Ascidiacea (Chordata, Tunicata) from Uruguay (SW Atlantic): checklist and zoogeographic considerations

The diversity of ascidians from the Southwestern Atlantic between 30°S and 40°S (southern Brazil, Uruguay and northern Argentina) remains as one of the poorest known of the West Atlantic. The objective of this work is to compile, analyze and discuss all published records of ascidians from Uruguay. They show the historical relevance of the studies performed by Herdman, Monniot F. and Monniot C. on ascidians collected at deep-sea stations by the HMS Challenger and the RV Atlantis II in the Argentine Basin. Total literature records include 38 ascidian species which are enumerated here for the first time. On the basis of the current knowledge, the ascidian fauna of Uruguayan waters encompasses: a) shallow-water species with temperate distribution (3 spp.); b) shelf and deep-sea species with Antarctic and Sub-Antarctic distribution (13 spp.); c) deep-sea species until now only collected off Río de La Plata (11 spp.); d) deep-sea species displaying a wide distribution (11 spp.). Only nine species have been recorded for the continental shelf; the remaining species were collected either from the slope (21) or the abyssal plain (5) or both deep-sea zones (3). Future research should be directed to record coastline and shelf species, assess the presence of exotic elements, and re-describe enigmatic species first described by Herdman (1882, 1886).


INTRODUCTION
Ascidians (Chordata, Tunicata) represent diverse and abundant components of benthic communities. They are present in all kinds of marine environments, with wide geographic and bathymetric distributions. Shenkar & Swalla (2011) estimated the global ascidian richness in about 3000 species, stressing the necessity of studies in areas still understudied and undersampled, such as the SW Atlantic. Investigations performed in the area in recent years have yielded new species (Lagger & Tatián 2013;Rocha et al. 2015) and further new data from redescriptions of poorly known ones (e.g. Maggioni et al. 2016). In the SW Atlantic, reports of invasive and cryptogenic species have also increased (Tatián et al. 2010;Schwindt et al. 2014;Skinner et al. 2016).
Ascidian diversity studies in this region have been mainly performed in the Patagonian shelf, South Argentina (e.g. Van Name 1945;Diehl 1977;Lagger & Tatián 2013;Taverna et al. 2018), and Brazil (e.g. Rocha & Costa 2005;Bonnet & Rocha 2011;Moreno et al. 2014). Rocha et al. (2012) presented a list of 461 species from shallow waters comprising the entire Atlantic Ocean. Despite these studies, the diversity of ascidians from the SW Atlantic coast between 30°S and 40°S (southern Brazil, Uruguay and northern Argentina) still remains comparatively less described, being the poorest known area in the region (Moreno et al. 2014).
In particular, faunistic knowledge of ascidians from Uruguayan waters mainly comes from the works of Herdman (1880;1881a;1881b;1886), Monniot C. & Monniot F. (1985a) and , who respectively reported the material collected by the HMS Challenger (1872-1876) and RV Atlantis II (1971) in the Argentine Basin. Therefore, most of these species come from the deep-sea; inner shelf ascidians from Uruguay have been poorly recorded. In fact, all six references summarized by ) (i.e., Millar 1969Juanicó & Rodríguez-Moyano 1976;Milstein et al. 1976;Cachés 1980;Obenat et al. 2001;Orensanz et al. 2002) for the inner shelf ascidian fauna correspond to unidentified or poorly documented records. Thus, as a first step for further knowledge of the scarcely known ascidian fauna of Uruguay, the aim of this work is to compile, analyze and discuss all the published records of ascidians from Uruguay as a basis for future research. The latter includes the analysis of ascidian aragonitic spicules as potential paleoecological indicators in the Uruguayan shelf (see e.g. Toledo et al. 2007;Sagular 2009;Shenkar & Swalla 2011;Sagular et al. 2017).

