Aplacophorans are vermiform marine molluscs that lack a shell but have a cuticle covered in calcareous spines or scales called sclerites. Major characteristics of this group include a reduced or non-existent foot, a simple radula, and the pericardium (a vestige of the coelom) associated with the reproductive system (Todt 2013). Until recently, the relative phylogenetic position of the two major clades of Aplacophora was unclear. However, phylogenomic studies (Kocot et al. 2011(Kocot et al. , 2019;;Smith et al. 2011) have strongly supported Aplacophora as a monophyletic clade consisting of two separate groups: Solenogastres and Caudofoveata.
Aplacophorans have been described from all oceans and depths. However, they are generally small animals that can go unnoticed in benthic samples. This is probably a major reason for the paucity of research on this group, and why many view these animals as rare (Todt 2013). Apart from difficulties in their collection, one of the major challenges to the study of solenogasters is that identification to the species, genus, and even family level requires the study of internal anatomical characters. Histology allows taxonomically informative internal anatomical structures, such as the radula, foregut glands and reproductive anatomy to be characterised to a level of detail that other methods cannot provide. To date, about 440 species of aplacophorans (298 Solenogastres) have been described, but the true number is estimated to be at least tenfold higher (Todt 2013). Although there are few living experts on Solenogastres, there has been a recent increase in taxonomic research on this clade by several active research groups located worldwide (e.g., Garcia-Alvarez et al. 2009;Saito and Salvini-Plawen 2014;Bergmeier et al. 2017Bergmeier et al. , 2019;;Ostermair et al. 2018;Passos et al. 2019;Pedrouzo et al. 2019;Cobo and Kocot 2021). Modern approaches are now combining molecular and morphological techniques when possible for a more precise taxonomic evaluation and identification (forger and Schrodl 2013; Bergmeier et al. 2016;Bergmeier et al. 2019).
There is a dramatic disparity in our understanding of aplacophoran species distribution and biodiversity (Todt 2013;Cobo and Kocot 2020), with many known from near the base of operations of experts on the group or marine stations (e.g., waters off Norway and Spain; Todt 2013; Pedrouzo et al. 2014). Around 30% of solenogasters are formally described from Antarctica (e.g., Salvini-Plawen 1978;Garcia-Alvarez et al. 1998, 2000;Garcia-Alvarez and Urgorri 2003;Zamarro et al. 2013). Salvini-Plawen (1978) studied this region extensively and published a monograph on this highly diverse fauna in which the current taxonomy of Solenogastres was established. While not aslarge as Antarctica, Australasia makes up a large, combined area with only a few aplacophoran species described to date. However, examination of material from just three deep-water surveys conducted in the Australian Bass Strait revealed 65 morphospecies (Scheltema 2001), a much higher aplacophoran diversity than previously believed. Even less is known about the aplacophoran fauna of New Zealand; only three species have been described to date: Neomenia naevata Salvini-Plawen & Paar-Gausch, 2004, Drepanomenia tenuitecta Salvini-Plawen, 2004 and Dorymenia quincarinata (Ponder, 1970), all of which are solenogasters (Ponder 1970;Salvini-Plawen, 2004;Salvini-Plawen and Paar-Gausch 2004). To improve our understanding of aplacophoran biodiversity in this region, samples from New Zealand waters need to be studied, identified, and described. The present study provides insight on the solenogaster biodiversity of the waters around New Zealand with new data on Dorymenia quincarinata and description of three new species of Proneomeniidae Simroth, 1893: Dorymenia tanifa n. sp., Dorymenia ancora n. sp.and Dor ymenia ludda n. sp.
This study includes the characterisation of six specimens (Table 1) selected from collections of the Museum of New Zealand Te Papa Tongarewa (NMNZ) and the National Institution for Water and Atmospheric research (NIWA); around 200 specimens in total. Based on the external appearance and sclerites, 38 specimens were tentatively identified as belonging to the family Proneomeniidae.
Of these, the specimens investigated were selected because of their designation as distinct morphospecies and for their good state of preservation (i.e., non-fragmented and limited desiccation/degradation of external morphology). External morphology was observed and photographed using an Olympus SZX-16 (Tokyo, Japan) dissecting microscope and imaged with an Olympus SC50 camera. Length measurements were made by following along the midline of the specimen using the scale bar as a reference. Sclerites were extracted from the anterior, ventral and posterior regions of the body by using a thin needle to scrape them onto a slide with 96% ethanol. Sclerites were air-dried and mounted with Araldite™ GY 502 10900 resin (Electron Microscopy Sciences, Hartfield, PA, USA)). Sclerite preparations were observed under an Olympus BX53 compound microscope with differential interference contrast (DIC) and imaged with an Olympus SC50 camera.
