Research Article Biogeography and Host-Specificity of Cyanobacterial Symbionts in Colonial Ascidians of the Genus Lissoclinum

Home , Didemnidae, Lissoclinum, Lissoclinum fragile, Lissoclinum patella, Prochloron

Systematics and Biodiversity (2020), 18(5): 496–509

Research Article Biogeography and host-specificity of cyanobacterial symbionts in colonial ascidians of the genus Lissoclinum

MIRIELLE LOPEZ-GUZMAN1, PATRICK M. ERWIN1, EUICHI HIROSE2 & SUSANNA LÓPEZ-LEGENTIL1 1Department of Biology & Marine Biology, and Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K. Moss Ln, Wilmington, 28409, NC, USA; 2Faculty of Science, University of the Ryukyus, Senbaru 1, Nishihara, 903-0213, Okinawa, Japan

(Received 19 November 2019; revised: 27 May 2020; accepted 28 May 2020)

Ascidians are known to harbour bacterial symbionts in their tunics. In particular, the ascidian genus Lissoclinum can host abundant and diverse cyanobacterial associates. Here, we determined the diversity and host-specificity of cyanobacteria inhabiting 28 ascidian samples corresponding to eight Lissoclinum species: L. bistratum, L. midui, L. patella, L. punctatum, and L. timorense from Japan, L. aff. fragile and L. verrilli from the Bahamas, and L. perforatum from Spain and Chile. Cyanobacterial symbionts were characterized using both partial 16S rRNA gene sequences and sequences obtained for the entire 16S-23S rRNA internal transcribed spacer region (ITS). We found that both host species and geographic location played a role in structuring ascidian-cyanobacterial symbioses. Broad biogeographic trends included the dominance of Prochloron symbionts in Japanese ascidians and the presence of a novel cyanobacterial lineage in L. aff. fragile hosts from the Bahamas. Within each geographic region, a high degree of host- specificity was observed, where similar symbionts were recovered from ascidian hosts across multiple collection locations. Further, our analysis revealed the existence of nine distinct Prochloron clades in Japan, some of which corresponded to particular host species and sampling sites. For L. aff. fragile, further differences were observed between cyanobacterial symbionts in ascidians from reef and mangrove habitats. Our results showed high host-specificity in ascidian-cyanobacterial symbioses characterized by cryptic diversity and structured by host identity, location and habitat.

Key words: cyanobacteria, photosymbionts, Prochloron, 16S rRNA, Synechocystis, Tunicate

Introduction in particular, between the colonial ascidian family Didemnidae (e.g. genera Lissoclinum, Didemnum, Ascidians (class Ascidiacea), commonly known as sea Trididemnum, Diplosoma; Kott, 2001) and cyanobacteria squirts, are sessile marine filter-feeding invertebrates in from the genera Prochloron (Prochlorales; e.g. Hirose the phylum Chordata and the subphylum Tunicata. et al., 2009) and Synechocystis (Chroococcales, e.g. Tunicates owe their name to the presence of an outer Lafargue & Duclaux, 1979;Lopez-Legentil et al., 2011; covering called a ‘tunic’, which is mainly composed of Munchhoff et al., 2007; Sybesma et al., 1981). cellulose (called ‘tunicine’ in tunicates). The tunic pro- € Symbiotic relationships among didemnid ascidians vides protection, adhesion to the substrate and can also and Prochloron cyanobacteria appear to be obligate and harbour symbiotic microorganisms, notably cyanobac- mutualistic, since Prochloron is vertically transmitted teria (e.g. Carpenter & Foster, 2002; Hirose, 2009; from parent to offspring (reviewed in Hirose, 2015) and Lopez-Legentil et al., 2011). Ascidians are the largest provides fixed carbon (Griffiths & Thinh, 1983; Pardy class of tunicates, with over 3,000 known species inhabiting the world’s oceans, including tropical, temperate, & Lewin, 1981) and possibly nitrogen (Pearl, 1984) to and polar habitats (Shenkar & Swalla, 2011). the ascidian host. Prochloron symbionts also contain the Accordingly, most tunicate-cyanobacterial associations genes necessary for mycosporine-like amino acid (Donia described to date have been reported for ascidians and, et al., 2011) and patellamide (Schmidt et al., 2005) biosynthesis (reviewed in Schmidt, 2015). Mycosporine- Correspondence to: Susanna Lopez-Legentil. E-mail: like amino acids absorb ultraviolet (UV) radiation, [email protected] which may confer protection to the ascidian against

