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

Three new species of Melanorivulus are described from the upper and middle Rio Paraná Basin, central Brazilian Cerrado. These species are members of the M. pictus species group, endemic to central Brazilian plateaus and adjacent areas, and are easily diagnosed by colour pattern characters, but their relationships with other congeners of the group are still uncertain. Melanorivulus proximus sp. n., from the middle Rio Aporé drainage, and M. nigromarginatus sp. n., from the Rio Corrente drainage, are possibly more closely related to other species endemic to streams draining the slopes of the Caiapó range, whereas M. linearis sp. n., from the upper Rio Pardo drainage, middle Rio Paraná Basin, is considered more closely related to M. egens, a species also endemic to this part of the Basin. This study corroborates the high diversity of species of Melanorivulus in the central Brazilian Cerrado plateaus repeatedly reported in previous studies, indicating once more that different species are often found restricted to short segments of the same river drainage. The intense habitat loss recorded in recent years combined to the high species diversity limited to specific Cerrado freshwater ecosystems, the veredas, indicates that species of Melanorivulus endemic to this part of the Brazilian Cerrado are highly threatened with extinction.


Introduction
The Cerrado savannas of central Brazil, with an area of about 2,000,000 km 2 , is among the 25 most important biodiversity hotspots of the world (Myers et al. 2000).The present study is directed to a group of small killifishes of the genus Melanorivulus Costa, 2006, with species reaching 45 mm of standard length (SL) or less, inhabiting the shallowest parts of the veredas, a typical Cerrado ecosystem comprising streams bordered by the buriti-palm (Mauritia flexuosa).About 50 valid species are presently placed in Melanorivulus, formerly considered as a subgenus of Rivulus Poey, 1860 (Costa 2011).Melanorivulus is similar to other South American aplocheiloid killifishes living in similar biotopes, such as Anablepsoides Huber, 1992, Atlantirivulus Costa, 2008, Cynodonichthys Meek, 1904, Laimosemion Huber, 1999, Rivulus Poey, 1860, which have slender body, short fins and long neural prezygapophyses on caudal vertebrae (Costa 1990).Species of Melanorivulus are distinguishable from species of those genera by the presence of black pigmentation concentrated on the whole margin of the caudal fin and on the distal margin of the dorsal and anal fins in females, ventral process of the angulo-articular short or rudimentary, and preopercular canal absent (Costa 2011).
A great diversity of species of Melanorivulus has been reported for the Cerrado region drained by the Rio Paraná Basin, the second largest river basin in South America.A total of 14 endemic species have been described for this region (Costa 1989(Costa , 2005(Costa , 2007a(Costa , b, 2008;;Nielsen et al. 2016;Volcan and Lanés 2017).These species belong to different and not closely related species groups (Costa et al. 2016): the M. punctatus group, containing slender small species, mainly diagnosed by the presence of oblique rows of red dots on the flank in males (e.g., Costa 2005); the M. pinima group, easily diagnosed by the reduction of black pigmentation on the head and humeral region in males, presence of longitudinal rows of red dots on the flank, and a longitudinally elongated white to light yellow mark above caudal spot in females (Costa 2007a), and the M. pictus group, diagnosed by a deeper body (i.e., body depth reaching about 25 % SL) and oblique red bars on the flank (Costa 2017).Eleven of the 14 endemic species are members of the last group, which are mostly concentrated in the rivers draining the Caiapó range and in the south adjacent areas of the middle Rio Paraná Basin (Costa 2005(Costa , 2012)).
The M. pictus group was first studied by Costa (1989), based on fish collections deposited in the Museu de Zoologia, Universidade de São Paulo, when M. apiamici (Costa, 1989), M. pictus (Costa, 1989) and M. vittatus (Costa, 1989) were described.In September 1994, efforts were directed to firstly sample typical Melanorivulus habitats along the Paraná River Basin, but based on the great morphological variability found among populations inhabiting close neighbouring areas, Costa (1995) concluded that all populations of this vast geographical area belong to a single polymorphic species, M. pictus.However, after more accurate field studies since 2004, numerous new species have been continuously described for the Paraná and other adjacent river basins (e.g., Costa 2005Costa , 2006Costa , 2012Costa , 2017;;Deprá et al. 2017).These studies have indicated that each species is limited to small areas and that different species may inhabit the same river drainage at different altitudes (Costa 2007b(Costa , 2017;;Volcan et al. 2017).However, some new species collected in recent years are still await formal descriptions.In this paper, three new species of the M. pictus group from the Paraná River Basin are described.

