HIGH THROUGHPUT AMPLICON SEQUENCING TO DETERMINE ZOOPLANKTON DIVERSITY IN THE RED SEA

Introduction Methods

Results

Discussion

Plankton nets (150 µm mesh, 0.5 m diameter) were used to assess 45 stations across 3 reefs in the southern/central Red Sea (Figure 1). Samples were stored in 100% ethanol and DNA extracted using a phenol:chloroform procedure.The 18S rRNA gene was amplified using 454 tagged primers (Amaral-Zettler et al., 2009) with unique barcodes. Flowgram data was processed using AmpliconNoise. Additional reference based chimera filtering using UCHIME with SilvaRef111 as a reference database. Subsequently sequences were clustered at 97% similarity into Operational Taxonomic Units (OTUs). Singletons and non metazoan sequences were removed from further analysis. Taxonomic assignments were created by comparison against the Silvamod database using LCAClassifier/CREST. Top 3 ranked OTUs by abundance classified against the NCBI database (env. sequences removed). Based on a Bray-Curtis dissimilarity matrix clusters were produced using a random walking method in InfoMap. The OTUs significant in characterizing each cluster were determined using ANOVA.

Coral reefs are known for harbouring some of the highest biodiversities on the planet. However, little is known about the planktonic community associated to coral reefs. The Red Sea, a narrow, semi confined sea combines high temperature, a deep basin, oligotrophic conditions and extensive coral reefs. The combination of parameters makes the Red Sea an ideal laboratory to understand how future climatic conditions may affect planktonic communities. Zooplankton studies have been limited to the northern regions and especially the Gulf of Aqaba. Due to similarities in morphology, especially amongst juveniles and larvae, the identification of zooplankton is complicated. A range of experts are thus required to undertake a taxonomically broad environmental study. This limits the spatial and temporal resolution of the study. The advent of high throughput sequencing technologies has meant that community DNA studies can be undertaken. This means that comprehensive studies in areas of complex taxonomy such as coral reefs are simplified. This study presents the results of a comprehensive molecular study of the diversity of zooplankton around three reef systems in the southern/central Red Sea.

John K. Pearman1, Mohsen M. El-Sherbiny2, Anders Lanzén3, Ali Al-Aidaroos2, Xabier Irigoien1 1.  Red  Sea  Research  Centre,  King  Abdullah  University  of  Science  and  Technology,  Thuwal,  23955-­‐6900,  Kingdom  of  Saudi  Arabia  2.  Department  of  Marine  Biology,  Faculty  of  Marine  Science,  King  Abdelaziz  University,  Jeddah  21589,  Kingdom  of  Saudi  Arabia  3.  Department  of  Ecology  and  Natural  Resources,  NEIKER-­‐Tecnalia,  Bizkaia  Technology  Park  Bldg.  812,  Derio  48160,  Spain  

Figure 1: Map illustrating the sampling points around the three different reef systems in the southern Red Sea.

Figure 2: Proportion of reads for phyla across all stations in the three reef systems. ‘Other’ accounts for unidentified metazoans and phyla that did not account for a proportion > 0.01 at least one station.

A total of 853,719 metazoan reads were obtained after denoising. 854 Metazoan OTUs were identified at 97% sequence identity. Arthropoda accounted for ~61 % of metazoan OTUs. Mollusca (~8 %), Chordata (~7 %) and Cnidaria (~7 %) also had significant numbers of OTUs. Across the majority of stations Arthropoda accounted for the highest proportion of reads. Peak proportions were 99 % (Figure 2). Arthropoda were dominated by Maxillopoda and especially the famailies Acartiidae and Calanidae (Table 1). Cnidarians dominated several stations. Peak proportion of 86 % at Farasan9. The most dominant OTU belonged to the Cnidaria, and specifically Muggiaea sp. AGC-2001. Liriope were also made a significant contribution to the proportion of Cnidarian reads Chordata and Ctenophora generally lower proportions, although reached peaks of 61 % and 41 % respectively. Chordata were dominated by OTUs relating to the tunicate genus Oikopleura Mollusca, although high numbers of OTUs, had low proportions of reads.

