Umbellula pomona sp. nov., a new sea pen from Mar del Plata Submarine Canyon (Cnidaria: Octocorallia: Pennatulacea)

Sea pens (Cnidaria: Anthozoa: Pennatulacea) constitute a distinctive group of colonial marine invertebrates. They inhabit the world`s oceans, from shallow to deep waters. Studies about this group in Argentina are scarce, and no species have been described in the area in over a decade. Based on samples collected in Mar del Plata Submarine Canyon at about 3000 m deep we describe a new species of sea pen, Umbellula pomona Risaro, Williams & Lauretta sp. nov. This is a spiculate Umbellula that differs from other species of Umbellula with sclerites, by the number, development and distribution of the autozooids in its terminal cluster, as well as the shape of its axis. Molecular data also distinguishes it from other known species. Of the forty-three described species approximately ten are considered valid for the genus Umbellula, four of them are registered for the South Atlantic Ocean and only three are described for the Antarctic region. Since sampling efforts in this area have been scarce, the number of species of sea pens from the region is likely to increase substantially in the coming years.


Introduction
The Mar del Plata Submarine Canyon is located at the continental margin of Argentina at about 38º S latitude. The geomorphology of the external shelf and the submarine canyon of the Argentinian continental margin are strongly infl uenced by the Malvinas Current, a branch of the Antarctic Circumpolar Current that runs towards the northeastern region of the Argentinian continental margin (Piola & Matano 2001). The Malvinas Current transports cold subantarctic water and collides with the Brazil Current that carries warm waters along the continental slope of South America. This collision generates one of the most energetic regions of the world ocean (Piola & Matano 2001).
Although submarine canyons are known to be hotspots of benthic biomass and are globally numerous, these environments are very poorly sampled (Del Río Iglesias et al. 2012), therefore it is very important to study their biodiversity. Based on the specimens collected during the expeditions to the canyon (2012 and 2013), several new records and new species of Cnidaria Verrill, 1865, Mollusca Linnaeus, 1758 and Echinodermata Bruguière, 1791 have already been published (Cerino & Lauretta 2013;Martinez et al. 2014;Farías et al. 2015;Signorelli & Pastorino 2015;Maggioni et al. 2016;Pastorino & Sánchez 2016;Pastorino 2016Pastorino , 2019Martinez & Penchaszadeh 2017;Pereira & Doti 2017;Bernal et al. 2018). Here, we describe a new species of Umbellula from Mar del Plata Submarine Canyon based on specimens collected between 2934 m and 3282 m deep, using both morphological and molecular data.
The general morphology of the specimens, the distribution of sclerites and the shape of transverse sections of the axis were studied by the naked eye and using a stereoscopic microscope. For the sclerites' shape we followed the nomenclature of Bayer et al. (1983). Scanning electron microscopy (SEM) was used to examine the shape and length of the sclerites from the tentacles, polyp body and rachis of the colonies and the shape of the axis in transverse section. All measures of the peduncle (when possible), rachis, polyp's body and tentacles were done with a digital caliper and correspond to the holotype. Photographs of the colonies were taken using a digital SLR Nikon D800 camera with a Nikkor 60 mm F2.8 macro lens. To separate and prepare the sclerites of rachis, polyp's body and tentacles to examine them by SEM, a portion of each tissue was cut and treated with diluted sodium hypochlorite (commercial bleach) for fi fteen minutes and then washed with distilled water. Finally, the residual of water was evaporated with ethanol 96% and the sclerites were placed on SEM tubs; for the axis sections, a portion of it was cut and all soft tissue was retired, then the same protocol of the sclerites was followed.

