A new species of Characidium (Characiformes: Crenuchidae) from the rio Doce basin, Brazil

A new species of Characidium is described from the tributaries of the upper and middle rio Doce basin, Minas Gerais, Brazil. The new species is distinguished from most congeners, except C. cricarense , C. hasemani , C. helmeri , C. kalunga , C. pterostictum , C. schubarti , C. summus , and C. travassosi by lacking scales in the area between the anterior limit of the isthmus and the anterior margin of cleithrum. The new species differs from the aforementioned species by a series of characters, including the presence of the adipose fin, 2 –4 scales between the anus and anal fin, two rows of dentary teeth, presence of the parietal branch of the supraorbital canal, 4 scale rows above the lateral line, and absence of two conspicuous inclined dark bands on each caudal-fin lobe. The new species further differs from most congeners with an unscaled ventral surface of the isthmus by the presence of 33– 34 pored scales on the lateral line and by the dark dashes on the caudal fin-rays not forming well-defined bands.


INTRODUCTION
The rio Doce is a southeastern Brazilian river basin that runs through two biodiverse hotspots, the Atlantic Forest and the Brazilian Savanna (Myers et al., 2000).The surrounding areas of the river, as well as the basin itself, have suffered tremendous environmental destruction, such as riparian deforestation, mining disasters, dam building, loss of native fish species, and the introduction of at least 29 nonnative fish species (Vieira, 2010;Salvador et al., 2018).More dramatically, a tailings dam in this basin burst severely recently, spreading toxic mud along the main river course and affecting wild communities as well as local human populations (Fernandes et al., 2016;Neves et al., 2016).The historical environmental alteration impacting the rio Doce basin has been intensely discussed for decades (e.g., Vieira, 2010;Pinto-Coelho et al., 2008;Latini, Petrere 2004;Fragoso-Moura et al., 2016;Sales et al., 2018;Vergilio et al., 2021), and the effects of the tailings dam disaster on the fish community have been assessed recently (e.g., Gomes et al., 2019;Ferreira et al., 2020;Macêdo et al., 2020;Weber et al., 2020;Vergilio et al., 2021).At least 11 species of fishes occurring in the basin are categorized as endangered, four of them endemic (ICMBio, 2018).
In contrast to the well documented environmental alterations of the rio Doce, part of the basin's biodiversity remains unknown (Sales et al., 2018).In the last two decades, 14 new species of fishes from the basin have been described, attesting broad advance in the knowledge of its fish fauna and resulting in more than 80 native species in the basin (Vieira, 2010;Salvador et al., 2018;Fricke et al., 2021).Recently, Sales et al. (2018) used genetic divergences to highlight the occurrence of hidden biodiversity within five genera of fish from the basin, along with cryptic and/or candidate species at least in four genera.Both cases implied the genus Characidium Reinhardt, 1867 as including unrecognized species.

