Zoosyst. Evol. 94 (2) 2018, 263-280 | DOI 10.3897/zse.94.24406 > PENSUFT. Jifit.. yee BERLIN Unrecognized biodiversity in a world’s hotspot: three new species of Melanorivulus (Cyprinodontiformes: Rivulidae) from tributaries of the right bank of the Rio Parana basin, Brazilian Cerrado Matheus V. Volcan!, Francisco Severo-Neto**, Luis Esteban K. Lanés!4 Instituto Pré-Pampa (IPPampa), Laboratorio de Ictiologia. Rua Uruguay, 1242, Bairro Centro, CEP 96010-630, Pelotas, Rio Grande do Sul, Brasil 2 Universidade Federal de Mato Grosso do Sul, Centro de Biociéncias, Cidade Universitaria, CEP 79070-700, Campo Grande, MS, Brasil 3 Instituto de Biociéncias, Letras e Ciéncias Exatas, Departamento de Zoologia e Botanica, Laboratorio de Ictiologia, Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP), Rua Crist6vdo Colombo, 2265, Jardim Nazareth, CEP 15054-000, Sao José do Rio Preto, SP, Brasil 4 Pontificia Universidade Catdlica do Rio Grande do Sul (PUCRS), Programa de Pés-Graduagdo em Zoologia, Laboratorio de Fisiologia da Conservacdo. Avenida Ipiranga, 6681 Pd.12, Bloco C, Sala 250 CP. 1429, CEP 90619—900, Porto Alegre, RS, Brasil http://zoobank.org/BCA6F 985-BE53-47C5-B64D-005 DAODD69BE Corresponding author: Matheus V. Volcan (matheusvolcan@hotmail.com) Received 12 February 2018 Accepted 6 April 2018 Published 18 April 2018 Academic editor: Peter Bartsch Key Words Cerrado killifish conservation diversity taxonomy Introduction Abstract The genus Me/anorivulus presents a wide geographical distribution in the Neotropical re- gion. Among Me/anorivulus, the M. pictus species group has currently 18 species distrib- uted in the Brazilian Cerrado, mainly in the upper Rio Parana basin, with some species occurrences in the upper Rio Araguaia and Tocantins. In the present study, we describe three new Melanorivulus species, belonging to the M. pictus species group from different drainages of the right bank of the Rio Parana basin in Mato Grosso do Sul state, Brazil. These new species are easily distinguished from the others by their unique colour pat- terns. Melanorivulus interruptus 1s distinguished from all species of the M. pictus group by the presence of oblique chevron-like red bars interrupted, mainly on the midline of the flank in males; while MZ ivinhemensis by the yellow colouration of the caudal fin with thin red bars arranged only in the median region of the fin in males. Melanorivulus amambaiensis 1s distinguished from all species of the M. pictus species group by having an orange anal fin or sometimes more reddish-orange with distal margin grey or dark grey and chevron-like bars along the body, distinctly branched ventrally forming an inverted Y-shape in males. The high diversity of the Melanorivulus species with high levels of endemism demands the development of conservation strategies to avoid the loss of their vulnerable habitats in the Cerrado biome. We expect presence of more species of the M. pictus species group also along the lower reaches of the tributaries of the Rio Parana. Additionally a dichotomic identification key of the MZ. pictus species group is provided. areas with many rare and endemic species and less than 30% remaining natural vegetation (Myers et al. 2000). It is Savanna biomes are distributed across tropical zones around the world. In South America, the Cerrado is the largest savanna biome, the second largest biome tn Brazil (smaller only than the Amazon), and represents about 23 % of Brazilian territory (Ratter et al. 1997). The Brazilian Cerrado is included in the world’s 25 main “hotspots”, one of the world’s most threatened biomes, mainly because of the replacement of natural vegetation by pastures and row crops (Ratter et al. 1997, Myers et al. 2000). The rivulid genus Me/anorivulus is a typical compo- nent of the Cerrado fauna, but presents a wide geograph- ical distribution in the Neotropical region, being also Copyright Matheus V. Volcan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 264 found in areas under the influence of the Chaco, Pantanal, Atlantic Forest and Amazon (e.g. Costa 2016, Costa et al. 2016, Nielsen 2017). However, most of the species are found in Brazil and occur in the Cerrado biome along several river basins drained mainly by the upper tributar- ies of the Rio Araguaia, Tocantins, Parana and Paraguay basins and the adjacent Cerrado-Amazon ecotone (Costa et al. 2016). Recent killifish inventories of the Brazilian Cerrado have revealed a high diversity of Melanorivulus species (e.g., Costa 2012, 2017, 2018, Volcan et al. 2017), indicating that species inhabiting the region have small geographical ranges, often restricted to short segments of a single river drainage (Costa 2017, Volcan et al. 2017). Despite the wide geographical distribution and high di- versity, little is known about the conservation, distribu- tion, biology and ecology of most Me/anorivulus species (Severo-Neto and Volcan 2018). Among Melanorivulus species, the M. pictus species group is distinguished from all other congeners, by the presence of a vestigial ventral process of the angulo-artic- ular (vs. process well-developed), curved first epibranchi- al (vs. approximately straight) and intense greenish blue or greenish golden to purplish blue flank above anal-fin base in males (vs. never similar colour pattern) (Costa 2007, 2008). The Melanorivulus pictus species group currently comprises 18 species (Volcan et al. 2017, Costa 2018), with 16 species occurring in the Rio Parana basin: M. apiamici (Costa, 1989) in the Rio Parana, M. egens (Costa, 2005), in the Rio SAo Domingos, M. formosensis (Costa, 2008) and M. nigromarginatus Costa, 2018 for the Rio Corrente, M. giarettai (Costa, 2008) for the Rio Araguari basin, M. /eali Costa, 2013 for the Rio Grande basin, M. nigropunctatus Volcan, Klotzel & Lanés, 2017 and M. ofaie Volcan, Klotzel & Lanés, 2017 for the Rio Verde basin, M. faucireticulatus (Costa, 2008), M. pic- tus (Costa, 1989) and M. vittatus (Costa, 1989) for the Rio Claro basin, M. polychromus Nielsen, 2016 for the Rio Sao José dos Dourados basin, M. rutilicaudus (Costa, 2005) for the Rio Verde basin, M. scalaris (Costa, 2005) for the Rio Sucurui basin, M. proximus Costa, 2018 for Rio Aporé and M. /inearis Costa, 2018 for the upper Rio Pardo basin. Two other species occur exclusively in the upper Rio Tocantins and Araguaia basin: M. planaltinus (Costa & Brasil, 2008) for the Rio Cocal and M. /itteratus (Costa, 2005) for the Salto stream, respectively. A previous analysis indicates that the most recent common ancestor of Melanorivulus probably occupied a region comprising the eastern Amazon savanna and the ecotone Amazon-Cerrado, and the current distribution 1s the result of a series of dispersal and vicariance events during the evolutionary history of the genus (Costa et al. 2016). In the present study, we describe three new spe- cies of Melanorivulus belonging to the M. pictus species group that are endemic to different drainages of the right bank of the Rio Parana basin in Mato Grosso do Sul state, Brazil. Additionally, we discuss the conservation, distri- bution patterns, and provide an identification key to the M. pictus species group. zse.pensoft.net Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... Material and methods In December 2016, a 12-day collection campaign was conducted to sample potential habitats for the occurrence of killifish species in the Mato Grosso do Sul state, Bra- zil, which included the largest river basins associated with Rio Parana. In order to define the sampling strategy, we analysed satellite images from Google Earth (earth.goo- gle.com) as well as locations of specimens vouched in