A new species of the cave-fish genus Lucifuga (Ophidiiformes, Bythitidae), from eastern Cuba

Abstract Recently, a barcoding study and a molecular phylogenetic analysis of the Cuban species of the cave-fish genus Lucifuga Poey, 1858 revealed the existence of different evolutionary lineages that were previously unknown or passed unnoticed by morphological scrutiny (i.e., cryptic candidate species). In the present study, Lucifuga gibarensis is described as a new species restricted to anchialine caves in the northeastern karst region of the main island. The species was earlier described as a variety of Lucifuga dentata, but since the name was introduced as a variety after 1960, it is deemed to be infrasubspecific and unavailable according to the International Code of Zoological Nomenclature Art. 15.2. The new species differs from L. dentata by pigmented eyes vs. eyes absent and lack of palatine teeth vs. present. Lucifuga gibarensis seems to be most similar to the Bahamian species L. lucayana by showing pigmented eyes, 13 or 14 precaudal vertebrae and ten caudal fin rays. However, differs from it by a larger size of the pigmented eye (1.1–1.9 vs. 0.9–1.0% SL) and number of posterior lateral line neuromasts (30–33 vs. 34–35).


Introduction
Lucifuga Poey, 1858 is a conspicuous genus of obligate cave-dwelling fishes, currently recognised with six species distributed in Cuba and Bahamas Møller et al. 2006Møller et al. , 2016; see comparative material). Another nominal species, Lucifuga inopinata Cohen and McCosker, 1998, from off Galapagos Archipelago belongs to another, yet undescribed, genus (Møller unpublished data).
Because of the characteristics of the habitats of Lucifuga species (caves, sinkholes and crevices) and the morphological modifications that they show in the evolutionary adaptations to the environment, the genus represents an iconic part of the fish fauna in Cuba. The scientific interest in these fishes, however, has been sporadic. Since the description of the genus and the two first Cuban species by Felipe Poey (1858), the studies dealing with the genus are very few and have mainly been dedicated to the discussions of morphological characters of taxonomic interest for the genus and species and the descriptions of new species (Gill 1863;Nalbant 1981;Díaz-Pérez et al. 1987a, 1987bDíaz-Pérez 1988); some aspects of feeding and reproductive system (Lane 1903;Eigenmann 1909;Thinès and Piquemal 1978;García-Debrás and Pérez 1999) and two studies that constituted the first approximation to the evolutionary relationships of the group based on a comparison of several morphological characters of the three species known at that time . Møller et al. (2006) found evidence for all Cuban and all Bahamian species representing two separate evolutionary lineages, but recently García-Machado et al. (2011) made a phylogenetic analysis of the Cuban species using mitochondrial and nuclear genes finding several new evolutionary lineages not identified previously by morphological analyses. It was also indicated that the separation in Cuban and Bahamian species as suggested by Møller et al. (2006) is no longer correct, since some of the new Cuban species are more closely related to Bahamian species than to other Cuban species. Their results also questioned the specific status of Lucifuga teresinarum Diaz, 1988, showing no difference to L. subterranea Poey, 1858 (see also Lara et al. 2010).
A controversial taxon has been Lucifuga dentatus var. holguinensis Díaz-Pérez, Nieto and Abio, 1987 from the Holguin province in eastern Cuba. It was suggested as a valid species name by Proudlove (2019), but the name has now been decided to be infrasubspecific and unavailable according to ICZN Art. 15.2, since it was introduced as a variety after 1960 (Fricke et al. 2019). In the present study, based on the molecular results of García-Machado et al. (2011) and from revisiting the morphological characters recently used to define species in the genus ), we present a new formal description of the species as Lucifuga gibarensis sp. nov.