MATERIALS AND METHODS
Ascidian records come from the Uruguayan coast and the Uruguayan Exclusive Economic Zone (URY EEZ, Figure 1), including shallow coastal areas, outer shelf, slope and abyssal plain. The last two mentioned areas belong to the Argentine Basin. A complex oceanographic system develops in the area, involving the confluence of warm and cold marine currents and the freshwater input of La Plata River. A general oceanographic, geomorphological and sedimentological outline of the zone can be found in Scarabino et al. (2016) and Hanebuth et al. (2018).
An inventory of species was compiled from papers, books chapters, meeting abstracts and theses.
The current status of the all species was verified through literature and the World Register of Marine Species webpage (www.marinespecies. org). The order of the species list followed the criteria adopted by the latter resource (Shenkar et al. 2018a).
For each species the following information is provided: 1) original combination and reference eventually followed by references on the synonymy (when extensive), 2) geographic distribution, 3) references and bathymetric distribution in Uruguayan waters and 4) observations, that may include the reference(s) that cited the species in its current combination, comments and/ or discussions on the status of the species or the Uruguayan records.
References regarding the geographic distribution of littoral species include: a) general/major revisions containing comprehensive treatment of these and/or b) works extending considerably (i.e. thousands of km) the range of a given species. In the case of deep-sea species, we considered all available references.
Only one record (station 242 of the cruise 60 of the RV Atlantis II) that is located some miles off this zone is included here considering the imminent enlargement of the UEEZ. Station 320 of the HMS Challenger falls exactly on the maritime boundary between Argentina and Uruguay and, therefore, such species are considered as part of the faunistic inventory of both countries. On the other hand, one species recorded as collected "off Montevideo" (i.e. Styela schmitti Van Name, 1945, see also Millar 1960 andMonniot F. 1983 who also indicated "off Montevideo" or "off Uruguay") was actually collected in shallow waters (at ca. 20 m) off Cabo San Antonio, Buenos Aires Province, Argentina (36°42'S, 53°23'W) and therefore it is not included here. In fact, that is also the type locality of Molgula platana Van Name, 1945 (Albatross station 2764), which was never referred from Uruguay. Full data and mapping of the stations of HMS Challenger and RV Atlantis II from Uruguayan waters are provided (Table 1, Figure 1).
Record not yet substantiated (mentioned in abstract).
Observations: Van Name (1945: 445) indicates that the original description of this species is incomplete and thus fails to permit a correct classification. Rocha and Lambert (pers. com. 2013) agree with Van Name´s comments and indicate this species as a nomen dubium until the type and especially topotypic material is re/described. This is not Aplidium flavum Huitfeld-Kaas, 1896, a synonym of Aplidium glabrum (Verrill, 1871) (Shenkar et al. 2018b).

Aplidium sp.
Observations: Monniot F. & Monniot C. (1976: 630-631) and Monniot C. & Monniot F. (1985a: 8) recorded undeterminable material (much contracted isolated individuals, not colonies) of Aplidium sp. from RV Atlantis II, cruise 60, sts. 237, 245, 246 and 259 collected between 993 and 3343 m. We did not include this record in the total count for the area nor in Table 1 because it is impossible to assess whether it corresponds to one of the previous Aplidium species or to a different one.
Observations: collected at HMS Challenger st. 320, which is the type locality of this species (Herdman 1886: 194-195) and recently found and redescribed off Mar del Plata at 308 m depth, i.e., relatively close to the type locality (Maggioni et al. 2016: 181-185). Van Name (1945) included this species tentatively in the genus Synoicum, a denomination that Maggioni et al. (2016) confirmed.
Aplidium incrustans Herdman 1886, also described from material of the HMS Challenger st. 320, has been referred by Van Name (1945) as a possible junior synonym of S. molle although stating that both species are poorly characterized. Maggioni et al. (2016) rejected this synonymy based on a detailed comparison of branchial sacs. They propose to maintain both entities as separate species. This is not Psammaplidium incrustans Herdman 1891 with type locality in Port Stephens, New South Wales, Australia, a junior synonym of Aplidium solidum (Herdman 1891) (Kott 2005 (Lesson, 1830) Holozoa cylindrica Lesson, 1830: 439. For synonymy see Kott (1969: 29) and Monniot C. & Monniot F. (1983: 36).
Record not yet substantiated (mentioned in abstract).

Culeolus anonymus x x
Minipera tacita x x

Protomolgula bythia x x x
Molguloides cyclocarpa x x

Molgula pyriformis x x
Fungulus perlucidus x x
Record not yet substantiated (mentioned in abstract).

References and bathymetric distribution in
"There are many similarities between S. flava and my specimens of S. glans. The most characteristic are: the arrangement of the tentacles, the dorsal tubercle, the undulated dorsal lamina and the uncommon endostyle. Considering these similarities and absence of information about the gonads of S. flava, we conclude that it would be possible to consider this species identical as well as to oblonga as to glans. Therefore S. flava remains as a dubious species and therefore we consider it as a nomen dubium".

