A new species of Anobothrus (Polychaeta, Ampharetidae) from the Weddell Sea (Antarctica), with notes on habitat characteristics and an updated key to the genus

Benthic samples were collected during two expeditions near the Antarctic Peninsula and in the South-Eastern Weddell Sea. During these studies, a new species of Ampharetidae Malmgren, 1867, Anobothrus konstantini Säring & Bick sp. nov., was found. Here we present a detailed description of this species. We used the traditional light microscope and scanning electron microscope (SEM) to identify and describe the diagnostic characters: a circular glandular band on segment 6; an elongate ridge between the notopodia on segment 12 and modifi ed notochaetae on this segment; 16 thoracic, two intermediate and ten abdominal segments. For the fi rst time, micro-computed tomography (microCT) was used for a species description of Anobothrus. Micro-CT provided information on the shape of the prostomium (Ampharete-type) and the arrangement of branchiae (four pairs in two rows, without a gap). In addition, we provide quantitative information on the environmental niche based on sediment parameters (chlorophyll a content, organic matter content, chloroplast equivalent, grain size) for the new Anobothrus species, relevant for, e.g., species distribution modelling. Finally, an identifi cation key for all Anobothrus species is provided.


Introduction
Polychaetes are one of the most speciose and dominant macrofaunal group of the Southern Ocean benthos (Clarke & Johnston 2003), and they are distributed in all substrates ranging from intertidal to abyssal depths (Schüller & Ebbe 2014). Despite comprehensive recent efforts, many species remain unknown. Many of the most abundant species in the Southern Ocean region belong to the hemi-sessile and tube-dwelling Ampharetidae (Schüller & Ebbe 2007. This family is one of the most abundant and species-rich among polychaetes, including so far more than 300 described species worldwide (Jirkov 2011;Bonifácio et al. 2015;Alalykina & Polyakova 2020; World Register of Marine Species, http://www.marinespecies.org). The taxonomy of Ampharetidae is complex and poorly resolved, with insufficient diagnoses. Major difficulties and confusion refer to different terminology and counting of segments and chaetigers following the prostomium. A conflict concerns the chaetae (paleae) of segment 2, which are excluded in the counts of chaetigers by some authors but included by others (Reuscher et al. 2009). The mode of counting needs to be defined to avoid uncertainties of the different counting expressions and misinterpretations. The terminology used in this work for counting segments, chaetigers, and uncinigers is shown schematically for a specimen of Anobothrus (Fig. 1).
Non-biological (seasonality of sea-ice extent, low bottom temperatures, currents, wind) and biological (seasonal primary production and nutrient pulses) parameters typical for polar systems shape the complexity of the benthic ecosystem in the Southern Ocean (Gutt et al. 2018). This study presents a detailed description of the abiotic parameters encountered at sites sampled for polychaetes to characterize the ecological niche, which builds a baseline for potential habitat modelling (Jansen et al. 2018) and species distribution modelling (Meißner et al. 2014) for the new species of Anobothrus.
The aim of this paper is to describe a new species of Anobothrus discovered during ecological studies in the Antarctic Peninsula area and in the Weddell Sea (Säring et al. submitted) including a revised key for all species of Anobothrus described worldwide. We show how the micro-CT method can help to describe diagnostic features that are otherwise difficult to recognize in poorly preserved individuals. We finally present the key environmental factors that characterize the habitat of this species.

