The diversity of coral reefs: What is at risk?

Transcription

1 1 The diversity of coral reefs: What is at risk? Laetitia Plaisance 1,2, M. Julian Caley 3, Russell E. Brainard 4 & Nancy Knowlton 1,2 1. Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, MRC 163, PO Box 37012, Washington, DC , USA 2. Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA , USA 3. Australian Institute of Marine Science, PMB No. 3, Townsville, QLD 4810, Australia. 4. NOAA Fisheries, Pacific Islands Fisheries Science Center, Coral Reef Ecosystem Division, 1125-B Ala Moana Blvd., Honolulu, HI USA

2 2 Tropical reefs shelter one quarter to one third of all marine species 1 but one third of the coral species that construct reefs are now at risk of extinction 2. Because traditional methods for assessing reef diversity are extremely time consuming, taxonomic expertise for many groups is lacking, and marine organisms are thought to be less vulnerable to extinction 3, most discussions of reef conservation focus on maintenance of ecosystem services rather than biodiversity loss 4. In this study involving the three major oceans with reef growth, we provide new biodiversity estimates based on quantitative sampling and DNA barcoding for species delimitation. We focus on crustaceans because they constitute almost half of the diversity sampled in this study and are the second most diverse group of marine metazoans 5. We show exceptionally high numbers of crustacean species associated with coral reefs (525 species living in an area equivalent to 6.3 m 2 ), a high prevalence of rare species (38% of the species were encountered only once), and highly restricted ranges (81% were found in only one locality). These characteristics render reef species vulnerable to extinction due to local habitat loss.

3 3 Estimates of reef species diversity range from ~600,000 to more than 9 million species worldwide 1,5,6. This diversity is concentrated in the Indo-Pacific Coral Triangle 7 and decreases with increasing distance from the Indo-Australian archipelago. Traditionally, large and well-studied reef-associated macrofauna, such as corals and fishes, have been used as surrogates in strategic biodiversity assessments 8 because they are comparatively easy to census and taxonomically relatively well known. However, these two groups represent just a tiny fraction of reef-associated diversity, and it is questionable whether the use of a few groups as surrogates for biodiversity assessment captures general patterns of diversity across all organisms 9. Here, we overcome these limitations using standardized sampling at seven localities one in the eastern Indian Ocean (Ningaloo, Western Australia), two in the western Pacific (Heron and Lizard Islands, Great Barrier Reef, Australia), three in the central Pacific (French Frigate Shoals, Northwestern Hawaiian Islands; the Northern Line Islands; and Moorea) and one in the Caribbean (Bocas del Toro, Panama) (Fig. 1). We used cytochrome oxidase I (COI), a DNA barcoding gene, to cluster individuals into operational taxonomic units (OTUs) because it has previously been shown to be effective in the taxa we encountered 10. To sample reef diversity, we removed invertebrates from either standardized volumes of dead coral heads (all localities except French Frigate Shoals, where head removal was prohibited) or settlement structures of approximately the same volume [French Frigate Shoals and Heron Island (the latter to compare with dead coral heads)]. In total, we obtained DNA barcodes for 4182 crustaceans (GenBank accession numbers: ****). Using the criterion of 5% similarity in COI sequences, we identified 509 unique OTUs in the Indo-Pacific and 16 in the Caribbean (Table 1). This threshold generally corresponds with boundaries between morphologically defined species in crustaceans 11 and in molecular studies is located on a plateau where the numbers of OTUs are

