 28 (2), 228–239. https://doi.org/10.1111/fog.12404. Chen W, Zhu S, Yang J, Li X, Li Y, Li J (2021) DNA barcoding reveals the temporal community composition of drifting fish eggs in the lower Hongshui River, China. Ecology and Evolution, 11 (16), 11507–11514. https://doi.org/10.1002/ece3.7943 . Kerr M, Browning J, Bønnelycke EM, Zhang Y, Hu C, Armenteros M, Murawski S, Peebles E, Breitbart M (2020) DNA barcoding of fish eggs collected off northwestern Cuba and across the Florida Straits demonstrates egg transport by mesoscale eddies. Fisheries Oceanography , 29 (4), 340–348. doi.org/10.1111/fog.12475 . Leyva-Cruz E, Vásquez-Yeomans L, Carrillo L, Valdez-Moreno M (2016) Identifying pelagic fish eggs in the southeast Yucatan Peninsula using DNA barcodes. Genome , 59(12), 1117–1129. https://doi.org/10.1139/ [email protected] . Lima MCCD, Lima SC, Savada CS, Suzuki KM, Orsi ML, Almeida FSD (2020) Use of DNA barcode in the identification of fish eggs in tributaries of the Paranapanema River basin. Genetics and Molecular Biology , 43 . https://doi.org/10.1590/1678-4685-GMB-2019-0352 . Cowen RK, Sponaugle S (2009) Larval dispersal and marine population connectivity. Annual review of marine science, 1(1), 443–466. https://doi.org/10.1146/annurev.marine.010908.163757 . Pauly D, Pullin RSV (1987) On the relationship between hatching time, egg diameter and temperature in marine eggs. Environ. Bioi. Fish. https://doi.org/10.1007/BF00004892 . Munro JL, Gaut VC, Thompson R, Reeson PH (1973) The spawning seasons of Caribbean reef fishes. Journal of Fish Biology , 5 (1), 69–84. https://doi.org/10.1111/j.1095-8649.1973.tb04431.x. Pattengill-Semmens CV (2002) The reef fish assemblage of Bonaire Marine Park: An analysis of REEF fish survey data. Proc. 53rd Gulf and Caribbean Fisheries Institute , 53, 591–605. Rivera Hernández JM, Shervette VR (2024) Queen Triggerfish Balistes vetula Age-Based Population Demographics and Reproductive Biology for Waters of the North Caribbean. Fishes, 9 (5), 162. https://doi.org/10.3390/fishes9050162 . van der Veer HW, Cardoso JF, Mateo I, Witte JI, van Duyl FC (2018) Occurrence and life history characteristics of tropical flatfishes at the coral reefs of Curaçao, Dutch Caribbean. Journal of Sea Research, 142 , 157–166. https://doi.org/10.1016/j.seares.2018.09.010 . Taylor M, Stephenson KA (2017) Impacts of Climate Change on Sea Temperature in the Coastal and Marine Environments of Caribbean Small Island Developing States (SIDS), Caribbean Marine Climate Change Report Card: Science Review 2017, pp 23–30. Ahlstrom EH & Moser HG (1980) Characters useful in identification of pelagic marine fish eggs. CalCOFI rep, 21, 121–131. Ivanova NV, Zemlak TS, Hanner RH, Hebert PD (2007) Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes , 7 (4), 544–548. https://doi.org/10.1111/j.1471-8286.2007.01748.x . Messing J (1983) New M13 vectors for cloning. In Methods in enzymology (Vol. 101, pp. 20–78). Academic Press. https://doi.org/10.1016/0076-6879(83)01005-8 . Kawakami T, Aoyama J, Tsukamoto K. (2010) Morphology of pelagic fish eggs identified using mitochondrial DNA and their distribution in waters west of the Mariana Islands. Environmental Biology of Fishes, 87, 221–235. https://doi.org/10.1007/s10641-010-9592-2 . Barden KP, Wittenrich ML, Cassiano EJ (2014) Candidate Species for Marine Ornamental Aquaculture: French Grunt, Haemulon flavolineatum: FA186/FA186, 3/2014. EDIS, 2014(4). https://doi.org/10.32473/edis-fa186-2014 . Maurer L, Dawson M, Boles L, Knight S, Stamper A (2020) Spawning of French grunts, Haemulon flavolineatum, in recirculating aquarium systems. PeerJ, 8, e9417. https://doi.org/10.7717/peerj.9417 . Colin PL, Clavijo IE (1988) Spawning activity of fishes producing pelagic eggs on a shelf edge coral reef, southwestern Puerto Rico. Bulletin of Marine Science , 43(2), 249–279. Ashaf-Ud-Doulah M, Islam SM, Zahangir MM, Islam MS, Brown C, Shahjahan M (2021) Increased water temperature interrupts embryonic and larval development of Indian major carp rohu Labeo rohita. Aquaculture International , 29, 711–722. https://doi.org/10.1007/s10499-021-00649-x . Marhaver KL, Chamberland VF, Vermeij MJA (2024) Coral Spawning Predictions, Southern Caribbean, 2024. CARMABI, Curacao. https://www.researchstationcarmabi.org/wp-content/uploads/2024/06/2024-CARMABI-Coral-Spawning-Predictions-FINAL-for-Printing-3-Page-A4-HR-compressed.pdf . Accessed 20 February 2025. Additional Declarations No competing interests reported. Supplementary Files AppendixI.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6314167","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449410637,"identity":"e18095b1-36c2-4beb-9024-31d73a8d1468","order_by":0,"name":"Timothy Patrick van Wagensveld","email":"data:image/png;base64,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","orcid":"","institution":"Royal Rotterdam Zoological \u0026 Botanical Gardens","correspondingAuthor":true,"prefix":"","firstName":"Timothy","middleName":"Patrick van","lastName":"Wagensveld","suffix":""},{"id":449410638,"identity":"5cff7ea7-b0c2-409c-ae2c-c5aa1e31c90e","order_by":1,"name":"Sander van Lopik","email":"","orcid":"","institution":"Royal Rotterdam Zoological \u0026 Botanical Gardens","correspondingAuthor":false,"prefix":"","firstName":"Sander","middleName":"van","lastName":"Lopik","suffix":""},{"id":449410639,"identity":"6315091c-5371-4489-974b-f16e9cae27bc","order_by":2,"name":"Jeroen Paul Ham","email":"","orcid":"","institution":"Royal Rotterdam Zoological \u0026 Botanical Gardens","correspondingAuthor":false,"prefix":"","firstName":"Jeroen","middleName":"Paul","lastName":"Ham","suffix":""}],"badges":[],"createdAt":"2025-03-26 16:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6314167/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6314167/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81937798,"identity":"b8afbcf5-9f9f-41ff-918c-e5371551f2d9","added_by":"auto","created_at":"2025-05-05 06:31:15","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":219689,"visible":true,"origin":"","legend":"\u003cp\u003eSample locations on the West coast of Bonaire. Marine park moorings from North to South: Bachelor Beach, Angel city and Pink