Eupera troglobia sp. nov.: the first troglobitic bivalve from the Americas (Mollusca, Bivalvia, Sphaeriidae) Author Simone, Luiz Ricardo L. https://orcid.org/0000-0002-1397-9823 Museu de Zoologia da Universidade de Sao Paulo; Cx. Postal 42494; 04218 - 970, Sao Paulo, SP, Brazil lrlsimone@usp.br Author Ferreira, Rodrigo Lopes https://orcid.org/0000-0003-3288-4405 Museu de Zoologia da Universidade de Sao Paulo; Cx. Postal 42494; 04218 - 970, Sao Paulo, SP, Brazil text Subterranean Biology 2022 2022-04-13 42 165 184 http://dx.doi.org/10.3897/subtbiol.42.78074 journal article http://dx.doi.org/10.3897/subtbiol.42.78074 1314-2615-42-165 9301CE7742B54F66A62B1E99949D6467 BA257F00A9ED5A07AC989F671CBA85A7 Eupera troglobia sp. nov. Figs 1-6 , 7-13 , 14-21 , 22-26 , 27-30 Material examined. Holotype . MZSP 155717. Paratypes MZSP 155716, 12 specimens , MNRJ 23647, 1 specimen , USNM, 1 specimen , all from type locality. Type locality. Brazil. Tocantins; Lagoa da Confusao , Casa da Pedra cave, 10°49'28.4"S , 49°37'16.5"W [Ferreira col., 3.viii.2021]. Diagnosis. Adult size ~4.5 mm. Lacking pigmentation in shell and soft parts. Shell very fragile, translucent, light yellow. Description. Shell (Figs 1 - 18 ). Adult shell ~4.5 mm. Equivalve; height ~80% of length; width ~60% of length. Walls thin, fragile, translucent. Anterior edge rounded, smaller than posterior edge; ventral edge rounded in medium specimens (Figs 7-9 ) to slightly ascendent in larger specimens (Figs 1-4 , 12 ); posterior edge almost straight in its middle level; dorsal edge weakly convex, almost straight. Color light yellow to light greenish yellow (Figs 1-6 ). Outer surface opaque. Sculpture of uniform concentric growth lines (Figs 1 , 2 , 12 , 13 ); ~15 per mm; each line alternating in height along its length (Fig. 13 ), but mostly 4-5 times taller than wide; interspaces ~10 times wider than each line. Growth lines usually continuous from anterior up to posterior hinge region (Figs 1 , 2 , 12 ). Umbo (um) slightly prosogyrate, central, protruding ~10% height beyond hinge level (Figs 1-4 , 8-9 ), occupying ~20% of dorsal edge. Hinge with small, blunt cardinal tooth (ca) in LV, ~1.5 times longer than tall, tip rounded (Figs 14-16 ), shallow correspondent socket in RV (Fig. 16 ); anterior (al) and posterior (pl) lateral teeth relatively equidistant from cardinal tooth (Figs 4-6 , 7-11 , 14-18 ), similar to each other, in both valves; located in anterior and posterior ends of hinge edge, in blunt angle preceding anterior ad posterior slopes; each lateral tooth ~4-times longer than wide, parallel to hinge edge; anterior pair of tooth usually with anterior small beak (Figs 14-15 : al, 17-18); both lateral teeth of LV slightly more ventral than lateral teeth of RV, encasing ventrally to them; both lateral teeth of RV with narrow socket lying dorsally for counterparts of LV (Figs 5 , 9 , 11 , 18 ). Inner surface glossy; scar of anterior adductor muscle (aa) occupying ~5% of entire inner valve surface, ~twice taller than wide, elliptic, slightly larger than scar of posterior adductor muscle (pa) (Figs 3-4 , 7-9 ). Pallial line continuous, simple, connecting both adductor muscle scars; relatively broad; located along ventral edge ~15% of total height distant from it. Inner surface possessing minute pits, possibly of aesthetes (Figs 10-11 : ae). Main muscle system (Figs 22 , 24 , 27 ). Anterior adductor muscle (aa) with elliptic transverse section, dorso-ventral height of slightly twice anterior-posterior width; located close to blunt angulation between dorsal and anterior shell edges. Posterior adductor muscle (pa) slightly smaller and located slightly more ventrally than anterior muscle. Pair of anterior pedal retractor muscles (ar) originated just dorsal to anterior adductor muscle in elliptic area equivalent to ~15% of that of anterior adductor muscle; running towards posterior relatively narrow, along ~20% of shell length, attached to adjacent visceral integument; insertion splaying in antero-dorsal foot base. No detectable protractor pedal muscle. Pair of posterior pedal retractor muscles (pr) originating similarly as anterior pedal muscle, but dorsally to posterior adductor muscle; running narrow anteriorly along ~50% of shell length, as central base to local visceral mass and attached to adjacent visceral integument; insertion splaying in postero-dorsal side of pedal base. Foot (Fig. 24 : ft). Cylindric, ~3-times longer than wide in contracted condition; ~half projected anteriorly beyond its base. Posterior region ventrally bulged, rounded. Anterior end bluntly tapering. Byssal furrow (bf) narrow, occupying ~1/4 of middle region of ventral foot surface. Byssus (by) as