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Systematics and Biodiversity Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tsab20 The end of an enigmatic taxon: Eudoxia macra is the eudoxid stage of Lensia cossack (Siphonophora, ) Mary M. Grossmann a b , Dhugal J. Lindsay a b & Allen G. Collins c a Yokohama City University , Yokohama , 236-0027 , Japan b Japan Agency for Marine–Earth Science and Technology (JAMSTEC) , Yokosuka , 237-0061 , Japan c National Museum of Natural History , Washington , DC , 20013-7012 , USA Published online: 03 Oct 2013.

To cite this article: Mary M. Grossmann , Dhugal J. Lindsay & Allen G. Collins (2013) The end of an enigmatic taxon: Eudoxia macra is the eudoxid stage of Lensia cossack (Siphonophora, Cnidaria), Systematics and Biodiversity, 11:3, 381-387, DOI: 10.1080/14772000.2013.825658 To link to this article: http://dx.doi.org/10.1080/14772000.2013.825658

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Research Article The end of an enigmatic taxon: Eudoxia macra is the eudoxid stage of Lensia cossack (Siphonophora, Cnidaria)

MARY M. GROSSMANN1,2, DHUGAL J. LINDSAY1,2 & ALLEN G. COLLINS3 1Yokohama City University 236-0027, Yokohama, Japan 2Japan Agency for Marine–Earth Science and Technology (JAMSTEC) 237-0061, Yokosuka, Japan 3National Museum of Natural History, 20013-7012, Washington, DC, USA (Received 15 March 2013; revised 25 June 2013; accepted 12 July 2013)

The greatest challenge in the study of the cnidarian order Siphonophora lies in the identification of all parts of the fragile colonies usually separated during net sampling. are usually established based on the morphology of at least one swimming bell of the polygastric stage, and a single genus, Eudoxia, has housed the description of free sexual eudoxid stages until these can be reliably linked to a polygastric stage. This genus, extensively used until the early 20th century, has progressively been emptied and, at the present day, contains only one form for which the identification of the polygastric stage has remained completely enigmatic: Eudoxia macra Totton, 1954. DNA barcoding techniques using the mitochondrial 16S gene allowed this eudoxid stage to finally be linked with its polygastric stage, Lensia cossack Totton, 1941.

http://zoobank.org/urn:lsid:zoobank.org:pub:F253E52B-C14D-4440-9BA5-EB9EAC6B42E0

Key words: DNA barcoding, Eudoxia macra, Lensia cossack, geographic divergence, mitochondrial 16S, Siphonophora

Introduction which may well be conspecific with Pyrostephos vanhoef- The greatest challenge in the study of the cnidarian or- feni Moser, 1925 (Grossmann et al., 2013). der Siphonophora lies in the identification of all parts of The genus Eudoxia (and its precursor Ersaea), exten- the fragile colonies usually separated during net sampling. sively used until the early twentieth century, has progres- The identification of different life stages of species under sively been emptied and, at the present day, contains only different names is not uncommon in the history of bino- one completely enigmatic form: Eudoxia macra Totton, mial . Within the Cnidaria, the Anthomedusae and 1954. The association of eudoxid and polygastric stages Athecata orders, and the Leptomedusae and Thecata orders is primarily done through morphological and distributional separated the medusoid and polypoid forms, respectively, similarities. Indeed, the phyllocyst of the bract of many of species presently regrouped into the and eudoxid stages resembles, in shape, the somatocyst of the Downloaded by [Smithsonian Institution Libraries] at 08:20 17 October 2013 Leptothecata. anterior nectophore of the polygastric stage (Totton, 1965; In the order Siphonophora, species are usually estab- Mapstone, 2009); and polygastric stages having nec- lished based on the morphology of at least one swimming tophores with marked ridges tend to have eudoxid stages bell of the polygastric stage, and a single genus, Eudoxia, with ridged bracts (personal observation: e.g. 5-ridged has housed the description of free sexual eudoxid stages Lensia, genera Diphyes, Eudoxoides, , etc.). Al- until these can be reliably linked to a polygastric stage. No though this identification of eudoxid stages has been per- other life stages have been described unless they could be formed successfully many times in the Mediterranean Sea ´ linked to a known polygastric stage (e.g. Russell, 1938; (e.g. Totton, 1932; Gamulin, 1966; Gamulin & Krsiniˇ c, 2000), where the diversity of Siphonophora is relatively D. Carre,´ 1972; C. Carre,´ 1979), except for the small post-larval physonect stage Mica micula Margulis, 1982, low, the extremely high biodiversity of Japanese waters (Fujikura et al., 2012) would not normally lend itself to this type of enterprise. Applying DNA barcoding techniques to the cnidarian or- Correspondence to: Mary M. Grossmann. E-mail: [email protected] der Siphonophora, the position of Eudoxia macra within the