Published online 25 Jun 2020 Biogeography and host-specificity 497 harmful UV exposure (Donia et al., 2011). Patellamides in the symbiosis remains unknown and may vary among are cytotoxic cyclic peptides that may defend the ascid- species, although evidence to date suggests that the ian colony against predation (Schmidt et al., 2005). cyanobacterium may be an obligate symbiont in at least Interestingly, these cyclic peptides also have a potential a few of these associations. Indeed, Acaryochloris-like use as anti-cancer treatments (Fu et al., 1998; Williams cells were observed in the inner tunic of L. fragile lar- & Jacobs, 1993). In return, Prochloron symbionts obtain vae, evidence of vertically transmission to the progeny a stable and protected habitat from potential predators (Lopez-Legentil et al., 2011). and environmental changes (Hirose & Maruyama, Most studies to date have revealed a high degree of 2004). Ascidian waste and in particular nitrogen (in the host-specificity between ascidians and cyanobacteria form of ammonia and urea, Goodbody, 1974; Markus & (Hirose et al., 2012;Lopez-Legentil et al., 2011; Parry Lambert, 1983) may also be recycled by Prochloron & Kott, 1988). In addition to host-specificity, slight dif- cells, since the cyanobacterium contains genes that code ferences in photosymbiont identity were also correlated for urease and urea transport and genes able to convert with geographic location in some cases (e.g. ammonia into glutamine (Donia et al., 2011). Lissoclinum fragile;Lopez-Legentil et al., 2011). On the Symbiotic associations with Synechocystis occur other hand, M€unchhoff et al. (2007) concluded that sym- mainly with didemnid ascidians from the genus biosis between didemnids and Prochloron sp. were inde- Trididemnum (Lafargue & Duclaux, 1979;Lopez- pendent of both host species and geographic origin and Legentil et al., 2011;M€unchhoff et al., 2007; Shimada attributed the lack of host-specificity to the low genetic et al., 2003; Sybesma et al., 1981), and occasionally the variation within the genus Prochloron. Although appar- genus Didemnum (Parry, 1984). Unlike the Prochloron- ently contradictory, different conclusions from these didemnid association, little is known about the role of studies may results from the varying resolution of the each partner in Trididemnun-Synechocystis symbioses. genetic marker utilized. M€unchhoff et al. (2007) However, Synechocystis symbionts are vertically trans- sequenced a fragment of the 16S rRNA gene, while mitted to the progeny, similar to Prochloron and indica- Lopez-Legentil et al. (2011) sequenced the same 16S tive of an obligate photosymbiosis (Hirose, 2015). In rRNA gene and the complete 16S-23S rRNA internal fact, although Synechocystis is the predominant sym- transcribed spacer (ITS) gene region. The ITS region is biont taxa in Trididemnum hosts, it can coexist with much more variable than the 16S rRNA gene and allows Prochloron cells in a few ascidian species (e.g. T. clin- for the genetic characterization and identification of ides, T. cyanophorum, and T. paracyclops; Hirose et al., closely related cyanobacterial species and strains (e.g. 2009;Lopez-Legentil et al., 2011;M€unchhoff et al., Erwin & Thacker, 2008). Thus, the conservative nature 2007, respectively). Coexistence of these two (or more) cyanobacteria may not be surprising (Kojima, & Hirose of the 16S rRNA gene may mask host-specific relation- et al., 2012), since both Synechocystis and Prochloron ships between didemnids and Prochloron, as observed share a common ancestor and similar cytological struc- in sponge-associated cyanobacteria (Erwin & tures (Shimada et al., 2003). The two cyanobacteria gen- Thacker, 2008). era also differ in the photosynthetic pigment In this study, we aimed to determine the host-specifi- composition, with Synechocystis containing chlorophyll city of ascidian-Prochloron associations and the influ- a and phycobilin proteins (Neveux et al., 1988) and ence of geographic location on host-symbiont structure. Prochloron species utilizing both chlorophyll a and b as To achieve our goal, we sequenced a fragment of the light-harvesting pigments and lacking phycobiliproteins mitochondrial gene cytochrome oxidase I (COI) to iden- (Lewin & Withers, 1975). tify host genotypes, and partial 16S rRNA and entire More recently, a third cyanobacterial genus with a ITS gene regions to characterize the cyanobacteria unique light-harvesting pigment (chlorophyll d) was inhabiting the tunic of these ascidians. We targeted spe- reported in association with the didemnid Lissoclinum cies in the didemnid genus Lissoclinum because this patella (Miyashita et al., 1996; Miyashita et al., 2003). genus is well known to harbour abundant and diverse These symbionts were related to the genus cyanobacterial symbionts: L. punctatum, L. midui, L. Acaryochloris and subsequently reported to form bio- timorense, and L. patella can harbour Prochloron films on the underside of Lissoclinum and Diplosoma (Behrendt et al., 2012; Hirose & Hirose, 2011; Hirose ascidians (Behrendt et al., 2012;K€uhl et al., 2005), et al., 2014;M€unchhoff et al., 2007; Ohkubo & form clusters within the lower section of Cystodytes del- Miyashita, 2012), while L. patella has also been found lechiajei tunic (Martınez-Garcıa et al., 2011) or be to harbour Acaryochloris spp. and Planktothricoides sp. embedded in the tunic matrix in Lissoclinum fragile (Behrendt et al., 2012; Ohkubo & Miyashita, 2012). (Lopez-Legentil et al., 2011). The role of Acaryochloris Furthermore, L. fragile and L. aff. fragile were found to 498 M. Lopez-Guzman et al. host Acaryochloris bahamiensis (Lopez-Legentil Science. The COI sequence for L. perforatum from et al., 2011). Chile (code C111R) was published in a previous study (Turon et al., 2016), sequences for all other samples have been deposited in GenBank (accession numbers Materials & methods MN586601 to MN586626). Sample collection Twenty-eight ascidian samples representing eight species of the genus Lissoclinum were investigated: L. aff. Cyanobacterial DNA extraction fragile, L. bistratum, L. midui, L. patella, L. perforatum, and sequencing L. punctatum, L. timorense and L. verrilli (Fig. 1). L. DNA extraction was performed on dissected tunic tissue aff. fragile exists in multiple colour morphs and the using the DNeasy Blood and Tissue Kit (Qiagen). The samples collected herein were red, pink and brown, primer set CYA359F and CYA23S1R (Iteman et al., while samples of this species analysed previously in 2000;N€ubel et al., 1997) was used to amplify the 30 Lopez-Legentil et al. (2011) were green. Samples were end of the 16S rRNA gene, the complete internal tran- collected from four different countries: Japan, the scribed spacer (ITS) gene region between the 16S and Bahamas, Spain, and Chile (Table 1; Fig. 2). Samples 23S rRNA genes, and a small fragment of the 23S were collected by snorkelling or SCUBA diving, imme- rRNA gene (16S-ITS-23S rRNA). Each PCR reaction diately fixed in 100% ethanol and stored at -20 C. Prior contained 0.5 mL of extracted DNA, 0.5 mL of the for- to DNA extraction, each sample was carefully separated ward and reverse primers (10 mM), 12.5 mL of MyTaqTM under a dissecting microscope into zooid and tunic frac- HS Red Mix (Bioline), and 11 mL of water to obtain a tions to maximize ascidian and cyanobacterial DNA final reaction volume of 25 mL. An initial denaturation yield, respectively. Tunic fractions including sections of step at 95 C for 2 min was followed by 35 amplification the cloacal cavity for L. bistratum and L. timorense cycles (denaturation at 95 C for 15 sec; annealing at ( 0.1 cm2) were devoid of epibionts. 50 C for 15 sec; and extension at 72 C for 20 sec), and a final extension at 72 C for 2 min in a MJ Research Ascidian DNA extraction and sequencing Peltier PTC-200 thermal cycler. Purified DNA was cloned in E. coli using the TOPO DNA extraction was performed on dissected zooids TA Cloning kit (Invitrogen, Life Technologies) accord- using the DNeasy Blood and Tissue Kit (Qiagen). PCR ing to the manufacturer’s instructions. Five to ten separ- amplification of a fragment of the mitochondrial ate clones per ascidian sample were screened by PCR Cytochrome c Oxidase subunit I (COI) gene was done using the primer sets LCO1490 and HCO2198 (Folmer using the plasmid primers T3 and T7 (included in the kit) and a total reaction volume of 25 mL: 0.5 mL of each et al., 1994) or Tun_forward and Tun_reverse2 TM (Stefaniak et al., 2009). PCR amplifications were per- primer (10 mM), 12.5 mL of MyTaq HS Red Mix, and 11.5 L of PCR water. The following PCR cycle param- formed with 1 to 2 mL of extracted DNA, 1 mL of the m eters were used: a single denaturation step at 95 C for forward and reverse primers (10 mM), 12 mL of MyTaqTM HS Red Mix (Bioline), and water to obtain a 2 min, followed by 35 amplification cycles (denaturation final reaction volume of 25 mL. For both primer sets, an at 95 C for 15 s; annealing at 50 C for 15 s; and extension at 72 C for 20 s), and a final extension step at initial denaturation step at 95 C for 1 min was followed by 35 to 40 amplification cycles of denaturation at 72 C for 2 min in a MJ Research Peltier PTC-200 thermal cycler. Sequencing for most samples was performed 95 C for 15 sec, annealing at 42 C for the Tun_forward/Tun_reverse2 primers or 47 C for the as described above for ascidian DNA sequencing; how- LCO1490/HCO2198 primers for 15 sec, and extension at ever, successful sequencing of a few cyanobacterial 72 C for 10 sec, followed by a final extension at 72 C products required an additional sequencing reaction with for 1 min. All PCR reactions were carried out in a MJ an internal primer (an equimolar mixture of the primers Research Peltier PTC-200 thermal cycler. PCR products CYA781Fa and CYA781Fb; N€ubel et al., 1997). All were purified using the QIAquick PCR Purification Kit sequences have been deposited in GenBank separately (Qiagen). Sequencing reactions were performed using by data set partition (see below, accession numbers for the BigDyeTM terminator v. 3.1 (Life Technologies) and the 185 sequences of the 16S rRNA gene fragment: the same primer set used during PCR amplification. MN596431 to MN596615; and for the 196 sequences of Automated sequencing was performed on a 3130xl the 16S-ITS-23S rRNA gene fragment: MN596616 Genetic Analyzer available at UNCW Center for Marine to MN596811). Biogeography and host-specificity 499

