Polysyncraton (Ascidiacea, Didemnidae): a re-examination of some specimens and descriptions of three new species

Polysyncraton Nott, 1892 is the second largest genus of didemnid ascidians; it has a wide distribution ranging from temperate to tropical waters. Seventy-one specimens of Polysyncraton from eight museum collections and recently collected samples were analyzed. This resulted in the description of three new species (P. cabofriense Oliveira & Rocha sp. nov. from Brazil, P. globosum Oliveira & Rocha sp. nov. from Australia and P. snelliusi Oliveira & Rocha sp. nov. from Suriname) and emended descriptions of three further species (P. amethysteum (Van Name, 1902), P. magnilarvum (Millar, 1962) and P. purou C. Monniot & F. Monniot, 1987).


Introduction
Currently, the genus is characterized by species with a large thorax, a large atrial languet, lateral thoracic organs usually located at the anterior region, more than three testis follicles (exceptionally three or less), five or less coils of the sperm duct and larvae that are usually large, gemmiparous and with more than six pairs of ectodermal ampullae (Monniot et al. 1991;Kott 2001). Although many morphological characteristics of Polysyncraton are similar to those of Didemnum, the unique combinations of characteristics shown by both genera allows their taxonomic separation. Moreover, a recent molecular study also suggested this separation, although the support of those clades was not strong (Oliveira et al. 2017).
The large number of species within Polysyncraton results in many species being differentiated by subtle morphological differences, which have not been described well in older literature. In an effort to complement the description of these species, many specimens (including types) from several localities around the world were re-examined and two new species were described, one from the Atlantic Ocean and one from Australia. Here we also describe a new species from recent collections in Brazil and another probably undescribed species that requires samples with larvae for a complete description.

Material and methods
A total of 71 samples representing 13 species of Polysyncraton was studied, some of them used for comparison (P. adelon , P. horridum Monniot F. & Monniot C., 2008, P. lacazei (Giard, 1872, P. louminae Monniot F., 1984 and P. trivolutum (Millar, 1960)). Those species are not included here because we did not find any further characters complementary to their original description.
Colonies in Polysyncraton can be slimy, soft or brittle in a gradient of spicule absence to high spicule density. In relation to surface texture, colonies can be smooth, reticulated or papillated in various arrangements (Monniot 1991;Kott 2001). There are usually many cloacas with a smooth or lobed rim. We used those characters in combination with zooid and larval characters to separate species. To obtain the spicules a piece of the colony (25 mm 2 ) was incinerated under an ethanol lamp flame and put it in bleach for 5-10 min. to remove the remaining tissue. For colonies with a low density of spicules, a piece of the colony was boiled in bleach until the tissue had been completely digested. After rinsing in water two or three times, the spicules were then stored in an microtube containing 70% ethanol. A drop of spicules from the storage vial was poured on a small piece of copper tape fixed to a SEM stub and covered in Quick Carbon Coater SC-701C for analysis under a JEOL JSM-6480-LV scanning electron microscope at Naturalis Biodiversity Center, Leiden. Part of the samples were covered in gold and analyzed under a JEOL JSM-6360-LV at the Universidade Federal do Paraná.
For all colonies with a high density of spicules, hydrochloric acid (HCl) was used to decalcify a small piece of the colony for easier dissection and study of zooids and larvae. Transverse sections of various thickness were cut from those pieces using a razor blade. The zooids and larvae were removed with microsurgery instruments under a Zeiss Discovery V8 MS-163 stereo microscope. They were stained with Harris hematoxylin and observed under a light microscope. The zooids and larvae were measured and photographed with a differential interference contrast (DIC) microscope (Zeiss Axio Scope) coupled to a camera (A1 AxioCam Mrc). Zooids and larval characters used to describe the species are illustrated in Fig. 1.
The large colony variation in the density of spicules and colouration in P. amethysteum (Van Name, 1902) pointed to the necessity of a molecular approach to better understand the limits of this variation. Total mitochondrial DNA was extracted from one piece of the colony (1 cm²) using Invitrogen® DNeasy & Tissue Kit by Life Technologies following the standard protocol. PCR amplification reactions followed the Platinum taq DNA Polymerase protocol with slight modifications. The PCR protocol consisted of European Journal of Taxonomy 519: 1-25 (2019) an initial denaturation step of 98 °C for 30 s, denaturation step of 98 °C for 5s followed by 60 cycles with an annealing temperature step of 45 °C for 5 s, extension step of 72 °C for 15 s, and a final extension step of 72 °C for 5 min. We used the tunicate-specific primers (Tun_for 5'-TCGACTAATCAT AAAGATATTAG-3' and Tun_rev2 3'-AACTTGTATTTAAATTACGATC-5') designed by Stefaniak et al. (2009). The sequences are available from BOLD (Table 1).
The phylogenetic reconstruction is based on the Maximum Likelihood (ML) and Bayesian Inference (BI). The ML tree was generated using the RAxML-HPC2 on XSEDE (8.2.10) (Stamatakis 2014), under the GTRCAT model (Stamatakis 2006) and 1000 nonparametric bootstrap replications. BI was conducted using MrBayes on XSEDE (3.2.6) (Ronquist et al. 2012) with a model partitioned by codon position in the protein-coding gene. The BI analysis was performed with 10 million generations for each run, sampling trees every 1000 generations, using Markov Chain Monte Carlo (MCMC). The first 25% of generations were discarded as burn-in (2.5 million generations) before the calculation of the consensus tree. Both ML and BI analyses were run through CIPRES Science Gateway ver. 3.3. (Miller

