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Comparison of Symbiodiniaceae diversities in different members of a Palythoa species complex (Cnidaria: Anthozoa: Zoantharia)—implications for ecological adaptations to different microhabitats

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Biodiversity and Conservation

Introduction

Zoantharians (Anthozoa: Zoantharia) belong to the phylum Cnidaria and can be dominant organisms in shallow coral reef areas (e.g.,  Burnett et al., 1994). In particular, the genus Palythoa is often among the most dominant benthos in coral reef areas (Irei, Nozawa & Reimer, 2011; Santos et al., 2016; Reimer et al., 2017a).

We recently reported on four putative Palythoa species (P. tuberculosa, P. sp. yoron, P. mutuki, and P. aff. mutuki) that form a species complex, and were observed to all occur within a narrow range of coral reefs in southern Japan (Mizuyama, Masucci & Reimer, 2018). For example, P. tuberculosa tends to occur across a wide range of habitats from shallow to deeper areas, from the intertidal zone to the mesophotic reef slope (Mizuyama, Masucci & Reimer, 2018), and has been reported from tropical to temperate regions (Reimer, Takishita & Maruyama, 2006). On the other hand, the other three Palythoa species appear to more restricted compared to P. tuberculosa in terms of their distribution and habitats within coral reefs. Palythoa mutuki is the second most dominant species in this genus in Okinawa and is often dominant at the reef edge, in surge channels, and in small bumps on reef flats (Irei, Nozawa & Reimer, 2011). Palythoa sp. yoron has yet to be formally described, but tends to occur on reef flats and backreef moats where it is exposed to strong water currents (Shiroma & Reimer, 2010). Although there is little published information on P. aff. mutuki, it has been observed near P. mutuki colonies on the reef flat (Mizuyama, Masucci & Reimer, 2018). Although molecular delineation of these Palythoa species groups was unsuccessful with molecular data, likely due to incomplete lineage sorting, they can be distinguished via morphological and reproductive data (Mizuyama, Masucci & Reimer, 2018). In addition, these Palythoa species display different microhabitat patterns within the coral reef, but it is still unclear how these species would have diversified under almost completely sympatric conditions.

Symbiodiniaceae endosymbiotic dinoflagellates are symbiotic with various metazoan phyla including Cnidaria (LaJeunesse et al., 2018). Many zoantharians maintain Symbiodiniaceae, similar to reef-building corals (Noda et al., 2017; Wee, Kurihara & Reimer, 2019). In the case of scleractinian corals, symbiotic relationships with Symbiodiniaceae are important for host survival in various environments (Baker, 2003), and can contribute to ecological divergence of coral host species (Winters et al., 2009). Previous molecular studies have reported that species composition of Symbiodiniaceae is closely related to host genotypes in corals (e.g.,  Bongaerts et al., 2010; Pinzon & LaJeunesse, 2011). Thus, information on the composition Symbiodiniaceae of the four Palythoa species above would also be helpful to understand their ecological divergence into different microenvironments within a reef. In particular, genotypic composition of symbiotic algae would be informative for understanding ecological divergence of these species because the genetic and/or community changes of microbiomes are expected to be faster than that of the hosts themselves (Torda et al., 2017), facilitating eco-physiological adaptation of holobionts into different microenvironments (e.g.,  Reimer et al., 2017b; Wee, Kurihara & Reimer, 2019). In this study, we aimed to (1) compare diversities of symbionts among the closely related Palythoa species P. tuberculosa, P. sp. yoron, P. mutuki and P. aff. mutuki, and (2) determine if diversities of symbionts explain eco-physiological adaptations to microhabitats of each species that entailed divergences among them (P. tuberculosa, P. sp. yoron and P. mutuki).

