Martell et al. Helgol Mar Res (2018) 72:12 https://doi.org/10.1186/s10152-018-0515-5 Helgoland Marine Research

ORIGINAL ARTICLE Open Access The illusion of rarity in an epibenthic jellyfsh: facts and artefacts in the distribution of Tesserogastria musculosa (Hydrozoa, Ptychogastriidae) Luis Martell* , Anne Helene S. Tandberg and Aino Hosia

Abstract Epibenthic and benthopelagic medusae are rarely collected by standard benthic or pelagic sampling methods, and many species are considered uncommon and geographically restricted. Peer-reviewed scientifc literature contains only two records of medusae belonging to the monotypic genus Tesserogastria Beyer, 1958 since their original description, both from the vicinity of the type locality in Osloford, contributing to an illusion of extreme rarity and restricted distribution. Our analysis of fresh samples and a thorough evaluation of all previous records of this taxon from both peer-reviewed scientifc sources and “gray” literature show that the species is both more common and widespread than suggested by the scant records in primary scientifc literature, and represents an example of an over- looked taxon in the epibenthos. High numbers of medusae of Tesserogastria musculosa Beyer, 1958 were collected at Rauneford in western Norway. New data, together with validated observations from fords in western and east- ern Norway as well as western Sweden, demonstrate that the species is much more common than is evident from published records. Data on the mitochondrial 16S ribosomal RNA and cytochrome oxidase I molecular markers for the species are provided for the frst time, as well as new observations on the morphology of living animals. Tesserogastria musculosa constitutes an example of a hydrozoan species with a misleading reported distribution, a situation likely to occur in all members of family Ptychogastriidae and other delicate epibenthic invertebrates. Sampling techniques specifcally targeting the epibenthos and careful processing of the samples are essential for correctly assessing the presence of the species, suggesting that the lack of records for this and other epibenthic medusae may in part be an artefact of the commonly used sampling methods. A comparison of molecular data for species and genus delimita- tion in Ptychogastriidae, presented here for the frst time, highlights the need for a thorough taxonomic revision of the family. Keywords: Hydromedusa, Trachylina, Soft bottom, DNA-barcoding, Benthic medusa

Background the families Cladonematidae, Olindiidae, Ptychogastrii- Epibenthic and benthopelagic medusae are an impor- dae, and Rhopalonematidae are morphologically adapted tant but easily overlooked component of neritic and oce- to life in a benthic habitat [2]. Epibenthic medusae can anic bottoms around the world. More than 45 species of be locally abundant, likely playing a signifcant role in hydromedusae live either attached to or closely associ- bentho-pelagic coupling [3] but, for the majority of the ated with the substrate [1], and many representatives of species, only a handful of records exist and information on their distribution, population dynamics and ecol- ogy is lacking. Te scarcity of records for epibenthic and *Correspondence: [email protected] benthopelagic medusae may be partially attributed to Department of Natural History, University Museum of Bergen, University methodological limitations. Sampling with traditional of Bergen, P.O. Box 7800, 5020 Bergen, Norway

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Martell et al. Helgol Mar Res (2018) 72:12 Page 2 of 13