The anterior and posterior regions of four specimens (NMNZ M.319677, NIWA 60415, NIWA 129496 and NIWA 28046) were histologically sectioned. They were decalcified in a stepwise fashion by placing tissue in a 15 ml Falcon tube with 3 ml of distilled water and 7 ml of 80% or 96% ethanol, depending on how they were originally preserved. The tissue was then soaked for 5 min. Using a pipette, 5 ml of the diluted ethanol was removed and replaced by 5 ml of distilled water for another 5 min soak. This was repeated two more times, after which all liquid was removed and replaced with a solution of 2 ml of 0.5 M ethylenediaminetetraacetic acid (EDTA), 2 ml of distilled water, and 1 ml of 10% formalin with Rose Bengal overnight to dissolve the sclerites completely. Specimens were then dehydrated in a stepwise fashion starting with a 5 min 10% formalin soak. For 30 min each step, they were then soaked once in 70% ethanol, once in 90% ethanol, twice in 95% ethanol, and three times in 100% ethanol with fresh ethanol for each successive soak. Specimens were then soaked in xylene for followed by a second soak in fresh xylene (10-15 min each soak). For infiltration, specimens were soaked in 3 parts xylene to 1 part paraffin for 30 min, 1 part xylene to 1 part paraffin for 30 min, 100% paraffin for 30 min, and two final soaks in 100% fresh paraffin for 1 h each. Infiltration was performed at a temperature of 56°C.
Samples embedded in paraffin were serially sectioned (5 µm) using a Leica RM2125 rotary microtome and stained using a combination of Weigert haematoxylin (Sigma-Aldrich, St. Louis, MO, USA) and Gomori's trichrome (Sigma-Aldrich, St. Louis, MO, USA). Slides were immersed in two successive washes of HistoChoice (Sigma-Aldrich, St. Louis, MO, USA) for 1S min each and gradually rehydrated using 2 min soaks in 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and DI water, respectively. Slides were soaked in Bouin's fixative at room temperature overnight or at 56°C for an hour. Slides were then rinsed for at least 2 min in tap water and 2 min in distilled water, or until the water ran clear, followed by soaking in haematoxylin for 10min. Slides were rinsed again in tap water, followed by distilled water until the water ran clear. Slides were then soaked in Gomori's trichrome for 15min immediately followed by a 15-30 s soak in 0.5% acetic acid for differentiation. Slides were again rinsed in distilled water until the water ran clear. The sections were dehydrated Table 1. Collection data from TheMuseum of New Zealand TePapaTongarewa (NMNZ) and theNew Zealand National Institute of Water and Atmospheric Research (NIWA).
174.noooo 0 E using successive 2 min soaks in 70% ethanol, 95% ethanol, 100% ethanol and HistoChoice. Once removed from HistoChoice and dried completely, sections were then mounted with Depex. Slides were observed under an Olympus BX53 compound microscope for manual lateral reconstructions. Reconstructions were made by following each internal structure and measuring their dimensions in each cross section. Using metric graph paper and a different colour for each structure, the x-axis was set as the thickness of each section (5 µm) and the y-axis was set as the height of each structure. The reconstruction was then scanned and digitised using Adobe Photoshop.
DNA was extracted from three samples (NIWA 60415, NIWA 129496, and NIWA 28046; Table 1) using the E.Z.N.A. Microelute DNA isolation kit. DNA concentration was measured using the Qubit 4.0 Fluorometer (Thermo Fisher, Waltham, MA, USA) using the dsDNA HS kit. PCR was performed using AMRESCO Hot Start Taq PCR Master Mix (VWR lnternationa, Radnor, PA, USA). The solenogaster-specific 16S primers 16Soleno-r and 16Soleno-f (Bergmeier et al. 2017) were used for NIWA 129496 and NIWA 28046. These failed to produce a PCR product for NIWA 60415 and so the solenogaster-specific 16S primers 16SbrH_Solenos (5'-CCGATTTGAACTCAGATCATGTAG-3') and 16SarL_Solenos (5'-CGACTGTTTAACAAAAA-CATTGCTC-3') were designed and attempted for NIWA 60415 as well. A master mix was prepared for each sample containing 12.5 µI of 2x AMRESCO Master Mix, 1 µI of each primer, and 9.5 µI of distilled water. For each sample, 24 µI of the master mix was combined with 1 µI of DNA. The protocol used for 16Soleno-r and 16Soleno-f was as follows: (1) preheat to 95°(; (2) 98°( for 30 s;(3) 98°( for 5 S, 50°( for 5 s x 40 cycles; (4) 72°C for 20 s; (5) 72°C for 1 min; and (6) hold at 10°C. For 16SbrH_Solenos and 16SarL_Solenos the protocol was: (1) preheat to 95° C; (2) 95°C for 2 min; (3) 95°C for 20 s, 60°C for 15 s, 72°C for 30 s x 40 cycles; (4) 72°C for 7 min. PCR success was determined with gel electrophoresis using 1x SB buffer at 120 V. Successful products were either directly purified using the Omega Biotek EZNA Cycle Pure Quick kit and eluted in 25 µI of elution buffer, or were gel purified using the Omega Bio-Tek MicroElute Gel Extraction kit and eluted in 30µI of elution buffer. Sanger sequencing was performed by GeneWiz and consensus sequences were assembled from forward and reverse reads using Sequencher (South Plainfield, NJ, USA). Uncorrected pairwise distance between Dorymenia lucida n. sp. (NIWA 129496) and Dorymenia quincarinata (NIWA 28046) 16S sequences (excluding sites containing gaps) were calculated in MEGA X. Despite using a typical sized piece of tissue for extraction, DNA recovered for Dorymenia ancora n. sp. was too dilute to visualise on a gel, most likely due to degradation, and PCR attempts were unsuccessful.