single, narrow, yellow thread. Mantle (Figs 22 , 26 , 27 ). Mantle lobe thin, translucent, thickened only in edges. Colorless. Edges of both lobes fused with each other in region of anterior adductor muscle, and in region posterior to middle level of ventral edge; fusion provided by inner fold. Mantle edge in non-fused region (Fig. 26 ) with narrow, flattened outer (of) and middle (mf) folds, no papilla or special structures detectable; inner fold (if) located in inner base of middle fold, with ~half of remaining folds height, as wide as tall. Pallial musculature (pm) thin, present in base of three folds. Fusion between posterior half of mantle edges (un) simple. Incurrent siphon (Fig. 27 : is) simple, cylindric, walls weakly muscular; distal edges simple, lacking papillae; length in retracted condition ~5% of shell length, ~twice longer than wide. Excurrent siphon (ex) similar to, but ~30% smaller than incurrent siphon; preserved inverted in several specimens. Siphonal musculature immersed in local mantel edges, lacking detectable bundles, neither producing pallial sinus in shell. Gill suspensory membrane (su) connected by cilia in posterior end of gill, membrane-like separating completely incurrent from excurrent chambers (Fig. 27 ). Pallial cavity (Fig. 22 ). Outer demibranchs (od) with ~1/4 of shell height in its middle region; tapering gradually towards anterior, up to middle level of inner demibranch dorsal edge; tapering subtly towards posterior; lamellae very narrow, with ventral curve covering small region of inner demibranch dorsal edge (Fig. 23 : od); dorsal connection with visceral mass via cilia (ci). Inner demibranchs (di) wide, area ~double as outer demibranchs; anterior region slightly wider than half of shell height, gradually tapering towards posterior up to certain distance from posterior adductor muscle; transversely folded; descendent lamella (Fig. 23 : id) simple, very narrow, free from ascendent lamella; ascendent lamella with ~70% of descendent lamella length; narrow food groove (fg) in inner demibranch ventral edge; inner demibranch connections with visceral mass and its counterpart (in posterior half - Fig. 24 ) via cilia (ci). Inner demibranch serving as marsupium of ~6-8 young specimens (Fig. 31 : yo), detailed below. Pair of palps (Figs 24 , 25 : pp) small (~half of anterior adductor muscle area), located just posterior to anterior adductor muscle; outer hemipalps (op) ~3-times longer than wide, 8-10 strong transverse folds, from edge to edge (even protruding beyond edges); tapering distally; folds ending before mouth area (mo), keeping smooth perioral area; inner hemipalp (ip) similar to outer hemipalp, but slightly smaller, usually placed close to anterior region of visceral mass. Visceral mass (Fig. 24 ). All visceral structures white. Stomach (st) occupying most of anterior half, disposed anterior-ventrally. Digestive diverticular (dg), lying along anterior region of stomach. Gonad and genital structures occupying anterior ~half of posterior half of visceral mass, covering posterior surface of stomach. Reno-pericardial structures occupying posterior half of posterior half of visceral mass, up to posterior adductor muscle. Details below. Circulatory and excretory systems (Figs 24 , 27 ). Heart occupying anterior half of reno-pericardial area. Ventricle (ve) large, as dorsal structure, totally surrounding intestine; wall thick. Anterior and posterior aortas initially running attached to adjacent intestine. Pair of auricles (au) connected to anterior region of ventricle, each one conic, running towards ventral and lateral; connecting to central region of gills. Kidneys (ki) as posterior half of reno-pericardial volume, connected anterior and dorsal surface of posterior adductor muscle; anterior region hollow, as urinary chamber; nephropore (ur) as single, small slit located in ventro-anterior surface of supra-branchial chamber; posterior region mostly filled by white, solid renal tissue. Digestive system (Fig. 24 ). Palps (pp) and mouth (mo) (Fig. 25 ) described above (pallial cavity). Esophagus (es) simple, narrow, running along ~20% of shell length from posterior region of anterior adductor muscle towards posterior and dorsal, inserting in anterior surface of stomach between its middle and dorsal thirds. Stomach (st) large, dorsally rounded, ventrally tapering towards ventral and anterior up to anterior region of foot base. No clear separation between intestine and style sac (ss). Duct to digestive diverticula (dd) located in center of both gastric lateral walls. Stomach inner surface simple, lacking chambers and large folds; gastric shield thin, located in postero-dorsal region. Intestine (in) subtly