Rediscovery of Obeliscus agassizi Pilsbry, 1906 (Gastropoda, Subulinidae, Obeliscinae), annotated checklist of species of Obeliscus Beck, 1837 and first description of the anatomy for the genus

ABSTRACT Obeliscus agassizi Pilsbry, 1906 was described from specimens collected in Brazil, by J. G. Anthony, during the Agassiz expedition, in 1865. The type locality of the species was given simply as Brazil, without further information. More than a century after its description, this species was rediscovered during an expedition to the Biological Reserve of Pedra Talhada, Alagoas/Pernambuco states, Brazil. In the present study we provide detailed description of the anatomy of the soft parts of O. agassizi specimens, collected during this expedition, besides more precise data concerning the species occurrence. This work presents the first anatomical description for the genus. An annotated checklist of Obeliscus Beck, 1837 species is also provided. The species of Obeliscus have been traditionally distinguished by shell characteristics, mainly the general shell shape, number of whorls proportionally to the shell size, shell ornamentation, the shape of the protoconch and aperture size and shape. The distinction between species is often subtle. Considering that shell traits may be not sufficient as single taxonomic characters, other evidence for species boundaries, especially anatomical characters, is needed. Presently, it is not possible to discuss the taxonomic meaning of the set of anatomical characters described for O. agassizi, at the intra-genus level, as there is no information on the anatomy of the soft parts of other species of Obeliscus. The comparative analysis of the genital system of O. agassizi, Neobeliscus calcareus (Born, 1780), Stenogyra terebraster (Lamarck, 1822) and Rectobelus birabeni (Hylton-Scott, 1946) pointed out anatomical characters with potential diagnostic significance for the genus Obeliscus. However, the establishment of a definitive differential diagnosis for this genus, based in both conchological and anatomical traits, depends on the analysis of a greater number of species of Obeliscus, as well as species of other genera of Obeliscinae Thiele, 1931.


INTRODUCTION
Subulinidae Fischer & Crosse, 1877 is a near pan-tropical family of land snails, including nine subfamilies, 81 genera and about 820 species Schileyko 1999). The systematics of this family has been entirely based on shell characteristics and the attempts to add new data about this family have been very scarce throughout the last century. Hence, Subulinidae figures as a neglected group of land snails, contrasting with its diversity.
Considering the lack of other criteria of discrimination besides the shell (available data on the internal anatomy of Subulinidae concern 30 genera and no more than 32 species [Schileyko 1999;Medeiros et al. 2013]), and taking into account that no full systematic revision of the family has been provided during more than a century, the description of the anatomy of the soft parts as well as the characterization of life history traits of subulinid species is a necessary challenge. Without these two sources of information, studies on ecology, evolution, phylogeny and conservation of subulinid species will remain in a state of impediment.
Obeliscinae Thiele, 1931 comprises 12 American, one Asian and three Pacific genera (Schileyko 1999). Among the 12 American genera, five were recorded in Brazil, including at least 13 endemic species Simone 2006). Only Neobeliscus calcareus (Born, 1780) (Pilsbry 1899, Stenogyra terebraster (Lamarck, 1822) (Schileyko 1999) and Rectobelus birabeni (Hylton-Scott, 1946) were characterized from an anatomical perspective. So far, all other species included in this subfamily are known only from their shells. More than a century ago,  stated that besides the information on the anatomy of N. calcareus given by him, nothing was known of the soft parts of the other genera. ZOOSYSTEMA • 2020 • 42 (12) The more recent revision of the family Subulinidae was performed by Schileyko (1999), who has provided new information on the anatomy of the soft parts for several genera. Schileyko (1999) illustrated the anatomy of the genital system of S. terebraster (new data) and of N. calcareus after . However, the author did not describe the anatomy of the other genera, including Obeliscus Beck, 1837, the type genus. Schileyko (1999) argued that the characters previously used to define Obeliscinae are not sufficient, proposing the maintenance of this subfamily until information on the anatomy of the soft parts of their representatives is available.
The genus Obeliscus Beck, 1837 includes about 16 species, distributed throughout Tropical South America and Greater Antilles. So far, none of these species was anatomically characterized. The diagnosis of the genus reviewed by  includes only shell characteristics and some fragmentary information on the radula of O. obeliscus, taken from the remains washed from a dry shell. After the work of , other additional five species of Obeliscus were described based on shells only (Vanatta 1918;Pilsbry 1944;Simone & Salvador 2016;Thach 2017).
Obeliscus agassizi Pilsbry, 1906 was described from specimens collected in Brazil by J. G. Anthony, during the Agassiz expedition. The type locality of the species was given simply as Brazil, without further information. More than a century after its description, this species was rediscovered by Maestrati et al. (2015) during an expedition to the Biological Reserve of Pedra Talhada, Alagoas/Pernambuco states, Brazil. In the present study we provided detailed description of the anatomy of O. agassizi, based on specimens collected during that expedition. An annotated checklist of Obeliscus species is also provided.

