Redescription and diagnoses of the genera Profundulus and Tlaloc (Cyprinodontiformes: Profundulidae), Mesoamerican endemic fishes

Abstract Until recently, the genus Profundulus was classified in two subgenera, Profundulus and Tlaloc, the sole members of the family Profundulidae. Newly discovered molecular data have been used to justify the elevation of these subgenera to genera. Yet morphological analyses to diagnose the two genera are lacking. The aim of this study is to provide a generic diagnosis and a taxonomic key to the species within the family Profundulidae based on morphology. The genus Tlaloc is diagnosed on the basis of five unique characters, among which are the prominent and oval-shaped mesethmoid, exceeding the posterior margins of the vomer; the anterior portion of the parasphenoid making contact with the mesethmoid and extend beyond the center of the mesethmoid; and a reduced autopterotic fossa. Profundulus is diagnosed here based on the following characters: the mesethmoid is small, crescent-shaped, and does not extend beyond the margins of the vomer; the anterior portion of the parasphenoid just contacting the mesethmoid and not extending beyond the center of the mesethmoid; a large autopterotic fossa. For each genus, description and distribution ranges are provided as well as a key for identification of the species.


INTRODUCTION
The family Profundulidae Hoedeman & Bronner, 1951 is a group of freshwater fishes with a restricted geographical distribution, extending from southern Mexico to Central America (Miller, 1955;Matamoros et al., 2012).It is one of the characteristic elements of the endemic fauna of Central America and the southern Mexico highlands (Miller, 1955), with most of the included species having a restricted distribution range and occurring only in a few adjacent river systems (Matamoros et al., 2012).The Profundulidae is one of the least speciose within the order Cyprinodontiformes (killifishes, pupfishes, and relatives), with only thirteen described species, compared to, for example, approximately 59 nominal species in the family Goodeidae, its sister-group (Nelson et al., 2016;Piller et al., 2022).The profundulids or Middle American killifishes are commonly known as escamudos (Lozano-Vilano, De La Maza-Benignos, 2016).
Until recently, the genus Profundulus was classified in two subgenera, Profundulus and Tlaloc, the sole members of the family Profundulidae (Miller, 1955(Miller, , 2009;;Parenti, 1981).Newly discovered molecular data have been used to justify the elevation of these subgenera to genera by Morcillo et al. (2016).In the absence of a diagnosis, the new clades were supported based on previously documented differences in both subgenera: presence or absence of a humeral spot at the base of the pectoral fin and the number of scales in the preorbital region and base of the caudal fin (Miller, 1955(Miller, , 2009)), as well as by a series of osteological characters of the axial and appendicular skeleton (González-Díaz et al., 2014).These morphological-osteological differences were based on a review of only six species.Our knowledge of the Profundulidae is growing rapidly, and the number of valid species has more than doubled since Miller's comprehensive revision of Profundulus in 1955.Five new species were described within the genus Profundulus and one in the genus Tlaloc.Additionally, Profundulus balsanus Ahl, 1935 was redescribed and recognized as a valid species (Jamangapé et al., 2016).
External morphological traits (meristic and morphometric) were once the primary sources of characters to distinguish the subgenera and species and to classify profundulid fishes (Miller, 1955(Miller, , 2009)).Different types of morphological characters were gradually introduced to killifish systematics (Parenti, 1981;Costa, 2006) and the morphological analysis of bones has been an important source of morphological characters for hypothesizing relationships among profundulids and cyprinodontoid sister families (Parenti, 1981;Costa, 1998;Ghedotti, Davis, 2013) and between the subgenera and some species of the Profundulidae (e.g., Uyeno, Miller, 1962;González-Díaz et al., 2014).
Profundulids are among the least studied Cyprinodontiformes, and the systematics of the family is still in its early stages, with genera and some species poorly defined, and few descriptions of their osteology (Lozano-Vilano, De La Maza-Benignos, 2016;Morcillo et al., 2016).Several recent molecular studies provide hypotheses on the relationship of profundulid taxa and confirm the monophyly of two previously proposed genera (or subgenera) (e.g., Doadrio et al., 1999;Morcillo et al., 2016;Calixto-Rojas et al., 2021).Molecular data support profundulid clades (genera) which have been scarcely described by morphological characters, highlighting the need to improve the osteological-morphological database for this group.Therefore, the aim of this study is to provide accurate descriptions of morphological characters, including external morphology of body, osteology, neuromasts (cephalic pores), and contact organs in Profundulus and Tlaloc, and to discover additional informative characters to diagnose and characterize both genera.A dichotomous key for the identification of the species of the family Profundulidae is presented.

