Quantitative species-level ecology of reef fish larvae via metabarcoding

  • Nature Ecology & Evolution 2306316 (2018)
  • doi:10.1038/s41559-017-0413-2
  • Download Citation
Published online:


The larval pool of coral reef fish has a crucial role in the dynamics of adult fish populations. However, large-scale species-level monitoring of species-rich larval pools has been technically impractical. Here, we use high-throughput metabarcoding to study larval ecology in the Gulf of Aqaba, a region that is inhabited by >500 reef fish species. We analysed 9,933 larvae from 383 samples that were stratified over sites, depth and time. Metagenomic DNA extracted from pooled larvae was matched to a mitochondrial cytochrome c oxidase subunit I barcode database compiled for 77% of known fish species within this region. This yielded species-level reconstruction of the larval community, allowing robust estimation of larval spatio-temporal distributions. We found significant correlations between species abundance in the larval pool and in local adult assemblages, suggesting a major role for larval supply in determining local adult densities. We documented larval flux of species whose adults were never documented in the region, suggesting environmental filtering as the reason for the absence of these species. Larvae of several deep-sea fishes were found in shallow waters, supporting their dispersal over shallow bathymetries, potentially allowing Lessepsian migration into the Mediterranean Sea. Our method is applicable to any larval community and could assist coral reef conservation and fishery management efforts.

  • Subscribe to Nature Ecology & Evolution for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


  1. 1.

    Bellwood, D. R., Hoey, A. S. & Hughes, T. P. Human activity selectively impacts the ecosystem roles of parrotfishes on coral reefs. Proc. R. Soc. B 279, 1621–1629 (2012).

  2. 2.

    McClanahan, T. R. et al. Critical thresholds and tangible targets for ecosystem-based management of coral reef fisheries. Proc. Natl Acad. Sci. USA 108, 17230–17233 (2011).

  3. 3.

    Hoegh-Guldberg, O. et al. Coral reefs under rapid climate change and ocean acidification. Science 318, 1737–1742 (2007).

  4. 4.

    Bellwood, D. R., Hughes, T. P., Folke, C. & Nyström, M. Confronting the coral reef crisis. Nature 429, 827–833 (2004).

  5. 5.

    Gardner, T. A., Cote, I. M., Gill, J. A., Grant, A. & Watkinson, A. R. Long-term region-wide declines in Caribbean corals. Science 301, 958–960 (2003).

  6. 6.

    Garpe, K. C., Yahya, S. A. S., Lindahl, U. & Öhman, M. C. Long-term effects of the 1998 coral bleaching event on reef fish assemblages. Mar. Ecol. Prog. Ser. 315, 237–247 (2006).

  7. 7.

    Campbell, L. M., Gray, N. J., Hazen, E. L. & Shackeroff, J. M. Beyond baselines: rethinking priorities for ocean -conservation. Ecol. Soc. 14, 14 (2009).

  8. 8.

    Cowen, R. K. & Sponaugle, S. Larval dispersal and marine population connectivity. Annu. Rev. Mar. Sci. 1, 443–466 (2009).

  9. 9.

    Cowen, R. K. in Coral Reef Fishes: Dynamics and Diversity in a Complex Ecosystem (ed. Sale, P. F.) 149–170 (Academic, London, 2002).

  10. 10.

    Doherty, P. J., Fowlert, T. & Fowler, T. An empirical test of recruitment limitation in a coral reef fish. Science 263, 935–939 (1994).

  11. 11.

    Armsworth, P. R. Recruitment limitation, population regulation, and larval connectivity in reef fish metapopulations. Ecology 83, 1092–1104 (2002).

  12. 12.

    Werner, F. E., Cowen, R. C. & Paris, C. B. Coupled biological and physical models: present capabilities and necessary developments for future studies of population connectivity. Oceanography 20, 54–69 (2007).

  13. 13.

    Llopiz, J. K. & Cowen, R. K. Variability in the trophic role of coral reef fish larvae in the oceanic plankton. Mar. Ecol. Prog. Ser. 381, 259–272 (2009).

  14. 14.

    Leis, J. M. Taxonomy and systematics of larval Indo-Pacific fishes: a review of progress since 1981. Ichthyol. Res. 62, 9–28 (2014).

  15. 15.

    Ko, H. L. et al. Evaluating the accuracy of morphological identification of larval fishes by applying DNA barcoding. PLoS ONE 8, e53451 (2013).

  16. 16.

    Limouzyparis, C., Mcgowan, M. F., Richards, W. J., Umaran, J. P. & Cha, S. S. Diversity of fish larvae in the Florida-Keys—results from SEFCAR. Bull. Mar. Sci. 54, 857–870 (1994).