4 4 relatively insensitive to the precise cut-off value chosen, both in a previous study 10 and at each of the localities in this study (data not shown). Settlement devices gave slightly lower numbers of species but overall similar geographic diversity patterns and similar assemblages of species when compared to dead coral heads from the same location (Table 1 and Methods). The observed numbers of species at the three Indo-west Pacific (IWP) localities were roughly two times higher than the numbers observed at three localities in the central Pacific (CP), which were in turn roughly four times higher than the number observed at the Caribbean locality. These patterns persist using two commonly used nonparametric biodiversity estimators 12 - ACE (Abundance Based Coverage Estimator) and Chao1 - that adjust the observed number of species upward by a factor whose size depends on the number of rare species in the sample. The estimators were calculated both using data from all sampling units or from a subset of six sampling units (the minimum for any locality) (Table 1, Figs. 2A and B). For the latter (which minimizes the effect of different numbers of samples), the rank order of diversity across localities was Lizard Island > Heron Island > Ningaloo > Northern Line Islands > Moorea > Hawaii > Panama (Fig. 2B), and the ranked estimates of regional richness, calculated by pooling all the samples from localities in each region and using ACE and Chao1, was (as expected) IWP > CP > Caribbean (Table 1). Rarefaction curves (Supplemental Figure 1) did not reach an asymptote at any locality, indicating that a large number of species remain to be sampled (even in Ningaloo and Heron Island where the sampling efforts were the highest, with more than 20 sample units collected). This study supports recent findings that many reef species have restricted ranges 13, in contrast to earlier assumptions. Previous analyses of endemism in fishes, corals, snails, lobsters and mantis shrimps suggested figures ranging from 7.2% (corals) to 53.6% (lobsters) 14,15. Recent molecular studies have shown that what were once

5 5 thought to be widespread species, are in fact complexes of cryptic species with much more localized distributions 16. In our study, 81% of the crustacean species were only found in one locality, 16% were found in two localities, 2.3% were found in three localities and 0.8% were found in four localities. An analysis based on the Bray-Curtis similarity index (Table 2), which measures similarity in community composition (0 means that the two communities are entirely different and 1 means they are identical) shows that only the two Great Barrier Reef localities (Lizard and Heron Islands) and two Central Pacific localities (the Northern Line Islands and Moorea) have similarity values > 0.1, and none of the values were higher than Overall, these figures are somewhat lower than similarity indices calculated for coral species from comparable depths in Indonesia, Papua New Guinea, Solomon Islands, Samoa and French Polynesia 17, where all pairwise values for sites from different regions ranged from just below.1 to.26). The relatively low community similarity between localities in part reflects the high prevalence of rare species at any locality. Nearly 40% of the species were singletons [i.e. the sequences by which they were characterized (that is, using the 5% threshold, see above) occurred just once], and an additional 14% were represented by only two specimens. Abundant species (represented by more than ten individuals) accounted for only 16% of the specimens. This pattern is characteristic of biodiversity surveys in tropical, extremely diverse environments 10,18,19 and points to the need for globally distributed standardized efforts to assess endemism and species numbers. Interestingly, in Hawaii s French Frigate Shoals, where we also sampled in non-forereef habitats (data not included in the estimates presented above), similarity indices across habitats were slightly higher (.30 and.28 between forereef and backreef and forereef and lagoon).

6 6 The entire planar area sampled for this study was only ~6.3 m 2. Yet on this very limited surface, we found a total of 525 crustacean species of which 412 were decapods and 168 brachyuran crab species. By way of comparison, the latter figure is almost 80% of the number of described brachyuran species from all European seas and 3.2% of the world s total 5. The finding of so many species in such a small area suggests that tropical crustacean diversity (and likely the diversity of reefs overall) has been seriously underestimated. For example, comparing our Caribbean diversity results to Small et al. s analyses 6 (based on the diversity found in a coral reef mesocosm derived from 5 m 2 of collections from the Bahamas) suggests that they should have found over four times the number of decapod and brachyuran species than they observed. Moreover, using our Caribbean samples to predict what we should have found in our Pacific samples based on area alone yields a number for crustaceans that is 12 times smaller than that observed (Pacific reef area is also more than ten times Caribbean reef area). Together this suggests that their estimates of million reef species, obtained by multiplying their estimated Caribbean diversity by 12 to obtain global reef diversity, could significantly underestimate the diversity of reefs worldwide, despite the fact that the most recent estimate of reef diversity was less than 600,000 species 5 and that of total marine diversity was less than 2 million species 20. Estimates of diversity based on extrapolations from small samples have many potential sources of error 21,22 and more extensive, quantitative sampling is clearly needed. Yet whatever the actual number of species living on reefs, past 23 and projected 24 losses of reefs represent an enormous threat to diversity of the world s oceans.

