Three new species of the killifish genus Melanorivulus from the central Brazilian Cerrado savanna (Cyprinodontiformes, Aplocheilidae)

Abstract Three new species are described from the Neotropical region comprising the Cerrado savannas of the central Brazilian plateaus, which is among the most important biodiversity centres in the world. These species are considered closely related to Melanorivulus dapazi from the same region, with which they share the presence of a rudimentary interarcual cartilage and a dark reddish brown distal margin on the male anal fin. The group comprising Melanorivulus dapazi and the three new species is here named as the Melanorivulus dapazi species group. Melanorivulus ignescens sp. n., from the upper Rio Araguaia basin, is distinguished from all other species of the Melanorivulus dapazi group by the anal-fin colour pattern in males; Melanorivulus flavipinnis sp. n. and Melanorivulus regularis sp. n. from the Rio Paraguai basin are distinguished from all other congeners of the Melanorivulus dapazi group by the colour pattern of the caudal fin and number of scales in the longitudinal series, respectively. All the new species are further unambiguously diagnosed by unique combinations of morphological characters, including meristic and morphometric data, and colour patterns. This study reinforces the importance of using live colour patterns to diagnose species and species groups of the genus Melanorivulus, but also indicates that osteological characters may be informative for species diagnosis. This study confirms the high diversity of species of Melanorivulus in the central Brazilian Cerrado plateaus already reported in previous studies, indicating that endemic species are often restricted to short segments of a single river drainage.


Introduction
The region comprising the Cerrado savannas of central Brazil has been considered among the most important biodiversity hotspots in the world (Myers et al. 2000), although many organisms endemic to this region were insufficiently sampled and poorly known until recent years . A typical component of the Cerrado fauna is the killifish genus Melanorivulus Costa, 2006, with species inhabiting the Veredas, a Cerrado ecosystem consisting of small streams running in shallow valleys, often exhibiting the buriti-palm Mauritia flexuosa along their banks (e.g., Costa 2007a; Oliveira et al. 2012). Probably as a consequence of small size, usually not surpassing 45 mm of total length, species of Melanorivulus occurring in this ecosystem were not represented in collections until recent years, with the great majority of the approximately 40 species occurring in the central Brazilian Cerrado being described only after 2005 (e.g., Costa 2012;Costa et al. 2016).
The greatest diversity among species of Melanorivulus endemic to the Cerrado is concentrated in the central-western Brazilian plateaus, which range in altitudes from 400 to 1,100 m above sea level (asl), in the Caiapó mountain range (Costa 2012). This area is drained by the upper tributaries of the Rio Araguaia, flowing north and belonging to the Amazonas-Tocantins river system, and the upper Paraguai and Paraná river basins, flowing southwest and south, respectively, and belonging to the Paraná-Paraguay-Uruguay river system. A total of 12 species have been recorded for this area, of which four are endemic to the Araguaia basin, one to the Paraguai basin, and seven to the Paraná basin (Costa 1989(Costa , 2005(Costa , 2006a(Costa , 2007a(Costa -b, 2008(Costa , 2012. During a recent expedition to this area, three new species were collected, one from the upper Araguaia basin and two from the Paraguai basin. All the three new species are considered to be closely related to M. dapazi, endemic to the Paraguai basin, by all sharing a rudimentary interarcual cartilage and a dark reddish brown stripe on the distal margin of the anal fin in males (vs. interarcual cartilage well-developed and never a similar stripe on the anal-fin distal margin; see Discussion below). This assemblage is hereafter called the M. dapazi species group and the three new species are herein described.

