Taxonomic review of Berthella and Berthellina (Gastropoda: Pleurobranchoidea) from Brazil, with description of two new species

Berthella currently comprises five valid species recorded from the western Atlantic, of which only Berthella agassizii (MacFarland, 1909) and Berthella stellata (Risso, 1826) have been recorded from Brazil. Two species of Berthellina occur in Brazil: Berthellina quadridens (Mörch, 1963) and Berthellina circularis (Mörch, 1863); however, based on a literature review and the anatomical study presented here, we only recognize B. circularis as occurring in the country. This study gives a detailed re-description of B. agassizii, B. stellata and B. circularis based on material from Brazil, and adds two new species to the genus, from the Brazilian coast, Berthella punctata sp. nov. and Berthellina ignis sp. nov. Berthellina circularis and B. quadridens are considered distinct species based on the denticulation of the radular teeth. B. quadridens presents the majority of teeth with two denticles, rarely three-four denticles, while in B. circularis from Brazil the radular teeth are highly denticulate with up to 17 denticles. Berthella and Berthellina do not form a monophyletic group, and cluster in a large polytomy within Pleurobranchidae. A detailed anatomical study is performed to improve the understanding of the evolutionary history of Pleurobranchidae. The comparison with available data on the odontophoric muscles of Pleurobranchoidea shows that Berthella and Berthellina present a pair of the muscle m10v that connects the mj with the ventral portion of the m5, while in Pleurobranchus and Pleurobranchaea the m10v connects the mj with the ventral portion of the m4; Berthella and Berthellina do not present the muscle m10d, which is present in Pleurobranchus and Pleurobranchaea. Based on these characters, Berthella seems to be more closely related to Berthellina than to Pleurobranchus.

In this study, we re-describe the species of Berthella and Berthellina from Brazil, with the aim of improving the taxonomical delimitation of these species.Most of them were described based on one or two preserved specimens, and subsequent records only expanded their geographical distribution, without adding information on their morphology.Moreover, two new species from Brazil are described, one from each genus.

MATERIAL AND METHODS
Descriptions of the external morphology were based on living and preserved specimens.Dissections and drawings of the digestive system, including intrinsic and extrinsic muscles of the odontophore, the reproductive system, the nervous system and the circulatory system were performed under a stereomicroscope with a drawing tube.
The radula and jaw were cleaned in 10% potassium hydroxide (KOH), subsequently rinsed in water and mounted for examination in a Jeol JSM-6390LV scanning electron microscope (SEM).
The nomenclature used to name the portions of the gill follows WILLAN (1983) and the one used for the odontophore muscles follows PONDER et al. (2008: 350-351, figs. 13.9-13.10),with additional information added by suffixes (d: dorsal; v: ventral).In the nervous system, when the nerve bifurcates, the number of the nerve is followed by letters, e.g., "a", "b".
In the lists of examined material, the number inside brackets indicates the number of specimens in each lot, followed by the number of specimens dissected.
Remarks.The name Cleantus has been considered a junior synonym of Berthella for a long time (WILLAN 1983).The authorship of the name, however, is questioned here.This name was propagated by GRAY (1847) as Cleanthus, with the letter "h", and the name Cleanthus Gray, 1847 was only listed in that paper, without any description or characterization.Therefore, the name/authorship of Cleanthus Gray, 1847 is not available, since it does not meet the requirements of Article 12.1 of the International Code of Zoological Nomenclature (ICZN 1999).According to the latter, a name needs to be accompanied by a description or a definition of the taxon that it denotes, or by an indication of it.Therefore, the author of Cleantus is LEACH (1852), who provided a description of the genus.
Mantle.Spicules not found, probably dissolved by fixation.Shell .Translucent white to opaque white; subquadrangular in outline; slightly convex profile; approximately two times longer than wide.Length 5.4 mm, width 2.7 mm (in preserved specimen with 6 mm in length); and, length: 5.3 mm, width 2.6 mm (in preserved specimen with 8 mm in length).Spire with 1.5 whorls.Protoconch smooth (Fig. 20).Lines of growth distinct; immediately after protoconch, with longitudinal sculptures transverse to lines of growth; sculptured portion corresponding 1/3 to 1/4 total length of last whorl (Fig. 20); anterior portion of last whorl smooth.Shell above heart, located anteriorly on left side of body.Shell covering approximately half of length of mantle.
Circulatory system (Fig. 14).Pericardium well developed in anterior portion of body (near cerebro-pleural ganglia).Blood flowing into auricle from gills, kidney and venous sinuses.Efferent branchial vessel connecting gill with the auricle.Auricle on right side, ventricle on left; auricle with thin wall; ventricle muscular.Blood gland small, creamy/pale orange covering left part of pericardium.Blood gland joined to aorta.
Reproductive system .Ampulla thin and elongated, about same width than deferent duct (Fig. 17); curved, but not convoluted.Spermoviduct branching into two ducts, oviduct leading to female gland mass and other duct leading to prostate .Prostatic portion of deferent duct convoluted (Fig. 17).Near deferent duct distal end it joins with elongated penial gland and narrows near into conical penis.Penial gland about two times thicker than deferent duct and approximately of same size of seminal receptacle .Penis (in preserved specimen with 8 mm length: length 0.36 mm; width 0.12 mm) (Fig. 15); completely contractible.