Beach\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6314167/v1/cf43b7516fd0a09fc36a8ad2.jpeg"},{"id":81939114,"identity":"2c98509c-fa90-4310-b974-dabe2f6b160f","added_by":"auto","created_at":"2025-05-05 06:39:15","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":470311,"visible":true,"origin":"","legend":"\u003cp\u003ePlankton net in the water collecting fish eggs, with the plastic bottle for egg collection on the left hand side of the net\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6314167/v1/4d6070ed1d5fa4683473ee7c.jpeg"},{"id":81939739,"identity":"ad7cc95f-c142-49c1-be0c-41982288d0e2","added_by":"auto","created_at":"2025-05-05 06:47:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226493,"visible":true,"origin":"","legend":"\u003cp\u003ePhotographs of our selected fish eggs that had successful DNA-barcoding results. a \u0026amp; b: \u003cem\u003eHaemulon\u003c/em\u003egenus, c – f: \u003cem\u003eRypticus saponaceaus\u003c/em\u003e, g \u0026amp; h:\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eScarus \u003c/strong\u003e\u003c/em\u003e\u003cem\u003etaeniopterus\u003c/em\u003e, i: \u003cem\u003eKatsuwonus pelamis\u003c/em\u003e, j \u0026amp; k: \u003cem\u003eHeteroconger longissimus\u003c/em\u003e, and l \u0026amp; m: \u003cem\u003eParaconger caudilimbatus\u003c/em\u003e. Two images were taken of each egg, with the focal point on the larvae (left side), and one on the oil globule(s) (right side). Images a – i were taken with a magnification of 40x, with scale bars set to 400 microns. Images j – m were photographed with 20x magnification and scalebars set to 900 microns\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6314167/v1/b1e6c18cfef0364392bafbce.jpg"},{"id":83859551,"identity":"8ec5cb95-9910-441d-aeba-0a4fd4af7d60","added_by":"auto","created_at":"2025-06-03 18:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1699862,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6314167/v1/8f23066b-1daf-49a5-8eec-7c49567a748e.pdf"},{"id":81937796,"identity":"9e5b75d8-bc30-4c8a-926e-fc3acf17042d","added_by":"auto","created_at":"2025-05-05 06:31:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25774,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixI.docx","url":"https://assets-eu.researchsquare.com/files/rs-6314167/v1/70e022d000609597ad4e9e08.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Revealing Reef Fish Eggs at the Species Level: Insights from DNA Barcoding Aligned with Egg Morphology and Spawning Times","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoral reef ecosystems are one of the most biodiverse and productive ecosystems on the planet, however, they are threatened by a variety of anthropogenic disturbances, including overfishing [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], pollution [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and climate change [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hence, the global coral cover has declined by 50% since the 1950s [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The loss of coral reefs negatively affects marine biodiversity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], food security [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and tourism [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, it is important to ensure the well-being of marine species by identifying critical habitats and critical phases in their life cycle.\u003c/p\u003e \u003cp\u003eReef fish are vital for the health of coral reefs due to their unique roles in the ecosystem. The grazing of algae overgrowth is such an example whereby the survival of juvenile corals increases, and overgrowth of existing corals is mitigated, thus improving the resilience of coral ecosystems [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e–\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Due to overexploitation, reef fish biomass has been negatively affected in the Caribbean, and it may take decades to recover [14–15]. In order to restore affected fish populations, it is necessary to support vulnerable species in their natural habitat, particularly during critical points in their life cycle e.g. spawning events. Yet, knowledge of many reef fish species and their spawning events remain critically absent.\u003c/p\u003e \u003cp\u003eFish have a variety of reproductive strategies including benthic egg layers, vivipary, mouthbrooding or pelagic reproduction. Pelagic reproduction, the simultaneous release of gametes directly into the water column, is the most common method of propagation in bony fish [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. After fertilization, buoyant or semi-buoyant eggs are taken by ocean currents to develop into larvae. The ability to capture and identify these pelagic fish eggs on a species-specific level provides important information on the reproductive biology of reef fishes, thus improving our knowledge on the critical reproductive life stage. However, morphological identification of fish eggs at a species level remains a challenging task. Therefore, researchers typically raise fish eggs into larvae to identify species [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Notably, larval rearing is complicated and often associated with high mortality during the first feeding stages due to insufficient knowledge on the nutritional requirements [18].\u003c/p\u003e \u003cp\u003eRecently, DNA-barcoding of pelagic fish eggs has become an emerging research method to identify species composition, critical habitats and seasonal spawning [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR17\" class=\"CitationRef\"\u003e19\u003c/span\u003e–\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It has been proven to be an effective method to identify species by sequencing Cytochrome-C oxidase subunit 3 (COI-3) of mitochondrial DNA. COI-3 is a commonly used gene for DNA-barcoding due to the relatively high mutation rate of this gene between closely related species. COI-3 codes of marine fish are available in The Barcode of Life Data System (BOLD). These sequences are provided by The Fish Barcode of Life Initiative (FISH-BOL). FISH-BOL is a collaborative international research effort that aims to efficiently study evolution and improve taxonomy by establishing a reference library. DNA-barcoding pelagic fish eggs is an effective method to study reproduction of broadcast spawning reef fish as the eggs do not actively move through the water column as opposed to fish larvae. Using fish larvae as a spawning indicator is less accurate as larvae can be up to weeks or months old when captured [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e26\u003c/span\u003e], meaning they could already have travelled long distances both in space and time. In addition, fish eggs in tropical waters have a relatively short period until hatching [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e27\u003c/span\u003e], Therefore, the origin of sampled eggs must be relatively close in proximity to the spawning time and location.