Material and methods
Specimens were captured with small dip nets (40 × 30 cm) and were euthanized soon after collection. Representative live specimens were kept alive for nearly 24 hours, photographed, and then euthanized. Euthanasia was conducted in a buffered solution of tricaine methanesulfonate (MS-222) at a concentration of 250 mg/l, for a period of about 10 minutes, i.e., until opercular movements ceased. Specimens were fixed in formalin for a period of 10 days, and then transferred to 70% ethanol. Collections were made with permits provided by ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade) and methods for euthanasia were approved by CEUA-CCS-UFRJ (Ethics Committee for Animal Use of Federal University of Rio de Janeiro; permit number: 01200.001568/2013-87). Material is deposited in Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro (UFRJ) and Coleção Ictiológica do Centro de Ciências Agrárias e Ambientais, Universidade Federal do Maranhão, Chapadinha (CICCAA).
Descriptions of colouration of living fish were based on photographs of both sides of individuals. Photographs were taken in small aquaria around 24 hours after collection. Additional direct observations were made with fish in small transparent plastic bottles just after collection. Measurements and counts follow Costa (1988). Measurements are presented as percentages of standard length (SL), except for those related to head morphology, which are expressed as percentages of head length. Fin-ray counts include all elements. Four specimens, two males and two males, were cleared and stained for osteological analysis using the methods presented in Taylor and Van Dyke (1985); the abbreviation C&S in lists of material indicates those specimens that were prepared for osteological examination. Terminology for osteological structures followed Costa (2006b), for frontal squamation Hoedeman (1958), and for cephalic neuromast series Costa (2001). Osteological characters used in species descriptions are those that show variability within Melanorivulus (e.g., Costa 2016). Herein, geographical localities involved terms popularly adopted in the local region to compose names of geographical accidents (e.g., rio, ribeirão) allowing more accurate identifications of localities in the field and avoiding common mistakes when tentatively translating them to English; following this reasoning, Rio Paraguai is used instead of Paraguay River. New species descriptions are listed according to their type localities, from north to south. dapazi and M. regularis); caudal fin, in males, without red bars and distinctive orange margin (vs. with red bars in M. regularis and M. flavipinnis, with broad bright orange band along the whole margin in M. dapazi); in females, ventral surface of the head with dark grey spots, often forming short stripe on the chin (vs. without dark grey spots in M. dapazi); caudal-fin short, its length 26.8-33.1% SL (vs. long, its length 34.1-38.7% SL in M. flavipinnis). Also distinguished from all other species of the M. dapazi group by having a constriction on the metapterygoid (vs. constriction absent).

Melanorivulus ignescens
Description. Morphometric data appear in Table 1. Body slender, sub-cylindrical anteriorly, slightly deeper than wide, compressed posteriorly. Greatest body depth at vertical just in front of pelvic-fin base. Dorsal and ventral profiles of trunk almost straight to slightly convex in lateral view; dorsal and ventral profiles of caudal peduncle nearly straight. Head moderately wide, sub-triangular in lateral view, dorsal profile nearly straight, ventral profile convex. Jaws short, snout weakly pointed in lateral view.
Dorsal and anal fins short, extremity slightly pointed in males, rounded in females. Caudal fin oval, slightly longer than deep. Pectoral fin rounded, posterior margin reaching vertical at 80-90% of length between pectoral-fin and pelvic-fin bases. Pelvic fin small, tip reaching between urogenital papilla and base of 1 st anal-fin ray in males, reaching between anus and urogenital papilla in females; pelvic-fin bases medially in close proximity. Dorsal-fin origin on vertical through base of 8 th anal-fin ray. Dorsal-fin rays 9-11; anal-fin rays 13-15; caudal-fin rays 30-31; pectoral-fin rays 13; pelvic-fin rays 5-6. No contact organs on fins.
Colouration. Males. Flank metallic green-blue to metallic light green, sometimes purple-blue above anal fin; oblique narrow orangish red bars irregularly arranged, often forming chevron-like marks anteriorly directed; horizontal rows of reddish orange dots on anteroventral part of flank, between bases of pectoral and pelvic fins; pale dark grey blotches on postorbital region mainly visible when fish is exposed to strong light. Dorsum light brown with black dots, venter white. Dorsal portion of head side light brown, ventral portion white; pale golden iridescence on opercular region. Jaws dark grey. Iris pale yellow, sometimes with dark brown bar on anterior and posterior portions. Dorsal fin light yellow with two or three oblique dark red bars on posterior portion of fin. Anal fin reddish orange in adult exemplars to yellowish orange in juveniles, basal portion bluish white, distal region becoming gradually dark red-brown, distal margin with high concentration of melanophores. Caudal fin light yellow, often with faint orange spots on middle portion; sometimes pale bluish posterior margin. Pectoral fin hyaline. Pelvic fin orange.
