The Resurrection of the Genus Bergia (Anthozoa, Zoantharia, Parazoanthidae)

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Systematics and Biodiversity

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The resurrection of the genus Bergia (Anthozoa, Zoantharia, Parazoanthidae)

Javier Montenegro, Martyn E. Y. Low & James Davis Reimer

To cite this article: Javier Montenegro, Martyn E. Y. Low & James Davis Reimer (2015): The resurrection of the genus Bergia (Anthozoa, Zoantharia, Parazoanthidae), Systematics and Biodiversity, DOI: 10.1080/14772000.2015.1101028

To link to this article: http://dx.doi.org/10.1080/14772000.2015.1101028

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Published online: 21 Dec 2015.

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Download by: [Ryukyu University] Date: 21 December 2015, At: 17:23 Systematics and Biodiversity (2015), 1À11

Research Article The resurrection of the genus Bergia (Anthozoa, Zoantharia, Parazoanthidae)

JAVIER MONTENEGRO1,3, MARTYN E. Y. LOW2 & JAMES DAVIS REIMER1,3 1Molecular Invertebrate Systematics and Ecology Laboratory, Graduate School of Engineering and Science, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 903-0213, Japan 2Lee Kong Chian Natural History Museum, Faculty of Science, National University of Singapore, 2 Conservatory Drive, Singapore 117377, Republic of Singapore 3Tropical Biosphere Research Center, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 903-0213, Japan (Received 5 May 2015; accepted 20 August 2015)

The genus Bergia was established by Duchassaing de Fonbressin and Michelotti in 1860, for two species, Bergia catelunaris and B. vialactea. Subsequently, in 1903 Duerden recognized these two species as conspecific, and used B. catelunaris in favour of B. vialactea, and transferred B. catelunaris to the genus Parazoanthus. However, over the last decade, it has been found that the genus Parazoanthus is actually polyphyletic and therefore it has gradually been divided and redefined. Based on phylogenetic analyses, Parazoanthus sensu stricto was recently limited to species which form associations with sponges, but it still comprised of three distinctive and monophyletic subclades. Of these clades, one Parazoanthus clade contains the type species for Parazoanthus, P. axinellae, while another clade was recently described as the genus Umimayanthus based on mitochondrial 16S-rDNA sequences. However, the other remaining Parazoanthus clade contains P. catenularis, the original type species of the genus Bergia. Based on a concatenated set of published data for five molecular markers (18S-rDNA, 28S-rDNA, ITS1/5.8S/ITS2-rDNA, 16S-rDNA and COI-DNA), this study confirms the monophyly of Bergia in the context of Parazoanthidae. The phylogenetic analyses strongly support the resurrection of the genus Bergia as a valid taxonomic unit. Key words: Bergia, Parazoanthidae, Parazoanthus, sponge association, Umimayanthus

Introduction on the same sponge two fairly distinct types of growth could be present, and he therefore considered these two The genus Bergia was established by Duchassaing de species as conspeciﬁc. Duerden (1903) synonymized both Fonbressin and Michelotti (1860) and deﬁned by having names, using Bergia catenularis in favour of Bergia via- “very short polyps forming a chain-like incrustation on

Downloaded by [Ryukyu University] at 17:23 21 December 2015 lactea (Low & Reimer, 2011). Additionally, Duerden the surface of sponges, with polyps arising from one (1903) synonymized Bergia with Parazoanthus Haddon another by stolons (propagules), not from a common and Shackleton, 1891 (family Parazoanthidae Delage & membrane, and originating from the upper or cephalic Herouard, 1901) after concluding that Bergia catelunaris part of the polyp” (p. 54 translated by Duerden, 1903) and possessed all the important anatomical diagnostic charac- by having “commensalistic and cateniform habits of colo- teristics that defined the genus Parazoanthus. nization” (p. 54 translated by Duerden, 1903). More recently, based on phylogenetic analyses, the Bergia was originally established to contain two spe- genus Parazoanthus has undergone further modifications. cies, Bergia catelunaris and Bergia vialactea, but after Sinniger, Montoya-Burgos, Chevaldonne and Pawlowski inspecting a large number of specimens, Duerden (1903) (2005) used mitochondrial DNA sequences and found that concluded that “it was very doubtful that any specific sep- the genus Parazoanthus was a polyphyletic group consist- aration can be maintained given the great variability on ing of several independent and well-supported subclades. habits according to age” (p. 26) and based on the fact that In following publications the genus was subsequently revised; Parazoanthus sensu stricto was defined as para- Correspondence to: Javier Montenegro. E-mail: jmontzalez@ zoanthids living on sponges, the genus Antipathozoanthus gmail.com Sinniger, Reimer & Pawlowski, 2010 was erected within