Nervous system (Fig. 18).Cerebral and pleural ganglia fused and so closely together,connective imperceptible.Eyes located laterally of cerebro-pleural complex; eyes borne upon very short optical nerves (no).Rhinophoral ganglia placed at bases of rhinophores, near cerebro-pleural ganglia; two main nerves leaving from rhinophoral nerves, runing until distal portion of rhinophores; rhinophoral nerves with many secondary nerves, perpendicular in relation to main nerves.Nerves leaving cerebro-pleural ganglia: cp1 inserting latero-ventrally; cp2 inserting dorsally into mantle; cp3 runing laterally, inserting into body wall, in right side nerve entering into mantle near base of gonopore (apparently on male portion) and, in left side, nerve entering into mantle near anterior portion of digestive gland; cp4 runs until most posterior portion of body, inserting into body wall; cp7 innervating latero-ventral side of body wall.Connective between visceral and cerebro-pleural ganglia evident, leaving posterior portion of right cerebro-pleural ganglia.Connective between buccal and cerebro-pleural ganglia leading from most anterior portion of cerebro-pleural ganglia in ventral view.Nerves leaving buccal ganglia: nb1 inserting into esophagus; nb2 inserting into salivary ducts; connective cerebro-pleuralbuccal shortly after nb2.Pedal commissure short, leaving from most anterior posterior of pedal ganglion.Pedal ganglia smaller than cerebro-pleural complex, in antero-posterior order: np1 inserting ventrally into oral veil; np2 inserting anteriorly into foot; np3 innervating foot; np4 inserting ventrally into foot and runs until most posterior portion of body.
Digestive system .Pharyngeal bulb not protruded.Mouth longitudinal, in middle of snout tip.Oral canal muscular just posterior to mouth , representing approximately 1/3 of pharyngeal bulb length.Muscle surrounding jaws (mj) strong, pair of large jaws located in its inner surface, mj originating in lateral and dorsal surfaces of oral canal, inserting into lateral and dorsal regions of buccal mass (Fig. 25).Jaws amber, lighter posteriorly; jaw of two plates surrounding radula inside buccal cavity (Fig. 27); elongated, reaching level of radula.Each jaw plate showing alternate rows formed by elongated and denticulate elements with slight cruciform lateral expansion (73 longitudinal and 26 transversal elements, in preserved specimen of 6 mm long; 69 longitudinal and 32 transversal elements, in preserved specimen of 11 mm long); elements consist on a main cusp with 3-6 denticles in each side, which could be of different sizes and not symmetric (Fig. 21).Pair dorsal jugal muscles (m1d) of buccal mass, inserting into m5, connecting to dorsal-anterior end of snout (Fig. 25).Pair m4, main dorsal tensor muscle of radula, reduced, originating in lateral region of cartilages, surrounding them ven-trally, inserting into subradular membrane .Pair m5, secondary dorsal tensor muscle of radula, large and broad, covering median portions of cartilage, extending up to dorsal region; originating in posterior surface of cartilages; inserting laterally in mj .Pair m7 absent.Pair m10d (dorsal) absent.Pair of m10v connecting mj with ventral portion of m5 (Fig. 26).Single auxiliary muscle m10a, ventral tensor muscle of radula, originating in anterior portion of oral canal, running in middle of buccal mass, inserting into radular sac (Fig. 26).Pair of strong retractor muscles originating in most posterior portion of m5 ; separated in its anterior third and jointed in its posterior portion, laying above anterior portion of digestive gland.Odontophore cartilage semicircle in outline (Fig. 28).Radula rectangular, two times longer than wide; formula 63 × 52.0.52 (from preserved specimen 11 mm length); 48 × 49.0.49(from preserved specimen 6 mm length).Radula lackinck rachidian tooth (Fig. 22); lateral plates smooth hook shaped, without denticles (Fig. 22); base of tooth enlarged (square) and concave (Fig. 23); innermost lateral tooth hook-shaped (Fig. 22); subsequent lateral plates hook-shaped, larger and more developed in center of rows (Fig. 23); outermost laterals teeth less developed (Fig. 24).Floor of Pharyngeal bulb greatly reduced, causing a slightly rotation in radula position.Absence of acid gland (Fig. 25).Esophagus sac-like tube, passing into voluminous stomach (Fig. 27).Salivary gland small and in front of digestive gland .Ducts of salivary glands entering pharynx musculature laterally to esophagus, opening into base of pharyngeal cavity between radula and jaw plates (Figs. 25,27); convoluted; without visible ampulla.Stomach passing ventrally into digestive gland, embedded in digestive gland until dorsal view (Fig. 26); internally, stomach with main groove from where depart others perpendiculars grooves (Fig. 27).Posteriorly, stomach passing into intestine; intestine passing in dorsal portion of digestive and hermaphrodite glands, opening laterally on body wall (Fig. 25); internally, intestine with longitudinal folds (Fig. 27).Glands: salivary, digestive, and hermaphrodite forming single aggregate .