Materials and methods
The morphological study of the Cuban Lucifuga species was based on the analysis of 214 individuals sampled from several localities covering most of its known distribu-tion areas (Fig. 1). Nine morphometric measurements were taken using a Vernier calliper (precision 0.05 mm) and eleven meristic counts (e.g., fin ray numbers, scales, etc.) were carried out using a Novel stereomicroscope (magnification 40 x maximum) and/ or radiographs. All morphometric measurements were weighted according to the standard length (SL) to avoid allometric effects. The number of vertebrae was counted using X-ray radiographs. Diagnosis. Body moderately elongated and compressed mainly from the abdomen to the caudal end. Snout with two nostrils: anterior nostril tube-shape and smaller, placed near to the upper lip; posterior nostril is a larger hole, placed ca. midway between snout and eyes cavity. The mouth is subterminal with the lower jaw only slightly shorter than the upper. Opercular spines absent. Seven branchiostegal rays.
The entire body is covered with small, rounded cycloid scales; fins naked except for scales on pectoral fin basis. Predorsal area and operculum scaled. Branchiostegal membranes, entire underside of the head, snout, interorbital areas and entire course of the cavernous cephalic system are naked. Origin of dorsal fin approximately above the tip of pectoral fins. Pelvic fin is subjugular with a single ray reaching ca. 1/3 to halfway to the anus. Pectoral fin behind the operculum, peduncle short and narrow. Lateral line with two series of sensory neuromasts: upper and anterior series extends from the head to a point ca. midway between dorsal and anal fin origins; and lower and posterior series extends from a point under and slightly in advance of the end of the upper series to the mid side from the caudal base.
There are three symmetric sensory canal series on each side of the head: supraorbital series with three pores (two anterior and one posterior): the anteriormost is at the snout rim, the second open between and above the nasal openings, and the posterior single pore is at the end of the lateral canal above the operculum. The infraorbital series with six pores (three anterior and three posterior): first pore is slightly below the anterior nasal opening, the other five pores (two anterior and three posterior) are along the edge of infraorbital rim. Finally, the mandibular series with six pores (three anterior and three posterior). The first pore is in the fold of skin between the lip and canal series, the second is at the side of the jaw tip on the lower lip, the third is at the anterior end of the mandibular series, the fourth to sixth posterior pores open ventrally along the mandibular series. There is also a large preopercular pore. Teeth are present on the premaxillae, dentaries and vomer; but are present or absent in palatines.
Sexual dimorphism. The male copulatory organ is completely integrated into a fleshy genital hood which projects posteriorly beyond the anus, the lateral end of the hood could be from broad to conical. A fleshy small conical papillae project from the middle of the distal margin of the hood and is enclosed by lateral earlike lobes. Penis is placed underneath the hood. Diagnosis. Dorsal fin rays 72-90; anal fin rays 58-72; pectoral fin rays 15-17, caudal fin rays 10; palatine teeth absent; rakers on anterior gill arch 17-19 (long gillrakers 3); occiput and area between lateral canal and preopercular canal scaled; diameter of pigmented eyes 1.1-1.9% SL; total vertebrae 50-53.

