Diversity and ecology of Symbiodinium in pocilloporid corals

The decline of coral reefs is well documented, yet a detailed understanding of the processes involved in the establishment, persistence, and ecology of the coral-dinoflagellate associations still remains largely unknown. The advent of molecular techniques has resulted in significant advances in understanding the molecular diversity present of symbiotic dinoflagellates from the genus Symbiodinium, but information concerning the functional, ecological, and biogeographical significance of this expanding symbiont diversity remains limited. This thesis therefore used molecular methodologies to uncover Symbiodinium diversity in Stylophora pistillata, Pocillopora damicornis, and Seriatopora hystrix at ecological scales, in response to thermal stress, and to long-term environmental shifts. In addition, all the molecular methods currently used in Symbiodinium research are critically reviewed to provide an important baseline for future studies. The application of ITS2-DGGE coupled with the integration of alternate speciation concepts and analyses showed great merit in assessing Symbiodinium diversity in S. pistillata, P. damicornis, and S. hystrix along a depth gradient (3 m to 18 m) at Heron Island on the Great Barrier Reef (Australia), and emphasizes that sampling regimes should focus on the role of symbionts within their functional habitat. S. pistillata associates with symbionts C78 or C35/a in shallow areas and C79 in the deep, but also harbors a generalist type C8/a that can be found at all depths. P. damicornis harbors C42/a in shallow areas while C33/a is generally found in deeper reef zones, although it is occasionally observed in the shallows. On the other hand, S. hystrix only harbors a single symbiont (C3/t) at all depths. The data from Chapter 2 therefore shows that closely related symbionts within a single clade can diverge rapidly under influence of ecological differentiation whereby each symbiont represents a separately evolving lineage that occupies a specific ecological niche. As such, closely related symbionts are likely to have evolved specializations that optimize performance within their environmental range. Previous studies have sought to explain the bleaching susceptibility of scleractinian corals as a function of the presence or absence of six major clades of Symbiodinium. In chapter 3 it is shown that sub-cladal types of clade C in S. pistillata differ in their response to thermal stress, and these differences are as large as those previously reported between different clades. Molecular (ITS2-DGGE) data is integrated with physiological measurements (PAM fluorometry, host protein, symbiont cell density) to investigate the response to stress (bleaching) and is directly related to fine-scale differences in symbiont-types. This suggests that the cladal distinction of Symbiodinium is insufficient to explain the highly variable responses commonly seen in reef-building corals. Furthermore, the results highlight that shifts in symbiont community within a host population are due to differential mortality rather than the uptake of novel symbionts. It therefore appears that changes in the thermal tolerance of corals by acquiring novel more resistant Symbiodinium to meet the challenges of global warming may be restricted, and as such cannot be expected to prevent large-scale reef degradation. Whilst most Symbiodinium cladal studies have focused on bleaching, Chapter 4 combines molecular (ITS2-DGGE) and physiological analyses (PAM fluorometry) to assess the flexibility of the coral–symbiont assemblages in S. pistillata, P. damicornis, and S. hystrix when faced with long-term shifts in key environmental conditions. To test this, a 32 month reciprocal transplant experiment was set-up on Heron Island, whereby corals were transplanted to a new light environment, i.e. shallow to deep, or deep to shallow. Although some host-symbiont combinations were able to shuffle sub-cladal symbiont types, almost all colonies reverted back to their original type within 7 to 12 months. Interestingly, transplanted colonies showed a broad acclimatory response by adjusting their physiological responses to those of the control colonies at the transplanted depth. However, those that persisted with sub-optimal symbiont types suffered disproportionate rates of mortality at the onset of additional stress (bleaching). This suggests that, despite their large acclimatory capacity, the holobiont was likely living at the limits of its tolerance range. As such, it appears that coral species cannot readily form a novel symbiotic unit by changing their symbiotic partner in response to prolonged periods of change, and it is therefore unlikely that they will be able to adjust their symbionts in an attempt to cope with changing global conditions. Finally, Chapter 5 reviews the different DNA markers (18S, 28S, ITS1, ITS2, cp23S) and screening methods (RFLP, LICOR, SSCP, DGGE, cloning-sequencing) currently applied to uncovering Symbiodinium diversity. Current rDNA markers are used to identify ancient or more recent evolutionary lineages and separate symbionts into broad groups such as clades, as well as uncovering fine-scale differences between ecologically different sub-cladal types. The 18S and 28S rDNA in combination with RFLP are appropriate for studies that focus on large groupings at the cladal level and may uncover broad biogeographical. However, studies on the ecology or physiology of host-symbiont associations clearly benefit from the capacity to detect the full level of variability present within a community (e.g. 28S-SSCP, ITS2-DGGE). Despite the suitability of the rDNA, it is an imperative that sequence information is combined with ecological data in order to accurately predict how each hostsymbiont combinations responds as a unit that is optimized to function within the range of its distribution. Symbiont types present in individual hosts are shown to be distinct cohesive groups that are not interchangeable on an ecological, functional and evolutionary scale. In summary, this thesis expands on the current knowledge of the role of Symbiodinium on coral reefs and introduces a number of novel concepts. It is imperative that effort and resources continue to be channelled to combine genetic and ecological studies on Symbiodinium, as they appear to be an important factor driving responses of their host. Although diversity assessments would benefit from the development of a single-copy molecular marker, it is important to continue utilizing current methodologies to increase our knowledge of coral-symbiont diversity if we are to understand and manage coral reefs in the short-term as environmental conditions continue to change