Morphology and distribution of the cave knifefish Eigenmannia vicentespelaea Triques , 1996 ( Gymnotiformes : Sternopygidae ) from Central Brazil , with an expanded diagnosis and comments on subterranean evolution

We present herein data on morphology and distribution of the cave knifefish Eigenmannia vicentespelaea Triques, 1996, from the São Domingos karst area, Central Brazil, comparing it to the epigean (surface) species, Eigenmannia sp., found in the same area (but not syntopic with E. vicentespelaea) and also with congeners from other localities. Collecting sites comprising epigean and subterranean stream reaches in São Domingos were sampled during the dry seasons of 1999, 2000, and 2001 using several methods. Preserved specimens of E. vicentespelaea (n=25, including holotype and paratype) and of Eigenmannia sp. (n=15) were compared with focus on morphometric characters, body pigmentation and eye condition. A combination of characters separates E. vicentespelaea from Eigenmannia sp. and other congeners: the length from the tip of the snout to the posterior of the anal fin base, ocular diameter: head length and pre-anal distance: head length proportions, and body pigmentation. A wider morphometric variation in E. vicentespelaea is described than that reported in the original description (based on two specimens).


Introduction
Electric fishes of the Order Gymnotiformes are an important component of the night-active ichthyofauna in South and Central America, corresponding to a monophyletic group based, among other characters, on the ability of producing and detecting weak electric fields (Alves-Gomes et al., 1995;Albert & Campos-da-Paz, 1998;Albert, 2001).These fishes are distributed between 35ºS, in Argentina, and 18ºN, in Mexico (Eigenmann & Allen, 1942;Bullock et al., 1979;Mago-Leccia, 1994), with some genera occurring almost exclusively in deep waters of the Amazon basin.The adaptations related to their predomi-nantly nocturnal activity favor the colonization of totally dark habitats such as those in the subterranean realm (defined as the network of interconnected subsoil spaces, with variable sizes, filled with water or air and characterized by permanent darkness-sensu Juberthie, 2000).
The only known troglobitic gymnotiform is E. vicentespelaea Triques, 1996, from the São Domingos karst area, upper Tocantins River basin, in Central Brazil (troglobites are defined as species restricted to subterranean habitats, usually showing specializations related to the evolution in isolation in this environment -troglomorphisms sensu Christiansen, 1962).This species was included in the E. virescens speciesgroup (sensu Albert, 2001), which comprises four species (E.virescens, E. trilineata, E. vicentespelaea, and an undescribed species) sharing the following character states: two or three longitudinal lines on either side of the body; lateral valvula cerebellum large; and anterior intermuscular highly branched.Triques (1996) based the description of E. vicentespelaea on two specimens collected in 1978 in São Vicente I Cave.The species diagnosis was based in the proportion of the ocular diameter in relation to the head length.This character has also been used to separate the epigean (surface) species, E. macrops, E. humboldtii and E. virescens, from Venezuela (Mago-Leccia, 1978), and also Sternarchorhynchus species from Brazil (Campos-da-Paz, 1997).
During a study on the epigean and subterranean ichthyofauna from São Domingos karst area, including the ecology and behavior of Eigenmannia spp.(Bichuette, 2003;Bichuette & Trajano, 2003), specimens of the cave E. vicentespelaea and an epigean species were collected, allowing a comparative study of their external morphology.The identification of the epigean species equivocal became because some character states (overlapping of OD:HL and SNL:HL proportions -R.Campos-da-Paz, pers.comm.)indicated that it would belong to the E. macrops species-group (sensu Albert, 2001), representing a new geographical record for this group (upper Tocantins River basin).However, the inclusion of the epigean species in the E. macrops species-group is doubtful since other character states, such as three longitudinal lines along the body, characteristic of the E. virescens species-group, are present in this species.For this reason, this epigean species from Domingos karst area is herein referred as Eigenmannia sp.
Preliminary observations on the large intrapopulational variability regarding eyes and body pigmentation in E. vicentespelaea, combined with the morphological similarity between the two Eigenmannia species from São Domingos karst area, raised some doubts about the diagnosis of E. vicentespelaea.
We present herein morphometric and distributional data on E. vicentespelaea, comparing it to the epigean species Eigenmannia sp. from the same area and congeners from other regions.An expanded diagnosis is proposed for the former, and the evolution in the subterranean environment is discussed.