Materials and methods
Specimens were captured with small dip nets (40 × 30 cm) and 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 field methods have been 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 observations just after collections, in small transparent plastic bottles.Type specimens were photographed live about 24 hours after collec tion.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.Osteological preparations followed Taylor and Van Dyke (1985); the abbreviation C&S in lists of material indicates those specimens that were cleared and stained for osteological examination.Terminology for osteological structures followed Costa (2006), for frontal squamation Hoedeman (1958), and for cephalic neuromast series Costa (2001).In lists of material, geographical features are written according to Brazilian Portuguese local use (e.g., córrego, ribeirão, rio), allowing more accurate identifications of lo calities in the field and avoiding common mistakes when tentatively translating them to English.
Description.Morphometric data appear in Table 1.Body relatively deep, subcylindrical anteriorly, slightly deeper than wide, compressed posteriorly.Greatest body depth at vertical just anterior to pelvic-fin base.Dorsal and ventral profiles of trunk slightly convex in lateral view, approximately straight on caudal peduncle.Head moderately wide, sub-triangular in lateral view, dorsal profile nearly straight, ventral profile convex.Snout blunt.Jaws short; teeth numerous, conical, irregularly arranged; outer teeth hypertrophied, inner teeth small and numerous.Vomerine teeth 3-5.Gill-rakers on first branchial arch 2 + 7-8.Dorsal and anal fins short, sharply pointed in males, rounded to slightly pointed in females.Caudal fin rounded, slightly longer than deep.Pectoral fin rounded, posterior margin reaching vertical at about 90 % of length between pectoral-fin and pelvic-fin bases.Pelvic fin small, longer in males, tip reaching between base of 2nd and 3rd anal-fin rays in males, reaching anus in females; pelvic-fin bases medially in close proximity.Dorsal-fin origin at vertical between base of 9th and 10th anal-fin rays.Dorsal-fin rays 9-11; anal-fin rays 13-15; caudal-fin rays 30-32; pectoral-fin rays 13-14; pelvic-fin rays 7.No contact organs on fins.Second proximal radial of dorsal fin between neural spines of 18 th and 20 th vertebrae; first proximal radial of anal fin between pleural ribs of 13th and 14th vertebrae; total vertebrae 30-31.
Colouration in life.Males.Flank metallic greenish blue to bright blue, with narrow oblique red bars between humeral region and posterior portion of caudal peduncle; bars irregularly arranged, forming chevron-like marks with angle varying in position on flank, often connected to short adjacent bars, forming Y-and X-shaped marks; bars with minute vertical extensions on each scale margin; dorsal portion of flank with oblique rows of red dots; anteroventral portion of flank with rows of red dots, often coalesced to form zigzag red marks.Dorsolateral portion of body, between posterior part of head and anterior part of flank, above humeral region, pale golden.Humeral region with horizontally elongated black spot.Dorsum light brown, venter white.Opercular region greenish golden with dark red reticulation on scale margins; suborbital region yellowish white; lower jaw dark grey.Iris pale yellow, with dark brown bar on anterior and posterior portions.Dorsal fin bluish white, sometimes yellowish on distal portion, with 4-5 transverse, narrow faint red or red stripes.Anal fin pale yellow, base and posterior portion bluish white with row of light red dots or short stripes.Caudal fin pale yellow to bluish white, with 5-6 narrow red or reddish orange stripes.Pectoral fin yellowish hyaline.Pelvic fin orangish pale yellow.
Females.Similar to males, except flank base colour pale greenish golden; dorsal and caudal fin bars dark grey; caudal fin base colour pale orangish pink; and pres-ence of black spot on dorsal portion of caudal-fin base and dark grey pigmentation concentrated on distal margins of dorsal and anal fins, anterior margin of pelvic fin and entire caudal-fin margin.

Colouration in alcohol.
Head and trunk pale brown, fins whitish hyaline; dark marks recorded for live specimens varying from dark brown to black.