Emended description
The specimen in NHM 1914.5.18.103 (Bermuda) has not been dissected because of the small size of the colony. It has a gelatinous and transparent tunic, without spicules, through which zooids could be seen on the colony surface without a pattern of organization. The specimen from the Atlantic coast of OLIVEIRA L.M. et al., Re-examination of specimens and three new species of Polysyncraton Florida (AMNH 314) has a brownish colour, is also small (3 cm in diameter and 1 mm thick), with several cloacal apertures and a spicule layer on the colony surface. The zooids are brownish and the atrial languet is bifurcated. The lateral thoracic organ is circular and protruding at the level of the 2 nd row of stigmata. The abdomen has a glandular tissue. The testis has three follicles surrounded by four coils of the sperm duct. Other specimens from the Gulf of Mexico (USNM 17364; Fig. 2; UF 1037, UF 1127 and UF 1749) have an atrial languet with a flat tip rather than bifurcated and testis with four follicles surrounded by five coils of the sperm duct. The glandular tissue in the abdomen has not been observed. Larvae were found in sample UF 1749 only. They look like those found in the specimens from São Paulo, Brazil. The specimens from Puerto Rico (AMNH 797 and AMNH 798) are similar in spicule density and zooid morphology to the samples from the Gulf of Mexico.
The colonies from Ghana (NHM 1959.5.27.23-24) are less than 3 cm in diameter. In contrast with the information given about this sample by Millar (1953), the larval trunk is 0.8 mm long. The colonies are pinkish-purple, gelatinous, the colony surface is smooth, shiny and marked with a spicule accumulation around the oral siphons. The zooids are 0.8-1.0 mm long. The oral siphon has six short triangular lobes. The atrial aperture is small. The atrial languet is bifurcated, sometimes with a flat tip. The lateral thoracic  (2019) organs are circular, not protruding and located in the anterior region of the thorax at the level of the 2 nd row of stigmata, close to the atrial aperture. The pharynx has six stigmata per half row. The abdomen is positioned horizontally in relation to the thorax. The secondary gut loop overlays the stomach. The testis is divided in five follicles surrounded by three coils of the sperm duct. The larvae are present on the colony surface. They are elliptical, gemmiparous, uncoloured, trunk measuring 0.8-1.0 mm. A tail with a diameter of 35 µm winds about halfway around the trunk. Three adhesive papillae are close together. The sucker of the central adhesive papilla is 100 µm in diameter. There are six elongated ectodermal ampullae on each side. The sensorial vesicle lays in the posterior region of the larval trunk. The ocellus is 40 µm long. The distance between ocellus and anterior region of the tail is 200 µm. Larva with two embryos: oozoid dorsal and blastozooid ventral.
The morphological characteristics of the zooids of the Brazilian specimens are very similar to those observed by Van Name (1902), although the variation in colony colour and spicule density is much larger in Brazil. The colour varies from purple to brick. The colonies are 3 to10 cm in diameter and 1 to 3 mm thick (Fig. 3). The atrial languet can be bifurcated or with a flat tip. The pharynx has seven to nine stigmata per half row. The lateral thoracic organ is circular and protruding at the level of the 2 nd row of stigmata. The testis has three to five follicles (even in zooids of the same colony), surrounded by three to five coils of the sperm duct. The larva is gemmiparous, with a trunk that is 0.8 to1.0 mm long. A tail 85 to 90 µm wide winds about three-quarters of the way around the trunk. The peduncles of the adhesive papillae are long, while the sucker of the central adhesive papilla is 63 to 80 µm in diameter. There are six or eight pairs of ectodermal ampullae, but four pairs in different stages of duplication were seen in immature larvae (Fig. 4c-f). The sensorial vesicle is in the mid or posterior dorsal region of the larval trunk. The ocellus is 56 to 60 µm long. The distance between the ocellus and the anterior region of the tail is 185 to 238 µm. The pharynx of the oozooid has 8, 7, 6, 6 stigmata on each half row. Lotufo (2002) claimed that Brazilian specimensof this species differ from the ones described by Van Name (1902) in the different arrangement of spicules and in the single cloaca per colony. However,  Table 1. OLIVEIRA L.M. et al., Re-examination of specimens and three new species of Polysyncraton the colonies studied by us showed a huge variation in colour, spicule density and cloaca number as also observed by Pérès (1949) and Millar (1953) in African specimens. Lotufo (2002) also mentioned having studied a sample from the type series deposited in the American Museum of Natural History, with very large larvae (1.5 mm trunk length), but we were not able to find and examine this sample in the museum collection. The only sample close to the type locality with available larvae was one from Florida (UF1749), with larvae whose trunk varies from 0.8 mm to 1.2 mm within the same colony. F. Monniot (2016) compared specimens from French Guiana with those from Bermuda and concluded that they belonged to the same species.