running posteriorly and dorsally after style sac end, flanking dorsal surface of foot base; short zigzag only in its middle level; intestinal length slightly larger than shell length; in pericardial region crossing directly, gradually directing ventrally and posteriorly up to posterior side of posterior adductor muscle, initially immersed in pallial edge tissue; after short distance running on supra-anal chamber (Fig. 27 ). Anus (an) simple, sessile, located between posterior and ventral surface of posterior adductor muscle; large anal papilla in middle of anal dorsal edge (Fig. 27 : an). Reproductive system (Fig. 24 ). Gonad white, solid, small, mostly located in lateral regions of stomach. Large hollow brood pouch as posterior 2/3 of genital structures, full of large embryos (em); brood pouch tapering towards ventral and posterior, opening in both sides in small orifice (ap) located in middle level of suprabranchial chamber. Central nervous system (Figs 24 , 27-29 ). Pair of cerebral ganglia (ce) located in region dorsal to mouth, each one ~1/20 of anterior adductor muscle size. Each ganglion (Fig. 28 : ce) elliptical, ~twice longer than wide; cerebral commissure (cc) wide, as long as each ganglion. Pair of pedal ganglia (Fig. 29 : pg) located in middle level of pedal base; both totally fused with each other, forming spheric mass; pair of statocysts (sy) very small, located in posterior side of pedal ganglia; both pedal ganglia slightly larger than one cerebral ganglion. Pair of visceral ganglia (Fig. 27 : vg) located slightly anterior to posterior adductor muscle central side; each ganglion fusiform, ~3 times longer than wide, located very close to each other, with pedal commissure very short; each visceral ganglion slightly larger than each cerebral ganglion; posteriorly single large nerve running towards posterior, ventrally to posterior adductor muscle (nv). Development. Large embryos found in gonadal brood pouch located inside visceral mass (Fig. 24 : em). Embryos coming out by genital orifice (Fig. 24 : ap), located in middle level of inner demibranchs. Both inner demibranchs serving as external branchial brood pouches (Fig. 31 : yo), becoming full of young specimens (yo) in their internal area between both lamellae. Young specimens with prodissoconch of ~0.2 mm (Fig. 20 ), growing up to teleoconch becoming ~3-times larger than prodissoconch (Figs 19 , 32 ), with ~0.7 mm. Prodissoconch almost plane, circular, smooth (Figs 19 , 20 ); teleoconch possessing only concentric undulations and growth lines (Figs 19 , 32 ); valves translucent (Fig. 32 ). Young intra-brood pouch specimen rounded, slightly flattened, dorsal region almost straight, umbos not protruding (Figs 19 , 32 ); shell lacking teeth in hinge and with almost no inner muscular scars (Fig. 21 ). Gross anatomy of these young specimens (Fig. 30 ) with very small adductor muscles (aa, pa), with anterior slightly more ventral; mantle lobes edges (mb) not fused with each other, lacking siphons; gill with only inner demibranch visible (id), relatively squared, possessing 7-8 transverse folds only; foot lacking visible byssal furrow. Material examined. Types . Measurements (in mm). Holotype MZSP 155717 (Figs 1-6 ): 4.3 by 3.5; Paratypes MZSP 153866: #7 (Figs 7-11 ): 2.3 by 1.9; #8 (Fig. 12 ): 4.5 by 3.6. Etymology. The specific epithet refers to the troglobitic mode of life of the animal, being an adjective in the feminine nominative singular. Habitat. Specimens of Eupera troglobia sp. nov. were only observed in the Casa de Pedra cave, and are possibly endemic to this cave (Fig. 33A, B ). The Casa de Pedra cave comprises a cave in limestone from the Couto Magalhaes Carbonatic Formation, associated with the Neoproterozoic basement of the Baixo Araguaia Supergroup, which, in addition to the limestones, presents subordinate phyllites, slates, metargilites, metarenites and quartzites ( Pereira and Morais 2012 ). The climate of the region is tropical, with two distinct periods: a dry season, between May and September, and a rainy season, between October and April, with a total annual rainfall around 1750 mm ( Martins et al. 2002 ). The cave has 1,038 meters of total length, with predominantly ellipsoidal conduits. There are few speleothems, in addition to thick allochthonous sediments on the cave floor. The cave is inserted in a limestone outcrop located close to the Lagoa da Confusao karstic lake (Figs 33C , 34A ), which overflows during the rainy season, flooding part of the flood plain surrounding it. In such periods (October to April), most cave conduits are completely filled with water. On the other hand, the cave becomes dry during the dry season, as few intermittent