Etymology.
From the Latin proximus (near, neighbour), referring to its distribution area at the same drainage as M. scalaris.
Description.Morphometric data appear in Table 2. Body relatively deep, sub-cylindrical anteriorly, deeper than wide, compressed posteriorly.Greatest body depth at vertical just anterior to pelvic-fin base.Dorsal and ventral profiles of trunk 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.Snout blunt.Jaws short; teeth numerous, conical, irregularly arranged; outer teeth hypertrophied, inner teeth small and numerous.Vomerine teeth 2-5.Gill-rakers on first branchial arch 1 + 8. Dorsal and anal fins short, tip slightly pointed in males, rounded in females.Caudal fin rounded, slightly longer than deep.Pectoral fin rounded, posterior margin reaching vertical just anterior to pelvic-fin insertion.Pelvic fin small, longer in males, tip reaching between urogenital papilla and base of 2nd anal-fin ray in male, reaching anus in females; pelvic-fin bases medially in close proximity.Dorsal-fin origin at vertical between base of 8th and 9th anal-fin rays.Dorsal-fin rays 10-11; anal-fin rays 13-15; caudal-fin rays 31-34; pectoral-fin rays 13; pelvic-fin rays 6-7.No contact organs on fins.Second proximal radial of dorsal fin between neural spines of 19th and 21st vertebrae; first proximal radial of anal fin between pleural ribs of 13th and 15th vertebrae; total vertebrae 30-32.
Scales small, cycloid.Body and head entirely scaled, except anterior ventral surface of head.Body squamation extending over anterior 25 % of caudal-fin base; no scales on dorsal and anal-fin bases.Frontal squamation E-patterned; E-scales not overlapping medially; scales arranged in regular circular pattern around A-scale without exposed margins.Longitudinal series of scales 30-33;     Females.Similar to males, except flank base colour pale greenish blue; dorsal and caudal fin bars dark grey; caudal fin base colour pale orangish pink; absence of black pigmentation on post-orbital and humeral regions; and presence of black spot on dorsal portion of caudal-fin base and dark grey pigmentation concentrated on distal margins of dorsal and anal fins, anterior margin of pelvic fin and entire caudal-fin margin.

Colouration in alcohol.
Head and trunk pale brown, fins whitish hyaline; dark marks recorded for live specimens varying from dark brown to black.
Distribution and conservation.Known only from two close small streams in the middle section of the Corrente River drainage, upper Paraná River Basin (Fig. 3).
Etymology.The name nigromarginatus (black margin), from the Latin, is a reference to the presence of a black margin on the anal in males.

Melanorivulus linearis sp. n.
http://zoobank.org/9312393A-94FD-4433-9818-88CC6F1666D9Figs 6-7, Table 3 Holotype.UFRJ 11678, male, 25.Diagnosis.Melanorivulus linearis is similar to M. egens, and distinguished from all other species of the M. pictus group by the presence of red chevron-shaped marks regularly distributed on the flank (vs.irregularly), absence of distinctive dark marks on humeral region (vs.presence), and absence of red dots on the anteroventral portion of flank (vs.presence).Melanorivulus linearis is distinguished from M. egens by the presence of red bars restricted to the dorsal portion of the caudal fin in males (vs.absence), presence of black bars on the caudal fin in females (vs.black dots); presence of a pale green spot on humeral region in males (vs.absence); and second proximal radial of the dorsal fin between neural spines of 18th and 19th vertebrae (vs. between neural spines of 19th and 21st vertebrae).3. Body relatively deep, sub-cylindrical anteriorly, deeper than wide, compressed posteriorly.Greatest body depth at vertical just anterior to pelvic-fin base.Dorsal and ventral profiles of trunk slightly convex in lateral view; dorsal and ventral profiles of caudal peduncle nearly straight.

Description. Morphometric data appear in Table
Head moderately wide, sub-triangular in lateral view, dorsal profile nearly straight, ventral profile convex.Snout blunt.Jaws short; teeth numerous, conical, irregularly arranged; outer teeth hypertrophied, inner teeth small and numerous.Vomerine teeth 3-5.Gill-rakers on first branchial arch 1 + 8. Dorsal and anal fins short, tip slightly pointed in males, rounded in females.Caudal fin rounded, slightly longer than deep.Pectoral fin rounded, posterior margin reaching vertical just anterior to pelvic-fin insertion.Pelvic fin small, longer in males, tip reaching between base of 2nd or 3rd 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 8th or 9th anal-fin ray.Dorsal-fin rays 10-11; anal-fin rays 13-15; caudal-fin rays 31-32; pectoral-fin rays 13-14; pelvic-fin rays 7.No contact organs on fins.Second proximal radial of dorsal fin between neural spines of 18th and 19th vertebrae; first proximal radial of anal fin between pleural ribs of 13th and 15th vertebrae; total vertebrae 30-31.