Materials & Methods

Specimens collection

Eighty-two colonies of three Palythoa species (P. tuberculosa, P. sp. yoron, and P. mutuki) were collected from a shallow fringing reef of Tokunoshima Island, Kagoshima, Japan (Figs. 1 and 2). Specimens of these three Palythoa species were collected in four different areas (Table 1, Fig. 2A): reef edge (Fig. 2B, 27.76998333N, 129.03988611E) for P. tuberculosa (Fig. 2C); reef flat 1 (Fig. 2D, 27.76997777N, 129.03925000E) for P. tuberculosa (Fig. 2E) and P. mutuki; reef flat 2 (Fig. 2F, 27.77195277N, 129.03843611E) for P. mutuki (Fig. 2G); and backreef moat (Fig. 2H, 27.76990833N, 129.03855833E) for P. tuberculosa and P. sp. yoron (Fig. 2I). To avoid collecting clones, we collected individuals from clearly different colonies while maintaining a set distance from each other of at least 1 m. In a previous study, even when closer to each other (within approximately 50 × 50 cm), no clones were observed in Zoanthus (Cnidaria: Anthozoa: Zoantharia) colonies (Albinsky et al., 2018). In addition, eighteen previously collected specimens of Palythoa species including 10 P. aff. mutuki specimens from Mizuyama, Masucci & Reimer (2018) were also examined in this study (Table 1).

Location of Tokunoshima Island and the sampling site (arrow in inset) for the Palythoa specimens in this study.

Figure 1: Location of Tokunoshima Island and the sampling site (arrow in inset) for the Palythoa specimens in this study.

Map data: GeoLite2 data created by MaxMind using the Generic Mapping Tools (GMT v5.4.5) software package. CC BY SA 4.0.
Landscape of the coral reef flat at the study site and in situ images of Palythoa species used in this study.

Figure 2: Landscape of the coral reef flat at the study site and in situ images of Palythoa species used in this study.

(A) Satellite image of the reef area obtained by Google Earth; (B) reef edge; (C) P. tubeculosa; (D) reef flat 1; (E) P. tuberculosa; (F) reef flat 2; (G) P. mutuki; (H) backreef moat; (I) P. sp. yoron. Map data: Google, Maxar Technologies. Scale bars in C, E, G, and I are 10 cm.

DNA extraction and PCR amplification

From each of these specimens, several polyps were cut with a surgical knife and DNA was extracted using DNeasy Blood and Tissue Kit (QIAGEN). DNA concentrations were checked by Qubit Fluorometer (ThermoFisher, Waltham, USA). Two molecular markers for genotyping symbiotic algae of Palythoa species were examined: nuclear internal transcribed spacer ribosomal DNA (ITS-rDNA) region including partial 18S–ITS1–5.8S–ITS2–partial 28S (primers zITSf: CCG GTG AAT TAT TCG GAC TGA CGC AGT and ITS4: TCC TCC GCT TAT TGA TAT GC, (Baillie, Belda-Baillie & Maruyama, 2000; appx. 700–750 bp) and plastid mini-circle non-coding region DNA (psbAncr) (primers 7.4-Forw: GCA TGA AAG AAA TGC ACA CAA CTT CCC and 7.8-Rev: GGT TCT CTT ATT CCA TCA ATA TCT ACT G, (Moore et al., 2003; appx. 800–850 bp). These regions were amplified according to the PCR thermal conditions in Wee, Kurihara & Reimer (2019). Amplified PCR products of symbionts were directly sequenced, and sequence data were manually checked based on the chromatogram files and low quality sites were removed at the 5′ and 3′ ends by BioEdit v.7.0.5.3 (Hall, 1999). Obtained sequences were deposited in the GenBank database (MN654128MN654306, Table 1).