MATERIAL AND METHODS
After capture, specimens were anesthetized with eugenol solution (1 ml/L), then fixed in 10% formalin, and finally preserved in 70% ethanol.The field studies did not involve endangered species.Counts and measurements were taken according to Buckup (1993a), Melo, Oyakawa (2015), andZanata et al. (2020).Measurements were taken with a digital caliper to the nearest 0.1 mm and expressed as percentages of standard length (SL), except subunits of head, which are given as percentages of head length (HL).In the list of paratypes, an asterisk indicates lots for which measurements were included in Tab. 1.In the lot MCNIP 4889, the LGC RD is the tissue voucher number deposited in the Laboratório de Genética e Conservação -LGC PUC Minas, and BOLD:ACS is the accession number to indicate the sample of the new species sequenced in Sales at al. (2018).Meristic data are given in the description, an asterisk indicates counts of the holotype, and the frequency of each count is given in parentheses.Counts of vertebrae, ectopterygoid teeth, branchiostegal rays, procurrent caudal-fin rays, caudalfin rays, epurals, and other osteological observations were made only in cleared and stained (cs) paratypes, prepared according to the method of Taylor, Van Dyke (1985).Dentary teeth were counted in cs specimens and were examined in the largest alcohol preserved specimens.One specimen was placed into an ethanol-based solution of alizarin (Springer, Johnson, 2000) for around 6 hours in order to stain the scales for a photo of the ventral squamation.The pattern of circuli and radii were observed on scales situated between the dorsal-fin base and lateral line, after being stained in alizarin.The pseudotympanum morphology was examined after the removal of the overlying skin, adipose tissue, and lateral-line nerve of alcohol-preserved specimens.Institutional abbreviations follow Fricke, Eschmeyer (2021).Comparisons with species not cited in the 'Comparative material examined' were based on the literature information (e.g., Agudelo-Zamora et al., 2020a,b;Armbruster et al., 2021;Buckup, 1992Buckup, , 1993a;;Gomes, 1947;Melo, 1967;Melo et al., 2016;2021a,b).
Scales cycloid; circuli absent on exposed portion of scales; up to 18 slightly divergent radii present on exposed portion of scales.Lateral line completely pored, with 33 (7) or 34* (25) scales; horizontal scale rows above lateral line 4 (30); horizontal scale rows below lateral line 4 (30).Scales along middorsal line between supraoccipital and origin  Humeral hiatus divided into anterior and posterior chambers by pleural rib of fifth vertebra (Fig. 3).Dorsal-fin rays ii,9* (30) or ii,10 (1); distal margin of dorsal fin nearly straight or somewhat convex.Adipose fin well developed.Pectoral-fin with 12-13 total rays; iii,7,ii (7), iii,8,i* (15), iii,8,ii (5) or iii,9,i* (4); second and third branched pectoral-fin rays usually longest; posterior tip of pectoral fin reaching or almost reaching pelvicfin origin in small specimens (approximately 30.0 mm SL) and pectoral-fin tip falling short of pelvic-fin insertion in larger specimens.Postcleithrum 1 absent.Pelvic-fin rays i,6,i (3), i,7 (3), i,7,i* (22); second to third branched pelvic-fin rays longest; posterior tip of pelvic fin falling short of anal-fin origin.Anal-fin rays ii,5 (2), ii,6* (27), ii6,i (3); posterior margin of anal fin slightly rounded, with second branched ray usually longest; last ray adnate (2).Caudal-fin rays i,9,8,i* (29).Dorsal procurrent caudal-fin rays 9 (2); ventral procurrent caudal-fin rays 7 (2).Total vertebrae 31 (2); precaudal vertebrae 17 (2); caudal vertebrae 15 (2).Supraneural bones 5 (2).Epural bones 2 (2).Uroneural bone 1 (2).Coloration in alcohol.Ground color of head and body pale brown (Fig. 1).Dorsal surface of head with dark patches of pigmentation, usually not forming conspicuous blotches; area between nares and slightly anterior to them usually clearer and area posterior to eyes darker.Dorsal