plankton nets generally does not reach the epibenthos or gear [11] as it is not possible to check if there are any the water column immediately above the bottom, while “jumps” of the bottom during the transect. specimens are easily damaged beyond recognition during Upon retrieving the sample, the cod-end was imme- collection with dredges or bottom trawls, and their frag- diately separated from the net-contents, and carefully ments are often discarded as planktonic contaminants. rinsed into a tub of seawater. Te net-contents were care- Te “mud jellyfsh” Tesserogastria musculosa, the sole fully washed into a separate tub with seawater and kept species in the genus and a member of the poorly known under a covering of water until processing. Te slow tow- family Ptychogastriidae, is one of the lesser-known spe- ing-speed and keeping the samples submerged in water cies of benthic hydromedusae. It was frst collected in at all times ensured that the many fragile hyper-benthic the mid-1950s by Beyer at Digerud in the Osloford [4]. species were collected intact. Te sample was slowly and Beyer later reported on T. musculosa’s local distribution repeatedly decanted over to a 500 μm sieve submerged in and abundance, suggesting that these medusae could be seawater. Tesserogastria musculosa medusae were indi- used as bioindicators of pollution in the area [5]. Sub- vidually handpicked from the sample over a light table sequent work published in 1971 by Hesthagen, one of with a broad-mouthed pipette prior to fxing the rest of Beyer’s students, resulted in a complete account of the the sample in 96% ethanol. In addition to the epibenthic morphology and behaviour of the species, and also con- sampling, a WP3 plankton net (780 μm mesh and non- stitutes the third and last record of the species published fltering cod-end) coupled to a CTD instrument (SAIV in a peer-reviewed scientifc journal and the last report sd200, frequency of measurement 1 s, parameters salinity, on the taxon in English [6]. Te presence of T. musculosa temperature, dissolved oxygen) was hauled from ca. 10 m has since been documented in Osloford—where it has, at above the bottom to the surface at a speed of 0.3 ms−1. times, been one of the most abundant components of the Medusae of T. musculosa were also searched for in this hyperbenthos—as well as in other Norwegian fords (e.g. plankton sample, and the accompanying environmental Fensford, Fanaford, Sogneford) [7–9], but these data data were used to characterize the sampled locality. are only available in technical reports, unpublished the- After collection, the medusae were brought to the ses, and personal observations included in the so-called laboratory and identifed to species level with the aid of “gray” literature. Te low accessibility of these sources specialized literature [4, 12]. Specimens were then fxed has led to a serious bias in the estimation of the current either in 4% formalin (for morphological analysis) or 96% and historical distribution of the species, resulting in its ethanol (for DNA barcoding), with the latter group of categorization as a rare taxon known exclusively from specimens photographically documented prior to fxa- Osloford. In this paper, we challenge this view by report- tion. Live and formalin-preserved specimens were exam- ing on the occurrence of high numbers of T. musculosa ined under a stereomicroscope (Olympus SZX16), and in Rauneford (western Norway) and critically evaluating their detailed morphology was observed using a com- the existing peer-reviewed and “gray” literature records pound microscope (Leica CTR 6000). A subsample of 50 for the species. We also provide the frst COI and 16S randomly chosen formalin-fxed individuals were meas- sequences for this taxon, and argue on the importance ured (bell height on the oral/aboral axis; from umbrella of careful sampling and processing of specimens for cor- margin to apex), and their sex determined before being rect assessment of the presence of this and other benthic deposited as vouchers in the University Museum of Ber- jellyfsh. gen (UMB, Norway, catalogue number ZMBN 123772). Te size distribution and sex ratio of T. musculosa in Methods Rauneford were determined, and the skewness coef- Live individuals of T. musculosa were collected in Raun- cient (g1) and associated standard error (SES) of the size eford, western Norway on February 10th 2017, using a frequency distribution calculated following Joanes and Rothlisberg and Pearcy (RP)-sledge [10] with a 500 μm Gill [13], skewness being detected whenever the absolute plankton net afxed horizontally over a thick rubber mat value of g1/SES is > 2 or < − 2. behind a steel sampling box. With the start position 60° For DNA barcoding, a ~ 1 mm3 tissue fragment was 16.950′ N, 5° 11.212′ E, and a towing-speed of 0.5 knots taken from the umbrella margin of three ethanol-fxed for an approximate of 20 min bottom-time in a southerly specimens. Tese samples were subsequently sent to the direction, we covered a roughly 600 m transect of sandy Canadian Centre of DNA Barcoding (CCDB) in Guelph mud at depths ranging between 142 (start) and 170 (end) for DNA extraction and sequencing according to their m. As the opening width of the sledge is 1 m, the sampled protocols [14]. Specimens were sequenced for both mito- area is ~ 600 m2, even though it should be taken into due chondrial cytochrome oxidase I (COI) and 16S ribosomal consideration that the RP-sledge is a semi-quantitative RNA markers. Te PCR primer pairs C_LepFolF/C_Lep- FolR (for COI), and 16SgaF/16SgaR (for 16S) were used in Martell et al. Helgol Mar Res (2018) 72:12 Page 3 of 13

PCR [15]. Te hydrozoan origin of the sequences was ver- were carried out for 500,000 generations. Trees were ifed by BLAST searches against the GenBank database sampled every 100 generations, discarding the 0.25% of of the National Center for Biotechnology Information. trees as burnin. High quality gene fragments resulting from assembly of To evaluate the previous assumption of spatial and forward and reverse sequences were labelled as barcode temporal rarity of T. musculosa, we estimated its distri- compliant according to the criteria of BOLDSYSTEM bution based on all published and peer-reviewed occur- and are available, together with voucher pictures and rence records and mentions in the scientifc literature, metadata, at Boldsystems.org (sample ID HYPNO_488 as well as in the “gray” literature (i.e. publicly available to 490) and GenBank (accession numbers MG700373 to documents with low accessibility, not subjected to peer- MG700375 for 16S, MG700376 to MG700378 for COI). review, and often published in a language other than An alignment was created independently for each of the English). We validated these records by (1) examining markers using MUSCLE [16] as implemented in MEGA the corresponding specimens deposited in the scien- v.7.0.26 [17], including the sequences of T. musculosa and tifc collections of the natural history museums of the those of all other available members of family Ptychogas- University of Bergen and the University of Oslo, and (2) triidae (see taxa and GenBank accession numbers in contrasting the information contained in each of them Table 1). Pairwise Kimura 2-parameter distances were against the original description of the species. Exam- calculated based on these alignments to estimate genetic ined material included the holotype (a single medusa diferentiation among species. mounted on a permanent slide) and 16 individuals des- A molecular phylogeny of the Ptychogastriidae was ignated as paratypes, all deposited in the Natural His- estimated independently for the above 16S and COI tory Museum of the University of Oslo (ZMO, catalogue alignments. Te sequences were analyzed using a phylo- number B 860 and B 861, respectively). In all, ca. 60 years genetic approach based on (a) maximum likelihood (ML) of issues (1958–2017) of scientifc and technical reports optimality criterion in PAUP* 4.0a [18] and (b) Bayesian from local, regional and national sources (e.g. NIVA– inference using MrBayes v. 3.2.6 [19]. Te best-ft model Norwegian Institute for Water Research, BIBSYS Brage) for each dataset was calculated using jModelTest (v. 2.1.5; were analyzed, and a comprehensive search for relevant [20]) with default settings and chosen using the Akaike unpublished thesis and dissertations from Scandinavian Information Criterion (AIC). For the 16S dataset, the universities was performed through online repositories best-ft model was TIM2 + I, while for the COI dataset (e.g. DUO/University of Oslo, BORA/University of Ber- the best ft model was GTR + G + I. In the ML analyses, gen; GUTEA/University of Gothenburg; NTNU Open; a maximum likelihood consensus tree was generated for UiT Open Research Data). Publicly available distribu- each marker in PAUP* by conducting a heuristic search tional and occurrence data were also compiled from the and bootstrapping with 200 replicates. In the Bayesian online repositories OBIS (Ocean Biogeographic Informa- analyses four parallel Markov chain Monte Carlo runs tion System) and GBIF (Global Biodiversity Information