Study area and sample collection
Twelve specimens of Anobothrus were collected from 8 of 16 sampled stations during two expeditions with the RV Polarstern. The tip of the Antarctic Peninsula (Drake Passage, Bransfield Strait, North-Western Weddell Sea) was explored during expedition PS 81 (22 Jan. -18 Mar. 2013, Gutt et al. 2013, while the Filchner Trough area in the South-Eastern Weddell Sea was investigated during PS 96 (6 Dec. 2015-14 Feb. 2016 (Table 1, Fig. 2; Säring et al. submitted). Water depth at the sampled stations ranged from 355 to 755 m.
Samples were collected with a MUC10 equipped with eight plexiglass core liners (inner diameter 94 mm, surface area 69.4 cm 2 ; Säring et al. submitted). For macrofaunal samples, sediments were sieved over a 500-µm mesh and fixed in a 4% formaldehyde-seawater solution (borax-buffered). More details on sediment core handling can be found in Säring et al. (submitted). For the comparison of spatial distribution, we calculated the total number of individuals per identified taxon per m 2 from the top until the bottom of the core.
For later morphological analyses, faunal samples were preserved in 70% ethanol. Environmental data from sediments (TOC = total organic carbon; Chla = chlorophyll a content; CPE = chloroplastic equivalent, grain size) were obtained from additional samples up to 5 cm depth within the same or additional MUC cores and have been published elsewhere (Veit-Köhler et al. 2018;Säring et al. 2021a, b;Vanreusel et al. 2021a, b). Here, we used the sediment layer 0-1 cm for the comparison of the environmental parameters associated with the new species. Data for salinity and temperature of bottom water were obtained from data collected by the CTD at the same stations (Schröder et al. 2013. Among the different regions, salinity varied from 34.45 psu in the North-Western Weddell Sea (station PS81-162-2) to 34.67 psu in the North Filchner Trough region (station PS96-017-3). The bottom temperature ranged from the lowest, -1.9°C, in the North-Western and South-Eastern Weddell Seas to 0.7°C in the Drake Passage.

Morphology
Specimens were examined using an Olympus SZH10 stereo microscope and an Olympus BH2 light microscope. Photographs were taken with an Olympus SZX10 stereo microscope, an Olympus BX51 microscope and an Olympus UC30 camera. Specimens were stained with methyl blue and ShirlastainA to visualize specific body regions and structures. The staining fades completely when the specimens are returned to ethanol. Three specimens were transferred through a graded ethanol series in acetone and critical point dried with a Leica EM CPD300. Two of them were attached to a stub and covered with gold palladium and used for scanning electron microscopy (SEM). Scanning electron microscopy was carried out using a Zeiss DSM 960A microscope. The anterior end of the third specimen was used for the analysis with the micro-CT machine (Xradia 410 Versa, X-ray Microscope). The newly collected  (Säring et al. 2021a;Vanreusel et al. 2021a). Mean and standard deviation of environmental parameters are given for each station. Sediment parameters: Chla = content of chlorophyll a; CPE = sum of chlorophyll a and phaeopigments; TOC% = total organic carbon; Silt & Clay % = grain size fraction < 63 µm; Sand % = grain size fraction > 63 and < 500 µm; Coarse Sand% = grain size fraction > 500 µm.
Anobothrus material was deposited in the Zoologische Sammlung, Universität Rostock (ZSRO, Zoological collection of Rostock University). The catalogue numbers are given below.
There is continuing confusion about the numbering anterior to the paleal segment (Day 1964;Parapar et al. 2012). We follow the opinion that the second segment is considered as the paleal segment; therefore, uncini begin on segment 6 = thoracic chaetiger 5 (Annenkova 1930;Eliason 1955;Uschakov 1965;Cazaux 1982;Orrhage 2001;Reuscher et al. 2009). Here we include the paleal chaetiger in our counts of thoracic chaetigers (thoracic chaetiger 1), as described by Reuscher et al. (2009). Furthermore, we use the term "intermediate segments", as introduced by Imajima et al. (2012), for segments with neuropodia formed as tori (similar to those in thoracic uncinigers) but lacking notopodia and notochaetae. Therefore, these segments were excluded from the abdominal segment count. Fig. 1 shows a schematic overview of the terminology used and the counting of segments, chaetigers and uncinigers.

Etymology
This species is dedicated to the brother of the first author (FS), Konstantin Zülske, who will be always a special part of her life.
Methyl blue staining pattern. Intensive staining of bases of noto-and neuropodia. Body uniformly spotted blue, without distinct pattern, but a circular glandular band on segment 6 (thoracic chaetiger 5, thoracic unciniger 1) becomes visible.