Remarks
Molecular analyses (ML and IB) also suggest the existence of only one valid species. Samples from Panama to south Brazil grouped in one clade, corresponding to P. amethysteum with a significant bootstrap value (95%) and posterior probability support (IB = 1) (Fig. 5). Although this clade seems to be split in two branches, one of them does not have enough bootstrap support. The intraspecific genetic divergence (p-distance) among haplotypes ranged from 0.7 to 9.8%, while the interspecific divergence with P. lacazei was ~22%, and with P. poro was ~27%. Based on genetic evidence and the lack of clear morphological separation in the samples examined, we consider that they all belong to P. amethysteum, a species with a large phenotypic variation.
The haplotype UPDID096-17 from Rio de Janeiro, in contrast, is a different species with 12.4 to 14.9 % distance from P. amethysteum, and will be commented on later (Polysyncraton sp.).

Etymology
The name refers to the region where the species was found.

Description
Living colonies are yellowish, with a diameter of 3 to 15 cm and a thickness of 2 to 3 mm. When preserved in formalin, colonies usually lose their colour and become cream coloured, sometimes white. They have an irregular shape, molding themselves to the substrate. The colony surface has several common cloacal apertures, each one with short lobes (Fig. 6a-b). The tunic is firm, brittle and smooth. Green algae were observed on the surface of some colonies. Spicules are distributed in the whole tunic, with relatively high concentrations on each side of the thorax at the level of the lateral thoracic organ. There are two kinds of spicules: stellate, 32 to 40 µm in diameter, with 6 or 7 blunt-tipped conical rays in transverse optical section; others are smaller (25 to 28 µm in diameter), with round and short rays (Fig. 6c).
Zooids are 0.8 to 1.0 mm long. The oral siphon has six small triangular lobes. The atrial aperture is wide, exposing most of the pharynx. The atrial languet is pointed and thin (Fig. 6d) or wide and rounded. In some specimens it was absent, probably lost during dissection. The lateral thoracic organs are circular, not protruding and localised between the first and second rows of stigmata on the border of the atrial aperture. The pharynx has seven stigmata in the first two half rows and six stigmata on the posterior two half rows. The muscular process is lacking. The testis is divided in three oval follicles surrounded by three coils of the sperm duct. The oocyte is single, measuring 0.6 mm.
The larvae are elliptical, gemmiparous, the trunk measuring 0.9 to 1.0 mm. A tail with a diameter of 95 µm winds about ¾ of a turn around the trunk. The three adhesive papillae show a short and wide peduncle. The sucker of the central adhesive papilla is 124 µm in diameter. There are 10 pairs of rounded ectodermal ampullae. The ocellus is 45 µm long and 346 µm distant from the anterior region of the tail.

