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Abyssal Rhachotropis (Eusiroidea, Amphipoda) from the Sea of Okhotsk
Author’s Accepted Manuscript Abyssal Rhachotropis (Eusiroidea, Amphipoda) from the Sea of Okhotsk A.N. Lörz, A.M. Jażdżewska, A. Brandt
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To appear in: Deep-Sea Research Part II Received date: 28 June 2017 Revised date: 29 August 2017 Accepted date: 26 September 2017 Cite this article as: A.N. Lörz, A.M. Jażdżewska and A. Brandt, Abyssal Rhachotropis (Eusiroidea, Amphipoda) from the Sea of Okhotsk, Deep-Sea Research Part II, https://doi.org/10.1016/j.dsr2.2017.09.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Abyssal Rhachotropis (Eusiroidea, Amphipoda) from the Sea of Okhotsk
Lörz, A.N. 1, Jażdżewska, A.M. ², Brandt, A. 3
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University of Hamburg, Centre of Natural History, Zoological Museum, Martin-LutherKing-Platz 3, 20146 Hamburg, Germany, Email: [email protected] ²Laboratory of Polar Biology and Oceanobiology, Department of Invertebrate Zoology and Hydrobiology, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha st., 90-237 Lodz, Poland 3 Senckenberg Research Institute and Natural History Museum, Department of Marine Zoology, Senckenberganlage 25, 60325 Frankfurt am Main, Germany Abstract The genus Rhachotropis has the widest horizontal and bathymetric distribution of all amphipod genera known worldwide. Characteristic species of Rhachotropis were collected at 3200 m depth in the Kuril basin of the Sea of Okhotsk. Morphological investigations revealed a species new to science, which is here described in detail. Juveniles were removed from the marsupium of the holotype; their morphological features were investigated via scanning electron microscopy and compared to the adults. The molecular barcode of the new species, Rhachotropis marinae, was obtained. Comparison with available Rhachotropis COI data revealed clear separation of the new species from all known species as well as from two other Rhachotropis species also collected during the SokhoBio expedition. The three Rhachotropis species from abyssal depths in the Sea of Okhotsk did not group together when their COI region was analysed with Rhachotropis sequences from other parts of the world. The number of described species of Rhachotropis is now increased to 62.
Keywords Amphipoda, Rhachotropis, new species, North Pacific, barcode
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1. Introduction Amphipoda, among other macrofauna, were sampled during the joint Russian-German expedition SokhoBio (Sea of Okhotsk Biodiversity Studies) on board of the R/V Akademik M.A. Lavrentyev in the Kurile Basin of the Sea of Okhotsk in summer 2015 (for a general introduction see Malyutina et al., this volume). The genus Rhachotropis S.I. Smith, 1883 (Eusiridae) contains 61 species (World Amphipoda Database, Horton et al. 2017). , is found in all oceans, and has the greatest bathymetric distribution (0—9460 m) of all amphipod genera (Lörz et al., 2012). Some Rhachotropis species are known to be very abundant locally (Cartes and Sorbe, 1999). A first investigation of the SokhoBio amphipod collection revealed characteristic specimens of a species of Rhachotropis new to science. This species, with a prominent protuberance on the head, is described here in detail. Two further species of Rhachotropis, one with smooth pereonites, the other with protuberant pereonites, were also collected at abyssal depths during the SokhoBio expedition. Since the proposal of the DNA barcoding concept by Hebert et al. (2003) the use of molecular methods in species recognition has become very popular and are often used to supplement morphological taxonomy (e.g. Hubert and Hanner, 2015; Jinbo et al., 2011; Seefeldt et al., in press and references therein). As the present material was suitable for DNA analyses we investigated the relationships of the three freshly collected Rhachotropis species via analysis of a cytochrome oxidase region (COI) and set them in context with COI sequences from Rhachotropis species from other parts of the world.
2. Material and Methods The detailed description of the study area is presented in the introduction of this volume (Malyutina and Brandt, this issue). Large amphipod specimens were immediately sorted on deck, fixed in -20° precooled 98% ethanol and later transferred to 96% ethanol. Amphipod samples collected deeper than 3000 m are regarded as “abyssal”, following the classification of abyssal amphipods by Barnard (1962). 2
2.1. Morphological description Specimens were examined and dissected under a Leica MZ12.5 stereomicroscope and drawn using a camera lucida. Small appendages (mouthparts, uropods, and telson) were temporarily mounted in glycerin and examined and drawn using a LeicaDM2500 compound microscope fitted with a camera lucida. The body lengths of specimens examined were measured by tracing individual’s mid-trunk lengths (tip of the rostrum to end of telson) using a camera lucida. All illustrations were digitally ‘inked’ following Coleman (2003, 2009). Two adult paratypes and 4 juveniles extracted from the marsupium of the holotype were dried, coated with gold-paladium and investigated via a scanning electron microscope LEO1525. In order to preserve the good condition of epibionts, the palp of maxilla 1 was selected for confocal laser scanning microscopy (CLSM). To gain auto fluorescence of the surfaces, the 405 nm laser lines were applied with emission filters set to 421–499 nm, the 488 nm laser lines to 489 –607 nm. The palp was scanned using a Leica DM2500 with a Leica TCS SPE at a resolution of 2480 x 2480 pixels with a 10x. The software package LEICA LAS X was used for recording the image from the scans. The image stacks were further processed infinalized in Adobe Photoshop CS5. Photos of material held at the Centre of Natural History (CeNak) were taken with a Canon EOS 5 Mark III with lens Canon MP-E65 macro mounted for stacking, the stacking programme software is Zerene Stacker 1.04 (setting P-max). Type material is held at the Zoological Museum University of Hamburg, CeNak. 2.2. DNA extraction and analyses DNA extraction from five individuals of the newly described species as well as ten additional individuals of Rhachotropis sp. from another station sampled during SokhoBio expedition (Tab. S1) was performed according to a standard phenol-chloroform method after Hillis et al. (1996). Air-dried DNA pellets were eluted in 100 μl of TE buffer, pH 8.00, stored at 4°C until amplification, and subsequently at -20°C for long-term storage. A fragment of Cytochrome Oxidase subunit I gene (COI; ca. 670 bp fragment) was amplified using standard LCO1490/HCO2198 primers (Folmer et al., 1994) with DreamTaq Green PCR Mastermix (Thermo Scientific) and reaction conditions following Hou et al. (2007). Sequences were obtained using BigDye sequencing protocol (Applied Biosystems 3730xl) by Macrogen Inc., 3
Korea. Sequences were edited using Geneious 10.1.2 leading to 15 sequences of 607 bp each. All sequences were deposited in GenBank with the accession numbers (Table S1). Relevant voucher information, taxonomic classifications, and sequences are accessible through the public data set “DS-RHSOKHO” on the Barcode of Life Data Systems (BOLD; www.boldsystems.org) (Ratnasingham and Hebert, 2007). To check the genetic relationships between the new species and the known Rhachotropis spp. distributed worldwide, 44 publicly available sequences (longer than 590 bp) of additional Rhachotropis specimens were retrieved from the BOLD database (Tab. S1). All sequences were aligned using the MAFFT v7.308 algorithm (Katoh and Stanley, 2013, Katoh et al., 2002) in Geneious 10.1.2, resulting in 560 bp alignment used for further analyses. The uncorrected p-distance and the Kimura 2-parameter (K2P) model (Kimura, 1980) were used to determine sequence divergence in MEGA V7.0.18 (Kumar et al., 2016). A NeighbourJoining (NJ) tree was built based on the uncorrected p-distance with both transition and transversion substitutions included and pairwise deletion chosen. Node support was inferred with a bootstrap analysis (1000 replicates). The COI sequence of an Antarctic Eusirus species mined from GenBank (accession number JQ412464) (Table S1) was used as the outgroup, since it is a closely related genus to Rhachotropis within the family Eusiridae.
3. Results Systematics Order AMPHIPODA Latreille, 1816 Suborder GAMMARIDEA Latreille, 1802 Family EUSIRIDAE Stebbing, 1888 Genus Rhachotropis S.I. Smith, 1883 Rhachotropis S.I. Smith, 1883: 222. Gracilipes Holmes, 1908: 526. Type species. Oniscus aculeata Lepechin, 1780 Rhachotropis marinae sp. nov. Syn. Rhachotropis grimaldi (Chevreux, 1887) by Gurjanova 1955 Fig. 11 (Figs 1–8, 9A) 4
Material examined. Holotype: ZMH K-46596, female 15.8 mm, SokhoBio expedition 2015, station 7-3, gear epibenthic sledge, sampling date 22. 07. 2015, 46°56.556'N 151°05.013'E, 46°56.791'N 151°04.860'E, 3299 m depth, Paratypes: ZMH K-46597, male 17.2 mm; ZMH K-46598 (on SEM Fig. 6); ZMH K-46599; ZMH K-46600 ; ZMH-K-46613, 7.8 mm(sex unknown); all paratypes have the same collection data as the holotype. Further material examined: 10 Rhachotropis specimens from SokhoBio station 9-7 (ZMH K46602-46611, Gen Bank accession numbers: MF409435-MF409449, Tab. S1). The holotype of Rhachotropis grimaldi (Chevreux, 1887) was further examined via photographs taken by Michele Bruni from the Museum of Monaco for this study.