7 7 Methods Summary All samples came from depths of 8-12 m. Crustaceans were collected by breaking dead coral heads into pieces or disassembling the settlement devices (ARMS). Preservation and DNA extraction, amplification and sequencing of the COI barcoding gene used standard protocols. Sequences were clustered into Operational Taxonomic Units (OTUs) using MOTHUR 25. We used ACE (Abundance-based Coverage Estimator) and Chao1 12 to estimate total diversity, using either all samples for each locality (which varied in number from 6 to 23) or a subset of 6 samples randomized a thousand times (to eliminate sample size biases). The Bray-Curtis similarity index was used to estimate the similarity in community composition within and between localities. In order to compare the effectiveness of dead coral heads and ARMS, both were collected at Heron Island. Analyses suggest that ARMS give somewhat lower absolute numbers of crustaceans than dead coral heads (Table 1). However, the patterns of diversity observed were as would be expected from longitudinal diversity gradients (Heron Island ARMS > French Frigate Shoals ARMS, Table 1). Moreover, the average Bray Curtis similarity index between ARMS and dead coral heads at Heron Island (.41) is comparable to that observed between randomized subsets of dead coral heads at Heron Island (.53); both of these values are within the range reported by Dornelas et al. 17 for within site similarity for corals (0.38 to 0.67) and much higher than any between locality similarity indices in our study (Table 2).

8 8 References: 1. Reaka-Kudla, M. The global biodiversity of coral reefs: a comparison with rain forests in Biodiversity II: understanding and protecting our biological resources (eds Reaka-Kudla, M., Wilson, D.E. & Wilson, E.O.) (Joseph Henry Press, Washington, D.C., 1997). 2. Carpenter, K.E. et al. One-third of reef building corals face elevated extinction risk from climate change and local impacts. Science 321, (2008). 3. McKinney, M.L. Is marine diversity at less risk? Evidence and implications. Diversity and Distributions 4, 3-8 (1998). 4. Moberg, F. & Folke, C. Ecological goods and services of coral reef ecosystems. Ecolog. Econ. 29, (1999). 5. Bouchet, P. The magnitude of marine biodiversity in The exploration of marine biodiversity: scientific and technological challenges (ed Duarte, C.M.) (Fundación BBVA, Bilbao, Spain, 2006). 6. Small, A., Adey, A. & Spoon, D. Are current estimates of coral reef biodiversity too low? The view through the window of a microcosm. Atoll. Res. Bull. 458, 1-20 (1998). 7. Hughes, T. P., Bellwood, D. R. & Connolly, S. R. Biodiversity hotspots, centres of endemicity, and the conservation of coral reefs. Ecol. Letters 5, (2002). 8. Bellwood, D. R. & Hughes, T. P. Regional-scale assembly rules and biodiversity of coral reefs. Science 292, (2001).