MATERIALS AND METHODS
The material consists of four specimens of Obeliscus agassizi, preserved in ethanol 70% (MZSP 133507;MZSP 133635), and deposited in the malacological collection of the Museu de Zoologia da Universidade de São Paulo (MZSP). All specimens were collected in the Pedra Talhada Biological Reserve, located in the municipalities of Quebrangulo in the state of Alagoas and of Lagoa do Ouro in the state of Pernambuco. Pedra Talhada is a federally administered biological reserve in North East Brazil, which contains a representative sample of the remaining Atlantic Rainforest ecosystem in the Serras of Guaribas, Pedra Talhada and Serra do Cavaleiro.
The snails were dissected immerse in ethanol and drawn under an Olympus stereo microscope, model SZX7As, equipped with camera lucida. Details of the shell were acquired through table top scanning electron microscope EDS. Shell specimens were analyzed without any preparation.
Identification was conducted based on the species original description provided by  as well as on the revision of the family Subulinidae provided by Schileyko (1999), images of the shells of type specimens available in the online collection database of the ANSP, and the catalogue by Simone (2006).
To provide an annotated checklist of the species of the genus Obeliscus an intensive research was performed using both the literature and collections databases of the Academy of Natural Sciences of Philadelphia, Bailey The search in these online databases allowed us to update the geographical distribution of some species. All museum depositits numbers were provided in the checklist, including information on type specimens. Museum deposit numbers were also referenced in the topic "other localities" when occurrence data were obtained from the databases consulted online. We also provided the website links for the species original descriptions, illustrations, museum deposits and bibliographic references cited in the checklist.
All the species are listed according to their current taxonomic status. The citations of the specimens from the collections databases are in accordance with the format proposed by Chester et al. (2019). Some information were sometimes not available in the databases, such as the number or the kind of available specimens (dry shell or ethanol preserved shell and soft parts).  1C). Embryonic whorls smooth, shining (Fig. 1A). Protoconch with c. 3 whorls. Boundary between protoconch and teleoconch well defined. Last whorls plicate below suture, nearly smooth elsewhere (Fig. 1B). Spire of c. 12 whorls, convex, very regularly increasing ( Fig. 2A). Sutures well marked, a little inclined (Fig. 1B, E). Teleoconch of c. 10 whorls, smooth, except for growth lines (Fig. 2D). Aperture somewhat oblique, ovate (Fig. 1C); inner lip simple, outer lip thin.

Head-foot (Figs 1D-F; 2B, C)
Head protruded. Head-foot almost transparent in living animals, color pale cream, with fine tessellation (Figs 1D-F, 2B). Foot broad, sole oval, edges rounded in fixed animals. Genital opening visible as a rounded area, without tessellation in fixed animals, and as a small fissure near head in living animals (Fig. 2B).
Mantle organs (Fig. 2C Circulatory and excretory systems (Fig. 2C) Pericardium located at columellar margin of posterior end of pallial cavity, c. 1/3 of the kidney length. Auricle small, triangular, located in middle length of kidney left margin. Ventricle near same size of auricle. Kidney occupying c. 1/5 of pulmonary cavity length, located along middle and left regions of transition pallial cavity and visceral mass. Kidney rather bilobed, with smaller lobe just anterior to auricle. Pulmonary vein running along columellar margin of pallial cavity, straight in posterior 4/5 portion of the mantle, curved in anterior 1/5 portion, converging to pneumostome region along with renal tube and rectum.

Visceral mass (Figs 2C, E)
Digestive gland brownish, located along inferior region of seventh visceral whorl, covering part of stomach at sixth whorl and filling entire visceral whorls posterior to stomach, including nepionic whorls (Fig. 2E). Rectum visible at eighth visceral whorl.

Genital system (Figs 3A-F)
Gonad located at the sixth visceral whorl, covered by the digestive gland (Fig. 3E: go). Albumen gland elongated, sheltering spermoduct under its columellar face. Carrefour lying at tran- sition between spermoviduct and albumen gland (Fig. 3B). Seminal receptacle as wide sinuous tube, lying at superior half of albumen gland (Fig. 3B: sr). Spermoviduct long, occupying first three shell whorls, running along columellar muscle (Fig. 3A, E). Autospermoduct situated at columellar face of spermoviduct ( Fig. 3A: at). Allospermoviduct situated at the opposite face, visible as a separated duct until reach the freeoviduct (Fig. 3A, E: al). Prostate with well defined acini, occupying columellar surface of spermoviduct (Fig. 3A, E: pt). Bursa copulatrix about as wide as its duct (Fig. 3E: bc), lying in transition between spermoviduct and free oviduct; length c. 1/2 of that of spermovioduct. Free oviduct c. 1/2 length of bursa copulatrix duct. Vagina c. 1/2 free oviduct length. Atrial retractor muscle well developed. Vas deferens with greater diameter at level of free oviduct, adhered to free oviduct wall at its more convoluted and thicker portion through fine muscle bundles. Vas deferens meeting gland with unidentified function after leaving prostate, running along entire free oviduct; vas deferens convoluted in median portion of free oviduct, showing greater diameter until basis of free oviduct, joining to penis. Penis long, club-shaped (Fig. 3A, C: pe). At the meeting point with the penial complex, the vas deferens forms a discrete bursa which lies on the phallus wall. Penial complex passing under right ommatophore and under esophagus. Penial retractor muscle (Fig. 3D: pm) originated in diaphragm region close to columellar muscle, inserted terminally in the epiphallus. illustrAtion. -Vanatta (1918: fig. 3).