Remarks
Only two species of Polysyncraton have been reported from Brazil, P. amethysteum with a distribution along the whole coastline and P. maurizelae Paiva et al., 2015 from Rocas Atoll (Paiva et al. 2015). Polysyncraton cabofriense sp. nov. differs from P. amethysteum by its colour, higher density of spicules, lobed cloaca and larvae with more pairs of ectodermal ampullae. Polysyncraton maurizeliae has orange colonies with smooth common cloacal apertures, zooids longer and with 12 stigmata in the 1 st row, and the lateral thoracic organ in the 3 rd row. Among other species from the Atlantic, P. louminae F. Monniot, 1984 has a colony of similar shape and spicules, but the colony is grey, zooids have longer oral siphons and two testis follicles surrounded by 3 to 5 coils of the sperm duct, and the larvae are not gemmiparous and have more pairs of ectodermal ampullae (F. Monniot 1984).

Distribution
Type locality: Brazil (Rio de Janeiro).

Etymology
The name refers to the shape of the colony.

Description
The species is characterised by its globular colonies, 3.5 cm at maximum length (Fig. 7a). The tunic is gelatinous and the colony surface is smooth. The spicules are distributed in low density, mainly on the colony surface. Just one cloacal aperture with a smooth edge was observed in each colony. The globular spicules are 20 to 30 µm in diameter and have numerous thin rays (16 to 18 rays in optical transverse section) (Fig. 7b).
The thorax has 12 conspicuous muscle bundles on each side (Fig. 7c). The oral siphon has six triangular lobes. The atrial languet is bifurcated but was lost in some thoraces when the zooids were dissected. The lateral thoracic organ is circular, not protruding, located at the level of the 1 st row of stigmata, in the middle of the thorax (Fig. 7c). The second gut loop does not overlay the stomach. The testis is divided in six follicles surrounded by two or three coils of the sperm duct (Fig. 7d), although the description by Millar (1963) mentioned up to eight follicles. Larvae are elliptical, the trunk 1.0 to 1.3 mm long (Fig. 7e). The tail winds about ¾ of the way around the trunk. At the left side of the larva, it is possible to see a wide membrane projecting from the sides of the tail (Fig. 7f). The three adhesive papillae with long and thin peduncles are close together and there are eight pairs of rectangular ectodermal ampullae. The   Fig. 8. Polysyncraton magnetae Hastings, 1931, type specimen (NHM 1930). a. Preserved colony. b. Cross section of the colony showing cavities found in the median region (arrows). c. Spicules. d. Cross section of a decalcified colony. Scale bars: a = 1 cm; c = 10 µm.
European Journal of Taxonomy 519: 1-25 (2019) sensory vesicle is in the central region of the larval trunk, with the ocellus and estatocyte close together. The larva is not gemmiparous.

Remarks
These Australian specimens, which were deposited at the Natural History Museum in London, have previously been identified as P. magnetae Hastings, 1931by Millar (1963. The sample NHM 1930.12.17.46 from the Great Barrier Reef, Stat. XXIII, R/V "Magneta", collected on 12 Mar. 1929 is part of the type series of P. magnetae and was re-examined (Fig. 8). Although also looking globular, the colony is not as solid as in P. globosum sp. nov. and has a high density of spicules in the tunic, with cylindrical rays of a larger diameter and in a smaller number than in P. globulosum (Fig. 8). The atrial languet in P. magnetae is almost imperceptible, short and pointed, and could be missed during dissection, although Hastings (1931) described it as large. Unfortunately, the larva of P. magnetae is not known for comparison with congeneric species. Kott (2001) also realised that Millar's identification was wrong and suggested that the NHM material belonged to P. meandratum F. Monniot, 1993. We do not agree with this suggestion because P. meandratum has thin and sheet-like colonies, an atrial languet larger than those observed in P. globosum sp. nov., less male follicles and a larva less than half the size of that of P. globosum sp. nov. (F. Monniot 1993).

Emended description
The specimen from Magadascar has a long and trifurcated atrial languet (Fig. 9a), while Millar's material from Mozambique has a short atrial languet (Millar 1962). The lateral thoracic organs are circular and not protruding, localised in the anterior half of the thorax at the level of the 1 st row of stigmata, close to the endostyle (Fig. 9b). The muscular process is longer than the abdomen, up to 1.4 mm in length and inserted in the middle of the esophageal peduncle. The abdomen is positioned perpendicularly to the thorax. The secondary gut loop does not overlay the stomach (Fig. 9a). The stoloniferous vessels are small and rounded, and a glandular tissue in the shape of a crescent lays inside the gut loop on the right side. The testis is slightly below the gut loop, divided in 10 to 12 follicles and surrounded by three to five coils of the sperm duct ( Fig. 9c-d). The larvae are elliptical, gemmiparous and the trunk is 1.4 mm long.
A tail with a diameter of 104 µm winds halfway around the trunk. The three adhesive papillae are close to each other, with short peduncles. The sucker of the central adhesive papilla is 133 µm in diameter and the peduncle is 401 µm long. There are 12 to 15 elongated ectodermal ampullae on each side of the larva body, 113 to 333 µm long, depending on the developmental stage of the larva (Fig. 9e). The sensorial vesicle (including ocellus and statocyte) is located in the mid-dorsal region of the larval trunk. The ocellus is 57 µm long and 436 µm distant from the anterior region of the tail.