Synonymy of S. flava with Styela nordenskjoldi
Michaelsen, 1898 (see above comments under S. squamosa) proposed by Kott (1969; F. 1980; 1983; 1994).  (1969), using the name Styela nordenskjoldi, and thus they rejected the synonymy proposed by that latter author for all the three species of Styela described by Herdman from off Río de La Plata.
Geographic distribution: worldwide in temperate zones (Barros et al. 2009). Observations: the record of Traustedt (1882), which forms the basis of the record of the species of Van Name (1945) for Uruguay, is most probably based on a failed introduction, likely the hull of a ship. Although S. plicata has some resistance to estuarine conditions (Barros et al. 2009), Montevideo harbor lies within an estuarine zone unsuitable for ascidians without any further record of this or any other ascidian species. This species should be carefully observed/monitored, since it was dispersed worldwide by vessels, as it holds many of the different features to become invasive (Barros et al. 2009;Pineda et al. 2013).
Observations: Rocha et al. (2012) listed a total of five Botryllus species for the Atlantic Ocean, being B. schlosseri until now absent in Brazil but present in Argentina (Amor 1964;Orensanz et al. 2002;Schwindt et al. 2014). Botryllus schlosseri is a "species complex" consisting of five genetically divergent clades (named from A to E) that should correspond to five distinct cryptic species (Bock et al. 2012;Yund et al. 2015). Clade A is globally widespread, while clade E has been identified only along the European coasts. The remaining clades B-D are geographically restricted to few European localities.
Clade A, the most common and widespread species, experiences recurrent long-distance dispersion (probably human-mediated) and is highly invasive (Bock et al. 2012). Recently, Brunetti et al. (2017) redescribed clade A in detail based on morphological data and associated it with a "DNA barcode".
Populations of B. schlosseri from South America display high gene diversity. A limited number of genotypes probably founded the Pacific Chilean populations, while the Atlantic Argentinean population was repeatedly colonized by new genotypes. The South and North American populations of B. scholsseri showed extensive dissimilarities, suggesting two distinct clades. All clades were supported by COI and 18S (Ben-Shlomo et al. 2010).
Observations:  accurately stated that Culeolus is one of the most poorly described genus of Ascidiacea due to the lack of recorded specimens and to the almost exclusive consideration of the external aspect, while the anatomy is much more useful. Moreover, the evaluation of incoming material has revealed the existence of intraspecific variability in characters such as the position of the gonads and the structure of the postero-ventral crest (Sanamyan K.E. & Sanamyan N.P. 2002;postero-ventral arc, according to Kott 1969). In this way, C. anonymus was originally described as two distinct populations ) based on its crest: one group with a continuous crest and the other with a crest consisting of separate papillae. Subsequent sampling, however, evidenced the existence of intermediate forms (Monniot C. & Monniot F. 1982 Genus Protomolgula Monniot, 1971 Protomolgula bythia Monniot, 1971 Protomolgula bythia Monniot F., 1971: 467, fig.  6 A, B. Geographic distribution: NE Atlantic; SE Atlantic; Guyana-Surinam Basin; Brazil Basin; Argentine Basin (Monniot F. 1971;Monniot C. & Monniot F. 1976a;1977b;1984;1985a Geographic distribution: SE Atlantic; Southern Ocean (Scotia Sea); Argentine Basin (Uruguay, lower continental slope) Monniot C. & Monniot F. 1982;1985a;Sanamyan K.E. & Sanamyan N.P. 2002).
Observations: the first description of this species was based on young specimens obtained at RV Atlantis II, cruise 60, st. 262 and therefore specific identification was not possible at the time (Monniot F.  Observations: Monniot F. & Monniot C. (1976: 630;652-655) did not agree with the identification of Kott (1969) who recorded this species from the Drake Passage. They supported the more bio-geographically anomalous record of Rodrigues (1966), as M. piriformis (sic) from Brazilian coast (São Paulo´s coast, ca. 24°S, 140 m). Lately, Monniot C. & Monniot F. (1983) and Monniot C. & Andrade (1983) stated that the correspondence of this species with the record of Rodrigues (1966) is uncertain as they noted differences in gonads, stomach and stigmata. Monniot C. & Monniot F. (1983) included in other species of Molgula several records of M. pyriformis performed in the Magellanic area for other authors (Michaelsen 1900;Kott 1969).
Record not yet substantiated (mentioned in abstract).
Observations: the taxonomy of this genus is in particular need of a revision (Turon et al. 2016).
A phylogenetic analysis based on 18S rDNA sequences showed that the former Sorberacea/ Hexacrobylidae are in fact highly modified carnivorous Molgulidae (Tatián et al. 2011).

