NEW RECORDS OF LEPTACANTHICHTHYS GRACILISPINIS AND MICROLOPHICHTHYS MICROLOPHUS (ACTINOPTERYGII: LOPHIIFORMES: ONEIRODIDAE) FROM THE SUBARCTIC ATLANTIC OCEAN, INCLUDING NEW LOPHIIFORM BARCODING DATA AND A RARE OBSERVATION OF A COPEPOD PARASITE IN CERATIOID ANGLERFISHES

In an ongoing effort to document and/or validate taxonomic identifications and monitor fishes in the subarctic Atlantic Oceans, including the production of pragmatic identification material, two new distributional records of ceratioid anglerfishes are presented: Leptacanthichthys gracilispinis (Regan, 1925) and Microlophichthys microlophus (Regan, 1925) of the dreamer family Oneirodidae. The former belongs to the relatively rarely observed “long-pectoraled” subgroup whereas the latter is a relatively common species distributed circumglobally in temperate and tropical waters. Both species were recorded for the first time off Greenland and the specimens could be expatriates although the diversity of deep-sea pelagic fishes in the subarctic Atlantic Ocean is not well known. In addition, molecular barcoding Cox1 DNA sequences of subarctic Atlantic lophiiform taxa are included, where the material was available, many being produced as part of the Greenland fishes barcoding program, a continuous effort to register and barcode all Greenland fish species. The program has currently barcoded 220 taxa of approximately 300 known fish species observed in Greenland waters, with the ceratioid anglerfishes constituting one of the most problematic fish groups in the region in terms of sampling, identification, and taxonomic assignments using integrative taxonomy. Taxonomic issues based on molecular OTUs** are reported for the genera Caulophryne, Cryptopsaras, and Dolopichthys based on Cox1 data. Finally, a relatively large copepod parasite in the family Pennellidae was found on L. gracilispinis and constitutes one of only two copepod parasites recorded on ceratioid anglerfishes.


INTRODUCTION
Anglerfishes of the suborder Ceratioidei are the deepsea component of the order Lophiiformes, otherwise associated with mostly shallow-water benthic frog-and toadfishes Orr 2007, Miya et al. 2010). Ceratioids exhibit extraordinary adaptations to the deepsea pelagic habitats. All females of the 169 ceratioid species (Bañón et al. 2019), with the exception of two species in the family Caulophrynidae and the monotypic Neoceratias spinifer Pappenheim, 1914, are mimetic aggressive anglers showing a modified first dorsal fin that have evolved into a long shaft (the illicium) with a distal bioluminescent bulb (the esca) (Lütken 1871, Bertelsen 1951, Pietsch 2009). The esca of metamorphosed females contains bioluminescent-producing bacteria Herring 1977, Munk et al. 1998) that have co-evolved with the host ceratioid species (Haygood et al. 1992). As a consequence, the co-evolving bacteria possess reduced genomes as compared to free-living relatives (Hendry et al. 2018). Both illicium and esca vary between the majority of species and therefore are important taxonomic characters (Parr 1927) and represent the basic knowledge needed to comprehend communication biology of these fishes. Bioluminescence in ceratioids is not fully understood although it is believed to function at least in attracting prey as well as in reproductive signalling (Pietsch 2009). However, as males are usually levels of magnitudes smaller than the females, and possess large olfactory organs, pheromones working within close range could be as important as the light for the latter function.
The extraordinary sexual reproductive system in ceratioids varies between deep-sea anglerfish lineages; • Males never attach to females, • Males attach temporarily, • Males are facultative or obligate "parasites".
When "parasitism" is noted, a fusion of the two sexes is taking place and the male is attached for life and nourished by the female (Pietsch 1976, 2005, Pietsch and Orr 2007. Males showing temporary attachment (or pre-fusion if facultative "parasites") have a "denticular apparatus" suitable for attachment and feeding (Bertelsen 1951).