Table 1:
Specimen list.
Specimen ID Location/Region Spiecies ID Date (m/d/y) Environment Accession no. of ITS Accession no. of psbA-F Accession no. of psbA-R
A01PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654209 MN654185
A02PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654210 MN654184 MN654134
A03PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654211
A04PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654212 MN654186 MN654135
A05PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654213 MN654187 MN654136
A06PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654214 MN654188
A07PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654215 MN654189 MN654137
A08PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654216 MN654190 MN654138
A09PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef edge MN654217
A11PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654218 MN654191 MN654139
A12PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654219 MN654192 MN654140
A13PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654220 MN654193
A14PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654221
A15PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654222
A16PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654223 MN654194 MN654141
A17PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654224
A18PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654225 MN654195 MN654142
A19PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654226 MN654198
A20PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Reef flat MN654227
A21PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654228 MN654169 MN654159
A22PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654229
A24PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654230 MN654143
A25PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654231
A26PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654232
A27PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654233
A28PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654234
A29PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654235
A30PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Jun 2, 2019 Backreef moat MN654236
B01PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654237
B02PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654238 MN654199 MN654144
B03PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654239
B04PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654240
B05PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654241 MN654145
B06PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654242 MN654170 MN654160
B07PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654243 MN654161
B08PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654244 MN654171 MN654162
B09PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654245
B11PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 2, 2019 Reef flat MN654246
B12PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654247 MN654172
B13PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654248
B14PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654249 MN654173
B15PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654250
B16PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654251
B17PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654252 MN654174 MN654163
B18PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654253 MN654200
B20PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654254
B21PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654255
B22PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654256
B23PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654257
B24PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654258 MN654175 MN654164
B25PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654176 MN654165
B26PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654259 MN654166
B28PmToKa Kaminomine/Tokunoshima Palythoa mutuki Jun 3, 2019 Reef flat MN654177 MN654167
C01PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654260
C02PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654261
C03PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654262
C04PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654263
C05PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654264
C06PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654265
C07PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654266
C08PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654267
C09PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654268
C10PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654269
C11PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654270
C12PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654271 MN654201 MN654146
C13PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654272
C14PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654273 MN654179 MN654147
C15PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654274 MN654180
C16PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654275 MN654202 MN654148
C17PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654276 MN654203 MN654149
C18PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 2, 2019 Backreef moat MN654277 MN654168
C19PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654278
C20PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654279 MN654204 MN654150
C21PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654280 MN654205 MN654151
C22PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654281 MN654206 MN654152
C24PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654282 MN654181
C25PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654283 MN654196 MN654153
C26PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654284 MN654154
C27PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654285 MN654207 MN654155
C28PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654286
C29PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654287 MN654208 MN654156
C30PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Jun 3, 2019 Backreef moat MN654288 MN654182 MN654157
159PamToKa Kaminomine/Tokunoshima Palythoa aff. mutuki July 28, 2010 In Mizuyama, Masucci & Reimer (2018) MN654300
233PamErYa Yakomo/Okinoerabu Palythoa aff. mutuki Jun 17, 2011 In Mizuyama, Masucci & Reimer (2018) MN654301
237PamErSu Sumiyoshi/Okinoerabu Palythoa aff. mutuki Jun 18, 2011 In Mizuyama, Masucci & Reimer (2018) MN654302
248PamToKa Kaminomine/Tokunoshima Palythoa aff. mutuki Jun 21, 2011 In Mizuyama, Masucci & Reimer (2018) MN654303
250PamToKa Kaminomine/Tokunoshima Palythoa aff. mutuki Jun 21, 2011 In Mizuyama, Masucci & Reimer (2018) MN654304 MN654183 MN654131
328PamOkTe Teniya/Okinawa Palythoa aff. mutuki Apr 5, 2012 In Mizuyama, Masucci & Reimer (2018) MN654305
364PamOkOk Oku/Okinawa Palythoa aff. mutuki Jun 25, 2012 In Mizuyama, Masucci & Reimer (2018) MN654306
2PtOkOd Odo/Okinawa Palythoa tuberculosa Aug 18, 2009 In Mizuyama, Masucci & Reimer (2018) MN654289 MN654158
39PtYoUk Ukachi/Yoron Palythoa tuberculosa Mar 4, 2010 In Mizuyama, Masucci & Reimer (2018) MN654290 MN654132
63PtErYa Yakomo/Okinoerabu Palythoa tuberculosa Mar 5, 2010 In Mizuyama, Masucci & Reimer (2018) MN654291 MN654133
100PtToKa Kaminomine/Tokunoshima Palythoa tuberculosa Mar 9, 2010 In Mizuyama, Masucci & Reimer (2018) MN654292 MN654128
15PyOkOd Odo/Okinawa Palythoa sp. yoron Sep 5, 2009 In Mizuyama, Masucci & Reimer (2018) MN654297 MN654130
51PyYoUk Ukachi(West)/Yoron Palythoa sp. yoron Mar 4, 2010 In Mizuyama, Masucci & Reimer (2018) MN654298
85PyErYa Yakomo/Okinoerabu Palythoa sp. yoron Mar 5, 2010 In Mizuyama, Masucci & Reimer (2018) MN654296 MN654197
105PyToKa Kaminomine/Tokunoshima Palythoa sp. yoron Mar 9, 2010 In Mizuyama, Masucci & Reimer (2018) MN654299 MN654178 MN654129
218PmOkOd Odo/Okinawa Palythoa mutuki May 4, 2011 In Mizuyama, Masucci & Reimer (2018) MN654294
77PmErYa Yakomo/Okinoerabu Palythoa mutuki Mar 5, 2010 In Mizuyama, Masucci & Reimer (2018) MN654293
280PmToKa Kaminomine/Tokunoshima Palythoa mutuki Oct 5, 2011 In Mizuyama, Masucci & Reimer (2018) MN654295
DOI: 10.7717/peerj.8449/table-1