  17. 17.

    Irisson, J., Paris, C., Gulgand, C. & Planes, S. Vertical distribution and ontogenetic ‘migration’ in coral reef fish larvae. Limnol. Oceanogr. 55, 909–919 (2009).

  18. 18.

    Evans, N. T. et al. Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding. Mol. Ecol. Resour. 16, 29–41 (2016).

  19. 19.

    Hubert, N., Delrieu-trottin, E., Irisson, J., Meyer, C. & Planes, S. Identifying coral reef fish larvae through DNA barcoding : a test case with the families Acanthuridae and Holocentridae. Mol. Phylogenet. Evol. 55, 1195–1203 (2010).

  20. 20.

    Hebert, P. D. N., Ratnasingham, S. & Waard, J. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270, S96–S99 (2003).

  21. 21.

    Ratnasingham, S. & Hebert, P. D. N. BOLD : the Barcode of Life Data System ( Mol. Ecol. Notes 7, 355–364 (2007).

  22. 22.

    Hubert, N., Espiau, B., Meyer, C. & Planes, S. Identifying the ichthyoplankton of a coral reef using DNA barcodes. Mol. Ecol. Resour. 15, 57–67 (2015).

  23. 23.

    Leray, M. & Knowlton, N. DNA barcoding and metabarcoding of standardized samples reveal patterns of marine benthic diversity. Proc. Natl Acad. Sci. USA 112, 2076–2081 (2015).

  24. 24.

    Qiu, X. et al. Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Appl. Environ. Microbiol. 67, 880–887 (2001).

  25. 25.

    Galan, M., Pagés, M. & Cosson, J. F. Next-generation sequencing for rodent barcoding: species identification from fresh, degraded and environmental samples. PLoS ONE 7, e48374 (2012).

  26. 26.

    Bucklin, A., Steinke, D. & Blanco-Bercial, L. DNA barcoding of marine metazoa. Ann. Rev. Mar. Sci. 3, 471–508 (2011).

  27. 27.

    Zhou, X. et al Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification. Gigascience 2, 4 (2013).

  28. 28.

    Deagle, B. E. et al. DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match. Biol. Lett. 10, 1789–1793 (2014).

  29. 29.

    Kiflawi, M., Belmaker, J., Brokovich, E., Einbinder, S. & Holzman, R. The determinants of species richness of a relatively young coral-reef ichthyofauna. J. Biogeogr. 33, 1289–1294 (2006).

  30. 30.

    Golani, D. & Bogorodsky, S. V. The fishes of the Red Sea—reappraisal and updated checklist. Zootaxa 2463, 1–135 (2010).

  31. 31.

    Genin, A., Lazar, B. & Brenner, S. Vertical mixing and coral death in the Red Sea following the eruption of Mount Pinatubo. Nature 377, 507–510 (1995).

  32. 32.

    Fine, M., Gildor, H. & Genin, A. A coral reef refuge in the Red Sea. Glob. Change Biol. 19, 3640–3647 (2013).

  33. 33.

    Hughes, T. P., Bellwood, D. R. & Connolly, S. R. Biodiversity hotspots, centers of endemicity, and the conservation of coral reefs. Ecol. Lett. 5, 775–784 (2002).

  34. 34.

    Brokovich, E., Einbinder, S., Shashar, N., Kiflawi, M. & Kark, S. Descending to the twilight-zone: changes in coral reef fish assemblages along a depth gradient down to 65 m. Mar. Ecol. Prog. Ser. 371, 253–262 (2008).

  35. 35.

    Taberlet, P., Coissac, E., Pompanon, F., Brochmann, C. & Willerslev, E. Towards next-generation biodiversity assessment using DNA metabarcoding. Mol. Ecol. 21, 2045–2050 (2012).

  36. 36.

    Leis, J. M. & Mccormick, M. I. in Coral Reef Fishes: Dynamics and Diversity in a Complex Ecosystem (ed. Sale, P. F.) 171–200 (Academic, London, 2002).

  37. 37.

    Paris, C. B. & Cowen, R. K. Direct evidence of a biophysical retention mechanism for coral reef fish larvae. Limnol. Oceanogr. 49, 1964–1979 (2004).

  38. 38.

    Ottosson, U., Sandberg, R. & Pettersson, J. Orientation cage and release experiments with migratory Wheatears (Oenanthe oenanthe) in Scandinavia and Greenland: the importance of visual cues. Ethology 86, 57–70 (1990).

  39. 39.