\u003c/p\u003e \u003cp\u003eIn the past, an elaborate study by Munro et al. [28] demonstrated the seasonal spawning for 35 species and provided spawning observations on 83 fish species in the Caribbean Sea. According to a large-scale survey, the number of fish species is estimated to be around 362 in Bonaire National Marine Park [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This illustrates the significant knowledge gap on the spawning patterns of many fish species, with minimal new research released on spawning patterns since then [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e30\u003c/span\u003e–\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In addition, studying the timing of reproduction and comparing it to past studies may elucidate the effects of global warming. During this study, DNA-barcoding was used on pelagic fish eggs to determine the corresponding species that spawned on the reefs of Bonaire. Moreover, the fish eggs were photographed before DNA-barcoding in order to document and provide morphological characteristics. Offering detailed illustrations of fish eggs can enhance their future identification without the need for DNA-barcoding. The establishment of a comprehensive fish egg database could prove cost-effective, and adopting this illustration-based method could serve as a potential protocol for developing such a database.\u003c/p\u003e \u003cp\u003eTherefore, the objectives of this pilot study were to elucidate if (1) fish eggs in this study area can be identified on a species level using DNA barcoding (2) whether there are morphological differences within a species, so that sampled eggs in the future can be identified based on photographs only, 3) whether our method is sufficient in creating a fish egg database and (4) to study the feasibility of making a year-round spawning calendar for reef fish using our method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Method","content":"\u003cp\u003eStudy area\u003c/p\u003e\u003cp\u003eBonaire is an island which is part of the Caribbean Netherlands in the Caribbean Sea located North of the coast of Venezuela. The average seawater temperature ranges from 26–29ᵒC annually [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This study was conducted on the fringing reefs of Bonaire on the leeward side (west coast) of the island. Bonaire’s reefs have been protected since 1979 when the marine park was established, whereby ongoing conservation, research and management efforts are undertaken by the marine national park ‘’Stichting Nationale Parken’’ (STINAPA). In addition, several no-dive sites and protected areas have been created.\u003c/p\u003e\u003cp\u003eSample collection\u003c/p\u003e\u003cp\u003eThe fish eggs were collected passively approximately 50 meters off the coast near the reef slopes at three different locations: Bachelor Beach: 12.12577° N, 68.28827° W; Angel City: 12.10302° N, 68.28824° W; Pink Beach: 12.06414° N, 68.28336° W (WGS84), (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Samples were collected in October 2022 and subsequently in October and November 2023. In order to collect eggs, floating plankton nets of 1,5 meters long with a circular opening with a diameter of 32 cm and a mesh size of 200 µm were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The net opening was weighted down to ensure a continuous opening. The nets were attached to the marine park moorings at approximately 1700 hr. The next day the plankton nets were collected in the morning around 0800 hr. At the rear of these plankton nets, plastic bottles were attached to collect the fish eggs.\u003c/p\u003e\u003cp\u003eA constant flow of fresh seawater was present by having a 200 µm mesh at the bottom of the bottle. In the morning the bottles with eggs were transported to the laboratory in a cooling box filled with seawater taken from the sampling locations to maintain a constant seawater temperature. The bottles were then emptied into a transparent jar in order to study the eggs.\u003c/p\u003e\u003cp\u003eMicroscopic photography\u003c/p\u003e\u003cp\u003eAfter settlement of debris, the eggs were sucked up from the surface layer in the jar using a pipette and analysed with an optic microscope (Euromex BioBlue BB.4260). Next, two photographs using different focal points were taken with a microscope camera (Amscope AF205). One photo is focussed on the oil globule, and the other on the larvae. Smaller eggs were photographed using 40x magnification and larger eggs were photographed using 20x magnification. Photograph settings were set to default modes on the Amscope software, except for ‘white balance’. Finally, after morphological study, each egg was individually placed in a 1,5-milliliter plastic Eppendorf tube with 96% denatured ethanol using a sterile pipette. The eggs were sampled for DNA-analysis in duplicates based on their morphological characteristics (oil droplet, diameter, chorion, perivitelline space, yolk segmentation) for DNA-barcoding [33].\u003c/p\u003e\u003cp\u003eDNA-barcoding\u003c/p\u003e\u003cp\u003e76 fish eggs were fixated in 96% ethanol and sent to Macrogen Europe for DNA-barcoding. The fish eggs were sorted in groups based on morphology. Only 41 samples yielded a successful amplification (≥ 97% similarity score in BOLD) of which thirteen are presented in this paper (full list of sequences available in Appendix I). Only morphological duplicates that have at least two successfully barcoded replicates are presented.