the Colecao Zoologica da Universidade Federal de Mato Gros- so do Sul. Field works were undertaken travelling the main highways and roads to reach access to previously selected areas; at every site we performed an active search for fishes. Fish samples were taken with a dip-net (D-shaped hand net, 60 cm x 40 cm, 2 mm mesh size), and then were euthanized with clove oil, fixed in situ with 4% formalde- hyde, and later transferred into 70% ethanol. The material was collected under IBAMA/ICMBio authorization (pro- cess number 56894-1). Morphological characters were obtained from speci- mens fixed in formalin after collection, and subsequent- ly transferred to 70% ethanol. Fish measurements were taken point-to-point with digital calipers to the nearest 0.1 mm on the left side of the specimen following Cos- ta (1995). Measurements are expressed as percentage of standard length (SL), except the head measurements, which are recorded as percentage of head length. Fin-ray counts include all elements. Scale count in the mid-lon- gitudinal series includes all scales between the upper attachment of the opercular membrane and the base of the caudal fin, excluding small scales posterior to the hypural plate. Numbers of vertebrae were recorded only from cleared and stained specimens (C&S), prepared ac- cording to Taylor and Van Dyke (1985). Frontal squama- tion nomenclature follows that described by Hoedeman (1958), and for cephalic neuromasts series Costa (2001). Descriptions of colour patterns were based on photo- graphs of both sides of live specimens photographed in the field, which were fixed in situ after photos, and individuals maintained in aquaria (not preserved). Institutional abrevia- tions are MCP (Museu de Ciéncia e Tecnologia da Pontificia Universidade Catolica do Rio Grande do Sul, Porto Alegre) and ZUFMS (Colecao Zoologica de Referéncia da Univer- sidade Federal de Mato Grosso do Sul, Campo Grande). Melanorivulus interruptus sp. n. http://zoobank. org/2B 106159-5398-44DC-B89C-7F 8CB6CE676A Holotype. MCP 53145, male, 32.3 mm SL, Brazil, Mato Grosso do Sul State, municipality of Campo Grande, first order stream, in the headwaters of Rio Pardo, 20°287117S, 54°29’25”W, altitude 589 ma.s.l.; M.V. Volcan & L.E.K Lanés, 13 Dec 2016. Paratypes. MCP 53146, 4 males, 21.6—-31.0 mm SL (1 C&S), 3 females, 22.5—29.0 mm SL, 1 juvenile, sex un- determined, 16.9 mm, all collected with the holotype. Zoosyst. Evol. 94 (2) 2018, 263-280 ZUFMS 5409, 7 females, 20.2-26.0 mm SL (3 C&S), same locality as holotype; F. Severo-Neto, T.R.F. Sinani & S. Ichikawa, 5 Aug 2017. Diagnosis. Melanorivulus interruptus is distinguished from all species of the M. pictus group, except M. nigromargina- tus, by the presence of oblique chevron-like bars interrupt- ed, mainly on the midline of the flank in males (vs. bars continuous, vestigial, irregular or presence of longitudinal rows of red dots on the side of the body; never bars inter- rupted on the midline of the body). It is distinguished from all other species in the M. pictus species group, except M. planaltinus, M. leali and M. pictus, by the presence of a rounded dorsal fin (vs. slight pointed or pointed dorsal fin). In addition, M. interruptus differs from M. amambaiensis, M. apiamici, M. egens, M. faucireticulatus, M. ivinhemen- sis and M. leali by a higher caudal fin ray count (32-33 vs. 28-31); from M. egens, M. faucireticulatus, M. leali, M. litteratus, M. rutilicaudus and M. formosensis by the position of the anal-fin origin between the pleural ribs of 15th and 16th vertebrae (vs. 13th and 15th vertebrae); from M. apiamici, M. egens, M. faucireticulatus, M. pictus, M. polychromus and M. proximus by the dorsal-fin origin at a vertical through the base of the 8th anal-fin ray (vs. verti- cal through base of 7th or 9th-10th anal-fin ray); from M. amambaiensis, M. egens, M. faucireticulatus, M. ivinhe- mensis, M. planaltinus, M. polychromus, M. nigropuncta- tus, M. ofaie and M. formosensis by the dorsal-fin origin between neural spines of 18th and 19th vertebrae (vs. 19th and 22th); from M. egens, M. faucireticulatus, M. leali, M litteratus, M. scalaris, M. vittatus, M. linearis and M. proxi- mus by the tip of pelvic fin reaching the urogenital papilla in males (vs. tip of pelvic fin reaching Ist to 4th anal fin ray). Another interesting diagnostic character is the presence of 8 rays in the pelvic fin in about 35% of the analysed spec- imens (vs. 5—7 rays, never 8 rays present in the pelvic fin). Description. Morphometric data are presented in Table 1. Males larger than females. Largest male examined 32.3 mm SL, largest female 29.0 mm SL. Dorsal profile slight- ly convex from snout to end of dorsal-fin base. Ventral profile convex from lower jaw to origin of anal-fin. Dor- sal and ventral profiles of caudal peduncle nearly straight. Body slender, approximately cylindrical and compressed. Greatest body depth at pelvic-fin base. Snout weakly pointed in lateral view. Jaws short. Short dorsal and anal fins. Dorsal-fin rays 9-10. Dor- sal fin rounded in males and females. Dorsal-fin origin on vertical through base of 8th anal-fin ray, and between neural spines of 18th and 19th vertebrae. Anal-fin rays 12-13. Anal fin slightly pointed in males and females. Origin of anal fin at vertical through pleural ribs of 15th- 16th vertebrae. Caudal fin rounded 32-33 rays. Pectoral fin rays 14. Pectoral fins rounded, with posterior margin reaching vertical at about 60-90% of length between pectoral-fin and pelvic-fin bases. Pelvic-fin rays 7—8. Pel- vic-fin posterior tip reaching vertical at anus to 3rd anal- fin ray. Pelvic-fin bases in close proximity. 265 Table 1. Morphometric data for the holotype and paratypes of Melanorivulus interruptus sp. nN. Holotype | Males (4) | Females (7) | 32.3 |21.6-31.0] 22.3-29.0 Standard length Percentages of standard length Body depth 20.8-24.5| 21.3-26.0 Caudal peduncle depth 13.3-14.8] 11.4-14.8 Predorsal length 75.0-77.1| 73.3-77.9 Prepelvic length 52.6-53.9| 51.8-56.3 Dorsal fin base length 11.9-13.7] 10.7-12.7 Anal fin base length 18.4-22.5] 17.8-19.4 Caudal fin length 26.8-29.4 Pectoral fin length 17.7-20.2 Pelvic fin length 8.5-10.4 Head length 26.2 |26.8-27.5| 24.9-27.7 Percentages of head length Head depth 77.9 |66.7-78.0] 66.1-76.2 Head width 76.8 |66.7-73.6| 74.7-82.1 Snout length 15.1-19.2 Lower jaw length 18.7-21.6 Eye diameter 28.6-31.9| 31.9-35.6 Scales cycloid. Body and head entirely scaled, ex- cept anterior ventral surface of head. Body squamation extending over anterior 15—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 with- out exposed margins; transverse row of scales anterior to H-scale. Longitudinal series of scales 30—32; transverse series of scales 8—9; scale rows around caudal peduncle 16. No contact organs on flank and fins. Cephalic neuromasts: supraorbital 3+3, parietal 1, an- terior rostral 1, posterior rostral 1, infraorbital 1+9-11+1, preorbital 2, otic 1, postotic 1-2, supratemporal 1, median opercular 1, ventral opercular 1, preopercular 2+4, man- dibular 2-3+1, lateral mandibular 1-2, paramandibular 1. Two neuromasts on caudal-fin base. Six branchiostegal rays. Gill rakers on first branchi- al arch 1+7. First epibranchial slightly curved. Total number of vertebrae 29-30, 13-14 precaudal vertebrae, 16-17 caudal vertebrae. Ventral process of angulo-artic- ular short, pointed. Vomerine teeth 1-3. Dermosphenotic present. Basihyal sub-triangular, greatest width 45-50% of length; basihyal cartilage 20-25% of total basihyal length. Second pharyngobranchial teeth absent. Colouration in life. Males (Figs 1 and 2). Flank light grey or light metallic blue; sometimes purple-blue