Table 1 Percentage distance­ a for mitochondrial 16S and COI between Tesserogastria musculosa and other ptychogastriids Ptychogastria polaris Ptychogastria polaris (NW) Glaciambulata (U) KY077292 MH407650 neumayeri (WS) KY421621

16S Ptychogastria polaris (NW) MH407650 0.236 Glaciambulata neumayeri (WS) KY421621 0.162 0.233 Tesserogastria musculosa (NW) MG700373 0.277 0.241 0.260 Ptychogastria polaris Ptychogastria polaris Ptychogastria polaris Glaciambulata (AP) KY072784 (JP) KY072787 (NW) MH407229 neumayeri (WS) KY426133

COI Ptychogastria polaris (JP) KY072787 0.266 Ptychogastria polaris (NW) MH407229 0.311 0.350 Glaciambulata neumayeri (WS) KY426133 0.219 0.246 0.347 Tesserogastria musculosa (NW) MG700376 0.241 0.258 0.335 0.269 a Calculated as Pairwise Kimura-2 parameter distances. Sampling localities for each specimen are indicated in parenthesis as either North Sea (NW), Japan Sea (JP), Antarctic Peninsula (AP), Weddell Sea (WS), or Unknown (U). GenBank accession numbers are underlined Martell et al. Helgol Mar Res (2018) 72:12 Page 4 of 13

Facility), and from the online collections catalogues of below 200 m depth were 34.93 ± 0.010, 8.58 ± 0.003 °C, the Natural History Museum London and the Royal Bel- and 7.81 ± 0.036 mg/L, respectively. gian Institute of Natural Sciences. Te average umbrella height of T. musculosa in Raun- eford ranged from 0.9 to 2.4 mm, with a mean ( SD) Results ± of 1.71 ± 0.37 mm. Te majority of the medusae were in A total of 260 individual medusae were collected in the intermediate size classes (1.5–1.9 mm). Te skew- Rauneford from an estimated sampled area of 600 m2, ness coefcient (g1/SES = − 0.95) indicated that the size corresponding to a density of approximately 0.43 ind/ distribution in Rauneford at the time of sampling was 2 m . Te sampled bottom consisted of sandy mud, with not signifcantly diferent from a symmetric/unimodal high amounts of organic degraded material. Our sam- frequency distribution (Fig. 1b). Mature female medu- ple was dominated by various fatfsh, holothurians and sae were readily recognized upon visual examination of Myxine glutinosa L, 1758. Apart from the macrofauna, the gonads through the umbrella, but the identifcation the main hyperbenthic taxa were T. musculosa and sev- of males and immature females required assessment of eral crustaceans (i.e. Mysidacea, Amphipoda and juvenile histological preparations. Te analysis of a subsample Decapoda), with Polychaeta and Ophiuroidea present in (n = 20) showed that all individuals larger than 1.5 mm large amounts in the heavy fraction remaining after the were mature, and the ratio of female to male in medusa decanting. Within Amphipoda (a taxon with a close rela- larger than this size approached 1:1. Te ratio of imma- tionship to the substrate similar to that of T. musculosa) ture to mature medusae was 1:3. Oedicerotidae was the dominating family, as would be Te morphology of the collected T. musculosa is typi- expected in a station with such soft, organic sediments. cal of the species, with a bell-shaped umbrella about Tis is a family that is known for being fragile, with long as high as broad (0.9–2.4 mm), ending in a small, blunt thin legs that often break of during sampling. Stratifca- apical projection surrounded by a circular depression tion of temperature, salinity, and oxygen concentration (Fig. 2a, b). Te otherwise thin umbrellar mesoglea is was pronounced in the surface waters above the sampling thickened at the margin in a belt of chordal cells, where locality, but conditions below ~ 130 m depth were rela- up to 350 tentacles are inserted at 3–5 levels without tively uniform (Fig. 1a). Mean values (± SD) of salinity, any apparent grouping. Most of the collected specimens temperature, and dissolved oxygen in the water column were completely devoid of tentacles, and only the drop- shaped scars left by shed tentacles remained (Fig. 2d, e).

