A new cave species of Rhamdia ( Siluriformes : Heptapteridae ) from Serra do Ramalho , northeastern Brazil , with notes on ecology and behavior

Rhamdia enfurnada, a new troglobitic (exclusively subterranean) catfish, is described from the Gruna do Enfurnado, Serra do Ramalho, southwestern Bahia State, middle São Francisco River basin. It differs from epigean congeners from the São Francisco River basin by the variable eye size, ranging from reduced to externally invisible (bilateral asymmetry in development of eyes is frequently observed), with a conspicuous orbital concavity (orbital diameter varying from 6.4 to 13.6% of head length in R. enfurnada and from 14.5 to 24.4% in the epigean species); by the dark pigmentation reduced due to a decrease in density of melanophores, and also variable. In comparison to other cave species, R. enfurnada presents a low degree of troglomorphism regarding development of eyes and pigmentation, indicating a relatively short time evolving in isolation in the subterranean environment: R. reddelli is described as lacking eyes and with almost no pigmentation; likewise, R. macuspanensis and R. laluchensis as having rudimentary eyes, covered with skin and not visible externally, and with reduced body pigmentation; R. zongolicensis is also almost devoid of dark pigmentation, but eyes are less reduced than in R. reddelli. R. quelen urichi, also does not present any trace of eyes externally, but the body coloration is described as pale yellowish brown. Rhamdia guasarensis has a concave dorsal profile of head and a complete absence of pigmentation and eyes. The population density (preliminarily estimated in 0.1-0.2 individuals m in average, and reaching 10 individuals m, in the dry season and size (several thousands of individuals) of R. enfurnada may be considered high for cave fish standards. Rhamdia enfurnada exhibited an increased midwater activity when compared to their typically bottom-dwelling epigean relatives; are mostly indifferent to light and moderately aggressive.


Introduction
The generally nocturnal, chemoriented Siluriformes present a high potential for life in subterranean habitats, characterized by the permanent darkness and frequent food limitation.Among catfishes, the generalist heptapterids include a significant number of exclusively subterranean species (troglobites) in the neotropics, such as the cave-dwelling Pimelodella kronei (Miranda Ribeiro, 1907), P. spelaea Trajano, Reis & Bichuette, 2004, Taunayia sp. and a new, undescribed genus, from Brazil, and several species from Mexico, Belize, Trinidad Island, and Venezuela (Weber et al., 1998;DoNascimiento et al., 2004).These troglobitic species differ in their degree of reduction of eyes and dark pigmentation, the most conspicuous characters related to the isolation in the subterranean environment (troglomorphisms), from species with only slightly reduced eyes and pigmentation, such as P. spelaea, to those totally anophthalmic and unpigmented, such as Taunayia sp. and a new heptapterid genus, passing through species showing intrapopulational variation in these characters (e.g., P. kronei).
Rhamdia catfishes are nocturnal, opportunistic carnivores, thus it is not surprising to find them in the subterranean environment.In fact, this genus is included among those found in caves throughout the world with the highest number of troglobitic derivatives, together with Trichomycterus (Siluriformes: Trichomycteridae; at least seven species), Sinocyclocheilus (Cypriniformes: Cyprinidae; seven species) and Lucifuga (Ophidiiformes: Bythitidae; five species) (Weber et al., 1998;Trajano & Bichuette, unpubl. data).
Rhamdia catfishes belonging to epigean (surface) species have been observed in several Brazilian caves, usually as isolated or few individuals (e.g., Trajano, 1991, for the upper Ribeira River valley, southeastern Brazil; Bichuette & Trajano, 2003, for São Domingos karst area, central Brazil; E. Trajano & M. E. Bichuette, pers.obs., for caves from Varzelândia and Cordisburgo, eastern Brazil).Most of these individuals were apparently in good condition and could have been feeding in the subterranean habitat, but there was no good evidence of reproduction in the caves -these records probably represent trogloxenes (organisms found regularly in subterranean habitats but which do not complete their life cycle in this environment -Trajano, 2003).So far, there was no indisputable record of reproductive, self-sustained subterranean populations of Rhamdia catfishes in Brazil, either troglophilic (belonging to species found both in epigean and subterranean habitats) or troglobitic (exclusively subterranean).