Etymology: This species is named for Marina Malyutina. Marina was one of the organisers of the expedition during which the new species was collected, and we greatly appreciate her outstanding contribution to peracarid taxonomy and systematics.
Diagnosis The new species is relatively easy to recognize due to a prominant protuberance on its head. It can be separated from other species of Rhachotropis by the combination of following characters: coxa 1 drawn out to anterior end of head, telson split half way, coxa 7 posteriorly drawn out and pointed, epimeral plate 3 entire posterior margin serrate. Description Antennae. Antenna 1 slightly shorter than antenna 2. Antenna 1 article 2 slightly shorter than article 1, 1.5 times as long as article 3; flagellum 12-articulate; accessory flagellum 2articulate. Antenna 2 peduncle article 3 and 4 subequal in length, several plumose setae on third and fourth article; flagellum 23-articulate. Mouthparts. Mandible with incisor process well-developed; lacinia mobilis denticulate; molar process conical; palp article 1 short, about one-third length of article 2, article 3 nearly as long as article 2, articles 2 and 3 with long slender setae. Maxilla 1 inner plate bearing 2 subterminal plumose setae; outer plate with 9 denticulate spines; article 1 of palp with long 5
setae at outer margin, article 2 of palp with several slender setae at tip. Maxilla 2 inner and outer plate subequal in length, margins bearing stout and slender setae; outer plate bearing 2 plumose setae; inner plate wider than outer plate. Maxilliped outer plate 2.5 times as long as inner plate, reaching half of article 2 of maxillipedal palp. Labrum circular in shape, two setose areas anteriorly. Hypopharynx setose distoanteriorly, outer lobes separated by broad gap. Pereopods. Coxa 1 drawn out reaching anterior end of head, coxa 1 posterior corner with strong hook. Gnathopods similar in shape, subchelate. Gnathopod 1 slightly smaller than gnathopod 2; basis bearing several small spines at anteroventral corner; ischium and merus with long setae at posteroventral corner; carpus lobe extending width of propodus, spines at terminal end of lobe; propodus widened, oval; dactylus slender, reaching end of palm. Coxa 2 subquadrate, posteroventral corner bearing small hook; gnathopod 2 basis 1.5 times as wide as basis of gnathopod 1; ischium to dactylus similar to gnathopod 1. Coxae 3 and 4 subquadrate, ventral margin with small hook on anterior and posterior corner. Pereopods 3 and 4 articles long and narrow; carpus, propodus and dactylus about same length.
Coxa 5 strongly lobate, anterior and posterior corner of ventral margin with insertion. Pereopod 5 as long as pereon; basis rectangular; merus slightly inflated, 1.3 times as long as carpus; propodus 1.5 times as long as carpus. Coxa 6 strongly lobate, posterior lobe extending further ventrally than anterior lobe, anterior and posterior corner of ventral margin with insertion. Pereopod 6 larger, but similar in shape to pereopod 5; basis with ridge. Coxa 7 posterior margin strongly drawn out. Pereopod 7 longer than pereopod 5 and 6; basis with rounded extension posteriorly; merus posteroventral angle produced; broken off at carpus. Pleopod 2 (right side) rami with 28 and 22 articles; rami 1.5 times as long as peduncle. Uropod 1 outer rami slightly shorter than inner rami; rami shorter than peduncle. Uropod 2 outer rami 0.2 times shorter than inner rami; peduncle shorter than outer rami and longer than inner rami. Uropod 3 inner rami slightly longer than outer rami, inner rami twice as long as peduncle. Telson three times as long as wide, cleft 45 %.
Remarks. Sexual differences: Male specimens (Fig. 1C) have longer antennae, more than 50 articles each, the third article is expanded anteriorly. The third epimeral plate is continuously
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serrate posteriorly in females, whereas males bear an interruption of the serration, with smooth sections towards the dorsal spine. Juveniles. Twenty four juveniles were counted in the marsupium of the holotype. Four were removed for scanning electron microscopy (Fig. 7 C—F). Several juveniles fell out while dissecting the right side for illustrating, but the majority were left undisturbed. Morphological differences between the juvenile and adult include juvenile lacking the protuberance on the head (Fig. 6 C, D versus adult Fig. 6 A, B), coxa 1 not bearing an insertion (Fig. 6 D versus adult Fig. 6 A, B), coxae 2—6 lacking “hooks” (Fig. 6 E, versus adult Fig. 6A, B), the accessory flagellum comprised of one article only (Fig. 6 F) (versus two articles in adults) and both antenna being shorter, only half the body length. Epibionts are dominant features of the mouthparts and gnathopods of the adults (Fig 8). 3.2. Molecular study Among the 15 sequences obtained in this study, 11 haplotypes were distinguished, forming three distinct groups. One group consisted of five sequences of the presently described species; the second two groups were made by individuals from additional stations. The divergence of the sequences within each group recognized was variable with the lowest value found for Rhachotropis sp. 2 (0.002 of both uncorrected p-distance and K2P), medium for R. marinae (0.014 both measures) and the highest for Rhachotropis sp. 1 (0.025 and 0.026 for uncorrected p-distance and K2P, respectively) (Tab. 1). Rhachotropis abyssalis, R. novazealandica, R. rossi, R. sp. A, R. sp. A1, R. sp B and R. sp 28 were all represented by single sequences. The NJ tree showed clear differentiation of all studied taxa, however, as many as half of them were singletons (Fig. 9). The groups of sequences from Okhotsk Sea remained as separate clades, but their positions in the tree are not well supported.
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Table 1. The intraspecific variation within Rhachotropis species calculated using uncorrected p-distance and Kimura 2-parameter (K2P). Values in column A were obtained when all sequences assigned to R. inflata were treated as a single unit; in column B the same values are calculated excluding one distinct sequence of R. inflata (R. inflata 1).
Rhachotropis aculeata (Lepechin, 1780) Rhachotropis chathamensis Lörz, 2010 Rhachotropis inflata (Sars, 1883) Rhachotropis inflata 1 Rhachotropis marinae sp. nov. Rhachotropis sp. 1 (smooth) Rhachotropis sp. 2 (bumpy)
p-distance A B 0.008 0.008 0.000 0.000 0.038 0.014 -------N/A 0.014 0.014 0.025 0.025 0.002 0.002
K2P A 0.008 0.000 0.042 -------0.014 0.026 0.002
B 0.008 0.000 0.014 N/A 0.014 0.026 0.002
4. Discussion 4.1 Morphological similarities The new species matches a species description by Gurjanova (1955) of Rhachotropis grimaldi (Chevreux, 1887). The type species of R. grimaldi was sampled in the Mediterranean at 510 m depth, Gurjanova (1955) illustrated a specimen from the Sea of Okhotsk at 2850 m, near Iturup Island, and lists several morphological differences. We are convinced that Gurjanova (1955) described a new species. She only had one specimen, and was lead by morphological similarities, such as the bumpy pereon, to believe her specimen was R. grimaldi. She did list several characters of her specimen that did not match the description of Chevreux’s specimen. Several authors have recorded R. grimaldi, including Ledoyer (1977), from 400 m near Marseilles (Mediterranean), Barnard (1916) from 500 m off South Africa (Atlantic).We have several specimens from 3200 m depth of the Okhotsk Sea, showing consistent morphological features and consistent morphological differences to R. grimaldi (Chevreux, 1887), see table 2. Our specimens of R. marinae new sp. from 3200 m depth of the Okhotsk Sea, show consistent morphological features and can be easily distinguished from R. grimaldi (Chevreux, 1887), see table 2.
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Table 2. Morphological differences between Rhachotropis marinae new sp. and R. grimaldi (Chevreux, 1887). R. marinae new sp. Protuberance on head
R. grimaldi (Chevreux, 1887) present
absent
Second urosomite posterior margin
smooth
spinose
Head length vs first three pereonites
longer
shorter
Antennular lobe
narrow, projected
short, rounded
Coxa 4 posterior margin
notched
smooth
Second epimeral plate
serrated
smooth
Many deep-sea Rhachotropis are reported to be blind, but careful examination of the preserved specimens of the new species collected at abyssal depths shows distinct oval eyes in both males and females. The eyes will be easily overlooked when specimens are preserved in ethanol for any length of time.