dams are present. A visit paid to the cave in August 2021, revealed the cave partially flooded, with most conduits inaccessible. The main entrance gallery was filled with water, which was still forming a small lake outside the cave (Fig. 34A ). Reaching the deepest areas inside the cave through the main entrance was impossible in that moment, but since there is another entrance in the middle of the cave (Figs 34B , 35A ), the inner portions were accessible (Fig. 35B ). Individuals of Eupera troglobia sp. nov. were found associated to a consolidated sediment deposit in a deeper portion of the cave (Figs 33D , 35C ). Many specimens were adhered to the sediments already exposed to the air (Fig. 35E, F ), while others were still under water (Fig. 35D, H, G ). However, it is important to note that the cave was still drying up, so all specimens would be exposed to the air. Considering that the cave remains out of water during at least three months a year, the individuals do survive during all this period somehow avoiding desiccation. The only known cave-restricted clam species, all belonging to the genus Congeria , from caves in the Dinaric Alps, also exhibit this behavior, presenting a notable tolerance to air exposure ( Jovanovic et al. 2016). Interestingly, in the case of all three Congeria species, only part of their populations becomes exposed during dry periods, and most part of the population remains underwater in such periods. Furthermore, even considering that Congeria specimens are able to tolerate air exposure for periods as 2 months, some individuals were observed still active, with their shells open and inhalant and exhalant syphons extruded ( Jovanovic et al. 2016). In the case of E. troglobia sp. nov. the single visit paid to the cave does not make it possible to form any hypothesis regarding the individuals' behavior along the air exposure (e.g., whether they remain active or not). Accordingly, it is highly recommendable that further studies investigate the biology and life cycle of this species. It is worth mentioning that Silva (2006) , in her report from the present cave, mentioned the presence of clams also associated to root masses pending from the cave ceiling during their survey (in the dry period). In that case, all specimens were also exposed to the air, and there were only few small ponds inside the cave, apparently devoid of clams. During the clam sampling in August 2021, some hydrochemical parameters were evaluated, both inside the cave and in the epigean lake (Lagoa da Confusao lake), which floods to the cave during rainy periods. The parameters inside the cave were quite distinct from those from the external lake: cave waters: temperature: 23.6 °C; pH 6.17; conductivity: 0.124 mS/cm; dissolved oxygen: 0.92 mg/L; TDS (total dissolved solids): 0.08 g/L; Salinity: 0.06‰; external lake: temperature: 28.1 °C; pH 7.14; conductivity: 0.017 mS/cm; dissolved oxygen: 10.35 mg/L; TDS (total dissolved solids): 0.011 g/L; Salinity: 0.01‰. It is noticeable the differences in temperature (lower inside the cave), pH (lower inside the cave), conductivity (higher inside the cave) and dissolved oxygen (much lower inside the cave). This certainly demonstrates that the species is not only able to survive in conditions quite distinct from those observed in surface waters, but also probably tolerates high levels of variation in hydrochemical parameters along the year, considering that the cave water originates from the lake flooding. Finally, it is also worth mentioning the number of embryos found in E. troglobia . Although in the literature, it is usual to find the term "embryo" referring to both the true embryos and the young, such stages are, in fact, distinct. True embryos (still in ontogenetic development) are those individuals found in the visceral marsupium. Those found in the inner demibranchs are called "young" , as they are already formed and the shell shows growth lines. In E. troglobia , there are a maximum of 10 young in each gill (~20 in total) and another 5-6 embryos in the visceral marsupium (on each side - 10-12 in total). Hence, the species presents around 30 immatures (considering both embryos and young). In the consulted literature, only the young specimens inside gills are considered. The other already studied Eupera species (all epigean), presented a considerably larger reported number. As an example, E. platensis had between 22 and 66 young specimens in gills ( Ituarte 1988 ); E. cubensis between 25 and 35 ( Heard 1964 ) and E. klappenbachi had between 24 and 62 ( Mansur and Veitenheimer 1975 ). Most cave-restricted species from several distinct groups usually have k-strategies ( Howarth 1983 ; Belles 