ISSN 1477-2000 print / 1478-0933 online C 2013 The Author(s). Published by Taylor & Francis. This is an Open Access article. Non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly attributed, cited, and is not altered, transformed, or built upon in any way, is permitted. The moral rights of the named author(s) have been asserted. http://dx.doi.org/10.1080/14772000.2013.825658 382 M. M. Grossmann et al.

family Quoy & Gaimard, 1827 and, more specif- Twenty-two specimens of seven species of the genus ically, amongst the non-ridged Lensia species is discussed, Lensia, and four Eudoxia macra gonophores were col- using both morphological and genetic data from specimens lected using plankton nets and preserved in 99.5% ethanol caught in south-east Asian and Antarctic waters. or frozen at –20◦C onboard after identification to species level (Table 1). Frozen specimens were transferred to 99.5% Material and methods ethanol before DNA extraction. For 15 specimens (access The formalin-seawater preserved material included four numbers KC78253434, 40–53), total DNA was extracted complete Eudoxia macra eudoxids, nine bracts and 14 using the Qiagen (Valencia, California, USA) DNEasy gonophores collected in 330 μm mesh IONESS (Kitamura Blood & Tissue kit, and a 623 bp segment of the mitochon- et al., 2001) tows in March 2006 off south-eastern Japan; drial 16S gene amplified and sequenced using ‘primer 1’ seven gonophores collected with a 1.13 m diameter fry net and ‘primer 2’ from Cunningham & Buss (1993) with the (330 μm mesh) between October and December 2010 off TaKaRa (Otsu, Shiga, Japan) ExTaq and BigDye kits. Se- the north-eastern coast of Japan; eight gonophores collected quencing was performed on an ABI 3130xl sequencer. For by MOCNESS (Wiebe et al., 1985) from the Sulu Sea in the other 11 samples, DNA extraction, amplification and November 2002 (Nishikawa et al., 2007); and two bracts sequencing were performed following the protocol estab- and four gonophores from MOCNESS tows in the Celebes lished in Collins et al. (2008). Sequence alignment was Sea, November 2002 (Nishikawa et al., 2007). Specimens performed manually using the Se-Al v.2.0a11 software of 23 species of Lensia, from the collections of the Japan (Rambaut, 2002). Intra- and inter-specific genetic variation Agency of Marine-Earth Science and Technology, were also was calculated as the Kimura 2 parameter (K2P) genetic examined. distance using MEGA version 5.05 (Tamura et al., 2011). A

Table 1. Characteristics of the samples sequenced for the present work. Downloaded by [Smithsonian Institution Libraries] at 08:20 17 October 2013 Eudoxia macra, the eudoxid stage of Lensia cossack 383

Figs 1–6. 1–4. Eudoxia macra: 1, 2. Lower view of bract (gastrozooid broken off in Fig. 1). 3. Right lateral view of gonophore. 4. Upper view of gonophore. 5, 6. Lensia cossack (right lateral view). Collected in Japanese waters (south of Kamogawa (1), east of Oshima Island (2), off north-eastern Japan (3, 4), and north of Okinawa (5, 6). Scale bar = 1 mm. Downloaded by [Smithsonian Institution Libraries] at 08:20 17 October 2013

Bayesian analysis was performed on MrBayes version 3.2.1 Results (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, The Eudoxia macra material conformed to that described 2003) under a General Time Reversible (GTR) model, with and illustrated by Totton (1954), being elongate, without 1 million generations, a sampling every 1000 generations, any marked teeth or ridges. The bracts (Figs 1, 2) were con- and a burn-in period of 5000 generations. The convergence ical, with a smooth transition between headpiece and neck of the data was verified using Tracer version 1.5 (Rambaut shield, about 2 mm tall. The distal margin showed a small, & Drummond, 2003). The consensus tree was analysed shallow central notch in some specimens. The hydroecium, in FigTree version 1.4.0 (Rambaut, 2007). A neighbour- reaching to 80% of the bract height (from distal edge of the joining analysis was performed using MEGA version 5.05 neck shield), was enclosed by large lateral flaps of the neck (Tamura et al., 2011), with complete gap deletion. Boot- shield. The phyllocyst, of variable size, was ovoid, extend- strap support was estimated over 1000 replicates. ing from the roof of the hydroecium nearly to the apex of Artwork was created from photographs of preserved the bract. The gonophores (Figs 3, 4) were elongate, 5 mm specimens using Adobe Illustrator CS5.1. 384 M. M. Grossmann et al.