9 9 9. Beger, M., McKenna, S. A. & Possingham, H. P. Effectiveness of surrogate taxa in the design of coral reef reserve systems in the Indo-Pacific. Conserv. Biol. 21, (2007). 10. Plaisance, L., Knowlton, N., Paulay, G. & Meyer, C. Reef-associated crustacean fauna: Biodiversity estimates using semi-quantitative sampling and DNA barcoding. Coral Reefs 28, (2009). 11. Costa, F. O. et al. Biological identifications through DNA barcodes: the case of the Crustacea. Can. J. Fish. Aquat. Sci. 64, (2007). 12. Hortal, J., Borges, P. A. V. & Gaspar, C. Evaluating the performance of species richness estimators: sensitivity to sample grain size. J. Anim. Ecol. 75, (2006). 13. Roberts, C. M. & Hawkins, J. P. Extinction risk in the sea. Trends Ecol. Evol. 14, (1999). 14. Reaka, M. R., Rodgers, P. J. & Kudla, A. U. Patterns of biodiversity and endemism on Indo-West Pacific coral reefs. Proc. Natl. Acad. Sci. U.S.A. 105, (2008). 15. Roberts, C. M. et al. Marine biodiversity hotspots and conservation priorities for tropical reefs. Science 295, (2002). 16. Meyer, C. P., Geller, J. B. & Paulay, G. Fine scale endemism on coral reefs: archipelagic differentiation in turbinid gastropods. Evolution 59, (2005). 17. Dornelas, M., Connolly, S. R. & Hughes T.P. Coral diversity refutes the neutral theory of biodiversity. Nature 440, (2006).

10 Coddington, J. A., Agnarsson, I., Miller, J. A., Kuntner, M. & Hormiga, G. Undersampling bias: the null hypothesis for singleton species in tropical arthropod surveys. J. Anim. Ecol. 78, (2009). 19. Bouchet, P., Lozouet, P., Maestrati, P. & Heros, V. Assessing the magnitude of species richness in tropical marine environments: Exceptionally high numbers of molluscs at a New Caledonia site. Biol. J. Linn. Soc. Lond. 75, (2002). 20. Chapman, A. D. Numbers of living species in Australia and the World, 2 nd ed. (Australia Biological Resources Study, Department of Environment, Water Heritages and the Arts, 2009). 21. Crawley, M. J. & Harral, J. E. Scale dependence in plant biodiversity. Science 291, (2001). 22. Guilhaumon, F., Gimenez, O., Gaston, K. J. & Mouillot, D. Taxonomic and regional uncertainty in species-area relationships and the identification of richness hotspots. Proc. Natl. Acad. Sci. U.S.A. 105, (2008) 23. Jackson, J. B. C. Evolution and extinction in the brave new ocean. Proc. Natl. Acad. Sci. U.S.A. 105, (2008). 24. Hoegh-Guldberg, O. et al. Coral reefs under rapid climate change and ocean acidification. Science 318, (2007). 25. Schloss, P. D. & Westcott S.L. Introducing mothur: A computational toolbox for describing and comparing microbial communities. (2009). 26. Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Marine Biol. Biotechnol. 3, (1994).

11 Hebert, P. D. N., Cywinska, A., Ball, S. L. & dewaard, J.R. Biological identifications through DNA barcodes. Proc. R. Soc. B 270, (2003).

12 12 Supplementary Information is linked to the online version of the paper at Acknowledgements We thank Florent Angly, Amy Hall, Christine Hoekenga, Javier Jara, Rob Lasley, François Michonneau, Megan Moews, Russell Moffitt, Shawn Smith and Molly Timmers for their assistance in the field, as well as the numerous helpers that have participated in the CReefs field trips in Australia. We are grateful to Amy Driskell, Genelle Harrison, Andrea Ormos and Lee Weigt for their assistance with DNA barcoding. We thank Kristin Hultgren, Ryuji Machida, Chris Meyer and Gustav Paulay for discussions about the different topics developed in this manuscript and Ei Fujioka for his help with the figures. We greatly appreciate the support by the Alfred P. Foundation and BHP Billiton of the Census of Marine Life CReefs project. Author Contributions L.P. conceived the study, sampled in the field, performed experimental work and analysis, and wrote the paper; J.C. contributed to the design of the study and organized and helped with fieldwork in Australia; R.B. contributed to the design of the study, developed the ARMS and the experiment in French Frigate Shoals; and N.K. conceived the study and helped with the writing of the paper. Author Information Reprints and permissions information is available at The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to L.P. (

