Checklist of ascidians (Chordata, Tunicata) from the southern Gulf of Mexico

Abstract This study is the first inventory of ascidians from shallow waters (0–25 m) of coastal and reef habitats in the southern Gulf of Mexico where ascidian diversity is poorly known. Sampled environments in 14 locations (38 sites) with 134 samples collected from 2015 to 2017 included coral reefs, coastal lagoons, mangroves, seagrass, ports, and artificial platforms. The 31 identified species comprise 19 genera and 13 families. Ten species are newly reported in the Gulf of Mexico: Ascidiapanamensis Bonnet & Rocha, 2011; Ecteinascidiastyeloides (Traustedt, 1882); Cystodytesroseolus Hartmeyer, 1912; Eudistomaaff.amanitum Paiva & Rocha, 2018; Eudistomarecifense Millar, 1977; Euherdmaniafasciculata Monniot, 1983; Euherdmaniaaff.vitrea Millar, 1961; Polycarpacartilaginea (Sluiter, 1885); Botrylloidesmagnicoecum (Hartmeyer, 1912) and Didemnumgranulatum Tokioka, 1954. Two new species will be described separately (Clavelina sp. and Pyura sp.). This study provides the first records for 26 species ascidians for the region as well as describes increased distributions of ten Atlantic species. Thus, our data provide a starting point for future ecological, experimental and taxonomic studies of ascidians of the Gulf of Mexico.


Introduction
The Ascidiacea is the most diverse class of tunicates with ca 3000 recognized species, with representatives found in all marine habitats (Shenkar and Swalla 2011). Local ascidian species diversity depends primarily on availability and diversity of hard substrates, as well as temperature and salinity (Lambert 2005), while population density depends on food availability (organic particles suspended in water; Monniot et al. 1991). Ascidians are active suspension filter-feeders and are key organisms at times when they contribute to the control of phytoplankton (Petersen and Riisgard 1992) and may reduce eutrophication or contaminant concentration (Naranjo et al. 1996, Draughon et al. 2010. Many species colonize most artificial substrates and thereby become among the dominant members of "fouling" communities (Carballo 2000). Among foulers there are species known for their invasion potential worldwide (Lambert 2005). Ascidians are also known because of the presence of bioactive metabolites with potential biomedical interest (Erba et al. 2001).
Ascidian diversity in the Gulf of Mexico includes records of 79 species in 15 families in the northern Gulf of Mexico (Carballo 2000, Cole and Lambert 2009, CONABIO 2016, Fortaleza and Lotufo 2018. The southern Gulf of Mexico, however, is much less known and, despite the ecological and biotechnological importance of ascidians, only includes nine reported species: Aplidium exile (Van Name, 1902); Polyclinum constellatum Savigny, 1816; Ecteinascidia turbinata Herdman, 1880;Eudistoma capsulatum (Van Name, 1902); Eudistoma hepaticum (Van Name, 1921); Stomozoa roseola (Millar, 1955); Botrylloides niger Herdman, 1886; Symplegma brakenhielmi (Michaelsen, 1904) and Symplegma viride Herdman, 1886(Van Name 1945Carballo 2000). Essentially there are no studies from Mexico and the seven reported species is far below the expected number considering the great diversity of suitable habitats. To fill this gap, here we provide an inventory of the coastal species of ascidians in reefs and other shallow habitats in the southern Gulf of Mexico, along the Yucatán Peninsula.

Material and methods
Samples were collected in 14 locations and 38 sites from 2015 to 2017 in coral reefs, coastal lagoons, mangroves, seagrass, ports and artificial platforms by free diving and SCUBA, in the states of Veracruz, Tabasco, Campeche, Yucatan and Quintana Roo (Fig. 1, Table 2). Specimens were anesthetized in menthol and fixed with 4% formaldehyde in seawater. External characters of ascidians provide little information for determining their taxonomy and therefore dissection is required, for which a list of multiple characters is available Monniot et al. 1991;. Dissection was carried out following Monniot and Monniot (1972) and internal structures were stained with Harris hematoxylin dye (see: https://bocasarts.weebly. com/tunicate-tools.html). Families and genera were identified following  for species of the Atlantic Ocean. Specimens were deposited in the Colección de Ascidias del Golfo de México (CAGoM), which is part of the collection of the Marine Invertebrates of Gulf of Mexico, National Autonomous University of Mexico (UNAM), Mérida, Yucatán. The resulting dataset has been uploaded to the Zenodo data repository (Alvarez et al. 2018).

Family
Global distribution. Jamaica (Goodbody 1984, Goodbody andCole 2006), Guadeloupe , Belize (Goodbody 2004;Goodbody and Cole 2006), Panama (Collin et al. 2005;, Venezuela (Goodbody 2004;Rocha et al. 2010), Mozambique (Monniot 1997). Remarks. Colonies with the largest number of zooids were found in coastal lagoons on wooden piers or mangrove prop roots as well as coral reefs far from the shore. This was the most common species in Chelém (an enclosed, very salty, lagoon), comprising two morphotypes. Some had orange zooids and others had transparent, uncolored, zooids with a ring of orange along the siphon rim. This species was never found in disturbed port areas.

Palomino-Alvarez.
Remarks. This is the only known west Atlantic species of the genus which has been reported from nine countries, both in tropical and subtropical regions . The species is very rare in the southern Gulf of Mexico (Table 1); it has been found in shallow waters of the northern Gulf of Mexico in Florida (Van Name 1945).