Elongated animal with a thick anterior region and pointed posterior end; thick pale yellow cuticle with hollow acicular sclerites; one type of scales along pedal groove. Mouth and atrium fused; polystichous radula with at least 8-9 teethperrow.Outlet of spawning duct unpaired. Ciliated mantle cavity. One pair of Table 2. Selection of the diagnostic characters for Dorymenia spedes. Distribution, habitus and anterior bodyregion.DP: digitiform projection (posterior). PF: number of pedal folds. DFG: dorsal foregut gland. Radula: radula type (a, b, or c).Teeth no.: number of teeth per row. +: present. -: absent. Empty cell: no data available. Data presented are based on descriptions of holotypes (Koren and Danielssen 1877;Kowalevsky and Marion 1887;Nierstrasz 1902Nierstrasz , 1908;;Heath 1911Heath , 1918;;Thiele 1913a;Ponder 1970;Salvini-Plawen 1978;Garcfa-Alvarez et al. 1998, 2000, 2003). (Thiele, 1913) Antarctic, 385 m 20-30x 25 a D. aistata Salvini-Plawen, 1978 D. discoveryi (Nierstrasz, 1908) Antarctic, 385 m Antarctic, 311-1437 m 70x4 25 X 2.25 Salvini-Plawen, 1978 Antarctic / S Pacific, 86-101 m 70x4 Salvini-Plawen, 1978 Antarctic, 75-549 m 92x4 a D. interposita Salvini-Plawen, 1978 Antarctic, 118 m 20 X 2.2 a D. Jonga (Nierstrasz, 1902) S Indian, 1150 m D. tricarinata (Thiele, 1913) Antarctic, 187-385 m 30 X 1.8 (Kowalevsky& Marion, 1887) Mediterranean, 20-60 m a D. weberi (Nierstrasz, 1902) S Indian, 694 m 18-37 X 0.75-2
Habitus. Animal elongated (13 mm long, 1 mm wide in middle body region) withthick andblunt anterior body region while posterior region is elongated with a rounded end (Figure lA). Pedal groove visible, pallial (= mantle) cavity and atrial opening both present as small slits. Rough outer appearance with inter-crossing sclerites embedded in cuticle. In 96% ethanol, animal appears off-white with yellow tint.
Mantle. Epidermal papillae present (25 µm). Thick cuticle with a maximum of 180 µmin anterior region. Acicular sclerites hollow, tapering to pointed end with different grades of curvature and size (Figure 1D-H) (100-200 µm long). Sclerites from pedal groove blunt and knife shaped (60-80 µm long; Figure 1H).
Pedal pit and groove. Pedal pit very small, extending posteriorly 50 µm (Figure 2A). Pedal glands 60 µm in width, 40 µm in height. Pedal groove with one fold that continues throughout length of animal and terminates before opening of mantle cavity.
Digestive system. Mouth forms immediately posterior to atrium and fuses into ventral atrium wall. Pharynx (100 µm long, 120-175 µm wide, 100-140 µm high) small and roundin anterior region (Figure 2A-2), surrounded by clusters of simple, circular subepithelial glands. Due to folding during sectioning or to maximise surface area during digestion, mid-region of pharynx apparent as two small branches in cross section until they form into one tubular structure at the anterior region of the radular apparatus. Approximately 300 µm into pharynx, second branch buds out of ventral wall of foregut and pharynx continues as two branches for approximately 200 µm before two branches fusetogether as terminus of pharynx (Figure 2A-3). Branches identical histologically. Polystichous radula with small central tooth and 8-9 short uniform teeth (maximum length= 17 µm) on each side.At least 20rowsofteeth.Radulafragmented in mid-regionof body,mostrows observed inside radular sac. Ventrolateral foregut glands present laterally at posterior end of radular apparatus (Figure 2A). Glands type-C (or Epimeniatype), approximately 130 µm wide and run laterally along midgut for 150 µm (Figure 2A-3). Midgut shortens into hindgut ventral to pericardium and opens into dorsal wall of pallial cavity.