with the five-ridged L. achilles placed as the outgroup: the seven-ridged group containing L. havock Totton, 1941 and L. multicristata (Moser, 1925); the multistriate group with L. exeter Totton, 1941 and L. grimaldi Leloup, 1933, and, finally, Lensia without marked ridges: L. campanella (Moser, 1925) and L. cossack Totton, 1941. Additionally, for the three species for which specimens from the Southern Ocean were obtained, the sequences formed distinct clades according to their geographic origin, and the intra-specific variation between samples from different geographic areas (intra-specific, inter-geographic) was on average six times higher than that between specimens from the same geo- graphic area (intra-specific, intra-geographic) (Fig. 7). Nu- cleotide differences between sequences varied from 9.5% to 21% for inter-species comparisons, from 8% to 12.2% for inter-geographic intra-species comparisons, and from 0% to 6.6% for intra-geographic intra-species comparisons. All Eudoxia macra sequences formed a statistically sup- Fig. 7. Quantile boxplot of the genetic distance (Kimura-2 param- ported clade containing all four Lensia cossack polygas- eter) between species (‘intra-genus’); between individuals of the tric stage specimens (Fig. 8). This clade was subdivided same species from different geographic locations (intra-specific, into two sub-groups, with lower posterior probability val- inter-geographic: ‘intra-species’); and between individuals of the ues, only one of which contained the L. cossack samples. same species from the same geographic location (intra-specific, intra-geographic: ‘intra-geogr.’). These two groups corresponded to small-scale geographic variations, the group containing the L. cossack samples be- ing from waters south of mainland Japan, while the two tall on average, without ridges or mouthplate but with large E. macra samples forming the second group were from longitudinal furrows, and a large bluntly pointed proximal off the north-eastern shores of the main island of Japan. projection directed towards the lower side. Small hydroecial The genetic distances between sequences from these two flaps bordered the flat hydroecial surface of the gonophore, groups were of 0.061 at their highest, well below the aver- at the proximal end, but were undeveloped at the distal (os- age inter-species distances observed for this group (Fig. 7). tial) end. The pedicular canal extended from the gonophore Microscope observations of the sequenced E. macra sam- surface to one side of the apex of the nectosac, as shown ples did not uncover sufficient morphological differences in Fig. 4); it gave rise to four radial canals which each to discriminate two distinct species. passed distally down the nectosac to the ostial region of the gonophore. The nectosac was 40% thinner and 15% shorter than the nectophore. Discussion Due to the morphology of Eudoxia macra, we consid- First described from the Indian and Atlantic Oceans ered Lensia species without marked ridges to be a possible (Totton, 1954), Eudoxia macra has since been found in polygastric stage of it. However, because the eudoxids of all tropical and temperate waters (Totton, 1965; Patriti, multistriate Lensia species have yet to be described, a cou- 1970; Alvarino,˜ 1980, 1981; Zhang, 1984; Daniel, 1985;

Downloaded by [Smithsonian Institution Libraries] at 08:20 17 October 2013 ple of representatives of this group were also included in the Alvarino˜ et al., 1990; Zhang & Lin, 1997; Kitamura, study. Following the results of a study using the mitochon- 2000; Gao et al., 2002; Zhang, 2005; Xu et al., 2008; drial COI gene (Ortman et al., 2010), seven-ridged Lensia Kitamura, 2009). In the present material from along the species were expected to be closely related, and L. achilles north-eastern and south-eastern shores of Japan, the bracts Totton, 1941 was chosen to represent the five-ridged Lensia, and gonophores, of similar size to those reported from the and placed as the outgroup. western Pacific (Zhang, 1984), were up to twice as long as A total of 26 mitochondrial 16S sequences were obtained, those described by Totton (1954) from the Indian ocean, of which four belonged to E. macra. All sequence data or the ones collected in the Sulu and Celebes Seas during have been deposited on GenBank (Table 1). The presence the present study. The samples collected south of Japan in of a barcoding gap between all but the multistriate Lensia March had undeveloped gonads, while samples collected species was verified (Fig. 7). The phylogenetic tree (Fig. 8) in May and June from similar locations possessed mature showed a perfect discrimination between the seven con- or spent gonads. No pigmentation was observed in any of sidered species (Bayesian posterior probabilities = 100%, the live material from Japanese waters. This could possibly neighbour-joining bootstrap ≥ 99%). Three main morpho- represent a geographic variation from the specimens from logically dissimilar Lensia subgroups could be discerned, the Atlantic and eastern coast of Africa described by Totton Eudoxia macra, the eudoxid stage of Lensia cossack 385

Downloaded by [Smithsonian Institution Libraries] at 08:20 17 October 2013 Fig. 8. Bayesian consensus tree based on the mitochondrial 16S gene. Bayesian posterior probabilities (top) and neighbour-joining bootstrap values (bottom) in%. Terminal posterior values, and those inferior to 95% not shown. Scale represents 0.2 substitutions per site.