DISCUSSION
Thirty-eight ascidian species have been recorded until know from Uruguayan waters. Only nine were recorded for the continental shelf; the rest were collected from the slope (21), the abyssal plain (5) or even both deep-sea zones (3). These records show the historical relevance of the determinations of Herdman, Monniot F. and Monniot C., who described the ascidians collected from deep-sea stations by the HMS Challenger and the RV Atlantis II in the Argentine Basin.
The present ascidian list reveals the following distribution patterns for the known species: Shallow water species with temperate distribution. Only two species of this group, Styela plicata and Botryllus schlosseri, have been identified as exotic, although many others are still unreported (Tatián & Scarabino pers. obs.). Shelf and deep-sea species with Antarctic/Sub-Antarctic distribution. This well-known pattern represents the northernmost distribution boundary of the species following the northern flow of several water masses belonging to the cold Malvinas Current (see figure 2). This group consists of 11 species (Table 1), some of them occurring deeper than their Sub-Antarctic areas (e.g. Kott 1969;Monniot C. & Monniot F. 1983;Carranza et al. 2007;2008;Scarabino et al. 2016). Monniot C. & Monniot F. (1978) and Monniot F. (1979) had already noted the affinity of the Argentine Basin deep-sea ascidian fauna (mostly based on the records here listed) with the Southern Ocean. Biogeographic studies (Ramos-Esplá et al. 2005;Tatián et al. 2005;Primo & Vázquez 2007) support the idea of the Scotia Arc functioning as a bridge between the Magellan region and the Antarctic. Deep-sea species only known off Río de La Plata. Eleven species belong to this group, including both solitary and colonial forms. However, it mainly contains species living on the upper slope (Table 1). In fact, only two abyssal species (Dicarpa spinifera and Minipera tacita) are at this moment considered as endemic from off Río de La Plata. At least one species may be synonymous with an Antarctic one (Styela flava) and several others described by Herdman are actually poorly described. Similarly, a high percentage of endemic deep-sea protobranch bivalves have already been reported from off Río de La Plata by Allen & Sanders (1997), although this has to be additionally assessed (Scarabino et al. 2016).

Deep-sea species having wide distributions.
Twelve species belong to this group, consisting almost entirely of solitary species. Most of them have been recorded only from the lower slope but some of them extend to the abyssal plain ( Table  1). The wide distribution of deep-sea benthic species is a well documented pattern (e.g. McClain & Hardy 2010 and references therein), although many of these may represent species-complexes (e.g. Brandt et al. 2012;Havermans et al. 2013). Bathystyeloides enderbyanus and Cnemidocarpa bathyphila are two of the most widespread species of deep-sea ascidians (Sanamyan K.E. & Sanamyan N.P. 2002;2006). The eventual role of the North Atlantic Deep Water circulation in maintaining the connectivity among at least Atlantic deep-sea populations must be addressed in the future.
Research on the Uruguayan ascidian fauna should be further directed to record coastline and shelf species in order to detect the presence of exotic ones, as well as to redescribe some enigmatic bathyal species first described by Herdman (1882;1886). Furthermore, it must be stressed the complete lack of molecular analysis for the deep-sea species recorded from the Argentine Basin currently recognized as world-wide distributed. Historia Natural (Uruguay) and by the Project "Caracterización del margen continental uruguayo" (ANCAP-Facultad de Ciencias, UdelaR, Uruguay); PEDECIBA and ANII-SNI; the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) under Grant PIP CONICET Nº 20130100508; and the Secretaría de Ciencia y Tecnología (SECyT), Universidad Nacional de Córdoba (Argentina) under Grants 30720130100645CB and MINCYT-MEC UR/11/05. We grateful to Rosana M. da Rocha for making available important literature as well as for her opinion (provided with G. Lambert) about the status of Psammaplidium flavum. We also thank Biodiversity Library Heritage for the same reason about literature. All our gratitude goes to Dr. Riccardo Brunetti, Tito Lotufo and an anonymous reviewer, who provided a meticulous revision of the MS. We also thank Graciela Fabiano and Orlando Santana for their support/ samples concerning introduced species in Port La Paloma (Uruguay) and C. Barranguet for kind correspondence.
This work is respectfully dedicated to the memory of Claude Monniot (1936Monniot ( -2008 and to our great friend and coauthor, Lobo Orensanz (1945Orensanz ( -2015. FS is especially grateful to Inés Pereyra for her permanent support and patience.