ISSN 0035-418 Revue suisse de Zoologie (March 2017) 124( 1 ): 167-182 Identification of the polyp stage of three leptomedusa species using DNA barcoding Peter Schuchert1*, Aino Hosia2 & Lucas Leclère3 1 Muséum d'histoire naturelle, C.P. 6434, CH-121I Genève 6, Switzerland 2 The Natural History Collections, University Museum of Bergen, Alléfaten 41, 5007 Bergen, Norway 3 Sorbonne Universités, UPMC Univ. Paris 06, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur- mer (LBDV), 181 chemin du Lazaret, F-06230 Villefranche-sur-mer, France * Corresponding author, E-mail: [email protected] Abstract DNA sequence data of hydromedusae and hydroids collected in the fjords near Bergen, Norway, permitted to connect three leptomedusae to three thecate hydroids with hitherto unknown life cycles. For all three species pairs, identical 16S and 18S sequences could be found. For comparisons, estimates of intraspecific variation of 16S sequences of other leptomedusa species were determined by comparing specimens collected at different localities. The sequence comparisons allowed us to conclude that Ptychogena crocea Kramp & Dumas, 1925 is the medusa stage of the hydroid Stegopoma plicatile (M. Sars, 1863), Earleria quadrata (Hosia & Pages, 2007) the medusa of Racemoramus panicula (G.O. Sars, 1874). and Cyclocanna welshi Bigelow, 1918 the medusa of Egmundella producta (G.O. Sars, 1874). Due to non-matching geographic distribution patterns of the medusa and hydroid phases, as well as the possibility that other related medusa species may have morphologically identical hydroids, the identities of Stegopoma plicatile and Racemoramus panicula are considered ambiguous. These nominal species likely refer to species complexes. Their names are therefore considered as partial synonyms of the medusa-based names and the latter should remain in use despite being more recent. Cyclocanna welshi and Egmundella producta are recognised as synonyms, and the species should from now on be referred to as Cyclocanna producta (G.O. Sars, 1874) n. comb. Keywords: Cnidaria - Hydrozoa - Leptothecata - marine - hydromedusae - hydroids - life cycles. yielded three leptomedusa species whose 16S and 18S INTRODUCTION sequences matched those of polyp stages collected in the The life cycles of many hydromedusae and hydroids same region. remain unknown because they are rare or difficult to Here we present a synthesis of these results with a re- cultivate. DNA barcodes, namely mitochondrial 16S evaluation of the life cycle and taxonomy of the three and COI sequences, have recently emerged as a useful Leptothecata species. tool to unravel hydrozoan life cycles through matching sequences obtained from medusae and hydroids. Two recent papers have thus identified the polyp stage of MATERIAL AND METHODS Oceania armata Kölliker, 1853 (Schuchert, 2016b) and Thecate hydroids were collected on various substrates connected the polyp Boreohydra simplex Westblad, by dredging. Leptomedusae were collected from various 1937 with the medusa Plotocnide borealis Wagner, 1885 depths and locations using plankton nets of different (Pyataeva et al., 2016). sizes (details on the new material are given in Appendix During the last decade, the authors have sampled 1). For depths below 70 m, a modified WP3 plankton net hydroids and medusae in Norway, mostly in the fjords with a non-filtering cod-end and 750 pm mesh size was near Bergen. The specimens were used for various used. For species identification we used Kramp (1959), taxonomic and systematic research, including DNA Cornelius (1995) and other works mentioned in the based phylogenetics and constructing barcode databases for the molecular identification of hydrozoans. A recent Taxonomy part. DNA extracts and 16S sequences were obtained as sampling in the deep waters of Korstjord and Raunefjord Manuscript accepted 08.02.2017 DOI: 10.5281/zenodo.322675 168 P. Schuchert, A. Hosia & L. Leclère given in Schuchert (2005, 2016b). Most 18S sequences 2009, 2015; Schuchert, 2005, 2016b; Zhang et al., were determined as described in Leclère et al. (2009), 2015), but for species-level relationships it is advisable except for three (marked with § in Appendix 1 ) which