Parazoanthidae for zoantharian species associated with re-established as a valid genus within the family antipatharians, and the genus Hydrozoanthus Sinniger, Parazoanthidae. Reimer & Pawlowski, 2010 was erected within the family Hydrozoanthidae for species associated with hydrozoans. Nevertheless, under this new deﬁnition Parazoanthus was Materials and methods still polyphyletic and consisted of three distinctive monophyletic clades, designated A, B and C in Sinniger et al. Sequences (2010). Subsequently, Montenegro, Sinniger and Reimer Previously published sequences of family Parazoanthidae, (2015) used sequences from the nuclear partial ribosomal including the genera Isozoanthus, Savalia, Bullagummi- internal transcribed spacer 1, 5.8S rDNA, and partial zoanthus, Mesozoanthus, Kauluzoanthus, Hurlizoanthus, internal transcribed spacer 2 (ITS1/5.8S/ITS2-rDNA), Zibrowius, Corallizoanthus, Kulamanamana, Antipatho- nuclear asparagine-linked glycosylation 11 (ALG11- zoanthus, Bergia, Umimayanthus and Parazoanthus, and DNA), mitochondrial 16S ribosomal (16S-rDNA), and sequences of family Epizoanthidae, genus Epizoanthus, mitochondrial cytochrome c oxidase subunit 1 (COI- Gray, 1867, were downloaded from GenBank for 18S- DNA), combined with morphological characteristics, and rDNA, 28S-rDNA, ITS1/5.8S/ITS2-rDNA, 16S-rDNA split clade B from Parazoanthus into the genus Umi- and COI-DNA. For each marker shorter or poor quality mayanthus (see Montenegro et al., 2015). Thus, currently sequences were eliminated and a total of 198 sequences the genus Parazoanthus consists of two clades, with the were used. type species of Parazoanthus, P. axinellae (Schmidt, For the genus Epizoanthus 22 sequences were used; six 1862), within clade A, and P. catenularis (Duchassaing sequences for 18S-rDNA, three sequences for 28S-rDNA, de Fonbressing & Michelotti, 1860), the type species of three sequences for ITS1/5.8S/ITS2-rDNA, seven sequen- the genus Bergia Duchassaing de Fonbressin &Michelotti, ces for 16S-rDNA and three sequences for COI-DNA. 1860, included inside clade C. Inside family Parazoanthidae, for genus Bergia 23 Within the family Parazoanthidae 12 genera are cur- sequences were used: one for 18S-rDNA, three for 28S- rently recognized as valid (Table 1). In this study we use rDNA, nine for ITS1/5.8S/ITS2-rDNA, six for 16S- previously published and publicly available sequence data rDNA, and four for COI-DNA; for Umimayanthus 54 for all genera from ﬁve phylogenetic markers (18S-rDNA, sequences were used: two for 18S-rDNA, two for 28S- 28S-rDNA, ITS1/5.8S/ITS2-rDNA, 16S-rDNA and COI- rDNA, 19 for ITS1/5.8S/ITS2-rDNA, 15 for 16S-rDNA, DNA) to demonstrate that the genus Bergia is a well-sup- and 16 for COI-DNA; for Parazoanthus 53 sequences ported monophyletic taxon, separate from Parazoanthus were used: four for 18S-rDNA, eight for 28S-rDNA, 14 and all other parazoanthid genera, and therefore should be for ITS1/5.8S/ITS2-rDNA, 19 for 16S-rDNA and eight

Table 1. Present classiﬁcation of family Parazoanthidae.

Diagnostic character(s) (Daly et al., 2007; Sinniger et al., 2010, Classiﬁcation 2013; Montenegro et al., 2015)

Zoantharia Gray, 1832 Clonal and soft-bodied polyps with two rows of marginal tentacles Downloaded by [Ryukyu University] at 17:23 21 December 2015 Macrocnemina Haddon & Shackleton, 1891 Fifth pair of mesenteries complete Parazoanthidae Delage & Herouard, 1901 Macrocnemic zoantharians with an endodermal sphincter muscle Antipathozoanthus Sinniger, Reimer & Pawlowski, 2010 DNA, grows exclusively on antipatharians Bullagummizoanthus Sinniger, Ocana~ & Baco, 2013 DNA, insertion/deletion pattern in 16S-rDNA V5 region Corallizoanthus Reimer in Reimer, Nonaka, Sinniger & Iwase, 2008 DNA, substrate speciﬁcity Hurlizoanthus Sinniger, Ocana~ & Baco, 2013 DNA, insertion/deletion pattern in 16S-rDNA V5 region Isozoanthus Carlgren, in Chun, 1903 No ring sinus, polyps solitary or weak coenenchyme Kauluzoanthus Sinniger, Ocana~ & Baco, 2013 DNA, insertion/deletion pattern in 16S-rDNA V5 region Kulamanamana Sinniger, Ocana~ & Baco, 2013 DNA, insertion/deletion pattern in 16S-rDNA V5 region Mesozoanthus Sinniger & Haussermann, 2009 DNA, absence of biological association Parazoanthus Haddon & Shackleton, 1891 Mesogleal lacuna and canal forming a ring sinus, substrate speciﬁcity (sponges) Savalia Nardo, 1844 Secretion of hard skeleton Zibrowius Sinniger, Ocana~ & Baco, 2013 Sand incrustation in the ectoderm, DNA insertion/deletion pattern in 16S-rDNA V5 region Umimayanthus Montenegro, Sinniger & Reimer, 2015 DNA, 14bp deletion C 9bp insertion in 16S-rDNA region The resurrection of Bergia 3