Remarks
The branchiae were lost in almost all specimens, branchiophores are apparently fused together and are not separated (compare Fig. 5B-D). Due to poor conservation, the segmental origin of branchiae could not be described in more detail. We suggest the following arrangement of branchiae of the anterior row: segment 2, branchiae in the middle position, segment 3, branchiae of outermost position, segment 4, innermost position, segment 5, branchiae in posterior position between innermost and middle branchiae of anterior transverse row. The holotype and paratypes from the South-Eastern Weddell Sea did not show any significant differences in diagnostic characteristics. Specimens of the additional material showed only minor differences to the diagnosis of the holotype and paratypes. Therefore, the additional material was used for the light microscopy ( Fig. 3A), drawing ( Fig. 4A-E, G-K) and the micro-CT (Fig. 5). However, we found one modification of one specimen from the additional material (ZSRO-P2658) when analysing the images from the micro-CT: one pair of small and fine paleae next to the regular large and thin paleae (Fig. 5B,  D). The small paleae are placed where newly formed chaetae are expected and may be a growing state (Tilic et al. 2015). However, the shape and form is different compared to the other paleae. The purpose of these paleae was not clearly clarified.
The presence of a reduced neuropodium on segment 5 (thoracic chaetiger 4) was presumed on one specimen using ShirlastainA because at the position of the thoracic neuropodia and of the same size as these, the same staining pattern was visible on this segment (Fig. 3E). However, uncini were not observed.  Due to the fixation in 4 % formaldehyde solution and the subsequent preservation in a 70% ethanol solution, no statement can be made about the pigmentation of fresh material. Bick sp. nov., A. bimaculatus Fauchald, 1972 andA. mancus Fauchald, 1972 differ from the other Anobothrus species with four pairs of branchiae and the presence of paleae, A. amourouxi Bonifácio, Lavesque, Bachelet & Parapar, 2015, A. anatarctica Monro 1939, A. glandularis (Hartmann-Schröder, 1965, A. gracilis (Malmgren, 1866), A. mironovi Jirkov, 2009, A. paleatus Hilbig, 2000, A. paleaodiscus Schüller & Jirkov, 2013 (Kinberg, 1867), A. patersoni Jirkov, 2009, A. pseudoampharete Schüller, 2008, A. rubropaleatus Schüller & Jirkov, 2013and A. wilhelmi Schüller & Jirkov, 2013, in having 11 instead of 12 thoracic uncinigers. Within this group, only A. paleatus has a glandular band with an elevated ridge on the fourth-to-last thoracic segment (thoracic segment 14, thoracic unciniger 9) the remaining eleven Anobothrus species, as well as A. konstantini Säring & Bick sp. nov., show a modification of the fifth-to-last thoracic segment. However, due to the difference in the number of segments (12 thoracic  The only two species with paleae on segment 2, four pairs of branchiae and 11 thoracic uncinigers are A. bimaculatus and A. mancus. However, A. bimaculatus is significantly larger (65 mm), has eyespots and has modified notopodia on segment 11, instead of on segment 12 as in Anobothrus konstantini Säring & Bick sp. nov. Anobothrus mancus is the only species with modified notopodia on segment 12, but segments 3 and 4 are fused, and notopodia are absent on segment 3. Furthermore, A. mancus is missing the circular glandular band on segment 6.

Distribution
The holotype and paratypes of Anobothrus konstantini Säring & Bick sp. nov. were found in shelf regions in the South-Eastern Weddell Sea (North Filchner Trough and South Filchner Trough). The additional material was sampled from shelf regions of the Antarctic Peninsula, in the Drake Passage and Bransfield Strait (Fig. 2, Table 1).

Ecology
The type material of Anobothrus konstantini Säring & Bick sp. nov. (1 holotype, 6 paratypes) was collected from soft sediments at water depths between 415 and 755 m from the South-Eastern Weddell Sea. This region is characterized by a high to constant ice cover and low bottom T (around -1.9°C, Säring et al. submitted: table 2;Schröder et al. 2016). The highest abundance was detected at one sampling site in the North Filchner Trough region (4 individuals per station), with low organic (TOC 0.2% ± 0.0) and the least fresh (Chla = 0.06 µg g -1 ± 0.01) material on the seafloor. This sampling site is described by a low amount of silt & clay (43.4%) compared to the higher amount of sand (49.1%). The remaining material (3 paratypes) was found at sites with higher silt & clay (> 63.2%) and lower sand (< 33.7%) content, and low Chla concentrations (< 0.19 µg g -1 ). Anobothrus konstantini Säring & Bick sp. nov. occurs in a high variety of sediments, from fine mud to coarser sandy substrates in regions with low amount of fresh material on the seafloor.
The localities for the additional material sampled adjacent to the Antarctic Peninsula, Drake Passage (2 specimens) and Bransfield Strait (3 specimens), are known for no or a variable ice-cover and bottom T up to 0.5°C (Säring et al. submitted: table 2;Schröder et al. 2013). Nevertheless, these localities for the additional material show similar environmental conditions as the sampling sites of the type material: low Chla concentrations (< 0.31 µg g -1 ) and TOC content (< 0.7%), and highly variable sediment substrates ( Table 1).