Molecular data
Small tissue fragments were obtained from the holotype of Umbellula pomona sp. nov. and one paratype, both preserved in 96% ethanol. The samples were preliminarily washed with deionized water, allowing removal of ethanol. Total genomic DNA was extracted following the salting-out method (Miller et al. 1988). Following previous published works (Dolan et al. 2013;Kushida & Reimer 2019) we amplifi ed two mitochondrial regions: mtMuts (France & Hoover 2002;Sánchez et al. 2003) andND2 (McFadden et al. 2004). The primer´s sequences and thermocycling profiles used to amplify each target gene are described in Table S1 (Supplementary File 1).
Amplifi cations were carried out in 20 μl reaction mixtures containing 1 × PCR buffer mix (the buffer includes dNTPs and MgCl 2 ), 0.2 μM of each primer, 1 U of Taq polymerase (MyTaq DNA Polymerase, Bioline), 4 μg of Bovine Serum Albumin (BSA), 2 μl containing < 10 ng template DNA, and brought to fi nal volume with dH 2 O. The PCR reactions were performed in a T100TM thermocycler (Bio-Rad, USA), with negative controls included to verify the absence of contamination. The PCR products size was confi rmed via 1.5% agarose gel electrophoresis. PCR products were purifi ed and sequenced in Macrogen, Korea.

Molecular analysis
We added our sequences to the data set used in Dolan et al. (2013) (Table 1), since we are dealing with a deep-sea species of Umbellula (which is the target genus of this work) and no new data on this genus was added in Kushida & Reimer (2019). All the sequences were downloaded from GenBank, although the mtMutS sequence of Anthoptilum sp. (KF313832) based on a primnoid (as noted by Kushida & Reimer 2019), so we did not include this sequence in our data matrix. The new sequences were edited using the software Geneious ver. 5.6.7 and checked using BLAST (Altschul et al. 1990) to rule out contamination and to confi rm gene identity. The concatenated data set consisted of 41 taxa and 1257 bp. Sequence alignment was performed using MAFT (Multiple Alignment using Fast Fourier Transform) ver. 7 (https://mafft.cbrc.jp/alignment/server/) using L-INS-i strategy and default parameters. Trees were built using Bayesian analysis (BA) and maximum likelihood (ML). To determine the evolution model (GTR+G and GTR+I+G for mtMuts and ND2, respectively), we implemented the Akaike information criterion (AIC) in PartitionFinder 2 software (Lanfear et al. 2016). Ellisellids were chosen as outgroup (Dolan et al. 2013;Kushida & Reimer 2019). The Bayesian tree was built using Mrbayes (Ronquist et al. 2012) on Cipres science gateway (Miller et al. 2010): number of runs: 2, number of chains: 4, number of generations: 100 000 000, chain sample frequency: 10 000. The fi rst 25% of each search was discarded (burninfrac = 0.25). To ensure that the Markov chains reached stationarity (effective sample size values over 200) we used the software Tracer ver. 1.7 (Rambaut et al. 2018). Maximum likelihood analyses were performed using PhyML ver. 3.1 (Guindon et al. 2010) for each individual gene and as a complete set. Bootstrap support was calculated based on 1000 rounds. Rachis long and slender. Axis quadrangular to round in cross section. Autozooids restricted to the distal terminus, usually forming an umbellulate cluster. Siphonozooids are present on the rachis at the base of the autozooids or on bare parts of the rachis. Sclerites either present in peduncle, rachis, and terminal cluster, or totally absent. When present, sclerites are rods, spindles or needles, three-fl anged round in cross-section.

Distribution
Cosmopolitan, from 250 m to over 6200 m deep (Williams 2011).

Remarks
Since sclerites were found in the rachis of U. pomona sp. nov., we added this character to the family diagnosis.

Remarks
Although in his revision of the family, Broch (1958) synonymized Umbellula dura Thomson & Henderson, 1906 with Umbellula durissima Kölliker, 1880 and Umbellula rosea Thomson & Henderson, 1906 with Umbellula thomsoni Kölliker, 1880, we consider that, based on the original descriptions and images of U. dura and U. rosea, those species have important morphological characters to compare with our specimens.

Differential diagnosis
Umbellula pomona sp. nov. is a spiculated Umbellula with three autozooids in its terminal cluster, a central well-developed polyp and two lateral, symmetric and smaller ones. It presents large sclerites in all its tissues as well as siphonozooids all along the rachis. Its central axis is circular in cross section all along its extension, and does not vary throughout the colony.