Two new species of troglomorphic catfishes, pertaining to the genera Trichomycterus (cited as Trichomycterus sp. 3 in Trajano, 2003) and Rhamdia, have been found in caves of the recently explored Serra do Ramalho karst area (southwestern State of Bahia).This area presents a great biospeological potential, with several large cave systems in the process of being explored and mapped, with remarkable terrestrial troglobites that include the first troglobitic Amblypygi recorded for Brazil (Charinus troglobius Baptista & Giupponi, 2003), a highly troglomorphic genus of gonyleptid harvestman (Giupponia chagasi Pérez & Kury, 2002) and a new genus of spider (Ochyroceratidae) with modified genitalia, under description (R.L. Baptista, pers. comm.).The new Rhamdia species was found in the Gruna do Enfurnado and is characterized by the large population size and the high individual variability in the degree of development of eyes and pigmentation.
We herein describe this new species, which represents the first troglobitic Rhamdia known from Brazil.Preliminary data on the ecology and behavior of these catfishes are also presented.

Material and Methods
Measurements and meristic data for the type series of R. enfurnada (20 specimens) were taken following Lundberg & McDade (1986) and Bockmann (1994), respectively.For comparison, the same variables were obtained from 19 specimens of epigean Rhamdia from the São Francisco River basin (see Comparative Material Examined).All measurements were taken on the left side of the specimens using a Mitutoyo digital caliper (0.1 mm precision).For the osteological study, two paratypes (99.6 and 58.9 mm SL) were cleared and stained using the method of Taylor & Van Dyke (1985).These two specimens were used for counting the number of vertebrae, ribs, branchiostegal rays and serrae of the pectoral-fin spine.In addition, the stomach contents of these two individuals were examined.The vertebrae counts included all free vertebrae posterior to the Weberian complex and the compound caudal centrum (counted as one).The degree of body pigmentation was evaluated by counting the number of melanophores in a 1mm 2 area below the dorsal fin, on the left side of three cave and three epigean specimens.
For comparisons of morphometric proportions, we used the two sample t-test (for parametric data) or the Mann-Whitney test (T test, when data was non-parametric), with á=5% (SigmaStat, version 2.0).Data from ecological study (July 2005)  Diagnosis.Rhamdia enfurnada differs from its epigean congener (Rhamdia cf.quelen) from the São Francisco River basin by the highly variable eye size, ranging from reduced to externally invisible, with an open orbital concavity (orbital diameter varying from 6.4 to 13.6% of head length in R. enfurnada and from 14.5 to 24.4% in the epigean species); eyes, when present, pigmented and covered by semi-opaque skin, orbital rim absent or poorly developed in most specimens, well-defined only in some small specimens; dark pigmentation reduced due to a decrease in density of melanophores; and iridorophores present and concentrated in the opercular region.In comparison to other troglobitic Rhamdia species, R. enfurnada exhibits a lower degree of troglomorphy regarding eyes and pigmentation, which are highly variable; Rhamdia reddelli is described as lacking eyes and with almost no pigmentation; likewise, R. macuspanensis and R. laluchensis as having rudimentary eyes, covered with skin and not visible externally, and with reduced body pigmentation; Rhamdia zongolicensis is also almost devoid of dark pigmentation, but eyes are less reduced than in R. reddelli.Rhamdia quelen urichi, from Trinidad, also does not exhibit any trace of eyes externally, but the body coloration is described as pale yellowish brown.Rhamdia enfurnada has a concave dorsal profile of the head and a complete absence of pigmentation and eyes.Variability in development of eyes and pigmentation as in R. enfurnada was only reported for Rhamdia laticauda typhla, from Belize.