Egg mass (Fig. 3).Egg mass forming a spiral ribbon with enlarged border with 1.5-2 turns in counterclockwise direction, containing numerous rows of many tiny eggs; usually white; 10 mm of diameter.White eggs surrounded by translucent matrix; diameter of eggs about 140 µm.Eggs disposed in longitudinal rows; many rows per thickness; each row presents about 35 eggs with 6-9 eggs per thickness.
Material examined.ZOOLOGIA 32 (6): 497-531, December 2015 these two species because Verrill's description lacked sufficient diagnostic characteristics to allow synonymy in the absence of the holotype of P. aurantica; thus, he preferred to consider P. aurantica as nomen dubium.
Besides that, P. aurantiaca has a problematic nomenclatural and taxonomic history.THIELE (1931) inferred that Pleurobranchopsis was a subgenus of Pleurobranchus, thus the new name combination Pleurobranchus aurantiaca (Verrill, 1900) became a homonymy of Pleurobranchus aurantiacus Risso, 1818, species described from the Mediterranean Sea.To solve this nomenclatural problem, THIELE (1931) created a new name for species from the Bermudas, Pleurobranchus verrilli Thiele, 1931.
Berthella agassizii and P. verrilli are well established as dis-tinct species, however we would like to emphasize the differences between these two species.In spite of Verrill's brief description, there are some characteristics that clearly distinguish these two species: in P. verrilli the gill is attached laterally along its entire length, while in B. agassizii we find 5-6 pinnae that are free from the body wall (Fig. 12); the mantle, gill and foot are orange in P. verrilli, whereas in B. agassizii the mantle is pink, the foot is white, and the gill with the upper pinnae are rosy (Figs.1-2); P. verrilli is relatively larger, between 32 mm to 36 mm, whereas B. agassizii measures 7 mm to 23 mm; and the possible egg mass find by VERRILL (1900) had numerous bright orange eggs in many rows, while the egg masses of B. agassizii are white (Fig. 3) (BANDEL 1976, present study).
Description.External morphology (29)(30)(31).Living specimens translucent white to brown, passing through rosy ; one dark pink spot, almost brown, in middle of mantle, which are surrounded by white patches (Figs.4-6); rhinophores, oral veil, gill and foot of same pattern color as mantle.Living specimens up to 13 mm in length; length of mature preserved specimens 6-9 mm; width 4 mm; length of foot 5-6 mm; width of foot 2 mm.Body oval and oblong.Mantle covered foot entirely.Mantle surface smooth in its middle; honeycomb appearance in mantle edge, larger honeycomb appearance in the posterior portion than anterior portion.Oral veil thin and trapezoidal connected with the head region (Fig. 30); laterally, oral tentacles with deep notch, corresponding almost its length.Rhinophores rolled joined at their bases, up to two-thirds of its length.Gill exposed laterally (Fig. 31); 2/5 length of body; main rachis smooth, without tubercles; alternate pinnae; simple pinnae; 17-22 pinnae; 7-13 pinnae free from body wall, attached by branchial membrane.Anal opening lying approximately above 5° pinnae (Fig. 31).Prebranchial pore opening beside main rachis.Genital aperture surrounded by collar (Fig. 31).Penis retractable.Foot slightly pointed at posterior end with elongated metapodial gland; metapodial gland 0.3 times foot length; anteriorly bilabiated, upper lip notched, smaller than lower one.Eyes localized just behind rhinophores.
Shell .Translucent white, with light golden tones in some parts of shell, visible according to incidence of light; subquadrangular in outline; slightly convex profile; ap-proximately two times longer than wide.Length 5.9 mm, width 2.9 mm (in preserved specimen with 8 mm in length); and, length: 6.8 mm, width 2.9 mm (in preserved specimen with 9 mm in length).Spire with 1.5 whorls.Protoconch smooth (Fig. 44).Lines of growth distinct; immediately after protoconch, with longitudinal sculptures transverse to lines of growth (Fig. 44); shell smooth after the last line of growth.Shell above heart, on right side of blood gland.Shell covering almost all visceral mass.
Circulatory system (Fig. 32).Circulatory system of B. punctata sp.nov.identical to the B. agassizii as described above.
Reproductive system .Ampulla very thick and elongated, eight to 17 times thicker than deferent duct ; curved, but not convoluted .Spermoviduct branching into two ducts, shorter and wider oviduct leading to female gland mass, other duct leading to prostate .Deferent duct glandular in 63% to 79% its length .Prostatic portion of deferent duct folds once to twice; near its distal end it joins with elongated and curved penial gland and narrows near into elongated penis.Penial gland four to five times thicker than deferent duct .Penis (Fig. 39) (in preserved specimen with 8 mm length: length 0.94 mm; width 0.3 mm); (in preserved specimen with 9 mm length: length 0.95 mm; width 0.4 mm); completely contractible.Bursa copulatrix and seminal receptacle join vaginal duct in its proximal portion 37).Elongated to rounded bursa copulatrix 37).Seminal receptacle stalked and bean-shaped; twice or same volume than bursa copulatrix (Fig. 37).Vagina not convoluted; about same diameter of deferent duct .Vaginal opening immediately ventral to penis.Genital aperture surrounded by fold.