Study area.
During the present study, specimens of E. vicentespelaea were found in the São Vicente I and São Vicente II caves, and specimens of Eigenmannia sp. in the Rio da Lapa and subterranean stream reaches of the São Mateus and Angélica caves.These localities are within the limits of the Terra Ronca State Park (13º30' -13º50' S, 46º0' -46º30'W), in São Domingos County, eastern State of Goiás, Central Brazil.The area lies in the Cerrado (the savannah-like Brazilian vegetation) phytogeographic domain (Ab'Saber, 1977), presenting a tropical semi-humid climate with 4-5 dry mo/yr (Nimer, 1979).São Domingos is a carbonate karst area characterized by the presence of continuous limestone outcrops belonging to the Bambuí Group and is crossed by several parallel streams running westward to join the Paranã River, a tributary of the upper Tocantins River, Amazon basin.After an epigean reach, each major stream enters into a cave through a sinkhole (Fig. 1), crossing hundreds to thousands of meters through subterranean conduits, and emerging to the surface through resurgence.

Materials and Methods
For the comparative study of the external morphology and morphometry of Eigenmannia species, different localities comprising both epigean and subterranean streams were sampled during the dry seasons of 1999, 2000, and 2001 using different collecting methods (e.g., hand-nets during snorkeling, electrofishing, and nets installed in the marginal vegetation of the epigean streams).The collected specimens were preserved in formalin and transferred to 70% alcohol for studies in the laboratory, where they were measured straight-line under a stereomicroscope with a dial caliper, 0.1 mm precision, on the left side.Measurements followed Mago-Leccia (1978), Triques (1996), and Campos-da-Paz (1997, 2000): LEA, length from the tip of the snout to the posterior of the anal fin base; HL, head length; SNL, snout length; OD, ocular diameter; PF, length of the pectoral fin; PPD, pre-pectoral distance; DSA, snout-anus distance; PAD, pre-anal distance.The following proportions were calculated (expressed in %): OD:HL, SNL:HL, PF:HL, HL:LEA, DSA:LEA and PAD:LEA.The degree of body pigmentation in preserved animals was categorized into two classes: evident pigmentation (P) with some coloration pattern (evident longitudinal stripes), and reduced pigmentation with faint longitudinal stripes -(RE).Three individuals of each species were cleared and stained (C&S) following the Taylor & Van Dyke (1985) method.All specimens were deposited in the ichthyological collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP), Brazil.
In order to test possible differences among the data set relative to the body size and the proportions between OD:HL and PAD:HL, non-parametric tests (Mann-Whitney and Wilcoxon) and ANOVA analysis (with a posteriori Dunn's test) were applied.The Spearman correlation test was applied in the comparison between LEA and OD values (Zar, 1996).Before each test, data were tested for homogeneity of variance (homoscedasticity) and normality.All analysis were made using Sigma Stat software (version 2.0), with á=0.05.

Results
Eigenmannia sp. was so far recorded in the epigean reach of Rio da Lapa, from the Terra Ronca Cave entrance (sinkhole of the stream -13º44'00"S 46º21'30"W) to about 100 m upstream, and in subterranean stream reaches of the Angélica (13º31'24"S 46º23'20"W) and São Mateus III (13º46'05.5"S 46º22'00"W) caves.The latter were always observed near the cave stream resurgences (where a subterranean stream reaches the surface).Apparently, the cave species, E. vicentespelaea, recorded only in caves of the São Vicente cave system (13º35'30"S 46º21'00"W, at the stream sinkhole in the São Vicente I Cave), has no contact with Eigenmannia sp., since the latter has not been found in epigean reaches of the São Vicente stream.
Morphometric data relative to the two studied species of Eigenmannia are shown in Table 1 (data from holotype and paratype not included) and a summary of differences between Eigenmannia species is presented in Table 2. Individuals smaller than 70 mm LEA (E.vicentespelaea) and 50 mm (Eigenmannia sp.) were not found.The LEA medians and the proportions between OD and HL and between OD and LEA, in both E. vicentespelaea and Eigenmannia sp., are shown in Figs. 2 and 3, respectively.The regression between OD and LEA in E. vicentespelaea is shown in Figure 4.
The OD:HL proportions varied between 17.6 and 24.4 % in most examined specimens of E. vicentespelaea (with one case of individual with no externally visible eyes -Fig.5), and between 19.7 and 28.3 % in Eigenmannia sp.OD:HL proportions in E. vicentespelaea are significantly smaller than the observed for Eigenmannia sp.(T = -418.0;p = 0.002).In addition, the eye size variation herein recorded for E. vicentespelaea is much wider than the proposed as diagnostic for this species, between 8.2 and 8.8 % (Triques, 1996).
Regarding coloration, specimens of E. vicentespelaea with reduced pigmentation -RE (91.3% of the sample) presented a translucent aspect when alive (Fig. 6).Only 8.7% of the examined E. vicentespelaea specimens showed body pigmentation similar to that of Eigenmannia sp.When preserved in alcohol, specimens of E. vicentespelaea showed a pale aspect, with no visible longitudinal stripes (Figs.5a-b).