Females. Side of trunk and head similar to males, but with paler colours. Ventral surface of head white, with dark grey spots often forming short stripe on chin. Dorsal fin pale yellow, with transverse series of grey spots; broad dark grey to black band on distal margin. Anal fin green-yellow, basal portion light blue with small red spots. Caudal fin pale yellow, with three or four dark grey bars, often interrupted; small black spot, smaller than pupil, on dorso-basal portion of fin overlapping anterior-most bar, more conspicuous in preserved specimens; broad dark grey to black band on whole fin margin.
Distribution. Known only from the type locality area, a small stream tributary to the Rio Bandeira, Rio das Garças drainage, upper Rio Araguaia basin, central Brazil, altitude approximately 520 m asl (Fig. 3).
Etymology. From the Latin, ignescens (becoming inflamed), an allusion to the orange anal fin in males. Diagnosis. Melanorivulus flavipinnis differs from all other species of the M. dapazi group by the presence, in males, of seven or eight narrow red bars on the caudal fin, irregularly shaped and sometimes interconnected (vs. five or six dark red-brown regularly shaped and never interconnected bars in M. regularis; four or fewer short rudimentary bars, sometimes absent, in M. dapazi; bars always absent in M. ignescens) and by the caudal fin, in females, being yellow on the middle portion and reddish orange on marginal region (vs. yellow to pale pink on the whole fin in the remaining species). Also distinguished from all other congeners of the M. dapazi group by the following combination of character states: 5-6 pelvic-fin rays (vs. 7 in M. dapazi and M. regularis); 30-32 scales in longitudinal series (vs. 35-37 in M. regularis); female caudal spot Description. Morphometric data appear in Table 2. Body slender, sub-cylindrical anteriorly, slightly deeper than wide, compressed posteriorly. Greatest body depth at vertical just in front of pelvic-fin base. Dorsal and ventral profiles of trunk almost straight to slightly convex in lateral view; dorsal and ventral profiles of caudal peduncle nearly straight. Head moderately wide, sub-triangular in lateral view, dorsal profile nearly straight, ventral profile convex. Jaws short, snout weakly pointed in lateral view. Jaw teeth numerous, conical, irregularly arranged, outer teeth larger and slightly curved, inner teeth straight.

Melanorivulus flavipinnis
Dorsal and anal fins short, tip slightly pointed in males, rounded in females. Caudal fin oval, longer than deep. Pectoral fin rounded, posterior margin reaching vertical at approximately 80-90% of length between pectoral-fin and pelvic-fin bases. Pelvic fin small, tip reaching between base of first and third anal-fin rays in males, reaching urogenital papilla in females; pelvic-fin bases medially in close proximity. Dorsal-fin origin on vertical through base of 8 th anal-fin ray. Dorsal-fin rays 9-10; anal-fin rays 14-15; caudal-fin rays 30-31; pectoral-fin rays 13; pelvic-fin rays 5-6. No contact organs on fins.