Nervous system (Fig. 38).Nervous system of B. punctata sp.nov.very similar to B. agassizii as described above, with the following exceptions: cp7 absent in cerebro-pleural ganglia.Nerves leaving buccal ganglia: nb1 inserting into esophagus; nb2 inserting into salivary ducts; nb3 leading to m5; connective cerebro-pleural-buccal shortly after nb3.Connective cerebro-pleural-pedal (ccpp) extremely short and it can only be seen after carefully dissecting.
Digestive system (Figs.[45][46][47][48][49][50][51][52].Digestive system of B. punctata sp.nov.very similar to B. agassizii as described above, with the following exceptions: Each jaw plate showing alternate rows formed by elongated elements without denticles with slight cruciform lateral expansion (18 elements transversal, in preserved specimen measuring 8 mm in length) ; anterior elements worn.Pair of m1va originating in posterior portion of oral canal (Fig. 51), running in the middle of buccal mass and inserting near radular sac.Pair of m1vb originating into dorsal portion of mouth and inserting into ventral portion of m5 (Fig. 50).Single auxiliary muscle m10a absent.Pair of strong retractor muscles originating in most posterior portion of m5 (Fig. 51); separated in middle of its anterior length and jointed in its posterior portion, laying above anterior por- tion of digestive gland.Radula rectangular; formula 72 × 38.0.38 (from preserved specimen 8 mm length); 65 × 47.0.47 (from preserved specimen 9 mm length).Radula lacking a rachidian tooth; innermost lateral tooth hook-shaped; subsequent lateral plates hook-shaped, larger and more developed in center of rows, base of tooth not enlarged (Fig. 47); outermost laterals teeth less developed (Fig. 48).Esophagus internally with longitudinal folds (Fig. 52).Salivary gland small and in front of digestive gland (Fig. 49).Ducts of salivary glands entering pharynx musculature laterally to esophagus, opening into base of pharyngeal cavity between radula and jaw plates (Fig. 50); convoluted; without visible ampulla.Voluminous stomach covering all dorsal portion of pharyngeal bulb (Figs.49-50); thin lines internally (Fig. 52); stomach passing dorsally into the digestive gland and surrounds the digestive gland .Posteriorly, stomach passes into intestine (Fig. 49); intestine long and thin and embedded in dorsal portion of the digestive and hermaphrodite glands and, opening laterally in body (Fig. 49); internally, intestine with longitudinal folds (Fig. 52).
Type  (Pruvot-Fol, 1953) and B. tamiu, mainly due to the pigmented spot in the middle of the mantle.
Mantle .Two types of spicules in mantle: linear, rod-like (length: 28 µm; thickness: 2.47 µm) (Fig. 68) and stellate with four-six rays irregular and in same plane or direct towards different plans (ray length: 2.88-20.18µm; ray thickness: 1.44-4.32µm) (Figs.69-70).Rod-like spicules calcareous; stellate spicules partially calcareous, but not entirely formed by calcium carbonate because in sodium hypochlorite they partially dissolve, it is also composed of an organic matrix not identified.
Shell (Figs. 9-10, 71-72).Two color types of shells were observed.First type, in small specimens up to 9 mm long alive, white and very fragile (Fig. 9); subquadrangular; covers all over dorsum; approximately two times longer than wide; length 3.2 mm, width 1.6 mm (in preserved specimen with 4 mm in length); and, length 2 mm, width 1.7 mm (in preserved specimen with 3.5 mm in length).Second type brown with first whorl translucent white (Fig. 10), not fragile as first type; convex, subquadrangular; covers approximately 70% of mantle; approximately two times longer than wide; length 5.7 mm, width 3.6 mm (with 16 mm long alive); and, length 8.8 mm, width 5.3 mm (in with 18 mm long alive).Spire with 1.5-1.7 whorls (Fig. 71).Protoconch smooth (Fig. 72).Lines of growth distinct; immediately after protoconch, with longitudinal sculptures transverse to lines of growth (Fig. 72); anterior portion of last whorl only lines of growth are recognizable (Fig. 71).
Circulatory system (Fig. 65).Circulatory system of B. stellata identical to the B. agassizii as described above.
Reproductive system (Fig. 66).Ampulla thick and elongated, about two-three times wider than deferent duct; curved, but not convoluted.Spermoviduct branching into two ducts, oviduct leading to female gland mass and other duct leading to prostate.Prostatic portion of deferent duct convoluted; not clearly differentiated from deferent duct.Near deferent duct distal end it joins with elongated penial gland and narrows near into conical penis.Penial gland about four-five times thicker than deferent duct.Penis (in specimen with 18 mm long alive: length 88 µm; width 73 µm); completely contractible.Bursa copulatrix and seminal receptacle join vaginal duct ZOOLOGIA 32 (6): 497-531, December 2015 in its ¾ distal portion.Rounded bursa copulatrix.Seminal receptacle stalked and elongated; representing 70% of length of bursa copulatrix.Vagina not convoluted; about same diameter or two times wider than deferent duct.Vaginal opening immediately ventral to penis.Genital aperture surrounded by fold.