Expanded diagnosis.
Eigenmannia vicentespelaea differs from the epigean Eigenmannia sp. from São Domingos karst area and other epigean congeners, by the following combination of characters: significantly reduced eyes, with ocular diameter usually between 17.6 and 24.4 % of head length, rarely with no externally visible eyes (OD:HL 19.7 to 28.3 % in Eigenmannia sp.); body pigmentation reduced, with a translucent aspect when alive; average body lengths significantly larger than that recorded for Eigenmannia sp. and smaller than that recorded for E. humboldtii and E. nigra.PAD:HL proportions significantly larger than in E. humboldtii, E. virescens and E. macrops.

Discussion
In view of the confused taxonomy and the disputed monophyly of the genus Eigenmannia, and the uncertain identification of the presently studied epigean species, a hypothesis of sister-group relationship between Eigenmannia sp. and E. vicentespelaea lacks phylogenetic support.On the other hand, these are the only members of the Clade F of Eigenmanninae (sensu Albert & Campos da Paz, 1998) found in the study area (Bichuette & Trajano, 2003).Thus, the most parsimonious geographic hypothesis (to be tested in a phylogenetic framework), which would involve no local extinctions of other species closer to any of the two above mentioned, is that of a sister-group relationship between them.
Several authors advocate a correlation between the time of isolation in the subterranean habitat and the degree of reduction of eyes and pigmentation (classical troglomorphisms), with populations isolated for shorter times showing less reduced and/or variable eyes and pigmentation than those homogeneously anophthalmic and unpigmented (Wilkens, 1982(Wilkens, , 1986;;Trajano, 1995).According to this hypothesis, E. vicentespelaea may represent a boundary case, as result of quite a short time in isolation, in view of the high degree of variability observed, mainly in relation to the eyes size.
It is not possible to know whether the troglobitic species is completely or only partially isolated from epigean species, because the latter was found in the same large river basin as E. vicentespelaea (the Paranã river basin, a tributary of the Upper Tocantins), but not in the same microbasins.In addition, there are records of quite large (see below) and well-fed specimens of Eigenmannia sp. in subterranean reaches from the Angélica and São Mateus streams, indicating that this typically epigean species is able to survive for a long time in the hypogean environment.Thus, in theory these fishes could join the exclusively subterranean E. vicentespelaea if they would find their way to the São Vicente stream basin.Therefore, E. vicentespelaea may be either the result of allopatric or parapatric differentiation.
The larger body sizes recorded for the specimens of Eigenmannia sp.captured in subterranean stream reaches may be related to a lower pressure of predation in this environment (corroborated by the record of potential predators in the epigean Rio da Lapa -Brycon sp.-see Bichuette & Trajano, 2003).This may be associated to a low interspecific competition in caves because it seems that food represented by prey living on the large amount of vegetal debris carried during floods into the studied caves is not limiting for these fishes in hypogean habitats (Bichuette, 2003).Therefore, cave specimens of Eigenmannia sp. have the opportunity to take food in rather abundance and live longer without being preyed upon, thus reaching larger sizes than the epigean ones.
The description of E. vicentespelaea was based on two specimens (Triques, 1996), preserved in alcohol since 1978, and its diagnosis was based solely on the OD:HL proportion.As expected, much wider variation in diagnostic characters and morphometric data was found when additional specimens were examined.Nevertheless, eyes size allied to pigmentation and body sizes may still be used to distinguish the two species of Eigenmannia from São Domingos karst area.