Haplotype network inference and phylogenetic estimation

Obtained sequences for ITS-rDNA, psbAncr forward and reverse regions were aligned, respectively. In order to discriminate taxa of Symbiodiniaceae, we extracted the ITS2 region utilizing SymPortal (Hume et al., 2019; https://symportal.org/) and performed BLASTN search against the nt database using the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for ITS-rDNA sequences. Haplotype network inference was performed for ITS-rDNA sequences using the alignment with TCS networks method (Clement et al., 2002) in PopART (Leigh & Bryant, 2015). Any columns in the alignment with gaps or ambiguous sites were automatically masked in the inference. The phylogenetic analyses were performed by MEGA version X (Kumar et al., 2018) and any loci with ambiguous (double peaks) sites and gaps was automatically deleted completely for calculation in order to avoid over/underestimation of genetic distance among each sequence. Molecular phylogenetic trees of each marker were constructed by maximum likelihood (ML) and neighbor joining (NJ) methods under the JC+G model for ITS-rDNA region and the JC model for psbAncr regions adopted by modeltest program within MEGA X. The significance of each node was tested by bootstrap test with 1,000 replications. Bayesian inference was performed using BEAST2 (Bouckaert et al., 2019) under default settings other than the clock model being changed to the relaxed log normal model, which showed the highest likelihood value according to the model comparison program compiled in BEAST2 (Drummond et al., 2006). Posterior probability (PP) on each branch was calculated summarizing four independent 10 million MCMC simulations.

Statistical analyses

To clarify the relationships between (1) symbiont lineages and host species, and (2) symbiont lineages and host microhabitats, Fisher’s exact test was conducted for the compositions of genotype for ITS-rDNA region and monophyletic clades for psbAncr forward and reverse regions. It should be noted that host microhabitat was restricted by host species for P. sp. yoron and P. mutuki, and thus we only targeted P. tuberculosa for these analyses (aim 2 above) When significance was detected in Fisher’s exact test, Cramér’s coefficient of association (V) was calculated to evaluate which factors (host species or host microhabitat) were strongly associated with each other.

Results

Sequence alignment

The total number of sequences of Symbiodiniaceae from specimens of the four Palythoa species obtained in this study was 98 sequences for the ITS-rDNA region (513–773 bp), 40 sequences for the psbAncr forward region (330–547 bp), and 41 sequences for the psbAncr reverse region (352–494 bp). As the primer set for psbAncr used in this study did not make a congruent contig, obtained sequences of forward regions and reverse regions were aligned separately (Noda et al., 2017). After alignment, a total of 449 sites with 5 parsimony informative (=PI) sites for the ITS-rDNA region, 260 sites with 94 PI sites for the psbAncr forward region, and 293 sites with 40 PI sites for the psbAncr reverse region were used for each phylogenetic estimation.