Description.Morphometric data in Table 1.Distribution of standard length classes relative to 121 individuals measured under anesthesia and released in July 2005 during ecological study presented in Fig. 2. Body elongated, its greatest depth at vertical passing by dorsal-fin origin (Fig. 1a).Dorsal profile anterior to dorsal fin slightly to distinctively convex, then descending to caudal peduncle, slightly concave between dorsal and adipose fins.Head depressed, broad and relatively short, trapezoidal in dorsal profile.Largest specimen of R. enfurnada, 218.6 mm SL, shows signs of age, including emaciation and arched backbone, probably being close to maximum size of species (Fig. 3).Mouth terminal, upper jaw slightly in advance of lower jaw.Rictal folds well developed, reaching or slightly surpassing posterior margin of orbit.Premaxilla with single band of small, conic teeth in 10 irregular tooth rows, its posterolateral corners rounded, not produced.Dentition of lower jaw similar to that of premaxilla, in six irregular tooth rows.Palatine and vomer edentulous.Maxillary barbel long, reaching origin of anal fin in smaller specimens, and between middle and tip of depressed pelvic fin in larger individuals.Mental barbels long, inner-mental barbel surpasses posterior border of branchial membrane, almost reaching pectoral-fin insertion; outer-mental barbel reaching tip of depressed pectoral fin.Inner mentalbarbel base inserted slightly in advance of outer mental-barbel base.Anterior nares tubular, near border of snout; posterior nares tubular (incomplete in some specimens) and short, with rounded openings; internarial length less than width between posterior nares.Eye variable in size (Fig. 4).Branchial membranes touching midventrally, but not overlapping (except in largest examined specimen, where membranes slightly overlap), and united to isthmus only anteriorly.Anus and urogenital papilla separated; urogenital papilla conspicuous and variable in size and shape, from short and triangular to elongate, located at midlength between bases of anal and pelvic fins.Dorsal fin with i,I,7 rays.Dorsal spine weakly developed, smooth, without serrae, comparatively short, its length 39.6-77.7% of dorsal-fin height.Adipose fin long (30.2-36.0%SL), its origin at vertical through middle of depressed pelvic fin, and its end slightly posterior to end of depressed anal fin.Adipose fin with slightly convex dorsal margin and conspicuous posterior free lobe.Caudal fin forked, ventral lobe slightly longer than dorsal; ventral lobe rounded in most individuals, pointed in some small specimens, dorsal lobe variable, pointed (in most small fish) to rounded.Dorsal lobe of caudal fin with i,7 and ventral lobe with i,8 principal rays.Anal fin with v,7-8 rays; its margin rounded in most specimens, pointed in some small fishes.
Pectoral fin with I,9 rays.Pectoral spine well developed, depressed, elliptical in transversal section, its anterior margin with 4-5 retrorse serrae distally and 7-13 small serrae proximally, posterior margin distinctly serrated (Fig. 5).First branched pectoral-fin ray longest, other branched rays decreasing in length posteriorly.Posterior cleithral process relatively long, strong, reaching one-third of depressed pectoral fin (Fig. 1c).Pelvic fin with i,5 rays, its origin at vertical through end of dorsal-fin base, pointed in smaller specimens, rounded in larger ones.
Skull roof (Fig. 6) with wide, elongated anterior cranial fontanel; mesethmoid contacts frontal posteriorly on either side of concavity formed by anterior fontanel; frontal broad, greatest width equal to length, meeting posteriorly along 1/4  of its length.Posterior cranial fontanel reduced to elliptical foramen near center of supraoccipital.Supraoccipital process short, its length equal to length of supraoccipital bone excluding posterior process.Infraorbital bone series comprising smaller orbital cavity.Branchiostegal rays six.