4.2.Juveniles Juveniles were taken from the marsupium of the holotype (see Fig. 6 C—F). The main morphological difference to the adults is being less spiny most likely due to allometry. We therefore do not interpret the smooth body of the juvenile as plesiomorphic state, but as pragmatic feature as a result of being cramped in the marsupium. 4.3.Epibionts These epibionts are characteristic features of Rhachotropis species collected in various areas and depths of the world’s oceans, e.g. on Rhachotropis chathamensis Lörz, 2010 in the South East Pacific, R. rossi Lörz, 2010 from the Ross Sea (Lörz, 2010) , R. macropus Sars, 1893 and R. aculeata (Lepechin, 1780) from the North Atlantic (pers. obs.) . The epibionts vary in density, but are mainly found on the mouthparts and gnathopods. While other families and genera of Amphipoda also bear the occasional epibiont, these are typical on the genus Rhachotropis. A further investigation of the host specificity is underway. 4. 4. Molecular investigation 9
The barcoding of Rhachotropis spp. from the Okhotsk Sea revealed the existence of at least three distinct species at the two stations studied. The samples were collected at similar abyssal depths, however, one of the stations (from which the new species is described) is situated in the Sea of Okhotsk itself, while the second station was situated on the Pacific side of Kuril Islands about 70 km away from the Bussol Strait. Further study of the Rhachotropis spp. from the area will reveal more about the distribution of these species. It is not yet known if these species will be recorded across the whole NW Pacific area or if they have restricted ranges (e.g. only to Okhotsk Sea or only to the open waters). The intraspecific variation of R. marinae sp. nov. is notable (Tab. 1), but falls well within the range commonly used for species delimitation in arthropods and particularly in Amphipoda. This value was set as 0.03 for both uncorrected p-distance and K2P (e.g. Hebert et al., 2003; Costa et al., 2007, 2009). The sequence divergence of the other two taxa recorded from Sea of Okhotsk is variable – very low in R. sp. 2, and rather high in R. sp. 1 (Tab. 1), however, these values also remain within the limits and allow us to treat each clade as a single species (Fig. 9). This study revealed high intraspecific variation within R. inflata. If all the sequences assigned to this species are treated as a single taxon, then the value of both p-distance and K2P exceeds the limit set for differentiating between species (e.g. Hebert et al., 2003; Costa et al., 2007, 2009) (Tab. 1). However, if the single sequence responsible for this great divergence is treated separately the resulting distance value allows us to consider thespecimens as a single taxon. This was already presented by Lörz et al. (2012) who studied the same sequence data. It is likely that the single sequence from Cornwallis Island (Tab. 3) belongs to another species closely related to R. inflata and was misidentified. It should be underlined that the Cornwallis Island specimen was initially used as an outgroup for a study of species belonging to another amphipod family (Epimeriidae), so the identification of the specimen would not have been the main objective of the authors (Schnabel and Hebert, 2003). The position of all studied species in the NJ tree is not well supported by bootstrap values and to a small degree can only be explained by geographic proximity (Fig. 9). Moreover, the three Rhachotropis taxa from the Okhotsk Sea do not group together. Lörz et al. (2012) studied the molecular affinity of several Rhachotropis species (used also in this study) and found that taxa from the same regions, but collected at different depths are very divergent. On this basis, it was proposed that in the case of this genus, depth has a greater influence on the phylogeny than the geography. The results of the present study do not support this concept, however the 10
present tree clearly has a different topology than the one presented by Lörz et al. (2012). What is more, the two taxa on which the depth-relation concept was proposed constitute the same branch (with medium strength bootstrap support). The three newly added Rhachotropis species, all coming from abyssal depths, do not group together, which would be expected if if depth was a key factor for divergence. It should be noted, however, that the trees were constructed using different methods and additional data were added to the new one, which might have influenced the results. It was already pointed out by Lörz et al. (2012), and should be underlined here, that there is still a need to use additional genes and additional Rhachotropis specimens from a wider variety of geographic locations and depths before we will be able to reconstruct the phylogeny of this worldwide distributed and common genus.
Acknowledgements The material from the Sea of Okhotsk was collected during the Russian-German SokhoBio expedition with the RV Akademik M.A. Lavrentyev with the financial support of the Russian Science Foundation (Project No. 14-50-00034) and sorted with the financial support of the PTJ (German Ministry for Science and Education), grant 03G0857A to Prof. Dr. Angelika Brandt, University of Hamburg. The molecular analysis was performed with University of Lodz internal funds. Marina Malyutina (Institute of Marine Biology, Vladivostok) kindly translated the Russian description of Gurjanova (1955) Rhachotropis grimaldi into English. Michèle Bruni (Museum of Monaco) took photographic images of the holotype of Rhachotropis grimaldi. Tammy Horton, Anne-Helene Tandberg and Wim Vader is thanked for critical comments on an earlier version of this paper. Nadine Dupérré (CeNak, Hamburg) helped with the camera system, Renate Walter (University Hamburg) handled the scanning electron microscope, Frank Friedrichs (University Hamburg) kindly supervised the use of the Confucal Laser Scanning Microscope. Saskia Brix (DZMB Hamburg) let the senior author use the facilities, including microscopes, of her office. The Zoological Museum Hamburg is thanked for the curation of the type specimens.
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Description of a New Species. PLoS ONE 7(3): e32365. doi:10.1371/journal.pone.0032365 Lörz, AN., 2015. An enigmatic Rhachotropis (Crustacea: Amphipoda: Eusiridae) from New Zealand. Zootaxa, 4006 (2), 383–391 Lowry, J.K., Springthorpe, R.T., 2005. New calliopiid and eusirid amphipods from eastern Australian waters (Crustacea : Amphipoda : Calliopiidae : Eusiridae). Proc. Biol. Soc. Wash., 118, 38–47. Malyutina, M., Brandt, A. 2017. Introduction to the SokhoBio (Sea of Okhotsk Biodiversity Studies) expedition" Malyutina, M.V., Chernyshev, A.V. Brandt, A. (this issue). Ratnasingham S., Hebert, P.D., 2007. BOLD: The Barcode of Life Data System (http://www. barcodinglife. org). Mol. Ecol. Notes, 7(3), 355-364. Schnabel K.E., Hebert P.D.N. 2003. Resource-associate divergence in the arctic marine amphipod Paramphithoe hystrix. Mar. Biol., 143, 851-857. DOI 10.1007/s00227003-1126-4 Seefeldt, M.A., Weigand, A.M., Havermans, C., Moreira, E., Held, C. in press. Fishing for scavengers: an integrated study to amphipod (Crustacea: Lysianassoidea) diversity of Potter Cove (South Shetland Islands, Antarctica). Mar. Biodiv., DOI 10.1007/s12526017-0737-9 Smith, S.I. 1883. List of Crustacea dredged on the coast of Labrador by the expedition under the direction of W. A. Stearns, in 1882. Proc. U.S. Nat’l. Mus.7, 218-222. Thurston, M.H., 1980. Abyssal benthic Amphipoda (Crustacea) from the East Iceland Basin. Bull. Br. Mus. Nat. Hist. (Zool.), 38 (1), 43–67. d’Udekem d’Acoz, C., Vader, W., Legeżyńska, J., 2007. On a diminutive Rhachotropis species from the North Sea, with a key to European Rhachotropis (Crustacea, Amphipoda, Eusiridae). Bollettino del Museo Civico di Storia Naturale di Verona, 31, 31–49. Watling, L., 1989. Classification system for crustacean setae based on the homology concept. In: Felgenhauer, B.E., Watling, L. & Thistle, A.B (Eds.), Functional morphology of feeding and grooming in Crustacea. A.A. Balkema, Rotterdam, pp. 214. 14
World Amphipoda Database (2017) Horton, T.; Lowry, J.; De Broyer, C.; Bellan-Santini, D.; Coleman, C. O.;
Daneliya, M.; Dauvin, J-C.; Fišer, C.; Gasca, R.; Grabowski, M.;
Guerra-García, J.
M.; Hendrycks, E.; Holsinger, J.; Hughes, L.; Jaume, D.; Jazdzewski,
K.; Just, J.;
Kamaltynov, R. M.; Kim, Y.-H.; King, R.; Krapp-Schickel, T.; LeCroy, S.;
Lörz, A.-
N.; Senna, A. R.; Serejo, C.; Sket, B.; Tandberg, A.H.; Thomas, J.; Thurston,
M.;
Vader, W.; Väinölä, R.; Vonk, R.; White, K.; Zeidler, W. (2017).. Accessed through:
World Register of Marine Species at http://www.marinespecies.org/aphia.php?p=taxdetails&id=489869 on 2017-05-29
15
Figure 1: Rhachotropis marinae new sp., A,B, holotype, female,15,8 mm, ZMH K-46596, A) dorsal view, B) lateral view; C) paratype, male, 17,2 mm, ZMH K-46597, lateral view. Scale bars: A,B)= 0,5 mm, C)= 1 mm.
16
Figure 2: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) first left maxilla, B) first right maxilla palp, C) outer plate first right maxilla, D) left mandible, E) second antenna, F) first antenna, G) right mandible, H) labrum, I) second maxilla, J) hypopharynx. Scale bars: A-D), G-J)= 0,5 mm, E,F = 0,5 mm.