\u003c/p\u003e\u003cp\u003eWhole genomic DNA was extracted from single fish eggs by using a DNA-isolation kit (Macrogen). Next, PCR-amplification was executed using the following primers; VF2_t1: 5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3‘ and FishF2_t1: 5‘- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’. With reverse FishR2_t1: 5’-TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’ and FR1d_t1: 5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’ [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and two M13 entailed primers for sequencing [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Sequence products ranged from 700 to 1700 base pairs of the COI-3 gene.\u003c/p\u003e\u003cp\u003eSequence products were then analysed using the Bold systems Identification V4 using animal identification setting on species level barcode records. Only samples with a \u0026gt; 97% similarity score in BOLD were taken into account.\u003c/p\u003e\u003cp\u003eSoftware\u003c/p\u003e\u003cp\u003eImages of the selected fish eggs were analysed using ImageJ software. The diameters of the chorion, oil globules, and the widest part of the perivitelline space were measured to provide morphological distinctions [33].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 3838 fish eggs were collected, of which 76 were subjected to DNA-barcoding. 13 eggs with morphological duplicates are presented in this paper (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In general, fish eggs exhibit a spherical shape and possess a transparent, smooth chorion. However, the eggs of \u003cem\u003eScarus taeniopterus\u003c/em\u003e deviate by being ellipsoid in shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDNA-barcoding\u003c/p\u003e \u003cp\u003eSix different taxa were identified (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); five on a species-level: \u003cem\u003eHeteroconger longissimus\u003c/em\u003e, \u003cem\u003eKatsuwonus pelamis\u003c/em\u003e, \u003cem\u003eParaconger caudilimbatus\u003c/em\u003e, \u003cem\u003eRypticus saponaceus\u003c/em\u003e and \u003cem\u003eScarus taeniopterus\u003c/em\u003e. Samples from the Haemulidae family were too closely related to distinguish on a species level based on COI-3 gene sequencing. In addition, \u003cem\u003eScarus taeniopterus\u003c/em\u003e reversed sequences had no matching result for both samples.\u003c/p\u003e \u003cp\u003eMorpholgy\u003c/p\u003e \u003cp\u003eThe two fish eggs from the \u003cem\u003eHaemulon\u003c/em\u003e genus exhibit morphological similarities. The eggs have a light-yellow oil globule and scattered melanophores on the embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The fish eggs were approximately 0,86 mm with oil globules 0,18\u0026ndash;0,19 mm in size. The perivitelline space has a width of 0,07\u0026ndash;0,12 mm (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis paper presents two duplicates of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esaponaceaus\u003c/em\u003e confirming that morphology can differ within a species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec \u0026ndash; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The fish eggs of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esaponaceus\u003c/em\u003e varied in size from 0,8\u0026ndash;0.9 mm in size with perivitelline spaces of 0,08\u0026ndash;0,12 mm. The oil globules with a round shape were 0,19\u0026ndash;0,21 mm in diameter. The odd shaped oil globules were 0,14\u0026ndash;0,15 mm in width and 0,27\u0026ndash;0,29 mm in length. The fish eggs are characterized by a distinct bright yellow oil globule and a white embryo. The fish eggs were collected in October and November 2022 and 2023. This study presents new spawning information on this species as there is no known information available of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esaponaceaus\u003c/em\u003e spawning during these months.\u003c/p\u003e \u003cp\u003eThe fish eggs of \u003cem\u003eS. taeniopterus\u003c/em\u003e have a characteristic ellipsoid shape with smaller yellow oil globules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). The duplicates are 1,2\u0026ndash;1,3 mm in length with small oil globules of 0,09 and 0,11 mm. The perivitelline space has a width of 0,22\u0026ndash;0,25 mm. The eggs were captured in November 2023 at two different locations (Pink Beach and Angel City).\u003c/p\u003e \u003cp\u003eThe egg of \u003cem\u003eK\u003c/em\u003e. \u003cem\u003epelamis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei) features a white, heavily pigmented embryonic body and a black oil globule located caudally to the embryo. The morphology resembled the eggs of the \u003cem\u003eHaemulon\u003c/em\u003e genus, but our results show that eggs with morphological similarities are not necessarily from the same taxa. The morphology matches the description by Kawakami et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEggs of \u003cem\u003eH\u003c/em\u003e. \u003cem\u003elongissimus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek) and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecaudilimbatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em) are notably larger than the other collected eggs, necessitating multiple oil globules for buoyancy. \u003cem\u003eH\u003c/em\u003e. \u003cem\u003elongissimus\u003c/em\u003e has less oil globules (13) than \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecaudilimbatus\u003c/em\u003e (40\u0026ndash;45). The duplicates of \u003cem\u003eH\u003c/em\u003e. \u003cem\u003elongissimus\u003c/em\u003e have the same amount of oil globules whereas \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecaudilimbatus\u003c/em\u003e have a different amount (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Both species are similar in diameter 2,3\u0026ndash;2,5 mm. The perivitelline spaces are wide. \u003cem\u003eH\u003c/em\u003e. longissimus has a width of 0,54\u0026ndash;0,62 mm. Whereas, \u003cem\u003eP. caudilimbatus\u003c/em\u003e has a width of 0,46\u0026ndash;0,58 mm. The oil globules for both species are small compared to the other eggs, ranging from 0,047\u0026ndash;0,12 mm. The egg shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej has brighter yellow oil globules compared to its counterpart in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek. Both species were captured in October 2023.