Color in alcohol.Specimens light gray to yellowish; base of dorsal fin, margin of adipose and dorsal fins slightly duskier, especially in darker fish; faint lateral stripe in darker fish, usually interrupted; melanophores concentrated on pseudotympanum in some specimens (not related to general degree of pigmentation); body coloration pattern otherwise homogeneous, pectoral, pelvic, anal, and caudal fins light colored.Melanophores concentrated on top of head and, in 11 out of 20 fish, also behind orbit, forming dark spot slightly larger than orbit.Density of melanophores in three cave and in three epigean specimens, respectively: 22.4, 28.8, and 30.4 melanophores per mm 2 in Rhamdia enfurnada, and 43.2, 46.4, and 51.2 melanophores per mm 2 in Rhamdia cf.quelen, from same river basin (MZUSP 54640).
Color in life.Light gray to yellowish, with melanophores concentrated on head region and margins of adipose and dorsal fins.Iridophores present, concentrated on opecular region, forming yellowish iridescent spot covering 2/3 of this region.
Etymology.From Gruna do Enfurnado, the name of the type locality, meaning shut, hidden, or isolated in a cave or cavern.thus the measured water temperatures were lower than expected since, due to the thermal isolation, the temperature in subterranean habitats usually follow the annual mean temperature.
Notes on ecology and behavior.Rhamdia catfishes were distributed throughout the cave, from shallow, pebble-bottomed reaches, where smaller fish concentrate and may attain densities of 10 individuals m -2 , to deep, slow-moving, soft-bottomed pools, where we found the larger specimens.The largest numbers of fishes were found at the distal permanent stream reach (around 150 individuals observed along the 600 m reach in July 2005 -Trajano & Bichuette, unpubl.data).A mark-recapture study is in progress aiming to estimate the population size, minimal extension of catfish movements and life cycle parameters.Examination of the stomach contents of the two cleared and stained individuals revealed a diet of invertebrates, mainly arthropods, including terrestrial items (fragments of Diplopoda and Ensifera fallen accidentally or washed into the water) and a great amount of detritus.
The catfish behavior was directly observed in the cave habitat.In addition, seven specimens were brought alive to the laboratory and kept collectively in a 100-l aquarium in a dark room.Like other troglobitic siluriforms (e.g., P. kronei, Taunayia sp., the new heptapterid genus from Chapada Diamantina, and the undescribed Trichomycterus species from Bodoquena and Serra do Ramalho, respectively Trichomycterus sp. 2 and 3 in Trajano, 2003), Rhamdia enfurnada exhibited an increased midwater activity when compared to their typically bottom-dwelling epigean relatives.Hiding places made of limestone blocks were available; the catfish exhibited variable cryptobiotic behavior, with smaller individuals hiding less frequently than the larger ones.The reduction of cryptobiotic habits is another difference in relation to epigean relatives shared by several unrelated troglobitic siluriforms (Trajano, 2003).
The catfishes from Gruna do Enfurnado reacted to sudden illumination with flashlights and noise with increased swimming activity, which indicates that these catfishes perceive these stimuli.In reaction to light experiments, using choice-chamber (as those described in Trajano & Gerhard, 1997, among others), these catfishes were mostly indifferent to light, i.e., they did not show significant preferences, either to the dark chamber or to the illuminated one (A.C.Barbosa, unpubl. data).
The catfishes from Gruna do Enfurnado were moderately aggressive under laboratory conditions: when two individuals met, apparently by chance (no spontaneous active pursuing was observed), usually the larger one chased away the smaller, in a quick pursuit, or the smaller fish rapidly avoided the other one, without pursuing.These interactions were usually observed during feeding with living Artemia, when increased activity enhanced the chances of meeting, and may be mostly related to competition for food.Nevertheless, as expected in view of the high population densities observed in the cave, in general the catfish seemed to be quite tolerant to the proximity of conspecifics, since no individual exhibited clear signals of stress, even after having been kept together for one and one-half years.Weak, size-related dominance was mostly displayed in relation to food and hidden places.