close to anal fin; 7-9 oblique narrow red bars anteriorly di- rected, often forming chevron-like marks irregularly arranged and usually interrupted in the midline of the body; commonly, the chevron-like bars are vestigial in the anterior region and begin at the end of the pectoral fin; well-defined, but interrupted, bars begin behind inser- tion of the pelvic fins. Inconspicuous dark pigmentation between postorbital region and area above midlength of pectoral fin, forming an irregular mid-lateral dark stripe; zse.pensoft.net 266 vertical rows of reddish dots on antero-ventral part of flank, between pectoral and pelvic fins. Dorsum yellow- ish-grey, venter whitish-grey. Dorsal portion of the head yellowish-grey; ventral portion yellowish-grey or pale golden without dots, marks or any pigmentation pattern; pale golden-greenish iridescence on opercular region. Jaws dark grey or brown. Iris pale yellow to pale brown, sometimes with dark brown bars on anterior and poste- rior portions. Dorsal fin yellow with four or five narrow red bars. Anal fin yellow, basal portion light blue-whit- ish, posterior portion pale blue with two or three faint red marks; distal region becoming gradually dark red- brown. Caudal fin yellow with five to seven red oblique Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... parallel bars, covered generally 1/3—1/2 of caudal fin on mid-dorsal portion, sometimes red bars inconspicuous; ventral portion light yellow without bars, distal ventral region becoming gradually dark red-brown with a not well delineated margin of orange-brown colour. Pecto- ral fin yellowish-hyaline. Pelvic fin yellow with posteri- or margin orange-brown. Females (Fig. 2). Generally flank similar to males, but with paler colours. Venter white. Dorsum and dorsal por- tion of head yellowish-grey; ventral portion of head yel- lowish-grey without spots and without any colouration pattern. Jaws yellowish-grey or brown; opercular region pale greenish-gold. Dorsal fin yellow, with three or four Figure 1. Me/anorivulus interruptus sp. n., MCP 53145, holotype, male, 32.3 mm SL, Campo Grande, Mato Grosso do Sul, Brazil. - aie a ‘ Le ‘ - _ a _ ; | _ | | Figure 2. Melanorivulus interruptus sp. n., MCP 53146, paratypes, living male (above) and female (below), Campo Grande city, Mato Grosso do Sul state, Brazil. zse.pensoft.net Zoosyst. Evol. 94 (2) 2018, 263-280 267 * M. amambaiensis * M. interruptus at ¥ AM. ivinhemensis M. apiamict M. egens M. faucireticulatus M. formosensis M. giarettai M. leali M. linearis M. litteratus M. nigromarginatus M. nigropunctatus M. ofaie M. pictits M. planaltinus M. polvchromus M. proxinis M. rutilicaudus AM. scalaris M. vittatus PE o ® @ oO e e A A r A A A a ¢ © Sd 1 @ ¢ Figure 3. Distribution of species of the Melanorivulus pictus group in the Rio Parana (grey) and Rio Araguaia and Tocantins (light grey) basins. Stars indicate the type localities of the new species. faint grey bars on posterior region forming a reticulated pattern. Anal fin yellow; basal portion light blue; distal re- gion becoming dark red-brown gradually; distal margin with high concentration of melanophores. Caudal fin pale yellow, with four or six grey bars usually interrupted in the inferior region (lower third of fin); a small irregularly shaped black spot on dorso-basal portion of fin, sometimes overlapping the first bar; faint grey margin. Pectoral fin hyaline or yellowish hyaline. Pelvic fin yellowish hyaline. Distribution. Melanorivulus interruptus is only known so far from a small stream, in the headwaters of the Rio Pardo, a tributary of the right margin of Rio Parana, state of Mato Grosso do Sul, Brazil (Fig. 3). Etymology. The name interruptus, from the Latin adjec- tive interrupted, referring to the presence of conspicuous oblique red bars forming chevron-like marks usually dis- rupted in the midline of the body in males. Habitat. The species was recorded in a typical Cerra- do area, inserted into the urban area of Campo Grande town at an altitude of 594 m a.s.l.. It was recorded in a small first order creek, but was only captured in bor- der areas with dense aquatic vegetation, mainly grasses (Fig. 4). The biotope, which is locally called “veredas”, a freshwater ecosystem comprising streams bordered by the buriti-palm, typically found in the Cerrado Biome, has moderate current speed. But, the place where the species was recorded is formed by small backwaters, with a clayey substrate and with a maximum depth of 10 cm. The species is encountered in areas exposed to the sun as well as in areas shaded by trees, mainly buri- tis-palm (Mauritia flexuosa). The region has a slightly undulating terrain and according to the classification of Koppen-Geiger has rainy summers and dry winters. Conservation. There are imminent threats to the popula- tion of M. interruptus: the stream is located in the Cam- po Grande urban area, which is developing rapidly around the area of the new species; there is a large real estate de- velopment alongside the stream that can cause significant impacts on the environment quality; the stream is crossed by a high traffic road and it is subject to accidents with potentially dangerous loads; besides, there are agricultur- al activities over its complete extension. Although larger collection efforts are likely to be undertaken in the area to better understand the species distribution pattern, MZ. inter- ruptus appears to have a very restricted distribution. In the adjacent watersheds distinct species occur (e.g. M. apiamici in the mouth of the Rio Pardo, 4 egens in the upper Rio Sao Domingos and M. nigropunctatus and M. ofaie in the Rio Verde — see Figure 4 with the distribution map of the M. pictus species group). Thus, according to the previously mentioned threats, a high degree of endemism and the ob- servations made in the field, M. interruptus was considered “Vulnerable”, following the criteria of IUCN (2012). The Species has an area of occupation of less than 2000 km/?, it occurs in only one locality and it suffers from a reduction in zse.pensoft.net 268 Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... Figure 4. First order stream (typical “veredas” habitat), tributary of headwaters of Rio Pardo, M. interruptus type locality. Melanorivulus ivinhemensis sp. n. http://zoobank.org/2531E407-3D5B-428D-9CD3-37AB7A 13600C Holotype. MCP 53147, male, 30.1 mm SL, Brazil, Mato Grosso do Sul State, municipality of Nova Andradina, first order stream of Rio Ivinhema, 22°11°53”S, 53°20’ 12”W, alti- tude 300 m a.s.1.; M.V. Volcan & L.E.K Lanés, 09 Dec 2016. Paratypes. MCP 53153, 6 males, 20.3—27.7-31.0 mm SL, 7 females, 18.5-29.7 mm SL (1 C&S), 2 juvenile, sex undetermined, 14.0-15.4 mm, all collected with the holotype. MCP 53148, 2 juvenile, sex undetermined, 14.3-16.9 mm, wetland in the boarder of BR 376 high- way, 22°21°22”S, 53°30’°32”W, altitude 252 m as.l.; M.V. Volcan & L.E.K Lanés, 08 Dec 2016. MCP 53149, 1 male, 18.6 mm SL, 2 females, 25.7—-30.4 mm SL, first order stream of Rio Ivinhema, 22°21°51”S, 53°39’°42”W, altitude 310 m as.l.; M.V. Volcan & L.E.K Lanés, 08 Dec 2016. ZUFMS 5365, 5 males, 21.8—29.5 mm SL (3 C&S), 3 females, 18.1—-20.4 mm SL, first order stream of Rio Ivinhema, 22°12717”S, 53°27°17”°W, altitude 317 m a.s.l.; T.R.F. Sinani & M.O. Bordignon, 17 Sep 2016. Diagnosis. Melanorivulus ivinhemensis is distinguished from all other species of the M pictus species group by having a yellow caudal fin with thin red bars arranged only in the median region of the fin in males (vs. red bars absent or red bars extending from the ventral or median region to the dorsal region of the caudal fin), except in M. rutilicaudus. Besides, it