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFish eggs, corresponding capture date, location and DNA-barcoding results. FW\u0026thinsp;=\u0026thinsp;Results forward primer, RV\u0026thinsp;=\u0026thinsp;Results reversed primer. Morphological characteristics are further presented in the second part of the table\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSpecies FW (% ID)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSpecies RV (% ID)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e29-10-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBachelor Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHaemulon\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eFlavolineatum (99.5) \u0026amp; aurolineatum (99.39)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eFlavolineatum \u0026amp; aurolineatum (100)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e29-10-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBachelor Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHaemulon\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eFlavolineatum (99)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eFlavolineatum (100) \u0026amp; aurolineatum (99.85)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e9-11-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRypticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.85)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.46)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e15-10-2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBachelor Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRypticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.85)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.46)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ee\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e9-11-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRypticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.48)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.46)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e9-11-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRypticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.85)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSaponaceus (99.54)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e14-11-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAngel City\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eScarus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eTaeniopterus (99.67)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo match\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e14-11-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eScarus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eTaeniopterus (98.14)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo match\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ei\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e17-10-2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBachelor Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eKatsuwonus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePelamis (99.83)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePelamis (98.48)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ej\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e29-10-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHeteroconger\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLongissimus (98.73)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLongissimus (98.42)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e29-10-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHeteroconger\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLongissimus (78.99)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLongissimus (98.46)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003el\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e29-10-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eParaconger\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaudilimbatus (100)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCaudilimbatus (100)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e \u003cp\u003e29-10-2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink Beach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eParaconger\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaudilimbatus (99.69)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCaudilimbatus (99.51)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEgg diameter (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOil globule diameter (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePerivetelline space (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eColour of chorion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSurface of chorion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYolk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eOil globule (n =)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eColour of oil globule\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMelanophores\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLight yellow/brown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.857\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLight yellow/brown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYellow/green\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYellow/green\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ee\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.296 L \u0026minus;\u0026thinsp;0.152 W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYellow/green\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.269 L \u0026minus;\u0026thinsp;0.14 W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYellow/green\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.3 L \u0026minus;\u0026thinsp;0,48 W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLigit yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2 L \u0026minus;\u0026thinsp;0.49 W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLight yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ei\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYellow/black\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ej\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.047\u0026ndash;0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.047\u0026ndash;0.121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLight yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003el\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.040\u0026ndash;0.113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLight yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.029\u0026ndash;0.