Using transcriptomics to delineate the status of Millepora species in the Caribbean Sea

1. Using transcriptomics to delineate the status of Millepora species in the Caribbean Sea Nikolaos V. Schizas, Dannise Ruiz Ramos, Ingrid Ortiz Gonzalez, Audrey Majeske, Ernesto Weil University of Puerto Rico, Mayagüez Department of Marine Sciences

2. Background The hydrocoral genus Millepora consists of 19 species distributed in warm waters around the globe 12 are found in the Indo-Pacific, 7 in the Atlantic/Caribbean

3. Caribbean species of Milleporaspp. Clockwise from the upper left: M. alcicornis, M. squarrosa, M. striata, and M. complanata

4. Background • Millepora is an important reef framework builder in some reefs • Millepora is a voracious plankton feeder, consuming up to 8 prey cm-2 day-1 • Milleporidsare superior space competitors over gorgonians • Millepora are susceptible to bleaching • Despite their abundance, geographical distribution and geological importance, the milleporids have seldom received attention in coral reef studies

5. Millepora visually dominate some reefs Milleporaovergrowing gorgonians (Photo:CCMA/NOAA)

6. Questions 1) Are there reliable morphological characters that separate the species? 2) Do the different species/morphotypes of Caribbean Millepora represent genetically distinct taxa? Photo credit- E. Weil

7. M. alcicornisbranching vs. encrusting vs. M. complanata M. alcicornisbranching M. alcicornisencrusting M. complanata MORPHOLOGY Ruiz Ramos, Weil, Schizas (2014). Zoological Studies.

8. Morphological Measurements g = Gastropore, d = Dactylopore, 1. Diameter of gastropore, 2. Diameter of dactylopore, 3. Distance among dactylopores, 4. Distances from gastroporeto nearest dactylopore, 5. Distances among gastropores. Ruiz Ramos et al. (2014). Zoological Studies

9. GOOD BAD Ruiz Ramos et al. 2014. Zoological Studies

10. BAD GOOD GOOD Ruiz Ramos et al. 2014. Zoological Studies

11. Molecular Methods Samples from 7 localities across the Caribbean were added for genetic analysis: Bocas del Toro (Panama), Grand Cayman, Mona (Puerto Rico - PR), Parguera (PR), Vieques (PR), Guadeloupe and Curaçao. One gene approach: mtCOI a variable marker in hydrozoans

12. Unresolved Gene genealogy – COI gene M. squarrosa RED = M. complanata BLUE = M. alcicornis(branching) GREEN = M. alcicornis (encrusting) Circle = M. striata Diamonds: haplotypes shared among the morphotypes. We cannot distinguish M. complanata from M. alcicornis and M. striata. Milleporasquarrosais sufficiently different, genetically.

13. Comparative transcriptomics 1) Presence/absence of transcripts 2) Differential expression 3) Sequence comparison

14. Study Design Ion Proton (P1 chip) – One transcriptome per chip 2 transcriptomes M. alcicornis vs. 1 transcriptomeM. complanata Test for consistency - 2 independent runs of M. alcicornis M. alcicornisbranching M. complanata

15. Ion Proton Chip (P1 chip) Data

16. Pre-processing pipeline Contamination-free sequences RNA-seq data is aligned to reference genomes Symbiodinium and Human genomes

17. Assembly pipeline M. alcicornisRNA-seq data 1 M. alcicornis RNA-seq data 2

18. Post-Assembly Analysis Pipeline • Bowtie (Aligner) – Aligns raw RNA-seq reads against assembled transcripts. • Can be used to count the number of reads that are well represented in the assembled transcriptome. • Can be used for visualization of reads alignment, information about read depth. • RSEM – Also uses Bowtie to estimates gene and isoform expression levels from RNA-Seq data for each sample). • Merge - Create a matrix using the expected fragment count data produced by RSEM across samples used for differential expression analysis. • EdgeR- Estimates differential gene expression across samples (pairwise comparison).

19. Differential Expression between replicated data sets –M. alcicornis Data from 253 putatively identified gene components suggests consistency between runs in Ion Proton 29 (11%) 224 (89%)

20. Most abundant transcripts from the 2 runs are consistent

21. Conclusions • Consistency between the two Ion Proton runs on M. alcicornis • Despite the high number of reads the resulting data sets were relatively small • Lack of a reference genome/transcriptome • We cannot explain the high contamination of rRNA(RiboMinus Kit–removes 12S and 18S rRNAs instead of the micropoly(A) Purist Kit which selects for mRNAs.

22. Photo credit- E. Weil

23. Continuing work… • We are adding paired-end Illumina data for M. alcicornis and M. complanata. • Generate high quality transcriptomes of M. striata and M. squarrosa. • Perform transplant experiments and examine gene expression patterns in “native” vs. transplanted M. alcicornis colonies.

24. Acknowledgments We thank the Seven Bridges Genomics: Sebastian Wernicke, Lu Zhang NemanjaIlic, and JelenaRadenkovic, for the bioinformatics analysis and guidance in this project. We thank the NIH Cancer Genome Facilities for allowing to use the Ion Proton and their facilities. The school of Arts and Sciences, University of Puerto Rico supported partially the travelling costs. Puerto Rico Sea Grant for providing funds for additional Illumina runs and training of students in NGS techniques