Ceratioid anglerfishes in the subarctic North Atlantic are, in general, rare occurrences with the majority of species registered and known only from a single or a few stray specimens (Jónsson and Pálsson 1999, Møller et al. 2010). Many more ceratioid species have been observed south of the Sub Polar Front (SPF), e.g., in the western central Atlantic (Pietsch 2002). The SFP is an important topographic and oceanographic species barrier between the temperate and subarctic North Atlantic (Sutton et al. 2013) although depth dependent (Vecchione et al. 2015). In fact, Sutton et al. (2017) defined the subarctic western Atlantic as a distinct mesopelagic eco-region that holds primarily cold-water biodiversity assemblages. New records of anglerfishes, and other mid-water taxa could, therefore, provide important information on distributional shifts although the rarity of ceratioids and insufficient knowledge of their true distributions at present makes any trends difficult to detect.
This study describes new records of Leptacanthichthys gracilispinis (Regan, 1925) and Microlophichthys microlophus (Regan, 1925) from the subarctic Atlantic, including a description of their morphologies. Barcoding Cox1 molecular data is included for all available subarctic oneirodid taxa with many newly constructed as part of the Greenland fishes barcoding program (Poulsen et al. 2018). Barcoding data indicate taxonomic issues in some ceratioid genera. In addition, a large crustacean parasite, rarely observed in ceratioid anglerfishes, is reported herewith. The new records and molecular data presented are part of a larger continuous taxonomic and monitoring effort off Greenland, a region that has witnessed many new species distributions the last 10 years, with the caveat that deep-sea pelagic waters are poorly known and difficult to monitor.

MATERIALS, METHODS
Material. One specimen of Leptacanthichthys gracilispinis was caught by R/V Pâmiut, Greenland Institute of Natural Resources (GINR), leg 6, haul 33, 23 September 2009, in the Davis Strait off south-western Greenland at 64.04°N, 057.37°W. It was deposited at the Zoological Museum University of Copenhagen (ZMUC) as ZMUC P922698 (tissue/field JYP#8126). Coll./ID J.Y. Poulsen. The bottom and fishing depth was within 942-950 m, with a bottom temperature of 3.74°C, although the actual catch depth is uncertain due to non-closing trawls. The specimen was barcoded (GLF140) as part of the Greenland Fishes (GLF) barcoding project (Poulsen et al. 2018) at BOLD * (Ratnasingham and Hebert 2007).
One specimen of Microlophichthys microlophus was caught also by R/V Pâmiut, leg 4, haul 14, 24 July 2017, on the Irminger Sea slopes off south-eastern Greenland at 61.57°N, 40.16°W. It was deposited as ZMUC P922698 (tissue/field JYP#1862). Coll./ID J.Y. Poulsen. The bottom and fishing depth was within 1454-1465 m and the bottom temperature was 3.45°C. This specimen was also caught during a routine survey with a non-closing Alfredo III trawl probing Greenland halibut abundances and the real catch depth uncertain. Barcode GLF319.
The specimens were X-rayed at the Australian Museum (AMS) and ZMUC and examined under stereomicroscopes. The morphological measurements were taken using a digital calliper to the nearest 0.1 mm and compared to all previous specimens recorded by Pietsch (1976Pietsch ( , 1978Pietsch ( , 2009 (Table 1).
The copepod parasite attached to L. gracilispinis ( Fig.  1B) was photo-identified as a taxon within the Pennellidae family, possibly Sarcotretes scopeli Jungersen, 1911 by Geoffrey A. Boxshall at the British Museum of Natural History (BMNH).

