A new species of Characidium (Characiformes: Crenuchidae) from coastal basins in the Atlantic Rainforest of eastern Brazil, with phylogenetic and phylogeographic insights into the Characidium alipioi species group

A new species of Characidium from southeastern Brazil is described based on morphological and molecular evidence from specimens collected between the rio Jucuruçu and rio Doce basins. The new species belongs to a group of species within Characidium with an unscaled area in the isthmus and is distinguished from these species, except C. alipioi, C. fasciatum, C. hasemani, and C. kamakan, by the greater distance (greater than 10% SL) and presence of 5-7 scales between the anus and the anal fin, and presence of 14 series of scales around the caudal peduncle. The species is distinguished from C. alipioi by having 4 series of scales above the lateral line (vs. 5 series) and greater distance between the anus and the anal fin; from C. fasciatum and C. kamakan, by the smaller body depth at the dorsal-fin origin, at the anal-fin origin, and at the caudal peduncle; from C. hasemani, by the short distances between the tip of the snout and the pelvic fin, the tip of the snout and the anal fin, and the tip of the snout and the tip of anal fin. The new species forms a presumably monophyletic group with C. alipioi and C. kamakan.


Introduction
Characidium is the most widespread and species-rich genus of crenuchid fishes. Currently, 69 valid species of Characidium are known, 13 of which were described during the last four years (Fricke et al., 2019). Monophyly of the group is supported by the presence of a dark spot on base of median caudal-fin rays, a feature secondarily lost in some species (Buckup, 1993a;Buckup, 1993b;Melo, Buckup, 2002). Most species inhabit fast flowing streams 2 e180121 [2] with rocky or sandy substrates. The genus has a broad distribution in South America, ranging from Panama, in Central America, to northeastern Argentina in southern South America (Fricke et al., 2019).

Material and Methods
Specimens analysed are deposited in the ichthyological collection of the Coleção Zoológica do Norte Capixaba (CZNC) of the Centro Universitário Norte do Espírito Santo, Universidade Federal do Espírito Santo, the Instituto Nacional da Mata Atlântica (formelly Museu de Biologia Professor Mello Leitão -MBML), the Museu Nacional, Universidade Federal do Rio de Janeiro (MNRJ), and the Museu de Zoologia da Universidade de São Paulo (MZUSP). The comparative data for Characidium fasciatum Reinhardt, 1867, also includes information obtained from the type specimens available from Buckup (Buckup, 1992).
Meristic and morphometric data were obtained from the left side of specimens, unless some damage was verified, with the use of a binocular stereomicroscope and a digital caliper with 0.01 mm precision. Counts and measurements were performed according to protocols previously established for crenuchid fishes (Buckup, 1993b;Melo, Oyakawa, 2015). We also counted number of scales in the series extending between anus and anal-fin base; the first scale of this series is usually small, pointy, and located just posterior to the anus, and the scale located immediately anterior to the anal-fin origin is usually U-shaped and surrounds the first anal-fin ray. In the text, all counts are followed by their frequency in parentheses; asterisk indicates the value for the holotype. We use the term "unscaled" to describe the absence of scales in the isthmus and chest area, instead of the term "naked", which has been used elsewhere.
Specimens were cleared and stained according to the method proposed by Taylor, Van Dyke (1985). Specimens were radiographed with the use of a Faxitron MX-20 Cabinet X-ray System. Cleared-and-stained (CS) specimens and radiographic images were used for skeletal counts, and confirmation of supernumerary anal-fin elements.
The four anterior-most vertebral centra modified into the Weberian apparatus were included in vertebral counts. The description of osteological characteristics followed the terminology from Weitzman (1962).