17
Figure 3: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) first gnathopod, B) second gnathopod. Scalebars: A= 0.5 mm, B = 1 mm.
Figure 4: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) pereopod 3, B) pereopod 4, C) pereopod 5, D) pereopod 6, E) pereopod 7. Scale bars: A-E)= 0,5 mm.
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Figure 5: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) maxilliped, B) second right pleopod, C) uropod 1, D) uropod 2, E) uropod 3, F) telson. Scalebars: A)= 1mm, B)= 0,5mm, C-F)= 1mm.
19
Figure 6: Rhachotropis marinae new sp., paratype, ZMH K-46598, 9 mm A) lateral habitus. B) second gnathopod, C) setation on edge of merus and carpus gnathopod2, D) setation at palm of gnathopod 2, D) dorsal view of pleosomites, F) lateral view of epimeral plates. Scalebars: A)= 1 mm, B)= 100 µm, C)= 10 µm, D)= 10 µm, E)= 200 µm, F)= 100 µm.
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Figure 7: Rhachotropis marinae new sp., A, B, paratype, ZMH K-46613, A) head and pereonites, B) coxa 5 (left) and coxa 4; C—F juvenile of holotype ZMH-K-46596, C) lateral habitus, D) head and coxa 1, E) coxa 7, 6, 5 (5 is right side), F) accessory flagellum. Scalebars: A= 200 µm, B= 100 µm, C= 200 µm, D= 30 µm, E= 20 µm, F= 10 µm.
21
Figure 8: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, epibionts on palp of maxilla 1, confucal laser scanning microscopy (CLSM) micrograph.
22
Figure 9: Neighbour-joining (NJ) tree of COI sequences of Rhachotropis (Tab. S1) based on uncorrected p-distances. Triangles indicate the relative number of individuals studied (height) and sequence divergence (width). The numbers in front of the nodes indicate bootstrap support (1000 replicates, only the values higher than 50% are presented). Below the photographs of three Rhachotropis species hitherto found in SokhoBio samples. A – R. marinae sp. nov. ZMH K-46599, B – Rhachotropis sp. 1 (smooth) ZMH K-46607, C – Rhachotropis sp. 2 (bumpy pereon) ZMH K-46602.
23
Table 3. Rhachotropis spp. and outgroup accession numbers in BOLD, GenBank and station data. Species
BOLD
GenBank
Area
Lat
Long
Depth
Rhachotropis abyssalis Rhachotropis aculeata Rhachotropis aculeata
AMPNZ095-09 WW865-08 WWGSL098-08
GU804296 FJ581880 FJ581881
Ross Sea St. Lawrence Gulf St. Lawrence Gulf
-76.19 47.90 48.39
176.30 -65.35 -59.55
3380 not recorded 150
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
WW851-08 WW850-08 WW105-07
FJ581882 FJ581883 FJ581884
St. Lawrence Gulf St. Lawrence Gulf St. Lawrence Gulf
50.82 50.82 49.92
-58.59 -58.59 -64.62
233 233 not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
WW129-07 WW459-08 WW458-08
FJ581885 FJ581886 FJ581887
St. Lawrence Gulf Cote-Nord Cote-Nord
51.14 50.25 50.25
-58.05 -66.70 -66.70
not recorded not recorded not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
BENTH312-08 BENTH313-08 BENTH314-08
JQ412470 JQ412471 JQ412469
Chukchi Sea Chukchi Sea Chukchi Sea
70.00 70.00 70.00
-168.40 -168.40 -168.40
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
CCNUN228-07 CCNUN150-07 CCNUN151-07
JQ412476 JQ412468 JQ412467
Somerset Island Resolute Resolute
72.77 74.68 74.68
-93.36 -94.86 -94.86
not recorded not recorded not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
CCNUN152-07 CCNUN178-07 CCNUN005-07
JQ412466 JQ412475 JQ412473
Resolute Devon Island Devon Island
74.68 74.67 75.76
-94.86 -91.70 -88.12
not recorded not recorded not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
RBGC036-03 DQ889127 WW024-07 JQ412472 GBCMA0080-06 AY271853
Resolute Beaufort Sea Resolute
70.90
-128.90
not recorded not recorded not recorded
Rhachotropis chathamensis Rhachotropis chathamensis Rhachotropis inflata
AMPNZ101-09 AMPNZ098-09 CCNUN620-08
GU804298 GU804300 JQ412491
New Zealand New Zealand Resolute
-43.80 -43.80 75.08
175.32 175.32 -94.86
418 418 not recorded
Rhachotropis inflata Rhachotropis inflata Rhachotropis inflata
CCNUN621-08 CCNUN622-08 CCNUN156-07
JQ412492 JQ412493 JQ412488
Resolute Resolute Resolute
75.08 75.08 74.68
-94.86 -94.86 -94.86
not recorded not recorded not recorded
Rhachotropis inflata Rhachotropis inflata Rhachotropis inflata
CCNUN157-07 CCNUN158-07 CCNUN159-07
JQ412497 JQ412499 JQ412495
Resolute Resolute Resolute
74.68 74.68 74.68
-94.86 -94.86 -94.86
not recorded not recorded not recorded
Rhachotropis inflata Rhachotropis inflata1 Rhachotropis novazealandica
RBGC038-03 JQ412486 GBCMA0081-06 AY271854 AMPNZ128-09 GU804309
Resolute Cornwallis Island New Zealand
-44.13
174.85
not recorded not recorded 520
Rhachotropis rossi Rhachotropis sp. 28 Rhachotropis sp. A
ANZR470-08 JF498593 GBCMA1154-08 EF989704 AMPNZ184-10 JF498594
Ross Sea California New Zealand
-76.59 36.33 -36.52
176.83 122.90 179.20
Rhachotropis sp. A1 Rhachotropis sp. B Rhachotropis sp. 2 (bumpy)
CMBIA625-12 AMPNZ102-09 ZMH K-46602
N/A California HM372956 New Zealand MF409443 Okhotsk Sea
32.73 -43.80 46.26
-117.34 175.32 152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46603
MF409442 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46604
MF409437 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46605
MF409449 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46606
MF409438 Okhotsk Sea
46.26
152.05
3371-3377
45 45 45
3210 300–700 5173 98 418
24
Rhachotropis sp. 1 (smooth)
ZMH K-46607
MF409441 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46608
MF409436 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46609
MF409447 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46610
MF409448 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46611
MF409439 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis marinae sp. nov.
ZMH K-46596
MF409440 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46597
MF409435 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46599
MF409444 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46598
MF409445 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46600
MF409446 Okhotsk Sea
46.94
151.08
3299
Eusirus sp.
ANZR028-08
-72.08
175.55
1620
JQ412464
Ross Sea
25
www.elsevier.com/locate/dsr2
PII: DOI: Reference:
S0967-0645(17)30247-3 https://doi.org/10.1016/j.dsr2.2017.09.020 DSRII4322
To appear in: Deep-Sea Research Part II Received date: 28 June 2017 Revised date: 29 August 2017 Accepted date: 26 September 2017 Cite this article as: A.N. Lörz, A.M. Jażdżewska and A. Brandt, Abyssal Rhachotropis (Eusiroidea, Amphipoda) from the Sea of Okhotsk, Deep-Sea Research Part II, https://doi.org/10.1016/j.dsr2.2017.09.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Abyssal Rhachotropis (Eusiroidea, Amphipoda) from the Sea of Okhotsk
Lörz, A.N. 1, Jażdżewska, A.M. ², Brandt, A. 3
1
University of Hamburg, Centre of Natural History, Zoological Museum, Martin-LutherKing-Platz 3, 20146 Hamburg, Germany, Email: [email protected] ²Laboratory of Polar Biology and Oceanobiology, Department of Invertebrate Zoology and Hydrobiology, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha st., 90-237 Lodz, Poland 3 Senckenberg Research Institute and Natural History Museum, Department of Marine Zoology, Senckenberganlage 25, 60325 Frankfurt am Main, Germany Abstract The genus Rhachotropis has the widest horizontal and bathymetric distribution of all amphipod genera known worldwide. Characteristic species of Rhachotropis were collected at 3200 m depth in the Kuril basin of the Sea of Okhotsk. Morphological investigations revealed a species new to science, which is here described in detail. Juveniles were removed from the marsupium of the holotype; their morphological features were investigated via scanning electron microscopy and compared to the adults. The molecular barcode of the new species, Rhachotropis marinae, was obtained. Comparison with available Rhachotropis COI data revealed clear separation of the new species from all known species as well as from two other Rhachotropis species also collected during the SokhoBio expedition. The three Rhachotropis species from abyssal depths in the Sea of Okhotsk did not group together when their COI region was analysed with Rhachotropis sequences from other parts of the world. The number of described species of Rhachotropis is now increased to 62.
