Three new species of Levinsenia Mesnil, 1897 (Annelida: Paraonidae) from shallow and deep waters of Southwestern Atlantic Ocean

Three new species of Levinsenia were collected during a benthic survey, from 10–3,000 m deep, in Espírito Santo Basin, off the southeastern Brazilian coast. These species are L. paivai sp. nov., L. blakei sp. nov. and L. lesliae sp. nov. Members of L. paivai sp. nov. are recognized by the presence of nine pairs of well-developed and heavily ciliated branchiae, those of L. blakei sp. nov. are characterized by the presence of three pairs of small branchiae, and those of L. lesliae sp. nov., by the absence of branchiae and presence of notopodial transitional chaetae. These new species are described herein and compared to the most similar congeners. These are the first new species of Levinsenia described from off the Brazilian coast.


Introduction
Among the annelids inhabiting soft substrates, individuals of the family Paraonidae Cerruti, 1909 [1] are among the more abundant in shelf and slope depths [2][3][4]. This family has extraordinarily high species richness in deep-water habitats [5,6] although a few species are also found in intertidal environments [7]. Paraonids are small burrowing worms, mostly between 2-4 cm long and 0.1-0.2 mm wide [2].
The family Paraonidae currently includes about 155 described species, with a worldwide distribution [8,9]. Due to the utilization of finer (0.5 mm mesh) sieves and the numerous projects focused on diversity in deep-sea environments [10], several new species have been described recently [11][12][13].
Strelzov [2], in a major review of the Paraonidae, noticed remarkable differences among specimens of the species assigned to Paraonis. The author kept P. fulgens (Levinsen, 1884) [21] and P. pygoenigmatica Jones, 1968 [25] in Paraonis, and split the remaining species among Sabidius   [26] and Tauberia Strelzov, 1973 [26], members of Sabidius having a prostomium with a trilobate anterior margin, while the animals belonging to Tauberia have cirriform notopodial postchaetal lobes. Tauberia was considered as a synonym of Levinsenia, according to the rules of the ICZN [5].
Members of Levinsenia are abundant in shelf and slope depths, although some species can also be found in shallow waters [27]. The external morphology of these animals is remarkably simple, in comparison to those belonging to the other genera in this family, and therefore there is a limited number of diagnostic characters [2], but all Levinsenia have: (1) prostomium lacking antenna; (2) presence of terminal sensory organ (palpode) on prostomium; (3) when branchiae are present, there are always more than 4 prebranchial segments; (4) pygidium tapering, with two anal cirri. Also, the use of modern equipment, such as the Scanning Electron Microscope (SEM), has been very useful in the search for new morphological characters, as well as the methyl green staining, which provided useful species specific staining patterns [16,18].
Specimens of Levinsenia have been found in all oceans. At least some species of the genus have been reported for polar regions, one for the Arctic Ocean and two for the Southern Ocean. In the Pacific, 12 species have been reported, seven for the northern Pacific and five for the southern. The Atlantic Ocean has the highest number of species of for the genus, with 10 species reported from the northern Atlantic and five from the southern [5]. Regarding the Brazilian coast, previous studies on paraonids registered the presence of six genera and 38 species, most of them from shallow waters [28]. Few systematic studies have been conducted on Brazilian paraonids [11,[29][30][31], so most (62%) of the previous records for the family in Brazil come from gray literature and papers with an ecological approach, in which specimens descriptions and illustrations are not given [28]. For this reason, most of these records are questionable.
The aim of this study is to describe three new species of Levinsenia based on specimens collected from off southeastern Brazil, state of Espírito Santo, between 19˚3'S 37˚44'W and 211 0'S 38˚28'W. Out of the 38 species of paraonids previously reported for the Brazilian coast, three belong to Levinsenia [28], although the reliability of this records is questionable, as discussed above. So the present paper raises to six the number of species of this genus occurring off Brazil.