Species delimitation was based on an integrative approach, where anatomic and color patterns were used in conjunction with DNA barcoding methods (Hebert et al., 2003). Muscular tissue from selected specimens, including sympatric and selected outgroup species, were used for DNA extraction following the salting out method (Miller et al., 1988). DNA quality was verified with standard agarose gel electrophoresis. DNA concentration was measured using a NanoDrop ND-2000 spectrophotometer. Partial sequences of the Cytochrome Oxidase Subunit I (COI) gene were amplified with Polymerase Chain Reaction (PCR) using primers FishF1 (5'-TCA ACC AAC CAC AAA GAC ATT GGC AC-3') and FishR1 (5'-TAG ACT TCT GGG TGG CCA AAG AAT CA-3') (Ward et al., 2005), and FishF6 (5'-TGT AAA ACG ACG GCC AGT ACY AAY CAC AAA GAY ATT GGC A-3') and FishR7 (5'-CAG GAA ACA GCT ATG ACC TAR ACT TCT GGR TGD CCR AAG AAY CA-3') which included M13 sequencing primer sequences appended (Jennings et al., 2019). The PCR protocol used the following thermal cycle: denaturation at 94°C/30s, primer hybridization at 50°C/45s, and nucleotide extension at 72°C/45s; this sequence was repeated 35 times in a loop, and the reaction was terminated with 72°C/10min and 4°C/5min. Quality of amplified products was verified with 1% agarose-gel electrophoresis. PCR products were purified using Exo-SAP (Handy et al., 2011) or PEG (Jennings, 2016;Lis, 1980), and bidirectionally sequenced following an automated Sanger method (Sanger et al., 1977;Otto et al., 2008), where a BigDyeTM Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) was used for labeling, purified again with ethanol precipitation and loaded on an automatic sequencer ABI3730xl (Applied Biosystems) at Fundação Oswaldo Cruz -FIOCRUZ.
The resulting chromatograms were aligned to a reference sequence using the Geneious v9.1 software and manually edited to ensure codon alignment and fine tune base calls. The sequences and associated data, including geospatial coordinates, sequence data, trace files, and primer details are available in the Barcode of Life Data Systems (BOLD Systems, http://boldsystems.org/; Ratnasingham, Hebert (2007)). Sample details are listed in Tab. 1, along with GenBank Accession codes.
The degree of genetic divergence among samples was evaluated from genetic distances using a maximum likelihood tree. The evolution model selected for calculation of distances was based on the on the jModelTest software (Posada, 2008), and the best model (HKY+G) was then used to generate a maximum likelihood tree through MEGA 7.0.26 software (Kumar et al., 2016). Specimens with less than 2% of genetic distance were considered conspecific following DNA barcoding standards for fish (Pereira et al., 2013). Tab. 1. List of specimens used to obtain molecular data in this study. Sample ID refers to the DNA Extract Collection of the Laboratório de Pesquisa em Biodiversidade Molecular, Museu Nacional (MNLM), which are also used to record specimen data in BoldSystems (http://www.boldsystems.org/). Voucher code refers to the whole-organism catalog numbers, and Specimen/Tissue core refers to tissue catalog number in the respective ichthyologic collection. Specimens from the paratype series are indicated by an asterisk. Description. In Tab. 2, morphometric data is summarized for holotype and paratypes. Characidium cricarense has elongate, fusiform body (Fig. 1). Greatest body depth at dorsal-fin origin. Dorsal profile in lateral view convex from snout to origin of dorsal fin; convex, posteriorly slanted at base of dorsal fin, almost straight between base of dorsal fin and adipose fin; posteriorly slanted at base of adipose fin, almost straight between adipose fin and caudal fin. Ventral profile in lateral view slightly concave between lower lip and base of pectoral fin, slightly straight from pectoral base to anal-fin origin, straight and slightly slanted at base of anal fin, straight below caudal peduncle. Pelvic-fin origin at vertical line about two scales posterior to dorsal-fin origin. Adipose fin at vertical line about two and a half scales posterior to anal-fin origin. Snout directed downwards, dorsally convex in lateral view (Fig. 1a). Mouth subterminal, aligned with or lower to ventral margin of orbit. Posterior tip of maxilla not reaching level of anterior margin of orbit. Orbit oval, slightly elongate along axis from tip of snout to parietal bone; margin of eye free. Nares widely separated; distance between nares larger than distance between posterior naris and eye. Margin of anterior naris expanded, forming circular rim; posterior naris with expanded margin on its anterior-most portion. Parietal branch of supraorbital laterosensory canal present, reaching parietal bone in larger specimens; frontal branch of supraorbital laterosensory canal present. Fontanel reduced, limited anteriorly by frontals, laterally by parietals and posteriorly by supraoccipital bones.

Coloration in alcohol.