for COI-DNA. For the remaining genera a maximum of Phylogenetic analyses two sequences per marker were used, whenever possible, The downloaded nucleotide sequences were initially resulting in a total of 34 sequences: 10 from 18S-rDNA, aligned using ClustalW v2.11 (Larkin et al., 2007). The two for 28S-rDNA, six for ITS1/5.8S/ITS2-rDNA, 16 for alignment was manually reﬁned using Se-Al v2.0a11 16S-rDNA and none for COI-DNA. Additionally, 12 (Rambaut, 2002) and the terminal regions in the align- sequences of undetermined parazoanthids associated to ments were trimmed. In this manner, ﬁve aligned datasets sponges were added to the alignment: two for 18S-rDNA, were generated; 1718 sites of 25 sequences for 18S- none for 28S-rDNA, three for ITS1/5.8S/ITS2-rDNA, rDNA, 780 sites of 18 sequences for 28S-rDNA, 934 sites four for 16S-rDNA and three for COI-DNA. For detailed of 53 sequences for ITS1/5.8S/ITS2-rDNA, 585 sites of information regarding sequences used, see Table 2.

Table 2. List of Zoantharia taxa and their associated GenBank data used in phylogenetic analyses. Sequences which were concatenated are indicated by Ã.

Phylogenetic markers

Taxa names 18S-rDNA 28S-rDNA ITS1/5.8S/ITS2-rDNA 16S-rDNA COI-DNA

Epizoanthidae Epizoanthus Epizoanthus paguricola KC218427Ã KJ483042Ã — AY995928Ã AB247347Ã Epizoanthus scotinus KC218425Ã KJ483043Ã GQ464899Ã GQ464870Ã — Epizoanthus incrustatus — GQ464929Ã GQ464894Ã GQ464865Ã — Epizoanthus arenaceus KC218428Ã — EU591538Ã AY995926Ã AB247348Ã Epizoanthus lindhali KC218426Ã — — EF687816Ã EF672677Ã Epizoanthus couchii KC218429Ã — — AB247343Ã — Epizoanthus vagus — — — AY995927Ã — Epizoanthus ramosus KC218430Ã —— —— Parazoanthidae Isozoanthus giganteus — GQ464931Ã GQ464896Ã GQ464867Ã — Savalia luciﬁca FS815 KC218398Ã —— —— Savalia savaglia HM044299Ã HM044298Ã EU346888Ã AY995925Ã — Savalia savaglia Red Spain — — — HQ110948 — Bullagummizoanthus emilyacadiaarum KC218403Ã — — KC218434Ã — Mesozoanthus fossii Chile2 — — — EF687821 — Mesozoanthus fossii Chile1 KC218409Ã — — EF687822Ã — Mesozoanthus fossii C3 — — EU591545Ã —— Kauluzoanthus kerbyi SH12 KC218404Ã — — KC218435Ã — Kauluzoanthus kerbyi SH14 — — — KC218436 — Ã Ã

Downloaded by [Ryukyu University] at 17:23 21 December 2015 Hurlizoanthus parrishi SH7 KC218402 — — KC218433 — Zibrowius ammophilus SH9 KC218407Ã — — KC218438Ã — Zibrowius ammophilus SH15 — — — KC218439 — Corallizoanthus tsukaharai KC218410Ã — EU035621Ã EU035625Ã, — EU035627 Kulamanamana haumeaae SH2 — — — KC218431Ã — Kulamanamana haumeaae SH3 — — — KC218432 — Kulamanamana haumeaae SH10 KC218399Ã —— —— Antipathozoanthus macaronesicus 18CV1 — — — HM130467Ã — Antipathozoanthus macaronesicus FS107 KC218411Ã —— —— Antipathozoanthus macaronesicus — — EU591556Ã, EU591552 — — Bergia Bergia cutressi — — EU418266, EU418264, EU828759Ã — EU418265Ã, EU418267 Bergia cutressi TOB44 — GQ464917Ã ——— Bergia catenularis NAV59 — — EU418289Ã ——

(continued) 4 J. Montenegro et al.

Table 2. (Continued )