Remarks
This species was first reported from Mozambique by Millar in 1961, but the original description was published later by him in 1962 (Millar 1962) with the following diagnostic characters: short atrial languet, testis with eight to ten follicles surrounded by 2½ coils of the sperm duct and larva with 15 pairs of ectodermal ampullae. The sample from Madagascar has a larger atrial languet and a larger number of testis follicles and coils of the sperm duct, but the larval characters are the same. Kott (1975) synonymised it with P. aspiculatum Tokioka, 1949, but later, C.  confirmed that Fig. 9. Polysyncraton magnilarvum (Millar, 1962), stained with hemalum. a. Zooid (arrow pointing at atrial languet). b. Ventral view of the anterior region of the thorax (arrow indicating the lateral thoracic organ). c. Testis follicles (arrow indicating one lobe). d. Abdomen (arrow indicates the sperm duct) (MNHN A2-2466). e. Larva (MNHN A2-2467). Scale bars: a = 0.2 mm; c-e = 0.1 mm.
European Journal of Taxonomy 519: 1-25 (2019) both species were valid and differentiated P. aspiculatum by its globular spicules, the larvae with a trunk length of 1 mm of trunk length and eight pairs of ectodermal ampullae.

Remarks
This species was described from French Polynesia by C. Monniot & F. Monniot (1987). All the characteristics of the specimens from Tanzania correspond to those in the original description (Fig. 10), thus enlarging the known distribution of this species.
Monniot & Monniot (2001) described P. vestiens from Palau as a different species from P. purou based on the smaller spicules, a wider atrial aperture and the absence of the muscular process in P. vestiens. However, in the specimens from Tanzania there is a variation in the size of the spicules and they are smaller (43 to 55 µm) than in specimens from Tahiti. The muscular process in P. purou is very small and not visible in some zooids ( Fig. 10a-b). Since the size of the atrial aperture is also a variable character that depends on the contraction of the zooid, there is doubt as to whether P. vestiens is a valid species.

Polysyncraton snelliusi
The name refers to the campaign vessel that collected the material.

Description
The colony is yellowish in preserved material, measuring 8 cm in diameter and 2 mm thick. It has an irregular shape, molding itself on top of a sponge. The colony surface has several common cloacal apertures with six to eight short lobes each (Fig. 11b) thoracic level. The tunic is firm, brittle and with many papillae associated with one of the lobes of the oral siphons. The zooids form circular systems around the cloaca and also linear systems along the cloacal channels, which are marked by a depression on the colony surface. They have a brown colour, leaving white circular or irregular patches on the colony surface (Fig. 11c). Two types of stellate spicules are distributed in the tunic. The larger ones (40 to 60 µm in diameter) have six rays in transverse optical section, but many other shorter rays among the main ones. Also, the rays are either blunt or more pointed. The smaller spicules (23 to 50 µm in diameter, a few to 60 µm), have seven to eight conical rays with round tips in transverse optical section, without intermediate rays (Fig. 11d).
The zooids are 1.0 mm long. The oral siphon is short (0.2 mm), with six long triangular lobes. The atrial aperture is wide, exposing almost all the pharynx, without an atrial languet. The pharynx stigmata formula is 12, 12, 11, 9. The lateral thoracic organs are circular, not protruding and localised at the level of the 2 nd row of stigmata. The muscular process is lacking. The esophageal peduncle is short and straight. The testis is divided in four follicles surrounded by three coils of the sperm duct. The secondary gut loop does not overlay the stomach (Fig. 11e).
The larvae are elliptical, not gemmiparous, with the trunk 0.8 to 0.9 mm long. A tail with a diameter of 116 µm winds halfway around the trunk. The sucker of the central adhesive papilla is 95 µm in diameter and the peduncle is 235 µm long. There are 12 to 14 elongated, thin ectodermal ampullae on each side of the larva, 105 to 120 µm long (Fig. 11f). The sensorial vesicle (including ocellus and statocyte) is slightly posterior to the mid-dorsal region of the larval trunk. The ocellus is 54 µm long and 361 µm distant from the anterior region of the tail.