Material and methods
The specimens were collected during a project ( 3,000 m) and two different canyons (canyon Rio Doce and canyon Watu Norte) (4 stations each, both 150-1,300 m) (Fig 1). Each of these four environments was sampled twice, in summer and again in winter. Samples were collected in three replicates at each station, using either a 294 L van Veen grab or a 125 L box-corer. The sediment was then sieved in a 0.5 mm mesh, fixed in 10% sea-water formalin, and preserved in 70% ethanol. Identifications were based on specific morphological characters. Specimens were examined using a stereomicroscope, compound light microscope and scanning electron microscope (SEM). For the SEM, specimens were dehydrated in a series of progressively stronger concentrations of ethanol (70-100%), then critical point dried (LEICA EM CPD300), coated with~35 nm of gold and examined and photographed at the Laboratório de Microscopia Eletrônica, Instituto de Biociências, Universidade de São Paulo (IB-USP). Also, some specimens were stained with methyl green, to investigate for species specific staining patterns.
Descriptions were made based on the holotypes and variation observed among paratypes is provided inside parentheses, after each corresponding measurement of the holotype. Complete specimens were measured; width was always taken at chaetiger 4. Maps were created using the Free and Open Source QGIS [32].

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code, from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:2-BBA7519-EFED-4B39-A67A-AE3CF11765FA. The electronic edition of this work was published in a journal with an ISSN and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.
Methyl green staining pattern. Prebranchial region staining limited to postchaetal areas; as solid band on branchial and 2-3 postbranchial chaetigers.
Habitat. Found in substrates with a high percentage of sand (63%), some amount of mud (29%) and a small percentage of pebbles (8%); water temperature 14-24˚C; between 28-150 m deep.
Individuals of L. paivai sp. nov. have branchiae much longer than those of members of L. flava, L. kantauriensis and L. tribranchiata, in which branchiae do not exceed 0.10 mm in length [2,10,16], while in members of L. paivai sp. nov. even the smallest pair of branchiae is longer than 0.10 mm (Table 1).
Individuals belonging to L. multibranchiata and L. pycnobranchiata have more than 20 pairs of branchiae [34,35], while members of L. paivai sp. nov. have only 6-9 pairs. Animals belonging to L. paivai sp. nov. also share similar morphology of branchiae with individuals of L. demiri. However, members of this latter species have strongly curved and slightly protruding modified spines [16], while among members of L. paivai sp. nov. such spines are slightly curved and strongly protruding.
Individuals of L. marmarensis also share with members of L. paivai sp. nov. similar number of pairs and morphology of branchiae, 7-8 pairs in members of L. marmarensis, 6-9 pairs in L. paivai sp. nov., ciriform in both. However, branchiae are ciliated among members of L. paivai

PLOS ONE
Three new Levinsenia from southwestern Atlantic sp. nov. and smooth in members of L. marmarensis. In addition, members of L. paivai sp. nov. have branchiae 0.13-0.36 mm long, progressively longer to posterior pairs, except for last two pairs, while in individuals of L. marmarensis the branchiae have a similar length throughout, all pairs 0.24 mm long. Furthermore, members of both species also differ in regards to the modified spines, which are strongly curved in the latter species [16]. Members of L. paivai sp. nov. differ from those of L. oculata in regards to the morphology of notopodial postchaetal lobes on prebranchial chaetigers, which are cirriform and progressively elongate among specimens of L. oculata, while they are tubercular and poorly developed in specimens of L. paivai sp. nov. [35].
Finally, specimens of L. oligobranchiata share with members of L. paivai sp. nov. similar distribution (6-9 pairs, among members of both species) and morphology of branchiae. However, those animals differ in branchial size, as among members of the former species branchiae are short and of even length, 0.18 mm long throughout, while individuals belonging to L. paivai sp. nov. have branchiae progressively longer, except for the last two pairs. They also differ in regards to the shape of notopodial postchaetal lobes, which are distinctly short to inconspicuous on posterior segments, in individuals of L. oligobranchiata [2], and well developed among those of L. paivai sp. nov.
Prebranchial chaetigers of specimens of Levinsenia paivai sp. nov. were observed in two different states; (1) when dorsal side of prebranchial chaetigers is not inflated, a longitudinal middorsal groove is formed (Fig 2A); (2) when dorsal side of prebranchial chaetigers is inflated, a dorsal crest is formed (Fig 2C). We believe this difference is due to the moment of fixation of the specimen. Probably the movements of an inflated anterior region would help the worm burrow through the sediment.