Phylogenetic markers

Taxa names 18S-rDNA 28S-rDNA ITS1/5.8S/ITS2-rDNA 16S-rDNA COI-DNA

Bergia catenularis — — — EU828757Ã — Bergia catenularis TOB37 — GQ464916Ã EU418292 — — Bergia puertoricense NAV58 — GQ464915Ã EU418312Ã —— Bergia puertoricense — — EU591584 EU828758, AB247351Ã AY995933Ã Bergia puertoricense FS211 KC218418Ã —— —— Bergia sp. Senegal — — EU591582Ã EF687820Ã EF672656Ã Bergia sp.5 Sulawesi — — — AY995934Ã EU591627Ã Bergia sp. Gyi12H — — — — KF499721Ã Umimayanthus Umimayanthus parasiticus — — — EU828756Ã, EF672663Ã AY995938 Umimayanthus parasiticus TOB47 — GQ464914Ã EU418306 — — Umimayanthus parasiticus 212 — — GQ848263Ã —— Umimayanthus parasiticus — — EU418306 — — Umimayanthus chanpuru 33J — — KR092680Ã KR092504Ã KR092594Ã Umimayanthus nakama 3J — — KR092643Ã KR092457Ã KR092579Ã Umimayanthus miyabi 70JR — — KR092646Ã KR092454Ã KR092573Ã Umimayanthus chanpuru 16J — — KR092678 KR092469 KR092609 Umimayanthus chanpuru NC Deep1 — — EU591578 EU591605 EU591623 Umimayanthus chanpuru NC Deep2 — — EU591580, EU591579 EU591609 EU591624 Umimayanthus nakama Japan1 — — EU591567 EU591608 EU591630 Umimayanthus nakama Japan2 — — EU591566 — — Umimayanthus nakama JDR2009 — — — — AB247352 Umimayanthus nakama 363JR — — KR092644 KR092458 KR092577 Umimayanthus nakama NIP155 — GQ464913 GQ464884 GQ464855 — Umimayanthus miyabi 179TF — — KR092645 KR092453 KR092570 Umimayanthus miyabi NC Shallow2 — — — EU591606 EU591625 Umimayanthus miyabi NC Shallow3 — — GQ848256, GQ848255 GQ848272 GQ848277 Umimayanthus miyabi NC Shallow1 — — EU591568 EU591607 EU591626 Umimayanthus sp. NC Shallow 383FS KC218419 — — — — Umimayanthus sp. Gyi5H — — — — KF499730Ã Umimayanthus sp. GYi7H — — — — KF499729Ã Umimayanthus sp. NC Deep 393FS KC218420 — — — — Downloaded by [Ryukyu University] at 17:23 21 December 2015 Umimayanthus sp. 47J — — KR092700 — — Parazoanthus Parazoanthus anguicomus — GQ464908Ã EU591574Ã, GQ464880 EF687827Ã, EF672660Ã GQ464851 Parazoanthus axinellae SPAM1 — GQ464907Ã ——— Parazoanthus axinellae Mediterranean — — EU591570Ã —— Parazoanthus axinellae PAU42453Ã — — AF398921Ã, AB247355Ã AY995935 Parazoanthus axinellae Ireland — — EU591571 — EF672659 Parazoanthus capensis SA262 — GQ464909Ã GQ464881Ã GQ464852Ã — Parazoanthus swiftii PAN9 — GQ464912Ã EU418332Ã —— Parazoanthus swiftii — — — EU828755, AB247350Ã AY995936Ã Parazoanthus swiftii FS197 KC218417Ã —— —— Parazoanthus swiftii AS3f — — — — KJ794176

(continued) The resurrection of Bergia 5

Table 2. (Continued )

Phylogenetic markers

Taxa names 18S-rDNA 28S-rDNA ITS1/5.8S/ITS2-rDNA 16S-rDNA COI-DNA

Parazoanthus swiftii 209b — — GQ848258 — — Parazoanthus aff. swiftii PER249 — GQ464911 GQ464883 GQ464854 — Parazoanthus aff. swiftii PER241 — GQ464910 GQ464882 GQ464853 — Parazoanthus darwini 03–290 — — — EU333748Ã — Parazoanthus darwini 04–348 — — EU333802Ã EU333751 — Parazoanthus elongatus NZ — — EU591564 EF687828 EF672662 Parazoanthus elongatus Chile — — EU591565Ã EF687829Ã EF672661Ã Parazoanthus elongatus FS222 KC218414Ã —— —— Parazoanthus aff. juanfernandezii CA128 — GQ464905Ã GQ464878Ã GQ464849Ã — Parazoanthus aff. juanfernandezii FRAPC1 — GQ464904 GQ464877 GQ464848 — Parazoanthus sp. hertwigi FS814 KC218415Ã — — — KC218397Ã Parazoanthus sp. 269 — — — HM130468Ã — Parazoanthus sp. 1401À1409 — — — HM130480Ã — Parazoanthus sp. 1401À1402 — — — HM130479Ã — Parazoanthus sp. 1401 — — — HM130478Ã — Parazoanthid sp. Parazoanthid sp.3 Sulawesi — — EU591575Ã AY995937Ã AB247354Ã Parazoanthid sp. Tasmania — — — EU591610Ã EU591620Ã Parazoanthid sp. Madagascar3 KC218421Ã — EU591576Ã EF687825Ã EF672664Ã Parazoanthid sp. 02À27 — — EU333810Ã EU333760Ã — Parazoanthid sp. AF052893Ã —— ——