Nervous system. Atrium 150 µm long, anterior to posterior (Figure 2A). Atrium contains bundles of numerous slender atrial papillae (Figure 2A-1) measuring 40-50 µm in length. Pair of large, stalked papillae extend from dorsal wall of atrium, another pair extending ventrally. Cerebral ganglion (SO µm long, 125 µm wide, 70 µm high) dorsal to anterior part of pharynx and ventral to foregut (Figure 2A). Pair of buccal ganglia (20-30 µm in diameter) form after start of radular apparatus and flanks radula. Pair of ventral ganglia observed in posterior region of radular apparatus. One large dorsoterminal sense organ (approximately 50 µm tall in cross-section) located dorsal to anterior region of pallial cavity (Figure 2B).
Gonopericardial system. Heart runs dorsally in the short, wide pericardium (370 µm long, 300 µm wide, 120 µm high) and ventral wall of the heart folds up dorsally (Figure 2B). Midgut extends dorsally in cross-section towards mid-posterior region of pericardium, pushing heart into two connected lobes. Two lobes centralise into one uniform tubular structure in terminal region of pericardium. Pericardioducts (400 µm long, 40 µm wide, 50 µm high) connect dorsolaterally to anterior region of spawning ducts and extend to terminus of the pericardium (Figure 2B). Paired seminal receptacles thick and located anterior to paired spawning ducts. Paired region of spawning duct approximately 350 µm long and circular in cross-section (120-130 µm in diameter) (Figure 2B-4). Posteriorly, two spawning ducts fuse into single, flattened duct ventral to rectum that extends approximately 140 µm and opens dorsally into anterior subsection of mantle cavity (Figure 2B-5). Pair of copulatory spicules run alongside posterior region of spawning ducts (Figure 2B-5), rounded in cross-section in their entirety (30 µm in diameter), extending ventrolaterally approximately 400 µm, opening into centre of pallial cavity (Figure 2B-5) and surrounded with relatively thick musculature (10-20 µm). Clusters of 15 abdominal spicules extend 125 µm posteriorly, located ventrally to copulatory spicules and mantle cavity (Figure 2B-5).
(100 µm thick, dorsoventrally) ventral to spawning duct. Outlet of spawning duct and rectum both open into dorsal wall of mantle cavity in its anterior region. Mantle cavity opens ventrally, lined with cilia. Copulatory spicules extend distally and terminate in mantle cavity (Figure 2B).
From Latin lucidus,a,um: full of light, shining.In reference to the yellow appearance of the animal.
Habitus. Animal thin, elongate (18 mm long, 1 mm in width) with slight curve anteriorly due to preservation. Body has no protuberances or keels. Anterior and posterior ends both rounded. Body surface appears smooth with sclerites tightly arranged in visible crisscross pattern. Atrio-buccal cavity and pallial cavity openings both visible as deep slits, pedal groove visible throughout entire length of body. Animal offwhite or light grey in colour in 800/4 ethanol (Figure 1C).
(10-12 µm long, 2-3 µm wide). Unlike most epidermal cells that are stained light blue, epidermal papillae are here stained dark pink. Cuticle thick, maximum thickness 120 µm in dorsal region and 60 µm in ventral region. Hollow acicular sclerites with different sizes and curvatures (90-210 µm long) (Figure 11).
Pedal pit approximately 125 µm long (Figure 3A) with pair of pedal glands (Figure 3A) extending posteriorly (60 µm long, 120 µm high, 180 µm wide). Pedal groove contains one to three folds. In the anterior region, one large middle fold flanked by two smaller folds. One fold present in posterior region of pedal pit.
Digestive system. Mouth opens at posterior end of atrium and continues as long, wide pharynx (135 µm long, 400-450 µm wide, 70-150 µm high) (Figure 3A). Epithelium folded and very glandular in anterior region of pharynx (Figure 3A-2). Foregut rounded in shape (90 µm long, 350 µm wide, 180 µm high) anteriorly then connects with the anterior region of midgut, posterior to radular region (Figure 3A-3). Radula polystichous with 24 teeth in each of 23 rows. Many rows of radula were fragmented but general structure still observable (Figure 3A-3). Unfragmented teeth measured up to 20 µm long with narrow base and pointed curved tip. Radular sac short and circular in cross section (SO µm long, 70-80 µm wide). Foregut glands open at both sides of radular apparatus (Figure 3A). Paired glands are type-C (Epimeniatype) and run ventrally along midgut for 400 µm (Figure 3A). Midgut forms into rectum ventral to pericardium. Rectum perfectly circular in cross section (200 µmin diameter) with thin musculature, opening into terminal dorsal wall of mantle cavity.