Figure 1. In situ pictures of the Lissoclinum ascidian species analysed in this study. Species name, colour morph (when appropriate) and sample location are indicated. Arrows indicate ascidians that may be difficult to detect. (A) Lissoclinum aff. fragile (brown), Bahamas; (B) Lissoclinum aff. fragile (red), Bahamas; (C) Lissoclinum aff. fragile (pink), Bahamas; (D) Lissoclinum verrilli, Bahamas; (E) Lissoclinum bistratum, Japan; (F) Lissoclinum midui, Japan; (G) Lissoclinum patella, Japan; (H) Lissoclinum punctatum, Japan; (I) Lissoclinum timorense, Japan; and (J) Lissoclinum perforatum, Chile (Photo credit: X. Turon). Scale bar: 1 cm. Table 1. Ascidians and cyanobacterial DNA sequences analysed in this study. Lopez-Guzman M. 500 16S 16S-ITS-23S Code Species Date Sample Location GPS Coordinates rRNA rRNA

1 Lissoclinum verrilli 10-Jun-08 As77 Sweeting's Cay Reef, Bahamas 2634004"N; 7753012"W NA NA 2 Lissoclinum verrilli 31-May-08 As52 Sweeting's Cay Reef, Bahamas 2638035"N; 7757044"W NA NA 3 Lissoclinum verrilli 3-Jun-08 As58 Great Stirrup Cay, Bahamas 2549031"N; 7753043"W NA NA 4 Lissoclinum aff. fragile 1-Jun-08 As95 Sweeting's Cay Reef, Bahamas 2638035"N; 7757044"W 5 5 5 Lissoclinum aff. fragile 3-Jul-10 As222 Sweeting's Cay Mangrove, Bahamas 2633008"N; 7751041"W 9 9 6 Lissoclinum aff. fragile 5-Jul-10 As240 Sweeting's Cay Reef, Bahamas 2633041"N; 7753003"W 8 8 7 Lissoclinum aff. fragile 8-Jul-10 As256 San Salvador, Bahamas 2435049"N; 7432037"W 11 11 8 Lissoclinum bistratum 13-Jul-11 LBJ1 Unarizaki, Iriomotejima 24250320'N; 123450540'E 6 6 Island, Japan LBJ3 88 9 Lissoclinum bistratum 27-Jul-11 ZLBY1 Yonehara, Ishigakijima Island, Japan 24270280'N; 124110410'E 8 8 ZLBY3 5 12 ZLBY5 89 ZLBY6 25 10 Lissoclinum bistratum 15-Jun-07 A41 Bise, Okinawajima Island, Japan 2442040"N; 12752050"E 10 10 11 Lissoclinum midui 23-Mar-12 M1 Shinri-hama, Kumejima 2621000"N; 12642043"E 9 9 Island, Japan M2 99 M3 10 10 12 Lissoclinum patella 25-Jul-14 LPK6 Kurimajima, Miyakojima 2443030"N; 12515017"E 7 7 Islands, Japan LPK7 55 LPK8 10 10 13 Lissoclinum punctatum 26-Oct-07 A15 Hirakubo, Ishigakijima Island, Japan 24 36 50"N; 124 19 00"E 9 9 0 0 al. et 14 Lissoclinum timorense 15-Jun-07 A42 Bise, Okinawajima Island, Japan 2442034"N; 12752038"E 8 8 15 Lissoclinum timorense 29-Aug-08 A100 Ara-hama, Kumejima Island, Japan 2618055"N; 12646024"E 10 10 16 Lissoclinum timorense 21-Mar-12 T1 Yobuko-hama, Tonakijima 2621029"N; 12708031"E 9 9 Island, Japan T2 99 T3 10 10 17 Lissoclinum perforatum 13-May-12 ZLPS1 Els Caials, Cadaques, 4217007"N; 0317046"E NA NA Catalonia, Spain 18 Lissoclinum perforatum 25-Sep-13 C111R Universidad Catolica del 2957058"S; 7121012"W NA NA Norte, Chile Total: 185 196 Location code (Figure 2), ascidian species, collection date, sample code and collection location and GPS position are indicated. 16S rRNA refers to partial 16S rRNA gene sequences ranging from 716 to 1,146 bp. 16S-ITS-23S rRNA gene sequences include the 3’ end of the 16S rRNA gene, the complete internal transcribed spacer (ITS) gene region between the 16S and 23S rRNA genes, and a small fragment of the 23S rRNA gene. Biogeography and host-specificity 501

Figure 2. Map showing the sampling sites of the Lissoclinum ascidians analysed in this study. Sites are as follow: (1), (2), (4) and (6) Sweeting’s Cay reef; (3) Great Stirrup; (5) Sweeting’s Cay mangrove; (7) San Salvador; (8) Unarizaki, Iriomotejima Island; (9) Yonehara, Ishigakijima Island; (10) Bise, Okinawajima Island; (11) Shinri-hama, Kumejima Island; (12) Kurimajima; (13) Hirakubo, Ishigakijima Island; (14) Bise, Okinawajima Island; (15) Ara-hama, Kumejima Island; (16) Yobuko-hama, Tonakijima Island; (17) Els Caials, Cadaques; and (18) Universidad Catolica del Norte. For further details see Table 1.