Fig. 1 Environmental data and size structure of Tesserogastria musculosa from Rauneford in February 2017. a Vertical profles of temperature (°C), salinity, and oxygen concentration (mg/l). b Size distribution of the umbrella height of a randomly chosen subsample of the population. g1 Skewness coefcient. SES Standard error of skewness. n number of medusae measured. Skewness is observed when the absolute value of = = = g1/SES is > 2 (population positively skewed) or < 2 (population negatively skewed) − Martell et al. Helgol Mar Res (2018) 72:12 Page 5 of 13

Te few tentacles available for examination were all fli- often extends beyond the bell margin before ending in form, blunt, circular in transversal section, with evenly- a square mouth with four simple lips. Eight rather nar- distributed nematocysts (slightly more abundant in the row radial canals connect the base of the manubrium to tip), and were devoid of any adhesive structure. Te the broad circular canal situated next to the conspicu- manubrium is four-lobed, cross-shaped in transversal ous nematocyst ring at the umbrellar margin (Fig. 2b). section (Fig. 2c), lacks mesenteries and a peduncle, and Te velum is broad and strongly muscular. Tere are

Fig. 2 Tesserogastria musculosa Beyer, 1958 from Rauneford, Western Norway. a, b General morphology of the medusae in lateral and aboral view. c Cross-section of manubrium. d, e Tentacle stump (black arrow) and appearance of the marginal rim. f Statocyst with statolith (white arrow). g Undischarged nematocyst capsules: stenotele (st), microbasic eurytele (eu), and atrichous isorhiza (is). h Manubrium of a female mature medusa. i Close-up of developing eggs in gonadic tissue. j Manubrium of male mature medusa. k Close-up of homogeneous gonad Martell et al. Helgol Mar Res (2018) 72:12 Page 6 of 13

eight short-stalked interradial statocysts, each with a Discussion single statolith (Fig. 2f). Te gonads are organized in Te distinctive morphology of the collected specimens eight masses forming four perradial pairs (each pair cor- prevented confusion with any other hydromedusa spe- responding to one manubrium lobe) and are evident in cies in the area and allowed for a straightforward iden- mature individuals: in females the eggs are arranged in tifcation of the animals as T. musculosa Beyer, 1958, 1–3 visible rows inside each gonadal mass (Fig. 2h, i), subsequently confrmed through a detailed comparison while in males the gonads appear homogenous (Fig. 2j, with the holotype of the species. Te morphology of the k). Tree distinct nematocyst types, distributed in the medusae is in close agreement with both the original umbrella margin and tentacles, were observed (Fig. 2g): description by Beyer [4] and the subsequent account by almost spherical stenoteles [(6–8) × (6–7) μm], tear drop- Hesthagen [6], except for the coloration pattern. Previ- shaped microbasic euryteles [(6–7) × (4–6) μm], and ous accounts on the coloration of T. musculosa include small atrichous isorhizas (1.5–2 μm). Te umbrella of the the presence of white to yellow epidermal spots located living specimens is translucent, with 8 interradial yellow at the perradial junctions of the radial canals with the spots, roughly square or rectangular in shape, situated at ring canal [6]. In the individuals from Rauneford, how- its margin, corresponding with the statocysts. Te distal ever, the entire ring canal and the distal parts of the radial portion of the radial canals is also pigmented, giving the canals were pigmented, with eight additional interradial appearance of faint and roughly-defned yellow triangles pigment patches not associated with the radial canals on the portion of the radial canals immediately above the present on the umbrella margin. A potential role of intersections of the circular canal and the radial canals. exumbrellar pigment spots as photoreceptors has been Te sequenced products of COI from three analyzed discussed previously for this species, since the medusae individuals were identical to each other, as were the 16S are known to spawn in response to strong illumination products. All sequences were free of gaps and had a fnal under the microscope in laboratory conditions [6], but length (after alignment and trimming) of 658 and 542 further research is needed to determine the degree of base pairs, respectively. When blasted against the Gen- interaction between light cues and ectodermal pigments Bank database, all T. musculosa sequences were shown to in T. musculosa. When examined alive under the direct be most similar to other ptychogastriid sequences, con- light of the microscope at diferent intensities, medusae frming the lack of contamination. Te genetic distances from both Rauneford (present study) and Osloford [6] between T. musculosa and the rest of the genera in fam- did not show any phototaxic response, suggesting that ily Ptychogastriidae are presented in Table 1, evidencing these bottom-living medusae may not depend on light the clear separation between the 16S and COI sequences for orientation and movement. It has been suggested that of T. musculosa and all other analyzed taxa. Preliminary the adult medusae permanently remain on the sea bot- comparisons of 16S and COI sequence data from the tom, with the possible exception of swimming as a fight three valid ptychogastriid genera are shown in Fig. 3, fur- response [6, 8]. ther confrming the separation of T. musculosa, but also Te number and position of the statocysts are diag- suggesting the non-monophyly of Ptychogastria polaris nostic characters separating Tesserogastria from all other Allman, 1878. genera in family Ptychogastriidae [12, 21]. However, In all, 57 previous records of T. musculosa from 30 some confusion exists regarding the exact position of localities were validated (Fig. 4, Table 2, Additional these structures in T. musculosa, as they have been alter- fle 1). Twenty-three diferent sources providing vali- natively described in scientifc accounts as adradial [6] or dated records were identifed, 87% of them from “gray” interradial [12]. Te original description of the species literature. Te known distributional range of the species does not include information on the position and number is expanded to include fords in the north-eastern sec- of statocysts because Beyer was unable to fnd any in for- tion of the Skagerrak strait (numerous localities inside malin-preserved animals, presumably due to the disinte- and around Osloford in eastern Norway, and Gullmar- gration of the statoliths in this fxative [4]. Nevertheless, ford in western Sweden), and the northernmost sector the statocysts from medusae from Rauneford fxated of the North Sea (the western Norwegian fords of Sog- with neutral-bufered formalin were evident even after neford, Fensford and Fanaford), demonstrating that the 6 months of preservation, allowing us to confrm that in presence of at times high numbers of T. musculosa has T. musculosa the statocysts are invariably interradial and been continuously documented for almost six decades in can be used as a reliable character separating Tesserogas- the North Sea region, particularly in the Osloford, from tria from both Ptychogastria and Glaciambulata. 1958 to 2017. For both the 16S and COI markers, the sequences gen- erated for this study show a large degree of diferentia- tion (> 20%) between T. musculosa and individuals of the Martell et al. Helgol Mar Res (2018) 72:12 Page 7 of 13