Etymology. This species is named after Dr. Paulo Cesar de Paiva, a well-known Brazilian polychaetologist, expert on systematics, benthic ecology and marine phylogeography of polychaetes, and also a great professor, who continuously inspires and contributes to the education of biologists, including both first authors of this paper. Comparative material examined. Levinsenia reducta (Hartman, 1965)  14 mm (0.12-0.14 mm)]; cylindrical terminal sensory organ (palpode), sometimes everted; cheek organs, eyes and median antenna all absent; patches of cilia present on ventral and lateral sides ( Fig  5C-5E); pair of nuchal organs as ciliated longitudinal slits. Anterior buccal lip with 2 longitudinal folds, posterior buccal lip with six longitudinal folds, extending to middle of chaetiger 1 (Fig 5E). Ventral mouth, saclike pharynx everted in some specimens.
Neurochaetae of prebranchial and branchial chaetigers all capillary. Postbranchial region with two types of neurochaetae: capillary and modified, strongly curved, stout spines, with expanded shaft and fringe on convex side, slightly protruding from neuropodia (Fig 5G). Modified spines first appearing on chaetiger 22. Prebranchial chaetigers with 12 capillary neurochaetae each, arranged in two rows; branchial chaetigers with 16 capillary neurochaetae each, arranged in three rows; postbranchial chaetigers with 4-7 modified spines, thick, strongly curved, with distinct fringe on convex side, arranged in posterior row, and three accompanying capillary chaetae slenderer than capillary chaetae of preceding regions, arranged in anterior row (Figs 5H and 6C). Pygidium unknown.
Methyl green stain. Staining pattern conspicuous, as a solid band per segment, on chaetigers 9-21.
Out of those, members of all L. acutibranchiata, L. kirbyorum, L. kosswigi, and L. reducta are readily distinguished from individuals of L. blakei sp. nov. in having more than 10 branchiate chaetigers [2,15,16,36], while those of the new species only have 4 branchiferous segments.
Specimens of L. antarctica are similar to members of L. blakei sp. nov. in having few branchiferous segments, only three among members of that species, and also in regards to the morphology of branchiae. However, these animals have seven prebranchial segments, while individuals of L. blakei sp. nov. have 8 or 9. In addition, the notopodial postchaetal lobes of the branchiate segments are cirriform among members of L. antarctica [2], while in specimens of L. blakei sp. nov. those lobes are tubercular.
Members of L. brevibranchiata differ from members of L. blakei sp. nov. in having six pairs of branchiae and also in the morphology of the modified spines, which are slightly curved, with no expanded shaft in those animals [2], instead of strongly curved, with expanded shafts, as in members of L. blakei sp. nov.
Individuals of L. canariensis, as said above, have more than seven prebranchial chaetigers. However, those animals have branchiae beginning far more posteriorly, at the posterior part of the body, which is a very unusual branchial distribution for members of this genus [37].
The first six chaetigers of specimens of Levinsenia blakei sp. nov. were observed in two different states; (1) when dorsal side of first six chaetigers is inflated, a dorsal crest is formed (Figs 5A and 6A); (2) when dorsal side of first six chaetigers is not inflated, a longitudinal mid-dorsal groove is formed (Fig 5B). We believe this difference is due to the moment of fixation of the specimen. As discussed above for L. paivai, this is probably due to burrowing movements.
Etymology. This species is named after Dr. James Blake, a very important researcher on biology and systematics of polychaetes during the last decades, continuously providing valuable contributions to the knowledge of several families of this group, including Paraonidae.
Methyl green stain. First five chaetigers weakly staining, only on postchaetal areas; on chaetigers 6-12, strong staining pattern, as a solid band on each segment.
Members of L. hawaiiensis are easily distinguished from specimens of L. lesliae sp. nov., as they have bifid modified neuropodial spines and three pygidial cirri, instead of a pair. Both these characters are unusual for members of Levinsenia and it is debatable whether the species belongs in this genus [10].
