Diversity and exibility of algal endosymbionts in globally-distributed large benthic foraminifera

Revealing the specificity and flexibility of the algal symbiont-host association is fundamental 29 for understanding how species can occupy a diverse range of habitats. Here we assessed the 30 global distribution of the algal symbiont diversity for three shallow-water species of large 31 benthic foraminifera (LBF) of the genus Amphistegina . Specifically, we investigated the role 32 of habitat and host identity on the diversity of algal biome. We found that species of Amphistegina form flexible symbiotic relationship with algal taxa, which are primarily shaped by their local habitat. These observations strongly suggest that the capacity of Amphistegina species to utilise a diverse array of available symbionts likely underpins the ecological success of these crucial calcifying organisms across their extensive 53 geographic range.

We found that species of Amphistegina form flexible symbiotic relationship with algal taxa, 50 which are primarily shaped by their local habitat. These observations strongly suggest that 51 the capacity of Amphistegina species to utilise a diverse array of available symbionts likely 52 underpins the ecological success of these crucial calcifying organisms across their extensive 53 geographic range. 54 Symbiont diversity is also closely linked to host identity and phylogeny. It has been shown 83 that in diatom-bearing LBF Amphistegina, the similarities and differences in lineages of 84 endosymbionts of four closely related species are consistent with what is known of their 85 evolutionary histories [18]. Similarly, dinoflagellate symbionts found in the Caribbean and 86 the Indo-Pacific show phylogenetic divergence, which is consistent with the phylogenetic 87 relationship within their LBF hosts in these two regions [19]. Some species of Amphistegina 88 show a stable and persistent algal symbiosis unaffected by water quality gradients [20], while 89 the composition of algal symbionts in diatom-bearing nummulitids changes over depth [21]. 90 The diversity of symbionts might also play a key role in thermal stress tolerances [22], and Here, we examined the diversity of algal symbionts (also referred to as algal biome) in 102 Amphistegina lobifera, A. lessonii and A. radiata within shallow habitats across the 103 Mediterranean, Red Sea, Indian and Pacific Oceans. Specifically, we investigate the influence 104 of host identity and local habitat on the composition of their symbiont communities. We 105 address the level of symbiont specificity (i.e. algal taxa that were only found in a given 106 species of Amphistegina) between the different host species, and evaluate whether these host-107 symbiont associations are partitioned between geographic regions and sites. We further 108 evaluate whether symbiont phylogenies are consistent with the evolutionary histories of host 109 species. 110 111

Phylogenetic analysis 113
Analysis of 18S rRNA of algal biome showed that specimens of Amphistegina predominately 114 host diatoms of the order Fragilariales, which was consistently abundant across sites and 115 species. On average, Fragilariales represented over 60% of identified diatoms. Diatoms from 116 the order Toxariales (class Mediophycea) were found to be abundant within specimens of A. 117 radiata collected from Micronesia (47.85 ± 6.65% of the identified taxa), which also showed 118 the lowest relative abundance of Fragilariales (42.01 ± 8.98%; Fig. 1a). However, Toxariales 119 was rare in all specimens of A. lessonii and absent in A. lobifera. Within Fragilariales, the 120 most common genus was Serratifera, followed by Nanofrustulum and Staurosira (Fig. 1b). 121 Other groups such as Bacillariophyceae were found to represent a substantial proportion of 122 the diatoms in A. lobifera, up to an average of 7.20 ± 3.70% in Sicily ( Fig. 1a; Supplementary 123 table 1). In contrast, A. lessonii was dominated by Fragilariales, and this order represented 124 nearly 100% of the algal assemblage in Zanzibar and Kimbe Bay (Fig. 1a). 125 126 Prevalence of amplicon sequence variants (ASV) was less than 70% among samples and 127 sample groups (Fig. 2). Within taxa, Fragilariales was the most common order, and 128 Serratifera represented a substantial proportion of taxa found across species and sites (Fig.  129 2). However, most ASVs were found in low relative abundance in only a few samples. 130 Phylogenetic analysis showed that there is no clear congruency between symbiont identity 131 and host relationship (Fig. 3), nonetheless it is worth noting that alpha diversity in A. lessonii 132 is consistently lower than in A. lobifera and A. radiata (Fig. 4). There was also a significant 133 difference in alpha diversity among sites and host species (Table 1). The highest average 134 diversity of ASVs was found in A. radiata samples collected from Kimbe Bay, whereas the 135 lowest diversity was consistently found in A. lessonii specimens ( Fig. 4; Supplementary table  136 2). Amphistegina lessonii was found to host a few phylotypes of diatoms of the genus 137 Nanofrustulum, in addition to the common genus Serratifera (Fig. 3). Additionally, we did 138 not identify a core algal biome utilising the 80% cut-off across species and sites analysed. 139 140