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHomogenous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLight yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTemporal patterns \u0026ndash; first results of spawning calendar\u003c/p\u003e \u003cp\u003eThe eggs belonging to the \u003cem\u003eHaemulon\u003c/em\u003e family were caught in October, 1 day after full moon (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eRypticus saponaceus\u003c/em\u003e eggs were caught in October and November, 6 days after full moon and 4 days before new moon. The two \u003cem\u003eS. taeniopterus\u003c/em\u003e eggs were both caught in November, 1 day after full moon. The \u003cem\u003eK. pelamis\u003c/em\u003e egg was caught in October at last quarter. \u003cem\u003eHeterconger longissimus\u003c/em\u003e and \u003cem\u003eP. caudilimbatus\u003c/em\u003e eggs were all caught in October, 1 day after full moon.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpawning calendar of collected and DNA-barcoded fish eggs. Eggs indicated in green were collected during waning moon phases, blue are eggs that were collected during waxing moon phases. Numbers indicate how many days after or before full moon or new moon the eggs were collected. The indicated number of eggs collected per species are presented per day relative to the moon phase. For example, 2 \u003cem\u003eHaemulon\u003c/em\u003e eggs were collected during the waning moon phase of October, 1 day after full moon. FM\u0026thinsp;=\u0026thinsp;full moon, HM\u0026thinsp;=\u0026thinsp;half moon, NM\u0026thinsp;=\u0026thinsp;New moon\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"19\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003e6\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003e7\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cem\u003eHM\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cem\u003e7\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u003cem\u003e6\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c18\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTaxa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommon name\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eNM\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003e6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003e7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cem\u003eHM\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e\u003cem\u003e7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e\u003cem\u003e6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHaemulon\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrunt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRypticus saponaceus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreater Soapfish\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOct/Nov\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScarus taeniopterus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrincess parrotfish\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNov\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKatsuwonus pelamis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkipjack tuna\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHeteroconger longissimus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrown garden eel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParaconger caudilimbatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMargintail conger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSpawning and Egg Morphology\u003c/p\u003e \u003cp\u003eThis study provides valuable insights into the reproductive spawning of five distinct fish families (Congridae, Haemulidae, Scaridae, Scombridae, and Serranidae) and the morphological characteristics of their eggs within the studied region. Eleven samples were identified at the species level, representing five distinct species: \u003cem\u003eH. longissimus\u003c/em\u003e, \u003cem\u003eK. pelamis\u003c/em\u003e, \u003cem\u003eP. caudilimbatus\u003c/em\u003e, \u003cem\u003eR. saponaceus\u003c/em\u003e, and \u003cem\u003eS. taeniopterus\u003c/em\u003e. Additionally, two samples were identified at the genus level (\u003cem\u003eHaemulon\u003c/em\u003e). The collection of fish eggs followed by DNA barcoding provides a comprehensive approach to studying fish reproduction. Eggs were collected using plankton nets, a cost-effective and efficient method. Prior to DNA barcoding, eggs were photographed to facilitate morphological characterization. Standardized criteria were applied to describe egg morphology, allowing for species differentiation and enabling comparisons with similar studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost eggs exhibited common features, including a spherical shape, a transparent and smooth chorion, except for \u003cem\u003eS. taeniopterus\u003c/em\u003e, which displayed an ellipsoid shape. Distinct morphological features such as oil globule colouration and the presence of melanophores on the chorion or embryonic bodies enabled species differentiation. Egg size and perivitelline space were also valuable indicators, with unusually shaped oil globules observed in \u003cem\u003eR. saponaceus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis study presents the first documentation of spawning for \u003cem\u003eR. saponaceus\u003c/em\u003e, \u003cem\u003eP. caudilimbatus\u003c/em\u003e, and \u003cem\u003eH. longissimus\u003c/em\u003e in the Caribbean Sea during October, as well as the first visual documentation of their eggs. Previously, only \u003cem\u003eK. pelamis\u003c/em\u003e and \u003cem\u003eHaemulon spp\u003c/em\u003e. had documented egg images. When comparing \u003cem\u003eK. pelamis\u003c/em\u003e images from Kawakami et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e36\u003c/span\u003e] with our findings, no morphological differences were observed, suggesting consistency across regions.