Figure 3: InfoMap clustering (edge value cufoff = 0.5) for Arthropoda based on a Bray Curtis distance matrix of the 1389F primer OTU table

Figure 4: InfoMap clustering (edge value cufoff = 0.5) for Chordata based on a Bray Curtis distance matrix of the 1389F primer OTU table

This genomic approach was able to show the high diversity (> 800 taxa) of metazoa associated to the coral reef. High diveristy of holoplankton in warm subtropical systems is expected (Rombouts et al., 2009) but the observed diversity also reflects the contribution of meroplankton larvae from the coral reef. Due to the variability in size, developmental stages (e.g. larval or adult) and varying gene copy numbers between and within zooplankton taxa the number or reads can not be assumed to correlate with abundance. As previously shown (Lindeque et al., 2013), the number of reads could approximate biomass. Molluscs have a high abundance of OTUs but a low proportion or reads, which could suggest the present of larval instead of adult stages. Larval stages are often hard to differentiate morphologically so genomic approaches may contribute to improve our knowledge of larval dispersal at the species level. The data suggests a high degree of connectivity between the reefs. Stations from different reefs clustered together suggesting a significant amount of horizontal transfer. Different phyla showed different clustering patterns. A presence of a cyclonic eddy (Raitos et al., 2013) approximately covering the 3 reefs suggests a mode of horizontal transport. Although there are currently limits to the level which genomic data can be assigned to a taxonomy this is likely to improve as more genomic data becomes available for type species. Molecular reference databases are lacking a large proportion of marine zooplankton skewing assignments towards known species. Calanus helgolandicus has not previously been recorded in the Red Sea. Morphological analysis of replica samples found other Calanoida, such as Undinula  vulgar  which  lacks  a  18S  rRNA  sequences  in  the  database. To take full advantage of expanding sequencing capabilities the construction of a definitive database linking morophological and genomic data will have to be created.

Amaral-Zettler, L. A., Mccliment, E. A., Ducklow, H. W. & Huse, S. M. 2009. A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PloS one, 4, e6372-e6372. Lindeque, P. K., Parry, H. E., Harmer, R. A., Somerfield, P. J. & Atkinson, A. 2013. Next Generation Sequencing Reveals the Hidden Diversity of Zooplankton Assemblages. PloS one, 8

Raitsos, D. E., Pradhan, Y., Brewin, R. J. W., Stenchikov, G. & Hoteit, I. 2013. Remote Sensing the Phytoplankton Seasonal Succession of the Red Sea. PloS one, 8. Rombouts, I., Beaugrand, G., Ibanez, F., Gasparini, S., Chiba, S. & Legendre, L. 2009. Global latitudinal variations in marine copepod diversity and environmental factors. Proceedings of the Royal Society B-Biological Sciences, 276, 3053-3062.

Results

Cluster analysis based on Bray Curtis dissimilarity matrices showed different spatial patterns for different taxa. 4 clusters were formed for the analysis of Arthropoda (Figure 3).The Arthropoda clusters were determined by variations in the proportions of various copepod OTUs. The largest cluster (red in Fig. 3) was defined by the presence of an OTU related to Acartia. The second cluster (green in Fig. 3) had high comparative proportions of a unclassified Calanidae. The Chordata subsection of diversity formed 8 distinct clusters.The two largest clusters (red and orange in Fig. 4) were characterized by the presence of an Oikopleura dioica OTU. Other Appendicularian OTUs defined the formation of the other clusters.

Top 3 Rank OTU Allith Al Qunfatah Farasan Top Blast Hit 1574 2 1 1 Muggiaea sp. AGC-2001 677 1 / / Calanus helgolandicus 706 / 2 2 Calanus helgolandicus 103 3 / / Liriope tetraphylla 72 / 3 / Acartia longiremis 44 / / 3 Subeucalanus pileatus

Table 1: Ranking of OTUs (only top 3) based on abundance for the three reefs. The taxonomic identity based on top Blast hit (excluding env. sequences) against the NCBI database

References

Email: john.pearman@kaust.edu.sa