Phylogenetic analyses gamma distributed rates among sites, and 1,000 boot- Ascidian COI sequences were aligned with ClustalW strap replicates. The 16S rRNA (data set 1) ML tree was constructed using the GTR G model and 100 implemented in Geneious v8 (Biomatters, Auckland, þ New Zealand; Kearse et al., 2012). Phylogenetic trees bootstrap replicates. ML analyses for the Prochloron and the best model of evolution for the COI sequences 16S-ITS-23S rRNA (data set 2) and the Bahamian 16S- ITS-23S rRNA (data set 3) included the K2 G and K2 were determined using MEGA v5 (Tamura et al., 2011). þ The neighbour-joining (NJ) tree was computed using the evolutionary models, respectively and 100 bootstrap rep- Tamura-Nei model, gamma distributed rates among sites licates. Additional ascidian COI and 16S rRNA cyano- and 1,000 bootstrap replications. A maximum likelihood bacterial sequences to build the trees were retrieved (ML) analysis was performed using the TN93 G from GenBank. model of evolution and 1,000 bootstrap replicates. þ Cyanobacterial sequences were aligned using MAFFT (Katoh et al., 2002) as implemented in Geneious. As Results above, phylogenetic trees and the best models of evolution were determined with MEGA. ML and NJ analyses Ascidian COI phylogeny were performed on three different data sets: 1. Partial A sequence fragment of the COI gene ranging from 532 16S rRNA gene sequences; 2. full 16S-ITS-23S rRNA to 600 bp was obtained for each sample. Phylogenetic sequence amplicons (including the 30 end of the 16S analyses revealed that Lissoclinum sequences formed a rRNA gene, the complete ITS gene region, and a small separate clade from other didemnid species of the gen- fragment of the 23S rRNA gene) from Prochloron era Diplosoma, Didemnum, and Trididemnum (Fig. 3). found in Japanese ascidians, and 3. full 16S-ITS-23S Each Lissoclinum species formed a distinct clade with rRNA sequence amplicons (same gene fragment as in bootstrap support values >50 and, when available, clus- 2.) from cyanobacteria found in Bahamian ascidians. All tered with reference sequences from GenBank (Fig. 3). NJ analyses were done using the Tamura-Nei model, Sequences obtained for L. bistratum from Japan formed 502 M. Lopez-Guzman et al.

Figure 3. Host phylogeny with partial COI gene sequences. Bold lettering represents sequences obtained in this study. Labels on terminal nodes indicate the ascidian species name and sample code (as in Table 1) or GenBank accession number. Location and country of sample provenance is in parenthesis. Tree topology was obtained from neighbour-joining (NJ) analysis with bootstrap support values located on the nodes (only values >50 are shown). Colour coded circles on tree nodes indicate bootstrap values from maximum-likelihood (ML) analysis. The Stolidobranchia species Styela plicata was used as an outgroup taxon. a clade with two well-supported sub-clades (bootstrap (bootstrap values >90 in all analyses). The three sam- values >89 in all analyses): one containing five samples ples analysed here for L. patella were collected in (ZLBY1, ZLBY3, ZLBY6, A41 and LBJ3), and one Okinawa (Japan) and formed a robust clade (bootstrap containing two samples (ZLBY5 and LBJ1, Fig. 3). The values 100 in all analyses) with sequences retrieved two sub-clades of L. bistratum did not strictly corres- from GenBank.¼ The sequence obtained from the one pond to collection location, as samples from the sample available for L. punctatum from Japan formed a Iriomotejima or Ishigakijima Islands were represented in distant clade (bootstrap >90) with all the L. verrilli both clades. Sequences of L. timorense were obtained sequences obtained here from the Bahamas plus a previ- from individuals collected from three different locations ously published L. verrilli sequence from a Panamanian within Japan and formed a single, well-supported clade sample (Fig. 3). Sequences of the Japanese L. midui Biogeography and host-specificity 503

Figure 4. Phylogeny of partial 16S rRNA gene sequences from cyanobacteria in Lissoclinum ascidians. Bold lettering represents sequences of cyanobacterial groups obtained in this study. Labels on terminal nodes indicate the total number of cyanobacterial sequences in a clade, followed by the species name, GenBank accession number, and sequence source or number of sequences obtained here and their location in parenthesis. Tree topology was obtained from maximum likelihood (ML) analysis with bootstrap support values located on the nodes (only values >50 are shown). Circles contain colours indicating the bootstrap support value for neighbour-joining (NJ) analysis. Black triangles indicate that sequences for ascidian photosymbionts are included in the clade. White triangles indicate no ascidian photosymbionts within these groups. Cyanobacterial group names as described in Shih et al. (2013) and the main text. formed a well-supported clade (bootstrap values 100 collected from Chile (C111R) and the one from Spain ¼ in all analyses). L. perforatum sequences obtained from (ZLPS1), nor L. verrilli from the Bahamas (As77, As52 samples collected from Chile and Spain formed a well- and As58). Partial cyanobacterial 16S rRNA gene supported clade (bootstrap values >90 in all analyses) sequences obtained in this study (data set 1, n 185) ¼ within a larger clade that included sequences of L. fra- ranged from 716 to 1,146 bp and were compared gile and L. aff. fragile from the Bahamas obtained to cyanobacterial 16S rRNA gene sequences retrieved herein and retrieved from GenBank. Finally, all L. fra- from GenBank to determine their taxonomic affiliation gile sequences formed a robust clade (bootstrap values (Fig. 4), with cyanobacterial group names following 100 in all analyses; Fig. 3). Shih et al. (2013). All 16S rRNA gene sequences ¼ obtained from Japanese Lissoclinum species formed a single clade with other Prochloron sequences obtained Cyanobacterial phylogeny from GenBank (Fig. 4) and a high bootstrap support Cyanobacterial sequences were obtained from six out of value (>90 in all analyses). The 16S rRNA gene the eight species of Lissoclinum species analysed: L. sequences from the cyanobacteria obtained from the bistratum, L. midui, L. punctatum, L. timorense, L. Bahamian L. aff. fragile represented a novel cyanobac- patella from Japan and L. aff. fragile from the terial lineage within Group C and, in particular, were Bahamas. No positive amplification for cyanobacterial most closely related to Group C3 (Fig. 4). Group C3 is DNA was obtained for the sample of L. perforatum comprised of common cyanobacterial genera such as 504 M. Lopez-Guzman et al.

Figure 5. Phylogeny of 16S-ITS-23S rRNA gene sequences obtained for Prochloron in Japanese ascidians. 16S-ITS-23S rRNA sequences include the 3’ end of the 16S rRNA gene, the complete internal transcribed spacer (ITS) gene region between the 16S and 23S rRNA genes, and a small fragment of the 23S rRNA gene. Clades A-I contain sequences obtained in this study and are in bold lettering. Labels on terminal nodes indicate clade name, number of sequences in each clade, ascidian host species, and sample code and location in parenthesis. Clade J contains sequences retrieved from GenBank for Trididemnum cyanophorum photosymbionts (accession numbers: JF506246, JF506247, JF506250, JF506252). Tree topology was obtained from maximum likelihood (ML) analysis with bootstrap support values located on the nodes (only values >50 are shown). Circles contain colours indicating the bootstrap support value for NJ analyses. Outgroup contains reference sequences from uncultured Synechocystis spp. (GenBank accession numbers: JF506237, JF506241, JF506243).