Nervous system. Atrium opening (65 µm long) oriented ventrally. Wide and rounded in cross-section (350-360 µm wide, 280-300 µm high) and continues internally for 80 µm. Approximately 35 digitiform papillae present around entire inside of atrium (Figure 3A-1). One pair of large, stemmed papillae present on ventral wall of atrium in posterior region. Papillae have narrow stem measuring approximately 200 µm in length and slightly thickening distally. Cerebral ganglion oval in cross-section (130 µm long, 130 µm wide, 100 µm high) (Figure 3A) and is dorsal to anterior region of the pharynx. Two buccal ganglia (60 µm long, 50 µm diameter) flank the radular region and anterior region of ventral foregut glands (Figure 3A). One pair of ventral nerve cords located ventrolaterally to pharynx and extend throughout entire anterior region of specimen. One dorsoterminal sense organ (approx. 30 µm high and 25 µm wide) located dorsally to anterior region of mantle cavity (not shown).
approximately 15 µm in length and spherical granulocytes 7 µm in diameter. Erythrocytes present throughout body but concentrated in mid-region around the midgut. Heart small (60 µm long, 100 µm wide, 20 high) and tubular, extending along the dorsal wall of the pericardium (80 µm long, 140-160 µm wide, 100-120 µm high) (Figure 38-4). Pericardioducts (25 µm high, 20 µm wide) connect dorsolaterally to spawning ducts (Figure 38) and extend posteriorly approximately 450 µm until looping up and back towards posterior region of pericardium (Figure 38). Spawning duct paired for most of its length (400 µm long, 40-64 µm high, µm wide), thin walled, and with thin layer of outer musculature. Spawning ducts fuse into single flattened duct, posteriorly, (Figure 38
anterior branches that present as pockets in crosssection (all three pockets measuring 70 µm in length, dorsoventrally), ventral to outlet of spawning duct (Figure 38). Pockets fuse into one simple cavity in terminal region of spawning duct (20 µm long, 120 µm wide, 150 µm high). Cavity lined with ciliated epithelium and opens dorsoventrally. Bundled copulatory stylets on each lateral side extend medially and terminate at anterior-most region of mantle cavity. Rectum opens into dorsal wall of posterior region of mantle cavity.
Tonita phonetic transcription of the Maori taniwha; mythological beings living in the sea or inland waters where giant waves are present. '.
-
200µm " E -• .I., "'" t ,. ,. (Figures 1 and4; Tables 123 4A). Pedal groove reaches and opens into mantle cavity and contains three folds, one large and medial, two smaller and lateral.
...
Mouth opens at posterior end of atrium and continues as a long, wide pharynx (600 µm long, 600 µm wide, 300 µm high). Pharynx has 40 µm thick wall with small folds and flattens into U-shape posteriorly. Mid-anteriorly, lateral walls of pharynx folded in medially resembling an anchor in cross-section (Figure 4A-1). Small clusters of subepithelial pharyngeal glands present in dorsal region. Pharynx fuses into ventral floor of midgut like an hourglass, with thin mid-section widening dorsally (pharynx) and ventrally (midgut; Figure 4A-2). Ventral foregut glands form laterally at anterior region of the radular apparatus. Glands are type-C or Epimeniatype (300 µm wide, 250 µm in height) and run laterally along the midgut. Total length unknown (Figure 4A-3). Radula polystichous with 45 rows and 28 teeth per row (Figure 4A-3), measuring up to 70 µm long with pointed and curved end. Midgut with regular constrictions, ventral to pericardium, emptying into rectum. Midgut flattened dorsally and curved upwards on its ventral side. Surrounding musculature thin with ciliated folds. Rectum rounded and opens into dorsal wall of pallial cavity (Figure 48).
Nervous system. Atrium short and wide (250 µm long, 250-600µm wide, 300-500 µm high) with approximately 20 digitiform papillae (80 µm long, 10 µm wide). Two pairs of large, stemmed papillae (100 µm long, 150 µmat the widest point) located in terminal region of atrium, one extending ventrally, the other extending dorsally. Cerebral ganglion dorsal to mid-region of pharynx and ventral to dorsal caecum. Oval in crosssection and verythin dorsoventrally due to compression during sectioning (200 µm wide, 50 µm high).Two small (approximately 20 µm) buccal ganglia 40 µm in length lateral to radula. Pair of ventral nerve cords present in posterior region of radular apparatus. Ventral nerve cords proceed ventrolaterally and terminate in midregion of spawning ducts (Figure 48).Four dorsoterminal sense organs penetrate the cutide above anterior-most part of mantle cavity, two in the mid-region (Figure 48-5), and one at terminal end.
Gonopericardial system. Erythrocytes long and without granulations. Disc-shaped granulocytes also present. Oocytes present in the gonad. Animal is a mature specimen with formed gonads with abundant oocytes. Pericardium small (900 µm long, 250 µm wide, 60-100 µm high) (Figures 48-4 and5). Heart runs along dorsal wall of pericardium and forms a drop shape halfway through the pericardium (Figures 48-4 and5). Pericardioducts (555 µm long, 50-90 µm in diameter) connect dorsally with spawning ducts in the middle region and loop up and back to anterior terminus of the pericardium (Figure 48-5). Pair of large seminal receptacles (800 µm long, 150-200 µm wide, 160-360 µm high) located anterior to spawning ducts. Spawning duct paired in its origin (Figure 48-4), paired region long (extending for approximately 1 mm), large in cross-section (400 µm wide, 150-300 µm high), thick walled, and flattened dorsoventrally (Figure 48). Paired spawning duct fuses into single, flattened duct posteriorly (245 µm long, 500-900 µm wide, 100-150 µm high), which opens ventrally into mantle cavity (Figure 48-6).