MATERIAL AND METHODS
The examined material is deposited in the fish collection of the Centro Interdiciplinario de Investigación para el Desarrollo Integral Regional, Oaxaca (CIDOAX); Colección Nacional de Peces, Universidad Nacional Autónoma de México, Mexico (CNPE-IBUNAM ); Field Museum of Natural History, Chicago (FMNH); Louisiana Museum of Natural Science, Louisiana (LSUMZ or LSU MNS); Universidad de Ciencias y Artes de Chiapas, Mexico (MZ-UNICACH).The list of the examined material includes the acronym of the collection and the catalog number, followed by the number of specimens, in parentheses the specimens cleared and stained (c&s), and by their standardlength range.
Osteological characters were obtained from specimens of the 13 profundulid species, cleared and double stained (bone alizarin and cartilage counter-stained with alcian blue), according to the technique described by Taylor (1967), with some modifications proposed by Taylor, Van Dyke (1985).The identification of bone elements was based on the bone nomenclature proposed by Gosline (1961), Parenti (1981), and Costa (1998).The terminology of fin rays and the count of vertebrae follows Arratia (2008) and Schultze, Arratia (2013).Based on the variation in meristic, morphometric, and osteological characters as well as the distribution of the species, a dichotomous key for the identification of the species of the family Profundulidae was established.
Diagnosis.Tlaloc, one of the two genera of the family Profundulidae, is diagnosed here by the following combination of characters: The mesethmoid is prominent and oval in shape, extending beyond the posterior margins of the vomer, encompassing the posterior medial extension and touching the lateral ethmoids (Fig. 2B).The anterior portion of the parasphenoid making contact with the mesethmoid and extend beyond the center of the mesethmoid.The autopterotic fossa is reduced (Fig. 2A).The dorsal margin of the interoperculum, with a long extension, is exceeding the edge of the bone (Fig. 3A).The ventral margin of the lacrimal is straight (Figs.4A-B).Tlaloc is further distinguished from Profundulus by having less than the basal half of the caudal fin densely scaled (except in T. portillorum) (vs.more than the basal half or more densely scaled) (Fig. 5A); by the absence of a humeral spot (vs.humeral spot present); by the origin of the dorsal fin positioned at a vertical line posterior to the origin of the anal fin (vs.origin of the dorsal fin positioned at a vertical line slightly anterior to the origin of the anal fin); by long epiotic processes, extending beyond the second vertebra (vs.short epiotic processes, not extending beyond the first vertebra).Abbreviations: de, dentary; pm, premaxilla; ra, retroarticular; mx, maxilla; pl, palatine; qu, quadrate; ar, articular; ms, mesopterygoid; io, interopercle; sy, sympletic; hy, hyomandibula; po, preopercle; op, opercle; so, subopercle.
Description.Morphometric data appear in Tab. 1. Body uniformly slender; head compressed, moderately large (22.4-36.7%).Mouth subterminal to terminal.In T. hildebrandi and T. portillorum, the lower jaw broad, heavy, and protruding so that the upper jaw is included, whereas in T. labialis and T. candalarius both equal in forward projection, or the lower jaw is included in the upper jaw.Greatest body depth in the vertical just posterior to pectoral fin (20.0-32.6%).The females are elongated, and the males are more robust than the females.Dorsal and anal fins located posterior to the half of body length.The branching pattern of the cephalic latero-sensory canals in Tlaloc is similar to the general pattern of the Cyprinodontiformes.The supraorbital series follows the Type II designation of Gosline (1949), with canals between pores 1-2a, 2b-4a, 4b-7; preopercular pores 6-7 (mode = 7); preorbital pores 3-5 (mode = 4); mandibular pores 4-5 (mode = 5); in addition, there are 2 to 4 rostral pores usually developed.
Coloration.The coloration varies strongly among individuals and ontogenetically.The skin of the head and body may show golden reflections, especially in the opercular region and mid flank.The form and coloration of the nuptial adult male and female are shown in Fig. 6.Unpaired fins (dorsal and anal) are orange in males, with irregular black markings on the dorsal fin, evident in Tlaloc candalarius.