Key to all species of Anobothrus Levinsen, 1844
The key accounts for the 23 species of Anobothrus Levinsen, 1844 considered valid, including the new species proposed here. It is modified after Bonifácio et al. (2015) and Alalykina & Polyakova (2020). Only one individual has been observed with a neuropodium-like structure on segment 5 but without uncini (Fig. 3E). The reduced neuropodium was detected on both sides of this segment. All other characters were identical to the remaining eleven specimens. It is possible that these reduced neuropodia were an artefact, or that they were not visible in the other specimens due to poor conditions. Additionally, three specimens with similar body shape and characters but with 12, instead of 11, thoracic uncinigers were found in the material studied but excluded here. These individuals have elevated notopodia with a dorsal ridge on the fifth-to last thoracic segment (thoracic unciniger 8) as it is described for A. patagonicus, but differ in the number of thoracic uncini: Anobothrus patatgonicus possesses up to 30 (Jirkov 2009) and the unidentified specimens 17-21. Due to poor conditions and damage to the anterior region we could neither verify a correct counting of segments nor a presence of a glandular band, or a fusion of segments 2 and 3. These specimens can be described elsewhere when more individuals in better quality are available.
Species of Anobothrus have one or several modifications on the fourth-, fifth-, or sixth-to-last thoracic chaetigers: elevated notopodia and / or glandular ridge between notopodia and / or modified notochaetae. Anobothrus konstantini Säring & Bick sp. nov. possesses these three characters on the fifth-to-last thoracic segment (segment 12, thoracic chaetiger 11, thoracic unciniger 7). Additionally, another glandular band was observed on segment 6 (thoracic chaetiger 5, thoracic unciniger 1) using ShirlastanA staining (Fig. 3D). This complete circular band on the anterior thorax of Anobothrus species is often not clearly visible (Jirkov 2009). Within Anobothrus this character is described as absent for A. fimbriatus Fiege, 2013 andA. dayi Imajima, Reuscher &Fiege, 2013, and is not mentioned in the description for A. mancus and A. pseudoampherete. However, based on the illustration in Schüller (2008), a modification of the notopodia on segment 8 (thoracic unciniger 3) may be assumed for the latter species and possibly be a hint of a circular band. In the literature, this band was compared with those from Melinnampharete, Eusamythella and Neosamytha (Desbruyeres 1979;Holthe 1986). However, the band is developed as a dorsal ridge in Melinnampharete, Eusamythella and Neosamytha, while in Anobothrus it is completely circular (Jirkov 2009). Glandular pores of this band were found on the dorsal side using a scanning electron microscope (Fig. 6B).
SEM micrographs are important and commonly used to detect not only epidermal structures, like pores, but also other hard-to-see characters. It is especially useful for small specimens and individuals in poor condition. Only using SEM, two rows of notochaetae were found on all thoracic chaetigers except for segment 3 (thoracic chaetiger 2), an anterior row with 3 shorter and a posterior row with 4 longer chaetae. A similar arrangement of notochaetae has been described for other Anobothrus species, e.g., A. amourouxi and A. wilhelmi (Schüller & Jirkov 2013;Bonifácio et al. 2015).
A micro-computed tomography (micro-CT) scanner can also be a useful tool for studying morphological characters (Faulwetter et al. 2013); three-dimensional imaging could give a boost to the development of virtual specimen collections, allowing rapid and simultaneous access to accurate virtual representations of type material. This paper explores the potential of micro-computed tomography (X-ray microtomography). In contrast to SEM, the advantage of micro-CT is that the examination of material is fast and gentle, the samples remain undamaged and are available for further investigations (Paterson et al. 2014). Micro-CT scanning is becoming a more widely used technique for the identification of new species, e.g., within the Trichobranchidae (Parapar & Hutchings 2015;Parapar et al. 2016a, b), and Cossuridae (Parapar et al. 2018b). Within the Ampharetidae this technique has only been utilized to examine the internal anatomy of Ampharete santillani (Parapar et al. 2018a). We used micro-CT scanning to obtain a closer insight of the anterior end, and were able to describe the prostomium and the arrangement of the branchiae (compare Fig. 5A-D). These characters were not visible using SEM or light microscopy, due to the bad condition of the specimens. Furthermore, we observed small paleae on the outer edge of the semicircular arrangement of the paleae (compare Fig. 5B). We could not clarify the purpose of these small paleae, which differed in shape and size to the remaining paleae. One assumption may be a growing state, based on the place where new paleae are expected (Tilic et al. 2015). To increase the image quality and reduce the examination time we freeze-dried our sample and cut off the posterior part. Due to the low number of individuals and poor condition, we did not consider a second micro-CT scan for this study.
Terebelliformia, including Ampharetidae, is one of the most species-rich groups in Polychaeta, with around 1100 described species and a notable ecological and morphological diversity Eilertsen et al. 2017;Horton et al. 2021).
In addition, currently generic relationships within the Ampharetidae and the relationships of species within a genus, such as in Anobothrus, have not yet been clarified (Reuscher et al. 2009). The morphological descriptions of Anobothrus species are challenging, due to the high variation of modifications and the presence of many morphologically similar, small-sized species. In recent years, genetic analysis has been a useful tool to identify many cryptic and pseudocryptic polychaete species and record a higher diversity than described by morphology alone (Nygren et al. 2018). In this study, we could not perform genetic analyses due to preservation in 4% formaldehyde solution. To still achieve a detailed and unambiguous description for quantitative aspects of this new species, we carried out a multidisciplinary approach: traditional light microscopy with methylene blue and ShirlastanA staining to identify macromorphology (e.g., appendages, glands, ciliary bands), SEM to detect micro-morphology (e.g., gland pores, structure of chaetae and uncini), and micro-CT for internal structures and external characters (e.g., paleae, branchial arrangement).