to include also a nuclear marker in order to exclude were retrieved by BlastN from transcriptome assemblies misleading effects of past introgressions or hybridisations (L. Leclère, C. Dunn, and C. Munro, unpublished data). on the mitochondrial markers (e. g. like in Miller et Alignments and phylogenetic analyses were performed al., 2012). We used the nuclear 18S gene sequence to as given in Schuchert (2005, 2016b). The aligned 16S complement the results obtained using the 16S data, as sequences were trimmed to the position of the 3’ ends they were available from other studies for a good number of the primers SHA/SHB which were used for this study of the species and samples used here. Although 18S is (Cunningham & Buss, 1993). The aligned 18S sequences not an ideal barcoding marker due its relatively low were trimmed to the shortest available sequence. interspecific variability, in the present data set the species Differences between sequence pairs were quantified were sufficiently well separated and the polyp-medusa using p-distances (uncorrected base-pair differences in matches were also seen in with the 18S sequences (Fig. 2, %, Collins et al, 2012) using the software Bioedit (Hall, Table 2), thus adequately complementing the 16S results. 1999). Neither 16S nor 18S resolves satisfactorily the phylo¬ In order to have a wider spectrum of species for genetic relationships at the family level. However, this comparisons, all suitable 16S sequences of leptomedusae was not the aim of this study and will be the subject of a (excluding Campanulariidae) found in the GenBank forthcoming publication using more markers and taxa (L. database were also included in the analyses (GenBank Leclère, unpublished data). accession numbers can be taken from Figs 1-2). The Campanulariidae form a distinct, separate clade (Leclère et al., 2009; Maronna et al., 2016) and were excluded 16S and 18S intraspecific variation from our analysis as they do not contribute additional The substitution rate of the mitochondrial 16S gene varies information. Additional sequencing of COI and 16S was considerably within the Leptothecata subclades (Leclère carried out at the CCDB (http://ccdb.ca/resources/) for et al., 2009; compare also Cunha et al. 2016 for COI). To some medusa specimens, with the sequences deposited quantify the extent of intraspecific variation within the in the BOLD database (Appendix 2). As these 16S leptomedusae, we calculated maximum p-distances for sequences represented the same haplotypes as given in available pairs of conspecific specimens from different Appendix 1, they were not included in the final analysis localities (Table 1). The observed values ranged from in order to avoid dense, unreadable phylogenetic trees. 0.34 to 1.18% and are lower than values observed for other hydrozoans [up to 4.4 % in Oceaniidae (Schuchert, Abbreviations 2016b); 5.5% in Corynidae (Schuchert, 2005); 19.6% BOLD The Barcode of Life Data System (see in Plumularia setacea (Schuchert, 2014), but the Ratnasingham & Hebert, 2007), Plumulariidae have a higher substitution rate (Leclère et http://boldsystems.org al., 2009) and P. setacea could be a species complex]. CCDB Canadian Centre for DNA Barcoding For the 18S sequence only four preliminary estimates for GenBank Genetic sequence database of the National the intraspecific divergences could be obtained (Table 2). Institute of Health, USA, These values allow evaluating sequence divergences http://www.ncbi.nlm.nih.gov/genbank other than perfect matches between polyp and medusa MHNG Muséum d'histoire naturelle de Genève, samples (Table 3). Switzerland Ptychogena crocea and Stegopoma plie at He RESULTS AND DISCUSSION 16S sequences obtained from three P. crocea medusae Maximum likelihood analyses (Fig. 3) and two samples of the hydroid Stegopoma 16S and 18S sequence data were used to obtain plicatile (Fig. 4), all collected in the vicinity of Bergen Maximum Likelihood trees (Figs 1-2) which graphically (Appendix 1), were identical. Likewise, also 18S and visualise inter- and intraspecific sequence divergences. COI sequences were identical (COI : GenBank JN109191, Comparing the length of the branches separating the BOLD