Keywords Amphipoda, Rhachotropis, new species, North Pacific, barcode
1
1. Introduction Amphipoda, among other macrofauna, were sampled during the joint Russian-German expedition SokhoBio (Sea of Okhotsk Biodiversity Studies) on board of the R/V Akademik M.A. Lavrentyev in the Kurile Basin of the Sea of Okhotsk in summer 2015 (for a general introduction see Malyutina et al., this volume). The genus Rhachotropis S.I. Smith, 1883 (Eusiridae) contains 61 species (World Amphipoda Database, Horton et al. 2017). , is found in all oceans, and has the greatest bathymetric distribution (0—9460 m) of all amphipod genera (Lörz et al., 2012). Some Rhachotropis species are known to be very abundant locally (Cartes and Sorbe, 1999). A first investigation of the SokhoBio amphipod collection revealed characteristic specimens of a species of Rhachotropis new to science. This species, with a prominent protuberance on the head, is described here in detail. Two further species of Rhachotropis, one with smooth pereonites, the other with protuberant pereonites, were also collected at abyssal depths during the SokhoBio expedition. Since the proposal of the DNA barcoding concept by Hebert et al. (2003) the use of molecular methods in species recognition has become very popular and are often used to supplement morphological taxonomy (e.g. Hubert and Hanner, 2015; Jinbo et al., 2011; Seefeldt et al., in press and references therein). As the present material was suitable for DNA analyses we investigated the relationships of the three freshly collected Rhachotropis species via analysis of a cytochrome oxidase region (COI) and set them in context with COI sequences from Rhachotropis species from other parts of the world.
2. Material and Methods The detailed description of the study area is presented in the introduction of this volume (Malyutina and Brandt, this issue). Large amphipod specimens were immediately sorted on deck, fixed in -20° precooled 98% ethanol and later transferred to 96% ethanol. Amphipod samples collected deeper than 3000 m are regarded as “abyssal”, following the classification of abyssal amphipods by Barnard (1962). 2
2.1. Morphological description Specimens were examined and dissected under a Leica MZ12.5 stereomicroscope and drawn using a camera lucida. Small appendages (mouthparts, uropods, and telson) were temporarily mounted in glycerin and examined and drawn using a LeicaDM2500 compound microscope fitted with a camera lucida. The body lengths of specimens examined were measured by tracing individual’s mid-trunk lengths (tip of the rostrum to end of telson) using a camera lucida. All illustrations were digitally ‘inked’ following Coleman (2003, 2009). Two adult paratypes and 4 juveniles extracted from the marsupium of the holotype were dried, coated with gold-paladium and investigated via a scanning electron microscope LEO1525. In order to preserve the good condition of epibionts, the palp of maxilla 1 was selected for confocal laser scanning microscopy (CLSM). To gain auto fluorescence of the surfaces, the 405 nm laser lines were applied with emission filters set to 421–499 nm, the 488 nm laser lines to 489 –607 nm. The palp was scanned using a Leica DM2500 with a Leica TCS SPE at a resolution of 2480 x 2480 pixels with a 10x. The software package LEICA LAS X was used for recording the image from the scans. The image stacks were further processed infinalized in Adobe Photoshop CS5. Photos of material held at the Centre of Natural History (CeNak) were taken with a Canon EOS 5 Mark III with lens Canon MP-E65 macro mounted for stacking, the stacking programme software is Zerene Stacker 1.04 (setting P-max). Type material is held at the Zoological Museum University of Hamburg, CeNak. 2.2. DNA extraction and analyses DNA extraction from five individuals of the newly described species as well as ten additional individuals of Rhachotropis sp. from another station sampled during SokhoBio expedition (Tab. S1) was performed according to a standard phenol-chloroform method after Hillis et al. (1996). Air-dried DNA pellets were eluted in 100 μl of TE buffer, pH 8.00, stored at 4°C until amplification, and subsequently at -20°C for long-term storage. A fragment of Cytochrome Oxidase subunit I gene (COI; ca. 670 bp fragment) was amplified using standard LCO1490/HCO2198 primers (Folmer et al., 1994) with DreamTaq Green PCR Mastermix (Thermo Scientific) and reaction conditions following Hou et al. (2007). Sequences were obtained using BigDye sequencing protocol (Applied Biosystems 3730xl) by Macrogen Inc., 3
Korea. Sequences were edited using Geneious 10.1.2 leading to 15 sequences of 607 bp each. All sequences were deposited in GenBank with the accession numbers (Table S1). Relevant voucher information, taxonomic classifications, and sequences are accessible through the public data set “DS-RHSOKHO” on the Barcode of Life Data Systems (BOLD; www.boldsystems.org) (Ratnasingham and Hebert, 2007). To check the genetic relationships between the new species and the known Rhachotropis spp. distributed worldwide, 44 publicly available sequences (longer than 590 bp) of additional Rhachotropis specimens were retrieved from the BOLD database (Tab. S1). All sequences were aligned using the MAFFT v7.308 algorithm (Katoh and Stanley, 2013, Katoh et al., 2002) in Geneious 10.1.2, resulting in 560 bp alignment used for further analyses. The uncorrected p-distance and the Kimura 2-parameter (K2P) model (Kimura, 1980) were used to determine sequence divergence in MEGA V7.0.18 (Kumar et al., 2016). A NeighbourJoining (NJ) tree was built based on the uncorrected p-distance with both transition and transversion substitutions included and pairwise deletion chosen. Node support was inferred with a bootstrap analysis (1000 replicates). The COI sequence of an Antarctic Eusirus species mined from GenBank (accession number JQ412464) (Table S1) was used as the outgroup, since it is a closely related genus to Rhachotropis within the family Eusiridae.
3. Results Systematics Order AMPHIPODA Latreille, 1816 Suborder GAMMARIDEA Latreille, 1802 Family EUSIRIDAE Stebbing, 1888 Genus Rhachotropis S.I. Smith, 1883 Rhachotropis S.I. Smith, 1883: 222. Gracilipes Holmes, 1908: 526. Type species. Oniscus aculeata Lepechin, 1780 Rhachotropis marinae sp. nov. Syn. Rhachotropis grimaldi (Chevreux, 1887) by Gurjanova 1955 Fig. 11 (Figs 1–8, 9A) 4
Material examined. Holotype: ZMH K-46596, female 15.8 mm, SokhoBio expedition 2015, station 7-3, gear epibenthic sledge, sampling date 22. 07. 2015, 46°56.556'N 151°05.013'E, 46°56.791'N 151°04.860'E, 3299 m depth, Paratypes: ZMH K-46597, male 17.2 mm; ZMH K-46598 (on SEM Fig. 6); ZMH K-46599; ZMH K-46600 ; ZMH-K-46613, 7.8 mm(sex unknown); all paratypes have the same collection data as the holotype. Further material examined: 10 Rhachotropis specimens from SokhoBio station 9-7 (ZMH K46602-46611, Gen Bank accession numbers: MF409435-MF409449, Tab. S1). The holotype of Rhachotropis grimaldi (Chevreux, 1887) was further examined via photographs taken by Michele Bruni from the Museum of Monaco for this study.
Etymology: This species is named for Marina Malyutina. Marina was one of the organisers of the expedition during which the new species was collected, and we greatly appreciate her outstanding contribution to peracarid taxonomy and systematics.
Diagnosis The new species is relatively easy to recognize due to a prominant protuberance on its head. It can be separated from other species of Rhachotropis by the combination of following characters: coxa 1 drawn out to anterior end of head, telson split half way, coxa 7 posteriorly drawn out and pointed, epimeral plate 3 entire posterior margin serrate. Description Antennae. Antenna 1 slightly shorter than antenna 2. Antenna 1 article 2 slightly shorter than article 1, 1.5 times as long as article 3; flagellum 12-articulate; accessory flagellum 2articulate. Antenna 2 peduncle article 3 and 4 subequal in length, several plumose setae on third and fourth article; flagellum 23-articulate. Mouthparts. Mandible with incisor process well-developed; lacinia mobilis denticulate; molar process conical; palp article 1 short, about one-third length of article 2, article 3 nearly as long as article 2, articles 2 and 3 with long slender setae. Maxilla 1 inner plate bearing 2 subterminal plumose setae; outer plate with 9 denticulate spines; article 1 of palp with long 5
setae at outer margin, article 2 of palp with several slender setae at tip. Maxilla 2 inner and outer plate subequal in length, margins bearing stout and slender setae; outer plate bearing 2 plumose setae; inner plate wider than outer plate. Maxilliped outer plate 2.5 times as long as inner plate, reaching half of article 2 of maxillipedal palp. Labrum circular in shape, two setose areas anteriorly. Hypopharynx setose distoanteriorly, outer lobes separated by broad gap. Pereopods. Coxa 1 drawn out reaching anterior end of head, coxa 1 posterior corner with strong hook. Gnathopods similar in shape, subchelate. Gnathopod 1 slightly smaller than gnathopod 2; basis bearing several small spines at anteroventral corner; ischium and merus with long setae at posteroventral corner; carpus lobe extending width of propodus, spines at terminal end of lobe; propodus widened, oval; dactylus slender, reaching end of palm. Coxa 2 subquadrate, posteroventral corner bearing small hook; gnathopod 2 basis 1.5 times as wide as basis of gnathopod 1; ischium to dactylus similar to gnathopod 1. Coxae 3 and 4 subquadrate, ventral margin with small hook on anterior and posterior corner. Pereopods 3 and 4 articles long and narrow; carpus, propodus and dactylus about same length.
