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

BackgroundThe 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

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

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

Milleporids are 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

Background

(Photo:CCMA/NOAA)

Millepora visually dominate some reefs Millepora overgrowing gorgonians

Photo credit- E. Weil

1) Are there reliable morphological characters that separate the species?

2) Do the different species/morphotypes of Caribbean Millepora represent genetically distinct taxa?

Questions

M. alcicornis branching vs. encrusting vs. M. complanata

Ruiz Ramos, Weil, Schizas (2014). Zoological Studies.

M. complanata

M. alcicornis branching M. alcicornis encrusting

MORPHOLOGY

Morphological Measurements g = Gastropore, d = Dactylopore, 1. Diameter of gastropore, 2. Diameter of dactylopore, 3. Distance among dactylopores, 4. Distances from gastropore to nearest dactylopore, 5. Distances among gastropores.

Ruiz Ramos et al. (2014). Zoological Studies

GOOD

BAD

Ruiz Ramos et al. 2014. Zoological Studies

GOOD

BAD

GOOD

Ruiz Ramos et al. 2014. Zoological Studies

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

Molecular Methods

Unresolved Gene genealogy – COI gene

RED = M. complanataBLUE = M. alcicornis (branching)GREEN = M. alcicornis (encrusting)Circle = M. striataDiamonds: haplotypes shared among the morphotypes.

M. s

quar

rosa

We cannot distinguish M. complanata from M. alcicornis and M. striata.

Millepora squarrosa is sufficiently different, genetically.

Comparative transcriptomics

1) Presence/absence of transcripts

2) Differential expression

3) Sequence comparison

Study Design

M. alcicornis branching M. complanata

Ion Proton (P1 chip) – One transcriptome per chip

2 transcriptomes M. alcicornis vs. 1 transcriptome M. complanata

Test for consistency - 2 independent runs of M. alcicornis

Ion Proton Chip (P1 chip) Data

M. alcicornis (run 1) M. alcicornis (run2)

M. complanata (not analyzed yet)

FASTQ Size (GB) 5.9 13.5 12.3

Total Reads 28,023,480 64,001,281

Read Length ~137 bp ~137 bp

Read Length Range

4-297 8-254

% GC 50 50

After quality trimmingTotal Reads

24,527,977 55,850,797

After removing Symbiodinium sp.FASTQ Size (GB)

5.2 GB 11.5 GB

% Reads Aligned to Symbiodinium

2.62% 2.33%

% of Reads Aligned to human

11.68 % 11.10 %

% of HQ Aligned Reads to human

5.35 % 5.21 %

Pre-processing pipeline

Symbiodinium andHuman genomes

RNA-seq data is aligned to reference genomes

Contamination-free sequences

Assembly pipelineM. alcicornis RNA-seq data 1

M. alcicornis RNA-seq data 2

• 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).

Post-Assembly Analysis Pipeline

Data from 253 putatively identified gene components suggests consistency between runs in Ion Proton

Differential Expression between replicated data sets –M. alcicornis

Similarly ExpressedDifferentially Expressed

224 (89%)

29 (11%)

Most abundant transcripts from the 2 runs are consistent

Transcript BLASTN functional annotation

M. alcicornis #1Normalized FPKM

M. alcicornis #1 rank

M. alcicornis #2Normalized FPKM

M. alcicornis #2 rank

comp22892_c0 PREDICTED: Strongylocentrotus purpuratusuncharacterized LOC100892564,misc_RNA

1566319.98 1 1689605.05 1

comp16419_c0 Millepora exaesa 18S ribosomal misc_RNA

287979.27 2 422834.99 2

comp38876_c0 PREDICTED: Hydra magnipapillata hypothetical protein LOC100207766, transcript variant 2

258682.54 3 209318.35 3

comp36161_c0 Bombyx mori gene for tRNAAsp

207115.61 4 176415.55 5

comp22881_c0 Homo sapiens RNA, 45S preribosomal5,ribosomal RNA

200387.66 5 125102.45 9

comp38810_c0 Tubulipora flabellaris 28S ribosomal RNA gene, partialsequence

142164.76 6 244234.79 3

comp37311_c0

Homo sapiens RNA, 45S preribosomal5,ribosomal RNA

130956.65 7 130956.65 6

comp39007_c0 Hydractinia symbiolongicarpus clone YU0089N10,complete sequence

111747.52 8 144085.95 7

comp36078_c1 Millepora sp. EK2011mitochondrion, partial

106730.5 9 127298.32 8

comp39538_c1 Millepora sp. AMN200828S, partial sequence

98143.14 10

comp39494_c0 Millepora sp. AMN200828S, partial sequence

89447.67 10

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.

Photo credit- E. Weil

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.

Acknowledgments

We thank the Seven Bridges Genomics: Sebastian Wernicke, Lu Zhang Nemanja Ilic, and Jelena Radenkovic, 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