Background color yellowish (Fig. 1). Black longitudinal stripe about half scale thick extending from tip of snout to base of caudal fin. Body with 8 to 13 triangular bars, wider dorsally, thinner ventrally; bars from opposite sides united across dorsal midline by a corresponding series of dorsal blotches; bars located posteriorly to pelvic fin extending to ventral surface of body. Humeral blotch partially merged into first bar. Dark spot present at base of middle caudal fin rays. All fins mostly hyaline, with scattered chromatophores along rays forming bands of variable intensity. Dorsal fin with three bands; main (intermediate) band dark, not reaching anterior-most margin of fin. In smaller specimens, caudal-fin pigmentation restricted to some scattered chromatophores at outer lobes, ontogenetically developing into conspicuous incomplete B-shaped pattern in larger specimens, formed by a posterior extension of the lateral stripe and diagonal bands across the dorsal and ventral portions of fin. Pectoral, pelvic and anal fins with dark, diffuse distal bands.  Color in life. Slightly translucent body, with olive-greenish or light-brownish background color on head and dorsum. Silvery background below lateral stripe. Dark chromatophore patterns as described for specimens preserved in ethanol.

Sexual dimorphism.
No hooks on fins or other sexually dimorphic features were observed externally on the examined specimens. Molecular delimitation. DNA barcodes based on the amplified 656bp (base pairs) partial sequence of COI mitochondrial gene were generated for a total of 36 specimens of Characidium cricarense (Tab. 1), encompassing specimens from the Jucuruçu basin (4 individuals), Itaúnas basin (6 individuals), São Mateus basin (11 individual), and Doce basin (15 individuals). All samples exhibit less than 2% of genetic divergence, which is the usually accepted upper limit of conspecific similarity (Pereira et al., 2013). Characidium cricarence received the Barcode Index Number (BIN) BOLD:ACI3743 through the BoldSystems automated algorithm (Ratnasingham, Hebert, 2013).
Geographical distribution. Characidium cricarense is known from the southern portion of Bahia State, northern portion of Espírito Santo State, and eastern portion of Minas Gerais State, where it is known from the rio Jucuruçu, rio Itanhém-Alcobaça, rio Mucuri, rio Itaúnas, and rio São Mateus drainages, extending to the rio Doce drainage in the central portion of the Espírito Santo State, Brazil (Fig. 3). The rivers from the northern portion of the distribution are within the coastal tablelands of the Barreiras sedimentary formation. These coastal tablelands correspond to a region of mild relief, with elevations usually not surpassing 150 m above sea level, formed by Cenozoic sediments, well-seen along the border between the States of Espírito Santo and Bahia (Sarmento-Soares, Martins-Pinheiro, 2013). On the right bank of the rio Doce, C. cricarense reaches the lower slopes of the Serra de Santa Teresa. According to a global biogeographic regionalization of freshwater systems, this geographic distribution belongs to the Northeastern Mata Atlântica ecoregion (Abell et al., 2008).
Ecological notes. Characidium cricarense inhabits lowland stretches of rivers, usually below 200 m above sea level, except in the Mucuri drainage, where it has been found in the upper rio Todos os Santos, above the city of Teófilo Otoni at about 500 m above sea level (G. M. Mattox, pers. comm.), and in the Jucuruçu drainage, in tributaries around the city of Palmópolis at about 300 and 600 m above sea level. Based on our field observations, individuals of this species usually occupy the edge of turbulent, moderately fast flowing rivers and streams (Fig. 4). Specimens aggregate along stretches less than 1.5 m deep, with transparent waters, occupying the waterfalls in rocky areas, and unconsolidated bottom over sandy, pebbles or gravel areas at the edge of the waterfall.
Etymology. The name refers to the rio Cricaré, where type locality of this fish is found, and is used as a Latinized geographic adjective.
Conservation status. Characidium cricarense occurs in coastal basins from Northeastern Mata Atlântica ecoregion (sensu Abell et al., 2008) along three Brazilian states: Bahia, Espírito Santo and Minas Gerais. The species inhabits turbulent waters in rapids, occupying the rocky areas as well as the unconsolidated bottom near the rapids. Even though a mining disaster occurred in Doce basin, where Characidium cricarense might occur, no specific threats to the species is detected in the whole area of distribution. Consequently, the conservation status of Characidium cricarense may be classified as Least Concern (LC), according to the International Union for Conservation of Nature (IUCN) categories and criteria (IUCN, 2017).