Digestive system (Figs.73-85).Digestive system of B. stellata very similar to B. agassizii as described above, with the following exceptions: Muscle surrounding jaws (mj) strong, welldeveloped (Fig. 79).Jaws light yellow, lighter posteriorly.Each  VAYSSIÈRE 1898, THOMPSON 1985, WILLAN 1984, GOSLINER & BERTSCH 1988).In the specimens from Brazil, the general color of translucent white was the only one found, the yellowish-brownish pigment in fact refers to the visceral mass and sometimes the shell.GOSLINER & BERTSCH (1988) noted that some specimens presented yellow tubercles; however, in the specimens from Brazil such tubercles are not present.
The shell of B. stellata was described as white or translucent by many authors (RISSO 1826, PEASE 1860, RISBEC 1928), while only ER.MARCUS (1957) described it as brown for B. tupala and BURN (1962) as pale fawn for B. postrema.In the specimens of the present study, there are two color types: in small live specimens, up to 9 mm long, the shell is white and very fragile (Fig. 9); in larger specimens the shell is brown, with the first whorl translucent white (Fig. 10) and not fragile.Also, there is a correlation between the length of the shell and its color: white shells are smaller than brown shells.Some intermediate shell color, as light gold, are rare.
The innermost lateral teeth of the radula vary considerably, sometimes in the same specimen; they can be smooth or bear one denticle at the base.GOSLINER & BERTSCH (1988) found all outermost lateral teeth with secondary denticles, as many other authors did (ER.MARCUS 1957, WILLAN 1984, Gosliner 1987apud GOSLINER & BERTSCH 1988).In most specimens studied here, the outermost lateral tooth has a secondary basal denticle.In only one specimen (MNRJ 30345, from Santa Catarina) the outermost tooth is smooth (Fig. 77).This fact was already reported by VAYSSIÈRE (1898) andRISBEC (1928).Therefore, the radula is also a really variable character within this species.
Despite the wide geographic distribution of B. stellata, there are only a few publications describing its reproductive system, fully or partially (e.g., ER. MARCUS 1957, BURN 1962, WILLAN 1984, EV. MARCUS 1984, GOSLINER & BERTSCH 1988).GOSLINER & BERTSCH (1988) studied the reproductive system of specimens from different localities and concluded that the duct of the seminal receptacle may join the vagina at the base of the bursa copulatrix or near the middle of the vaginal duct.In specimens from Brazil, the bursa copulatrix and the seminal receptacle join the vaginal duct at its ¾ distal portion.ER.MARCUS (1957) described the reproductive system of B. tupala without mentioning the seminal receptacle, which led GOSLINER & BERTSCH (1988) to suggest that the description of the arrangement of the reproductive organs was wrong.We agree with this assumption, since B. tupala was originally described based on specimens from Brazil, as the material studied here, and we found the seminal receptacle in all specimens dissected, as expected.BURN (1962) also described only one allosperm receptacle and that is most likely an error.
Berthella tupala is the only species originally described from Brazil among the synonymies of B. stellata.The original description of B. tupala, as well as the description in the present work, agrees with the descriptions of B. stellata (e.g., BURN 1962, WILLAN 1984, EV. MARCUS 1984, GOSLINER & BETSCH 1988).The wide geographical distribution of B. stellata is quite questionable.The characters traditionally used to distinguish species of Berthella, external morphology, radula, jaw and reproductive system, are not as useful as previously supposed.Based on available descriptions of B. stellata, we cannot conclude whether we are dealing with more than one species or whether there are cryptic species involved, but we believe that molecular data and more detailed descriptions might help this process.In this paper we describe more characters than those used traditionally, since they might turn out to be useful for comparison with further descriptions of specimens of different localities.

Berthellina Gardiner, 1936
Berthella auct.non Blainville, 1824: VAYSSIÈRE, 1896: 115. Berthellina GARDINER, 1936: 198.Type species by original designation: Berthellina engeli Gardiner, 1936. Description (adapted from WILLAN 1983, 1987, THOMPSON 1970, EV. MARCUS 1984).Body elliptical or convex; internal shell between one-quarter and one fifth the body length, or absent; pedal gland never present; gill rachis smooth; anus at posterior end of gill membrane; radula teeth elongate, lamelliform, with denticles on distal section of posterior edge; jaw elements smooth or denticulate with forward denticles.Pair m4, main dorsal tensor muscle of radula, well developed, originating in lateral region of cartilages, surrounding them ventrally, inserting into subradular membrane.Pair m5, secondary dorsal tensor muscle of radula, covering median portions of cartilage, extending up to dorsal region; originating in posterior surface of cartilages; inserting laterally in mj.Pair m7 absent.Pair m10d (dorsal) absent.Pair m10v (ventral), protractor muscle of odontophore, connected posterior portion of canal oral with ventral portion of m5.Single auxiliary muscle m10a, ventral tensor muscle of radula, originating in anterior portion of oral canal, running in middle of buccal mass and inserting into radular sac.Pair of strong retractor muscles originates in most posterior portion of m5 and radular sac.