67 sequences for 16S-rDNA, and 446 sites of 34 sequen- bootstrap replicate trees were constructed using the same ces for COI-DNA. Alignment masks were generated using method. For ML and BI phylogenetic reconstructions, GBlock v0.91 (Castresana, 2000) to eliminate ambigui- TOPALi v2 (Milne et al., 2009, http://www.topali.org/) ties, poorly aligned positions and divergent regions; the was used to test for the best-fitting model for each gene configuration was set to allow small final blocks, gap posi- partition. The following models were obtained and imple- tions between final blocks and less strict flanking positions mented in all ML and most BI analyses (see exceptions for 16S-rDNA and ITS1/5.8S/ITS2-rDNA. Following below): K80(010010)CI for 18S-rDNA, TrNef(012230)C GBlock editing, five masked alignments were obtained; ICG for 28S-rDNA, K80(010010)CICG for ITS1/5.8S/ 1712 sites of 25 sequences for 18S-rDNA, 737 sites of 18 ITS2-rDNA, K80(010010)CG for 16S-rDNA and TrNef

Downloaded by [Ryukyu University] at 17:23 21 December 2015 sequences for 28S-rDNA, 541 sites of 53 sequences for (010020)CG for COI-DNA; for BI analyses SYMCICG ITS1/5.8S/ITS2-rDNA, 437 sites of 67 sequences for 16S- was used for 28S-rDNA and HKYCI for COI-DNA, as rDNA, and 444 sites of 34 sequences for COI-DNA. The indicated by TOPALi. generated Gblock alignment masks were used to construct ML trees were built using Geneious with the plugin for a concatenated alignment. Any missing data, including PhyML v3.0 (Guindon & Gascuel, 2003) following the gaps, were replaced with “N”. The concatenated align- models and parameters as indicated by TOPALi, with ment consisted of 3871 positions and 48 sequences (see eight gamma-categories of substitution rates. 1000 boot- Table 2 for details on sequences used in the concatena- strap replicates were calculated using the same settings. tion). All alignments are available in the Dryad repository BI trees were built using Geneious with the plugin for (http://datadryad.org) with doi:10.5061/dryad.7b76q MrBayes v3.2.2 (Huelsenbeck & Ronquist, 2001) follow- Phylogenetic reconstructions were performed for each ing the models and parameters as indicated by TOPALi, genetic marker using Neighbor joining (NJ), Maximum with eight gamma-categories of substitution rates. MCMC likelihood (ML), and Bayesian inference (BI). NJ chains were run for 5,000,000 generations with the tem- trees were constructed with the program Geneious v8.1 perature for the heated chain set to 0.2. Chains were sam- (Kearse et al., 2012, http://www.geneious.com) with the pled every 200th generation. Burn-in was set to 1,750,000 HasegawaÀKishinoÀYano genetic distance model generations (35%) for all alignments, at which point the (HKY; Hasegawa, Kishino, & Yano, 1985); 1000 Average Standard Deviation of Split Frequency 6 J. Montenegro et al.