One pair of single elongated copulatory spicules (180 µm long) oval in cross-section (Figure 48-5). Spicules surrounded by musculature (20-50 µm thick) and extend ventrolaterally into mantle cavity.
Mantle cavity long, narrow, in posterior region (660 µm long, 50-150 µm wide, 110-200 µm high), wider in middle region (625 µm long, 150-450 µm wide, 200-400 µm high) where it has folded and slightly ciliated epithelium (Figure 48-6). Two diverticula in anterior dorsal region and several posterior diverticula located ventrolaterally.
From Latin ancora: support or refuge. In reference to the shape of the anterior pharynx.
The two genera that make up the family Proneomeniidae, Proneomenia Hubrecht, 1880 and Dorymenia, are distinguished solely by the presence of copulatory stylets in Dorymenia (Garcia-Alvarez and Salvini-Plawen 2007; Garcia- Alvarez et al. 2009;Scheltema and Schander 2000). 0orymenia tanifa n. sp., 0oryme nia ancora n. sp., and 0orymenia lucida n. sp. all have a pair or two pairs of copulatory stylets, placing them in this genus. Within 0orymenia there are currently 25 described species, the majority of which are from the Antarctic, while 0. peroneopsis Heath, 1918 and 0. sarsii (Koren & Danielssen, 1877) are from the North Atlantic Ocean, 0. vagans (Kowalevsky & Marion, 1887) is from the Mediterranean Sea, 0. longa (Nierstrasz, 1902) and 0. weberi (Nierstrasz, 1902) are from the South Indian Ocean, 0. acuta Heath, 1911 is from the North Pacific, and 0. quincarinata was described from New Zealand (Nierstrasz 1902;Ponder 1970;Garcia-Alvarez and Salvini-Plawen 2007).
Considering the geographical distribution, and in view of the combination of internal characters and habitus, the species described here differ substantially from all the other described species of the genus (Tables 23), as well as from each other, enough to determine that they constitute new species.
Of note, in Solenogastres haemolymph cells or haematocytes are commonly present as free-floating redstained cells that vary in shape (i.e., rod-like, conical, or tear-drop shaped) and granulations (Salvini-Plawen 1997). Garcia-Alvarez, Urgorri and Salvini- (2000) were the first to use the term 'erythrocytes' in reference to solenogaster blood cells, with detailed specification on granulations and cell shape (Garcia-Alvarez, Urgorri and Salvini-Plawen 2000), and its usage in reference to haematocytes quickly became commonplace in solenogaster research. Garcia-Alvarez et al. (2000) and Garcia-Alvarez and Urgorri (2003) grouped some 0orymenia species based on the radular structure and shape of the teeth, but not all the species were considered. In addition, some of the radular characteristics are unknown for some species (Table 2). Therefore, following their classification, we simplify the possible radula types of 0orymenia, none of which are exclusive of other types: (a) radula with uniform teeth (that can have long or short bases), with a curved apical tip; (b) radula with elongated teeth; (c) radula with one or two medial teeth. We focus a large part of the discussion on the radula due to its importance (Scheltema et al. 2001), although other characters were also considered. The radula of 0. ancora n. sp. is formed by short, uniform teeth with a curved end, similar to the radulae of most 0orymenia species but clearly distinguishable from 0. harpagata Salvini-Plawen, 1978, 0. tricarinata (Thiele, 1913), 0. cristata Salvini-Plawen, 1978, 0. troncosoi Garcia-Alvarez, Urgorri & Salvini-Plawen, 1993 and 0. profunda Salvini-Plawen, 1978, all with radulae with middle teeth. The radula of 0. ancora n. sp. also differs from that of 0. acutidentata Salvini-Plawen, 1978, 0. singulatidentata, 0. paucidentata, 0. parvidentata and 0. quincarinata, all with radulae with elongated teeth. From the remaining 0orymenia species, 0. ancora n. sp. differs in the combination of internal characters (Table 2) and is easily differentiated from all but one thanks to the number of dorsoterminal sensory organs. 0orymenia ancora n. sp. has four dorsoterminal sensory organs, which was also reported for 0. menchuescribanae Garcia-Alvarez et al., 2000. Nevertheless, these species differ in the number of radular teeth (45 teeth per row in 0. ancora n. sp., up to 100 teeth per row in 0. menchuesaibanae), the erythrocytes (long and not granulated in 0. ancora but elongated drops without granules and granulocytes in 0. menchuescribanae), and the absence of abdominal spicules in the new species.
