Multi-gene incongruence consistent with hybridisation in Cladocopium (Symbiodiniaceae), an ecologically important genus of coral reef symbionts

Abstract

Coral reefs rely on their intracellular dinoflagellate symbionts (family Symbiodiniaceae) for nutritional provision in nutrient-poor waters, yet this association is threatened by thermally stressful conditions. Despite this, the evolutionary potential of these symbionts remains poorly characterised. In this study, we tested the potential for divergent Symbiodiniaceae types to sexually reproduce (i.e. hybridise) within Cladocopium, the most ecologically prevalent genus in this family. With sequence data from three organelles (cob gene, mitochondrion; psbA^ncr region, chloroplast; and ITS2 region, nucleus), we utilised the Incongruence Length Difference test, Approximately Unbiased test, tree hybridisation analyses and visual inspection of raw data in stepwise fashion to highlight incongruences between organelles, and thus provide evidence of reticulate evolution. Using this approach, we identified three putative hybrid Cladocopium samples among the 158 analysed, at two of the seven sites sampled. These samples were identified as the common Cladocopium types C40 or C1 with respect to the mitochondria and chloroplasts, but the rarer types C3z, C3u and C1# with respect to their nuclear identity. These five Cladocopium types have previously been confirmed as evolutionarily distinct and were also recovered in non-incongruent samples multiple times, which is strongly suggestive that they sexually reproduced to produce the incongruent samples. A concomitant inspection of next generation sequencing data for these samples suggests that other plausible explanations, such as incomplete lineage sorting or the presence of co-dominance, are much less likely. The approach taken in this study allows incongruences between gene regions to be identified with confidence, and brings new light to the evolutionary potential within Symbiodiniaceae.

Keywords: Approximately unbiased test; Cladocopium; Hybridisation; Incomplete lineage sorting; Incongruence length difference test; Next generation sequencing; Symbiodinium; Symbiont.

Figure 1. Stepwise analyses performed per site to identify incongruences in Cladocopium.

Figure 2. Pairwise tanglegrams for site BHB.

Red branches with bolded taxa labels indicate incongruent samples, as identified by the tree hybridisation analysis (executed in Dendroscope 3.0 (Huson & Linz, 2016)). Branch labels are ML bootstrap values (1,000 replicates for cob, 100 for ITS2 and psbA^ncr). (A) cob vs. ITS2: found incongruent by ILD and AU tests. (B) psbA^ncr vs. ITS2: found incongruent by ILD and AU tests. (C) cob vs. psbA^ncr: found congruent by ILD and AU tests.

Figure 3. Short selections of raw sequence data for incongruent sample BHB146 and related sequences (polymorphisms in bold).

In organellar gene regions (A) and (C), BHB146 groups with samples BHB104 and BHB105 (Cladocopium type C42a, C1v). In the nuclear gene region (B), BHB groups with BHB122 and BHB149 (Cladocopium type C1#).

Figure 4. Pairwise tanglegrams for site BSP.

Red branches with bolded taxa labels indicate incongruent samples, as identified by the tree hybridisation analyses (executed in Dendroscope 3.0 (Huson & Linz, 2016)). Branch labels are ML bootstrap values (1,000 replicates for cob, 100 for ITS2 and psbA^ncr). (A) cob vs. ITS2: found incongruent by ILD and AU tests. (B) psbA^ncr vs. ITS2: found incongruent by ILD and AU tests. (C) cob vs. psbA^ncr: found congruent by ILD and AU tests.

Figure 5. Short selections of raw sequence data for incongruent sample BSP343 and related sequences (polymorphisms in bold).

(A) In the nuclear ITS2 region, BSP343 groups with samples BSP383 and BSP387; point mutations at base pairs 23 and 238 (available in Data Availability) identify it as Cladocopium type C3u. (B) In the organellar psbA^ncr region, BSP343 groups with BSP386 and BSP388, as a variant of Cladocopium type C40. The cob gene was invariant in this case.

Figure 6. Short selections of raw sequence data for incongruent sample BSP364 and related sequences (polymorphisms in bold).

(A) In the nuclear ITS2 region, BSP364 groups with samples BSP362 and BSP373 (Cladocopium type C3z). (B) In the organellar psbA^ncr region, BSP364 groups with BSP344 and BSP366, as a variant of Cladocopium type C40. The cob gene was invariant in this case.

Figure 7. Average proportions of different background sequence populations for samples identified as C3z (eight samples) and C40 (13 samples) from site BSP.

(A) For C3z population, proportion of sequences that were C3z vs. non-C3z sequences (i.e. background sequences), averaged over all eight samples. (B) Average proportion of background sequences for C3z samples, identified using the database of Franklin et al. (2012) and GenBank BLAST search. Sequences followed by stars (* or **) indicate novel sequences and are named according to their most closely related sequence in the databases. Parentheses indicate the total number of samples (out of eight) that the background sequence appeared in. Rare sequences (<3% average individual abundance) are clustered together. The red box indicates the background sequence with the organellar identity of the putative hybrid (BSP364). (C) For C40 population, proportion of sequences that were C40 vs. non-C40 sequences (i.e. background sequences), averaged over all 13 samples. (D) Average proportion of background sequences for C40 samples. Parentheses indicate the total number of samples (out of 13) that the background sequence appeared in. The red box indicates the background sequence with the nuclear identity of the putative hybrid. Other details as per (B).

Figure 8. Predictions under incomplete lineage sorting.

(A) General pattern expected for ILS. A single ancestral population with polymorphism in both the psbA^ncr and ITS2 regions is present before a speciation event. After speciation, the ITS2 polymorphism fails to segregate, while through stochastic processes the C40 polymorphism is eliminated and leads to incongruence between nuclear and chloroplast genes. (B) The process of ILS that would be required for this example. The ITS2 region fails to segregate after speciation; despite the extensive presence of C40 alleles, a small subpopulation of symbionts with dominant C3z alleles is maintained (weak dashed blue line) in the C40 population and both are recovered in present-day sampling, at the same site, as pure C3z populations.