RESULTS
Oneirodidae Gill, 1878 Leptacanthichthys Regan et Trewavas, 1932Leptacanthichthys gracilispinis (Regan, 1925 ( Table 1, Fig. 1A-E) Description. (Based on specimen ZMUC P922698): First radial shorter than second and pectoral-fin lobe long, narrow, longer than longest fin ray (dorsally positioned); short and broad suboperculum; pharyngobranchials II and III showing 8 long slender teeth; frontals relatively straight; sphenotic spines large, appearing vertical when viewed from anterior, with posterior slant when viewed from side; lower jaw broken at symphysis although small symphysial spine present ventrally, larger knob present dorsally; maxillomandibular ligament 9.8 mm (15.8% SL); posterior part of lower jaw showing "Leptacanthichthys" configuration: long mandibular spine present, shorter quadrate spine and angular bone without distinct spine (Fig. 1); bases of mandibular and quadrate spines broken off left and right sides, however, spines still present although rotated compared to their correct positions; multiple teeth in lower jaw, highly variable in size, some fang-like and almost straight; fewer upper jaw teeth, much smaller, less variable in size, fang-like teeth absent; vertebral column strong and ossified; large neural spines present, anterior four particularly large; haemal spines present, anterior two large; pre-caudal vertebrae 3; caudal vertebrae 17; ribs, epipleurals and epineurals absent (Fig. 1C); caudal fin rays 9: 2 simple + 4 bifurcated + 3 simple; pterygiophore of illicium 3.2 mm (5.2% SL), wider than illicium, originating between anterior part of frontals; illicium slender, especially at midpoint, and flexible; esca with pigmented streak on dorsal margin and single posterior compressed appendage containing no bioluminescent dark tissue (Fig. 1D). Reproductive state: Ovaries 6 mm in lengths, flaccid, suggesting spent or previous larger eggs present now gone; minute eggs present, largest 0.1 mm diameter in ovary walls along its entire length.
Microlophichthys Regan et Trewavas, 1932Microlophichthys microlophus (Regan, 1925 ( Table 1, Fig. 2A-E) Description. (Based on specimen ZMUC P2395464): Sphenotic spines present, large symphysial spine present ventrally, no discernible spine dorsally on lower jaws; quadrate spine robust; opercle notched posteriorly; pectoral fin lobe large, approximately equal in length to fin rays or slightly shorter, that are situated somewhat dorsoposterior on lobe; dorsal fin in advance of anal-fin origin; teeth on lower jaws variable in size on whole jaw; teeth on premaxillaries in multiple rows, variable in size only in the anterior parts; skin smooth, although most skin lost on specimen; subopercle distally expanded and rounded; esca with appendage without bioluminescent tissue, red bulb under bioluminescent tissue in esca (Fig.  2D); stomach content white mass, items indiscernible. Morphological characters similar to those described by Pietsch (2009) and not included for further descriptions (Table 1). Reproductive state: Ovaries 6.2 mm in lengths, red coloured, no eggs visible. Cox1 DNA barcoding sequences. In order to support a more comprehensive comparison of Cox1 barcodes of ceratioids in general, I included here all subarctic Atlantic taxa that have barcoding data available. Ceratioid observations in the subarctic regions are based on the presently reported study, Pietsch (2009), Møller et al. (2010, Jónsson and Pálsson (2013), and the Global Biodiversity Information Facility (Anonymous 2012). All specimens included for the Cox1 barcode in this study are metamorphosed females. A Kimura-2-Parameter (K2P) (Kimura 1980) model was employed for the analysis of the dataset consisting of 55 specimens including a total of 22 subarctic Atlantic distributed taxa ( Table 2). The resulting topology is shown in Fig. 3. Three taxonomic results based on Cox1 barcodes related to the genera Dolopichthys, Cryptopsaras, and Caulophryne are discussed below. Besides that, the Cox1 barcoding results are not discussed further due to the inclusion of only subarctic Atlantic taxa and the focus on molecular taxonomy of ceratioids in the subarctic Atlantic.