0. tanifa n. sp. also has short radular teeth with curved apical tips. Therefore, as with 0. ancora n. sp., it can be easily differentiated based on the radula from 0. harpagata, 0. tricarinata,0. cristata,0. troncosoi,0. profunda,0. acutidentata,0. singulatidentata,0. paucidentata,0. paNidentata and 0. quincarinata. This species also has characteristic copulatory spicules, formed by four long bound spicules that differentiate it from all described 0orymenia species. 0orymenia ludda n. sp. has a radula with medial teeth, which makes it similar to 0. harpagata, 0. tricarinata, 0. aistata, 0. troncosoi and 0. profunda. The new species has a unique medial tooth, making it different from 0. harpagata, 0. tricarinata and 0. cristata, which have two medial teeth. 0. lucida n. sp. and the other two species with a unique medial tooth (0. troncosoi and 0. profunda) have the same number of teeth per row (Table 2). Nevertheless, 0. lucida is easily differentiated from them in the shape and size of the copulatory stylets, which are simple and rounded in cross-section, while in 0. troncosoi and 0. profunda they have a distal end with a four-point star shape. In addition, 0. lucida n. sp. differs from 0. troncosoi in size (0. /ucida n. sp. is smaller. 12 x 1 mm while 0. troncosoi reaches 22 x 12 mm), pedal folds (three in 0. /ucida, one single fold in 0. troncoso,), sclerites (curved and up to 170 µm length in the new species; mostly straight and up to 210 µmin 0. troncoso,) and in the abdominal spicules (in the new species there are 15 abdominal spicules; the number of abdominal spicules in 0. troncosoi was not specified but the length of the folds bearing the abdominalspicules is approximately 260 µm according to the original description of the species; Garcia-Alvarez et al. 1998).
Finally, there are clear differences between the three new 0orymenia species described here. They differ in size (0. ancora n. sp. is much larger, measuring 32 mm x 2.5 mm than 0. lucida n. sp. is 13 mm x 1 mm while 0orymenia tanifa n. sp. is intermediate, measuring up to 18 mm x 1 mm), habitus (0. ancora n. sp. is pale yellow while 0. lucida n. sp. has a bright yellow appearance and 0. tanifa n. sp. is a soft white to light grey colour), radular structure (0. ancora n. sp. has 28 uniform narrow-based teeth with a curved apical tip per row and 45 rows, 0. lucida n. sp. has a smaller radula with a middle tooth and 18-20 teeth per row and 20 rows and 0. tanifa has 24 uniform teeth with a slightly curved apical tip in each of 23 rows), pallial cavity (0. lucida n. sp. has a simple pallial cavity with no diverticula, 0. tanifa n. sp. lacks diverticula but the pallial cavity is ciliated and 0. ancora n. sp. has two anterior diverticula and several posterior diverticula that run ventrolaterally), number of dorsoterminal sense organs (0. ancora n. sp. has four while 0. lucida n. sp. and 0. tanifa n. sp. only haveone), the presence/absence of abdominal spicules (0. lucida n. sp. has clusters of abdominal spicules while 0. ancora n. sp. and 0. tanifa n. sp. do not) and the copulatory stylets (formed by a bundle of four spicules that are long and rounded in crosssection in 0. tanifa n. sp.; rounded, simple, long spicules in 0. lucida n. sp., and oval, short spicules in 0. ancora n. sp.). Further study of the sclerites of the new species (especially 0. tanifa n. sp., as the sclerites were dissolved and unavailable for examination) would be desirable as it has been shown that they can be useful to distinguish species of this family (Scheltema et al. 2000;Pedrouzo et al. 2014). However, with the available information, it is possible to use the sclerites to observe differences in these three species, especially when distinguishing those of 0. ancora (D. ancora has shorter sclerites (40-140 µm) with a more prominent hollow cavity) than the other species where sclerites could be studied). (Ponder, 1970) (Figure S; Tables 123 Diagnosis. Animal with five longitudinal ridges that run along entire length of body. Visible anterior and posterior slits connected by the pedal groove. Polystichous radula with 2 central teeth and 40 teeth per row. Outlet of spawning duct paired. Seminal receptacle not represented as a sac. With copulatory stylets.
Distribution. Dorymenia quincarinata is known from eastern New Zealand at least as far north as Wellington and at least as far south as Christchurch at depths between 14 and 238 m.