Remarks.GARDINER (1936) proposed the genus Berthellina, based on B. engeli, to include species with lamellate radular teeth.VAYSSIÈRE (1896) presented his classification scheme for Pleurobranchoidea and described each genus and subgenus of it.However, according to GARDINER (1936), the species called Berthella by VAYSSIÈRE (1896) in fact represents Berthellina.The general features of Berthellina that VAYSSIÈRE (1896) pointed out were: polygonal elements of the jaw, usually without lateral denticles, and lamellar radular teeth with serrate edges at the upper half of the teeth.
Shell (Fig. 101).White, decalcified and fragile; two times longer than wide; yellowish periostracum.Convex; subquadrangular in outline (Fig. 101).Length 4.3 mm, width 2.7 mm (in preserved specimen with 22 mm in length).Spire with 1.5 whorls.Protoconch smooth.Lines of growth distinct; immediately after protoconch, with longitudinal sculptures transverse to lines of growth; anterior portion of last whorl only lines of growth are recognizable.Shell above heart, on right side of blood gland (Fig. 91).
Circulatory system (Fig. 93).Pericardium well developed in anterior portion of body (near cerebro-pleural ganglia).Blood flowing into auricle from gills, kidney and venous sinuses.Efferent branchial vessel connecting gill with the auricle.Auricle on right side, ventricle on left; auricle with thin wall; ventricle muscular.Blood gland small, creamy covering left part of pericardium.Blood gland close or joined to aorta; it covers partially ventricle.
Reproductive system .Ampulla elongated, two times wider than deferent duct; curved, sometimes slightly convoluted (Fig. 95).Spermoviduct branching into two ducts, long oviduct leading to female gland mass, other duct leading to prostate (Fig. 95).Prostate globular and granular; five-seven times wider than deferent duct.Deferent duct not convoluted, narrowing into not cuticular penis and penial gland (Fig. 95).Penis semi-internal, never completely internal when retracted (Fig. 94).Penial gland elongated, not-convoluted.Penis half-moon shape (in preserved specimen with 21 mm length: length 1.7 mm; in preserved specimen with 25 mm length: length 1.8 mm).Seminal receptacle stalked .Rounded bursa copulatrix; seminal receptacle two times more volume than seminal receptacle (Fig. 96).Vagina not convoluted; two times wider than deferent duct.Vagina opening immediately ventral to penis.Genital aperture surrounded by wide flap (Fig. 94).
Nervous system (Fig. 97).Nerve ring above oral canal.Cerebral and pleural ganglia fused.Eyes located latero-centrally of cerebro-pleural complex; eyes borne upon very short optical nerves.Rhinophoral ganglia placed at bases of rhinophores, near cerebro-pleural ganglia; two main nerves leaving from rhinophoral nerves, runing until distal portion of rhinophores; rhinophoral nerves with many secondary nerves, perpendicular in relation to main nerves.Nerves leaving cerebro-pleural ganglia: cp2 bifurcating near origin, both cp2a and cp2b in-serting dorsally into mantle; cp6 inserting dorsally into mantle; cp3 runing until median portion of body wall, near anterior portion of digestive gland in left side, inserting into mantle laterally.Connective between visceral and cerebro-pleural ganglia well developed, which has two main nerves.Connective between buccal and cerebro-pleural ganglia leading from ventral view of cerebro-pleural ganglia, in the most anterior portion of ganglion.Nerves leaving buccal ganglia: nb1 bifurcating near origin, both nb1a and nb1b inserts into esophagus; nb2 inserting into salivary ducts; connective cerebro-pleural-buccal shortly after nb1; nb3 inserting into m4; nb4 inserting into radula sac; nb5 inserting into retractor muscles of buccal bulb.Connective cerebro-pleural-pedal (ccpp) extremely short and can only be seen after carefully dissecting.Pedal commissure short.Pedal ganglia smaller than cerebro-pleural complex: np1 inserting ventrally into oral veil; np2 inserting anteriorly into foot; np3 innervating foot; np4 inserting ventrally into foot and runs until the most posterior portion of body.In the right side of pedal ganglion, just after the np2, there is a connective that leads to genital ganglion.