Barcoding, haplotype network and phylogenetic trees

As the result of BLAST searches, all query sequences of the ITS-rDNA region (n = 98) were confirmed as belonging to the genus Cladocopium. Seventeen ITS-rDNA unique sequences (=genotypes) were observed in TCS network, with most of the sequences belonging to one of major three ITS-rDNA genotypes (Fig. 3, Table S1). No significant clade was detected for the ITS-rDNA phylogenetic tree (Fig. S1). Summarizing these ITS-rDNA genotypes from the viewpoint of host species, P. tuberculosa possessed mainly Genotype01 (n = 20) followed by Genotype02 (n = 7), and P. sp. yoron also possessed mainly Genotype01 (n = 20) followed by Genotype03 (n = 8) (see details in Table S1). On the other hand, P. mutuki possessed mainly Genotype02 (n = 13) with a few Genotype01 (n = 3) and Genotype03 (n = 2). Although the number of specimens examined was smaller (n = 6) than those the other species, P. aff. mutuki also possessed mainly Genotype01 (n = 5).

Haplotype network tree constructed with nuclear ITS-rDNA region alignment using TCS networks method.

Figure 3: Haplotype network tree constructed with nuclear ITS-rDNA region alignment using TCS networks method.

Scale represents number of sequences with circle sizes proportional to haplotype frequency. Colors represent Palythoa species: red, P. tuberculosa; yellow, P. sp. yoron; blue, P. mutuki; green, P. aff. mutuki.

In contrast, phylogenetic trees generated from psbAncr regions had a higher resolution. Two monophyletic clades were well supported by bootstrap values and posterior probability in both forward (Fig. 4 clf1, ML = 100, NJ = 100, PP = 1 and clf2, ML = 100, NJ = 100, PP = 1) and reverse trees (Fig. 5 clr1 and clr2, ML = 100, NJ = 100, PP = 1). Summarizing these Symbiodiniaceae lineages from the viewpoint of host species, P. tuberculosa inhabiting the reef edge possessed clf1/clr1 lineage (n = 7∕5) and one specimen inhabiting at the backreef moat possessed clf2/clr2 lineage. Palythoa sp. yoron inhabiting at the backreef moat possessed mainly clf1/clr1 (n = 9∕13), however, approximately one third of specimens (n = 5) possessed other lineages. On the other hand, P. mutuki inhabiting the reef flat possessed mainly clf2/clr2 (n = 8∕8) other than two specimens that possessed clf1/clr1. Unfortunately, as most of P. aff. mutuki specimens were not amplified by this primer set, we could only obtain phylogenetic information on one specimen which possessed the same lineage as P. sp. yoron (C24ToKa-PF) for the forward region and clr1 for the reverse region.

Molecular phylogenetic tree of Symbiodiniaceae of Palythoa species using mitochondrial psbAncr forward region.

Figure 4: Molecular phylogenetic tree of Symbiodiniaceae of Palythoa species using mitochondrial psbAncr forward region.

Bootstrap values of maximum likelihood (ML) and neighbor joining (NJ) methods, and posterior probability (PP) are shown more than 70% for ML and NJ, and more than 0.95 for PP at the nodes, respectively. Scale bars indicate substitutions per site. Colored letters and colored diagrams represent Palythoa species and their habitats, respectively: red, P. tuberculosa; yellow, P. sp. yoron; blue, P. mutuki; green, P. aff. mutuki; circle in pink, reef edge; triangle in purple, reef flat; square in orange, backreef moat.
Molecular phylogenetic tree of Symbiodiniaceae of Palythoa species using mitochondrial psbAncr reverse region.

Figure 5: Molecular phylogenetic tree of Symbiodiniaceae of Palythoa species using mitochondrial psbAncr reverse region.

Bootstrap values of maximum likelihood (ML) and neighbor joining (NJ) methods, and posterior probability (PP) are shown more than 70% for ML and NJ, and more than 0.95 for PP at the nodes, respectively. Scale bars indicate substitutions per site. Colored letters and colored diagrams represent Palythoa species and their habitats, respectively: red, P. tuberculosa; yellow, P. sp. yoron; blue, P. mutuki; green, P. aff. mutuki; circle in pink, reef edge; triangle in purple, reef flat; square in orange, backreef moat.