Coxa 5 strongly lobate, anterior and posterior corner of ventral margin with insertion. Pereopod 5 as long as pereon; basis rectangular; merus slightly inflated, 1.3 times as long as carpus; propodus 1.5 times as long as carpus. Coxa 6 strongly lobate, posterior lobe extending further ventrally than anterior lobe, anterior and posterior corner of ventral margin with insertion. Pereopod 6 larger, but similar in shape to pereopod 5; basis with ridge. Coxa 7 posterior margin strongly drawn out. Pereopod 7 longer than pereopod 5 and 6; basis with rounded extension posteriorly; merus posteroventral angle produced; broken off at carpus. Pleopod 2 (right side) rami with 28 and 22 articles; rami 1.5 times as long as peduncle. Uropod 1 outer rami slightly shorter than inner rami; rami shorter than peduncle. Uropod 2 outer rami 0.2 times shorter than inner rami; peduncle shorter than outer rami and longer than inner rami. Uropod 3 inner rami slightly longer than outer rami, inner rami twice as long as peduncle. Telson three times as long as wide, cleft 45 %.
Remarks. Sexual differences: Male specimens (Fig. 1C) have longer antennae, more than 50 articles each, the third article is expanded anteriorly. The third epimeral plate is continuously
6
serrate posteriorly in females, whereas males bear an interruption of the serration, with smooth sections towards the dorsal spine. Juveniles. Twenty four juveniles were counted in the marsupium of the holotype. Four were removed for scanning electron microscopy (Fig. 7 C—F). Several juveniles fell out while dissecting the right side for illustrating, but the majority were left undisturbed. Morphological differences between the juvenile and adult include juvenile lacking the protuberance on the head (Fig. 6 C, D versus adult Fig. 6 A, B), coxa 1 not bearing an insertion (Fig. 6 D versus adult Fig. 6 A, B), coxae 2—6 lacking “hooks” (Fig. 6 E, versus adult Fig. 6A, B), the accessory flagellum comprised of one article only (Fig. 6 F) (versus two articles in adults) and both antenna being shorter, only half the body length. Epibionts are dominant features of the mouthparts and gnathopods of the adults (Fig 8). 3.2. Molecular study Among the 15 sequences obtained in this study, 11 haplotypes were distinguished, forming three distinct groups. One group consisted of five sequences of the presently described species; the second two groups were made by individuals from additional stations. The divergence of the sequences within each group recognized was variable with the lowest value found for Rhachotropis sp. 2 (0.002 of both uncorrected p-distance and K2P), medium for R. marinae (0.014 both measures) and the highest for Rhachotropis sp. 1 (0.025 and 0.026 for uncorrected p-distance and K2P, respectively) (Tab. 1). Rhachotropis abyssalis, R. novazealandica, R. rossi, R. sp. A, R. sp. A1, R. sp B and R. sp 28 were all represented by single sequences. The NJ tree showed clear differentiation of all studied taxa, however, as many as half of them were singletons (Fig. 9). The groups of sequences from Okhotsk Sea remained as separate clades, but their positions in the tree are not well supported.
7
Table 1. The intraspecific variation within Rhachotropis species calculated using uncorrected p-distance and Kimura 2-parameter (K2P). Values in column A were obtained when all sequences assigned to R. inflata were treated as a single unit; in column B the same values are calculated excluding one distinct sequence of R. inflata (R. inflata 1).
Rhachotropis aculeata (Lepechin, 1780) Rhachotropis chathamensis Lörz, 2010 Rhachotropis inflata (Sars, 1883) Rhachotropis inflata 1 Rhachotropis marinae sp. nov. Rhachotropis sp. 1 (smooth) Rhachotropis sp. 2 (bumpy)
p-distance A B 0.008 0.008 0.000 0.000 0.038 0.014 -------N/A 0.014 0.014 0.025 0.025 0.002 0.002
K2P A 0.008 0.000 0.042 -------0.014 0.026 0.002
B 0.008 0.000 0.014 N/A 0.014 0.026 0.002
4. Discussion 4.1 Morphological similarities The new species matches a species description by Gurjanova (1955) of Rhachotropis grimaldi (Chevreux, 1887). The type species of R. grimaldi was sampled in the Mediterranean at 510 m depth, Gurjanova (1955) illustrated a specimen from the Sea of Okhotsk at 2850 m, near Iturup Island, and lists several morphological differences. We are convinced that Gurjanova (1955) described a new species. She only had one specimen, and was lead by morphological similarities, such as the bumpy pereon, to believe her specimen was R. grimaldi. She did list several characters of her specimen that did not match the description of Chevreux’s specimen. Several authors have recorded R. grimaldi, including Ledoyer (1977), from 400 m near Marseilles (Mediterranean), Barnard (1916) from 500 m off South Africa (Atlantic).We have several specimens from 3200 m depth of the Okhotsk Sea, showing consistent morphological features and consistent morphological differences to R. grimaldi (Chevreux, 1887), see table 2. Our specimens of R. marinae new sp. from 3200 m depth of the Okhotsk Sea, show consistent morphological features and can be easily distinguished from R. grimaldi (Chevreux, 1887), see table 2.
8
Table 2. Morphological differences between Rhachotropis marinae new sp. and R. grimaldi (Chevreux, 1887). R. marinae new sp. Protuberance on head
R. grimaldi (Chevreux, 1887) present
absent
Second urosomite posterior margin
smooth
spinose
Head length vs first three pereonites
longer
shorter
Antennular lobe
narrow, projected
short, rounded
Coxa 4 posterior margin
notched
smooth
Second epimeral plate
serrated
smooth
Many deep-sea Rhachotropis are reported to be blind, but careful examination of the preserved specimens of the new species collected at abyssal depths shows distinct oval eyes in both males and females. The eyes will be easily overlooked when specimens are preserved in ethanol for any length of time.
4.2.Juveniles Juveniles were taken from the marsupium of the holotype (see Fig. 6 C—F). The main morphological difference to the adults is being less spiny most likely due to allometry. We therefore do not interpret the smooth body of the juvenile as plesiomorphic state, but as pragmatic feature as a result of being cramped in the marsupium. 4.3.Epibionts These epibionts are characteristic features of Rhachotropis species collected in various areas and depths of the world’s oceans, e.g. on Rhachotropis chathamensis Lörz, 2010 in the South East Pacific, R. rossi Lörz, 2010 from the Ross Sea (Lörz, 2010) , R. macropus Sars, 1893 and R. aculeata (Lepechin, 1780) from the North Atlantic (pers. obs.) . The epibionts vary in density, but are mainly found on the mouthparts and gnathopods. While other families and genera of Amphipoda also bear the occasional epibiont, these are typical on the genus Rhachotropis. A further investigation of the host specificity is underway. 4. 4. Molecular investigation 9
The barcoding of Rhachotropis spp. from the Okhotsk Sea revealed the existence of at least three distinct species at the two stations studied. The samples were collected at similar abyssal depths, however, one of the stations (from which the new species is described) is situated in the Sea of Okhotsk itself, while the second station was situated on the Pacific side of Kuril Islands about 70 km away from the Bussol Strait. Further study of the Rhachotropis spp. from the area will reveal more about the distribution of these species. It is not yet known if these species will be recorded across the whole NW Pacific area or if they have restricted ranges (e.g. only to Okhotsk Sea or only to the open waters). The intraspecific variation of R. marinae sp. nov. is notable (Tab. 1), but falls well within the range commonly used for species delimitation in arthropods and particularly in Amphipoda. This value was set as 0.03 for both uncorrected p-distance and K2P (e.g. Hebert et al., 2003; Costa et al., 2007, 2009). The sequence divergence of the other two taxa recorded from Sea of Okhotsk is variable – very low in R. sp. 2, and rather high in R. sp. 1 (Tab. 1), however, these values also remain within the limits and allow us to treat each clade as a single species (Fig. 9). This study revealed high intraspecific variation within R. inflata. If all the sequences assigned to this species are treated as a single taxon, then the value of both p-distance and K2P exceeds the limit set for differentiating between species (e.g. Hebert et al., 2003; Costa et al., 2007, 2009) (Tab. 1). However, if the single sequence responsible for this great divergence is treated separately the resulting distance value allows us to consider thespecimens as a single taxon. This was already presented by Lörz et al. (2012) who studied the same sequence data. It is likely that the single sequence from Cornwallis Island (Tab. 3) belongs to another species closely related to R. inflata and was misidentified. It should be underlined that the Cornwallis Island specimen was initially used as an outgroup for a study of species belonging to another amphipod family (Epimeriidae), so the identification of the specimen would not have been the main objective of the authors (Schnabel and Hebert, 2003). The position of all studied species in the NJ tree is not well supported by bootstrap values and to a small degree can only be explained by geographic proximity (Fig. 9). Moreover, the three Rhachotropis taxa from the Okhotsk Sea do not group together. Lörz et al. (2012) studied the molecular affinity of several Rhachotropis species (used also in this study) and found that taxa from the same regions, but collected at different depths are very divergent. On this basis, it was proposed that in the case of this genus, depth has a greater influence on the phylogeny than the geography. The results of the present study do not support this concept, however the 10
present tree clearly has a different topology than the one presented by Lörz et al. (2012). What is more, the two taxa on which the depth-relation concept was proposed constitute the same branch (with medium strength bootstrap support). The three newly added Rhachotropis species, all coming from abyssal depths, do not group together, which would be expected if if depth was a key factor for divergence. It should be noted, however, that the trees were constructed using different methods and additional data were added to the new one, which might have influenced the results. It was already pointed out by Lörz et al. (2012), and should be underlined here, that there is still a need to use additional genes and additional Rhachotropis specimens from a wider variety of geographic locations and depths before we will be able to reconstruct the phylogeny of this worldwide distributed and common genus.