half of head darker than ventral half, except in a few more darkly pigmented specimens.Dorsal half of head with sparsely distributed melanophores of with patches of dark pigmentation bordered by clear areas; a dark stripe present from anterior margin of snout to anterior margin of eye, less conspicuous in largest specimens examined; an inconspicuous dark blotch posterior to eye, somewhat aligned with stripe of snout, but usually with its anterior portion slightly elongated ventrally.Ventral half of head with variable pigmentation; melanophores sparsely distributed and not forming spots or completely absent in a few specimens; skin covering laterosensory canal on infraorbital 3, 4 and 5 usually without dark pigmentation, resulting in pale curved line posterior to eye.Ventral surface of head with sparsely distributed melanophores; isthmus pale or with a few melanophores.Opercle mostly dark, with pale bordering membranes.Humeral region with rounded humeral blotch, somewhat merged with midlateral stripe and imperceptible in some specimens.A continuous narrow (approximately half scale wide) dark stripe extending from humeral blotch to end of caudal peduncle; stripe usually more conspicuous anterior to last blotch or bar on caudal peduncle.Dorsal half of body usually with up to three horizontal series of dark spots formed by concentration of melanophores on dorsal half of exposed portion of scales.Occurrence, position and form of blotches or bars on flanks highly variable; variation apparently not ontogenetically influenced or sexually dimorphic.Majority of specimens with vertically elongate irregular blotches or bars, without sharply defined borders (Figs.1A-E); blotches or bars usually not solidly dark, resulting in chain-like pattern.Blotches with variable height, usually not crossing entire body, restricted to its dorsal half in some specimens, limited to central portion or ventral half of body in others.When present, bars number up to 10, variable in form and position, inclined or not; some specimens with bars divided in two inconspicuous branches (MZUSP 104694, 30.0 mm SL;MCNIP 3014, 51.1-43.3 mm SL).Number of bars not necessarily equal on contralateral sides of specimen; one side may have bars while other side displays diffuse blotches; bars not connected dorsally to each other.Bars present in small (MZUSP 104679, 19.7-24.3mm SL) and large (MCNIP 1993, 51.8 mm SL) specimens.A few specimens with homogeneous coloration on body (MZUSP 109301, 40.0-42.5 mm SL; MCNIP1026, 28.4 mm SL), with melanophores equally distributed or concentrated on border of scales and without conspicuous dark blotches or bars on flanks (Fig. 1F).Basicaudal spot usually well marked; spot poorly visible in some specimens (MZUSP 109301, 40.0-42.5 mm SL).Ventral surface of body usually with melanophores sparsely distributed throughout, more conspicuous posterior to anus; some specimens with preanal region completely pale.All fin rays display dark pigmentation, represented mainly by black dashes, more evident on dorsal and caudal fins.Dorsal fin with dark pigment on basal portion of rays and interradial membranes; some specimens with dark dashes arranged in one inconspicuous band at midlength of fin and others with up to three series of dashes restricted to rays.Caudal fin usually with conspicuous dark dashes on rays, loosely organized in up to three series following contour of fin; some specimens with caudal fin homogeneously pigmented and without well-defined dark dashes.Anal fin usually devoid of dark pigmentation in smaller specimens; fin of medium and large Leonardo Oliveira-Silva, Sérgio A. Santos, Maridiesse M. Lopes and Angela M. Zanata specimens with inconspicuous concentration of melanophores on its anterior half or with a series of dashes at its midlength.Pectoral and pelvic fins with dark dashes on dorsal surface of rays, not forming bands, or somewhat homogeneously darkened.Adipose fin usually with melanophores on its central portion.