Relationships among symbiont genotype/lineages, host species and host microhabitats

From the results of Fisher’s Exact test, significant differences were detected in all combinations, i.e., ITS-rDNA genotype and host species (p < 0.01), psbAncr forward lineages and host species (p < 0.01), psbAncr reverse lineages and host species (p < 0.01), and ITS-rDNA genotype and host microhabitats for P. tuberculosa (p < 0.05) (Table 2). In other words, it was shown that Symbiodiniaceae lineages and host species were not independent, nor were Symbiodiniaceae lineages and host microhabitats for P. tuberculosa. The effective dose calculated by Cramér’s coefficient of association (V) was largest between host species and psbAncr forward/reverse lineages (V = 0.786, V = 0.682, respectively), and moderate for the other combinations (host species and ITS-rDNA genotypes, V = 0.477; host microhabitats and ITS-rDNA genotypes).

Table 2:
Composition of genotype for ITS-rDNA sequences and monophyletic clades for psbAncr sequences of Symbiodiniaceae from Palythoa species used in this study and microenvironments of host habitats.
Significances were tested by Fisher’s Exact Test and V value represents Cramer’s coefficient of association.
Symbiodiniaceae genotype (ITS-rDNA) Symbiodiniaceae lineage (psbAncr forward region) Symbiodiniaceae lineage (psbAncr reverse region)
Genotype01 Genotype02 Genotype03 clf1 clf2 clr1 clr2
Host species P. tuberculosa 20 7 0 13 1 13 2
P. sp. yoron 20 1 8 10 0 14 1
P. mutuki 3 13 2 2 8 2 8
P. aff. mutuki 5 1 0
Total 48 22 10 25 9 29 11
p < 0.01, V = 0.477 p < 0.01, V = 0.786 p < 0.01, V = 0.682
Host habitats of P. tuberculosa Reef edge 8 0
Reef flat 7 2
Backreef moat 2 4
Total 17 6
p < 0.05, V = 0.508
DOI: 10.7717/peerj.8449/table-2

Notes:

P. aff. mutuki was removed from statistical analyses of psbAncr region due to low numbers of specimens.

Discussion

Symbiodiniaceae genotype/lineage and host species

The development of molecular markers such as psbAncr that have higher resolution than commonly used 18S or ITS ribosomal DNA markers has helped unveil a more detailed picture of the genetic diversity of Symbiodiniaceae (Takishita et al., 2003; LaJeunesse & Thornhill, 2011; LaJeunesse et al., 2018) (but see also Hume et al., 2019 who utilized intragenomic variation of ITS2 to resolve genetic delineations). Accordingly, host species biodiversity has been discovered from the initial observation of differences of Symbiodiniaceae phylotypes in some cnidarian species (e.g., gorgonian Eunicea flexuosa, Prada et al., 2014; scleractinian coral Seriatopora hystrix, Warner, Van Oppen & Willis, 2015).

From the results of Mizuyama, Masucci & Reimer (2018), none of the four molecular markers utilized could clearly delineate four Palythoa species, although they could delineate two closely related species groups composed of P. tuberculosaP. sp. yoron and P. mutukiP. aff. mutuki. These previous results seem to be reflected in the results in the current study of Symbiodiniaceae genotypes of ITS-rDNA and lineages of psbAncr regions. Palythoa tuberculosa and P. sp. yoron mostly shared the same symbiont genotype (Genotype01); nevertheless, they also partially shared the other genotypes with P. mutuki (Genotype02 and Genotype03). With regard to psbAncr lineages, even though the delineation of species groups between P. tuberculosaP. sp. yoron and P. mutuki were shown more clearly, they were not divided completely. The situation requires further investigation via obtaining more P. aff. mutuki specimens’ psbAncr sequences. Unfortunately, in the current study, despite much searching, we could not find large numbers of P. aff. mutuki on the reef in Tokunoshima Island, even though they were previous sampled for Mizuyama, Masucci & Reimer (2018). We do not know what happened to P. aff. mutuki colonies, but they may have been strongly affected by the bleaching events of 2016 and 2017 observed in southern Japan (Masucci et al., 2019).