Discussion
The monophyly of Rhamdia is not yet established.Bockmann (1998) placed two representative species of the Heptapteridae, R. quelen and R. laticauda, in a paraphyletic sequence, with the latter as sister-group to Pimelodella.Perdices et al. (2002), analyzing mitochondrial DNA, elucidated the relationship of Mesoamerican Rhamdia.In that paper, the monophyly of three trans-Andean taxa, R. guatemalensis, R. laticauda and R. cinerascens, was strongly supported.Those authors also proposed that the representatives of R. quelen from Mesoamerican are phylogenetically distinct from South American individuals (opposite of that proposed by Silfvergrip, 1996).The inclusion of Rhamdia enfurnada in this genus was based on its general appearance, the reduced posterior fontanel and the short supraoccipital process (Eigenmann & Eigenmann, 1890).
Rhamdia enfurnada possesses typical reductive character states in common with the other exclusively subterranean species and populations of the genus.Such troglomorphisms, which distinguish these catfishes from the epigean ones found in the São Francisco River basin (Rhamdia cf.quelen -MZUSP 54640 and 37979), include the reduction of the eyes (orbital diameter in relation to head length, 6.4-3.6% in R. enfurnada vs. 14.5-24.4% in the examined epigean specimens) and of the dark pigmentation as expressed by the low density of melanophores (22.4-30.4melanophores per mm 2 in R. enfurnada vs. 43.2-51.2melanophores per mm 2 in epigean specimenscomparison of densities of melanophores in epigean and cave specimens indicates that the weaker pigmentation in R. enfurnada was due, at least in part, to an evolutionary reduction in density of melanophores), both characters highly variable in the studied population.
The shorter maxillary barbel in R. enfurnada than in Rhamdia cf.quelen is rather unexpected since an emphasis on non-visual sensory organs is predicted for subterranean animals.In fact, the elongation of structures bearing chemo-and mechanosensory organs is common among troglobites.This is the case of the Mexican troglobitic Rhamdia species, R. reddelli, R. zongolicensis, R. laluchensis and R. macuspanensis, which have maxillary, inner-mental and outer-mental barbels longer than those of the epigean closest relative, R. laticauda (Weber et al., 2003).On the other hand, like R. enfurnada, Pimelodella kronei tended to have maxillary barbels shorter than the epigean putative sister-species, P. transitoria.In this case, it has been suggested that agonistic encounters, frequently leading to barbel injuries, could explain a considerable number of individuals with regenerating barbels in the cave species (Trajano & Britski, 1992).Rhamdia enfurnada is much less aggressive than P. kronei, hence this explanation does not seem to apply to the former.For the moment, we do not have any explanation for the apparently shortened barbels in R. enfurnada.
In comparison to other troglobitic Rhamdia species, R. enfurnada has a lower degree of troglomorphy regarding eyes and pigmentation, which are highly variable.Rhamdia reddelli is described as lacking eyes (with only one specimen showing a tiny eye spot) and with almost no pigmentation (Miller, 1984); likewise, R. macuspanensis and R. laluchensis as having rudimentary eyes, covered with skin and not visible externally, and with reduced body pigmentation (Weber & Wilkens, 1998;Weber et al., 2003).Rhamdia zongolicensis is also almost devoid of dark pigmentation, but the eyes are less reduced than in R. reddelli (Wilkens, 1993).Rhamdia quelen urichi (as referred by Weber et al., 1998, andRomero &Paulson, 2001), from Trinidad, also does not have any trace of eyes externally, but the body coloration is described as pale yellowish brown (Norman, 1926).In our opinion, the described differences do support a species status for this taxon, which should be referred as R. urichi.Rhamdia guasarensis, the only troglobitic species from South America, is particularly specialized, characterized, among others, by a concave dorsal profile of head and widened cutaneous pores of the cephalic laterosensory system, which may be related to sensorial compensation mechanisms, in addition to a complete absence of pigmentation and eyes (DoNascimiento et al., 2004).Variability in development of eyes and pigmentation as in R. enfurnada was only reported for Rhamdia laticauda typhla, from Belize ["Reduction of eyes to various degrees is evident… Some individuals have very small, deep pits each with a small black spot at the bottom….A few individuals have larger eyes (about one-half … the normal eye size of R. laticauda laticauda…]"… "There is considerable variation in body coloration…, with some individuals being almost white and others tending to be a dark brown."- Greenfield et al., 1982).No author mentions iridescence in these species, thus the presence of iridophores may be unique to R. enfurnada.