Etymology
The species is named after the birthplace of the fi rst author (JR), Pomona (Río Negro, Argentina). The word 'pomona' is used as a noun in opposition. The colony looks rugous in all its extension, especially the terminal cluster of autozooids. The color of the polyps, rachis and the peduncle is white or light yellow (preserved). The holotype is 214 mm in length. It has a terminal cluster of three autozooids with tentacles, one central and larger polyp growing on the distal-most region of the rachis, and two smaller but well-developed lateral polyps growing at the base of the central one (Fig. 2). The central polyp is 25.3 mm in length and the two lateral polyps' lengths are 3.0 mm and 2.6 mm. The tentacles of the two types of polyps are larger than its body, the measures are 12.3 mm for the central autozooid, and 1.1 mm and 1.7 mm for the lateral ones. The axis is 178 mm in length and it is circular in cross-section, approximately 0.74 mm in diameter. The rachis is approximately 0.8 mm in diameter in the middle zone between the peduncle and the terminal cluster. The peduncle is 8.68 mm in length, and its appearance is soft and smooth. The autozooids of the cluster grow with a kind of orientation determining a 'dorsal' region where the axis inserts and a 'ventral'  region towards which the polyps come together (Fig. 2). The siphonozooids are numerous and resemble small white dots, and are distributed all along the rachis from the base of the autozooids to the middle of the rachis. These polyps are circular and inconspicuous; and have a diameter of about 393 ± 93 μm (299-486 μm, N = 36).

Variability
Of the three paratypes (A, B and C), one (paratype C) is signifi cantly smaller than the others, so we consider it could be a juvenile (Fig. 8). The color of the three colonies is white or light yellow when preserved, all of them have three autozooids with the same grade of development as the holotype (as it can be seen in Fig. 2B) and its central axis is round in all its extension (Fig. 3). The large paratypes (A and B) lack their peduncle. Their general aspect is just like the holotype, they are rugous in all their length because of the presence of conspicuous sclerites in the autozooids and rachis (Fig. 2B). The total length of the large paratypes is 134 mm and 231 mm, their central autozooids are 36 mm and 34 mm long (length of the tentacles: 20 mm and 12 mm, length of the columns: 16 mm and 22 mm, respectively) while the dimensions of the lateral ones are 4.8 mm and 5.6 mm in one paratype and 6.4 mm and 7 mm in the other. Their rachis are 97 mm and 200 mm in length and their diameters are 0.9 mm and 2.2 mm at thier widest sections. Their axis' diameter is 0.83 mm and 1.3 mm. Their siphonozooids are 313 μm and 370 μm (mean) in diameter and look like the siphonozooids of the holotype. Finally, the sclerites of these paratypes look alike and have similar sizes as the sclerites of the holotype. The juvenile paratype's (C) total length is 110 mm, its central autozooid is 7.9 mm in length (column and tentacles are 4.4 mm and 3.4 mm long, respectively), and the length of the lateral ones is 2.9 mm (column and tentacles are 1.4 and 1.5 mm in length, respectively). The peduncle's length is 4.6 mm and the rachis' 95 mm, while its diameter is 0.41 mm at its widest section. Its central axis is 0.35 mm in diameter. Finally, the siphonozooids of this paratype are tiny spots with the same aspect and distribution as those on the holotype and the largest paratypes.