specimens HYPN0 314 & HYPN0 286). These samples allows rapid identification of identical or highly results indicate that the hydroid identified as Stegopoma similar sequences found for polyp and medusa stages plicatile is the polyp stage of P. crocea. In the 16S tree, its (Figs 1-2, highlighted in red), as well as the visualisation congener Ptychogena lactea appeared as a sister-species, ot the intraspecific distances seen in a few other species although well separated from P. crocea. Other members (highlighted in blue). of the Laodiceida clade (Maronna et ai, 2016), such as The mitochondrial 16S gene sequence tends to be a Modeeria rotunda, clustered nearby. reliable marker for Hydrozoa (Miglietta et al., 2007, Ptychogena crocea is a distinct and conspicuous medusa DNA barcoding and life cycles of leptomedusae 169 KY363951 Melicertum octocostatum Norway FJ550510 Melicertum octocostatum Norway -FN424119 Stegella lobata polyp Antarctica -FJ550472 Staurodiscus gotoi New Zealand -2ËJ FJ550476 Modeeria rotunda polyp Mediterranean 1JN714674 Modeeria rotunda polyp Gulf of Cadiz PA KJ866187 Ptychogena lactea North Atlantic 21 KT809322 Ptychogena lactea Greenland FJ550513 Stegopoma plicatile polyp Norway 100 73 KKYY336633994549 SPtteygcohpoogmenaa pclriocacteiale Npoorlwypa yN orway Ptychogena crocea KY363953 Ptychogena crocea Norway KY363958 Ptychogena crocea Norway -KY363948 Staurophora mertensii Norway AY512515 Melicertissa spe. Guam ' KY363967 Laodicea undulata Mediterranean 95 KY363963 Laodicea undulata Sweden FJ550471 Laodicea undulata Mediterranean 100 FJ550496 Hebella venusta polyp Honduras LN810549 Clathrozoon wilsoni polyp Okinawa 100 I-FJ418645 Helgicirrha malayensis South China Sea FJ418655 Helgicirrha brevistyla South China Sea AY285162 Eugymnanthea japonica ‘ KY363968 Helgicirrha cari Mediterranean KY363965 Eutima gracilis Sweden FJ550514 Eutima curva New Zealand 100 IKY363937 Eirene viridula Mediterranean 98 LFJ550502 Eirene viridula English Channel KT266605 Blackfordia virginica Brazil AY787897 Octophialucium indicum New Zealand -HM053546 Sugiura chengshanense South China Sea 89 J EU305469 Aequorea victoria USA 1_I AY512518 Aequorea forskalea USA 1 nr>l iKsY\r3-6 3n9 40 A* equo-rea -spec.r E-ngIl —an-d1 FJ418649 Eirene kambara South China Sea 93 -FJ418648 Eirene hexanemalis South China Sea "FJ418650 Eirene lacteoides South China Sea ' FJ418652 Eirene pyramidalis South China Sea -KT266638 Rhacostoma atlanticum Brazil -I KY363964 Eutima gegenbauri Sweden lFJ550515 Eutima gegenbauri Mediterranean 90r FJ418653 Eutima krampi China Sea ' FJ418654 Eutima levuka South China Sea _ioo_| KY363946 Eutonina indicans Norway L KY363938 Eutonina indicans NE Pacific _TKT266613 Eucheilota maculata Brazil "1kY363942 Eucheilota maculata English Channel FJ550501 Eucheilota maculata English Channel ' KY363950 Tima bairdii Norway .1 KY363961 Eucheilota menoni New Zealand 1 FJ550493 Eucheilota menoni New Zealand KY363960 Eucheilota menoni New Zealand KP776776 Eucheilota menoni polyp Bay of Biscay -FJ550460 Calycella syringae polyp English Channel iQQi KY363945 Opercularella lacerata polyp Norway FJ550509 Opercularella lacerata polyp Norway _imr FJ550473 Mitrocomella niwai New Zealand -KX355404 Mitrocomella brownei English Channel T KY363966 Phialella quadrata polyp Chile If FJ550474 Phialella quadrata polyp New Zealand /fi KY363969 Phialella quadrata medusa English Channel 97 KY363962 Phialella quadrata polyp English Channel AY789834 Campanulina pumila polyp USA (misidentification) AY512517 Tiaropsidium kelseyi USA Pacific FJ550511 Racemoramus panicula polyp Norway KY363957 Earleria quadrata Norway . . ,_ 1 KY363952 Earleria quadrata Norway EOrlCTIO gUOUTOtO I KY363956 Earleria quadrata Norway JN714650 Racemoramus panicula polyp Gulf of Cadiz JN714648 Racemoramus panicula polyp Gulf of Cadiz JN714649 Racemoramus panicula polyp Alboran Sea loo j KU512889 Egmundella producta polyp Norway Cyclocanna producta T KY363954 Cyclocanna welshi Norway KY363941 Cyclocanna welshi Norway -I KY363949 Mitrocomella polydiademata Norway '0 KY363939 Mitrocomella polydiademata Scotland ‘ KY363947 Halopsis ocellata Norway ' KY363955 Cosmetira pilosella Norway 0.1 100JKY363943 Tiaropsis multicirrata Norway ^.1550468 Tiaropsis multicirrata Iceland Fig. 1. 