13 13 Figure Legends Figure 1: Sampling localities in the Indo-Pacific and Caribbean. Figure 2: Estimated diversity values for seven sampled localities using the Abundance-based Coverage Estimator (ACE) and Chao1 (+/- lower and higher bound of 95% confidence interval). A- Estimated diversity based on all samples. B- Comparable analysis restricted to six samples from each locality (in order to minimize the effect of different numbers of samples), randomized a thousand times. (FFS corresponds to French Frigate Shoals, Hawaii). Table legend Table 1: Sampling details and results for each locality and localities combined. Presented are numbers of sampling units [dead coral heads or settlement devices (ARMS, see Methods)]; estimated total planar area sampled for each locality; the numbers of DNA sequences analyzed; the numbers of taxa (OTUs) for all crustaceans, all decapods, and brachyuran crabs; the numbers of crustacean singletons (see text) (percentages in parenthesis); and for crustaceans the ACE (Abundance-based Coverage Estimator) and Chao1 estimated diversity values (see text) [based both on all samples and just six samples per locality (in parentheses, to eliminate biases caused by unequal sample numbers)]. The IWP and CP columns show the results for the localities of the Indo-West Pacific combined (Ningaloo, Lizard Island and Heron Island) and the Central Pacific combined [Moorea, Northern Line Islands and French Frigate Shoals (FFS), Hawaii] respectively. Table 2: Bray-Curtis similarity indices for all pairwise comparisons between localities. FFS corresponds to French Frigate Shoals, Hawaii.

14 14 Methods Sampling New sampling locations included localities in the Indian Ocean (Ningaloo, Western Australia), the western Pacific Ocean (Lizard and Heron Islands, Great Barrier Reef, Australia), the central Pacific [French Frigate Shoals (FFS), Northwestern Hawaiian Islands] and the Caribbean (Bocas del Toro, Panama). Additionally, we included published diversity results from the Northern Line Islands and Moorea (French Polynesia) in the central Pacific 10. Similar-sized dead coral heads (diameter ~30 cm, the footprint or planar reef area per head ~ 15 2 = 707 cm 2 ) from the family Pocilloporidae in the IWP and from three genera (Eusmilia, Porites and Agaricia) to span as much diversity as possible in the Caribbean, where Pocillopora does not occur, were used as standardized samples and were collected on the reef at a depth of 8 to 12 meters. Dead coral heads were collected and processed following the method described in Plaisance et al. 10 with the exception that the heads were bagged before detaching from the bottom. The invertebrate community was extracted from the dead heads by breaking them into small pieces. At FFS, no dead coral heads were collected because of the policies of the Papahanaumokuakea Marine National Monument; instead autonomous reef monitoring structures (ARMS) were deployed in 2006 and retrieved a year later. The ARMS are small, long-term collecting devices designed to mimic, to some degree, the structural complexity of a coral reef. They consist of nine stacked PVC layers, half entirely open and subdivided into four compartments (the footprint or planar reef area per ARMS ~ 529 cm 2 ). During their retrieval, a mesh was placed around them to prevent escapes. When they were disassembled, each layer was scanned carefully in order to collect all mobile invertebrates that had settled on them.

15 15 In order to compare the effectiveness of dead coral heads and ARMS, both were collected at Heron Island. Analyses suggest that ARMS give somewhat lower absolute numbers of crustaceans than dead coral heads (Table 1). However, the patterns of diversity observed were as would be expected from longitudinal diversity gradients (Heron Island ARMS > French Frigate Shoals ARMS, Table 1). Moreover, the average Bray Curtis similarity index between ARMS and dead coral heads at Heron Island (.41) is comparable to that observed between randomized subsets of dead coral heads at Heron Island (.53); both values are within the range reported for within site similarity for corals of 0.38 to 0.67 by Dornelas et al. 17 and much higher than any between locality similarity indices in our study (Table 2). Molecular analysis Crustaceans were fixed in 95% ethanol and a tissue sample was preserved for DNA analysis for one to ten individuals per morphospecies per sample unit (coral rubble or ARMS). Genomic DNA was extracted from ethanol-preserved tissues (most commonly a leg) using standard proteinase-k digestion followed by phenol chloroform extraction on the AutoGenprep 965 (Autogen). Standard PCR amplification using primers described in Folmer et al. 26 and automated sequencing techniques were used to sequence in both directions part of the mitochondrial COI gene used for DNA barcoding 27. Statistical analysis We used a 5% sequence dissimilarity threshold with the furthest neighbor clustering method for species discrimination (see Plaisance et al. 10 for detailed justification of this threshold). Sequences were clustered into Operational Taxonomic Units (OTUs) using MOTHUR 25. We used ACE (Abundance-based Coverage Estimator) and Chao1 non-parametric estimators 12 to estimate total diversity, using