Sexual dimorphism and contact organs.
There is little sexual dimorphism in species of the genus Tlaloc.Males are slightly larger than females; the largest specimen recorded was a male from Tlaloc hildebrandi (111.49mm SL), whereas the maximum size recorded in a female was in Tlaloc labialis (101.93 mm SL).The shape of the anal fin, however, shows a marked sexual difference: the anterior anal rays of the male are not greatly longer than the posterior ones, giving the distal margin of the fin an evenly rounded edge.However, in the female, since the medial rays, from about the sixth to the tenth, are much longer than either the anterior or the posterior rays, the distal margin of the fin is lobate when expanded.Males have contact organs or spinules, articulated with the lateral surfaces of the anal fin rays with some of them conspicuously long; this is the most notable, though not striking difference between males and females of all Tlaloc species (Fig. 7).

Geographical distribution.
Restricted to the Atlantic slope of Middle America (Fig. 8).From the border between Oaxaca and Chiapas, Mexico, to the center of Honduras, with the exception of Tlaloc portillorum, this is located on both slopes of Honduras, Atlantic and Pacific.

Diagnosis.
Profundulus is diagnosed here based on the following characters: The mesethmoid is small, crescent-shaped, and does not extend beyond the margin of the vomer (Figs.2C-D); the vomer is greatly broadened anteriorly, lacks lateral processes and is in contact with the lateral ethmoids; the anterior portion of the parasphenoid is just in contact with the mesethmoid and does not extend beyond the center of the mesethmoid; the autopterotic fossa is large (Fig. 2D); the dorsal margin of the interoperculum, with a short extension, does not exceed the edge of the bone (Fig. 3B); the ventral margin of the lacrimal slightly concave (Figs.4C-D).Profundulus is further distinguished from Tlaloc in having more than the basal half of the caudal fin densely scaled (except in P. kreiseri) (vs.less than the basal half of caudal fin densely scaled) (Fig. 5B); by a humeral spot (except in P. adani) (vs.humeral spot absent) (Fig. 5B); by the origin of the dorsal fin positioned at a vertical line slightly anterior to the origin of the anal fin (vs.origin of the dorsal fin positioned at a vertical line posterior to the origin of the anal fin); by short epiotic processes, not extending beyond the first vertebra (except in P. parentiae) (vs.long epiotic processes, extending beyond the second vertebra).
Description.Morphometric data appear in Tab. 2. Body rather robust, elongate; head compressed, large (24.1-35.3%); the interorbital broad (38.2-58.7%)and typically concave or nearly flat.Ascending premaxillary process short and broad and bluntly rounded at the tip.Mouth subterminal, lower jaw broad, heavy and protruding so that upper jaw is included.Dorsal fin rounded in both males and females with its basal length short (11.1-19.2%).Anal fins of males rounded, slightly elongated in females.Caudal peduncle relatively short (11.1-23.2%).Median hypural plate divided into subequal parts by an open groove.
Coloration.The form and coloration of the nuptial adult male are shown in Fig. 5B.Most of the body dark, often with irregular brown-dark spots on the scales, on the sides of the body on to the caudal fin.A golden yellow blotch covers the operculum and reaches the base of the pectoral fin.
Sexual dimorphism and contact organs.The sexual dimorphism is not very evident in species of the genus Profundulus.However, the males are slightly larger than females; the largest specimen recorded was a male from P. guatemalensis (83.9 mm SL), while the maximum size recorded in a female was in P. adani (77.9 mm SL).In males and females of all species of Profundulus, the dorsal and the anal fins have a similar morphology.Like Tlaloc (Fig. 7) the males of Profundulus have contact organs or spinules, articulated with the lateral surfaces of the anal fin rays with some of them conspicuously long; this is the most notable, though not striking difference between males and females of all Profundulus species.