(1954: 118), which were described to have a manubrium zone, and primarily in the upper 500 m of the water column with a “deeply pigmented tip”. (Margulis, 1971; Zhang, 2005). Based on the sequences of a fragment of the 16S ribo- Although only two non-ridged Lensia were successfully somal RNA gene, Eudoxia macra formed a well-supported sequenced, Lensia campanella and the remaining three clade with the non-ridged Lensia cossack (Fig. 8). The valid species, L. asymmetrica Stepanjants, 1970, L. meteori latter were about 4.5 mm tall, without marked ridges (Figs 5, (Leloup, 1934) and L. subtilis (Chun, 1886) all have known 6), and without the characteristic apical twist observed in eudoxid stages, described by C. Carre´ (1968), Pugh & Pages` preserved L. campanella. The somatocysts were of the same (1997), Gamulin & Krsiniˇ c´ (2000), and Totton (1932), re- general shape as the phyllocysts of E. macra bracts. Both E. spectively. A eudoxid stage associated with Lensia cossack macra and L. cossack are present in all oceans, if not very was briefly described and illustrated by Daniel (1985), but common in the Pacific, mostly in the tropical to subtropical the illustration does not resemble Eudoxia macra, and, as 386 M. M. Grossmann et al.

the listed characters are insufficient to assign it to another stage of the stauromedusa Haliclystus antarcticus Pfeffer, species, this record is considered a nomen nudum. 1889 (Miranda et al. 2010), and an intensive study of the Daniel (1985) tentatively assigned Eudoxia macra to Sul- Leptothecata family Sertulariidae using this gene allowed culeolaria chuni (Lens & van Riemsdijk, 1908). However, both cryptic species complexes and potential synonymies rearing experiments of the latter by Claude Carre´ (1979) to be identified (Moura et al., 2011). For the first time, DNA showed this species released only the gonophores of the barcoding using the mitochondrial 16S gene was success- cormidia, the bracts and gastrozooids remaining attached to fully applied to the taxonomic identification of cnidarians the stem. Additionally, Sulculeolaria cormidia differ mor- of the order Siphonophora. This technique allowed the eu- phologically from Eudoxia macra, the bracts being flattened doxid stage known as Eudoxia macra to finally be linked as in Chuniphyes, curled around the stem so as to form a with its polygastric stage, Lensia cossack. As the database cone, with marked basal teeth. No phyllocyst has so far been of well-characterized medusozoan samples associated with described in Sulculeolaria bracts, while figure 4 in C. Carre´ the mitochondrial 16S gene grows, we expect further im- (1979) shows two lateral bracteal canals. The gonophores provement in this group’s systematics. display clear ostial teeth (Vogt, 1854; C. Carre,´ 1979). The three genetically and morphologically dissimilar groups found within the genus Lensia corresponded to Acknowledgements those previously observed using the mitochondrial COI We are grateful to the reviewers for critical and constructive gene (Ortman et al., 2010), and may, once a sufficiently comments on the manuscript and nomenclature employed. representative genetic database is established, serve as a We also thank Drs Katsunori Fujikura and Hiroyuki Ya- solid base for a splitting of the genus Lensia in order to mamoto of the BioGeos Research team, JAMSTEC, for obtain new, monophyletic genera. Another important result their support, and Dr Jun Nishikawa of the Atmosphere and from the present study was the quantification of intra- Ocean Research Institute of Tokyo University for his help specific distances observed amongst taxa from different in collecting many of the samples. This study is a contri- geographic regions, and the separation of the sequences bution to the Census of Marine (CMarZ), an of a given species into distinct clades according to their ocean realm field project of the Census of Marine Life, geographic origin, and this even at a regional scale. Small- and the International Network for Scientific Investigations scale geographic differences may also explain the higher of Deep-Sea Ecosystems (INDEEP). This work was par- genetic distance (0.12) found between the two Japanese tially funded by Japan Society for the Promotion of Science L. achilles samples. Indeed, although both were sampled (JSPS) KAKENHI, grant number 24248032, and MEXT in Sagami Bay, KC782554 was collected between 400 and Grant-in-Aid for Scientific Research on Innovative Areas 600 m, a depth at which waters of northern origin such TAIGA (20109003). AGC acknowledges the Smithsonian as the Oyashio Intermediate Water (Senjyu et al., 1998) Laboratories of Analytical Biology, where some of the se- periodically intrude into the Bay, while KC782553 was quences were generated. collected between 750 and 800 m, a more hydrographically stable layer. The depth of the inter-geographic divergences observed in L. achilles, L. havock and L. multicristata, with References K2P genetic distances between 0.16 and 0.25, and between ALVA R I NO˜ , A. 1980. The relation between the distribution of zoo- 8.0% and 12.2% nucleotide differences, were similar to plankton predators and anchovy larvae. CalCOFI Reports 21, the inter-specific variations found between the multistriate 150–160. Lensia, and those between L. campanella and L. cossack, ALVA R I NO˜ , A. 1981. . In: BOLTOVSKY, D., Ed., At-

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