Fig. 3 Phylogenetic hypothesis of Ptychogastriidae. Phylogenetic hypothesis based on mithocondrial 16S (a) and COI (b). For both markers the topology of the ML and Bayesian trees was identical. Numbers above the branches indicate the posterior probability in the Bayesian analysis and percentage of bootstrap support in the corresponding ML tree, respectively. Branches in black indicate high support values, while grey branches represent low support values. Members of Rhopalonematidae are included as outgroups following [3]. GenBank accession numbers are provided for all sequences

other two species in the family for which genetic data are Japan [3]. Our P. polaris individuals from Hjelteford are available (i.e. Glaciambulata neumayeri Galea et al., 2016 again clearly diferent from both the Antarctic and Japa- and P. polaris), highlighting the value of these two mark- nese specimens, and the preliminary phylogenetic analy- ers as DNA-barcodes for this species. At genus level, all sis suggests that the taxonomy of the Ptychogastriidae, three ptychogastriid genera show a similarly large degree and in particular the hypothesis of monophyly of genus of diferentiation, which is comparable to the distances Ptychogastria, needs to be revised, as the intraspecifc observed by Grange et al. between individuals identifed degree of diferentiation within this genus appears com- as P. polaris from the Antarctic Peninsula and the Sea of parable to the degree of diferentiation between genera. It Martell et al. Helgol Mar Res (2018) 72:12 Page 8 of 13

Fig. 4 Validated records of Tesserogastria musculosa, with a detailed view of the Osloford

is unlikely that DNA sequences from either the holotype species in the type locality at any given time of the year or the paratypes of T. musculosa will be available in the [6]. near future, since in both cases the specimens have been Te high numbers of medusae in some localities along subjected to fxation and long-term storage in formalin. Osloford allowed Beyer to analyze the local distribution Te 16S and COI sequences provided here for T. muscu- of the species. Tis lead him to suggest that T. musculosa losa constitute thus a useful tool for identifcation, as well is an indicator of non-polluted bottoms, with the num- as the frst molecular evidence of the relationships of the ber of individuals rapidly increasing towards the open genus with both Glaciambulata and Ptychogastria. sea [5]. Later on, the consistent patterns of decreasing Tesserogastria musculosa was a common component of abundance towards the inner parts of the ford caused the epibenthic community in the sampled locality, with Beyer and Indrehus to advocate the species as an indica- the estimated density (ca. 0.5 ind/m2) similar to what has tor of oceanic and unpolluted conditions in the area [9]. been reported for other ptychogastriid medusae in boreal Despite this general pattern, the population has fuctu- waters (0.01–0.91 ind/m2 for P. polaris in northeast ated during the recent decades, with a dramatic popu- Greenland, and 0.01–0.76 ind/m2 for the same species lation reduction in the inner ford starting in the 1960s in the Barents Sea) [22, 23]. Te density of T. musculosa and continuing until the mid-1990s [9]. Remarkably high was however considerably lower than the maximum den- numbers of medusae were observed again in 1996 [26], sities reported for ptychogastriid medusae in Antarctic and the species has since been recorded rather consist- waters (up to 13 ind/m2 for P. polaris in Antarctic fords) ently in Osloford until as recently as 2012 [27], but with- [3]. In Digerud and neighbouring localities, T. musculosa out information on population density or abundance. has reached maximum values of > 10 ind/m3, being the High densities of ptychogastriid medusae in soft sedi- most abundant organism in at least one sampling sta- ments have been linked to high productivity in subant- tion [5], while subsequent observations in Osloford have arctic fords [3, 28], and to the accumulation of organic shown that the species is common and, at times, highly and inorganic debris in the seafoor of relatively isolated abundant in soft bottoms [9, 24–26]. Te medusae from deep environments [4, 29]. Rauneford’s sediment char- Rauneford and Osloford shared similar size ranges acteristics and oceanographic processes (e.g. enhanced and unimodal distribution of bell height, but the mean benthic productivity, vertical fux, trapping of detritus) umbrella height in this study (1.71 mm) was higher than are likely to provide a suitable habitat for epibenthic the mean height (0.96–1.03 mm) reported for the same medusae in an analogous way to the observed abundance Martell et al. Helgol Mar Res (2018) 72:12 Page 9 of 13