Following Perdices et al. (2002), the troglomorphic Rhamdia species found in caves of Mexico and Belize belong to the trans-Andean laticauda and guatemalensis clades, which forms a monophyletic group with cinerascens clade.Thus, differences between R. enfurnada (that probably belong to the cis-Andean quelen group) and these species, such as those concerning the pectoral spine (serrae present in both margins of the pectoral spine vs. strong serrae present in the posterior margin of the pectoral spine and anterior margin smooth), the maxillary barbel (relatively longer in R. enfurnada) and mithocondrial DNA are likely to reflect different epigean ancestry.Willkens (1986) proposed a correlation between the degree of morphological reduction and the time of subterranean evolution based on the neutral mutation model (Wilkens, 1988(Wilkens, , 2005)).From this, it may be concluded that R. enfurnada, which presents highly variable eyes and body pigmentation, has been isolated in the subterranean environment for a relatively short time when compared to other cave Rhamdia species (except R. laticauda typhla).On the other hand, R. enfurnada exhibits a reduction in the cryptobiotic habits and an important midwater and surface activity, which are considered apomorphic, advanced traits for troglobitic catfishes (Trajano & Bockmann, 1999).
The population density (preliminarily estimated in 0.1-0.2individuals m -2 in average, and reaching 10 individuals m -2 , in the dry season (E.Trajano and M.E.Bichuette, unpubl.data) and size (several thousands of individuals) of R. enfurnada may the considered high for cave-fish standards (Trajano, 2001a).This may be related to the large amounts of organic matter as detritus deposited by flash floods during the rainy season and providing an abundant source of food directly for the catfishes (one examined stomach had a considerable amount of detritus) and for their prey.During a two-day visit, in July 2005, ca.120 fishes were captured and measured, of which 100 were marked and released.This represents quite a large number for a troglobitic fish, only comparable to the armored catfish, Ancistrus cryptophthalmus (Loricariidae), among the studied Brazilian cave siluriforms.On the other hand, contrary to the observed for Ancistrus (Trajano, 2001b), the few data on population density of epigean Rhamdia catfishes point to much lower numbers (0.032-0.041 individuals m -2 , L.F. Duboc, unpubl.data).
It noteworthy that the distribution of size classes, shown in Fig. 2, is not normal, but skewed toward larger size/age classes.The same tendency was observed for Pimelodella kronei (Trajano & Britski, 1992), but not for P. speleae, which exhibited a normal distribution of classes of size (Trajano et al., 2004).Age distributions skewed toward larger classes were also observed for specialized troglobitic amblyopsid fishes (Culver, 1982), and is evidence of a K-selected life cycle (precocial lifestyle, sensu Balon 1981Balon , 1999)), a common trait of troglobites (Culver, 1982).Rhamdia enfurnada is among the largest troglobitic fishes in the world.The Serra do Ramalho karst area is not protected by law.Nowadays, the accelerated extraction of the original vegetation for cotton and soybean cultivation represents a main threat.Thus, there is reason for major concern about the future of this endemic species (despite the large population size).A proposal of a conservation unit including the Gruna do Enfurnado system is urgently needed to assure its protection.

Fig. 2 .
Fig. 2. Distribution of standard length (mm) classes relative to 121 individuals of R. enfurnada.Data collected in July 2005.