16S maximum likelihood phylogenetic tree of leptomedusan species obtained with PhyML (GTR+G+I model) and based on 605 bp positions of the mitochondrial 16S gene. Node-support values are bootstrap values of 100 pseudoreplicates (shown only if > 70%). For more details see text and Appendix 1. Samples based on the polyp stage are indicated, all others are medusa samples. Highlighted in red are matching medusa and polyp stage samples. Highlighted in blue are samples used to estimate intraspecific variability (Table 1). 170 P. Schuchert, A. Hosia & L. Leclère FJ550595 Melicertum octocostatum Norway KY363981 Melicertum octocostatum Norway AY789778 Stegella lobata polyp Antarctica FJ550574 Hebella venusta polyp Honduras 99j KY363982 Earleria quadrata Norway _ . , KY363973 Racemoramus panicula polyp Norway LOrlGTIO QUQOrOtQ too I KY363974 Opercularella lacerata polyp Norway FJ550594 Opercularella lacerato polyp Norway FJ550531 Tiaropsis multicirrata Iceland AF358079 Tiaropsidium kelseyi USA too KY363990 Phialella quadrata English Channel KY363988 Phialella quadrata polyp Chile AY789777 Campanulina pumila polyp USA (misidentification) too KY363976 Egmundella producta polyp Norway 85 Cyclocanna producto 98 KY363984 Cyclocanna welshi Norway — KY363977 Halopsis ocellata Norway 97 J- KY363979 Mitrocomella polydiademata Norway T_f FJ550521 Mitrocomella brownei English Channel FJ550536 Mitrocomella niwai New Zealand 99^EU305493 Eutima sapinhoa FJ418674 Eugymnanthea japonica South China Sea 100 FJ550599 Eutima curva New Zealand I ^71_FF J418675 Eutima krampi South China Sea FJ418676 Eutima levuka South China Sea KY363986 Eutima gracilis Sweden 92 — FJ550600 Eutima gegenbauri Mediterranean 100 KY363971 Eutonina indicans NE Pacific ri 85 KY363975 Eutonina indicans Norway ~ KY363980 Tima bairdii Norway KT722394 Eucheilota maculata Brazil FJ418677 Helgicirrha brevistyla South China Sea [Ç KY363989 Helgicirrha cari Mediterranean FJ418668 Helgicirrha maloyensis South China Sea KT722425 Rhacostoma atlanticum Brazil FJ418673 Eirene pyramidalis South China Sea — FJ550522 Octophialucium indicum New Zealand AF358077 Aequorea victoria USA I AF358076 Aequorea aequorea USA KY363972 Aequorea spec. English Channel I KT722387 Blackfordia virginica Brazil If FJ418670 Eirene kambara South China Sea FJ418671 Eirene lacteoides South China Sea KY363978 Staurostoma mertensii Norway I AF358075 Melicertissa spec. Guam 87[_j KY363985 Laodicea undulata Sweden 100 KY363970Laodicea undulata Mediterranean KY363987 dathrozoon wilsoni polyp Okinawa FJ550535 Staurodiscus gotoi New Zealand FJ550540 Modeeria rotunda polyp Mediterranean 701_I FJ550598 Stegopoma plicatile polyp Norway Ptychogena crocea 100 KY363983 Ptychogena crocea Norway 0.1 Fig. 2. 18S maximum likelihood phylogenetic tree of leptomedusan species obtained with PhyML (GTR+G+I model) and based on 1665 bp positions ot the nuclear 18S gene. Node-support values are bootstrap values of 100 pseudoreplicates (shown only if > 70%). For more details see text and Appendix 1. Samples based on the polyp stage are indicated, all others are medusa samples. Highlighted in red are matching medusa and polyp stage samples. Highlighted in blue are samples used to estimate intraspecific variability (Table 2). DNA barcoding and life cycles of leptomedusae 171 Table 1. Maximal observed intraspecific p-distance values (%) of 16S sequences ot Leptothecata with a medusa stage. distance species and localities_ Eirene viridula Villetranche KY363937 - English Channel FJ550502 0.51 Eucheilota maculata Brazil KT266613 - English Channel FJ550501 0.51 Eucheilota menoni New Zealand FJ550493 - Bay of Biscay K.P776776 0.34 Eutima gegenbauri Mediterranean FJ550515 - Sweden KY363952 0.84 Laodicea undulata Sweden K.Y363963 - Mediterranean FJ550471 0.50 Mitrocomellapolydiademata Norway KY363949 - Scotland KY363939 0.17 Phialella quadrata New Zealand FJ550474 - Chile KY363966 1.18 Tiaropsis multicirrata Iceland FJ550468 - Norway KY363943 0.84 Table 2. Selected examples of maximal observed intraspecific p-distance values (%) of the 18S marker. distance species and localities__ Eutonina indicans Norway KY363975 NE Pacific KY363971 0.24 Laodicea undulata Sweden KY363985 - Mediterranean KY363970 0 Melicertum octocostatum Norway, intrapopulation, KY363981 - FJ550595 0.12 Phialella quadrata English Channel KY363990 - Chile KY363966_ 0 Table 3. Observed minimal and maximal p-distances in %, for details of the used species and samples see Appendix 1. 