life history trAits of Obeliscus
Although there is no study on the life history of species of Obeliscus, fragmentary data on reproduction can be found in the taxonomic literature. Some information given by Moricand (1836: 11, pl. 2, fig. 28) and reaffirmed by Pilsbry (1906: 246) on O. obeliscus indicate that this species is ovoviviparous, releasing non-calcified eggs formed by a thin and transparent membrane containing shelled embryos. About Obeliscus swiftianus (Pfeiffer, 1852),  affirmed: "…none of the specimens seen contains eggs, such as are commonly seen in Opeas. It is probably viviparous, like all Obelisci." Since this author mentioned only oviparity and viviparity, discriminating viviparous species as those containing shelled embryos in the spermoviduct, we believe that this species is in fact ovoviviparous. Ovoviviparity and egg retention seem to be widespread reproductive modes in subulinids Baker 1927;Batts 1957;Araújo & Keller 1993;Schileyko 1999;Carvalho et al. 2009; Pilate 2013) and may be common to obeliscine snails (Moricand 1836; Pilsbry & Vanatta 1899;Pilsbry 1899. tAxonoMic reMArks Schileyko (1999) estimated about 10 species of Obeliscus. In the present work, we listed 16 species of Obeliscus, nine of them recorded in Brazil. Among the species occurring in Brazil, six are endemic to this country.
The species of Obeliscus have been traditionally distinguished by shell characters, mainly the general shell shape, number of whorls proportionally to the shell size, shell ornamentation, the shape of the protoconch, and aperture size and shape. The distinction between species is often subtle. Considering that shell features may be not sufficient as single taxonomic characters, other evidences of species boundary, especially anatomical characters, is needed.
Obeliscus agassizi and O. sylvaticus were found by  cohabiting in the same collecting site. This fact, along with the close resemblance in their shells, indicates the need to provide a differential diagnosis for these species, through a more exhaustive anatomical characterization. Currently, the two species can be differentiated mainly by the shape of the shell aperture. Obeliscus sylvaticus has a more slender shell with a smaller aperture, which is also quadrangular in shape, while the shell aperture of O. agassizi is somewhat oblique and ovate in shape. The strength of shell traits to delimit species of Obeliscus should be evaluated in the future, with the accumulation of information on the anatomy of the soft parts and with the use of molecular markers.
Presently it is not possible to evaluate the taxonomic significance of the various morphological traits of the soft parts for the Obeliscinae genera as there is information only on four species from four genera. The data on the anatomy of the genital system of O. agassizi (present study), Neobeliscus calcareus provided by Pilsbry (1899, Stenogyra terebraster provided by Schileyko (1999) and Rectobelus birabeni provided by Hyton-Scott (1946), did not allow a comprehensive comparative analysis, but some anatomical differences among the representatives of these four genera are worth of mentioning. Obeliscus agassizi differs from N. calcareus in the site of insertion of the vas deferens into the penial complex, the site of insertion of the bursa copulatrix duct into the free oviduct, the site of insertion of the penial retractor muscle and the size of the bursa copulatrix duct relative to the size of the free oviduct. Obeliscus agassizi differs from R. birabeni in the site of insertion of the vas deferens into the penial complex, the site of insertion of the penial retractor muscle and the size of the bursa copulatrix duct relative to the size of the free oviduct. Obeliscus agassizi differs from S. terebraster in the site of insertion of the bursa copulatrix into the free oviduct and in the site of insertion of the vas deferens into the penial complex.
Considering the anatomical characteristics common to the genera Obeliscus, Neobeliscus, Stenogyra, some traits are possible diagnostic characters for this subfamily. This concerns characters of the genital system, i.e., atrium and vagina short; long, club-shaped penial complex and terminal penial retractor muscle; of the circulatory and excretory systems, i.e., venation faint; of the respiratory system, i.e., pulmonary vein unbranched; and of the excretrary system, i.e., kidney more than double the length of the pericardium, and kidney length contained about 31/2 times in that of the mantle. The genus Rectobelus Baker, 1927, share with the genera Obeliscus, Neobeliscus, Stenogyra the presence of short atrium and vagina, and long, club-shaped penial complex. This genus must to be reexamined since there is no description of the circulatory and excretory systems. The penial complex of R. birabeni is not long and club-shaped as observed for the other three genera.
This preliminary analysis gives an idea of potential diagnostic characters, but it is still necessary to evaluate if they are consistent in examining a great number of species from these and the other Obeliscinae genera, along with the intra generic variability on these traits.
Author's contributions SD conceived and designed the study, performed anatomical and taxonomic analysis, original illustrations and wrote this manuscript. LRLS edited the plates and co-wrote this manuscript. LFCO performed shell SEM analysis and revised this manuscript. CL collected the studied specimens and revised this manuscript. PM collected the studied specimens and co-wrote this manuscript.