Genus
Global distribution. Southern Gulf of Mexico (described herein) and Panama (Paiva and Rocha 2018). Remarks. Records of E. clarum have been found in mangroves and to a depth of 20 m in coral reefs (Goodbody, 2000). We found specimens mainly in coral reefs and near shore in places with strong anthropogenic impact.
Global distribution. United States (Van Name 1921), Bermudas (Van Name 1902, 1945Berrill 1932;Monniot 1972), Belize (Goodbody 2000), Panama (Collin et al. 2005;, Bonaire (Millar 1962), Venezuela and Caribbean Islands (Millar 1962;Goodbody 1984), Tobago (Cole 2012), Guadeloupe (Monniot 1983), Senegal (Pérès 1949), and French Polynesia (Monniot and Monniot 1987a). Remarks. Colonies are small and found beneath rocks and on bivalve shells. Global distribution. United States (Van Name 1921, 1945, Bermuda (Monniot 1972;Van Name 1902, 1945), Virgin Islands (Van Name 1921, 1945, and Belize (Goodbody 2000). Remarks. Zooids of some samples have a third opening at the base of the thorax through which fecal pellets are expelled. While colonies appeared healthy, this third opening may have been due to body wall rupture caused by obstruction of the atrial canal by incubating larvae in the atrial cavity or excess sediments in the water. The appearance of third siphons may be induced by experimental injuries in a few solitary ascidians (Jeffery et al. 2015).

Family Stomozoidae
Remarks. Colonies were found on dead coral and between large rocks. The tunic is very firm and dark purple, similar to colonies from the Red Sea and Madagascar.  Remarks. Although this species is common in many Caribbean countries, we found it in only one location.
Remarks. We found colonies in a single location growing on algae. Lissoclinum fragile is reported from tropical and subtropical regions where it is very common in marinas on artificial substrates, which suggests that is has been extensively introduced while the original geographical distribution remains unknown.

Remarks.
With a global distribution, D. granulatum is known to rapidly colonize artificial substrates (Oren and Benayahu 1998), so it may be widely introduced, but its origin is unknown. In the southern Gulf of Mexico colonies were found on corals, rocks and algae near shore.

Family Euherdmaniidae Ritter, 1904 Genus Euherdmania Ritter, 1904
Euherdmania fasciculata Monniot, 1983 Material examined. CAGoM-00471, Pro 1, 13 m, 26-05-2015, leg. L. Palomino-Alvarez. Remarks. Morphological patterns of colony shape, zooids completely embedded in the tunic, conspicuous musculature throughout the body, number of siphon lobes, number of stigmatal rows, and the testis position correspond with E. vitrea by Millar (1961), but the absence of the developed gonads and larvae prevent confirmation of this species. Also, the tunic is dark red colored while all colonies collected in Brazil are uncolored.

Fig. 4P
Material examined. CAGoM-0125, B10, 11 m, 17-06-2015, leg. L. Palomino-Alvarez. Remarks. This is the first record of B. magnicoecus in the Gulf of Mexico. General characteristics agree with descriptions by Brunetti (2010), but caecum size in the Mexico specimens is larger.
Global distribution. According to Brunetti (2010) the records from South Africa have characteristics in agreement with the type and are trustful (Michaelsen 1934;Millar 1955). Recently a new record from French Guiana (Monniot 2016) has been published. A more detailed study of the species in the type locality should be performed to permit the revision of other records in the Atlantic (Guadeloupe - , Belize -Goodbody 2004. Remarks. Botrylloides niger is among the species considered common and abundant in tropical waters (Rocha et al. 2010) with a widespread geographical distribution, where it attaches to natural and artificial substrates (Sheets et al. 2016 -as B. nigrum). We found it beneath rocks, on coral reefs, in ports and lagoons.

Genus Microcosmus Heller, 1877
Microcosmus exasperatus Heller, 1878 Remarks. Can be very common on mangrove prop roots in the Caribbean Sea (Panama - Rocha el al. 2005, Venezuela -Rocha et al. 2010). Here we found it mainly in harbors, with > 20 specimens found in Sisal harbour, suggesting that it was introduced.

Discussion
With this first checklist from the southern Gulf of Mexico, we list 31 species, five in the order Phlebobranchia, 19 Aplousobranchia and seven Stolidobranchia. Ascidians found in the southern Gulf of Mexico comprise 24% of the species, 24% of the genera and 60% of the families of ascidians that have been found throughout the Gulf of Mexico (Van Name 1945;Abbott 1951;Carballo 2000;Lambert et al. 2005;Cole and Lambert 2009). Also, they comprise 7% of the species, 22% of the genera and 53% of the families reported from the Atlantic Ocean . Styelidae and Polycitoridae are the most species-rich families.
The number of species we found is surprisingly less than expected and that have been reported from other regions of the Caribbean, including Belize, Bocas del Toro (Panama), Jamaica and Guadeloupe, while similar to the number of species found in Cuba, Curaçao and Puerto Rico . A possible explanation for fewer species is simply variation in sampling effort among studies, or that greater diversity has been found in association with mangrove roots in some of those studies. Mangroves are less common in the southern Gulf of Mexico surveyed here and therefore they were sampled less often in this study. In the present study we visited eight coral reefs, where ascidians are mainly found beneath pieces of dead coral and in crevices and pits where they are not easily found. Sampling effort was somewhat greater in the two sites with more species (Madagascar and Bajo 10). Four sites were in harbors or associated with marinas and urban construction, one of which had many species (Progreso). Harbors are known as entrances for exotic species, among which only C. roseolus, P. constellatum Table 3. World distribution of the ascidian species found in the present survey in southern Gulf of Mexico.
Gulf of Mexico remains to be tested. Also, increasing sampling effort will certainly uncover more species.