Multivariate analysis of diversity of diatoms among species and sites 141
There was a significant difference between the algal community of A. lessonii, A. lobifera, 142 and A. radiata among sites (PERMANOVA(site x species): R2 = 0.199, Pseudo-F= 8.92; p<0.01; 143 Table 2). However, group dispersions analysis also revealed that homogeneity of variances 144 was uneven, indicating that reported p-values should be treated with a high degree of caution. 145 Species provided little overall explanatory value (R2 = 0.09; Pseudo-F=16.84; Table 2), 146 which is reflected in the extensive overlap of algal biomes found in each of the three species 147 (Fig. 5a). Algal biomes of A. radiata were more constrained. Site provided the highest overall 148 explanatory value (R2 = 0.31; Pseudo-F=7.58; Table 2) and was also significant within each 149 species (A. lobifera R2 = 0.53, A. lessonii R2 = 0.66, A. radiata R2 = 0.62; Table 2). Despite 150 algal biomes being more distinct between sites than between species, variability within sites 151 was highly uneven (Figs. 5b-q). Sites contained algal biomes with low variability shared by 152 all species (i.e. Lord Howe Island; Fig. 5j  suggests that Amphistegina likely evolved with a preference for this group of diatoms, 180 although the origin of this association remains unclear. Species of the genus Serratifera were 181 found in every specimen and at all sites, however the overall diversity of all diatom 182 phylotypes within species and specimens is higher than previously thought ( Fig. 3;  Through the assessment of algal biome diversity in these three species of the genus 218 Amphistegina, we demonstrate that symbiont communities are dictated to a certain degree by 219 site but are not constrained by species identity or phylogenetic relationships. While patterns 220 of alpha diversity are partly informed by species identity, levels of flexibility are mostly 221 shaped by site. This confirms the role of the symbiont community as an important interface 222 between the host and the local environment (e.g. [22,25]). Symbiont communities respond 223 differently to differing conditions, and the high variability within sites reveals that a wide 224 array of symbiont communities remains viable within most sites (Fig. 7). However, the algal 225 biome is more constrained in some sites than others. For example, A. lobifera populations 226 that occur at the edge of their geographic distribution tend to have a highly variable algal 227 biome, with high variability between specimens from the same site (e.g. Greece, Ningaloo 228 Reef, Sicily; Fig. 7d, n, p), whereas in the core of their distribution a consistent algal biome 229 across specimens is more common (e.g. Great Barrier Reef, Indonesia, Kimbe Bay; Fig. 7c, 230 g, i). This allows speculations that novel invading species are at an advantage to increased 231 environmental tolerance given by a pool of different endosymbionts. In contrast, A. lessonii 232 not only showed lower alpha diversity of algal phylotypes than other Amphistegina species, 233 but also a more conserved algal biome among the sites analysed. As a result, we were unable 234 to find a universal core algal biome across all Amphistegina species and sites analysed, and 235 only a local-scale species-specific core biome was detected, further supporting the hypothesis 236 that the composition of the biome is largely driven by site [20]. Ultimately, our results 237 suggest that local microhabitat, and the environmental factors associated with it, are likely to 238 impose the strongest influence on both the algal phylotype available, and how hosts acquire 239 their algal symbionts. geography and environmental conditions of sites. In contrast, A. radiata is usually found in 304 regions on the reef slope. For this study, samples were collected from shallow areas of the 306 reef slope (<10 m water depth) by snorkelers or SCUBA divers following previously 307 described methods [27]. Briefly, pieces of reef rubble containing the targeted species were 308 collected, scrubbed, and specimens picked out and placed in 96% ethanol or air-dried for 309 further analysis. All specimens were collected from the same rubble sample.  Briefly, forward and reverse sequences lacking adaptors and primer sequences were checked 354 for quality, trimmed, and filtered to remove low-quality sequence reads. Quality score cut-off point was determined based on quality of both forward and reverse sequence reads, 356 maintaining the recommended overlap for merging the sequences. The DADA2 method was 357 utilised for barcoding filtering, de-replication, chimeric identification and removal, and 358 merging pair-end reads. DADA2's error model automatically filters out singletons, removing 359 them before the subsequent sample inference step. Sample inference was performed using the 360 inferred error model. Afterwards, an ASV We calculated diversity indices such as Chao 1 [56], which we used to project estimates of 385 taxonomic richness within each specimen (i.e., alpha diversity), and Simpson index that 386 combines evenness and richness of a given specimen [57]. Indices were calculated using 387 ASVs. We compared significant differences in diversity indices among species and sites by 388 performing a rank sum Kruskal-Wallis test. Prevalence, which is the percentage of specimens 389 where a given ASVs is detected, was calculated only for ASVs classified as 'Fragilariales'. 390 Differences within and between species and sites were analysed through a two-way 391 Permutation Multivariate ANOVA (PERMANOVA) using weighted-UniFrac resemblance 392 matrices [58] to account for presence/absence, but also abundance of ASVs between samples.                               Total abundance (log10-transformed) plotted against prevalence of Fragilariales genera in all samples in Amphistegina lessonii, A. lobifera, and A. radiata.

Figure 3
Phylogenetic tree of algal biome community associated with Amphistegina lessonii, A. lobifera and A. radiata across all sites. Dendrogram represents the 892 algal taxa with a relative abundance of at least 1% summed across all samples. Bars represent relative abundance of each phylotype (i.e. ASVs) identi ed in A. lessonii (red), A. lobifera (green), and A. radiata (blue).    Specimens of (A) Amphistegina lessonii, (B) A. lobifera, and (C) A. radiata collected from the same habitat in Kimbe Bay, Papua New Guinea. Scale bars represent 1 mm in A and B, and 2 mm in C. Note that specimens were preserved in 96% ethanol, and therefore symbiont pigment colour shown here does not represent natural coloration.

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