1992 ), due to the relatively stable environments that subterranean habitats usually present. Among the reproductive adaptations related to such strategy, there are a reduced number of offspring, increased offspring body size, parental care, among others. The reduced number of embryos compared to some epigean Eupera species, associated to the proportional large size of the young observed in the visceral marsupium of E. troglobia , probably represent another adaptation to the cave environment, confirming the cave-restricted status of this species. Figures 1-6. Eupera troglobia holotype MZSP 155717 shell (L 4.3 mm), right valve in left column, left valve in right column 1 outer right view 2 outer left view 3 inner left view 4 inner right view 5 inner left-slightly ventral view 6 inner right-slightly ventral view. Figures 7-13. Eupera troglobia shell SEM images of paratypes 153866 7 specimen #7, both valves connected, opened ~120°, ventral view 8 #7, left valve, inner left view 9 #7, right valve, inner left view 10 #7, left valve, detail of hinge region, inner left view 11 #7, right valve, detail of hinge region, inner right view 12 specimen #8, left valve, outer left view 13 same, detail of surface on middle region of ventral edge. Scale bars: 500 µm ( 7, 12 ), 300 µm ( 8 ), 200 µm ( 10, 13 ). Figures 14-21. Eupera troglobia shell SEM images of paratypes 153866 14 specimen #8 (part damaged), valves opened ~40°, ventral view 15 same, higher magnification 16 same, higher magnification, region of cardinal tooth 17 specimen #9 (part damaged), valves opened ~60°, ventral view, mainly showing hinge of left valve 18 same, right valve 19 shell of young specimen extracted from gill's marsupium, left valve, outer left view 20 same, detail of umbo showing prodissoconch in its center, left-slightly dorsal view 21 another intra marsupial specimen (part damaged), right valve, inner ventral-slightly left view, focus on hinge, part of left valve still attached by ligament. Scale bars: 500 µm ( 14, 17, 18 ), 300 µm ( 15 ), 100 µm ( 19 ), 50 µm ( 16, 21 ), 30 µm ( 20 ). Figures 22-26. Eupera troglobia anatomical drawings 22 whole right view, right valve and part of right mantle lobe removed 23 gill, transverse section in its middle level 24 whole right view, right gill removed, visceral structures seen as in situ if region was transparent, peripherical structures with only topology indicated 25 palp region, ventral view, hemipalps slightly deflected 26 mantle edge, transverse section in middle level of ventral edge. Scale bars: 0.5 mm. Figures 27-30. Eupera troglobia anatomical drawings 27 peri-siphonal, posterior region, right view, right gill and mantle lobe removed, siphons longitudinally sectioned, visceral structures seen as in situ 28 transition between palps and esophagus, right view, with concern to cerebral ganglia 29 pedal ganglia, dorsal view 30 gross anatomy of young specimen from gill brood pouch, right view, right mantle lobe removed. Scale bars: 0.5 mm. Figures 31-32. Eupera troglobia development, paratype MZSP 153866 pregnant specimen (2-3) 31 whole right view, shell and part of right mantle mole removed, young specimens (yo) seen in marsupium of inner demibranch by translucency 32 young specimen extracted from marsupium, right view. Scale bars: 1 mm. Figure 33. Location of the Casa de Pedra cave A South America, with Brazilian states highlighted B Tocantins state with Lagoa da Confusao municipality highlighted in white lines C aerial view from the Lagoa da Confusao region, where the urban area and the karst lake are visible; red star indicating the location of the Casa de Pedra cave D Casa de Pedra cave; red star indicating the location of Eupera troglobia sp. nov. Figure 34. Lagoa da Confusao karst area A limestone outcrops close to the Lagoa da Confusao karst lake. The dark blue indicates the floodplain (not flooded in the moment of the photograph) and the dark blue indicates those flooded areas. The arrow indicates the main entrance of the Casa de Pedra cave B outcrop where the Casa de Pedra cave is located; 1. indicates the main entrance of the cave; 2. Upper entrance. Figure 35. Casa de Pedra cave A secondary upper entrance of the cave B cave chamber that was partially flooded C area inside the cave where specimens of E. troglobia sp. nov. were found D detail of the consolidated sediment indicating the area where submerged specimens of E. troglobia sp. nov. were found E E. troglobia sp. nov. specimens in situ exposed to the air F same, detail of an air exposed specimen, with a harvestman ( Eusarcus sp.) near it G in situ submerged specimens of E. troglobia sp. nov. H location of a submerged specimen of E. troglobia sp. nov.