Coloration in life.
Ground color slightly olivaceous dorsally and white ventrally (Fig. 4).Conspicuous black stripe on snout continuous with narrow black midlateral stripe on flanks; golden line extending along dorsal portion of black midlateral stripe.Parts of iris, infraorbitals, and opercle with silvery and golden hues.Black rounded and vertically elongate blotches distributed along midlateral line of body of some individuals (Fig. 4A), with that coloration less conspicuous in others (Fig. 4B).Pigmentation on fins similar to that of preserved specimens.Geographical distribution.Characidium krenak is known to occur in tributaries of the upper and middle rio Doce basin, Minas Gerais State, Brazil (Fig. 5).

Ecological notes.
Characidium krenak was collected in clear and dark water tributaries at 600 to 900 m above sea level.It inhabits slow to turbulent flowing streams, with rocky, sandy or clayish substrate (Figs.6A, C).Those environments are in different degrees of preservation, ranging from sites with intact riparian vegetation (Figs.6A, B,  E) to areas highly impacted by direct anthropogenic interference, such as mining and deforestation for agriculture, eucalyptus plantations and livestock (Figs.6C, D, F).
Etymology.The specific name krenak honors the Krenák indigenous people (also known as Aimorés, Grén or Krén), that nowadays inhabit a small area on the left margin of the rio Doce.The Krenák were victims of constant massacres in the past and are currently impacted by severe environmental alterations.A noun in apposition.
Conservation status.The rio Doce basin has been considered highly impacted in the last decades due to a series of anthropogenic activities.Mining is one of the most striking, and occurs throughout most of the known range of C. krenak.One of the major recent catastrophes reported in the rio Doce basin was a burst of the oredisposal dam in November 2015, in the municipality of Mariana, Minas Gerais State, with incalculable impacts on the whole aquatic biota that critically endanger all of its biodiversity (Reis et al., 2019).Though the known range of occurrence of C. krenak does not include the portion of the main channel of the rio Doce impacted directly by the aforementioned burst of the dam, the species is present in tributaries that have been impacted indirectly.Deforestation due to agriculture and livestock ranching generate New Characidium from the rio Doce basin different environmental impacts in streams where C. krenak occurs.Such streams drain the municipalities of Alvorada de Minas and Conceição do Mato Dentro and include córrego Ponte Nova, ribeirão das Pedras, córrego Antonieta, ribeirão Passa Sete, Córrego Vargem Grande, córrego Vermelho, córrego Trote Velho and other tributaries of the rio Santo Antônio.In particular, streams from the rio Santo Antônio sub-basin near Conceição do Mato Dentro are more likely to suffer from agricultural or ranching impacts than are the tributaries of the rio Piranga sub-basin.Siltation can be observed in some tributaries of the rio Santo Antônio sub-basin (Fig. 6E), in the surroundings of urban areas and downstream from ongoing mining projects.Dam construction is also particularly damaging to the rheophilic fauna (Agostinho et al., 2008;Hrbek et al., 2018), and such enterprises exist within the area of occurrence of C. krenak (ANA 2015;fig. 10).Although one of the authors (SAS) has witnessed a decline in abundance of C. krenak in recent years, no adequate information is available to make direct or indirect assessments of extinction risk based on the distribution and/or population status of the species.Therefore, we recommend that C. krenak be classified as Data Deficient (DD), according to the guidelines published by the International Union for Conservation of Nature Standards and Petitions Committee (IUCN, 2019).
Remarks.The new species shares all putative synapomorphies of the Clade C1 proposed by Buckup (1993b), including the absence of postcleithrum 1, the naked area of ventral squamation and the reduced cranial fontanel limited antero-laterally by the parietals.However, in C. krenak the bordering of the cranial fontanel is variable, being limited anteriorly by the frontals in one cs specimen examined herein and by the parietals in the other.According to Buckup (1993b), the size of the fontanel is variable among species included in the Clade C1, but no intraspecific variation was cited by the author.Such variation is almost always absent in original descriptions of species of Characidium, possibly due to the small number of conspecific specimens typically examined for the feature.The recent description of C. onca Melo, Ribeiro & Lima (i.e., 2021), provides an exception, and those authors report two specimens in which the cranial fontanel is limited antero-laterally by the parietals and posteriorly by the supraoccipital in two specimens, and one specimen in which the fontanel extends anteriorly to contact the frontals (Melo et al., 2021b).This variation is similar to that observed here in C. krenak.Examination of this feature in a large number of conspecific specimens is desirable in future species descriptions in order to provide an overview of its variation within Characidium.
Two other species of Characidium are known to occur in the rio Doce basin: C. cricarense and C. cf.timbuiense (Sales et al., 2018;Malanski et al., 2019) Conversely, the occurrence of C. timbuiense in the rio Doce has yet to be confirmed, and will depend on a detailed study that clearly delimits the species taxonomically and geographically.Aside from the distinct ventral squamation that C. krenak and C. timbuiense share (see diagnosis), the former differs from the congener by the presence of 4 scale rows above the lateral line (vs.5), lacking of a conspicuous dark band crossing the proximal third to midlength of all dorsal-fin rays and membranes (vs.possessing such a band) and possessing conspicuous dark dashes on the caudal-fin rays that do not form continuous bands, or uniformly pigmented caudal-fin rays (vs.possessing dark bands or blotches on the caudal fin formed by dark pigmentation on the rays and membranes).
The new species possesses morphological adaptations (e.g., streamlined body, distal portion of the four first pectoral-fin rays protruded beyond the margin of the interradial membrane and scaleless isthmus) present in congeners that inhabit fast-flowing water environments, as described originally in C. cf.timbuiense (Buckup et al., 2000) and recorded later in various congeners [e.g., C. iaquira (Zanata et al., 2020), C. kamakan (Zanata, Camelier, 2015) and C. nambiquara (Zanata, Ohara, 2020)].These modifications possibly provide adherence, resistance and strength to avoid being swept downstream in the fast-flowing water (Buckup et al., 2000).So far, C. krenak is known from tributaries of the upper and middle rio Doce basin on the Serra do Espinhaço, where it occurs most commonly in middle-to fast-flowing habitats.The literature mentions this region as poorly known despite its species richness, and as capable of harboring several endemic and/or endangered species (Alves et al., 2008;Santos, Britto, 2021).The presence of a newly discovered and apparently endemic species of Characidium in the rio Doce highlights the need to include specimens from this region in other taxonomic studies.Furthermore, the extensive anthropogenic activities that have altered the environment of this basin potentially threaten the endemic fish species that occur there and highlight the need for focused conservation policies.