Digestive system .Mouth transversal in middle of snout tip.Oral canal muscular just posterior to mouth, representing approximately 1/3 of pharyngeal bulb length; internally rough .Muscle surrounding jaws (mj) strong, pair of large jaws located in its inner surface , mj originating in lateral and dorsal surfaces of oral canal, inserting into lateral and dorsal regions of buccal mass.Jaws amber, lighter posteriorly; jaw of two plates surrounding radula inside buccal cavity; elongated, reaching level of radula.Each jaw plate showing alternate rows formed by elongated elements with a cruciform lateral expansion (104 longitudinal and 70 transversal, in preserved specimen of 12 mm long and 109 longitudinal and 64 transversal, in preserved specimen of 21 mm long) ; each element with 1-5 small denticles, but some elements could be not symmetric; anterior elements worn.Pair of dorsal jugal muscles m1d (Fig. 107), inserting into m4, connecting to dorsal-anterior end of oral canal.Pair m4, main dorsal tensor muscle of radula, well developed, originating in lateral region of cartilages, surrounding them ventrally, inserting into subradular membrane .Pair m5, secondary dorsal tensor muscle of radula, covering median portions of cartilage, extending up to dorsal region; originating in posterior surface of cartilages; inserting laterally in mj .Pair m7 absent.Pair m10d (dorsal) absent.Pair m10v (ventral), protractor muscle of odontophore, connecting posterior portion of oral canal with ventral portion of m5 (Fig. 108).Single auxiliary muscle m10a, ventral tensor muscle of radula, originating in anterior portion of oral canal, running in middle of buccal mass and inserting into radular sac (Fig. 108).Pair of strong retractor muscles originates in most posterior portion of m5 and radular sac (111)(112); separated in 1/2 its total length and jointed in its posterior portion, laying above anterior portion of digestive gland (Fig. 108).Odontophoral cartilages subquadrangular in outline (Fig. 110).Radula amber; formula 108 × 144.0.144 (from a preserved specimen with 21 mm length) and 56 × 107.0107 (from a preserved specimen with 12 mm length).Rachidian tooth absent.Lateral teeth long and slender with subtermial denticles .Innermost lateral tooth with 4-8 irregular denticles (Fig. 104).Subsequent lateral plates larger and more developed in center of rows with 10-17 irregular denticles (Fig. 105).Outermost laterals teeth less developed up to 10 denticles (Fig. 106).Aperture of acid gland located between jaw plates .Duct of acid gland thin (same width as salivary duct) (Fig. 107); runs between stomach (until median portion of stomach) and hermaphrodite gland when it clearly distinguishes two species.In this paper, the radula of some specimens of B. quadridens mounted by Evelinae Marcus were analyzed (MZSP 121401, MZSP 121402), probably the same material studied by EV.MARCUS (1976) and EV.MARCUS (1984) (see discussion in material and methods section), and it was observed that the majority of teeth have two denticles (Figs. 113,115), rarely three or four (Fig. 114).This pattern was not observed in the material of Berthellina from Brazil, since their teeth are highly denticulate, with up to 17 denticles .Furthermore, B. circularis has fewer denticles in jaw elements, 1-5 forward denticles , than B. quadridens, 1-8 forward denticles.Given what was discussed above we follow EV.MARCUS (1984) in considering that only B. circularis occurs in Brazil and B. quadridens does not.The latter is restricted to the Caribbean Sea and the Pacific coast of Panama, contradicting the authors who recorded it from Brazil (e.g., EV. MARCUS & ER. MARCUS 1969, EV. MARCUS 1972, 1979, VALDÉS et al. 2006, GARCÍA et al. 2008, RIOS 2009, PADULA et al. 2012).
Mantle .Mantle covered by sparse spicules.Two types of spicules in mantle: linear, rod-like (length: 120-130 µm; thickness: 5-10 µm) (Fig. 123) and stellate, with linear rays (ray length: 27.19-30.0µm; ray thickness: 10 µm) (Fig. 124); stellate spicules difficult to see how many rays it have, some with four rays, because many of them broken into mantle (Fig. 125).Rod-like spicules calcareous; stellate spicules partially calcareous, but not entirely formed by calcium carbonate because in sodium hypochlorite breaks itself, it is also composed of an organic matrix not identified.
Shell (Fig. 126).Shell white to brownish, decalcified, flattened and fragile; two times longer than wide; subquadrangular in outline.Length 4.7 mm, width 2.6 mm (in preserved specimen with 17 mm in length).Spire with 1.5 whorls.Protoconch smooth.Lines of growth distinct; immediately after protoconch, with longitudinal sculptures transverse to lines of growth; anterior portion of last whorl only lines of growth are recognizable.Shell located in middle to posterior portion of body (Fig. 126).
Circulatory system (Fig. 118).Circulatory system of B. ignis sp.nov.identical to the B. circularis as described above.
Nervous system (Fig. 122).Nervous system of B. ignis sp.nov.very similar to B. circularis as described above, with the following exceptions: Nerves leaving cerebro-pleural ganglia: cp6 inserting dorsally into mantle, nerve very short; cp4 runing until most posterior portion of body, inserting into body wall, only in the left ganglion of cerebro-pleural ganglia, in the right ganglion absent; cp5 inserting dorsally into mantle.Connective between visceral and cerebro-pleural ganglia short.Nerves leaving buccal ganglia: nb1 bifurcating near origin, both nb1a and nb1b inserting into esophagus; nb2 inserting into salivary ducts; cerebro-pleural-buccal shortly after nb1; nb3 inserting into m4; nb4 inserting into radula sac; nb5 inserting into retractor muscles of buccal bulb; nb6 inserting into duct of acid gland.Pedal ganglia smaller than cerebro-pleural complex: np1 inserting ventrally into oral veil; np2 and np3 was not observed, nervous system small and difficult to analyze, it could exist; np4 inserting ventrally into foot and runs until the most posterior portion of body.