Table 2 Validated previous records of Tesserogastria musculosa Beyer, 1958 summarized by reference References Locality Region/country Collection date­ a Abundance Type of record/language

[4] Digerud (type locality) and Skagerrak/Norway May 1952; June 1949, 1952, > 4000 Medusae Published scientifc article/ several others in Osloford and 1953 English [5] Steilene, Gåsöy, Lysakerford, Skagerrak/Norway January and October 1962, Up to > 1000 ind/m3 Published scientifc article/ and Helvik; Osloford August 1965 English [6] several stations inside Skagerrak/Norway September and December N/D Published scientifc article/ Osloford 1966, April 1967 English [7] Fensforden and Sognef- North Sea/Norway N/D N/D Technical report/Norwegian jorden [8] Fanaford North Sea/Norway April, June, September and Abundant Thesis dissertation/Norwegian December 1979 [9] several stations inside Skagerrak/Norway August 1981 to 1993. Common Technical report/Norwegian Osloford Unknown dates in 1983–1986 [24] Gråøyrenna; Osloford Skagerrak/Norway May 1973 > 1500 Medusae Published scientifc article/ English [25] Gråøyrenna and Elle; Oslof- Skagerrak/Norway December 1974, June 1975 Abundant Thesis dissertation/Norwegian jord [26] Granerøstøa; Osloford Skagerrak/Norway June 1996 Abundant Technical report/Norwegian [27] Elle; Osloford Skagerrak/Norway September 2012 Presence only record Technical report/Norwegian [39] Elle and Gråøyrenna; Oslof- Skagerrak/Norway September 2000 Presence only record Technical report/Norwegian jord [43] Osloford Skagerrak/Norway N/D Several medusae Museum catalogue/French [44] Rauer Skagerrak/Norway November 1997 Presence only record Technical report/Norwegian [45] Elle; Osloford Skagerrak/Norway September 2001 Presence only record Technical report/Norwegian [46] Vesthullet; Osloford Skagerrak/Norway September 2002 Presence only record Technical report/Norwegian [47] Elle and Steilene; Osloford Skagerrak/Norway September 2003 and 2004 Presence only record Technical report/Norwegian [48] Steilene, Gråøyrenna, and Skagerrak/Norway September 2005 Presence only record Technical report/Norwegian Vesthullet; Osloford [49] Gråøyrenna; Osloford Skagerrak/Norway September 2008 Presence only record Technical report/Norwegian [50] Gråøyrenna; Osloford Skagerrak/Norway September 2009 1 Medusa Technical report/Norwegian [51] Spro; Osloford Skagerrak/Norway October 1962 N/D Museum catalogue/English [52] Essvik, and Kiuben; Gullmar- Skagerrak/Sweden October 1964 N/D Museum catalogue/English ford [53] Digerud; Osloford Skagerrak/Norway N/D N/D Museum catalogue/English N/D no data a Exact position (latitude, longitude) of the sampled sites are not always available in the original sources. Sampling was conducted in all cases with a Beyer’s 50 cm epibenthic closing net or a slightly modifed version of the same of P. polaris in subantarctic fords [3], Ptychogastria mechanisms behind these remain poorly understood asteroides (Haeckel 1879) in Mediterranean canyons [29], [31, 33]. Episodes of bloom-and-bust have never been and T. musculosa in Osloford [4]. reported for benthic ptychogastriid hydromedusae and Contrary to the interannual variation, signifcant sea- these organisms may not be part of the subset of medu- sonal population fuctuations have not been observed for sozoan species with life-cycle attributes that predispose T. musculosa in its type locality, with high numbers of them to bloom [34]. Existing evidence appears to sug- medusae collected throughout the year in seasonal sur- gest that epibenthic medusae in genera Ptychogastria and veys [6]. In West-Norwegian Fanaford, however, strong Tesserogastria do not form blooms with abrupt periods seasonality in T. musculosa was reported by Kaartvedt, of presence in the environment followed by completely who observed the highest abundances in June and much disappearance, and instead maintain rather stable large fewer specimens in April, September and December [8]. populations in habitats such as the bottom of Boreal, Unpredictable, sporadic and episodic population fuc- Arctic and Antarctic fords, and deep submarine can- tuations are widely documented for planktonic jelly- yons [3, 6, 22, 23]; although the observed variations in the fsh [30, 31] and some benthic hydrozoans [32], but the abundance of T. musculosa in Fanaford [8] could instead Martell et al. Helgol Mar Res (2018) 72:12 Page 10 of 13