(ASDOSF) values were <0.01. Analyses using ML and BI genera inside the family Parazoanthidae, as well as from were carried out for the concatenated alignment, where the genus Epizoanthus (see Fig. 1). gene partitions were set for ML in RAxML v8 (Stamata- kis, 2014), and for BI as indicated by TOPALi per each Global distribution. Reported from the northern Atlantic marker in MrBayes. Ocean, but our analyses also show unidentified specimens from Sulawesi (EU591627 Sinniger & H€aeusserman, 2009; AY995934, Sinniger et al., 2005) and Taiwan Results (KF499721, Reimer, Irei, Fujii, & Yang, 2013) in the Pacific Ocean. Systematics Family Parazoanthidae, Delage & Herouard, 1901 Remarks. Bergia spp. may be confused with Umimayan- Bergia Duchassaing de Fonbressin & Michelotti, 1860 thus spp., which is morphologically similar but geneti- A resurrected genus. cally clearly distinct. For GenBank sequences belonging to genus Bergia please see Table 2. ZooBank.org LSID: urn:lsid:zoobank.org:act:ABD328EE- F211-4023-AE50-C6F9B87A04D0 Type species: Bergia catenularis Duchassaing de Fon- Individual gene partition phylogenies bressin & Michelotti, 1860, by subsequent designation by ML trees of the individual gene partitions are shown in Duerden (1903). Caribbean Antilles, Atlantic Ocean. Supplementary Data 1 (see supplemental material online). Epizoanthus paguricola was used as outgroup, except in Description: Small polyps forming a chain-like pattern the ITS1/5.8S/ITS2-rDNA analysis in which E. arena- connected over the sponge surface, or scattered polyps ceous was the outgroup. connected under the sponge surface. Bergia includes three In the 18S-rDNA analysis (Supplementary Data 1, see species, the type species B. catenularis, B. puertoricense supplemental material online), the sequences of Bergia (West, 1979) (comb. nov.), and B. cutressi (West, 1979) puertoricense, Umimayanthus spp. and Parazoanthus spp. (comb. nov.), all of which formerly belonged to genus formed a poorly supported clade (ML <50%, NJ D none, Parazoanthus. Until recently B. cutressi was included BI <0.95). Bergia puertoricense was basal to genus Umi- within Epizoanthus, and recently moved by Swain et al. mayanthus and Parazoanthus (ML <50%, NJ D none, (2015) to genus Parazoanthus. In Swain and Wulff (2007) BI <0.95). The Umimayanthus clade was weakly sup- B. catenularis was found to be associated with sponges of ported as sister to Parazoanthid sp. Madagascar3 and the genera Cribrochalina Schmidt, 1870; Neopetrosia De genus Parazoanthus (ML <50%, NJ D none, BI <0.95). Laubenfels, 1949; Petrosia Vosmaer, 1885; and Xesto- Parazoanthus was a monophyletic clade with moderate spongia De Laubenfels, 1932; B. puertoricense was found support (ML D 77%, NJ D 81%, BI D 1.00). to be associated with sponges belonging to the genera In the 28S-rDNA analysis (Supplementary Data 1, see Svenzea Alvarez, Van Soest & Rutzler,€ 2002; Hymeniaci- supplemental material online), all Parazoanthidae sequen- don Bowerbank, 1858; Agelas Duchassaing de Fonbressin ces formed a clade with very strong support (ML D & Michelotti, 1864; and Stromatospongia Hartman, 1969; 100%, NJ D 100%, BI D 1.00). Savalia savaglia formed B. cutressi was associated with Calcifibrospongia Hart- Downloaded by [Ryukyu University] at 17:23 21 December 2015 the sister group to genus Bergia, although with very weak man, 1979; Cribrochalina Schmidt, 1870 and Xestospon- nodal support (ML <50%, NJ D none, BI D none). The gia De Laubenfels, 1932. genus Bergia was a monophyletic group with very strong In vivo images of colonies of B. catenularis and B. support (ML D 97%, NJ D 97%, BI D 1.00). Bergia puertoricense are shown in supplementary data 2 (see cutressi was basal to a well-supported clade composed of online supplemental material, which is available from the B. puertoricense and B. catenularis (ML D 100%, NJ D article’s Taylor & Francis Online page at http://dx.doi. 100%, BI D 1.00). Genus Parazoanthus was paraphyletic org/10.1080/14772000.2015.1101028); images of B. to the inclusion of Umimayanthus. The monophyly of cutressi can be found in Morphbank (http://www.morph Umimayanthus (ML <50%, NJ D 57%, BI <0.95) and bank.net/), image accession numbers 474287 and 474286. the internal relationships within Parazoanthus were only weakly supported. Diagnosis. Bergia can be distinguished from all other In the ITS1/5.8S/ITS2-rDNA analysis (Supplementary zoantharians including Parazoanthus spp., Umimayanthus Data 1, see supplemental material online), all Parazoanthi- spp. and Epizoanthus spp. by a unique deletion of 60 bp dae sequences formed a well-supported clade (ML D (from position 133 to 192 in our alignment) and several 99%, NJ D 100%, BI D 1.00). Genus Parazoanthus was consecutive base substitutions in the 16S-rDNA region. paraphyletic to the inclusion of Umimayanthus and Ber- These characters clearly separate this genus from all other gia. A clade composed of sequences of P. anguicomus, P. The resurrection of Bergia 7

Fig 1. Molecular characterization of the resurrected genus Bergia. Summary of deletions, insertions and base substitutions that charac- terize the different Bergia species along the region 16S-rDNA. The black box in the 16S-rDNA region indicate the 60 bp-long deletion that characterizes the resurrected genus Bergia.

axinellae and P. capensis (ML D 78%, NJ D 100%, BI ML D 68%, NJ D 81%, BI >0.95; ML D 96%, NJ D <0.95) formed the sister group to the remaining Para- 97%, BI D 0.98, respectively). zoanthus, Umimayanthus and Bergia sequences. A second In the 16S-rDNA analyses (Supplementary Data 1, see Parazoanthus clade was formed with P. elongatus and P. supplemental material online), all Parazoanthidae sequen- aff. juanfernandezii (ML D 99%, NJ D 100%, ces were grouped together with strong support (ML D

Downloaded by [Ryukyu University] at 17:23 21 December 2015 BI D 1.00), which formed the poorly supported sister 77%, NJ D 100%, BI D 0.99). All sequences belonging to group to a well-supported clade composed of P. swiftii genus Bergia were in a well-supported clade (ML D 98%, and P. darwini (ML D 100%, NJ D 100%, BI D 1.00). NJ D 71%, BI D 1.00) and formed the sister group to Together these formed the sister group to Umimayanthus Parazoanthus, Umimayanthus and Antipathozoanthus and Bergia clades. Bergia cutressi, B. puertoricense and macaronesicus (albeit nodal support was lacking for this B. catenularis sequences formed a monophyletic group sister-group relationship). The monophyly of Parazoan- with strong support (ML D 88%, NJ D 55%, BI D 1.00); thus was only poorly supported (ML <50%, NJ D 78%, the sequences of B. cutressi were contained within a sin- BI <0.95). Umimayanthus was polyphyletic but support gle clade (ML D 99%, NJ D 100%, BI D 1.00), sister to a was lacking for the monophyly of either of the clades as clade formed of B. puertoricense and B. catenularis (ML well as for their interrelationships. D 100%, NJ D 100%, BI D 1.00). Umimayanthus formed In the COI-DNA analyses (Supplementary Data 1, see a poorly supported paraphyletic assemblage which supplemental material online) all sequences belonging to included unidentiﬁed parazoanthid species from Madagas- family Parazoanthidae were grouped together in a single car, Sulawesi and Bergia sp. Senegal (ML <50%, NJ D clade with strong support (ML D 100%, NJ D 100%, none, BI D 0.83). Within this assemblage, U. chanpuru, BI D 1.00). The sequences belonging to Bergia were con- U. miyabi and U. nakama each formed well-supported tained in a single clade with moderate support (ML D monophyletic groups (ML D 98%, NJ D 98%, BI D 0.99; 74%, NJ D 73%, BI <0.95). The genera Parazoanthus 8 J. Montenegro et al.