Synechococcus and Leptolyngbya, and includes many Overall, there was a high degree of host-specificity. sequences obtained from marine invertebrate photosym- Clade A contained Prochloron sequences from all five bionts. One cyanobacterial sequence from a sample of samples of L. timorense (T1-T3, A42, A100) collected L. bistratum (LBY6) formed a well-supported clade from three different locations in Japan (Yokubo-hama, (bootstrap values >90) with a sequence from the genus Ara-hama and Bise; Fig. 5). Other sequences obtained Planktothricoides and other uncultured cyanobacteria for L. timorense (samples T1-T3, A42) were grouped in found in sediment and a sulfidic spring (Fig. 4). Clade F and originated from two islands in Japan The 163 sequences obtained to build the Prochloron (Yobuco-hama and Bise; Fig. 5). The Prochloron sym- 16S-ITS-23S rRNA phylogenetic tree (data set 2) ranged biont sequences obtained for L. bistratum formed four from 507 to 1,522 bp, and formed 9 distinct and well- clades (Clade B, C, D, and E). Clade B contained supported symbiont clades (Clades A to I; Fig. 5), some Prochloron sequences obtained from a single sample of of which corresponded to particular species and sam- L. bistratum (ZLBY5), Clade C contained sequences pling sites. The Prochloron ITS gene region was 5 from two samples (LBJ1, ZLBY5), Clade D from four times more variable than the Prochloron 16S rRNA samples (A41, ZLBY1, ZLBY3, ZLBY6), and Clade E gene portion sequenced here, averaging 5.6% divergence from all samples except LBJ1, spanning the 3 locations between clades (compared to 1.2% for the 16S rRNA). where this species was collected (Fig. 5). Photosymbionts Biogeography and host-specificity 505

(Clade G and H), both of which contained cyanobacteria sequences from all three samples (M1-M3; Fig. 5). Finally, Prochloron sequences obtained for L. punctatum (A15) were distributed in Clade A (7 sequences) and Clade I (2 sequences). Phylogenetic analyses based on 33 16S-ITS-23S rRNA cyanobacteria sequences ranging from 1,552 to 1,721 bp for the Bahamian ascidian host L. aff. fragile (data set 3) and sequences retrieved from GenBank for L. fragile formed 3 symbiont clades (Fig. 6). Sequences for L. fragile photosymbionts formed a well-supported clade (>90 bootstrap support), while the majority of cyanobacteria sequences obtained from the three L. aff. fragile samples (As95, As240, As256) collected from reefs (Sweeting’sCayReefandSan Salvador) were closely related and clustered together (Fig. 6). Similarly, all cyanobacteria sequences obtained from the L. aff. fragile host (As222) collected from a mangrove were clustered together in a well-supported clade (bootstrap values >70 for both analyses; Fig. 6).

Discussion Cyanobacterial symbionts were present in the Japanese species Lissoclinum bistratum, L. timorense, L. midui, L. punctatum, L. patella and the Bahamian L. aff. fragile, and were not detected in L. perforatum from Chile and Spain, and L. verrilli from the Bahamas. 16S rRNA gene sequences showed that the Japanese ascidians hosted Prochloron cyanobacteria, while the Bahamian ascidian hosted a novel cyanobacterial lineage previously retrieved from L. fragile (also from the Bahamas; Lopez-Legentil et al., 2011) but of unknown cyanobacterial affiliation. ITS sequences exhibited higher vari- ability than 16S gene sequences and revealed fine-scale patterns of host-specificity and location effects. 16S- ITS-23S rRNA sequences from Prochloron symbionts formed 9 well-supported and distinct clades (Clades A to I), some formed by sequences from a particular spe- Figure 6. Phylogeny of 16S-ITS-23S rRNA gene sequences cies and location (e.g. Clades B, G, H) and others by obtained for cyanobacteria in Lissoclinum aff. fragile from the multiple species or locations (e.g. Clade A, E). 16S- Bahamas.16S-ITS-23SrRNAsequencesincludethe3’ end of the ITS-23S rRNA sequences from L. aff. fragile symbionts 16S rRNA gene, the complete internal transcribed spacer (ITS) gene region between the 16S and 23S rRNA genes, and a small fragment collected from reefs clustered separately from those of the 23S rRNA gene. Bold lettering represents sequences obtained obtained from a mangrove habitat. in this study. Terminal nodes contain the ascidian host species name, Lissoclinum species are often found in association code (Table 1)andcollectionhabitat(reefvs.mangroves).Tree with cyanobacterial symbionts, in particular the genera topology was obtained from maximum likelihood (ML) analysis with Prochloron or Synechocystis (Erwin et al., 2014; Hirose bootstrap support values located on the nodes (only values >50 are shown). Circles contain colours indicating the bootstrap support value et al., 2012; Kott, 2001;Lopez-Legentil et al., 2011). for NJ analyses. All sequences retrieved from GenBank for L. fragile However, in this study, no photosymbionts were are of an uncultured cyanobacterium. detected by PCR amplification in two of the Lissoclinum species investigated: the Caribbean L. ver- for L. patella (LPK6-LPK8) were also grouped within rilli and L. perforatum collected in Chile and Spain. In Clade E (Fig. 5). Prochloron sequences retrieved from L. an earlier study from the Caribbean, Hirose et al. (2012) midui were distributed in two closely related clades reported both Prochloron and Synechocystis 506 M. Lopez-Guzman et al. photosymbionts in the tunic surface of L. verrilli from within the same clade (Clade E), but were not specific to Panama forming a facultative association. The authors the species. Similarly, some cyanobacterial sequences for collected individuals from the species that were exposed the remaining two ascidian species (L. punctatum and L. to the light and reported a greenish pigmentation in their timorense) were grouped together in a mixed clade (Clade samples (see Fig. 1 in Hirose et al., 2012). The samples of A), while the others formed host-specific clades (Clade I L. verrilli utilized in the present study were brown and and Clade F, respectively). Thus, most Prochloron white (Fig. 1D), and were collected on the underside of sequences were specific to a given host species, though rocks. The lack of light exposure in these habitats likely some are shared among species, suggesting different explains the absence of cyanobacterial symbionts in these modes of symbiont acquisition. samples and further supports the facultative nature of pho- Species-specific photosymbionts are likely acquired tosymbionts in L. verrilli. To the best of our knowledge, through vertical transmission (reviewed in Hirose, no previous study has investigated the presence of micro- 2015), while others may be harvested from the sur- bial symbionts in L. perforatum. This ascidian species is rounding seawater (M€unchhoff et al., 2007) to maintain mostly white with some orange streaks (Fig. 1J) and com- symbiont genetic diversity (Donia et al., 2006). The monly found in overhangs, thus not directly exposed to mode of symbiont transmission has been studied for all the light. As for the L. verrilli samples analysed here, the Lissoclinum species investigated herein, and suggests a habitat preference of L. perforatum may explain the lack genetic basis for transmission mode reflected in the of photosymbionts, although further samples need to be symbiont phylogeny (Fig. 5, Clades A to I). L. puncta- analysed to confirm the lack of symbionts found herein. tum harbours Prochloron cells (Clades A and I) intracel- In addition to differences in habitat preference, overall lularly in the tunic and extracellularly in the variation in ascidian coloration has also been correlated peribranchial and common cloacal cavities; however, with symbiont composition (e.g. Hirose et al., 2014; Prochloron cells were not present in the gametes or the Lewin & Cheng, 1975). Here, no Acaryochloris baha- embryo and were only acquired when the hatching lar- miensis photosymbiont was detected in our brown, red or vae passed through the common cloacal cavity (Kojima pink L. aff. fragile samples, while Lopez-Legentil et al. & Hirose, 2010). Similarly, Prochloron symbionts in L. (2011) reported A. bahamiensis as the main photosym- timorense (Clades A and F) and L. bistratum (Clades B, biont in green color morphs of L. aff. fragile collected in C, D, and E) were only acquired when the larvae the same area and habitat. hatched into the common cloacal cavity in which many Phylogenetic analyses revealed that all except for one Prochloron cells occurred (Hirose & Nakabayashi, of the 16S rRNA sequences obtained here belonged to 2008; Hirose et al., 2006, respectively). However, unlike two well defined and well-supported cyanobacterial for L. punctatum, in L. bistratum and L. patella (Clade clades. The first clade was exclusively composed of E), Prochloron cells were exclusively distributed in the Prochloron sequences, and the second consisted of all peribranchial and cloacal cavities and absent in the tunic sequences from L. aff. fragile and other marine inverte- (Hirose, 2015). Interestingly, in L. midui, Prochloron brate symbionts, yet no cultured cyanobacterial species. symbionts form unique genetic clades (Clades G and A single cyanobacterial sequence most closely related to H), are extracellular, and are exclusively found in the Planktothricoides raciborskii was obtained from one tunic (Hirose & Hirose, 2011). Phylogenetic analyses sample of L. bistratum (Ishigakijima Island, Japan), pos- based on the COI gene revealed that this species occu- sibly representing a transient species or denoting a fac- pied a basal position relative to the other 4 Lissoclinum ultative association. Further analyses of the Prochloron species from Japan (Hirose & Hirose, 2011; here), more clade utilizing the ITS gene region revealed a high closely related to the aposymbiotic L. perforatum from degree of host-specificity between ascidians and their Chile and Spain, and L. aff. fragile from the Bahamas. cyanobacterial symbionts. For instance, the two well- The high degree of host-specificity observed here for L. supported clades of L. bistratum obtained in the ascidian midui and its cyanobacterial symbionts further confirms COI phylogeny hosted two different cyanobacterial com- that Prochloron cells are transmitted vertically to the munities (Clades B-C, and Clades D-E). Ascidian sam- progeny (Hirose & Nozawa, 2020). Finally, L. verrilli is ples ZLBY5 and LBJ1 had Prochloron symbionts from known to form facultative associations with Prochloron, Clade B and C, while photosymbionts for all the other where the cyanobacterial cells are loosely distributed on L. bistratum samples formed Clade D and E. Host- the colony surface (Hirose et al., 2012; Hirose, 2015). specificity was also observed for L. midui, with all Here, no Prochloron sequences were obtained from our Prochloron sequences obtained here forming two distinct samples supporting the facultative nature of the associ- clades that were unique to this ascidian species. All ation and the likely acquisition of these symbionts by Prochloron sequences obtained for L. patella grouped horizontal transmission. Biogeography and host-specificity 507

