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The Pennsylvania State University The Graduate School GENOMIC AND TRANSCRIPTOMIC INSIGHTS ON THE ORBICELLA SPECIES COMPLEX A Dissertation in Biology by Ana María González Angel © 2019 Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2019 The dissertation of Ana María González Angel was reviewed and approved* by the following: James Marden Professor of Biology Chair of Committee Mónica Medina Professor of Biology Dissertation Advisor István Albert Research Professor of Bioinformatics Stephen W. Schaeffer Professor of Biology Associate Department Head of Graduate Education *Signatures are on file in the Graduate School iii ABSTRACT In this dissertation I explore different aspects of the ecological speciation and genomics of marine organisms. First, I review the literature and explore different factors, particularly, depth, that isolate marine populations. These factors can be sufficiently strong to enhance divergence among populations, and so, lead to reproductive isolation. I discuss numerous examples but focus on the well documented case of Orbicella, a Caribbean coral genus that is environmentally segregated and has evolved at least two mechanisms of reproductive isolation, temporal isolation and gamete recognition. In Chapter 2, I explore the genomes of these corals to infer how different their protein coding ortholog groups are and if gene content is reflected in the differentiation in these taxa. Overall, I found the genomes of Orbicella sister species are extremely similar and other factors may be responsible for their differentiation such as gene expression, gene silencing, differential transcription factor activity or SNP presence. In Chapter 3, I study the temporal isolation by assessing the gene expression profiles of two sister Orbicella species during and after the moment of spawning, which occurs only one time per year. Although their genomes are very similar, gene expression profiling suggests these species use their genetic toolkit very differently. Minimal overlap was found in the differentially expressed genes (DEG) involved in the spawning behavior, and the ones that do not overlap have different identities and putative functions while many others are remain uncharacterized and unknown. Further studies including more timepoints are need to address the rhythmicity of the DEG putatively responsible for the allochronic assortative mating (or timing of gamete release) that occurs in Orbicella. Other aspects of the reproductive barriers such as gamete recognition and hybrid inviability also require attention. iv Overall, the studies of the genomic and cellular elements responsible for the prezygotic isolation in Orbicella are still nascent and warrant more work to unravel their complexity and consequences in ecology and evolutionary history of this group. v Table of Contents List of Figures………………………………………………………………………..viii List of Tables ............................................................................................................... x Acknowledgements ..................................................................................................... xi Chapter 1, Ecological Speciation in Corals…………………………………………..1 Abstract ................................................................................................................ 1 Introduction ........................................................................................................ 2 Biodiversity in the Ocean ..................................................................................... 3 Speciation in the Ocean ........................................................................................ 5 Ecological Speciation .......................................................................................... 5 Adaptation Across Gradients in the Sea ............................................................... 7 Depth as a Driver of Ecological Speciation in Coral Reefs …………. ...……….9 Mechanisms of reproductive isolation among populations living in different habitats ……...……………………………………………………...............15 Spawning timing………………………………………………………………...15 Sperm-egg recognition systems…………………………………………………18 Evolutionary Genomics of the Coral Speciation Process……………………….21 Conclusion………………………………………………………………………23 References………………………………………………………………………24 Chapter 2, Gene Orthology assessment in Orbicella………………………………...41 vi Abstract………………………………………………………………………….41 Introduction……………………………………………………………………...41 Methods……………………………………………………………...…………..43 Results…………………………………………………………………………...44 Discussion……………………………………………………………………….52 Conclusion…………………………………………………….…………………54 References…………………………………………………….…………………55 Chapter 3, Transcriptional insights of temporal isolation in broadcast spawning corals Abstract………………………………………………………………………….59 Introduction……………………………………………………………………...60 Methods………………………………………………………………………….65 Results .................................................................................................................. 69 Discussion ............................................................................................................ 73 Conclusions .......................................................................................................... 77 References………………………………………………………………………. 77 Chapter 4, Conclusion ................................................................................................. 84 Appendix A: Cyphastrea genome assembly supplementary information…………… 88 Appendix B: Lists of summarized (padj <0.05) gene ontology groupings in the genomes of Orbicella ……………………………………………………………………………99 vii Appendix C: Lists of transcripts present in gametes and parental samples………….109 Appendix D: Lists of Differentially Expressed Genes in Orbicella annularis and O. franksi …………………………………………………………………………………………………..118 viii LIST OF FIGURES Figure 1-1: O. annularis, O. franksi, and O. faveolata. Photos are courtesy of Mónica Medina. …………………………………………………………………………………….10 Figure 1-2: We retrieved ortholog protein sequences from protein models from the genomes of four symbiotic cnidarians (O. faveolata, Acropora digitifera, Stylophora pistillata, and Exaiptasia pallida) and one asymbiotic cnidarian (Nematostella vectensis) using blast bidirectional best hit (BBHs) (Altschul et al.,1990). We did protein alignments and curation with ClustalW (Thompson et al.,1994) and Gblocks (Castresana, 2000). We built protein distance matrices using Hamming dissimilarities algorithm implemented in Ugene (Okonechnikov et al.,2012) (Fig 1). The heatmaps of the protein distances between different reproductive proteins in five Cnidarians are depicted. Green colors represent closer distances (fully conserved proteins equal to 1), while red colors represent more distant relationships (equals a value of 0.05). Grey indicates sequence absence. A) Comparison among proteins from sister Orbicella species B) Comparison of O. faveolata, Acropora digitifera, Stylophora pistillata, and Exaiptasia pallida genomes and Nematostella vectensis..................................23 Figure 3.1: Sample collection time points sequenced in this study and experimental design. The orange boxes around the sampling time points for each species show the transcriptome comparisons we present here. The horizontal arrow at the bottom signals time progress and spawning occurred on September 14 2014...............................................................................69 ix Figure 3-2: Heatmaps showing the differentially expressed genes in O. annularis and Orbicella franksi. A) Orbicella franksi data B) O. annularis data. The samples in batches of four correspond to the biological replicates during either spawning or post-spawning. There are 117 in O. franksi and 48 DEG in O. annularis. Colors indicate expression such that yellow represents upregulation respect to blue hues and log2 fold change of Z scores. ..................74 Figure 3-3: Venn Diagrams showing the overlap in differentially gene expression profiles in three species during spawning and after spawning. Comparisons were based on UNIPROT/SWISSPROT identifier codes. Note than only the transcripts that had an annotation are plotted here (73/117 known to be DEG in O. franksi, 25/48 known to be DEG in O. annularis, 198/206 known to be DEG in Acropora millepora).................................76 x LIST OF TABLES Table 1-1: Cases of ecological segregation in marine invertebrates. The segregation column refers to Depth (D), Latitudinal gradients (L), Habitat (H), Host Preference (HP), or Intertidal height (I). Most studies consider mesophotic environments as habitats beginning at 30 meters of depth, but sometimes this varies depending on author’s criteria. For more details on mesophotic description, see (Laverick, et al.,2017))............................................................................................................................ ..7 Table 2-1: Metrics of ortholog groups found in the genomes of A. cervicornis, A. palmata, O. annularis, O. faveolata, and O franksi ………………………………….48 Table 2-2: Quantitative overlap in orthologous groups...............................................49 Table 2-3: Species-specific ortholog groups in O. franksi. Annotations were obtained by aligning to NCBI, BLAST P program, non-redundant database. The bold sequences are the description sequence of the orthologous group and it is also the one that corresponds to the annotation boxes..............................................................................53