indicate that seasonal dynamics difer widely among pop- adopting a more careful handling approach of the sam- ulations of this species in diferent habitats. ples [40]. For a relatively easy-to-identify species distributed Te population assessment of benthic ptychogastriid in the well-studied vicinities of active marine biological medusae has only recently become feasible thanks to stations in both Osloford and Rauneford, T. muscu- improved sampling techniques and in situ observations losa has a puzzlingly low number of records in the pub- by divers or ROVs, resulting in the description of new lished, peer-reviewed scientifc literature. Subsequently, species and several new records for the already-known it has been generally considered a rare and geographi- ones [3, 12]. Other multidisciplinary approaches (e.g. cally restricted species. Based on our data and a thor- sediment traps) have also been successfully used for the ough review of the “gray” literature, this perception is collection of well-preserved ptychogastriid medusae in most likely incorrect, and T. musculosa in fact appears to remote locations such as submarine canyons [29], and be a relatively common and widely distributed compo- thus represent a potential source of valuable informa- nent of the epibenthos in several fords along the North tion for this taxon. Ptychogastriid medusae were rarely Sea coast. Te lack of published records for T. musculosa reported before the late 1990s [22, 23], partly due to the does not result from the rarity of the species, neither can difculties of sampling deep marine environments, but it be attributed to undersampling of its habitat, as numer- also because the medusae, often damaged beyond recog- ous studies have been conducted on the benthic fauna of nition, were readily considered planktonic contaminants. the areas surrounding both the Marine Biological Station Te standard processing of benthic samples with sieves in Drøbak and the Espeland Marine Biological Station in often results in the gelatinous species getting extruded Rauneford since their founding in 1894 and mid-1950s, and destroyed, leading to them remaining unreported. respectively [35, 36]. Te lack of records for T. musculosa and other benthic More than with other epibenthic animals, our ability and benthopelagic medusae [41] may thus be an artefact to detect the presence of T. musculosa—and most likely of the commonly used sampling techniques. other gelatinous epibenthos—appears to depend on a A second issue contributing to the perceived rarity of combination of the chosen sampling gear and a careful T. musculosa is the poor accessibility of the bulk of the processing technique. In the original description, Beyer records, which consist of technical reports, unpublished stressed the importance of the sampling gear in fnding T. theses, and museum collection catalogues. Many basic musculosa in Osloford, stating that his specially designed and applied studies in biogeography, phylogenetics, and sledge, known as the Beyer sledge, consistently yielded ecology rely on species distribution data compiled exclu- catches of T. musculosa [4]. In the localities where abun- sively from the published scientifc literature but, for dant medusae have been caught with sledges, sampling some species, such as T. musculosa, these data are incom- with plankton net hauls from above the bottom has never plete and may contain serious biases. While the use of yielded a single specimen, either in Osloford [4] or Raun- unverifed anecdotal occurrences and unpublished obser- eford and the surrounding fords [37]. Beyer suggests vations for the assessment of the current and historical that “the species has probably been caught many times in ranges of rare species can certainly lead to large errors of grab and Mortensen dredge samples, but has then been omission and commission [42], for some taxa, such as T. disregarded together with the plankton inevitably caught musculosa, inclusion of records from the “gray” literature in these apparatus”, thus highlighting the importance of is crucial for a realistic estimation of their distribution. gentle sampling, adequate sample processing techniques, Common complaints against the inclusion of “gray” liter- and correct identifcation for observing this species [4]. ature data in distributional analyses include the low qual- Prior to the current study, all specimens of T. musculosa ity and low accessibility of the sources. While the latter is ever collected have been obtained with Beyer’s epibenthic also an issue in the case of T. musculosa (e.g. all records sledge (illustrated in [38]) with a 50 cm closing plankton are in languages other than English and several unpub- net mounted on a steel toboggan, designed for the pur- lished documents are not available online), the quality of pose of catching organisms on or immediately above the the data they contain is in general quite high: all observa- bottom [5, 6, 8, 9, 27, 39]. In the current study, a slightly tions were made by trained professionals working in the less gentle and much larger Rothlisberg and Pearcy (RP) feld of marine biology/ecology, in many cases in collabo- sledge was used, but this was compensated for by the ration with the original author of the species or with one careful processing of the samples, as well as the very of his students, using established and well documented slow transect and retrieval speed. It is not surprising that sampling protocols. careful processing of samples will result in higher num- In the particular case of T. musculosa, reviewing and bers of species found: in Kiel Bay, Remane found > 300 including occurrence records from “gray” literature con- new species after modifying his sampling techniques and siderably alters the perception of its distribution and Martell et al. Helgol Mar Res (2018) 72:12 Page 11 of 13