The cyanobacterial sequences obtained here from all Collection of Bahamian samples was conducted with the Bahamian specimens of L. aff. fragile belonged to a novel permission of the Government of the Bahamas. cyanobacterial lineage that was unrelated to common didemnid photosymbionts (Prochloron and Synechocystis). The clade consisted exclusively of uncul- Disclosure statement tured cyanobacteria retrieved from a range of sources, No potential conflict of interest was reported by including the ascidian L. fragile (Lopez-Legentil et al., the author(s). 2011), other invertebrate hosts (sponges, corals), and bio- films. No further information regarding phylogenetic position was discernible for this clade; although it was most closely related to the cyanobacterial Group C3. Group C3 Funding is formed by Synechococcus and Leptolyngbya species Research trips to the Bahamas were funded by a grant (Shih et al., 2013), however, this relationship was poorly from the US National Science Foundation (OCE supported (bootstrap values <50). Interestingly, analysis 0550468) to Joseph R. Pawlik (UNCW, USA). of the 16S-ITS-23S rRNA gene region for symbionts Additional funding was provided by the Spanish within this novel cyanobacterial clade revealed a high Government grant CTM2017-88080 (AEI/EDER, UE). degree of host-specificity related to the habitat where each ascidian sample was collected (mangrove vs. reef). Mangrove habitats are characterized by turbid and nutrient-rich waters, while reef environments typically exhibit ORCID transparent and nutrient-poor waters. Both light exposure Susanna Lopez-Legentil http://orcid.org/0000-0001- and nutrient availability are important factors for the 8737-6729 physiology of cyanobacterial symbionts. For instance, Prochloron cells isolated from ascidians in light-exposed References and shaded environments exhibited different rates of Alberte, R. S., Cheng, L., & Lewin, R. A. (1986). photosynthesis and respiration (Alberte et al., 1986). Photosynthetic characteristics of Prochloron sp./ascidian Thus, differences in the environmental factors that charac- symbioses. I. Light and temperature responses of the algal terize these two habitats may be sufficient to select for symbiont of Lissoclinum patella. Marine Biology, 90, different photosymbiont strains. 575–587. https://doi.org/10.1007/BF00409278 Behrendt, L., Larkum, A. W. D., Trampe, E., Norman, A., In summary, we investigated 8 species of Lissoclinum Sørensen, S. J., & K€uhl, M. (2012). Microbial diversity of for their cyanobacterial symbionts. Two species had no biofilm communities in microniches associated with the photosymbionts, one (L. verrilli) representing aposymbi- didemnid ascidian Lissoclinum patella. The ISME Journal, otic colonies of species known to form facultative associ- 6, 1222–1237. https://doi.org/10.1038/ismej.2011.181 ations with Prochloron and Synechocystis (Hirose et al., Carpenter, E. J., & Foster, R. A. (2002). Marine cyanobacterial symbioses. In A. N. Rai, B. Bergman, & U. 2012). Six species hosted photosymbionts: the five Rasmussen (Eds.), Cyanobacteria in symbiosis. Springer. Japanese species harboured Prochloron symbionts and Donia, M. S., Fricke, W. F., Partensky, F., Cox, J., Elshahawi, one Bahamian species (L. aff. fragile) harboured a novel S. I., White, J. R., Phillippy, A. M., Schatz, M. C., Piel, J., cyanobacterial lineage. Contrary to previous studies Haygood, M. G., Ravel, J., & Schmidt, E. W. (2011). (Hirose et al., 2009;M€unchhoff et al., 2007), we found a Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis. high degree of host-specificity between ascidian hosts and Proceedings of the National Academy of Sciences of the cyanobacterial symbionts using a highly variable marker United States of America, 108, E1423–E1432. https://doi. (ITS gene region) that revealed cryptic diversity struc- org/10.1073/pnas.1111712108 tured by host identity and habitat. This study highlights Donia, M. S., Hathaway, B. J., Sudek, S., Haygood, M. G., the importance of using sufficiently variable genetic Rosovitz, M. J., Ravel, J., & Schmidt, E. W. (2006). Natural combinatorial peptide libraries in cyanobacterial markers to investigate the evolutionary relationships symbionts of marine ascidians. Nature Chemical Biology, 2, between cyanobacteria and their ascidian hosts. 729–735. https://doi.org/10.1038/nchembio829 Erwin, P. M., Pineda, M. C., Webster, N., Turon, X., & Lopez-Legentil, S. (2014). Down under the tunic: bacterial Acknowledgements biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians. The ISME Journal, 8, Xavier Turon (CEAB-CSIC, Spain) kindly provided 575–588. https://doi.org/10.1038/ismej.2013.188 some of the samples analysed in this study. Melissa Erwin, P. M., & Thacker, R. W. (2008). Cryptic diversity of Smith (scientific illustrator, UNCW) did Fig. 2. the symbiotic cyanobacterium Synechococcus spongiarum 508 M. Lopez-Guzman et al.