Distribution and ecology
A total of twelve individuals was found from the sampled shelf regions ( (Fauchald 1972;Hilbig et al. 2000;Jirkov 2009;Imajima et al. 2013), whereas A. amourouxi was described from the North Eastern Atlantic (Bonifácio et al. 2015 However, nothing is known about the habitat of the species of Anobothrus in terms of grain size or food availability parameters so far. We found no specimens of A. konstantini Säring & Bick sp. nov. at sites with higher fresh food input (Chla) and organic carbon (TOC), such as in the northwestern Weddell Sea, or in predominantly silty sediments (Table 1). Only general functional traits of Ampharetidae are known from the literature (Jumars et al. 2015: supplemental table A). According to this information, all genera within the Ampharetidae are characterized as discretely motile, tube-dwelling, surface-deposit feeders that use their tentacles to feed on microorganisms and particles. Combined with information on its general functional traits, we can assume that A. konstantini Säring & Bick sp. nov. has a preferred habitat with lower silt and higher sand content in the sediment and a lower content of fresh detritus on the surface of the sediment in the Southern Ocean.
This study is part of a larger ecological study (Säring et al. submitted) with a set of different environmental parameters, in which 857 polychaetes from 31 families were collected. Thirty-nine specimens were identified as Ampharetidae (4.5%), twelve of which belong to Anobothrus konstantini Säring & Bick sp. nov. It seems that the Ampharetidae have a somewhat opposite distribution to that of other deposit feeders, such as Maldanidae and Paraonidae, which are mostly subsurface feeders and are very abundant in the North-Western Weddell Sea, whereas they are less abundant in the other four regions (Säring et al. in prep.).
Combining taxonomic studies with the quantitative description of environmental parameters and / or functional traits can contribute to a better understanding of species distribution and provide the basis for species distribution modeling (e.g., Meißner et al. 2014). Most species descriptions, especially for small invertebrates, only include information on depth range and geographic distribution. Describing a new species including quantitative information about its habitat, as we do here, allows quantitative relationship analysis and can be used to predict species distributions in hard-to-reach regions or for changing habitats such as those expected in the Southern Ocean (Jansen et al. 2018).