is distinguished from all M. pictus species by the origin of the anal fin at a vertical through pleural ribs of 15th-18th vertebrae zse.pensoft.net (vs. 13-15 vertebrae), except in M. amambaiensis, M. planaltinus, M. scalaris, M. polychromus, M. ofaie, M. interruptus and M. nigropunctatus, and by the greater snout length in males (16.1—21.3 vs. 11.4-16.3), except in M. amambaiensis, M. apiamici, M. interruptus, M. vittatus, M. polychromus and M. nigropunctatus. Distin- guished from M. planaltinus, M. rutilicaudus, M. inter- ruptus by a lower caudal fin ray count (29-31 vs. 32-34) and from M. amambaiensis by a higher caudal fin ray count (29-31 vs. 25—28). Distinguished from M. fau- cireticulatus, M. giarettai, M. interruptus, M. leali, M. vittatus and M. formosensis by a short basihyal cartilage 10-15% of total basihyal length (vs. 20-25%). From M. interruptus, M. vittatus, M. ofaie, M. nigropunctatus, M. proximus, M. linearis and M. nigromarginatus it is dis- tinguished by a smaller dorsal fin base length in females (8.9-10.4% vs. 10.5—14.8%). Distinguished from M. gi- arettai, M. proximus, M. linearis, M. nigromarginatus and M. planaltinus by lower number of gill rakers on the first branchial arch (1+7 vs 1-2+8), and from the M. pictus by the dorsal-fin origin on a vertical through base of 8th or 9th anal-fin ray (vs. on vertical through base of 7th anal-fin ray). Description. Morphometric data are presented in Table 2. Females usually larger than males, largest female 30.4 mm SL, largest male examined 30.1 mm SL. Dorsal pro- file slightly convex from snout to end of dorsal-fin base, straight on caudal peduncle. Ventral profile convex from lower jaw to origin of anal-fin, approximately straight to end of caudal peduncle. Body slender, approximately cy- lindrical and compressed, greatest body depth anterior of Zoosyst. Evol. 94 (2) 2018, 263-280 Table 2. Morphometric data for the holotype and paratypes of Melanorivulus ivinhemensis sp. n. Males (n=9) | Females (n=9) Standard length 30.1 18.6-29.5 18.5-30.4 Percentages of standard length Body depth 19.3-23.5 | 19.1-23.1 Caudal peduncle depth 12.4-14.3 | 11.7-14.0 Predorsal length 74.0-79.0 | 75.1-79.4 Prepelvic length 51.8-55.5 53.3-58.5 Dorsal fin base length 9.3-12.4 8.9-10.4 Anal fin base length 17.2-23.3 16.3-20.9 Caudal fin length 24.9-31.0 26.6-31.6 Pectoral fin length 18.4-20.8 | 17.5-20.7 Pelvic fin length 9.1-12.2 | 85-104 Head length 25.1-28.0 | 23.8-28.2 Percentages of head length Head depth ALG 60.5-73.6 61.7-70.8 Head width 81.1 70.5-76.2 72.3-82.0 Snout length 17-3 16.1-21.3 12.9-20.6 Lower jaw length 20.6 18.3-22.0 16.8-21.6 Eye diameter 29.6-37.0 29.1-34.4 pelvic fin base, in the pelvic fin base or at the anus. Snout slightly pointed. Jaws short. Short dorsal and anal fins. Dorsal-fin rays 7-9. Dor- sal fin rounded or slightly pointed in males, rounded in females. Dorsal-fin origin on a vertical through base of 8th or 9th anal-fin ray, and between neural spines of 20th and 22th vertebrae. Anal-fin rays 12—14. Anal fin slightly pointed in males and females. Origin of anal fin at a ver- tical through pleural ribs of 15th-18th vertebrae. Caudal fin oval shaped, deeper than long, 29-31 rays. Pectoral fin rays 11-13. Pectoral fins rounded, with posterior margin reaching vertical at about 60-90% of the length between pectoral-fin and pelvic-fin bases. Pelvic-fin rays 6—7, one individual with one single pelvic fin. Posterior tip of pel- vic fin reaching vertical between anus to 2nd anal-fin ray. Position of pelvic-fin bases variable, in close proximity, or with bases separated by a great distance, similar to the size of the base of the pelvic fin. Scales small, cycloid. Body and head entirely scaled, except anterior ventral surface of head. Body squamation extending over anterior 15—25% of caudal-fin base. No scales on dorsal and anal-fin bases. Frontal squamation E and F-patterned or with two patterns, one on each side in one specimen. E-scales generally not overlapping me- dially. In one specimen E-scales marginally overlapped. Transverse row of scales anterior to H-scale; scales ar- ranged in regular circular pattern around A-scale. A-scale usually without exposed margins, four specimens with posterior margin exposed, overlapping the B-scale. Lon- gitudinal series of scales 29-31; transverse series of scales 8-9; scale rows around caudal peduncle 16. No contact organs on flank and fins. Cephalic neuromasts: supraorbital 3+3, parietal 1, anterior rostral 1, posterior rostral 1, infraorbital 1+11- 12+1, preorbital 2, otic 1, postotic 1-2, supratemporal 1, median opercular 1, ventral opercular 1—2, preopercular 269 2+4—6, mandibular 3—4+1, lateral mandibular 1—2, para- mandibular 1. Two neuromasts on caudal-fin base. Six branchiostegal rays. Gill rakers on first branchial arch 1+7. First epibranchial slightly curved. Total number of vertebrae 29-31, 14 precaudal vertebrae, 15-17 cau- dal vertebrae. Ventral process of angulo-articular short, pointed. Vomerine teeth 2—5. Dermosphenotic present. Basihyal sub-triangular, greatest width 45—50% of length; basihyal cartilage 10-15% of total basihyal length. Sec- ond pharyngobranchial teeth absent. Colouration in life. Males (Fig. 5). Metallic green- ish-gray flank, purple-blue close to anal fin; red dots on anterior flank area, sometimes few red dots on the antero- ventral region; one to three oblique narrow bars between the medial pectoral fin portion and close to the pelvic fin base, sometimes one or two of these bars forming chev- ron-like marks with an angle on the ventral region of the flank; 7-9 oblique narrow red bars anteriorly directed, forming chevron-like marks with an angle on midline of flank, and generally positioned posteriorly to the pelvic fin base on the caudal peduncle. Two parallel and oblique dark brown bars often interconnected by a brown-dark horizontal line between postorbital and posterior opercu- lar region, sometimes forming a broad and well-defined mid-lateral dark stripe between postorbital region and area above the pectoral fin at mid length. Yellowish-grey dorsum with small black dots, venter white. Dorsal por- tion of the head yellowish-grey sometimes with small black dots; ventral portion white; golden iridescence on opercular region. Dark grey jaws varying to pale yellow- ish-grey. Iris pale yellow. Dorsal fin yellow with three or five oblique narrow red bars on posterior portion of fin. Anal fin yellow or orange-yellow, sometimes with a dis- tal dark margin, basal portion light metallic blue-whitish, posterior portion pale blue with two or three faint reddish oblique bars. Caudal fin yellow or hyaline-yellowish, pre- senting three to six vertical or slightly oblique parallel red or red brownish bars in the central region. Pectoral fin yellowish hyaline. Pelvic fin yellowish hyaline or orange and hyaline, sometimes with a distal dark margin. Females (Fig. 6). Flank similar to males. Dorsum and dorsal portion of head greenish-gray with small black dots; ventral portion of head white, with black marks, sometimes inconspicuous; pale golden iridescence on opercular re- gion. Jaws pale yellowish-grey. Iris pale yellow, sometimes with dark brown bars on anterior and posterior portions. Dorsal fin yellowish-orange, with horizontally elongated dark brown to black spots or bars in the medial region; dark grey narrow margin delineating entire dorsal fin. Anal fin yellowish-orange, basal portion pale blue with two inter- rupted reddish bars, posterior portion pale blue with two or three reddish oblique bars, distal region becoming grad- ually dark reddish brown on marginal border, distal mar- gin with high