among sponge hosts. Molecular Ecology, 17, 2937–2947. and host ascidians. Systematics and Biodiversity, 10, https://doi.org/10.1111/j.1365-294X.2008.03808.x 435–445. https://doi.org/10.1080/14772000.2012.735716 Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. Hirose, E., Uchida, H., & Murakami, A. (2009). (1994). DNA primers for amplification of mitochondrial Ultrastructural and microspectrophotometric characterisation cytochrome c oxidase subunit I from diverse metazoan of multiple species of cyanobacterial photosymbionts invertebrates. Molecular Marine Biology and Biotechnology, coexisting in the colonial ascidian Trididemnum clinides 3, 294–299. (Tunicata, Ascidiacea, Didemnidae). European Journal Fu, X., Do, T., Schmitz, F. J., Andrusevich, V., & Engel, of Phycology, 44, 365–375. https://doi.org/10.1080/ M. H. (1998). New cyclic peptides from the ascidian 09670260802710269 Lissoclinum patella. Journal of Natural Products, 61, Iteman, I., Rippka, R., Tandeau De Marsac, N., & Herdman, 1547–1551. https://doi.org/10.1021/np9802872 M. (2000). Comparison of conserved structural and Goodbody, I. (1974). The physiology of ascidians. Advances regulatory domains within divergent 16S rRNA-23S rRNA in Marine Biology, 12, 121–149. spacer sequences of cyanobacteria. Microbiology, 146, Griffiths, D., & Thinh, L. (1983). Transfer of 1275–1286. https://doi.org/10.1099/00221287-146-6-1275 photosynthetically fixed carbon between the prokaryotic Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002). MAFFT: green alga Prochloron and its ascidian host. Marine and anovelmethodforrapidmultiplesequencealignmentbasedon Freshwater Research, 34, 431–440. https://doi.org/10.1071/ fast Fourier transform. Nucleic Acids Research, 30,3059–3066. MF9830431 https://doi.org/10.1093/nar/gkf436 Hirose, E., & Nakabayashi, S. (2008). Algal symbionts in the Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, larval tunic lamellae of the colonial ascidian Lissoclinum M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., timorense (Ascidiacea, Didemnidae). Zoological Science, Duran, C., Thierer, T., Ashton, B., Meintjes, P., & 25, 1205–1211. Drummond, A. (2012). Geneious Basic: An integrated and Hirose, E. (2009). Ascidian tunic cells: morphology and extendable desktop software platform for the organization functional diversity of free cells outside the epidermis. and analysis of sequence data. Bioinformatics (Oxford, Invertebrate Biology, 128, 83–96. https://doi.org/10.1111/j. England), 28, 1647–1649. https://doi.org/10.1093/ 1744-7410.2008.00153.x bioinformatics/bts199 Hirose, E. (2015). Ascidian photosymbiosis: Diversity of Kojima, A., & Hirose, E. (2010). Transfer of prokaryotic algal cyanobacterial transmission during embryogenesis: symbionts from a tropical ascidian (Lissoclinum punctatum) Photosymbiosis and embryogenesis. Genesis (New York, N.Y.: colony to its larvae. Zoological Science, 27, 124–127. 2000)), 53 121–131. https://doi.org/10.1002/dvg.22778 https://doi.org/10.2108/zsj.27.124 Hirose, E., Adachi, R., & Kuze, K. (2006). Sexual Kojima, A., & Hirose, E. (2012). Transmission of reproduction of the Prochloron-bearing ascidians, cyanobacterial symbionts during embryogenesis in the coral Trididemnum cyclops and Lissoclinum bistratum, in reef ascidians Trididemnum nubilum and T. clinides subtropical waters: seasonality and vertical transmission of photosymbionts. Journal of the Marine Biological (Didemnidae, Ascidiacea, Chordata). The Biological Bulletin, Association of the United Kingdom, 86, 175–179. https:// 222,63–73. https://doi.org/10.1086/BBLv222n1p63 doi.org/10.1017/S0025315406013002 Kott, P. (2001). The Australian Ascidiacea. Part 4. Hirose, E., & Hirose, M. (2011). A new didemnid ascidian Aplousobranchia (3), Didemnidae. Memoirs of the Queensland Lissoclinum midui sp. nov. from Kumejima Island Museum, 47,1–407. (Okinawa, Japan), with remarks on the absence of a Kuhl,€ M., Chen, M., Ralph, P. J., Schreiber, U., & Larkum, common cloacal system and the presence of an unknown A. W. D. (2005). A niche for cyanobacteria containing organ. Zoological Science, 28, 462–468. https://doi.org/10. chlorophyll d. Nature, 433, 820. https://doi.org/10.1038/ 2108/zsj.28.462 433820a Hirose, E., Iskandar, B. H., & Wardiatno, Y. (2014). Lafargue, F., & Duclaux, G. (1979). Premier example, en Photosymbiotic ascidians from Pari Island (Thousand Atlantique tropical, d’une association symbiotique entre une Islands, Indonesia). ZooKeys, 422,1–10. https://doi.org/10. ascidie Didemnidae et une cyanophyceee Chroococcale: 3897/zookeys.422.7431 Trididemnum cyanophorum nov. sp. et Synechocystis Hirose, E., & Maruyama, T. (2004). What are the benefits in trididemni nov. sp. Annales de l’Institut Oceeanographique, the ascidian-Prochloron symbiosis? Endocytobiosis and 55, 163–184. Cell Research, 15, 51–62. Lewin, R. A., & Cheng, L. (1975). Associations of microscopic Hirose, E., Neilan, B. A., Schmidt, E. W., & Murakami, A. algae with didemnid ascidians. Phycologia, 14, 149–152. (2009). Enigmatic life and evolution of Prochloron and https://doi.org/10.2216/i0031-8884-14-3-149.1 related cyanobacteria inhabiting colonial ascidians. In P. M. Lewin, R. A., & Withers, N. (1975). Extraordinary pigment Gault & H. J. Marler (Eds.), Handbook on Cyanobacteria composition of a prokaryotic alga. Nature, 256, 735–737. (pp. 161–189). Nova Science Publishers, Inc. https://doi.org/10.1038/256735a0 Hirose, E., & Nozawa, Y. (2020). Convergent evolution of the Lopez-Legentil, S., Song, B., Bosch, M., Pawlik, J. R., & vertical transmission mode of the cyanobacterial obligate Turon, X. (2011). Cyanobacterial diversity and a new symbiont Prochloron distributed in the tunic of colonial Acaryochloris-like symbiont from Bahamian sea-squirts. ascidians. Journal of Zoological Systematics and PLoS One., 6, e23938. https://doi.org/10.1371/journal.pone. Evolutionary Research. https://doi.org/10.1111/jzs.12370 0023938 Hirose, E., Turon, X., Lopez-Legentil, S., Erwin, P. M., & Markus, J. A., & Lambert, C. C. (1983). Urea and ammonia Hirose, M. (2012). First records of didemnid ascidians excretion by solitary ascidians. Journal of Experimental harbouring Prochloron from Caribbean Panama: genetic Marine Biology and Ecology, 66,1–10. https://doi.org/10. relationships between Caribbean and Pacific photosymbionts 1016/0022-0981(83)90023-0 Biogeography and host-specificity 509

