INTRODUCTION
In the phylum Nemertea, about 1280 species are currently recognized as valid (Kajihara et al., Reference Kajihara, Chernyshev, Sun, Sundberg and Crandall2008). While the majority of the members are marine benthic forms, approximately 100 species have been reported as pelagic ranging from the epipelagic to bathypelagic zones. These pelagic forms belong exclusively to the Hoplonemertea, which, under the currently accepted systematic scheme, comprises the Monostilifera and the Polystilifera; the latter is further subdivided into the Reptantia and the Pelagica (Brinkmann, Reference Brinkmann1917a). There are only two named (Wheeler, Reference Wheeler1934; Korotkevich, Reference Korotkevich1961; Crandall & Gibson, Reference Crandall and Gibson1998; Chernyshev, Reference Chernyshev2005) and a few unnamed (Crandall, Reference Crandall2006) pelagic monostiliferans; these are restricted to Antarctic waters. The rest of the pelagic species comprise the polystiliferous Pelagica, suggesting that the pelagic life style has evolved more than once in this phylum.
Three different authors have published taxonomic monographs of the Pelagica (Brinkmann, Reference Brinkmann1917b; Coe, Reference Coe1926; Korotkevich, Reference Korotkevich1955), and the taxon now contains 99 named species (Gibson, Reference Gibson1995; Maslakova & Norenburg, Reference Maslakova and Norenburg2001; Kajihara & Lindsay, Reference Kajihara and Lindsay2010). However, their rare occurrence as well as the difficulty in preparing taxonomically meaningful specimens has been hampering the systematics of the Pelagica. Maslakova & Norenburg (Reference Maslakova and Norenburg2001) pointed out that 51 out of the 98 species known at that time had been described based upon a single specimen. Little is thus known about the intraspecific variation of characters, identity, distribution and biology for most of the species in this taxon.
During a research cruise in Sagami Bay, Japan, two specimens of pelagic nemerteans were procured, from which morphological and DNA sequence data were obtained. Histological examination of one of the specimens revealed that it belongs to Protopelagonemertes beebei Coe, Reference Coe1936. In this paper we report the morphology of the obtained material, together with a comparison of DNA sequences previously reported for a congeneric individual from off California, USA, discussing the species identity of P. beebei.
MATERIALS AND METHODS
Two specimens of Protopelagonemertes beebei were caught by an ORI33 net that was towed to a maximum depth of 1300 m and an altitude of 200 m above the seafloor, at 35°08.94′N 139°17.19′E in Sagami Bay, Japan, on 13 March 2010 during the research cruise KT10-2 (Chief Scientist: S. Nishida, University of Tokyo) of the RV ‘Tanseimaru’, operated by the Japan Agency for Marine–Earth Science and Technology (JAMSTEC). One specimen (JAMSTEC sample number 1100021510) was fixed in RNAlater® (Ambion) before being sent for RNA extraction to Dr Sónia Andrade at Professor Gonzalo Giribet's laboratory in the Department of Organismic and Evolutionary Biology, Museum of Comparative Zoology, Harvard University, Cambridge, MA, USA. The other specimen was sliced on its lateral body edge in vivo; the lateral body piece was fixed and preserved in 99.5% EtOH (JAMSTEC sample number 1100021511), while the rest of the body was fixed in 5% formalin–seawater (JAMSTEC sample number 1100021512). The latter was secondarily fixed in Bouin's fluid, pre-stained by 5% acid fuchsin in 70% EtOH, dehydrated by 100% EtOH, cleared in xylene, and embedded in paraffin wax (m.p. 56–57°C). Sections were cut to a thickness of 8 µm and stained according to Mallory's trichrome method (Gibson, Reference Gibson1994). DNA was extracted from the EtOH-preserved