Colouration.
Males.Flank metallic greenish blue, sometimes purplish blue above anal fin, with oblique narrow red bars between humeral region and posterior portion of caudal peduncle; bars regularly arranged, forming chevron pattern directed anteriorly, with angle on flank midline or above it; bars with minute vertical extensions on each scale margin; dorsal portion of flank with few red dots; anteroventral portion of flank without red marks; pale green spot on humeral region.Dorsum light brown, venter white.Side of head light brown on dorsal portion, yellowish white on ventral portion to pale golden on opercle; melanophores dispersed, not forming distinct marks on post-orbital region; lower jaw dark grey.Iris pale yellow, sometimes with dark brown bar on anterior and posterior portions.Dorsal fin light yellow, with four to six oblique red bars through whole fin.Anal fin yellowish orange, basal portion purplish white with six or seven short red bars, distal margin black.Caudal fin light yellow, with six to eight narrow red bars extending between dorsal and middle portions of fin; fin margin dark grey.Pectoral fin hyaline.Pelvic fin light yellow with narrow black margin.Caudal peduncle depth 15.9 14.4-16.212.9-14.1  Females.Similar to males, except flank base colour pale greenish golden; no distinct marks on humeral region; dorsal and caudal fin bars dark grey to black; caudal fin base colour pale white; absence of pale green spot on humeral region; and presence of triangular black spot on dorsal portion of caudal-fin base and dark grey pigmentation concentrated on distal margins of dorsal and anal fins, anterior margin of pelvic fin and entire caudal-fin margin.
Distribution.Known only from the type locality, upper section of the Rio Pardo, middle Rio Paraná Basin, central Brazil (Fig. 3).
Etymology.From the Latin, linearis (consisting of lines), an allusion to the red oblique lines regularly arranged on the flank in males.