Martınez-Garcıa, M., Koblızek, M., Lopez-Legentil, S., & Schmidt, E. W., Nelson, J. T., Rasko, D. A., Sudek, S., Eisen, Anton, J. (2011). Epibiosis of oxygenic phototrophs J. A., Haygood, M. G., & Ravel, J. (2005). Patellamide A containing chlorophylls a, b, c, and d on the colonial and C biosynthesis by a microcin-like pathway in ascidian Cystodytes dellechiajei. Microbial Ecology, 61, Prochloron didemni, the cyanobacterial symbiont of 13–19. https://doi.org/10.1007/s00248-010-9694-6 Lissoclinum patella. Proceedings of the National Academy Miyashita, H., Ikemoto, H., Kurano, N., Adachi, K., Chihara, of Sciences, 102, 7315–7320. https://doi.org/10.1073/pnas. M., & Miyachi, S. (1996). Chlorophyll d as a major 0501424102 pigment. Nature, 383, 402–402. https://doi.org/10.1038/ Shenkar, N., & Swalla, B. J. (2011). Global diversity of 383402a0 Ascidiacea. Public Library of Science One, 6, e20657. Miyashita, H., Ikemoto, H., Kurano, N., Miyachi, S., & https://doi.org/10.1371/journal.pone.0020657 Chihara, M. (2003). Acaryochloris marina gen. et sp. nov. Shih, P. M., Wu, D., Latifi, A., Axen, S. D., Fewer, D. P., (cyanobacteria), An oxygenic photosynthetic prokaryote Talla, E., Calteau, A., Cai, F., Tandeau de Marsac, N., containing chl d as a major pigment. Journal of Phycology, Rippka, R., Herdman, M., Sivonen, K., Coursin, T., Laurent, 39, 1247–1253. https://doi.org/10.1111/j.0022-3646.2003. T., Goodwin, L., Nolan, M., Davenport, K. W., Han, C. S., 03-158.x Rubin, E. M., … Kerfeld, C. A. (2013). Improving the M€unchhoff, J., Hirose, E., Maruyama, T., Sunairi, M., Burns, coverage of the cyanobacterial phylum using diversity-driven B. P., & Neilan, B. A. (2007). Host specificity and genome sequencing. Proceedings of the National Academy of phylogeography of the prochlorophyte Prochloron sp., an Sciences of the United States of America, 110, 1053–1958. obligate symbiont in didemnid ascidians. Environmental https://doi.org/10.1073/pnas.1217107110 Microbiology, 9, 890–899. https://doi.org/10.1111/j.1462- Shimada, A., Yano, N., Kanai, S., Lewin, R. A., & Maruyama, 2920.2006.01209.x T. (2003). Molecular phylogenetic relationship between two Neveux, J., Duclaux, G., Lafargue, F., Wahl, M., & Devos, L. symbiotic photo-oxygenic prokaryotes, Prochloron sp. and (1988). Pigments of some symbiotic cyanobacteria. Vie Synechocystis trididemni. Phycologia, 42, 193–197. https:// Milieu, 38, 251–258. doi.org/10.2216/i0031-8884-42-2-193.1 N€ubel, U., Garcia-Pichel, F., & Muyzer, G. (1997). PCR Stefaniak, L., Lambert, G., Gittenberger, A., Zhang, H., Lin, primers to amplify 16S rRNA genes from cyanobacteria. Applied and Environmental Microbiology, 63, 3327–3332. S., & Whitlatch, R. B. (2009). Genetic conspecificity of the https://doi.org/10.1128/AEM.63.8.3327-3332.1997 worldwide populations of Didemnum vexillum Kott, 2002. Ohkubo, S., & Miyashita, H. (2012). Selective detection and Aquatic Invasions, 4, 29–44. https://doi.org/10.3391/ai.2009. phylogenetic diversity of Acaryochloris spp. that exist in 4.1.3 association with didemnid ascidians and sponge. Microbes Sybesma, J., Duyl, F. C., & Bak, R. P. M. (1981). The and Environments, 27, 217–225. https://doi.org/10.1264/ ecology of the tropical compound ascidian Trididemnum jsme2.me11295 solidum. III. Symbiotic association with unicellular algae. Pardy, R. L., & Lewin, R. A. (1981). Colonial ascidians with Marine Ecology Progress Series, 6, 53–59. https://doi.org/ prochlorophyte symbionts: evidence for translocation of 10.3354/meps006053 metabolites from alga to host. Bulletin of Marine Science, Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., 31, 817–823. & Kumar, S. (2011). MEGA5: molecular evolutionary Parry, D. L. (1984). Cyanophytes with R-phycoerythrins in genetics analysis using maximum likelihood, evolutionary association with seven species of ascidian from the Great distance, and maximum parsimony methods. Molecular Barrier Reef. Phycologia, 23, 503–505. https://doi.org/10. Biology and Evolution, 28, 2731–2739. https://doi.org/10. 2216/i0031-8884-23-4-503.1 1093/molbev/msr121 Parry, D. L., & Kott, P. (1988). Co-symbiosis in the Turon, X., Ca~nete, J. I., Sellanes, J., Rocha, R. M., & Lopez- Ascidiacea. Bulletin of Marine Science, 42, 149–153. Legentil, S. (2016). Ascidian fauna (Tunicata, Ascidiacea) Pearl, H. W. (1984). N2 fixation (nitrogenase activity) of subantarctic and temperate regions of Chile. Zootaxa, attributable to a specific Prochloron (Prochlorophyta)- 4093, 151–180. https://doi.org/10.11646/zootaxa.4093.2.1 ascidian association in Palau, Micronesia. Marine Biology, Williams, A. B., & Jacobs, R. S. (1993). A marine natural 81, 251–254. product, patellamide D, reverses multidrug resistance in a Schmidt, E. W. (2015). The secret to a successful relationship: human leukemic cell line. Cancer Letters, 71, 97–102. lasting chemistry between ascidians and their symbiotic https://doi.org/10.1016/0304-3835(93)90103-G bacteria. Invertebrate Biology: A Quarterly Journal of the American Microscopical Society and the Division of Invertebrate Zoology/Asz, 134, 88–102. https://doi.org/10. 1111/ivb.12071 Associate Editor: Ana Riesgo