subsample using a standard phenol–chloroform protocol (Sambrook, Reference Sambrook and Sambrook2001). Partial sequence of cytochrome c oxidase subunit I gene (COI) was amplified using universal primers LCO1490 and HCO2198 (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994). A hot start polymerase chain reaction (PCR) was performed using an Applied Biosystems 2720 Thermal Cycler (Life Technologies), in a 20-µl reaction volume containing 1 µl of template total DNA (approximately 10–100 ng) and 19 µl of premix made with 77-µl deionized water, 10-µl Ex Taq Buffer (TaKaRa Bio), 8-µl dNTP (each 25 mM), 2-µl each primer (each 10 µM), and 0.5-µl TaKaRa Ex Taq (5 U/µl, TaKaRa Bio). Thermal cycling conditions comprised an initial denaturation at 94°C for 1 minute; 35 cycles of denaturation at 95°C for 15 seconds, annealing at 45°C for 30 seconds, and elongation at 72°C for 1 minute, and a final elongation at 72°C for 7 minutes. The PCR product was purified according to the method of Boom et al. (Reference Boom, Sol, Salimans, Jansen, Wertheim-Van Dillen and Van der Noordaa1990) with some modifications (Kobayashi et al., Reference Kobayashi, Ohta, Katoh, Kahono, Hartini and Katakura2009). Both strands were sequenced with a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) following the manufacturer's protocol, using the same primer set as the initial PCR amplification. Sequencing was performed with an Applied Biosystems 3130 DNA Analyzer (Life Technologies). Chromatograms and sequence data were analysed using a PHRED/PHRAP/CONSED software package (Gordon et al., Reference Gordon, Abajian and Green1998).
RESULTS
Protopelagonemertidae Brinkmann, Reference Brinkmann1917b, p. 178.
Bathynemertidae Brinkmann, Reference Brinkmann1917a, p. 4.
Bathynemertidae Brinkmann, Reference Brinkmann1917b, p. 7.
NOTE
Maslakova & Norenburg (Reference Maslakova and Norenburg2001) erroneously attributed the authority of the family name to Coe (Reference Coe1926), but the name was established in a note added in proof by Brinkmann (Reference Brinkmann1917b) when he replaced Bathynemertidae Brinkmann, 1917 with it, as its type genus Bathynemertes Brinkmann, 1917 turned out to be a junior homonym of Bathynemertes Laidlaw, 1906.
Protopelagonemertes Brinkmann, Reference Brinkmann1917b, p. 178.
Bathynemertes Brinkmann, Reference Brinkmann1917a, p. 4.
Bathynemertes Brinkmann, Reference Brinkmann1917b, p. 7.
TYPE SPECIES
Bathynemertes hubrechti Brinkmann, 1917, fixed by monotypy (Brinkmann, Reference Brinkmann1917b).
Protopelagonemertes beebei Coe, Reference Coe1936. (A) Photograph taken in life on-board the RV ‘Tanseimaru’, head, ventral view; (B) photograph taken in life, dorsal view, about 2.5 cm in body length; (C) transverse section through brain region, showing body-wall components; (D) transverse section through pyloric region, arrowhead indicating body-wall musculature rudimentarily developed on lateral side; (E) transverse section showing oesophagus about to separate from rhynchodaeum, arrowhead indicating circular muscles around rhynchodaeum. (A, C–E) JAMSTEC sample number 1100021512; (B) unregistered specimen used for RNA extraction.
Protopelagonemertes beebei Coe, Reference Coe1936, JAMSTEC sample number 1100021512, transverse sections. (A) Transition zone from oesophagus to stomach; asterisk indicating muscles posteriorly leading to proboscis insertion; (B) posterior stomach region, showing mid-dorsal vessel exposed to rhynchocoel lumen; (C) anterior portion of proboscis anterior chamber, showing 22 proboscis nerves (black arrowheads), small, white arrowheads indicate additional nerve swellings which make the proboscis appear to have 25 nerves; (D) posterior portion of proboscis anterior chamber, showing 19 proboscis nerves (black arrowheads).