concentration of melanophores. Caudal fin orange, sometimes orange and hyaline in central portion, with three to six dark brown to black bars, sometimes bars formed by dense, vertically elongated spots, the rays and zse.pensoft.net 270 Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... Figure 5. Colour pattern variation of male paratypes of M. ivinhemensis sp. n., MCP 53153, Nova Andradina municipality, Rio Ivinhema, Mato Grosso do Sul state. inter-spaces between them pigmented; dark brown to black circular or sub triangular spot on dorso-basal portion of the caudal fin; dark grey margin. Pectoral fins hyaline. Pelvic fins orange-yellow, with a reddish-brown anterior margin. Distribution. Melanorivulus ivinhemensis is so far only known from first order streams and small wetlands asso- ciated with both margins of the lower course of the Rio Ivinhema, Rio Parana basin (Figure 3). zse.pensoft.net Etymology. The name ivinhemensis is a reference to the occurrence of the new species in the Rio Ivinhema basin. Habitat notes. Melanorivulus ivinhemensis was recorded in marginal areas of small first order streams and in wet- lands completely exposed to the sun on a slightly undu- lating terrain at altitudes ranging from 267 to 315 ma.s.l. (Fig. 7). One wetland was deep (about 70 cm of maximum depth), with muddy substratum and turbid water (Fig. 7b), Zoosyst. Evol. 94 (2) 2018, 263-280 eo 7al. Figure 6. Female paratypes of M. ivinhemensis sp. n., MCP 53153, Nova Andradina municipality, Rio Ivinhema, Mato Grosso do Sul state. Figure 7. Sampling sites of Melanorivulus ivinhemensis sp. n. (a) Wetland associated with a small drainage, direct tributary of Rio Ivinhema, type locality, (b) wetland along BR 376 highway, (c) small drainage, direct tributary of Rio Ivinhema. zse.pensoft.net 272 Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... while in the other localities the species was recorded in Shallow areas, not exceeding 20 cm depth, in crystal- line waters and with a clayey substrate (Fig 7a, c). Eight fish species were recorded co-occurring with M. ivinhe- mensis: Aphyocharax dentatus Eigenmann & Kennedy, 1903, Pyrrhulina australis Eigenmann & Kennedy, 1903, Hyphessobrycon anisitsi (Eigenmann, 1907), Hyphesso- brycon eques (Steindachner, 1882), Serrapinnus kriegi (Schindler, 1937), Cichlasoma dimerus (Heckel, 1840), Hoplerythrinus unitaeniatus (Spix & Agassiz, 1829) and Moenkhausia sanctaefilomenae (Steindachner, 1907). Conservation status. The new species 1s endemic of Rio Ivinhema and was recorded in four different areas, in a range of about 25 km along both sides of the river and there is a large density of potential environments for its occurrence along this stretch. It has been recorded 1n ar- eas with no major impacts — although under intense agri- cultural activity — and therefore there 1s no evidence that the species 1s threatened with extinction. Melanorivulus amambaiensis sp. n. http://zoobank.org/6E2F9D08-66CB-4B41-866E-3A BOF878EFAB Holotype. MCP 53150, male, 27.9 mm SL, Brazil, Mato Grosso do Sul State, near of municipality of Navirai, first order stream of Rio Amambai, margin of BR 487 high- way, 22°57711”S, 54°26’52”W, altitude 332 m as; M.V. Volcan & L.E.K Lanés, 14 Dec 2016. Paratypes. MCP 53151, 4 males, 20.9-25.9.0 mm SL (1 C&S), 10 females, 22.8-28.7 mm SL (3 C&S), all collected with the holotype. ZUFMS 5497, 1 male, 21.6 mm SL, 4 females, 22.2—22.9 mm SL, all collected with the holotype. Diagnosis. The presence of an orange or orange-red anal fin with grey or dark grey distal margin (vs. no similar colour pattern) and chevron-like red bars in inverted Y-shape in the flank of males (vs. no similar pattern) distinguishes the M. amambaiensis from all other species of the M. pictus spe- cies group. Additionally, the new species is distinguished by the lower caudal fin ray count (25—28 vs. 29-34), ex- cept M. faucireticulatus; by the lower body depth in males (29.4-21.6 mm SL vs. 21.8—26.5), except from M. vittatus, M. polychromus, M. nigropunctatus, M. interruptus and M. ivinhemensis, by a lower caudal peduncle depth in males (12.4-13.5 mm SL vs. 13.5—-16.8 mm SL), except in M polychromus, M. nigropunctatus, M. interruptus and M. ivinhemensis. Females are distinguished from M. apiamici, M. faucireticulatus, M. giarettai, M. planaltinus, M. rutil- icaudus, M. scalaris, M. nigropunctatus, M. proximus, M. linearis, M. nigromarginatus and M. formosensis by the lower body depth (19.1-21.5 mm SL vs. 21.6—26.0 mm SL); and from M. apiamici, M. egens, M. faucireticulatus, M. giarettai, M. planaltinus, M. nigropunctatus, M. line- aris, M. nigromarginatus and M. ofaie by a shorter pre-dor- sal length (74.5—-76.6 mm SL vs. 76.4-83.3 mm SL). zse.pensoft.net Table 3. Morphometric data for the holotype and paratypes of Melanorivulus amambaiensis sp. n. Holotype | Males (n=5) | Females (n=11) Standard length vais) 20.9-25.9 |24.0-28.7 Percentages of standard length Body depth 21.6 20.4-20.9 |19.1-21.5 Caudal peduncle depth 129 12.4-13.5 |11.7-13.7 Predorsal length 76.1 74.9-79.1 |74.5-76.4 Prepelvic length 56.8 53.6-55.3 [52.0-54.5 Dorsal fin base length 12.3 10.1-12.0 |9.3-11.4 Anal fin base length 21.4 19.1-21.4 |18.5-20.7 Caudal fin length 27.5 28.1-30.7 |26.5-28.6 Pectoral fin length yea 17.9-21.0 |17.3-20.1 Pelvic fin length Lis 9.4-12.0 8.5-11.6 Head length 26:5 26.2-26.9 |24.6-27.6 Percentages of head length Head depth alee 61.6-69.5 |62.4-71.4 Head width 75.7 70.5-72.4 |70.1-79.3 Snout length Reyes 16.7-17.8 |15.2-20.6 Lower jaw length 20.6 18.3-21.3 |18.0-21.0 Eye diameter 28.7 28.5-32.1 |28.7-33.1 Description. Morphometric data are presented in Table 3. Females larger than males, largest female examined 28.7 mm SL, largest male 27.9 mm SL. Dorsal profile slight- ly convex from snout to end of dorsal-fin base, straight on caudal peduncle. Ventral profile weakly convex from lower jaw to operculum. Straight from operculum to or- igin of pelvic-fin and in the caudal peduncle. Body slen- der, cylindrical and compressed, greatest body depth at origin of pelvic-fin base. Snout blunt. Jaws short. Short dorsal and anal fins. Dorsal-fin rays 8-9. Dorsal fin slightly pointed in males, rounded in females. Dor- sal-fin origin on a vertical through base of 8th or 9th anal- fin ray, and between neural spines of the 20th and 21th vertebrae. Anal-fin rays 12—14. Anal fin slightly pointed in males and females. Origin of anal fin at a vertical through pleural ribs of 15th—1 6th vertebrae. Caudal fin oval shaped, longer than deep, 25—28 rays. Pectoral fin rays 11-13. Pec- toral fins rounded, with posterior margin reaching vertical at about 70-90% of length between pectoral-fin and pel- vic-fin bases. Pelvic-fin rays 6—7. Posterior tip of pelvic fin reaching a vertical at slightly anterior to the anus to Ist anal-fin ray. Pelvic-fin bases in close proximity. Scales small, cycloid. Body and head entirely scaled, except anterior ventral surface of the head. Body squama- tion extending over anterior 15—20% of caudal-fin base. No scales on dorsal and anal-fin bases. Frontal squamation E and F-patterned or with two patterns, one on each side (present in one specimen). E-scales not overlapping me- dially. Transverse row of scales anterior to H-scale; scales arranged in regular circular pattern around A-scale. A-scale without exposed margins. Longitudinal series of scales 27-31; transverse series of scales 8-9; scale rows around caudal peduncle 16. No contact organs on flank and fins. Cephalic neuromasts: supraorbital 3+3, parietal 1, an- terior rostral 1, posterior rostral 1, infraorbital 1+9-11+1— 2, preorbital 2, otic 1, postotic 1-2, supratemporal 1, Zoosyst. Evol. 