The copepod parasite attached to L. gracilispinis ( Fig.  1B) was photo-identified as a taxon, within the family Table 1 Morphometric and meristic data of Leptacanthichthys gracilispinis and Microlophichthys microlophus, caught off south-western and south-eastern Greenland respectively during the presently reported study (ZMUC specimens), compared to specimens examined by Pietsch (2009

DISCUSSION
A few morphometric characters of the new subarctic record ZMUC P922698 (Leptacanthichthys gracilispinis) show values outside previously reported, such as the mandibular-and quadrate spine lengths (Table 1). However, morphometric values are generally close to the ranges noted by Pietsch (1978). Similarly, the distinct illicium and esca show similar morphology to what has been reported for other specimens of L. gracilispinis: the illicium is very thin compared to other ceratioids and the esca have only one large appendage without bioluminescent tissue (Fig. 1E). A peculiar feature of L. gracilispinis is the long pectoral fin lobe showing the fin rays on the dorsal margin (Fig. 1E). This is a unique configuration of the pectoral fins and is only found in a small fourgenera subgroup of dreamers in the family Oneirodidae (see Pietsch 1978): L. gracilispinis; Puck pinnata Pietsch, 1978; Ctenochirichthys longimanus Regan et Trewavas,   The table contains only valid names of fishes, which may sometimes differ for names listed in individual papers; the Cox1 DNA sequences were either determined for the presently reported study (GLF records), downloaded from public accessible BOLD barcoding projects, or granted use from unpublished barcoding projects; metadata included if known; the taxonomic identification of ceratioids is difficult, hence the identifiers are included for specimens if known.  fig. 3 of Bañón et al. 2019). The L. gracilispinis shows an uncorrected DNA sequence distance of 97% to several species within the Oneirodidae (data not shown), supporting a close association of longpectoraled dreamers within this family as already noted from morphology (Pietsch 1974, 1978, Pietsch and Orr 2007 and mitogenomic data (Miya et al. 2010). A currently unresolved phylogenetic relation of the longpectoraled genus Puck to Thaumatichthys as found by Miya et al. (2010) needs verification with additional taxa. The latter study also found multiple non-coding regions in the mitogenomes of oneirodids-characters proved very useful in elucidating phylogenetic relation (Poulsen et al. 2013). New unpublished mitogenomic data (J.Y. Poulsen data, data not shown) confirms this feature in some oneirodids.
The new subarctic record of Microlophichthys microlophus off south-eastern Greenland shows most characters within the ranges noted by Pietsch (2009) and are therefore not given much discussion. However, head length and measurements of the jaws fall outside the ranges noted in Pietsch (2009; table 1, fig. 2). The Greenland specimen is relatively large compared to previous observations (Table 1) and the small differences could be due to allometric growth changes. The Cox1 barcode shows the Greenland specimen to have 100% DNA sequence similarity to specimens from the Mid-  (Poulsen 2015a), and even though the Greenland specimen is 62 mm SL and therefore relatively large compared to presently described material, L. gracilispinis can apparently grow to 103 mm SL (Pietsch 2009). However, a status as expatriate of L. gracilispinis in the subarctic Atlantic is by no means substantiated, as the pelagic deep-sea realm in this region is poorly known due to a variety of factors (Bertelsen and Krefft 1965, Poulsen 2015b, Poulsen et al. 2018. The four long-pectoraled taxa show circumglobal distributions, although not observed in the Indian Ocean as yet (Pietsch 2009). Leptacanthichthys gracilispinis is the most commonly observed of the four long-pectoral anglerfishes and indications are that it is widely distributed in the North Atlantic. The new record of M. microlophus off Greenland is unsurprising as it is relatively common in all major oceans and the temperate Atlantic (Pietsch 2009).