Protopelagonemertes beebei Coe, Reference Coe1936, JAMSTEC sample number 1100021512, transverse sections. (A) Stylet basis, indicated by asterisk; (B) mid-dorsal vessel and lateral vessels in caudal region; (C) caudal vessel; (D) lateral nerve cord in stomach region; (E) ovary.
Protopelagonemertes beebei Coe, Reference Coe1936, p. 101, figures 25, 34; Coe, Reference Coe1945, p. 151, text-Figure 2; Coe, Reference Coe1954, p. 234, pl. 8, figure a, pl. 9, figure r, text-Figure 4A–C.
Planktonemertes beebei: Korotkevich, Reference Korotkevich1955: p. 65.
?Protopelagonemertes sp. 544: Thollesson & Norenburg, Reference Thollesson and Norenburg2003, p. 408.
?Bathynemertes hubrechti: Wheeler, Reference Wheeler1934, p. 281, figures 58, 59.
MATERIAL EXAMINED
JAMSTEC sample number 1100021512 (on-board sample number ORI100313-2b), female, serial transverse sections, 130 slides.
SEQUENCE
A total of 616 base pairs of COI sequence has been deposited in EMBLE/DDBJ/GenBank (Accession #AB587263).
EXTERNAL FEATURES
In life, the body is uniformly orange (Figure 1A, B), more or less cross-sectionally rounded, tapering at head and tail, about 2.5 cm in length, 7 mm in maximum width. There are no tentacles or fins.
BODY WALL, MUSCULATURE AND PARENCHYMA
The ciliated epidermis, 50–60 µm in thickness at the brain region (Figure 1C), is almost uniform in thickness throughout the body. The connective tissue basement membrane has a deep cup-like structure (Figure 1C), varying in thickness throughout the body; it is especially thick on the ventral side in the posterior part of the body, reaching 140 µm in thickness. The body-wall musculature consists of an outer circular and an inner longitudinal muscle layer (Figure 1C); the body-wall muscle layers are reduced laterally in the anterior and posterior part of the body (Figure 1D), whereas in the middle part of the body these are normally developed. The presence/absence of a diagonal muscle layer was not ascertained in the cross-sections. Pseudostriated muscle fibres were not found. Dorsoventral muscles are well developed, consisting of strands of more than ten muscle fibres, running between intestinal diverticula (Figure 1D). The parenchyma is poorly developed, only filling the spaces between the intestinal lateral diverticula, the rhynchocoel, and the body-wall inner longitudinal muscle layer in the posterior region of the body.
ALIMENTARY SYSTEM
The oesophagus opens into the ventral wall of the rhynchodaeum (Figure 1E). The oesophagus is histologically divided into two portions. The anterior portion, 760 µm in length and 320 µm in width, is characterized by a less glandular, non-ciliated epithelium about 20 µm in thickness, surrounded by longitudinal muscle fibres. The posterior portion is 780 µm in length and 600 µm in diameter, with ciliated epithelium about 50 µm in thickness, containing acidophilic glandular cells (Figure 2A); it is sparsely surrounded by circular muscle fibres. The stomach wall is dominated by basophilic glandular cells (Figure 2A). The pylorus opens to the dorsal wall of the intestine, roughly at 1/3 of the body length. The intestinal caecum and its diverticula extend anteriorly beyond the brain (Figure 2A). The lateral diverticula of the intestine and its caecum are well developed, filling up much of the body space and meeting above the rhynchocoel (Figure 1D). The diverticula are so tightly packed in the body, and the branching of the diverticula is so complicated, that precise counts of the numbers of the diverticula of the intestine and its caecum were impossible.
PROBOSCIS APPARATUS
The rhynchodaeal opening is situated at the tip of the head. The rhynchodaeum is surrounded by circular muscles throughout its length (Figure 1E). The origin of the muscles leading to the proboscis insertion is in the pre-cerebral region, unlike monostiliferans in which these muscles are derived post-cerebrally. The proboscis insertion is situated at the level of brain; muscles derived inward from the body-wall longitudinal muscle layer in the pre-cerebral region run posteriorly along the rhynchodaeum and oesophagus (Figure 2A) before the muscles participate in the proboscis insertion.