Phylogenetic analysis
Both phylogenetics reconstructions (i.e., BA and ML) agree in the basic topology of the trees (for simplicity we only show BA). Both type specimens of U. pomona sp. nov. were grouped together with low support values within the same group (possibly because we only have one gene sequence for each species). Umbellula spp. were recovered in two clusters, Umbellula clade I including most of the included Umbellula species and a second one (Umbellula clade II) including only U. monocephalus, Umbellula pomona sp. nov. and Umbellula sp. 2 from Dolan et al. (2013) (Fig. 9).
As currently defi ned, our specimens agree with the current set of characters assigned to Umbellula. They have a long, slender rachis, a cluster of autozooids on their distal-most part and numerous siphonozooids all along the rachis, in addition to conspicuous, rod-or needle-like sclerites. Chunella Kükenthal, 1902  and Amphiacme Kükenthal, 1902 are other similar genera, but have several polyp clusters disposed in intervals along the rachis and do not have sclerites (Kükenthal 1915).
From a morphological point of view, there are two major species groups in the genus Umbellula, based on the presence/absence of sclerites. Up to date, of all described species, fi ve are spiculate (of which only three are probably valid) whose morphology needs to be compared: U. thomsoni, U. durissima, U. dura, U. rosea and U. monocephalus. None of them share the diagnostic set of characters (arrangement of the polyp cluster, form of the central axis and sclerite sizes) of our specimens (Table 2). Umbellula pomona sp. nov. has a reduced number of polyps, forming a terminal cluster with one central autozooid and two smaller, lateral ones growing from its base, a cylindrical central axis, and the sizes of thier sclerites range from 1446 μm to 269.1 μm, depending on location in the tissues. Umbellula thomsoni has been reported with three to ten autozooids in its terminal cluster growing like a fl ower or an umbrella, with a similar grade of development among them. The axis of this species differs in its shape in cross section, as near the cluster it is quadrangular, while it becomes cylindrical near the peduncle. Its sclerites vary from approximately 1300 μm (pinnules and tentacles) to less than 300 μm (rachis and body wall of the polyps) (Kükenthal 1915;Williams 1990). Umbellula monocephalus has only one polyp in the distal region of the rachis (Pasternak 1964;Grasshoff 1972;Tiefenbacher 2001). Umbellula durissima has   Ocean / 3300 * Some authors consider U. dura as a synonym of U. durissima and U. rosea as a synonym of U. thomsoni (Broch 1958) numerous polyps forming an apical cluster of three autozooids, one terminal and well developed, two lateral and two or three less developed ones (without tentacles) below the principal cluster. Its central axis is cylindrical in all its length. The sclerites vary in length from 2800 μm (body wall of the polyps and tentacles) to approximately 200 μm (rachis), additionally, this species does not have siphonozooids along the rachis, but they are grouped between the autozooids in the terminal cluster (Kölliker 1880;Thomson & Henderson 1906;Kükenthal 1915). Umbellula dura has three autozooids growing at the same level forming a kind of circle in which the central autozooid is smaller than the other laterals in juvenile states, but in more developed states have six autozooids in total, four large and two very small, all disposed in three sets. Its central axis is cylindrical in cross section and the sclerites vary in length from 1500 μm to 300 μm in the autozooids and from 140 μm to 100 μm in the rachis, also, in these tissue has X-shaped sclerites (Thomson & Henderson 1906), that are absent in U. pomona sp. nov. Umbellula rosea is the more similar species to U. pomona sp. nov., but it can be differentiated because although it has a central terminal polyp and two pairs of lateral autozooids below it, the axis of U. rosea is quadrangular in cross section throughout (Thomson & Henderson 1906) while the axis of U. pomona sp nov. is cilindrycal in all its extention. In addition, Kükenthal (1915: 54), and Thomson & Henderson (1906: 5) describe the presence of small, thick, oval sclerites in the peduncle of U. rosea, which are absent in U. pomona sp. nov. Finally, some paratypes of U. rosea have just a few sclerites in their autozooids' tissue of around 250 μm long (Thomson & Henderson, 1906) while U. pomona sp. nov. has very numerous and conspicuous sclerites in these tissues, from 1446 μm to 543 μm long. Some paratypes of U. pomona sp. nov. have oocytes, which confi rms that these are adult specimens. In consequence, we describe here a new species for our specimens. A summary of all diagnostic characters is presented in Table 2.
Umbellula pomona sp. nov. inhabits the deep region of Mar del Plata Submarine Canyon, at a depth of about 3000 m. Sixty-four sampling stations were established during three expeditions, from 200 m to 3500 m deep, over 150 specimens of sea pens were collected but only four specimens of the new species were found. This low number of specimens is not rare because in the study area, many species report a very low abundance. Many deep-sea invertebrate species from Mar del Plata Submarine Canyon have been registered / described based on a few (and even only one) specimens (Pastorino 2016;Lauretta & Penchaszadeh 2017;Martinez et al. 2019). Since sampling efforts in this area have been almost nonexistent until a few years ago, the number of species of sea pens (and other invertebrates) from Mar del Plata Submarine Canyon is likely to increase substantially in the coming years.