16S 18S species or species pairs within Ptychogena crocea (KY363953, KY363959, KY363958) 0 one sample within Stegopoma plicatile (FJ550513, KY363944) 0 one sample Ptychogena crocea - Stegopoma plicatile 0 0 within Earleria quadrata (KY363952, KY363957, KY363956) 0.17 - 0.34 one sample within Racemoramuspanicula (JN714650, FJ550511, JN714649, JN714648) 0 - 0.39 one sample Earleria quadrata - Racemoramus panicula 0-0.17 0 within Cyclocanna welshi (KY363954, KY363941) 0.17 one sample within Egmundella producta KU512889 one sample one sample Cyclocanna welshi - Egmundella producta 0 - 0.17 0 and easily identifiable. Its intensively orange-yellow Modeeria rotunda has a reddish-brown manubrium and manubrium and gonad pouches makes it very visible characteristic interradial subumbrellar pockets giving the in plankton samples, even young stages (Fig. 3D). In impression that the manubrium is partly sunken into the addition to its yellow-orange colour, the characteristic mesogloea. lateral perradial stomach diverticula (gonad pouches, Chromatonema rubrum has relatively small, white Fig. 3B) are very large and not attached to the subumbrella. lateral gonad pouches and an orange to brown-reddish Preserved material can nevertheless be confused with manubrium (Kramp, 1919, 1920). In addition, the Modeeria rotunda (Quoy & Gaimard, 1827) and cnidomes of the species differ: while P crocea has 14- Chromatonema rubrum Fewkes, 1882, which also occur 15 pm long microbasic mastigophores, C. rubrum and in deep waters of the North Atlantic (see Kramp, 1919, Modeeria rotunda have microbasic euryteles (19-20 pm 1920, 1959; Russell, 1953; Edwards, 1973; Bleeker & and 25 pm; Russell, 1940). van der Spoel, 1988; Cornelius, 1995; Schuchert, 2001; The congener Ptychogena lactea A. Agassiz, 1865, which also occurs in the NE Atlantic, has gonads on thinner Pagès et al., 2006). 172 P. Schuchert, A. Hosia & L. Leclère Fig. 3. Ptychogena crocea Kramp & Dumas, 1925, living medusae, except C, from Korsfjord, Norway. (A) MHNG-INVE-94101, lateral view, bell diameter 23 mm height 14 mm. (B) Same specimen as in A, close up of gonads. (C) Same specimen as in A, nematocysts. (D) Younger individual, used to obtain DNA isolate 1163. (E) Same as D, close up of bell margin seen from oral side, showing tentacles, tentacle stumps and several cordyli. Fig. 4. Stegopoma plicatile (M. Sars, 1863), preserved sample MHNG-INVE-69614 (yielding DNA isolate 803) from Korsfjord, Norway, 650 m. (A) Whole colony. (B) Hydrotheca. (C) Branch with gonotheca (arrow). ¿ DNA barcoding and life cycles of leptomedusae 173 m m 2 . 0 174 P. Schuchert, A. Hosia & L. Leclère and more numerous lateral diverticula that are attached The related medusa Modeeria rotunda has polyps with to the subumbrella. Their colour differs also from hydrothecae identical to those of Stegopoma plicatile, P. crocea, being white, or with a peachy or greenish tint. but with a colony that remains stolonal according to our More details and illustrations of these two Ptychogena current knowledge (Edwards, 1973; Cornelius, 1995; species will be presented in a forthcoming publication (P. Schuchert, 2001). In the northern Atlantic Ocean, there Schuchert, unpublished data). are two further related medusa species with unknown Ptychogena crocea is a deep-water medusa with a rather polyp stages that potentially have a Stegopoma-like polyp restricted distribution and has so far been reported mostly stage: Ptychogena lactea and Chromatonema rubrum. along the Norwegian coast (Kramp & Dumas, 1925; Naumov (1969) attributed a “Cuspidella”-like hydroid Kramp, 1933, 1961; Rees, 1952; Hosia & Barnstedt, (Bouillon et al., 2006) to Ptychogena lactea because he 2007), but there are also records of single specimens found them to have a similar distribution in the Arctic from the Cape Verde Islands and from the Gulf of Maine Sea. This is, however, not a convincing argument and (Léon et ai., 2007). However, these two records need to the polyp of P. lactea must be considered unknown. be confirmed by new samples as at least the one from the Ptychogena lactea is primarily an Arctic species and Cape Verde had an atypical colour and could have been the distribution of Stegopoma plicatile (Schuchert, Chromatonema rubrum. 