The rhynchocoel is about 90% of the body length, with walls composed of a wickerwork of circular and longitudinal muscle fibres, up to 200 µm thick in the stomach region (Figure 2B). No pseudostriated muscle fibres were found in the rhynchocoel wall.
The anterior chamber consists of a glandular epithelium with distinct conical papillae, an outer circular muscle layer, a longitudinal muscle layer containing proboscis nerves, a thin inner circular muscle layer, and a delicate endothelium. There appears to be 22 or more proboscis nerves anteriorly (Figure 2C), and 19 posteriorly (Figure 2D); in some sections, the number of the proboscis nerves appears to be up to 25 due to the presence of small additional nervous bulges similar in shape and position to actual proboscis nerves. However, unlike true proboscis nerves, these cannot be traced antero-posteriorly with certainty (Figure 2C). The middle chamber contains a curved stylet basis, which was not well preserved in serial cross-sections (Figure 3A). Histologically, the posterior chamber can be divided into two regions: the anterior region consists of a glandular epithelium, an outer longitudinal muscle layer containing up to 10 bundles of proboscis nerves, a circular muscle layer, and an endothelium; the posterior region lacks longitudinal muscles, and is filled with basophilic substances.
BLOOD VASCULAR SYSTEM
A pair of cephalic vessels meet each other above the rhynchodaeum. Posteriorly, each cephalic vessel forks into two (lateral and medial) branches after passing through the cerebral ring. Each lateral branch leads to a respective lateral vessel; the medial branches on both sides run backward between the rhynchocoel and stomach, before fusing with each other to form a mid-dorsal vessel. The mid-dorsal vessel runs under the rhynchocoel for about 300 µm, and then enters the rhynchocoel wall (Figure 2B). It remains within the rhynchocoel wall for 3 mm, and is located just beneath the rhynchocoel wall further posteriorly. Near the caudal end, the lateral vessels merge with the mid-dorsal vessel anterior to the posterior nervous commissure (Figure 3B, C); from the portion where the three vessels merge, a caudal vessel extends further posteriorly for about 170 µm (Figure 3C) before terminating blindly above the anus.
NERVOUS SYSTEM
The brain has an outer neurilemma, but no inner neurilemma. The dorsal and ventral commissures are 100 µm and 140 µm in diameter, respectively. Numerous nerve fibres lead forward from the brain; these are branched anteriorly, running around the rhynchodaeum, mostly innervating the epidermis in the pre-cerebral region. The lateral nerve cords are situated far from the body wall (Figure 1D); they send fibres dorsally and ventrally in the intestinal region, but their ultimate fate cannot be traced with certainty in the light microscopy. An accessory lateral nerve is present anteriorly (Figure 3D); it can be traced back to the 1/3 of the body length. There are no mid-dorsal nerve, neurochord cells, neurochords, and lateral-nerve-cord muscles.
EXCRETORY SYSTEM
This is absent.
SENSORY SYSTEM
No apical organ, cerebral organs, band-shaped organs, or eyes were found.
GLANDULAR SYSTEM
Neither cephalic glands, nor postero-lateral glandular organs were found.
REPRODUCTIVE SYSTEM
The specimen examined is female. Eighteen pairs of ovaries are arranged in a row on each side, distributed from the pyloric region to the level of the posterior end of the rhynchocoel. Each ovary is tubular, 300 µm in diameter, curved above the lateral nerve cord (Figure 3E), containing about 25 oocytes of about 100 µm in diameter. No open gonopore was observed.
REMARKS
One of the noticeable characteristics of the present material is that the oesophagus opens into the rhynchodaeum, instead of opening separately as in the majority of the Pelagica. A united mouth-rhynchostomodaeal opening has only been reported in Planktonemertes agassizii Woodworth, Reference Woodworth1899, Mononemertes scarlata Coe, Reference Coe1945, Plenanemertes rubella Coe, Reference Coe1954, and the species of the genus Protopelagonemertes.