commonness. Furthermore, due to the hyperbenthic hab- in Ptychogastriidae, presented here for the frst time, itat and the gelatinous morphology of the medusa, the shed further insight into the diversity and distributional species is unlikely to be collected or recorded by the most range of epibenthic trachyline medusae and highlighted commonly used pelagic or benthic sampling methods. the need for a thorough taxonomic revision within the A similar methodological bias applies to other epiben- family. thic and benthopelagic medusae, which remain a poorly Additional fle known group [37]. Targeted sampling with suitable gear and processing protocols, or using ROVs and other opti- Additional fle 1. Validated records of Tesserogastria musculosa Beyer, cal platforms, is required to establish a better under- 1958. List of all the records of Tesserogastria musculosa Beyer, 1958 vali- standing of diversity and ecology of ptychogastriids and dated for this study and used to generate Fig. 4. other gelatinous benthos. In addition to updating our knowledge of the distri- Authors’ contributions bution of the species, the reconstruction of the taxo- LM and AH conceived the study; LM, AH, and AHST collected samples and nomic history of T. musculosa also allowed us to clarify performed the experiments; AH and AHST secured funding and provided the existing confusion regarding its date of description. resources; LM wrote the frst draft of the manuscript. All authors read and approved the fnal manuscript. Although the paper by Beyer was actually published in 1958, some authors have mistaken the date of this pub- lication as 1959 [2, 12, 21] probably based on a series of Acknowledgements The authors thank the crew of RV Hans Brattström and Christine Østensvig for copies of Vol. 6 of the Nytt Magasin for Zoology printed their help in collecting the medusae and the staf at the Espegrend Marine in that year. Te error has become widespread in the Biological Station for providing facilities. Thanks are also due to the staf and peer-reviewed scientifc literature, although it has not curators of the Natural History Collections of the University Museum in Bergen and the Natural History Museum of the University of Oslo for providing loans completely permeated the “gray” literature produced and access to relevant material, to Prof. Endre Willassen for commenting on in Norway. Beyer himself reported the date as 1958 in the phylogenetic methods and results, and to the anonymous reviewers for subsequent works, as did his students and collabora- their useful comments on the manuscript. tors (e.g. Hesthagen clearly states that the species was Competing interests frst described by Beyer in 1958) [6]. An enquiry into the The authors declare that they have no competing interests. publications of the University of Oslo (publisher of Nytt Availability of data and materials Magasin for Zoology) has confrmed that the correct date All data generated or analyzed during this study are included in this published of publication is 1958, the correct date for the genus is article and its Additional fle. Tesserogastria Beyer, 1958, and the correct name of the Consent for publication species is Tesserogastria musculosa Beyer, 1958. Not applicable.

Ethics approval and consent to participate Conclusions Not applicable. Tesserogastria musculosa is not restricted to its type locality; freshly collected samples and validated obser- Funding The present study was funded by the Norwegian Taxonomy Initiative projects vations from fords in western and eastern Norway as 70184233/HYPNO (LM and AH) and 70184235/NorAmph (AHST). The funder well as western Sweden demonstrates that the species is had no role in study design, data collection, data analysis, data interpretation, more common and widespread than is evident from pub- or writing of the manuscript. lished records. Our current ideas about the restricted distributions of other epibenthic medusae need to be Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- reassessed, as they are likely to be similarly biased, and lished maps and institutional afliations. further sampling at additional localities will likely pro- vide more records of these organisms, including T. mus- Received: 17 March 2018 Accepted: 20 June 2018 culosa. Sampling techniques specifcally targeting the epibenthos and careful processing of the samples are essential for correctly assessing the presence of epiben- References thic medusae, and the lack of records for these organ- 1. Larson RJ, Matsumoto GI, Madin LP, Lewis LM. Deep-sea benthic and isms may in part be an artefact of the commonly used benthopelagic medusae: recent observations from submersibles and a sampling methods, but thorough validation and reassess- remotely operated vehicle. Bull Mar Sci. 1992;51:277–86. 2. Bouillon J, Gravili C, Pagès F, Gili JM, Boero F. An introduction to hydrozoa. ment of previous records, including those in “gray” litera- Mém Mus Natl d’Hist Nat. 2006;194:1–592. ture, are also necessary to estimate the real distributional 3. Grange LJ, Smith CR, Lindsay DJ, Bentlage B, Youngbluth MJ. High area of delicate epibenthic invertebrates. Te compari- abundance of the epibenthic trachymedusa Ptychogastria polaris Allman, son of molecular data for species and genus delimitation Martell et al. Helgol Mar Res (2018) 72:12 Page 12 of 13

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