Discussion
Studies on systematics of Melanorivulus have consistently demonstrated the importance of colour pattern characters both to diagnose species and to support monophyletic groups (Costa 2016).According to recent phylogenetic analyses (Costa 2016;Costa et al. 2016), colour pattern characters highly corroborates groups that are supported by other morphological characters, as well as by molecular data.However, the relatively low variability of mor-phometric, meristic and osteological characters among species of the M. pictus group makes colour pattern characters essential source to diagnose species and to estimate their relationships, since molecular data are not still available for most species.Consequently, the new taxa herein described exhibit colour patterns characters that in combination easily allow their recognition as new species, but their relationships are still unclear.
Melanorivulus proximus is the second species recorded for the Rio Aporé drainage.Melanorivulus scalaris also occurs in the Aporé drainage, but in altitudes between about 740 and 800 m asl, whereas M. proximus is here reported for altitudes between about 440 and 540 m asl.The veredas of this drainage were first sampled in 1994, but specimens here recognised as belonging to M. proximus were then identified as M. pictus (Costa, 1995; see Introduction above for historical context).Costa (2005) described Rivulus scalaris Costa, 2005 (= M. scalaris) based on material collected in the Ribeirão São Luiz, upper Rio Sucuruí drainage.Specimens collected in the middle section of the Rio Aporé drainage were then tentatively identified as M. scalaris and listed as additional material (non-types).Costa (2007) recorded R. scalaris to the Rio da Prata floodplains, upper Rio Aporé drainage, in a plateau area where the upper Ribeirão São Luiz and the Rio da Prata are in contact.However, the taxonomic status of the middle Aporé populations was not clarified until now.
The frequent occurrence of irregularly interconnected chevron-shaped red marks on the flank in males of M. proximus suggests that it is closely related to M. scalaris, in which this colour pattern is always present (Fig. 8).However, the pointed anal fin in males and the strongly pigmented reticulation on the head side in females, suggest that M. proximus is more closely related to species endemic to neighbouring drainages that exhibit these derived character states, comprising M. faucireticulatus from the Claro and Verde river drainages (Costa 2007b: figs 1-2) and M. rutilicaudus from the Rio Verde drain-age (Costa 2005: Figs 8-9).In large adult specimens of M. scalaris the anal fin tip is not pointed (Fig. 8) and the caudal fin is pale yellow in females (Fig. 9).
Previous studies indicate that the Sucuruí, Aporé, Corrente, Verde and Claro river drainages, which drain the south-eastern slope of the Caiapó range and flow directly to the Rio Paranaíba as part of the upper Rio Paraná Basin, concentrates a great diversity of species of Melanorivulus (Costa 2005(Costa , 2007a(Costa , b, 2008)).These species are often members of clades endemic to Caiapó range drainages, including those belonging to the upper Rio Araguaia Basin, on its northern slope (e.g., Costa 2006).However, characters supporting phylogenetic relationships of M. nigromarginatus, from the Rio Corrente drainage, with other species of the Caiapó range drainages are ambiguous.
Melanorivulus nigromarginatus is easily distinguished from all other species endemic to the Caiapó range drainages by the presence of a black marginal band on the anal fin in males (Fig. 4), suggesting that it may be more closely related to M. egens (Costa 2005: fig. 11) and M. linearis (Fig. 6), which also have similar black anal-fin margin, but are endemic to tributaries of the middle section of the Rio Paraná (Fig. 3).Contrastingly, the presence of a distinctive dark humeral spot in M. nigromarginatus suggests that it may be more closely related to other species occurring in other Caiapó range drainages (e.g., M. faucireticulatus, M. formosensis, M. proximus, M. rutilicaudus, M. scalaris, M. vittatus).All these species share the presence of a distinct humeral blotch varying from dark red to black (Figs 1-2, 4-5), whereas this derived condition is not present in M. egens and M. linearis (Figs 6-7).In addition, the presence of orangish pink pigmentation on the caudal fin in females that occur in M. nigromarginatus (Fig. 5), M. proximus (Fig. 2), M. faucireticulatus (Costa 2007b:   The present study once more reports the occurrence of different species of Melanorivulus inhabiting separate sections of the same river drainage as already described in previous studies (Costa 2007b(Costa , 2017;;Volcan et al. 2017;Fig. 3).Recently, Costa (2017) compared this distributional pattern to that reported for other vertebrates occurring in the Cerrado, which is explained to be correlated with Miocene topographical reorganization causing geographical isolation of ancestral populations in plateaus and peripheral depressions (Prado et al. 2012;Guarnizo et al. 2016).Although estimates of divergence time among lineages of the M. pictus group are not still available, this paleogeographical scenario could explain the present distribution of distinct species of Melanorivulus at different altitudinal zones of river drainages.
Costa (2012) reported a strong process of habitat loss in the rivers draining the Caiapó range as a result of the quick expansion of agriculture land use in areas previously occupied by natural vegetation.In recent years, the veredas have often been extirpated after diversion of their water sources for plantation irrigation, as well as widespread deforestation, which has reached their margins when water flow persists.Considering the great diversity of endemic species of Melanorivulus inhabiting the Veredas of the Caiapó range and the continuous extirpation of Vereda habitats, this study supports the endangered status of species inhabiting this region.

2
, otic 1, post-otic 1, supratemporal 1, median opercular 1, ventral opercular 1, pre-opercular 2 + 4, mandibular 3 + 1, lateral mandibular 1-2, paramandibular 1. Colouration.Males.Flank metallic light green, with narrow oblique red bars between humeral region and posterior portion of caudal peduncle; bars irregularly arranged, forming chevron pattern directed anteriorly, usually fragmented, with angle on flank midline or above it; bars with minute vertical extensions on each scale margin; dorsal portion of flank with oblique rows of red dots; anteroventral portion of flank with rows of red dots.Dorsum light brown, venter white.Side of head light brown on dorsal portion, yellowish white on ventral portion to pale golden on opercle; broad dark grey to black postorbital stripe, continuous to humeral black blotch; lower jaw dark grey.Iris pale yellow, with dark brown bar on anterior and posterior portions.Dorsal fin light yellow, with four to six oblique faint red bars.Anal fin light yellow to orange, basal portion greenish white with five or six orangish red spots, distal margin black.Caudal fin light yellow, with six to eight narrow orangish red bars extending on entire caudal fin, except its ventral-most portion.Pectoral fin hyaline.Pelvic fin light yellow to orange narrow black margin.
fig.2), and M. rutilicaudus(Costa 2005: fig.9), reinforces the hypothesis of close relationships.On the other hand, probably M. egens and M. linearis from the middle Rio Paraná Basin are closely related species, sharing the presence of red chevron-shaped