Planktonemertes agassizii has a flattened, translucent body (Woodworth, Reference Woodworth1899; Coe, Reference Coe1926), unlike the somewhat rounded, opaque body of the present specimens. Mononemertes scarlata differs from the present material in possessing intestinal lateral diverticula not meeting above the rhynchocoel (Coe, Reference Coe1945). Plenanemertes rubella is not a well-described species, but it has 14 proboscis nerves (Coe, Reference Coe1954), while our specimen has 19–22 nerves in the proboscis.
The present material can be classified in the genus Protopelagonemertes by having: (1) an opaque body without tail fin and/or tentacles; (2) well developed intestinal lateral diverticula filling up much of the body space and meeting above the rhynchocoel; (3) a rhynchocoel wall composed of interwoven circular and longitudinal muscle fibres; and (4) a common pore of the rhynchodaeum and the mouth.
In addition to P. beebei, two other congeners have so far been described from the world oceans (Figure 4): P. hubrechti (Brinkmann, 1917), reported from North Atlantic (Brinkmann, Reference Brinkmann1917a, Reference Brinkmannb; Coe, Reference Coe1936), South Atlantic (Wheeler, Reference Wheeler1934) and Antarctica (Wheeler, Reference Wheeler1940); and P. joculatori Van der Spoel, Reference Van der Spoel1988 from the Banda Sea (Van der Spoel, Reference Van der Spoel1988). These species are similar in body structure and have been distinguished solely by the number of the proboscis nerves (see Discussion).
Map showing localities from which specimens of the genus Protopelagonemertes have been recorded. Sources: 1, Brinkmann (Reference Brinkmann1917b); 2–5, Wheeler (Reference Wheeler1934); 6, 7, Coe (Reference Coe1936); 8, Wheeler (Reference Wheeler1940); 9, 10, Coe (Reference Coe1954); 11, Van der Spoel (Reference Van der Spoel1988); 12, Thollesson & Norenburg (Reference Thollesson and Norenburg2003); 13, present study.
The new morphological findings in the present study are: (1) the presence of a caudal vessel; and (2) the absence of postero-lateral glandular organs; the existing literature is uncertain about these characters (Coe, Reference Coe1936, Reference Coe1954).
GEOGRAPHICAL DISTRIBUTION
Protopelagonemertes beebei Coe, Reference Coe1936 was originally described based on a single female specimen taken at a depth of about 1646 m, 32°12′N 64°36′W, off Nonsuch Island, Bermuda, north-west Atlantic (Coe, Reference Coe1936). A second female was collected at 27°10′N 117°02′W, off the coast of Baja California, north-east Pacific, by a trawl lowered to 3200 m depth (Coe, Reference Coe1954). One male individual was obtained in a net drawn from 7200 m depth at 45°24′N 154°11′E, off Simushir Island, Kuril Islands, north-west Pacific, expanding the species' geographical range more than 7000 km westward (Coe, Reference Coe1954). The present study constitutes the fourth record of P. beebei, representing the first record of a female from the north-west Pacific (Figure 4). However, if the species eventually turns out to be a junior synonym of P. hubrechti, then the species shows an almost cosmopolitan distribution (see Discussion).