    Pineda, J., Porri, F., Starczak, V. & Blythe, J. Causes of decoupling between larval supply and settlement and consequences for understanding recruitment and population connectivity. J. Exp. Mar. Biol. Ecol. 392, 9–21 (2010).

  40. 40.

    Dibattista, J. D. et al. A review of contemporary patterns of endemism for shallow water reef fauna in the Red Sea. J. Biogeogr. 43, 423–439 (2016).

  41. 41.

    Matasuura, K. & Tyler, J. C. Resultats DES Campagnes Musorstom Vol. 17 (ed. Séret, B.) 173–208 (Museum National d’Histoire Naturelle, Paris, 1997).

  42. 42.

    Turan, C. & Yaglioglu, D. First record of the spiny blaasop Tylerius spinosissimus (Regan, 1908) (Tetraodontidae) from the Turkish coasts. Mediterr. Mar. Sci. 12, 247–252 (2011).

  43. 43.

    Clayton, D. Replication of animal mitochondrial DNA. Cell 28, 693–705 (1982).

  44. 44.

    Chu, H. T. et al. Quantitative assessment of mitochondrial DNA copies from whole genome sequencing. BMC Genomics 13, S5 (2012).

  45. 45.

    Munwes, I., Geffen, E., Friedmann, A., Tikochinski, Y. & Gafny, S. Variation in repeat length and heteroplasmy of the mitochondrial DNA control region along a core-edge gradient in the eastern spadefoot toad (Pelobates syriacus). Mol. Ecol. 20, 2878–2887 (2011).

  46. 46.

    Blaser, M., Bork, P., Fraser, C., Knight, R. & Wang, J. The microbiome explored: recent insights and future challenges. Nat. Rev. Microbiol. 11, 213–217 (2013).

  47. 47.

    Cowles, T. in Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation (eds Seuront, L. & Strutton, P. G.) 31–49 (CRC Press, Boca Raton, 2003).

  48. 48.

    Wiebe, P. H. et al. New development in the MOCNESS, an apparatus for sampling zooplankton and micronekton. Mar. Biol. 87, 313–323 (1985).

  49. 49.

    Leis, J. & Carson-Ewart, B. M. (eds) The Larvae of Indo-Pacific Coastal Fishes: An Identification Guide to Marine Fish Larvae (Brill, Leiden, 2000).

  50. 50.

    Richards, W. J. Early Stages of Atlantic Fishes: An Identification Guide for the Western Central North Atlantic (CRC Press, Boca Raton, 2005).

  51. 51.

    Ivanova, N. V., Zemlak, T. S., Hanner, R. H. & Hebert, P. D. N. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes 7, 544–548 (2007).

  52. 52.

    Klöppel, A., Brümmer, F., Schwabe, D. & Morlock, G. Detection of bioactive compounds in the mucus nets of Dendropoma maxima, Sowerby 1825 (Prosobranch Gastropod Vermetidae, Mollusca). J. Mar. Biol. 2013, 283506 (2013).

  53. 53.

    Khalaf, M. Fish fauna of the Jordanian coast, Gulf of Aqaba, Red Sea. J. King Abdulaziz Univ. Mar. Sci. 15, 23–50 (2004).

  54. 54.

    Hensley, D. A. Two new flatfish records from the Red Sea, an Indopacific samarid (Samariscus inornatus) and the European plaice (Pleuronectes platessa). Isr. J. Zool. 39, 371–379 (1993).

  55. 55.

    Russell, B. C. & Golani, D. A review of the fish genus Parascolopsis (Nemipteridae) of the western Indian Ocean, with description of a new species from the northern Red Sea. Isr. J. Zool. 39, 337–347 (1993).

  56. 56.

    Ben-Tuvia, A. A review of the Indo-West Pacific congrid fishes of genera Rhynchoconger and Bathycongrus with the description of three new species. Isr. J. Zool. 39, 349–370 (1993).

  57. 57.

    Randall, J. E. & Golani, D. Review of the moray eels (Anguilliformes: Muraenidae) of the Red Sea. Bull. Mar. Sci. 56, 849–880 (1995).

  58. 58.

    Kimura, S., Golani, D., Iwatsuki, Y., Tabuchi, M. & Yoshino, T. Redescriptions of the Indo-Pacific atherinid fishes Atherinomorus forskalii, Atherinomorus lacunosus, and Atherinomorus pinguis. Ichthyol. Res. 54, 145–159 (2007).

  59. 59.

    Golani, D. Upeneus Davidaromi, a new deep water goatfish (Osteichthyes, Mullidae) from the Red Sea. Isr. J. Zool. 47, 111–121 (2001).