and Umimayanthus were not supported as monophyletic sp.3 Sulawesi and Parazoanthid sp. Madagascar3 (ML D groups, but U. nakama, U. miyabi and Umimayanthus sp. 70%, BI D none); within this genus, only the sister-group JDR 2009 formed a clade (ML D 64%, NJ D 64%, relationship between U. nakama and U. miyabi received BI <0.95), within which U. miyabi formed a moderately strong nodal support (ML D 94%, BI D 1.00). All Para- supported clade (ML D 64%, NJ D 70%, BI <0.95). zoanthus sequences formed another clade with weak support (ML D 77%, BI <0.95), and were sister to the Bergia and Umimayanthus clades (ML D 86%, BI D 1.00). Phylogeny of the concatenated dataset The phylogenetic reconstruction using the alignment con- catenation of the 18S-rDNA, 28S-rDNA, ITS1/5.8S/ITS2- Discussion rDNA, 16S-rDNA and COI-DNA is shown in Fig. 2.The The 16S-rDNA region, despite being relatively slowly concatenated sequence of Epizoanthus paguricola was evolving in comparison with the ITS1/5.8S/ITS2-rDNA used as outgroup. All Parazoanthidae sequences formed a region in Anthozoa (Huang, Meier, Todd, & Chou, 2008; well-supported clade (ML D 100%, BI D 1.00). The Shearer, van Oppen, Romano, & Worheide,€ 2002), has monophyly of Bergia was strongly supported (ML D 99%, previously proven to be very useful in studying the phy- BI D 1.00), although its sister-group relationship to Umi- logeny and taxonomy of the family Parazoanthidae mayanthus was only poorly supported. Bergia catenularis (Sinniger et al., 2010) in general, and for the genus Umi- and B. puertoricense were sisters with strong support (ML mayanthus in particular (Montenegro et al., 2015). In this D 97%, BI D 1.00). Umimayanthus was paraphyletic to study, all Bergia spp. sequences had a unique 60 bp-long the inclusion of Parazoanthid sp. Tasmania, Parazoanthid deletion that clearly distinguished this genus from all

Isozoanthus giganteus 100 Parazoanthid sp 02-27 \\ Mesozoanthus fossii 100 Antipathozoanthus macaronesicus \\ Zibrowius ammophilus Parazoanthid sp. 100 Savalia savaglia Savalia lucifica Kulamanamana haumeaae Hurlizoanthus parrishi Bullagummizoanthus emilyacadiaarum Corallizoanthus tsukaharai

Kauluzoanthus kerbyi Parazoanthidae 58 \\ Bergia sp. Senegal Bergia sp. Gyi12H 87 Bergia sp.5 Sulawesi Bergia 99 97 Bergia catenularis Bergia puertoricense 53 Bergia cutressi 94 Umimayanthus miyabi Umimayanthus nakama 60 Umimayanthus sp GYi7H 70 Umimayanthus parasiticus Umimayanthus Parazoanthid sp. Madagascar3 Umimayanthus chanpuru Umimayanthus sp. Gyi5H 86 78 Parazoanthid sp. Tasmania Parazoanthid sp.3 Sulawesi 57 Parazoanthus sp hertwigi FS814 Downloaded by [Ryukyu University] at 17:23 21 December 2015 67 56 Parazoanthus elongatus Parazoanthus aff. juanfernandezii Parazoanthus sp. 269 95 69 Parazoanthus anguicomus 77 Parazoanthus capensis 75 Parazoanthus axinellae Parazoanthus 78 Parazoanthus darwini Parazoanthus sp. 1401-1409 Parazoanthus sp. 1401 Parazoanthus sp. 1401-1402 Parazoanthus swiftii Epizoanthus ramosus 99 Epizoanthus vagus Epizoanthus couchii Epizoanthus arenaceus Epizoanthus incrustatus Epizoanthidae Epizoanthus lindhali Epizoanthus scotinus Epizoanthus paguricola