DISCUSSION
Identity
The implications of the present study are that: (1) the two species Protopelagonemertes beebei and P. hubrechti might be synonymous; or (2) at least a subset of the individuals reported as P. hubrechti cannot be distinguished morphologically from the present material herein identified as P. beebei. Including the third congener P. joculatori Van der Spoel, Reference Van der Spoel1988, members of the genus Protopelagonemertes resemble each other, and have been distinguished almost entirely by the number of the proboscis nerves (e.g. Coe, Reference Coe1936; Van der Spoel, Reference Van der Spoel1988): 36 in P. joculatori; 19–21 (varying within a single specimen) in P. beebei (Coe, Reference Coe1936); and 29 in P. hubrechti (Brinkmann, Reference Brinkmann1917a, Reference Brinkmannb). No further information on the variability of this character was provided with respect to the additional two specimens identified as P. beebei from the north-east Pacific, off Baja California, and the north-west Pacific, off the Kuril Islands, respectively (Coe, Reference Coe1954). On the other hand, it has been documented in the succeeding reports of the individuals referred to as P. hubrechti that the number of proboscis nerves was 22, 24, 25 and 26 in the four specimens from the south-east Atlantic, off South Africa (Wheeler, Reference Wheeler1934), 26, 27–29 and 30 in the three specimens from the north-west Atlantic, off Nonsuch Island, Bermuda (Coe, Reference Coe1936), and 30 in a specimen from Antarctica (Wheeler, Reference Wheeler1940). The number of proboscis nerves in the present material from Sagami Bay, Japan, varies from 19 to 22 (or, seemingly up to 25) within a single individual, making the variation of this character completely continuous between what have been reported for P. beebei and P. hubrechti. Accumulating morphological and molecular data from the same individuals in future studies is a prerequisite for clarifying the identity of these two species, especially with regards to whether or not the proboscis nerve number can be used to distinguish between species.
The genetic distance found by Kimura's (1980) 2-parameter method between the mitochondrial COI sequences from the present material and that of an unidentified specimen of the genus Protopelagonemertes (Thollesson & Norenburg, Reference Thollesson and Norenburg2003) collected 160 km off Point Conception, California, USA (GenBank Accession # AJ436927) was 5.6% (33-nucleotide difference out of 616 base pairs), implying that these are closely related, and may even be conspecific, even though they were collected from localities more than 9000 km apart. Incidentally, Chen et al. (Reference Chen, Strand, Norenburg, Sun, Kajihara, Chernyshev, Maslakova and Sundberg2010) reported 5.8% intraspecific genetic distance in the benthic cephalotrichid palaeonemerteans from the north-eastern Pacific.
Morphology
The intact epidermis that remained on almost the entire body of the examined specimen enabled us to conclude definitively that it lacks ‘postero-lateral glands’ (Roe & Norenburg, Reference Roe and Norenburg2001), a pair of specialized glandular regions found in 32 named species in the Pelagica (Maslakova & Norenburg, Reference Maslakova and Norenburg2001). So far these glands have been reported as absent in Nectonemertes pelagica and Phallonemertes murrayi (Maslakova & Norenburg, Reference Maslakova and Norenburg2001), as well as two unidentified specimens, one in the genus Crassonemertes and one in Nectonemertes (Roe & Norenburg, Reference Roe and Norenburg2001) The presence/absence of these glands is unknown for more than half the members of the Pelagica. If the postero-lateral glands have been acquired only once in the lineage of the Pelagica, then, given that the latter is monophyletic (Thollesson & Norenburg, Reference Thollesson and Norenburg2003), protopelagonemertids are relatively ‘primitive’ within this clade, unless they have lost these glands secondarily.
ACKNOWLEDGEMENTS
H.K. is grateful to Mrs Junko Sato for her assistance in preparing histological sections and taking digital images; and to Mr Osamu Hashiguchi for providing Figure 1D, which was produced by the digital microscope VHX-1000 (KEYENCE, Japan). D.J.L. thanks Dr Shuhei Nishida, Ocean Research Institute, University of Tokyo, for his efforts as chief scientist during the RV ‘Tanseimaru’ cruise (KT10-02) during which the specimens were collected, as well as the participating students, staff, crew and ship captain. This study is a contribution to the Census of Marine Zooplankton (CMarZ), a Census of Marine Life ocean realm field project, and was conducted under the umbrella of the Japan National Regional Implementation Committee (Japan NRIC). This study was partially supported by a Grant-in-Aid for Young Scientists for both H.K. (No. 30360895) and T.K. (No. 22770072).