Members of L. uncinata, in addition to the absence of branchiae, closely resemble those of L. lesliae sp. nov. in having tubercular notopodial postchaetal lobes on anterior chaetigers, and on the beginning of the modified spines, chaetiger 16 among members of L. uncinata, chaetiger 14 in specimens of L. lesliae sp. nov. Members of these species differ, however, because individuals of L. uncinata lack notopodial transitional chaetae and their neuropodial modified spines strongly protrude from neuropodia and are slightly curved [2,36], while among members of L. lesliae sp. nov. those spines barely protrude from parapodia and are strongly curved, with expanded shafts.
The first five chaetigers of specimens of Levinsenia lesliae sp. nov. were observed in two different states; (1) when dorsal side of first five chaetigers is not inflated a longitudinal mid-dorsal groove is formed (Figs 7A and 8A); (2) when dorsal side of first five chaetigers is inflated, a dorsal crest is formed (Fig 7B). We believe this difference is due to the moment of fixation of the specimen and reflects the burrowing movements of the worm, as discussed for both the species described above.
Although Levinsenia lesliae sp. nov. was found in a wide bathymetric range there is a considerable difference in their distribution between summer and winter samples. In summer samples, specimens were found between 20-1,053 m deep, even though samples were taken until 3,000 m deep. During the winter, specimens have a wider bathymetric range (20-3,020 m deep), being noticeable that only in two, out of the eight slope transects, individuals of L. lesliae sp. nov. were found below 1,054 m. Furthermore, few specimens were found in those deeper stations, totaling only 21 individuals collected below 1,054 m ( Table 2). A careful morphological examination was performed among individuals from shallower (until~1,000 m deep) and deeper (below~1,000 m deep) stations, but no significant difference was observed.
Etymology. This species is named after Leslie Harris, for all her contributions to the knowledge of polychaetes. An extraordinarily kind person, who made possible for the first author to visit the Polychaete Collection of the Natural History Museum of Los Angeles County.

Discussion
The three new species of Levinsenia described in this paper showed different patterns of bathymetric distribution. Individuals of L. paivai sp. nov. have a shallow and restricted bathymetric range, not exceeding 150 m deep. A wider bathymetric range was observed for members of L. blakei sp. nov., which were found between 300-1,400 m, and even greater among members of L. lesliae sp. nov., which were collected between 20-3,020 m. Members of this latter species also showed the highest abundance, with 3,250 specimens collected during summer and 1,819 during winter; these animals were found in all the four sampling areas, with a remarkably high abundance at a particular spot in canyon Watu Norte, from which 2,749 specimens were obtained in summer and 1,449 in winter.
Recently, due to the modern techniques available, especially SEM, new morphological characters have been added to the genus Levinsenia, such as the patches of ciliation on the prostomium; notopodial sensory pores; bands of cilia across the dorsum, connecting branchiae within pairs; and details of pubescence on modified neurochaetae [18]. In addition to these characters, methyl green staining patterns seem to be a consistent species specific character [15,18], showing consistent differences between members of the three species on the distribution of chaetigers stained with a solid band.
Methyl green staining has become an useful tool for the systematics at species level in several groups of polychaetes, such as Spionidae, Capitellidae, Cirratulidae, Sabellidae and Terebelliformia [39][40][41][42][43]. We suggest a similar search for new morphological characters and new techniques, such as the use of methyl green, should be carried out for the other genera of Paraonidae.
This study is also important due to the area sampled, and when it was made. The region was strongly impacted by the collapse of a mining damn inland in Brazil, which released over 50 million cubic meters of mining tailings into Rio Doce, by the end of 2015 [44]. Those mining tailings ended up in the Atlantic through the estuary of Rio Doce, spreading all over the bottom of Espírito Santo basin [44], precisely the area investigated for this study. As the collections for this study were made before that event, it is a possible that the species described herein have become locally extinct, making it fundamental at this point to have a better knowledge of the fauna previously occurring in the area, to properly evaluate the impacts of that disaster. Decreases in diversity and species eveness of macrofaunal assemblages have already been observed following the accident [45].