Greenland waters are considered too cold for ceratioid reproduction, based on comprehensive data by Bertelsen (1951) and Pietsch (2009). However, several ceratioid species are regularly observed off south-western and south-eastern Greenland (J.Y. Poulsen, personal observation): Ceratias holboelli Krøyer, 1845; Oneirodes eschrichtii Lütken, 1871; Oneirodes macrosteus Pietsch, 1974;Chaenophryne longiceps Regan, 1925;Lophodolos acanthognathus Regan, 1925; and Melanocetus johnsonii Günther, 1864. The species Linophryne coronata Parr, 1927 appears to be more common off Iceland than Greenland (Bertelsen 1976, Bañón et al. 2006, Jónsson and Pálsson 2013, although this knowledge is based on few specimens only. The remaining 31 species registered in the subarctic waters of the North Atlantic are, on the contrary, rare occurrences, known only from one or a few stray specimens (Møller et al. 2010, Jónsson andPálsson 2013). In fact, 2009 witnessed yet another rare ceratioid specimen off south-western Greenland, the sixth specimen of Linophryne bicornis Parr, 1927 known,  Poulsen, presented in Møller et al. (2010) with barcode GLF096 and caught at a bottom temperature of 3.64°C. Leptacanthichthys gracilispinis, M. microlophus, and L. bicornis are the only three new ceratioid records that have been observed in the region for over a decade, owing to an increased sampling and taxonomic effort onboard including integrative taxonomy. Yearly fluctuations in warm North Atlantic drift waters are potentially a factor concerning observations of ceratioids in this region (Gaemers and Poulsen 2017).
The large volume pelagic deep-sea water masses off the Labrador Sea (south-western Greenland) and the Irminger Sea (south-eastern Greenland) remain poorly studied, and deep-sea pelagic fishing efforts would certainly result in many new discoveries in these regions considering that benthic distributions are continuously being extended (Poulsen et al. 2018) and new recent pelagic species discovered (Poulsen 2015b). Future fish records in these regions should elucidate the subarctic north-western ecoregion as delimited by Sutton et al. (2017). Few ceratioid species with males attached (Pietsch 2009), such as, for example, Linophryne lucifer Collett, 1886 observed in 1967 by Jónsson (1967) in the Denmark Strait off southeastern Greenland (Bertelsen 1976(Bertelsen , 1986, have been observed north of the SPF. Free-living male ceratioids seem restricted to the tropical and subtropical zones between 40°N and 40°S (Bertelsen 1951, Pietsch 1974, 2009. No attached males or signs of previous attachment were found on the new records. The L. gracilispinis specimen caught off Greenland showed only tiny eggs present and clearly not close to reproducing at the time of capture. Contrary to the yearly observations of some ceratioid species off Greenland, these results support that reproduction is not taking place at these latitudes. Online databases dedicated to taxonomic assignments of fish species based on barcoding DNA fragments have shown valuable in several respects concerning the distribution, morphology, taxonomy, and, as a consequence, the species diversity of fishes (Ward et al. 2009, Bañón et al. 2013, Barros-García et al. 2018. This study provides barcoding data of all ceratioid species sampled off Greenland for more than a decade and is intended to support comparisons for future discoveries. All records in the Greenland fishes barcoding project are freely available in BOLD (prefix GLF, Poulsen et al. 2018). However, the BOLD database should be used with great caution concerning Cox1 ceratioid comparisons at present, as many species show almost identical DNA sequences indicating taxonomic identifications problematic. This is evident in, for example, the family Oneirodidae, a difficult group of fishes in which damage is often present in speciesdefining characters, and therefore difficult to identify. Future works using Cox1 comparisons should provide and validate identifications and metadata associated with records used (Table 2). Although several ceratioid species recorded from the subarctic Atlantic is missing for their Cox1 barcode in the presently reported study, many are rare and no tissues available, three taxonomic issues can be noted from the barcoding results in the presently reported study (Fig. 3). Thorough morphological examinations of these three genera are beyond this study.