94 (2) 2018, 263-280 243 Figure 8. Colour pattern variation of male paratypes of MZ. amambaiensis sp. n., MCP 53151, small stream tributary of Rio Amam- bai, Mato Grosso do Sul state. median opercular 1, ventral opercular 1—2, preopercular 2+4—5, mandibular 2—3+1, lateral mandibular 1—2, para- mandibular 1. Two neuromasts on caudal-fin base. Six branchiostegal rays. Gill rakers on first branchi- al arch 1+7-8. First epibranchial slightly curved. Total number of vertebrae 30-31, 13-14 precaudal vertebrae, 17 caudal vertebrae. Ventral process of angulo-articu- lar short, pointed. Vomerine teeth 2-3. Dermosphenotic present. Second pharyngobranchial teeth absent. Colouration in life. Males (Fig. 8). Flank light metallic blue-whitish, sometimes purplish above anal fin; numer- ous oblique narrow red bars irregularly arranged, forming reticulated chevron-like marks anteriorly directed with a branching on the lower portion in inverted Y-shape, some- times with a branching on the upper and lower portions in X-shape; between these bars one to four red dots oblique- ly distributed mostly on dorsal portion of flank; reddish dots on anteroventral part of flank, sometimes forming rows, mostly between bases of the pectoral and the pel- vic fin; one to three oblique reddish-brown bars between the postorbital region and the pectoral fin; reddish-brown spots on the humeral region. Dorsum metallic grey or yellowish grey, venter white. Dorsal portion of head yellowish-grey; ventral portion white, without marks or any pigmentation pattern; golden iridescence on oper- cular region. Jaws grey to pale yellowish-grey. Iris pale yellow. Dorsal fin yellowish-hyaline with three or four oblique narrow brownish-red or red bars; basal portion pale yellow. Anal fin orange or orange-red, basal portion pale blue with reddish dots forming reticulated pattern, posterior portion pale blue sometimes with two or three reddish oblique bars, distal region grey or dark grey on the marginal border. Caudal fin hyaline or yellowish-hy- zse.pensoft.net 274 Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... Figure 9. Colour pattern variation of females of 1 amambaiensis (not preserved), sp. n., small stream tributary of Rio Amambai, Mato Grosso do Sul state. aline, yellowish-orange on ventral portion; four to seven narrow oblique brownish-red or red bars cover 2/3 of cau- dal fin, absent in ventral portion. Pectoral fin hyaline, or yellowish-hyaline. Pelvic fin orange or yellowish-orange, sometimes with orange-brown anterior margin. Females (Fig. 9). Flank similar to males, but more ev- ident reddish-brown or brown spots between pectoral and pelvic fins. Numerous oblique narrow red bars irregularly arranged, forming reticulated chevron-like marks ante- riorly directed, sometimes with a branching on the lower portion in inverted Y-shape. Dorsum and dorsal portion of head greenish-gray with small black dots; ventral portion of head white, with dark grey spots; golden iridescence on opercular region. Jaws greyish-brown. Iris pale yellow, sometimes bordered with grey. Dorsal fin yellowish-or- ange or yellowish hyaline, with three reddish-brown nar- row horizontal bars forming a reticulated pattern. Anal fin orange-yellow, basal portion pale blue with reddish dots forming a reticulated pattern, posterior portion pale blue with two or three reddish oblique bars, distal region be- coming gradually dark reddish brown on the marginal bor- der. Caudal fin yellowish-orange, with three to seven grey or dark grey bars; small elongated black spot on dorso-bas- al portion of the fin, sometimes overlapping the most ante- rior bar. Pectoral fins hyaline. Pelvic fins yellowish-orange, sometimes with reddish-brown anterior margin. Distribution. The species is only known from its type lo- cality, a small first order drainage and direct tributary of Rio Amambai, state of Mato Grosso do Sul, Brazil (Fig. 3). zse.pensoft.net Etymology. The name amambaiensis is a reference to the occurrence of the new species in the Rio Amambai basin. Habitat notes. Melanorivulus amambaiensis was record- ed in a small natural drainage, direct tributary of the Rio Amambai, with depth not exceeding 50 cm and with low flow in an area totally exposed to the sun, parallel to BR 487 highway (Fig. 10). The region has a smoothly undu- lating terrain and the area of occurrence Is at an altitude of 327 ma.s.l.. The drainage has a clayey substrate, which gives an orange colour to the water when disturbed. Only Gymnotus aff. carapo Linnaeus, 1758 was registered co-occurring with VM. amambaiensis. Conservation status. The species is known only from its type locality. It seems to be a micro endemic of the Rio Amambai basin, since other species occur in the adjacent basins. Part of its original area was fragmented by BR 487 and there is high frequency traffic of ve- hicles on this highway. Consequently, the species is subject to accidents with potentially dangerous loads, which present a threat to the population of M. amam- baiensis. In addition, the surrounding region ts largely degraded by agricultural activities that also threaten its habitat quality. Thus, M@. amambaiensis was accounted as “Vulnerable” according to the IUCN (2012) stand- ards. The species has an area of occurrence of less than 2000 km7?, occurs in only one locality and suffers from a reduction in its area of occupation and in the quality Zoosyst. Evol. 94 (2) 2018, 263-280 275 Key to Melanorivulus pictus species group from the Rio Parana, Tocantins and Araguaia basin. 1 1 20 Red pigmentation forming chevron-like bars in the body of Males ..................cscccscceeceeecsecssecsconeeecsseessecusteuseecceeseseeenes 2 Red pigmentation in the flank arranged in irregular lines to form vermiculate color pattern In MaleS ............ccceceeeeeeee es UE AA hse ee APT MUNIN «17 en 2 Boy BOM oR a) ASE Nee Les PO Man are BL. tee PO ed MS oe es On 9 cn ee Melanorivulus giarettal PelVicatitmore sents 1S SVOMme rete ls ita eaten clare de uF ac Bape l eis vnja 6 Val ie Srmetapyenitn i Aa hae cei na ans Ud Raat ge aie a tae Seba 3 Absence or extreme reduction of pelvic fin, 5-7 vomerine teeth ..............cecceeeeeee cece eeeeeeeeeeeeaeees Melanorivulus planaltinus 5-rayssigithe.pelwic-tin, LOsfaysuintthes pectoral finv2z-verlebrae..2. ris oe Lees 6 oe we Melanorivulus apiamici 6-8 rays in the pelvic fin, 11-14 rays in the pectoral fin, 29-32 vertebrae .......... cc cccccceece cece cece eee ee eeseeeseeeeeeesaeeeeeeeaeees 4 REC: DabS resent wisthie vam Cle WHDIY, (At8+ See 8s eck DP en poten, ceca rere el Mt an 8 Pe ote oe Bee 0 De oer BM Um en ee me tee 5 Red ‘tar svalsSenceuiivinievCaliCial Ni tui has acest eeels ee le a eases spede es Asatte eae Melanorivulus egens 25-28 caudal fin ray, anal fin orange or orange-red in MAleS .......... cece eeeee cece eeeee eee eeeeeeeeee tees Melanorivulus amambaiensis 28-34 caudal fin ray, anal fin yellow, light yellow to orange or yellowish-hyaline in Males ...............:cccceeeeeeeeeeeeeeeeeeeeeees 6 7 scales in transversal series, oblique rows of red dots on flank, more concentrated on caudal peduncle, forming chev- COT ISIC GS, SONI ce Tt ee PF tee De AGL e tate a WR nce OE OPE SITET te ae a Pe eee ON: ST Melanorivulus leali 8-10 scales in transversal series, oblique red bars forming chevron-like bars along the body, with forward-pointing ver- tex;-generally inthe westerior PORMCHrOl Tne tay as Reese Maat Se SaaS ES Sh wecre Doce ae es a pees eae aA 7 Chevron-like red bars interrupted on midline of flank, fragmented or vestigial in the body of adult males................... 