Remarks
Eight species of Polysyncraton have been reported from the Atlantic Ocean (Rocha et al. 2012;Paiva et al. 2015) and none of them look like the one described herein. Polysyncraton snelliusi sp. nov. can be distinguished from P. cabofriense sp. nov. by the absence of papillae on the colony surface and the presence of gemmiparous larvae in the latter. Among other species with papillae on the colony surface, P. echinatum Kott, 2001 has a gelatinous tunic, stellate spicules without secondary rays and smaller larvae (trunk 0.6 mm long), with four subdividing ectodermal ampullae on each side; P. meandratum F. Monniot, 1993 has globular spicules with flat-tipped rays, a bifurcated atrial languet and larvae with eight pairs of ectodermal ampullae; P. pseudorugosum F. Monniot, 1993 has stellate spicules with sharp pointed rays and deeper common cloacal canals, seven testis follicles and smaller larvae with eight pairs of ectodermal ampullae.

Remarks
The colony is orange in situ, measuring 3 cm in diameter and 1 to 2 mm thick. The tunic is firm and gelatinous. The cloacal aperture has five lobes. The spicules are distributed in the colony surface only and are more concentrated around the oral siphon of each zooid.  (2019) of rays, 15 to 25 µm in diameter. The zooids are orange. The oral siphon is short, with six triangular lobes. The atrial aperture is wide, exposing almost all the pharynx, with 9, 9, 8, 8-7 stigmata per half row. The atrial languet is large and bifurcated. The lateral thoracic organ is circular, not protruding, located at the level of the 1 st row of stigmata. The muscular process is shorter than the abdomen, inserted in the middle of the esophageal peduncle. The testis is divided in four follicles surrounded by four coils of the sperm duct. Larvae were not found.
In the phylogenetic reconstruction (Fig. 5), this species (UPDID096-17) is separate from P. amethysteum, from which it differs by the colour of the colony, lobed cloaca and the position of the lateral thoracic organ. Lotufo (2002) reported orange specimens from Rio de Janeiro and Espírito Santo, but we were able to examine the sample from Angra dos Reis and concluded that Lotufo's sample (ColBio Tun 1245) actually belongs to the genus Leptoclinides Bjerkan, 1905. This species differs from P. cabofriense sp. nov., by the colour of the colony, the abundance, shape and position of the spicules, and in having more follicles and coils of the sperm duct. Given the single colony found and lack of larvae we prefer not to name it, although characters suggest that it is an undescribed species.

Distribution
Brazil: Rio de Janeiro (this study).

Discussion
Didemnidae is one of the largest families in Ascidiacea and its interspecific differentiation is usually achieved by the combination of characters, among which larval characters are usually very important. For Polysyncraton, the spicule density, distribution, shape and size, the size of the lateral organ, the number of testicular follicles and coils of the sperm duct, the size of the larva, the gemmiparous condition and the number of ectodermal ampullae are known as the most important characters to differentiate species (Kott 1962;F. Monniot 1984). We have also observed specific differences in the colour and shape of the colony (sheet-like or globose) and the cloaca rim (smooth or lobed).
Unfortunately, formalin fixation is routine in protocols of ascidian preservation (C. Monniot & F. Monniot 1972), even though samples are maintained in ethanol for long term preservation in museum collections.
Thus, genetic studies are only possible with samples specifically collected for this purpose and we were only able to use genetic evidence to delimit species from samples recently collected along the Brazilian coast. This genetic study was useful to demonstrate that P. amethysteum has a large distribution range along the West Atlantic and the Gulf of Mexico, with a lot of variation in colour, spicule density, number of testicular follicles and sperm duct coils, and larva size.
Most of the diversification in Polysyncraton is found in the Indo-Pacific, with Australia being the beststudied country (Kott 2001). Genetic studies with the goal to understand the evolution of this genus will have to focus on the biodiversity in this region. Even though the Pacific is the most species-rich region, we were able to identify a new species (Polysyncraton globosum sp. nov.) from Australia, which has almost 45% of all Polysyncraton species currently known.
Considering that only three species of Polysyncraton were previously known from the tropical West Atlantic (P. amethysteum, P. louminae and P. maurizelae), the two new species from this region here described, and another possibly undescribed species, represent a significant contribution. It is important to also emphasize that sampling efforts in the West Atlantic and Gulf of Mexico are still not comprehensive and other species are expected to be discovered.