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 inhab- iting 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) bio- synthesis (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 ISSN 1477-2000 print / 1478-0933 online # The Trustees of the Natural History Museum, London 2020. All Rights Reserved. https://dx.doi.org/10.1080/14772000.2020.1776783 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