Symbiodiniaceae genotype/lineage and microhabitat of host species

From the results of the phylogenetic analyses, three microhabitats were not exclusively allocated in distinct Symbiodiniaceae genotypes or monophyletic clades, but the ratios of different genotypes were significantly different for P. tuberculosa. Regarding P. tuberculosa, Symbiodiniaceae Genotype01 was mostly detected on the reef edge and reef flat, while Genotype02 was mainly observed in the backreef moat. Although there were not enough samples to conduct statistical examinations of P. sp. yoron and P. mutuki due to their habitat specificity, Genotype02 and clf2/clr2 were detected mainly on the reef flat, while Genotype01 and clf1/clr1 were observed from all three environments.

It has been reported that zoantharian species with different symbiotic genotypes show species-specific photosynthetic responses against seawater temperature and p CO2 (Graham & Sanders, 2016; Reimer et al., 2017b; Wee, Kurihara & Reimer, 2019). Although the four Palythoa species in this study occurred sympatrically on one reef, the environmental conditions in a reef can be quite different according to small-scale geographical features. Seawater temperatures on reef flats frequently reach near 40 °C (Achituv & Dubinsky, 1990). In enclosed reefs, seawater temperatures and p CO2 show higher variations than those in exposed reefs (Suzuki, Nakamori & Kayanne, 1995; Fitt et al., 2001). Thus, the relationship between Symbiodiniaceae and host Palythoa species may change among different microhabitats in a reef area, facilitating ecological divergence of Palythoa species within a narrow geographic range.

Although a previous molecular study could not distinguish the boundaries among these Palythoa species (Mizuyama, Masucci & Reimer, 2018), it is suggested by our results that these species are ecologically divergent, and physiological differences within Symbiodiniaceae species may contribute to their ecological adaptation. In fact, Howells et al. (2012) reported that Cladocopium C1 in Acropora tenuis showed different physiological responses between northern and southern populations in the Great Barrier Reef. Considering that Cladocopium contains various species distinguished by differences of only a few bp in the ITS2 maker (Thornhill et al., 2014), meta-barcoding analyses via next-generation sequencing would be necessary to further understand the detailed relationship between Symbiodiniaceae and Palythoa species complex.

Conclusions

We succeeded in obtaining genotypic data of Symbiodiniaceae from four putative Palythoa species and detected micro-scale geographic variations of the symbiotic algae among these species within a single coral reef. Our results suggest that ecological divergence among Palythoa species may be related to differences in Symbiodiniaceae diversities among microhabitats, even within a narrow reef area. More powerful genetic data such as that generated by next-generation sequencing could provide us with additional understanding on how neighboring Palythoa species have co-evolved with Symbiodiniaceae among the different microhabitats in a reef.

Supplemental Information

Composition of genotypes for ITS-rDNA sequences of Simbiodiniaceae from 4 Palythoa species

DOI: 10.7717/peerj.8449/supp-1

Molecular phylogenetic tree of Symbiodiniaceae of Palythoa species using sequences of the nuclear ITS-rDNA region

Shaded boxes represent three main genotypes occupying most sequences from four Palythoa species. Bootstrap values of maximum likelihood (ML) and neighbor joining (NJ) methods, and posterior probability (PP) are shown more than 70% for ML and NJ, and more than 0.95 for PP at the nodes, respectively. Scale bars indicate substitutions per site. Colored letters and colored diagrams represent Palythoa species and their habitats, respectively: red, P. tuberculosa; yellow, P. sp. yoron; blue, P. mutuki; green, P. aff. mutuki; circle in pink, reef edge; triangle in purple, reef flat; square in orange, backreef moat.

DOI: 10.7717/peerj.8449/supp-2

The alignment files of sequences for ITS-rDNA, 98 sequences; psbAncr forward region, 40 sequences; psbAncr reverse region, 41 sequences

DOI: 10.7717/peerj.8449/supp-3

The chromatogram files for ITS-rDNA region (forward)

DOI: 10.7717/peerj.8449/supp-4

The chromatogram files for ITS-rDNA region (reverse)

DOI: 10.7717/peerj.8449/supp-5

The chromatogram files for psbAncr forward region

DOI: 10.7717/peerj.8449/supp-6

The chromatogram files for psbAncr reverse region

DOI: 10.7717/peerj.8449/supp-7