16 16 either all samples for each locality (which varied in number from 6 to 23) or a subset of 6 samples randomized a thousand times (to eliminate sample size biases). Both estimators use the number of rare species (for Chao I, the numbers of species occurring once and twice; for ACE, the number of species that occur from once to ten times) to adjust upward from the observed number of species. Individual-based rarefaction curves for each locality were also plotted. The Bray-Curtis similarity index was used to estimate the similarity in community composition within and between localities; we also compared our values with the same indices calculated for coral slope communities found in supplemental Table 2 of Dornelas et al. 17

17 17 Locality Nature of sample Lizard Island Dead Coral Dead Coral Heron Island Ningaloo All IWP ARMS Coral + ARMS Dead Coral Coral + ARMS N. Line Islands Dead Coral Moorea FFS All CP Panama Dead Coral ARMS Coral + ARMS Number of samples Estimated planar area (m 2 ) Number of sequences Dead Coral All Locations Coral + ARMS Number of crustacean OTUs Number of decapod OTUs Number of brachyuran OTUs Number (%) of crustacean singletons 40 (31.5) 32 (27.6) 17 (22.3) 49 (30.6) 41 (29.7) 130 (36.6) 34 (40) 17 (27.9) 12 (22.2) 64 (35.6) 5 (31.3) 199 (37.9) Chao 1 estimate for full sample (randomized 6 sample subset) 192 (112) 202 (111) 94 (79) 238 (112) 215 (111) 528 (137) 150 (72) 77 (81) 65 (65) 267 (123) 19 (19) 781 (139) ACE estimate for full sample (randomized 6 sample subset) 212 (134) 229 (114) 102 (86) 316 (118) 193 (113) 516 (149) 175 (84) 102 (78) 67 (67) 300 (128) 20 (20) 746 (152)

18 Lizard Is Heron Is Ningaloo Moorea Line Is FFS Heron Is 0.24 Ningaloo Moorea Line Is FFS Panama

19 French Frigate Shoals Bocas del Toro Panama Kilometers The Line Islands Lizard Island Moorea, French Polynesia Ningaloo Reef Heron Island 0 1,000 2,000 3,000 Kilometers

20 a ACE Chao1 Es mated number of species b Lizard Is. Heron Is. Ningaloo Line Is. Moorea FFS Panama

21 Supplementary Figure 1 This figure shows the individual-based rarefaction curves for the seven localities investigated depicting the number of species recorded as a function of the number of individuals sequenced. The curves did not reach an asymptote at any site, indicating that a large number of species remain to be sampled. The rank order of diversity using rarefaction curves was similar to that provided by the diversity estimators ACE and Chao1 (the only difference being one switch in rank, with the central Pacific Northern Line Islands being more diverse than the Indian Ocean Ningaloo site; this could be an artifact of the greater geographic area covered in the Northern Line Islands samples). (PDF; 344 KB)

22 Supplemental Figure 1: Individual-based rarefaction curves for the seven localities investigated (FFS corresponds to French Frigate Shoals, Hawaii) depicting the number of species recorded as a function of the number of individuals sequenced Number of species Lizard Is. Heron Is. Ningaloo Moorea Line Is. FFS Panama Number of individuals sequenced