\u003c/p\u003e \u003cp\u003eEggs of the \u003cem\u003eHaemulon\u003c/em\u003e genus closely resembled images from Barden et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], particularly in melanophore distribution. However, embryonic bodies in our samples appeared more pigmented, potentially due to variations in microscope and camera equipment. Documented spawning times for \u003cem\u003eHaemulon\u003c/em\u003e species include peak reproductive activity for \u003cem\u003eH. sciurus\u003c/em\u003e in Bermuda during May\u0026ndash;June, with lunar periodicity, while \u003cem\u003eH. flavolineatum\u003c/em\u003e exhibits year-round spawning, peaking in March [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. No \u003cem\u003eS. taeniopterus\u003c/em\u003e eggs with advanced embryonic development were captured, suggesting that \u003cem\u003eS. taeniopterus\u003c/em\u003e spawn early in the morning, as previously described by Colin and Clavijo [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Eggs of \u003cem\u003eR. saponaceus\u003c/em\u003e exhibited morphological variation within the species, emphasizing the need for additional sampling. The unusual egg shapes may be attributed to elevated sea temperatures during the pilot study, as thermal stress has been associated with developmental deformities [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The \u003cem\u003eK. pelamis\u003c/em\u003e egg highlights that morphologically similar eggs may originate from different species. Consequently, when establishing a fish egg database, it is essential to document morphological features, as visual identification alone may be insufficient.\u003c/p\u003e \u003cp\u003eChallenges in DNA-barcoding\u003c/p\u003e \u003cp\u003eDNA barcoding success was limited due to transportation and storage constraints. However, strong sequence overlap with the Barcode of Life Database (BOLD) supports the accuracy of species identification for most samples. Only sequences with \u0026ge;\u0026thinsp;97% similarity to BOLD entries were included, except for \u003cem\u003eS. taeniopterus\u003c/em\u003e, which exhibited\u0026thinsp;\u0026ge;\u0026thinsp;97% overlap with the forward primer but lacked alignment with reverse primer database entries, introducing uncertainty regarding sample identity. On-site DNA barcoding is recommended to enhance sequencing success rates. Additionally, COI-3 sequences were insufficient to distinguish \u003cem\u003eHaemulon\u003c/em\u003e species at the species level, suggesting that COI-3 may not be suitable for fine-scale identification within this genus.\u003c/p\u003e \u003cp\u003ePhotography\u003c/p\u003e \u003cp\u003ePhotographing fish eggs from multiple angles presented technical challenges. Eggs were placed on a microscope slide with a droplet of seawater, however, the oil globules always caused them to float upright with the oil globules on top, complicating multi-angle imaging. Removal of seawater using a pipette sometimes repositioned the eggs, enabling additional perspectives. However, standardizing this process remains challenging and requires further methodological refinement.\u003c/p\u003e \u003cp\u003eFish Egg Dispersal and Spawning Ground Identification\u003c/p\u003e \u003cp\u003eThe precise dispersal patterns of fish eggs remain unclear due to the absence of surface current data for hindcasting, making it difficult to pinpoint spawning locations. Despite this limitation, our findings contribute valuable insights by mapping egg distribution patterns and defining a reproductive timeframe for the \u003cem\u003eHaemulon\u003c/em\u003e genus, \u003cem\u003eK. pelamis\u003c/em\u003e, \u003cem\u003eH. longissimus\u003c/em\u003e, \u003cem\u003eS. taeniopterus\u003c/em\u003e, \u003cem\u003eP. caudilimbatus\u003c/em\u003e, and \u003cem\u003eR. saponaceus\u003c/em\u003e. The feasibility of establishing a fish egg database using the methods presented in this study is promising. However, additional research is required to expand species representation and include samples from diverse geographic regions to assess intraspecific morphological variation. A comprehensive fish egg database and improved spawning documentation will support conservation efforts and the development of sustainable fishery policies. More broadly, this approach can be applied to the identification of other marine organisms that begin their life cycle in the planktonic stage, such as corals, echinoderms, and jellyfish.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings confirm that DNA barcoding is an effective tool for species-level identification of fish eggs. However, we anticipate that the ability to sequence samples locally on Bonaire would significantly enhance our current success rate of approximately 54%. Additionally, local sequencing capabilities would allow for an increase in sample size, improving the robustness of our dataset.\u003c/p\u003e \u003cp\u003eRegarding our second research objective, our results indicate that morphological variation in fish eggs can occur within a single species. Further research is required to determine in which species these variations exist and to what extent they manifest.\u003c/p\u003e \u003cp\u003eThe feasibility of establishing a fish egg database (third research objective) appears promising. However, the presence of slight morphological differences among duplicates underscores the need for additional investigation. Moreover, eggs exhibiting morphological similarities do not always belong to the same species. Therefore, while it is feasible to develop a fish egg database based on photographic records, we emphasize the importance of including detailed morphological characteristics. Solely relying on visual examination for species identification is insufficient. Nevertheless, this study demonstrates the effectiveness of egg collection, photography, and the documentation of morphological traits. To ensure consistency and comparability in the database, we recommend that future researchers utilize the same microscope and camera equipment.