8 Chevron-like red bars well defined, generally not interrupted in the body of maleS.................cccceeeeeeeeeeeeeeeeeeeeeeeeeaeeees 10 Dorsal fin rounded in males, 14 pectoral fin rays .......... ccc cec cece ccc eeeeeeeeeee ees eeeeeeeeeeeeeeeeseeeeeeeenees Melanorivulus interruptus Dorsal fin pointed or slightly pointed in males, 12-13 pectoral fiN rays 20.2... eee cec cee ce cee ee cee e cee eeeeeeceeeeeeeaeeeeeeeseeeeaeeeas ) Frontal squamation E-patterned, pectoral fin hyaline and pelvic fin light yellow in males... Melanorivulus nigromarginatus Frontal squamation F-patterned, pectoral and pelvic fin orange In maleS.............cccccceeee eee Melanorivulus nigropunctatus Red -Darspresenkoniy- Onamidcerat- Cauca THY WG MIALGS =s2.tiac, bento eeeeents 10 we Pane, SONNY aE PORE ae FT ea, owt 11 Caudal fin with dorsal and middle portions presenting vertical red Dar ............cccccec cece cece cece cece eeeeeeee esse eeseeseeeeaeeees 12 10-12 dorsal fin rays, 32-34 caudal fin rays, 34-35 scales in longitudinal series, 10 scales In transversal series.......... ssstedteonh beast isaac coasts sskesd saath PE lastest toed hs retested dat ballads ta seeeoe coca et ee E ed Melanorivulus rutilicaudus 7-9 dorsal fin rays, 29-31 caudal fin rays, 29-31 scales in longitudinal series, 8-9 scales in transversal series............ er etd AMUN teh cad kL MeE wreaks nn RUA Lea se ae ls Seis | hae es Rl 1 ea cade le 2 Sgt shies tee Melanorivulus ivinhemensis Dorsal fin origin on vertical of 7th ray of anal fin, 11-12 anal fin ray, dorsal fin rounded................. Melanorivulus pictus Dorsal fin origin on vertical of 7-1Oth ray of anal fin, 12-14 anal fin ray, dorsal fin pointed or slightly pointed........ 13 6 pelvic fin rays, dorsal fin origin between neural spines of vertebrae 21th-22th, absence of red color in dorsal fin...... I a RARE SEE yA DE REPS A ih 5 SERN EEL Sirk bh REA RACE Ache k S8 ERA Rt CEL a hOB AON NR ER it Melanorivulus polychromus 6-7 pelvic fin rays, dorsal fin origin between neural spines of vertebrae 18th-21th, red pigmentation present in dorsal IN Decoy acigidaaeclt te oenate ets nseass eat ea eel Sigel EEN AAS septs nd etal le AREA estan Epa LAA E EE SLi Savaedidtacl ap diet eee tases et ted RAULL CAR peg 14 EFOnMtalascu atiialioMve= Pater mech. £1 20 a Saree BON I Dee, conte ano eee cians eer oe ee en ee ee 15 ROR aleselalhatOr gr POeWlOh MOO vcs fxcckieyy, SAE haat tes Sey PL A ect eee anil ge ey PAR ae a Melanorivulus litteratus Broad sub-basal red strip on dorsal fin in males, black reticulate pattern in ventral portion of the head of females, oe OCC NCR CHM (VSGAYES: «+h cx Bkaae ABnastag sae xshseelan Sr See Atises svn nctee tien toamtlancdseacksebaa hn Sualh dot hesuaccetfeuce = Melanorivulus faucireticulatus No distinctive broad sub-basal red strip on dorsal fin in males, sometimes black pigmentation present in ventral portion of the head of females never forming an reticulate pattern, 30-34 caudal fin rayS 2.0... 00. ccc cece eee cece ee eeee ees eeeeeeeeeeeees 16 One cilltraker-orvupper Winrar hrs toGAnel) ALARCON; itcna Wet on eee th Rl a hel a Pa area eae I aN cs Bie eet nh Wao 2 ae ole nee, 17 WWe-Silerakerseon wp perl inbcornirstiaramctial-arcitcantscunte cnn eee foeet csr rpech iat iabedd Secs Melanorivulus proximus Red dots present on the anteroventral portion of flank, distinctive dark marks on humeral region in males............... 18 Red dots absent on the anteroventral portion of flank, absence of dark marks on humeral region in males................06 nh sac aad a ns Ae OS 4 ee ROR na st ese sR ok Pe SR A ac dd Pn RE OA i ZR Melanorivulus linearis Flank greenish blue to metallic blue, with oblique red bars forming narrow chevron-like bars, posterior tip of pelvic-fin reachifie VEEiiCallat-anuS-tO "LSPA Mataysen tiene. agen h eelde le) mete tsb El else) semece Meg ha areds om eek sean nt ods Boek 19 Flank intense metallic blue with broad red chevron-like oblique bars, posterior tip of pelvic-fin reaching vertical at 1st to rah dgpsl a fcl Pil ai Gch ae, sie een Oma ee eo, Rae eae en ee es ee eC cion See ee EE Seas Aer ek ce on a RE 20 8 scales in transversal series, origin of anal fin at vertical through pleural ribs of 14th-15th vertebrae, chevron-like red bars with a branching on the upper portion forming Y-shaped red marks in males..............c006 Melanorivulus formosensis 9-10 scales in transversal series, origin of anal fin at vertical through pleural ribs of 15th-16th vertebrae, chevron-like red bars in males never forming Y-shaped red MAarKS ............cccccceeceecceeceeeseeseeeeecesseeceectsaeeeeceeseecseesees Melanorivulus ofaie Small red squares in the flank aligned to form broad red chevron-like oblique bars in males, 12-13 pectoral fin rays, 2 WOO INS TEST ot aoe LM no ohW dey Be ol UPI nla alee tink Cems, Madey Grae, beclag OtTS load Won Minde lene hele Unc Leet, Pete Melanorivulus scalaris Broad chevron-like oblique red bars in males not forming squares, 14 pectoral fin rays, 3-4 vomerine teeth................. B Aeeiscee sna N thee ate sc WO creme es eta alah RA een a 2 28 cement Scan sae sae oe ae eee Ge oes oe RN Melanorivulus vittatus zse.pensoft.net 276 Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... type locality of M. amambaiensis. Discussion congeners, however with well defined, homogeneous and structured colour patterns in each species (Fig. 11). The Relationships among species belonging to the M. pictus _ relatively low variability of character states among spe- group are not clear. Usually there is a high overlap inmer- cies of the M. pictus group makes colour pattern char- istic, morphometric and osteological characters among acters an essential source to diagnose species and to es- zse.pensoft.net Zoosyst. Evol. 94 (2) 2018, 263-280 Figure 11. Males of different species of the M. pictus group recorded in the region of the right bank of Rio Parana. A) M. apiamici; B) M. egens; C) M. nigropunctatus; D) M. ofaie; E) M. scalaris. Photos by Matheus Volcan. timate their relationships, since molecular data are still not available for most species (Costa, 2018). The only molecular study corroborates species and groups delim- itations according to colour patterns (Costa et al. 2016). Costa (2016) discussed the importance of using live colour pattern characters to diagnose species and spe- cies groups of Melanorivulus. According to Costa (2016, 2017) patterns involving the caudal fin contain a high zse.pensoft.net 278 Volcan, M.V. et al: Unrecognized biodiversity in a world’s hotspot: three new species... Figure 12. Variation of colour pattern of the caudal fin of males of different species of the Me/anorivulus pictus group from Rio Parana, Brazil. A) M@. amambaiensis, B) M. apiamici, C) M. egens, D) M. interruptus, E) M. ivinhemensis, F) M. nigropunctatus; G) M. ofaie; H) M. scalaris. concentration of phylogenetically informative characters, useful to delimit most species of the M. zygonectes, M. pinima and M. dapazi group. The same is also valid to distinguish species of the M. pictus group (Costa, 2018). The three new species present distinct and unique col- our patterns, mainly in relation to the pigment and arrange- ment of the chevron-like bars along the body, the colour pattern of the fins, as well as the position and shape of the bars in the caudal fin. Males of M. ivinhemensis, for exam- ple, have a yellow caudal fin, with vertical bars arranged only in the median portion of the fin, M. interruptus has a yellow caudal fin with diagonal red bars, sometimes ves- tigial and inconspicuous, extending from the median to the dorsal region, while males of M amambaiensis have a hyaline or yellowish-hyaline caudal fin with yellowish-or- ange