Digestive system .Digestive system of B. ignis sp.nov.very similar to B. circularis as described above,

DISCUSSION
Based on the present detailed anatomical study, we conclude that the circulatory and nervous systems do not provide useful characters for species differentiation.For example, between Berthellina, the only difference found in the nervous system was in the nerves that extend from the cerebro-pleural ganglia, cp4 and cp5, which are present in B. ignis sp.nov., but not in B. circularis (Figs. 97,122).ORTEA et al. (2014) proposed that the network of subepidermal fibers could be used to characterize Bertellina species, but did not describe or illustrate how to differentiate species based on this feature.In B. circularis and B. ignis sp.nov., such network of subepidermal fibers is present, but we have not been able to distinguish a general pattern.
The radula and jaw elements, when mentioned, are the only features of the digestive system that appear in older descriptions.In search for new characters, we have analyzed the odontophoric muscles and have uncovered a general pattern in the position and presence/absence of extrinsic and intrinsic odontophoric muscles for Berthella and Berthellina.As already observed for Pleurobranchaea (ALVIM et al. 2014) and Pleurobranchus (pers.obs.), the odontophoric muscles are useful characters, particularly to distinguish among genera, and in some cases species, and we believe that they should be considered in future descriptions.
Berthella and Berthellina present the pair of the muscle m10v connecting the mj with the ventral portion of m5, al-though the pair of the muscle m10v connects the mj with the ventral portion of m4 in Pleurobranchus (pers.obs.) and Pleurobranchaea (ALVIM et al. 2014).The pair of the muscle m10d is absent in Berthella and Berthellina; in contrast, Pleurobranchus and Pleurobranchaea present the pair of m10d (ALVIM et al. 2014, pers. obs.).Based on these characters, Berthella seems to be more closely related to Berthellina than to Pleurobranchus.These new data may help improve the understanding of the evolutionary history of Pleurobranchidae.
Among the species of Berthella analyzed in the present study, some differences were observed concerning the odontophoric muscles.Berthella agassizii follows the same pattern already reported for other pleurobranchids, with the presence of a single auxiliary muscle m10a (pers.obs.), a ventral tensor muscle of the radula, which originates in the anterior portion of the oral canal, running in the middle of the buccal mass and inserting into the radular sac.However, the auxiliary muscle m10a is absent in B. stellata and B. punctata sp.nov., which only present one pair of m1v.This difference is outstanding and it can be the starting point to understand the evolutionary history of the genus and to recover natural groups, since Berthella is currently known as paraphyletic (MARTINOV & SCHRÖDL 2009).The anatomical study of the odontophoric musculature of more species of Berthella is necessary to understand if these patterns are present in other species.
In Brazil, B. punctata sp.nov., B. circularis and B. ignis sp.nov.are restricted to the Tropical Atlantic, in Tropical Northwestern Atlantic provinces, North Brazil Shelf and Tropical Southwestern Atlantic (SPALDING et al. 2007).Berthella punctata sp.nov., B. circularis and B. ignis sp.nov.were not observed in the Warm Temperate Southwestern Atlantic province, despite exhaustive collecting in Rio de Janeiro, São Paulo and Santa Catarina.One possible explanation for species with this distribution was suggested by COELHO & RAMOS (1972), for Decapoda, in which those species are adapted to warm waters, where the seasonal variation of temperature is reduced.Taxonomic review of Berthella and Berthellina from Brazil, with description of two new species ZOOLOGIA 32 (6): 497-531, December 2015 de Amparo à Pesquisa do Estado do Rio de Janeiro), through project E-26/110.325/2014 and project E-26/110.068/2014.

53 54 55 56 59 58 57 60 the
jaw elements of B. punctata sp.nov.lackdenticles(onecusp),whileRISBEC (1928: fig.68)described the jaw elements of P. caledonicus as having one or two cusps; fourth, the radular formulae differ in both species (B.punctata sp.nov.72 × 38.0.38, in a 8 mm long preserved specimen; P. caledonicus 130 × 150.0.150).Additionally, RISBEC (1928) mentioned the presence of a mucus gland on the left side of the pharyngeal bulb near the mouth of P. caledonicus, which was not observed in B. punctata sp.nov.A future study of specimens of P. caledonicus from New Caledonia is necessary to elucidate the correct generic allocation of P. caledonicus.VALDÉS et al. (2006) illustrated a morphotype named as Berthella sp. from Puerto Rico that closely resembles B. punctata sp.nov. in the external morphology and coloration.A detailed comparison between specimens from Brazil and Puerto Rico is not possible because there is no data available on the internal anatomy in VALDÉS et al. (2006).Pleurobranchus stellatus Risso, 1826: 41; Mazzarelli, 1891: 73, figs.1-5.Pleurobranchus pellucidus Pease, 1860: 24; Risbec, 1928: 63.Synonymized by GOSLINER & BERTSCH (1988).