\u003c/p\u003e \u003cp\u003eFinally, our results indicate that this approach can be successfully employed to develop a spawning calendar for reef fish (research objective 4), much to the like of coral reproduction calendars [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Despite a limited number of sampling days, we documented reproductive activity in six different taxa, three of which had no previously recorded spawning information. Continued year-round sampling and an increased number of DNA-barcoded fish eggs will further refine and expand upon the preliminary spawning calendar presented in this study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eAll fish egg collections have been conducted with consent as per the guidelines of the National Parks Foundations, STINAPA, Bonaire, Caribbean Netherlands. Only appointed mooring lines were used for passive fish egg collection. The Basel declaration was signed online by the authors and was referred to as ethical guidance in our use of collected fish eggs.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to Participate declaration\u003c/h2\u003e \u003cp\u003enot applicable\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financially supported by The World Wide Fund for Nature - Dutch Caribbean (project number 202379) and Stichting Koninklijke Rotterdamse Diergaarde \u0026ndash; Rotterdam Zoo.\u003c/p\u003e \u003cp\u003eCompeting interests\u003c/p\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003cp\u003eData availability Statement\u003c/p\u003e \u003cp\u003eAll data supporting the findings of this study are available within the paper and its supplementary Information. All raw data, regarding forward and reverse primer sequences are presented in this paper and have been included in Appendix I, along with original reference describing the microsatellites used in this study\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.V.L drafted the collection methodology. All authors were involved in collecting fish eggs in the field and field equipment maintenance. T.V.W. drafted the manuscript and analysed the data together with J.H. Both J.H and S.V.L. contributed to the manuscript with valuable feedback and input.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe gratefully acknowledge grants from The World Wide Fund for Nature \u0026ndash; NL (WWF-NL) and Stichting Koninklijke Rotterdamse Diergaarde (Rotterdam Zoo). The Dutch Caribbean Nature Alliance (DCNA) generously supported the project by providing workspace and project guidance. Permission to work in the national marine park of Bonaire was granted by STINAPA. In addition, much appreciation goes out to their dedicated marine park rangers for their assistance and guidance at sea. Lastly, we are very grateful to our passionate students and volunteers.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShantz AA, Ladd MC, Burkepile DE (2020) Overfishing and the ecological impacts of extirpating large parrotfish from Caribbean coral reefs. 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CARMABI, Curacao. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.researchstationcarmabi.org/wp-content/uploads/2024/06/2024-CARMABI-Coral-Spawning-Predictions-FINAL-for-Printing-3-Page-A4-HR-compressed.pdf\u003c/span\u003e\u003cspan address=\"https://www.researchstationcarmabi.org/wp-content/uploads/2024/06/2024-CARMABI-Coral-Spawning-Predictions-FINAL-for-Printing-3-Page-A4-HR-compressed.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 20 February 2025.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bonaire, DNA-barcoding, fish eggs, fish spawning, fish egg morphology","lastPublishedDoi":"10.21203/rs.3.rs-6314167/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6314167/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study utilized DNA-barcoding to identify pelagic reef fish eggs on a species-specific level around Bonaire. Eggs were collected at three different sites (Bachelor Beach: 12.12577\u0026deg; N, 68.28827\u0026deg; W; Angel City: 12.10302\u0026deg; N, 68.28824\u0026deg; W; Pink Beach: 12.06414\u0026deg; N, 68.28336\u0026deg; W; WGS84). Furthermore, egg morphology was described using established criteria, revealing common features such as a spherical shape, transparent and smooth chorion, notable differences such as oil globule colour, and the presence of melanophores. This study provides valuable insights into the reproduction and the morphology of eggs of five distinct fish species and a single genus. In duplicates, we identified eggs belonging to 5 different taxa; \u003cem\u003eHaemulon\u003c/em\u003e genus, \u003cem\u003eRypticus saponaceus\u003c/em\u003e, \u003cem\u003eParaconger caudilimbatus\u003c/em\u003e, \u003cem\u003eHeteroconger longissimus\u003c/em\u003e, and \u003cem\u003escarus taeniopterus.\u003c/em\u003e A single identification was made for \u003cem\u003eKatsuwonus pelamis.\u003c/em\u003e All eggs were collected during October and November in 2022 and 2023. The long-term goal of this study is to establish a fish egg database for the Caribbean including photographic and genetic information, and a fish spawning calendar. Further research is needed and ongoing to uncover other species and to study whether morphology within species and regions vary.\u003c/p\u003e","manuscriptTitle":"Revealing Reef Fish Eggs at the Species Level: Insights from DNA Barcoding Aligned with Egg Morphology and Spawning Times","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 06:31:10","doi":"10.21203/rs.3.rs-6314167/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f465721-0157-4884-8e72-5c2e9a8640c1","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T18:38:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 06:31:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6314167","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6314167","identity":"rs-6314167","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}