Acknowledgements The material from the Sea of Okhotsk was collected during the Russian-German SokhoBio expedition with the RV Akademik M.A. Lavrentyev with the financial support of the Russian Science Foundation (Project No. 14-50-00034) and sorted with the financial support of the PTJ (German Ministry for Science and Education), grant 03G0857A to Prof. Dr. Angelika Brandt, University of Hamburg. The molecular analysis was performed with University of Lodz internal funds. Marina Malyutina (Institute of Marine Biology, Vladivostok) kindly translated the Russian description of Gurjanova (1955) Rhachotropis grimaldi into English. Michèle Bruni (Museum of Monaco) took photographic images of the holotype of Rhachotropis grimaldi. Tammy Horton, Anne-Helene Tandberg and Wim Vader is thanked for critical comments on an earlier version of this paper. Nadine Dupérré (CeNak, Hamburg) helped with the camera system, Renate Walter (University Hamburg) handled the scanning electron microscope, Frank Friedrichs (University Hamburg) kindly supervised the use of the Confucal Laser Scanning Microscope. Saskia Brix (DZMB Hamburg) let the senior author use the facilities, including microscopes, of her office. The Zoological Museum Hamburg is thanked for the curation of the type specimens.
11
References Barnard, J.L., 1963. South Atlantic Abyssal Amphipods Collected by R. V. Vema. Abyssal Crustacea, Research Series. Scr. 1, 1–78. Bellan-Santini, D. , 2006. Rhachotropis species (Crustacea: Amphipoda: Eusiridae) of hydrothermal vents and surroundings on the Mid-Atlantic Ridge, Azores Triple Junction Zone. J. Nat. Hist. 40 (23-24), 1407–1424. Brandt, A, Alalykina, I., Fukumori, H., Golovan, O., Kniesz, K., Lavrenteva, A., Lörz, AN., Malyutina, M., Philipps-Bussau, K., Stransky, B. (this volume) First insights into macrofaunal composition of bathyal Sea of OkhotskCartes, J.E., Sorbe, C. , 1999. Deep-water amphipods from the Catalan Sea slope (western Mediterranean): bathymetric distribution, assemblage composition and biological characteristics. J. Nat. Hist. 33(8), 113–1158. Chevreux, E., 1887. Crustaces amphipodes noveaux dragues par l’Hirondelle, pendant sa campagne de 1886. Bull. Soc. Zool. France 12, 566-580. Coleman, C.O., 2003. "Digital inking": How to make perfect line drawings on computers. Organism, Diversity and Evolution, Electronic Supplement, 14, 1–14. (http://senckenberg.de/odes/03-14.htm, accessed May 2009) Coleman, C.O., 2009. Drawing setae the digital way. Zoosyst.and Evol., 85 (2), 305–310. http://dx.doi.org/10.1002/zoos.200900008 Costa F.O., deWaard J.R., Boutillier J., Ratnasingham S., Dooh R.T., Hajibabaei M., Hebert P.D.N. 2007. Biological identifications through DNA barcodes: the case of the Crustacea. Can. J. Fish. Aquat. Sci., 64, 272–295. Costa F.O., Henzler C.M., Lunt D.H., Whiteley N.M., Rock J. 2009. Probing marine Gammarus (Amphipoda) taxonomy with DNA barcodes. Syst. Biodivers., 7, 365– 379. Dahl, E., 1959. Amphipoda from depths exceeding 6000 m. Galathea reports, 211–240. Folmer O., Black M., Hoeh W., Lutz R., Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. & Biotech., 3 (5), 294-299. Gurjanova E., 1951. Bokoplavy Morei SSSR i Sopredel’nykh Vod. (Amphipoda-Gammaridea). Izd. Akademia Nauk SSSR, Moscow & Leningrad. 12
Gurjanova, E., 1955. Novye vidy bokoplavov (Amphipoda, Gammaridea) iz servernoi chasti Tixogo Okeana. Zool. Inst. Akad. Nauk SSSR, Trud. 18, 166-218. Hebert PD, Ratnasingham S, deWaard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B, 270, S96–S99. Hillis, D.M., Mable B.K., Moritz, C., 1996. Applications of molecular systematics. In Hillis, D. M, Moritz C. and Mable B. (eds), Molecular Systematics. Sunderland, MA: Sinauer Associates, pp. 515-543. Hubert, N., Hanner, R. 2015. DNA Barcoding, species delineation and taxonomy: a historical perspective. DNA Barcodes, 3, 44–58. Jinbo, U., Kato, T., Ito, M. 2011. Current progress in DNA barcoding and future implications for entomology. Entomol. Sci., 14 (2), 107-124.Katoh, K., Misawa, K., Kuma, K., Miyata, T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res., 30 (14): 3059-3066. doi: 10.1093/nar/gkf436 Katoh K., Standley D.M. 2013. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol., 30 (4), 772-780. doi: 10.1093/molbev/mst010 Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 16, 111-120. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol. & Evol., 33(7), 1870-1874. Lepechin, I., 1780. Tresoniscorum species descriptae. Acta Acad. Scient. Imp. Petrop. Lörz, AN., 2010 Deep-sea Rhachotropis (Crustacea: Amphipoda: Eusiridae) from New Zealand and the Ross Sea with key to the Pacific, Indian Ocean and Antarctic species. Zootaxa, 2482, 22–48. Lörz, AN., Linse, K., Smith, P.J., Steinke, D., 2012. First Molecular Evidence Underestimated Biodiversity of Rhachotropis (Crustacea, Amphipoda), with
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Description of a New Species. PLoS ONE 7(3): e32365. doi:10.1371/journal.pone.0032365 Lörz, AN., 2015. An enigmatic Rhachotropis (Crustacea: Amphipoda: Eusiridae) from New Zealand. Zootaxa, 4006 (2), 383–391 Lowry, J.K., Springthorpe, R.T., 2005. New calliopiid and eusirid amphipods from eastern Australian waters (Crustacea : Amphipoda : Calliopiidae : Eusiridae). Proc. Biol. Soc. Wash., 118, 38–47. Malyutina, M., Brandt, A. 2017. Introduction to the SokhoBio (Sea of Okhotsk Biodiversity Studies) expedition" Malyutina, M.V., Chernyshev, A.V. Brandt, A. (this issue). Ratnasingham S., Hebert, P.D., 2007. BOLD: The Barcode of Life Data System (http://www. barcodinglife. org). Mol. Ecol. Notes, 7(3), 355-364. Schnabel K.E., Hebert P.D.N. 2003. Resource-associate divergence in the arctic marine amphipod Paramphithoe hystrix. Mar. Biol., 143, 851-857. DOI 10.1007/s00227003-1126-4 Seefeldt, M.A., Weigand, A.M., Havermans, C., Moreira, E., Held, C. in press. Fishing for scavengers: an integrated study to amphipod (Crustacea: Lysianassoidea) diversity of Potter Cove (South Shetland Islands, Antarctica). Mar. Biodiv., DOI 10.1007/s12526017-0737-9 Smith, S.I. 1883. List of Crustacea dredged on the coast of Labrador by the expedition under the direction of W. A. Stearns, in 1882. Proc. U.S. Nat’l. Mus.7, 218-222. Thurston, M.H., 1980. Abyssal benthic Amphipoda (Crustacea) from the East Iceland Basin. Bull. Br. Mus. Nat. Hist. (Zool.), 38 (1), 43–67. d’Udekem d’Acoz, C., Vader, W., Legeżyńska, J., 2007. On a diminutive Rhachotropis species from the North Sea, with a key to European Rhachotropis (Crustacea, Amphipoda, Eusiridae). Bollettino del Museo Civico di Storia Naturale di Verona, 31, 31–49. Watling, L., 1989. Classification system for crustacean setae based on the homology concept. In: Felgenhauer, B.E., Watling, L. & Thistle, A.B (Eds.), Functional morphology of feeding and grooming in Crustacea. A.A. Balkema, Rotterdam, pp. 214. 14
World Amphipoda Database (2017) Horton, T.; Lowry, J.; De Broyer, C.; Bellan-Santini, D.; Coleman, C. O.;
Daneliya, M.; Dauvin, J-C.; Fišer, C.; Gasca, R.; Grabowski, M.;
Guerra-García, J.