2001) also matches its occurrence. The morphologically In contrast, the hydroid Stegopoma p/icati/e has a similar Chromatonema rubrum is a rare oceanic medusa, very wide, circumglobal distribution (Vervoort, 1972; but has a much wider distribution (Kramp, 1919, 1959; Edwards, 1973; Cornelius, 1995; Calder, 2012). It Bleeker & van der Spoel, 1988). It occurs in deep waters is widespread in the Arctic and reaches as far south (406-1750 m; Bleeker & van der Spoel, 1988). To our as Sweden and into deep waters off Brittany. It has knowledge, the medusa has not been found in coastal also been reported in Boreal and Arctic regions of the waters of Norway, but only far off the continent (Kramp, Pacific and scattered points in the Southern Hemisphere 1919; Fraser, 1974; Bleeker & van der Spoel, 1988; and Antarctica. The type material came from Norway. Licandro et al., 2015). Even if the discussed two medusae Although the species has been described repeatedly, do not occur in coastal waters of Norway, they remain the reproduction of S. plicatile remained unknown for candidates for having a Stegopoma-like hydroid. So do a long time until Schuchert (2001) reported for the first the other Ptychogena medusae known from outside the time that it releases a medusa. This was also observed region of the NE Atlantic: P. antarctica Browne, 1907, for one of the current samples (MHNG-INVE-69614) P. californica Torrey, 1909, and P. hyperborea Kramp, which produced medusae of about 2 mm in diameter with 1942. four perradial tentacles, small interradial bulbs and 1-3 When describing Stegopoma plicatile, Sars (1863) had cordyli per quadrant. There were no ocelli or statocysts material from four localities reaching from near Bergen, present. The stomach was distinctly cruciform in cross- through Tromso, to the northern tip of Norway in the section and lacked the yellow colour. Unfortunately, it Barents Sea. To our knowledge, no lectotype has been was not possible to cultivate the medusae for more than selected, and thus a more precise type locality cannot be two days. given. Stegopoma plicatile was also designated by Totton We can conclude from our sequence analyses that (1930) as the type species of the genus. the hydroid from the vicinity of Bergen identified as While it is clear that Ptychogena crocea medusae are Stegopoma plicatile is the polyp stage of Ptychogena produced by hydroids referable to Stegopoma plicatile, crocea. The taxonomic consequences, however, are it is still possible that even the type material of the latter more difficult to assess. The markedly different dis¬ was composed of several species. We therefore refrain tribution patterns of the endemic, medusa-based species from fully synonymising the two names and applying Ptychogena crocea and the widely distributed polyp- the senior name Stegopoma plicatile to the medusa based Stegopoma plicatile strongly suggest that the Ptychogena crocea or suggesting a new combination of latter is most probably a complex of cryptic species. the two names. Synonymising the two names would lead The morphology of the Stegopoma hydroids is rather to a confusing situation in which an endemic medusa simple and poor in diagnostic features (see Cornelius has the name of a circumglobally distributed hydroid 1995; Schuchert, 2001), and it is likely that other and which is likely a species complex. In the synonymy medusae related to Ptychogena crocea might have nearly given below, Stegopoma plicatile is thus taxonomically identical hydroids. Several Leptothecata are known to only considered as a partial synonym. A similar situation have indistinguishable or very similar hydroid stages, was found for the medusa treated in the following. but distinct medusae [e. g. “Cuspidella”-type