Remarks. Dorymenia quincarinata was originally described based on gross anatomy observed via dissection of two specimens that were poorly preserved and was assigned to the genus Proneomenia (Ponder 1970). It was subsequently redescribed and moved to the genus Dorymenia by Salvini-Plawen (2004). Here, new material was examined using histology to supplement the description of this species. Sample NIWA 28046-A measures 30 x 2 mm and is a light yellow when preserved in 96% ethanol, which is consistent with the original description (Ponder 1970). Neither the original description nor the re-description to Dory menia quincarinata (Salvini-Plawen 2004) include data on cuticle thickness. NIWA 28046-A has an average cuticle thickness of 220 µm. Ponder (1970) mentions the organisation of the 'vestibulum' ( = atrium) but leaves out detailed information of any atrial papillae other than the presence of closely packed villi. Based on the new material, there are at least five slender papillae that extend 70 µm into the atrium (Figure SA). In addition to two ventral papillae (70 µm long and 80 µm wide), there is a pair of large papillae (130 µm long and 180 µm at its widest point) extending ventrally from the dorsal wall of the atrium (Figure SB). The vascular system and haemolymph cells were not described for this species previously. In the specimen NIWA 28046-A, the heart (180 µm long dorsoventrally, 230 µm wide) runs along the dorsal wall of the large pericardium (500 µm long dorsoventrally, 1010 µm wide). Haemolymph cells consist of three main types: smooth and elongated erythrocytes, small spherical erythrocytes, and small spherical granulocytes.
In regard to the reproductive system, Ponder (1970) describes the spawning ducts as having 'separate openings into the cloacal chamber', which is not a characteristic of Proneomeniidae and has raised questions about its taxonomic classification (Salvini-Plawen 2003). The paired spawning duct (Figure SF) of sample NIWA 28046-A is 600 µm dorsoventrally and 700 µm wide and opens dorsally into the mantle cavity as an unpaired outlet (Figure SD, E), unlike the original description (Ponder 1970). The pair of copulatory stylets is perfectly circular in cross section (100 µm in width and height), has thick surrounding musculature, and is located ventrolaterally to the spawning ducts.
The nervous system was also excluded from the original description. In NIWA 28046, there is a small cerebral ganglion dorsal to the anterior region of the pharynx. A pair of small lateral nerve cords extends from the radula to the posterior region of the spawning duct. There is one dorsoterminal sense organ (80 µm wide and 60 µm high) dorsal to the midregion of the pallial cavity.
We were able to sequence a fragment of 16S from D. quincarinata (NIWA 28046; NCBI Accession OP548502) and 0. lucida n. sp. (NIWA 129496; NCBI Accession OPS48501). The uncorrected pairwise distance between these sequences was 13.3%.
Considering the thick cuticle (>50 µm), the presence of epidermal papillae, the layered organisation of hollow acicular sclerites, the presence of a polystichous radula, and the Epimeniatype foregut glands, all the analysed specimens belong to the family Proneomeniidae. Within Proneomeniidae, two genera exist: Proneome nia and Dorymenia. These two genera are distinguished from one another solely by the presence of copulatory stylets in the genus Dorymenia (Garda-Alvarez and Salvini-Plawen 2007;Garcia-Alvarez et al. 2009). Although this characteristic is related to the state of development of the specimens, it has proven to be useful when differentiating between the existing species and those included in this work.
The importance of the radula to the differentiation and grouping of Dorymenia species has been noted by several authors (Salvini-Plawen 1978;Garcia-Alvarez et al. 2000, 2003;Scheltema and Schander 2000). Consequently, in this work, we emphasise the importance of this character. Phylogenetic analyses exploring evolutionary relationships within Dorymenia would be valuable in evaluating the taxonomic utility of this character. A thorough characterisation of sclerites and copulatory spicules has also been argued to be important for classifying Dorymenia species (Scheltema and Schander 2000; Pedrouzo et al. 2014). Nevertheless, information on these structures is not always available. Sclerites are usually verysimilar among Dory menia species and to observe any taxonomicallyinformative differences, it is necessary to studyseveral individuals of the same species. The correct characterisation of copulatory stylets and abdominal spicules, as well as radulae, requires obtaining the structures by digesting the tissues in addition to studying them through serial sections. This needs several individuals of the same species, which is often not possible when it comes to solenogasters. Thus, even though the usual diagnostic characters (habitus, pedal folds, presence/absence and number of dorsoterminal sensory organs, presence/absence of abdominal spicules, presence/absence of dorsal foregut gland, etc.: Tables 2,3) are enough to classify Dorymenia species, a larger effort on the study of the characters addressed as important here, along with genetic data, would bring a better understanding of the genus and the differences between the species within it.
As discussed above, there has been limited sampling which has caused a lack in knowledge of the distribution of solenogasters. For New Zealand, only three solenogaster species were described previously (Neomenia naevata, Drepanomenia tenuitecta and Dorymenia quin carinata). With the new species described here, we are adding to this number. These three species are described from specimens from two large collections totalling around 200 specimens. Based on the habitus and sclerites we identified 38 specimens as Proneomeniidae. Although in this family, as demonstrated here, the study of the internal anatomy is essential to distinguish between species, we tentatively classified them into approximately 15 morphospecies (including the three species described here). As for the rest of the specimens in the collection, we have distinguished as many as 37 other morphospecies from different groups within 'Cavibelonia', Neomeniamorpha and Pholidoskepia. However, because of the small size and homogeneous external aspect of many of these putative species, some may constitute multiple, cryptic species. In condusion, as is usual for solenogasters from understudied locations, the actual number of species is high, and it is likely that most of them are new to science.