M.; Hendrycks, E.; Holsinger, J.; Hughes, L.; Jaume, D.; Jazdzewski,
K.; Just, J.;
Kamaltynov, R. M.; Kim, Y.-H.; King, R.; Krapp-Schickel, T.; LeCroy, S.;
Lörz, A.-
N.; Senna, A. R.; Serejo, C.; Sket, B.; Tandberg, A.H.; Thomas, J.; Thurston,
M.;
Vader, W.; Väinölä, R.; Vonk, R.; White, K.; Zeidler, W. (2017).. Accessed through:
World Register of Marine Species at http://www.marinespecies.org/aphia.php?p=taxdetails&id=489869 on 2017-05-29
15
Figure 1: Rhachotropis marinae new sp., A,B, holotype, female,15,8 mm, ZMH K-46596, A) dorsal view, B) lateral view; C) paratype, male, 17,2 mm, ZMH K-46597, lateral view. Scale bars: A,B)= 0,5 mm, C)= 1 mm.
16
Figure 2: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) first left maxilla, B) first right maxilla palp, C) outer plate first right maxilla, D) left mandible, E) second antenna, F) first antenna, G) right mandible, H) labrum, I) second maxilla, J) hypopharynx. Scale bars: A-D), G-J)= 0,5 mm, E,F = 0,5 mm.
17
Figure 3: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) first gnathopod, B) second gnathopod. Scalebars: A= 0.5 mm, B = 1 mm.
Figure 4: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) pereopod 3, B) pereopod 4, C) pereopod 5, D) pereopod 6, E) pereopod 7. Scale bars: A-E)= 0,5 mm.
18
Figure 5: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, A) maxilliped, B) second right pleopod, C) uropod 1, D) uropod 2, E) uropod 3, F) telson. Scalebars: A)= 1mm, B)= 0,5mm, C-F)= 1mm.
19
Figure 6: Rhachotropis marinae new sp., paratype, ZMH K-46598, 9 mm A) lateral habitus. B) second gnathopod, C) setation on edge of merus and carpus gnathopod2, D) setation at palm of gnathopod 2, D) dorsal view of pleosomites, F) lateral view of epimeral plates. Scalebars: A)= 1 mm, B)= 100 µm, C)= 10 µm, D)= 10 µm, E)= 200 µm, F)= 100 µm.
20
Figure 7: Rhachotropis marinae new sp., A, B, paratype, ZMH K-46613, A) head and pereonites, B) coxa 5 (left) and coxa 4; C—F juvenile of holotype ZMH-K-46596, C) lateral habitus, D) head and coxa 1, E) coxa 7, 6, 5 (5 is right side), F) accessory flagellum. Scalebars: A= 200 µm, B= 100 µm, C= 200 µm, D= 30 µm, E= 20 µm, F= 10 µm.
21
Figure 8: Rhachotropis marinae new sp., holotype, female, 15,8 mm, ZMH K-46596, epibionts on palp of maxilla 1, confucal laser scanning microscopy (CLSM) micrograph.
22
Figure 9: Neighbour-joining (NJ) tree of COI sequences of Rhachotropis (Tab. S1) based on uncorrected p-distances. Triangles indicate the relative number of individuals studied (height) and sequence divergence (width). The numbers in front of the nodes indicate bootstrap support (1000 replicates, only the values higher than 50% are presented). Below the photographs of three Rhachotropis species hitherto found in SokhoBio samples. A – R. marinae sp. nov. ZMH K-46599, B – Rhachotropis sp. 1 (smooth) ZMH K-46607, C – Rhachotropis sp. 2 (bumpy pereon) ZMH K-46602.
23
Table 3. Rhachotropis spp. and outgroup accession numbers in BOLD, GenBank and station data. Species
BOLD
GenBank
Area
Lat
Long
Depth
Rhachotropis abyssalis Rhachotropis aculeata Rhachotropis aculeata
AMPNZ095-09 WW865-08 WWGSL098-08
GU804296 FJ581880 FJ581881
Ross Sea St. Lawrence Gulf St. Lawrence Gulf
-76.19 47.90 48.39
176.30 -65.35 -59.55
3380 not recorded 150
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
WW851-08 WW850-08 WW105-07
FJ581882 FJ581883 FJ581884
St. Lawrence Gulf St. Lawrence Gulf St. Lawrence Gulf
50.82 50.82 49.92
-58.59 -58.59 -64.62
233 233 not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
WW129-07 WW459-08 WW458-08
FJ581885 FJ581886 FJ581887
St. Lawrence Gulf Cote-Nord Cote-Nord
51.14 50.25 50.25
-58.05 -66.70 -66.70
not recorded not recorded not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
BENTH312-08 BENTH313-08 BENTH314-08
JQ412470 JQ412471 JQ412469
Chukchi Sea Chukchi Sea Chukchi Sea
70.00 70.00 70.00
-168.40 -168.40 -168.40
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
CCNUN228-07 CCNUN150-07 CCNUN151-07
JQ412476 JQ412468 JQ412467
Somerset Island Resolute Resolute
72.77 74.68 74.68
-93.36 -94.86 -94.86
not recorded not recorded not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
CCNUN152-07 CCNUN178-07 CCNUN005-07
JQ412466 JQ412475 JQ412473
Resolute Devon Island Devon Island
74.68 74.67 75.76
-94.86 -91.70 -88.12
not recorded not recorded not recorded
Rhachotropis aculeata Rhachotropis aculeata Rhachotropis aculeata
RBGC036-03 DQ889127 WW024-07 JQ412472 GBCMA0080-06 AY271853
Resolute Beaufort Sea Resolute
70.90
-128.90
not recorded not recorded not recorded
Rhachotropis chathamensis Rhachotropis chathamensis Rhachotropis inflata
AMPNZ101-09 AMPNZ098-09 CCNUN620-08
GU804298 GU804300 JQ412491
New Zealand New Zealand Resolute
-43.80 -43.80 75.08
175.32 175.32 -94.86
418 418 not recorded
Rhachotropis inflata Rhachotropis inflata Rhachotropis inflata
CCNUN621-08 CCNUN622-08 CCNUN156-07
JQ412492 JQ412493 JQ412488
Resolute Resolute Resolute
75.08 75.08 74.68
-94.86 -94.86 -94.86
not recorded not recorded not recorded
Rhachotropis inflata Rhachotropis inflata Rhachotropis inflata
CCNUN157-07 CCNUN158-07 CCNUN159-07
JQ412497 JQ412499 JQ412495
Resolute Resolute Resolute
74.68 74.68 74.68
-94.86 -94.86 -94.86
not recorded not recorded not recorded
Rhachotropis inflata Rhachotropis inflata1 Rhachotropis novazealandica
RBGC038-03 JQ412486 GBCMA0081-06 AY271854 AMPNZ128-09 GU804309
Resolute Cornwallis Island New Zealand
-44.13
174.85
not recorded not recorded 520
Rhachotropis rossi Rhachotropis sp. 28 Rhachotropis sp. A
ANZR470-08 JF498593 GBCMA1154-08 EF989704 AMPNZ184-10 JF498594
Ross Sea California New Zealand
-76.59 36.33 -36.52
176.83 122.90 179.20
Rhachotropis sp. A1 Rhachotropis sp. B Rhachotropis sp. 2 (bumpy)
CMBIA625-12 AMPNZ102-09 ZMH K-46602
N/A California HM372956 New Zealand MF409443 Okhotsk Sea
32.73 -43.80 46.26
-117.34 175.32 152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46603
MF409442 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46604
MF409437 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46605
MF409449 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 2 (bumpy)
ZMH K-46606
MF409438 Okhotsk Sea
46.26
152.05
3371-3377
45 45 45
3210 300–700 5173 98 418
24
Rhachotropis sp. 1 (smooth)
ZMH K-46607
MF409441 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46608
MF409436 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46609
MF409447 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46610
MF409448 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis sp. 1 (smooth)
ZMH K-46611
MF409439 Okhotsk Sea
46.26
152.05
3371-3377
Rhachotropis marinae sp. nov.
ZMH K-46596
MF409440 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46597
MF409435 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46599
MF409444 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46598
MF409445 Okhotsk Sea
46.94
151.08
3299
Rhachotropis marinae sp. nov.
ZMH K-46600
MF409446 Okhotsk Sea
46.94
151.08
3299
Eusirus sp.
ANZR028-08
-72.08
175.55
1620
JQ412464
Ross Sea
25