Phylogenetic position of Umbellula pomona sp. nov.
In all the phylogenetic reconstructions made, both specimens of U. pomona sp. nov. were grouped together (with low support, since we could only amplify one different gene for each specimen  Dolan et al. (2013), 'Umbellula clade II' was characterized by species with sclerites and a round axis, characters also presents in U. pomona sp. nov., which supports this clade from the morphological data. If both clades were to be separated in two genera, 'Umbellula clade I' would retain the generic name Umbellula (since it includes the type species of the genus) and a new genus would have to be proposed for 'Umbellula clade II'. Pending a complete revision of the nominal species assigned to Umbellula, we prefer to be conservative and include our species within Umbellula until a such revision is done and a decision based on molecular and morphological data can be made.

Valid species within Umbellula
Up to date, there are 43 nominal species within Umbellula (Williams 1995;Cordeiro et al. 2019;this paper). At least nine species were recognized as probably valid by Williams (1995)  Umbellula pellucida Kükenthal, 1902 No North Indian Ocean Kükenthal 1902Kükenthal , 1915Broch 1958;Williams 1995 Umbellula spicata Kükenthal, 1902 No Indian Ocean Kükenthal 1902Kükenthal , 1915Broch 1958;Williams 1995 Umbellula monocephalus Pasternak, 1964 Broch (1957) in his revision of the family considered U. thomsoni and U. leptocaulis Kölliker, 1880 as the same species. In this work, we consider the valid species according to the revision made by Broch (1957) and López-González & Williams (2011). Clearly, a complete revision of Umbellula spp. is needed, probably using both morphological and molecular data. Table 3 shows the valid species of Umbellula with their known distributions considered in this paper.

On the distribution of Umbellula spp. of the South Atlantic Ocean (SAO)
Umbellula is a cosmopolitan genus ( Table 3). Specimens of this group have been reported from all over the world, from the equator to the poles. Reports of Umbellula in the South Atlantic are limited; according to the papers made by Broch (1958) and Williams (1995), only four species were reported in the SAO: U. durissima, U. monocephalus, U. thomsoni and U. lindahli, of which only U. thomsoni and U. lindahli correspond to the southwestern Atlantic Ocean (SAO), where our specimens are located. The diversity of the group in Antarctic waters is also low, with three species reported (U. durissima, U. lindahli and U. carpenteri) (Kölliker 1880;Broch 1958;López-González & Williams 2011). As mentioned before, the conditions in the sampled area are strongly infl uenced by the Malvinas Current and the Brazil-Malvinas Confl uence, which may explain the presence of Antarctic species in the SAO deep sea. Given the depths where our specimens came from (2934 m and 3282 m), it is possible that this record is (or is near to) the southern limit distribution of the species, since in the confl uence area at that depth the North Atlantic Deep waters are present (see Voigt et al. 2013), which run southward from the northern hemisphere and in the confl uence area divide the Circumpolar Deep Water vertically in two, staying at a depth of 2000-3000 m. A similar situation has also been proposed in the case of the gastropod Theta lyronuclea (Clarke, 1959), previously reported from the North Atlantic Ocean and recently found in the same station as U. pomona sp. nov. (Sánchez & Pastorino 2020).

Data availability
The datasets generated during the current study are available from the corresponding authors on reasonable request.