ventral portion and irregular brownish-red bars. This is always combined with an orange or orange-red anal fin with a dark grey margin (Fig. 12). Likewise, the colour and the arrangement pattern of vertical bars on the caudal fin, as well as the black spot in the caudal peduncle are highly variable in shape and size among females of the different species of the MZ. pictus species group (Fig. 13) and also are informative for species identification. It is hypothesized that M. interruptus is closely related to M. linearis as judged by the absence of distinctive dark zse.pensoft.net marks on the humeral region, and the absence of red dots on the anteroventral portion of the flank. With MZ egens it shares the E-pattern of the frontal squamation, the yellow unpaired fins and the absence or reduction of red bars or red pigmentation at the base of the anal fin in males. Females of these three species also present all unpaired fins yellow with a dark grey distal margin as well as hyaline pectoral fins. Melanorivulus ivinhemensis shares some character- istics with M. rutilicaudus and seems to be more closely related to this species. Mainly this is because both have the unpaired fins coloured yellow and red bars are present only in the middle of the caudal fin in males, a unique pattern among the species of the M. pictus species group. On the other hand, relationships between VM. amambaiensis and the other species of the M. pictus species group are not clear because of the unique colour pattern. Chevron-like red bars with inverted Y-shaped red marks and an orange or orange-red anal fin with a dark grey distal margin in males are not encountered in any other species of the group. Species of the M. pictus group generally present a high degree of endemism, occurring in small stretches of ba- sins that drain the upper Rio Parana and the upper Rio Araguaia and Tocantins (See Figure 3 distribution map of M. pictus species group) and are known for a few sites only (e.g. Costa 2005, 2007, 2008, 2018, Volcan et al. Zoosyst. Evol. 94 (2) 2018, 263-280 3 279 Figure 13. Variation of colour pattern of the caudal fin of females of different species of the Me/anorivulus pictus specis group from Rio Parana, Brazil. A) M. amambaiensis, B) M. apiamici;, C) M. egens; D) M. interruptus, E) M. ivinhemensis, F) M. nigropunctatus; G) M. ofaie; H) M. scalaris. 2017). This pattern is observed for different Melanoriv- ulus species groups (e.g. clade M. zygonectes and M. dapazi), where species are restricted to small portions of hydrographic basins in a similar way (Costa 2016, 2017). This micro endemism is extremely concerning from the conservation perspective. The loss and/or degradation of a small stretch of a river basin may result in the extinc- tion of a species, as in the case of the vulnerable popula- tions of M. amambaiensis and M. interruptus, which are known only from their type localities. Most species of the M. pictus group are reported for highlands, mainly in the headwaters of the main tributary drainage on the right bank of the Rio Parana (Costa 2012, 2018, Volcan et al. 2017). On the other hand, perhaps caused by insufficient sampling, there is a large gap of records in lower areas, downstream the headwaters (Fig- ure 3). Here, only M. apiamici is recorded, in lower areas associated to the main channel of the Rio Parana. From the availability of potentially suitable habitats, we would expect the presence of representatives of the Melanoriv- ulus pictus species group also along the lower reaches of most of the tributaries of the Rio Parana. Costa (2017) suggests that the high species diversity in the Cerrado is correlated with the topography, which generated geographical isolation of populations and may explain the present distribution of distinct species of Mel- anorivulus along different altitudinal zones of river drain- ages. However, in addition to topography, it is observed that many of the species are restricted to small watersheds, sometimes in similar ranges of altitude, as in the case of M. linearis and M. interruptus that both are recorded in the headwaters of the Rio Pardo or M. nigropunctatus and M. ofaie, both occur on opposite sides of the Rio Verde. Thus, besides the topography, micro watersheds can also constitute barriers, suggesting that their restricted areas of distribution are a consequence of the low vagility of these species. We can assume a similar situation of high diversity and putative undiscovered species in other areas and basin tributaries of the upper Rio Parana. These drain large areas of the Cerrado, mainly downstream from the headwaters of the main drainage formations of the upper Rio Parana, where there is insufficient sampling of rivulid fish. Comparative material All from Brazil, Mato Grosso do Sul state. Melanoriv- ulus nigropunctatus: Holotype. MCP 50017, male, 24.1 mm SL, stream tributary of left margin of Rio Verde, zse.pensoft.net 280 20°02’29’S, 53°9°42”W; B. Klotzel, 29 Dec 2013. Para- types. MCP 50018, 5 males, 17.9—23.6 mm SL (1 C&S), 2 females, 20.7—22.7 mm SL (1 C&S), all collected with the holotype. Melanorivulus ofaie: Holotype. MCP 50019, male, 30.5 mm SL, stream tributary of right margin of Rio Verde, 20°25’27"S, 52°56’34"W; B. Klotzel, 29 Dec 2013. Paratypes. MCP 50022, 5 male, 19.9 mm SL (1 C&S), 6 females, 17.3—24.3 mm SL (2 C&S), collected with the holotype. MCP 50020, 3 males, 17.2—23.3 mm SL, 4 fe- males, 16.2-19.3 mm SL, stream tributary of right mar- gin of Rio Verde, 20°01’50”S, 53°15’14"W.; B. Klotzel, 11 Jul 2013. MCP 50021, 4 juveniles, sex undetermined, 12.8-15.6 mm SL, stream tributary of right margin of Rio Verde, 20°02711”S, 53°15’26”W; B. Klotzel, 11 Jul 2013. ZUFMS-PIS 4736, 4 male, 21.5—23.3 mm SL, 3 females, 17.3—20.1 mm SL. same locality as holotype; B. Klotzel, 11 Jul 2013. Melanorivulus apiamici: ZUFMS 5500, 4 males 20.2—24.2 mm SL, 1 female, 20.8 mm SL, topo- types, Bataguassu, 21°43’32”S, 52°15’41"W; M.V. Vol- can & L.E.K. Lanés, 12 Dec 2016. Melanorivulus egens: MCP 53152 3 males 17.8—20.6 mm SL, 1 female, 17.5 mm SL, topotypes, Camapua, 19°19’25”S, 53°21’06"W; M.V. Volcan & L.E.K. Lanés, 12 Dec 2016. Melano- rivulus rossoi. ZUFMS PIS 5431, 5 males 18.1—21.0 mm SL, 4 females, 18.5—19.8 mm SL, Campo Grande, 20°40°08’S, 54°45’20"W; F. Severo-Neto, 18 Jun 2015. Melanorivulus scalaris: ZUFMS 2114, 6 males 14.1—23.6 mm SL, 3 females, 17.7—22.1 mm SL, topotypes, Costa Rica, 18°53’57”S, 52°58’04’W; O. Froehlich, M. Casaro, N.C Penatti & L.S. Inocéncio, 31 Mar 2004. Melanoriv- ulus punctatus: ZUFMS 5254, 5 males, 17.5-18.9 mm SL, 5 females, 16.1—-17.8 mm SL, Corumba, 19°34’36”S, 57°01’ 10" W; F. Severo-Neto, 17 Dec 2015. Acknowledgements This study was supported by Instituto Chico Mendes de Protec¢éo da Biodiversidade (ICMBio) and funded by Fundacao Grupo Boticario de Protecao a Natureza as part of the project “Peixes Anuais dos Campos Sulinos”. The man- uscript benefited from corrections and suggestions made by Wilson Costa, Marcelo Loureiro and Dalton Nielsen. References Costa WJEM (1995) Pearl killifishes: The Cynolebiatinae. Systematics and Biogeography of a Neotropical annual fish subfamily (Cyprino- dontiformes: Rivulidae). TFH Publications, Neptune City. Costa WJEM (2001) The neotropical annual fish genus Cynolebias (Cyprinodontiformes: Rivulidae): phylogenetic relationships, tax- onomic revision and biogeography. Ichthyological Exploration of Freshwaters 12: 333-383. Costa WJEM (2005) Seven new species of the killifish genus Rivulus (Cyprinodontiformes: Rivulidae) from the Parana, Paraguay and up- per Araguaia river basins, central Brazil. 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