  60. 60.

    Golani, G. & Lerner, A. A long-term study of the sandy shore icthyofauna in the northern Red Sea (Gulf of Aqaba) with reference to adjacent mariculture activity. Raffles Bull. Zool. 14, 255–264 (2007).

  61. 61.

    Baranes, A. & Golani, D. An annotated list of deep-sea fishes collected in the northern Red-Sea, Gulf-of-Aqaba. Isr. J. Zool. 39, 299–336 (1993).

  62. 62.

    Herler, J., Bogorodsky, S. V. & Suzuki, T. Four new species of coral gobies (Teleostei: Gobiidae: Gobiodon), with comments on their relationships within the genus. Zootaxa 3709, 301–329 (2013).

  63. 63.

    Khalaf, M. A. & Kochzius, M. Community structure and biogeography of shore fishes in the Gulf of Aqaba, Red Sea. Helgol. Mar. Res. 55, 252–284 (2002).

  64. 64.

    Randall, J. E. & van Egmond, J. in Results of the ‘Oceanic Reefs' Expedition to the Seychelles (1992–1993) Vol. 1 (ed. van der Land, J.) 1–70 (Nationaal Natuurhistorisch Museum, Leiden, 1994).

  65. 65.

    Freinschlag, M. & Patzner, R. A. Shrimp-gobies in the southern Gulf of Aqaba (Red Sea). Zool. Middle East 55, 41–46 (2012).

  66. 66.

    Fricke, R., Golani, D., Appelbaum-Golani, B. & Zajonz, U. New record of the spiny pufferfish, Tylerius spinossissimis (Regan, 1908), from Israel, Gulf of Aqaba, Red Sea (Actinopterygii : Tetraodontiformes: Tetraodontidae). Acta Ichthyol. Piscat. 46, 115–118 (2016).

  67. 67.

    Pietsch, T. W. & Grobecker, D. B. Frogfishes of the World: Systematics, Zoogeography, and Behavioral Ecology (Stanford Univ. Press, Stanford, 1987).

  68. 68.

    Brokovich, E. The Community Structure and Biodiversity of Reef Fishes at the Northern Gulf of Aqaba (Red Sea) with Relation to their Habitat. MSc thesis, Tel Aviv Univ. (2001).

  69. 69.

    Blecher-Gonen, R. et al. High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein–DNA interactions and epigenomic states. Nat. Protoc. 8, 539–554 (2013).

  70. 70.

    Dondoshansky, I. & Wolf, Y. Blastclust (NCBI Software Development Toolkit) (NCBI, Bethesda, 2002).

  71. 71.

    Zerbino, D. R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008). 

  72. 72.

    Standley, K. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

  73. 73.

    Price, M. N., Dehal, P. S. & Arkin, A. P. Fasttree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).

  74. 74.

    Matsen, F. A., Kodner, R. B. & Armbrust, E. V. pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC Bioinformatics 11, 538 (2010).

  75. 75.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  76. 76.

    R Development Core Team R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, 2011).

  77. 77.

    Hufbauer, R. A., Rutschmann, A., Serrate, B., Vermeil de Conchard, H. & Facon, B. Role of propagule pressure in colonization success: disentangling the relative importance of demographic, genetic and habitat effects. J. Evol. Biol. 26, 1691–1699 (2013).

  78. 78.

    Iwasaki, W. et al. MitoFish and MitoAnnotator: a mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol. Biol. Evol. 30, 2531–2540 (2013).

  79. 79.

    Palumbi, S. in Molecular Systematics 2nd edn (eds Hillis, D.M., Moritz, C. & Mable, B.K.) 205–247 (Sinauer Associates, Sunderland, 1996).

  80. 80.

    Baldwin, C. C., Mounts, J. H., Smith, D. G. & Weigt, L. A. Genetic identification and color descriptions of early life-history stages of Belizean Phaeoptyx and Astrapogon (Teleostei: Apogonidae) with comments on identification of adult Phaeoptyx. Zootaxa 2008, 1–22 (2009).

  81. 81.

    Hebert, P. D. N., Penton, E. H., Burns, J. M., Janzen, D. H. & Hallwachs, W. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc. Natl Acad. Sci. USA 101, 14812–14817 (2004).

  82. 82.

    Ivanova, N. V., Dewaard, J. R. & Hebert, P. D. N. An inexpensive, automation-friendly protocol for recovering high-quality DNA. Mol. Ecol. Notes 6, 998–1002 (2006).

  83. 83.