0.04 ML / NJ

Fig 2. Maximum-likelihood tree obtained using the concatenated and masked alignments of 18S-rDNA, 28S-rDNA, ITS1/5.8S/ITS2- rDNA, 16S-rDNA, and COI-DNA. Values at branches represent bootstrap ML values over 50%. Bayesian posterior probabilities over 0.95 are represented by thicker branches. For GenBank accession numbers see Table 2. The resurrection of Bergia 9

other genera within the family Parazoanthidae, and from 2013). Bergia has similar polyp diameters and tentacle genus Epizoanthus (Fig. 1). Additionally, in the phyloge- numbers to Umimayanthus, which are both smaller/less netic analyses of the 16S-rDNA region Bergia formed an numerous than in Parazoanthus. The genus Bergia has independent and well-supported monophyletic clade. polyp diameter ranges of 1À1.5 mm and tentacle numbers Our results support the hypothesis made by Swain and from 20À24, while Umimayanthus has polyp diameter Wulff (2007) about the paraphyletic status of genus Epi- ranges 0.16À2.3 mm and 14À22 tentacles. On the other zoanthus in relation with B. cutressi and give a congeneric hand, Parazoanthus species have polyp diameters of 2À8 explanation to the convergent symbiotic association of B. (»10) mm and 24À48 tentacles (Montenegro et al. 2015). catenularis and B. cutressi with sponges of genus Cribro- Bergia species also have similar marginal muscle arrange- chalina (C. dura and C. vasculatum) and Xestospongia ments to those found in Parazoanthus spp. (branchiform (X. muta and Xestospongia spp.), as indicated by Swain endodermal, to the exception of B. cutressi, Swain et al., and Wulff (2007). Additionally, our phylogenetic analyses 2015). At the moment Bergia is the only parazoanthid support the recent decision by Swain et al. (2015) to trans- genus that has been reported in the literature to be associ- fer B. cutressi from the genus Epizoanthus to Parazoan- ated with the sponge genera Svenzea, Petrosia, Xestospon- thus clade C sensu Sinniger et al. (2010), which is gia, Cribrochalina, Calcifibrospongia, Stromatospongia recognized in this study as genus Bergia. (Swain & Wulff, 2007;Swain,2009). Nevertheless these The phylogenetic position of the genus Bergia in the spongeÀBergia associations should only be used as a family Parazoanthidae remains somewhat unclear, as its guide, rather than as a definitive ecological pattern, given position was variable and/or poorly supported. Neverthe- that some of the reports were extracted from the literature, less, the monophyly of the genus Bergia was very clear in where the sponge species were identified by experts but all analyses, except in the ITS1/5.8S/ITS2-rDNA analysis the Bergia specimen descriptions were incomplete (Swain where Bergia sp. Senegal was presented as a single long & Wulff, 2007). branch. However, Bergia sp. Senegal sequences clearly belong to the resurrected genus Bergia based on the results of our phylogenetic reconstructions of 16S-rDNA Conclusions and COI-DNA (Supplementary Data 1, see supplemental The results of this study bring to an end the polytomic char- material online), the presence of the 60 bp-long deletion acter of the genus Parazoanthus with the recognition of in 16S-rDNA (Fig. 1), and the results of our concatenated Bergia as a valid genus inside the family Parazoanthidae. phylogenetic analysis (Fig. 2). Concatenated phylogenetic The next logical step in Parazoanthidae research is to focus analyses have been shown to be useful at reducing the on the several remaining potentially undescribed species effect of conflicting signals resulting from different within the genera Bergia and Umimayanthus,manyof genetic markers (Garamszegi & Gonzalez-Voyer, 2014), which are present as single, unique sequences in Sinniger et as could be the case in this study with Bergia sp. Senegal, al. (2010), Montenegro et al. (2015), and in this study. Fur- which was clearly supported as part of Bergia in the phy- thermore, as the identity of host sponges in the Caribbean logenetic analyses of regions 16S-rDNA and COI-DNA, region has proven to be an important characteristic for the but not in the ITS1/5.8S/ITS2-rDNA analyses. Overall, taxonomic determination of sponge-associated zoantharians, the results of this study corroborate and support past con- more research on the identification of the associated host clusions from Sinniger et al. (2010). sponges should be undertaken (Swain & Wulff, 2007). Downloaded by [Ryukyu University] at 17:23 21 December 2015 In Sinniger et al. (2010) the genus Bergia (as ‘clade C’) was described as “highly divergent compared with the other Parazoanthus …” (p. 12) and “branching at different posi- Acknowledgements tions within Parazoanthidae in both COI and mt 16S-rDNA trees” (p. 12). Considering our phylogenetic results as well The first author thanks all the members of the MISE labo- as previous literature, we conclude that the genus Bergia is ratory for their support. Dr Tohru Naruse (University of a valid genus within Parazoanthidae, despite showing some the Ryukyus) is thanked for his initial suggestions on the morphological and ecological convergences with the genera status of Bergia. The first author is very grateful to Parazoanthus and Umimayanthus as mentioned by both Dr Andrea Waeschenbach at the Natural History Museum Duerden (1903) and Sinniger et al. (2010). Bergia species (London, UK) for significantly improving the quality of establish symbiotic interactions with sponges as do Para- this study with very accurate comments and zoanthus and Umimayanthus, and also have overlapping recommendations. distributions with Parazoanthus in the Atlantic Ocean (Montenegro, 2015)andwithUmimayanthus in the Pacific Ocean based on two records of unidentified Bergia speci- Disclosure statement mens from Sulawesi (Sinniger & H€ausserman, 2009; Sin- No potential conflict of interest was reported by the niger et al., 2005) and Taiwan (KF499721, Reimer et al., authors. 10 J. Montenegro et al.

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