hydroids producing medusae of the genera Cosmetira Forbes, 1848 or Mitrocomella Haeckel, 1879, “Campanili ina” - Earleria quadrata and Racemoramus panicula type hydroids giving rise to medusae of the genera Eucheilota McCrady, 1859 and Eutonina Hartlaub, 1897 Earleria quadrata medusae (Fig. 5, GenBank number (Cornelius, 1995)]. KY363957) and a Racemoramus panicula hydroid DNA barcoding and life cycles of leptomedusae 175 (GenBank FJ550511) from the same region proved to While Earleria quadrata is only known very locally from have identical 16S (Fig. 1) and 18S sequences (Fig. 2; deep waters of the Korsljord south of Bergen (Flosia & KY363982, KY3639731). Several medusae of Earleria Pages, 2007), the hydroid Racemoramus panicula has quadrata could be examined and they showed low a very wide distribution. It occurs circumglobally in 16S sequence variation of up to 2 bp differences per temperate to tropical regions, at depths of 20-5200 m about 590 bp total length (Fig. 1, Table 3). This is (Schuchert, 2001; Calder, 2012). This extensive range compatible with intraspecific variation observed in other made Calder (2012) doubt whether the observations leptomedusan species (Table 1). Interestingly, three represent a single species, prompting him to exclude 16S sequences derived from Racemoramus panicula records from the Pacific Ocean and attribute them to from the Gulf of Cadiz and the Alboran Sea (identified R. denticulata (Clarke, 1907), a species which so far had by C. Moura, JN714648 through JN714650) were often been regarded as conspecific with R. panicula. The very similar to the Norwegian samples, one of them type locality of R. panicula is the Oslotjord (Norway) in (JN714648) even identical to both the medusa Earleria a depth of 91-110 m (G. Sars, 1874). quadrata (KY363957) and the hydroid derived sequence Among the Campanulinidae, Racemoramus has a quite unique colony structure, consisting of an unbranched (FJ550511 ). Earleria quadrata is unmistakable among Norwegian stem from which groups of “Campanulina"-like leptomedusae due to its intensively deep-purple hydrothecae originate at intervals (Fig. 6 and Cornelius, pigmented manubrium with white lips (Fig. 5). The 1995; Schuchert, 2001; Calder, 2012). Calder (2012) numerous open statocysts (1-3 between each of the up therefore proposed to keep it distinct from the genera to 40 tentacles) with 6 or more concretions in a single Campanulina van Beneden, 1847 and Opercularella row are also rather characteristic. Earleria quadrata is Flincks, 1868. No life cycle of any Racemoramus species endemic to the coastal waters of Norway and the genus is known so far, although Rees & Rowe (1969) found currently comprises six species (Schuchert, 2017). The that Racemoramus panicula from Sweden releases a polyp stage was hitherto only known for the Californian medusa. Earleria corachloeae, the only hitherto known Earleria corachloeae Widmer, Cailliet & Geller, 2010. hydroid of the genus Earleria, has branched colonies Fig. 5. Earleria quadrata (Hosia & Pages, 2007), living medusa from Korsfjord, one individual in a catch of four used to obtain DNA isolate 1162. 1 Resequencing the sample of R. panicula used to obtain the 18S sequence FJ550596 showed that it had two possible Fig. 6. Racemoramus panicula (G. O. Sars, 1874), sample sequencing errors. Because the new sequence is also longer MHNG-1NVE-48748 from Korsfjord after DNA and the genus name has meanwhile changed, a new Gen- extraction, schematic drawing of part of main stem and Bank submission was made (KY363973). some side-branches (some broken off). 