Colouration. Males. Flank metallic green-blue to metallic light blue, sometimes purple-blue above anal fin; oblique narrow orangish red bars irregularly arranged, often forming chevron-like marks anteriorly directed; short light red stripe on humeral region; horizontal rows of reddish orange dots on antero-ventral part of flank, between bases of pectoral and pelvic fins; pale dark grey blotches on postorbital region mainly visible when fish is exposed to strong light. Dorsum light yellowish-brown with black dots, venter white. Dorsal portion of head side light brown, ventral portion white; pale golden iridescence on opercular region. Jaws dark grey. Iris pale yellow, sometimes with dark brown bar on anterior and posterior portions. Dorsal fin light yellow with seven or eight narrow oblique red bars, often forming reticulate pattern on distal portion of fin. Anal fin pale blue on its proximal half, with faint oblique red bars, light yellow in its distal half, distal region becoming gradually dark reddish brown on marginal border, distal margin with high concentration of melanophores. Caudal fin bright yellow, more intensely pigmented on dorsal and ventral portions, with seven or eight narrow red bars, irregularly shaped and sometimes interconnected. Pectoral fin yellowish hyaline. Pelvic fin light blue with orangish brown anterior margin.
Females. Side of trunk and head similar to males, but with paler colours. Ventral surface of head white, with dark grey spots often forming short stripe on chin. Dorsal fin pale yellow, with oblique grey bars; broad dark grey to black band on distal margin. Anal fin green-yellow, basal portion light blue with small red spots. Caudal fin pale yellow on middle portion, reddish orange on marginal region, with five to seven dark grey bars, often interconnected; small black spot, smaller than pupil, on dorso-basal portion of fin overlapping anterior-most bar, conspicuous only in preserved specimens; broad dark grey to black band on whole fin margin.
Distribution. Known only from the type locality, a small stream tributary to the Rio Anhumas, Rio São Lourenço drainage, Rio Paraguai basin, central Brazil, altitude approximately 420 m asl (Fig. 3).
Etymology. From the Latin, flavipinnis (yellow fins), referring to the bright yellow colouration of the caudal fin in males.

Melanorivulus regularis sp. n.
http://zoobank.org/6DE3B93B-9257-4557-B4C1-20B4D2BD05FE Fig 6,   Description. Morphometric data appear in Table 3. Body slender, sub-cylindrical anteriorly, slightly deeper than wide, compressed posteriorly. Greatest body depth at vertical just in front of pelvic-fin base. Dorsal and ventral profiles of trunk almost straight to slightly convex in lateral view; dorsal and ventral profiles of caudal peduncle nearly straight. Head moderately wide, sub-triangular in lateral view, dorsal profile nearly straight, ventral profile convex. Jaws short, snout weakly pointed in lateral view. Jaw teeth numerous, conical, irregularly arranged, outer teeth larger and slightly curved, inner teeth straight.
Colouration. Males. Flank light metallic blue; oblique narrow orange-red bars irregularly arranged, often forming chevron-like marks anteriorly directed; horizontal rows of reddish orange dots on antero-ventral part of flank, between bases of pectoral and pelvic fins; dark brown pigmentation concentrated on postorbital, overlapped by black dots on superficial layer of skin. Dorsum light yellowish-grey, venter white. Dor- sal portion of head side light brown, ventral portion white; pale golden iridescence on opercular region. Jaws dark grey. Iris pale yellow to pale brown. Dorsal fin pale yellow with four or five narrow red bars on posterior portion of fin. Anal fin orangish-yellow, basal portion white, posterior portion pale blue with two or three faint red oblique bars; distal region becoming gradually dark red-brown, distal margin with high concentration of melanophores. Caudal fin pale blue to pale yellow, with five or six dark red-brown regularly shaped bars, ventral portion light yellow without bars, ventral margin orangish-brown. Pectoral fin yellowish-hyaline. Pelvic fin pale blue with brown anterior margin. Females. Side of trunk and head similar to males, but with paler colours. Ventral surface of head white, with dark grey spots often forming short stripe on chin. Dorsal fin pale yellow, with three or four bars on posterior region; broad dark grey to black band on distal margin. Anal fin pale yellow, basal portion light blue. Caudal fin pale yellow, with four or five dark grey bars; small black spot, slightly smaller than pupil, on dorso-basal portion of fin; broad dark grey to black band on whole fin margin.
Distribution. Known only from the type locality, Ribeirão da Sobra, an upper tributary of the Rio Itiquira, Rio Paraguai basin, central Brazil, in altitude about 750 m asl (Fig. 3).