Lucifuga gibarensis
Description. Meristic and morphometric characters are given in Tables 1, 2. Body moderately elevated behind the head, with a slight depression in the interorbital region (Figs 2, 3). Eyes pigmented (similar to the condition present in L. spelaeotes and L. lucayana ). Anterior gill arch with three elongate rakers and 14-16 low dentigerous pads. The areas between lateral canal and preopercular canal, and the occiput are scaled (Fig. 2). Caudal fin free (not fused with dorsal and anal fins). In the lateral line series of sensory neuromasts, the upper and anterior count with 13-15, the lower and posterior with 30-35. Teeth are present on the premaxillae (5-7 rows), dentaries (6 or 7 rows) and vomer (2 or 3 rows in two separate patches). Palatines without teeth.
Coloration. Uniformly brown or light brown, with lighter fins and naked parts on the head. Nevertheless, one juvenile specimen (ZMUC P771732) was very pale, but still with tiny dark pigment dots (Fig. 3b).
Distribution and habitat. Lucifuga gibarensis shows a very restricted known distribution, in a lithographically isolate karst patch of caves at the north of Gibara municipality, Holguín province, without any overlap with other Cuban species of the genus (Fig 1; García Hernández et al. 2016). It is ca. 800 km away from the nearest L. dentata, L. subterranea and L. simile distribution areas. The distance to the Bahamian species on Little Bahama Bank (L. lucayana) and Great Bahama Bank (L. spelaeotes) is ca. 650 km and 240 km, respectively. The location area is composed by three caves (Aguada de Macigo, Tanque Azul and Cueva El Baga) located near to the shore ca. 3-15 km from each other (Corella et al. 2000, Dietz 2015). The Aguada de Macigo cave is the type-locality with an emergent large doline, ca. 22 m deep and salinity of 16 ppt. According to Díaz-Pérez et al. (1987b), the individual designated as holotype was caught at 12 m depth.
Etymology. The specific epithet refers to the village of Gibara, where the three caves inhabited by this species are located. We do not follow variety epithet used by Díaz-Pérez et al. (1987b), since the L. gibarensis better describes the narrow distribution of the species near the village Gibara instead of the entire region Holguin.
Genetic distances. Among Cuban species, García-Machado et al. (2011) have demonstrated that L. gibarensis [at that time as L. dentata var. holguinensis] is not phylogenetically close to L. dentata by showing a large mitochondrial DNA divergence of 30.5% (16.5% with cytochrome b gene) as well as several diagnostic nucleotide variations at nuclear genes. In contrast, L. gibarensis is phylogenetically closely related to other two lineages of undescribed species of Lucifuga from Cuba (named Lucifuga sp. 3 and L. sp. 4) (García-Machado et al. 2011). However, genetic distance to both Bahamian species is not yet known.
Comparisons. Based on external appearance, Lucifuga gibarensis sp. nov. resembles the Cuban species L. dentata (from which it was designated as variety, see Díaz et al. 1987b) and L. simile. Nonetheless, it differs in several characters: e.g., number of caudal fin rays (10 vs. 8), diameter of the pigmented eyes (1.1-1.9 vs. 0.0-0.2% SL), lack of palatine teeth vs. present and scaled occiput vs. naked or weakly scaled occiput. It also differs in dorsal and anal fin rays mean number (fewer than L. dentata and more than L. simile) ( Table 1).
Lucifuga gibarensis sp. nov. also resembles L. subterranea in the lack of palatine teeth and the scaled occiput, but it differs in the body moderately elevate behind the head vs. little elevated (see maximum height in Table 1), number of pectoral fin rays (15-17 vs. 10-13), number of caudal fin rays (10 vs. 8), the diameter of the pigmented eyes (1.1-1.9 vs. 0.0-0.3% SL) and in the number of rakers on the anterior gill arch 17-19 vs. 12-17 (Table 1).
Finally, Lucifuga gibarensis resembles both Bahamian species in the head profile, the number of caudal fin rays (10), the occiput scales (similar to L. spelaeotes and less scaled than L. lucayana) and in the presence of relatively large pigmented eyes (Table  1). With L. lucayana it also shares the lack of palatine teeth. It differs in the number of pectoral fin rays (15-17 vs. 17-18 in L. lucayana and 17-20 in L. spelaeotes); and diameter of pigmented eye is larger than in L. lucayana (1.1-1.9 vs. 0.9-1.0% SL).
Remarks. It has been demonstrated that L. gibarensis is not phylogenetically close to L. dentata. The estimate of mtDNA genetic divergence between these two lineages is huge (P = 30.5%) and several diagnostic nucleotide changes at the intron 4 of calmoduline gene and intron 1 of the homeodomain EVX gene were described Designation as a variety of L. dentata, was wrong as judgment, given the sharp differences observed at three major morphological characters: palatine teeth; number of caudal fin rays; and degree of pigmentation in the eyes. Particularly, the number of caudal fin rays (10) and pigmented eyes were realised in L. spelaeotes description (Cohen and Robins 1970), and recognised as diagnostic characters to distinguish the Cuban and Bahamian species at that time (Cohen and Robins 1970;Møller et al. 2006).
As a result of the present study, we describe a new species, Lucifuga gibarensis, which is supported by morphology and molecular phylogenetic analysis (García-Machado et al. 2011). We found unique diagnostic characters that distinguish this species from all the species described so far. Díaz-Pérez et al. (1987b) identified this taxon as a variety of Lucifuga dentata (L. dentata var. holguinensis), and recognised the presence of 10 caudal fin rays and pigmented eyes (characters distinguished by Cohen and Robins (1970) as important to separate L. spelaeotes from the two Cuban species known at that time), but underestimated the taxonomic relevance of these characters and avoid them. They also underrated the absence of palatine teeth vs. present in L. dentata, a         useful taxonomic character to distinguish Lucifuga species (see Poey 1858; Møller et al. 2006). Furthermore, Møller et al. (2006) pointing out that the Bahamian species differing from all four Cuban species formerly known by having higher caudal fin rays number (10 vs. 8), larger pigmented eyes diameter (0.7-1.8 vs. 0.0-0.3% SL), higher vertebrae number (50-55 vs. 45-48), and higher pectoral fin rays number (17-20 vs. 10-17) supporting the hypothesis that Bahamas and Cuba are represented by two different evolutionary lineages (see also . However, the new Cuban species L. gibarensis, shared a similar combination of these characters with Bahamian species apart from low number of pectoral fin rays in L. gibarensis. Based on these characters, our results do not support that lineages are confined to only one Archipelago.
With the available knowledge, species with reduced or completely absence of eyes and 8 fin rays are only found in western Cuba; but species having pigmented eyes and 10 caudal fin rays are found in both archipelagos. Detailed phylogenetic studies including all Atlantic Lucifuga spp. will be crucial to clarify the phylogeographic relationships between the Cuban and Bahamian members of this genus.

Identification key to species of Lucifuga
The current key is based on a small number of samples. Measures that overlapping in range were only used when it helps distinguishing between two species. We also use several exemplars from Juanelo Piedra and Luis Piedra caves which are near to El Cajio cave (ca. 2 km) the type-locality referred by Poey (1858). Lucifuga dentata is the most abundant and widely distributed Lucifuga species in Cuba. It is found in caves from median-southern karts from central (Matanzas province) to the western part of the island (Guanahacabibes Peninsula). Its distribution is not continuous, with the most important gap between western Havana and Guanahacabibes, Pinar del Río (Hernández et al. 2016). Remarks. We examined specimens of L. simile from the two known localities: the type-locality Grieta Punta de Guana cave (Nalbant, 1981) and La Pluma Cave (Díaz-Pérez et al. 1987a). This species was also reported from El Tunel cave in Quivican, southern Havana, living in sympatry with L. dentata (Díaz-Pérez et al. 1987a). However, this later report need verification.