176 P. Schuchert, A. Hosia & L. Leclère that correspond with the traditional diagnosis of the genus Campanulina van Beneden, 1847 as proposed by Bouillon et al. (2006). While it is very probable that Sars’ material of Race- moramus panicula is the polyp stage of Ear 1er ia quadrata and the latter is thus a junior synonym, the hydroid Racemoramus panicula as used today - even in the restricted sense of Calder (2012) - is most likely a species complex. The disparate distributions of the medusa and the hydroid stage support this hypothesis. Therefore, we prefer to conclude more precisely that the medusa of Earleria quadrata has a polyp stage that corresponds to the morphology of Racemoramus panicula, but that both names are better not fully synonymised until a world-wide molecular genetic study of Racemoramus permits delimiting the different populations and their distributions. We should thus continue to use the name of the medusa for this species for the time being. As mentioned above, polyps of Racemoramus panicula Fig 7. Cyclocanna welshi, lateral view, width about 10 mm, from the Gulf of Cadiz and the Mediterranean had 16S living medusa one day after capture, the bell is inverted sequences which were identical or very similar to the and has shrunken considerably as it is usual for sensitive Norwegian medusa E. quadrata. This extends the likely hydromedusae. Details: go = gonad, ma = manubrium, distributional range of this medusa close to that of the rt = short type of tentacle, st = statocyst, tb = bulb of morphologically similar Mediterranean medusa Earleria large tentacle type. antoniae (Gili et al., 1998). It is therefore advisable to molecularly assess the species status of these two Earleria species to exclude the possibility of them specimens only (Kramp, 1926, 1961; Cornelius, 1995). representing morphologically divergent populations of It occurs in cool, deep waters of the North Atlantic. The the same species. type locality is off Virginia, USA, in 0-140 m depth. The taxonomic consequences at the family level are The polyp Egmundella producta, formerly known as discussed in the next section. Lovenella producta, has been redescribed and revised recently (Schuchert, 2016a). It is also rather rare, but its known distribution in the Atlantic matches that of Cyclocanna welshi and Egmundella producta C. welshi. Records from the Pacific Ocean (Fraser, 1937) The two available samples of the medusa Cyclocanna must either be referred to Egmundella gracilis Stechow, welshi yielded 16S haplotypes differing only in one bp 1921 or another, as of yet unnamed species (Schuchert, position. One of them was identical to the 16S obtained 2016a). troni the sample of Egmundella producta polyp described The matching distributions of both medusa and hydroid and figured by Schuchert (2016a). Likewise, the two 18S permit synonymizing the names. The new name must sequences were identical for polyp and medusa. thus be Cyclocanna producta (G.O. Sars, 1874) n. comb., Cyclocanna welshi is a highly distinctive medusa. Its as Sars’ species name is older. The genus Egmundella four radial canals and the gonads along them make a Stechow, 1921 cannot be synonymised with Cyclocanna sharp bend shortly before reaching the circular canal and Bigelow, 1918, as the type species of Egmundella is continue running along the circular canal before giving Egmundella gracilis Stechow, 1921 and its life cycle-as into it, resulting in a pattern reminiscent of a swastika well as that of any other congener - remains unknown. (Fig. 7). Moreover, there are two types of tentacles: All the other Egmundella species must thus remain in four large perradial tentacles with large bulbs situated at this genus until more is known about their medusae or the marginal ends of the radial canals, and many short, gonophores. papilliform tentacles between these. There are eight Kramp (1933) placed Cyclocanna welshi in the family open statocysts, each with a linear array of concretions. Mitrocomidae Haeckel, 1879 and subsequent authors The genus is monospecific. The collected samples were continued to do so, primarily on account of the open all reliably identifiable, but deteriorated rapidly so that statocysts and the absence of ocelli or cirri. According no satisfactory photos could be made. The specimens to the diagnosis given in Bouillon et al. (2006), the appeared to be less pigmented than otherwise described hydroids of this family are of the “Cuspidelld"-Xype. (Bigelow, 1918; Kramp, 1926), but this is likely due to With the identification of the hydroid of Cyclocanna as them being smaller (younger) stages. an “Egmundella”-iypo, the family diagnosis clearly needs Cyclocanna welshi is a rare medusa known from a few a revision. The genus Earleria is also currently placed