Etymology. From the Latin, regularis (regular), a reference to the caudal fin bars in males, regularly shaped and arranged on fin.

Discussion
Morphological characters indicate that all three new species here described are more closely related to M. dapazi than to other congeners, with these four species comprising the M. dapazi group. In all species of this group, the interarcual cartilage is rudimentary, nearly equal in size to the adjacent cartilage at the tip of the first epibranchial (Fig. 7). In other species of Melanorivulus, the cartilage is well-developed, larger than first epibranchial cartilage, and around one fourth the length of the first epibranchial (e.g., Costa 2016: fig. 4). In addition, species of the M. dapazi group share the presence of a dark red-brown distal margin on the male anal fin (Figs 1, 4 (Costa 1989(Costa , 2010Costa et al. 2015).   fin. In addition, in both species the spot on the basal portion of the female caudal fin is inconspicuous in live fish (Figs 2 and 5) and poorly visible in preserved specimens. In M. dapazi and M. regularis, there are seven pelvic-fin rays and the female caudal spot is conspicuous and delimited in live (Fig. 8) and preserved specimens, conditions considered plesiomorphic for Melanorivulus (Costa, 2016). The unique pigmentation pattern on the ventral surface of the head in females that is shared by M. flavipinnis, M. ignescens, and M. regularis (Fig. 9), may be indicative of close relationships among these three species. Costa (2016) discussed the importance of using live colour pattern characters to diagnose species and species groups of Melanorivulus, showing high congruence with molecular data. In that study, particular attention was given to patterns involving the caudal fin, which contained a high concentration of phylogenetically informative characters, useful to delimit most species of the M. zygonectes group. Concordantly, the present study shows that colour patterns documented from live fish is an accurate tool to recognise species of the M. dapazi group (see key for species identification above).  Osteological characters have been used to infer relationships among species groups of Melanorivulus and for diagnostic purposes (e.g., Costa 2016; this study). The present study shows that osteological characters may be also useful to diagnose single species. The unique shape of the metapterygoid recorded for M. ignescens, with a constriction in its middle portion (Fig. 10), and the unique arrangement of teeth on the fourth ceratobranchial in M. flavipinnis, exhibiting a median gap (Fig. 11), are not present in other congeners. In addition, M. regularis differs from other species of the M. dapazi group by having 32 vertebrae (vs. 29-31 in other species). Thus, although checking osteological characters in larger specimen samples is often not practicable, it is recommendable that osteology be included in taxonomical studies of Melanorivulus to complement species diagnoses. Recent killifish inventories in the area of the central Brazilian plateaus drained by the upper tributaries of the Araguaia, Paraná and Paraguai river basins have revealed an unexpected high diversity of species of the genus Melanorivulus (e.g., Costa 2012). The present study confirms this high diversity, indicating once again that species inhabiting the region have small geographical ranges, often restricted to short segments of a single river drainage. For example, among species endemic to the Paraguai basin, M. regularis was found in a single locality of the Rio Itiquira drainage, at approximately 750 m asl, whereas the present field survey indicated that at altitudes around 450 m asl of the same drainage, the only species found was M. dapazi, which also occurs in similar altitudes of the neighbouring areas included in the Rio Correntes drainage (Costa 2005). On the other hand, M. flavipinnis here described from the Rio São Lourenço drainage at approximately 420 m asl, is substituted by M. cyanopterus (Costa, 2005), at altitudes of approximately 250 m asl (Costa 2005). The last species is a member of the M. punctatus group, distantly related to the M. dapazi group and geographically widespread in the lower Paraguai river basin . Recent studies with other vertebrates occurring in the Cerrado indicate that high species diversity in the region is correlated with the topographical reorganization during the Miocene, which generated geographical isolation of ancestral populations in plateaus and peripheral depressions (Prado et al. 2012;Guarnizo et al. 2016). This paleogeographical scenario may explain the present distribution of distinct species of Melanorivulus along different altitudinal zones of river drainages.