DISCUSSION
This study based on morphological characters supports relationships found between genera of Profundulidae as proposed in molecular analyses.Recently Morcillo et al. (2016) resurrected the genus Tlaloc, based on molecular evidence, recognizing the monophyly of the group.Monophyly of Tlaloc is supported in this study by five unique morphological characters: the mesethmoid is prominent and oval in shape, protruding from the posterior margins of the vomer, encompassing the posterior medial extension and making contact with the lateral ethmoids; the upper portion of the parasphenoid makes contact with the mesethmoid and does extent beyond the medial part of this last bone; the autopterotic fossa is reduced; the ventral margin of the lacrimal is straight; the dorsal margin of the interoperculum, with a long extension, exceeds the edge of the bone.
Profundulus was erected and diagnosed by Hubbs (1924), based on following diagnostic characters: The lateral rims of the genital aperture of the adult female are scarcely pronounced, surrounding not more than the first anal ray; the anal fin in the adult male is lower, instead of higher, than in either the young or the adult female.Miller (1955) in a review study of Profundulus (Cyprinodontidae) include these two traits and provided three new characters to characterize Profundulus: The shape of the premaxillary process, the nature of the hypural plate (divided into subequal parts by an open groove), and the number of gill rakers.Parenti (1981) distinguished Profundulus (representing the family Profundulidae) from all other cyprinodontoids by a large autopterotic fossa and a high number of gill rakers on the anterior arm of the first arch (14-23).With the reclassification of Profundulus in two genera, Profundulus and Tlaloc (Morcillo et al., 2016), the diagnostic character of a large autopterotic fossa is now limited to Profundulus.We here add five characters to distinguish Profundulus from Tlaloc: The mesethmoid is small, crescent-shaped, and does not exceed the margins of the vomer; the vomer is greatly broadened anteriorly, lacks lateral processes and is in Redescription of the genera Profundulus and Tlaloc contact with the lateral ethmoids; the upper portion of the parasphenoid, just in contact with the mesethmoid, does not extend beyond the medial part of this last bone; the dorsal margin of the interoperculum, with a short extension, does not exceed the edge of the bone; the ventral margin of the lacrimal is slightly concave.Miller (1955) erected two subgenera based on the presence in Profundulus or absence in Tlaloc of conspicuously embedded scales in the preorbital region, the basal half or more of the caudal fin densely scaled, and the presence of a humeral spot in Profundulus or absence in Tlaloc.These characters were found in the analysis of only five described species.Although with most species these characters allow the separation of genera; in our review, including the 13 species currently described, none of these characters functioned as unique characters to diagnose genera.González-Díaz et al. (2014) described eight osteological differences between the subgenera Profundulus and Tlaloc (seven of the skull and one of the axial skeleton), based on the analysis of only six species.Again, we observed only two characters to be considered of generic significance: the mesethmoid (small vs. large; our analyses corroborate the findings that were previously described by Uyeno, Miller, 1962), and the dorsal margin of the interoperculum (long vs. short extension).The form of the vomer (Y-shaped) was described by Costa (1989) as a character that defines the family Profundulidae; this character was described by González-Díaz et al. (2014) as a triangular-shaped character in Profundulus and Y-shaped in Tlaloc.We however observed Y-shaped vomers in both genera (not triangular) (Figs.2B-C), so we agree with the description of Costa (1998).
The current study provides sufficient empirical evidence to confirm the separation and diagnosis of the genera Tlaloc and Profundulus.Analysis of morphologicalosteological characters support the clades previously defined by molecular data.The comparative morphology has played an important role in the reconstruction of the evolutionary history and classification of cyprinodontiform fishes, often providing useful phylogenetic information at different taxonomic levels.For this reason, it would be important to incorporate this information to assess alternative hypotheses among members of the family Profundulidae and other groups within the Cyprinodontoidei suborder.

FIGURE 1 |
FIGURE 1 | Morphometric characters, based on sketches of Profundulus in lateral view.Morphometric measurements in the Material and Methods section.

FIGURE 5 |
FIGURE 5 | Male general morphology and caudal fin squamation, life colour patterns in: A. Tlaloc labialis; and B. Profundulus punctatus.Solid arrow points to the squamation of the caudal fin, dashed arrow points to the humeral spot.

FIGURE 7 |
FIGURE 7 | Bony spinules in fin rays of male Tlaloc labialis, indicated by arrow (MZ-UNICACH 6740, 75.7 mm SL), articulated with the lateral surfaces of the anal fin rays.

FIGURE 8 |
FIGURE 8 | Geographical distribution of species of the genera Tlaloc and Profundulus in southern Mexico and Central America.

TABLE 1 |
Morphometric data of Tlaloc and Profundulus species.Asterisk mark the number of specimens analyzed, in parenthesis the average values.

TABLE 2 |
Meristic data of Tlaloc and Profundulus species.Asterisk mark the number of specimens analyzed, in parenthesis the modal values.