Two specimens identified as Dolopichthys longicornis Parr, 1927, the only species in the genus noted from the subarctic Atlantic (Møller et al. 2010, Jónsson andPálsson 2013), show identical Cox1 DNA sequences to several specimens identified as Dolopichthys karsteni Leipertz et Pietsch, 1987 that were collected from different Atlantic localities such as the Mid-Atlantic Ridge and Georges Bank (Table 2). However, Pietsch (2009) noted no presence of either species in the subarctic Atlantic. I leave these results as presented for future studies employing barcoding Cox1 data and note that Dolopichthys allector Garman, 1899 from the North Atlantic Ocean was found to be closely related to D. karsteni (Fig. 3).
Cryptopsaras couesii Gill, 1883 show two relatively divergent Operational Taxonomic Units (OTUs) with an uncorrected distance of 5.4% (Kenchington et al. 2017). A total of 37 substitutions are observed in 688 base pairs of the Cox1 fragment of which only two are not observed in the third codon positions (one synonymous C-T transition and one non-synonymous A-G transition in first codon positions). Seven of the 37 substitutions are transversions and three are non-synonymous. This variation between C. couesii specimens is reminiscent of substitutional variations observed in mid-water and deep-sea taxa delimited also from solid morphological characters (Byrkjedal et al. 2011, Poulsen et al. 2016. Unfortunately, no Cox1 barcode of a C. couesii specimen off Greenland could be included, as this species has not been observed for more than a decade in the region, contrary to Ceratias holboelli Krøyer, 1845 that is caught annually (J.Y. Poulsen, personal observation, Fig. 3).
Caulophryne jordani Goode et Bean, 1996 andCaulophryne pelagica (Brauer, 1902) show highly similar DNA sequences, based on four specimens barcoded from the Atlantic and Pacific Oceans (Table 2). There is no structure in the few substitutions present indicating one OTU (Fig. 3). The few differences observed are mostly associated with fragment end-regions that could be caused by primer attachment artefacts in the replication process during the PCR amplification and sequencing. These primer attachment sites (usually about 20 base pairs in the end regions) are unfortunately not always removed before the Cox1 DNA sequences are uploaded to the BOLD repository (J.Y. Poulsen, personal observation). This is an issue when comparing Cox1 barcodes if not assessing the locations of the variations observed. Regardless, and in respect to other ceratioid OTUs (Fig.  3), the variation between C. jordani and C. pelagica is not supporting two distinct OTUs (see Miya et al. (2010) for specimen identifications).
A plethora of crustacean parasites are found in association with deep-sea fishes (Klimpel et al. 2001) although these types of modified parasites have only been observed once in ceratioid anglerfishes (Prokofiev 2014). Therefore, the copepod parasite attached to L. gracilispinis off Greenland is the second external parasite recorded in deep-sea anglerfishes (Fig. 1A, 1C) with a hydroid (Hydrozoa) Hydrichthys pietschi, reported on the skin of Ceratias holboelli by Martin (1975). The parasite, presently reported from L. gracilispinis, is a copepod in the family Pennellidae and likely represents the species Sarcotretes scopeli (see G.A. Boxshall, personal comment) that has also been recorded from various other mid-water fishes. Sarcotretes scopeli is a common fish ectoparasite in the North Atlantic showing a low host-specificity (Boxshall 1998). It is reminiscent of the parasite shown by Prokofiev (2014) on Chaenophryne melanorhabdus Regan et Trewavas, 1932, although no identification was provided. Copepod parasites are regularly observed in deep-sea fishes (Boxshall 1998). The observation of only few copepod parasites observed in ceratioids is noteworthy (T. Pietsch, personal comment), although demersal species are observed much more frequent to be parasitized than meso-and bathypelagic species in the Arctic (Klimpel et al. 2006). Relatively large mesoparasitic copepod parasites (families Pennellidae and Sphyriidae), featuring an internal holdfast and the majority of the body protruding outside the fish (Piasecki and Avenant-Oldewage 2008), are common observations on various fishes off subarctic Greenland, across phylogenetically independent fish lineages (J.Y. Poulsen, personal observation).