    Ward, R. D., Zemlak, T. S., Innes, B. H., Last, P. R. & Hebert, P. D. N. DNA barcoding Australia’s fish species. Phil. Trans. R. Soc. B 360, 1847–1857 (2005).

  84. 84.

    Sade, A. R. et al. The Israel National Bathymetric Survey: northern Gulf of Aqaba/Eilat poster. Isr. J. Earth Sci. 57, 139–144 (2008).

Download references


We thank M. McGrouther from the Australian Museum, P. L. Munday from James Cook University, J. Herler from the University of Vienna and P. Borsa from Universitas Udayana for providing tissue samples for this study, the staff of the Inter-University Institute for Marine Sciences in Eilat, Israel, and the Marine Science Station of The University of Jordan and Yarmouk University for their help in conducting the research. This study was supported by the United States–Israel Binational Science Foundation (BSF grant 2008/144 to M.K. and C.B.P.), the Israeli Ministry of the Environment (grant 111-51-6 to M.K. and R.H.), the Angel Faivovich Foundation (to R.S.) and by the Nancy & Stephen Grand Israel National Center for Personalized Medicine. Field sampling was supported in part by the World Bank, as part of the Red Sea–Dead Sea Water Conveyance Study Program.

Author information

Author notes

    • Rachel Armoza-Zvuloni

    Present address: Dead Sea and Arava Science Center, Yotvata, Israel

  1. Naama Kimmerling, Omer Zuqert and Gil Amitai contributed equally to this work.


  1. Marine Biology Program, Eilat Campus, Department of Life Sciences, Ben-Gurion University, Eilat, Israel

    • Naama Kimmerling
    •  & Igal Berenshtein
  2. Interuniversity Institute for Marine Sciences, Eilat, Israel

    • Naama Kimmerling
    • , Tamara Gurevich
    • , Rachel Armoza-Zvuloni
    • , Irina Kolesnikov
    • , Igal Berenshtein
    • , Asaph Rivlin
    • , Moti Ohavia
    • , Roi Holzman
    •  & Moshe Kiflawi
  3. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    • Omer Zuqert
    • , Gil Amitai
    • , Sarah Melamed
    •  & Rotem Sorek
  4. The Nancy and Stephen Grand Israel National Center for Personalized Medicine (INCPM), Weizmann Institute of Science, Rehovot, Israel

    • Shlomit Gilad
    •  & Sima Benjamin
  5. Department of Ocean Sciences, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL, USA

    • Claire B. Paris
  6. Department of Zoology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    • Roi Holzman
  7. Department of Life Sciences, Ben-Gurion University, Eilat, Israel

    • Moshe Kiflawi


  1. Search for Naama Kimmerling in:

  2. Search for Omer Zuqert in:

  3. Search for Gil Amitai in:

  4. Search for Tamara Gurevich in:

  5. Search for Rachel Armoza-Zvuloni in:

  6. Search for Irina Kolesnikov in:

  7. Search for Igal Berenshtein in:

  8. Search for Sarah Melamed in:

  9. Search for Shlomit Gilad in:

  10. Search for Sima Benjamin in:

  11. Search for Asaph Rivlin in:

  12. Search for Moti Ohavia in:

  13. Search for Claire B. Paris in:

  14. Search for Roi Holzman in:

  15. Search for Moshe Kiflawi in:

  16. Search for Rotem Sorek in:


M.K., C.B.P., R.S. and R.H. designed the study. N.K., I.K., I.B., A.R. and M.O. performed the field sampling of larvae. N.K., O.Z., G.A., T.G., R.A.-Z., I.K., S.M., C.B.P., M.K. and R.H. processed the field samples and collected data for the COI database. O.Z., G.A., S.G., S.B. and R.S. performed and analysed the high-throughput sequencing. O.Z., N.K., M.K., R.H. and R.S. analysed the data. M.K., R.H., R.S., O.Z. and N.K. wrote the paper. All authors contributed to writing the manuscript through comments and discussions.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Roi Holzman or Moshe Kiflawi or Rotem Sorek.

Supplementary information

  1. Supplementary Information

    Supplementary Table 1, Supplementary Figure 1

  2. Life Sciences Reporting Summary

  3. Supplementary Table 2

    Metagenomic and ecological features of larvae samples

  4. Supplementary Table 3

    Gulf of Aqaba/Red Sea fish and their available COIs

  5. Supplementary Table 4

    Occurrence and abundance of classified larvae

  6. Supplementary Data 1

    Fasta file of all reference COI barcodes in our set

  7. Supplementary Data 2

    Fasta file of all COI-mapped reads in our set