FIGURE 2 |
FIGURE 2 | Ventral squamation of Characidium krenak (MZUSP 112265, 36.9 mm SL, paratype) showing the lack of scales on a region of the isthmus not extending posterior to the anterior margin of the cleithrum (naked portion restricted to area anterior to the dotted line).

FIGURE 4 |
FIGURE 4 | Lateral views of uncatalogued aquarium specimens of Characidium krenak, showing the variable color patterns in life.A. Distinct black, rounded and vertically elongate blotches distributed along midlateral line of body.B. Inconspicuous blotches on midlateral line of body.All from Brazil, Minas Gerais State, Conceição do Mato Dentro, rio Santo Antônio sub-basin, rio Doce basin.

FIGURE 5 |
FIGURE 5 | Map of the rio Doce basin, Brazil, showing the type locality (red star) and collection sites (yellow circles) of Characidium krenak from rio Piranga and rio Santo Antônio sub-basins; each symbol may represent more than one locality.

FIGURE 6 |
FIGURE 6 | Sampling localities of Characidium krenak, Brazil, Minas Gerais State, rio Santo Antônio sub-basin, rio Doce basin: A. Unnamed stream tributary of the right bank of córrego Ponte Nova; B. Ribeirão das Pedras, a tributary of the right bank of rio do Peixe, near its confluence with Córrego Vermelho; C. Unnamed stream, before the meeting with córrego Ponte Nova; D. Córrego Escadinha, at right bank of ribeirão das Pedras, with deforested riparian vegetation for livestock activities; E. Córrego Vargem Grande, undergoing siltation process due to anthropogenic activities; F. Mining activities around the streams that flow into rio do Peixe, tributary of the rio Santo Antônio sub-basin, area of occurrence of C. krenak.

TABLE 1 |
Morphometric data of holotype and paratypes of Characidium krenak, range includes the holotype.N = total number of specimens examined; SD = Standard deviation.

Agudelo-Zamora HD, Tavera J, Murillo YD, Ortega-Lara A. The
New Characidium from the rio Doce basin collecting permits, Tiago Pessali for his help in the loan of the lots from MCNIP, to Instituto Estadual de Florestas (IEF-MG), Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA), and Instituto Chico Mendes de Conservação da Biodiversidade (ICMBIO) for providing the collecting permits.LOS and SAS are funded in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001.