Discussion
Characidium cricarense is a member of the group of species without scales in the area of the isthmus. All species of Clade C1, and some of its immediate outgroups, have unscaled isthmus (Buckup, 1993a). Among species without scales in the isthmus, C. cricarense shares a large distance from the anus to the anal-fin origin (> 10% SL). This character is restricted to a small group of congeners, which includes Characidium alipioi, C. cricarense, C. kamakan, and C. hasemani. Characidium alipioi, C. cricarense and C. kamakan are distributed in Atlantic rainforest coastal streams between the Pardo drainage in Bahia and the São João drainage in Rio de Janeiro (Leitão, Buckup, 2014;Zanata, Camelier, 2015). These species are morphologically similar, and have similar pigmentation patterns, including a B-shaped set of caudal bands, and the clearer area in front of the main dark band of dorsal fin. Although a formal phylogenetic analysis was not carried out herein, the combination of characteristics that include the pigmentation pattern, unscaled isthmus but scaled chest, and long distance from the anus to the anal-fin origin with little imbricated, aligned scales, might diagnose a putative species clade that we call the C. alipioi species group. The number of species of the C. alipioi group may increase in the future, because recent studies (Serrano et al., 2019) have demonstrated the presence of cryptic species in the Paraíba do Sul drainage that are morphologically similar 9 e180121[9]  to C. alipioi. Characidium hasemani is distributed in northern Brazil and Guyana (Fricke et al., 2019), and has very distinct pigmentation pattern, including double dark bands in caudal fin and the middle band in the dorsal fin reaching its anteriormost margin. Further research is required to evaluate the hypothesis of close phylogenetic relationship between C. hasemani and the C. alipioi species group.
Among members of the C. alipioi group, C. cricarense may be distinguished from C. kamakan by the smaller depth of the body at all three body depth landmarks (Tab. 3). Adults of C. kamakan may be diagnosed by the conspicuous reticulated pattern formed by patches of pigment in the scales (Zanata, Camelier, 2015), but this reticulate pattern may be absent in younger specimens. C. cricarense may be distinguished from C. alipioi by the smaller number of scales between the lateral line and the predorsal series (4 vs. 5 series in C. alipioi). A few specimens of C. cricarense have five dorsolateral series of scales above the lateral line, but this count is by far infrequent among examined specimens. Additionally, C. cricarense has a greater distance, and 1 or 2 additional scales between the anus and anal-fin origin than C. alipioi.
Characidium cricarense occurs in six separate coastal drainages: rio Jucuruçu, rio Itanhém-Alcobaça, rio Mucuri, rio Itaúnas, rio São Mateus and lower part of rio Doce. The low morphologic and genetic divergence among these isolated populations is congruent with a geologically recent common origin. Considering that currently there is no direct freshwater connection between these drainages, and that during the Last Glacial Maximum (LGM) the sea level was at about 130 meters below the present baseline (Mahiques et al., 2010;Lambeck et al., 2014), it is hypothesized that there was some connectivity between the six basins, perhaps through deltaic areas that extended over the continental shelf, which was exposed between southern Bahia and northern Espírito Santo during the LGM. This hypothesis is corroborated by the wide expanse of the continental shelf in this area, forming the so called Abrolhos Bank, and the existence of northwardly directed paleochannels of a delta development from the rio Doce, which extended the river mouth to approximately 50 km to the north of the modern one (Dominguez et al., 1987;Rossetti et al., 2015). The fluvial erosion of the Barreiras Formation in southern Bahia carved valleys during epochs of lower sea level (Bittencourt et al., 1979). Subsequent submersion of the continental platform has produced the current isolation of these valleys, which represent headwaters of formerly larger river basins. Additionally, the recent transgressive phase of sea level fluctuation formed drowned lakes (e.g. Lagoa de Juparanã) and lowlands around the lower Doce basin. These lakes and lowlands may have facilitated freshwater connectivity to the São Mateus basin. We have not detected the presence of C. cricarense in the rio Peruípe basin. This basin is located between the rio Itanhém-Alcobaça and the rio Mucuri basins. The lack of records in the rio Peruípe basin may be due to poor sampling efforts, and further research is required to confirm the apparent absence of C. cricarense in that basin. The DNA barcode sequences revealed only minor divergence (less than 1%) among the various populations of C. cricarense. The largest haplotype divergence was recorded in the rio Doce basin, which has two main lineages of mitochondrial haplotypes (Fig. 2). The genetic divergence, however, is well below the accepted level of 2% divergence within conspecific lineages (Pereira et al., 2013), and we were not able to identify significant morphologic variation among specimens of rio Santa Maria do Rio Doce drainage and other tributaries of rio Doce.
The DNA barcode similarity between C. cricarense and C. alipioi corroborates our hypothesis that the species with a large distance between the anus and the anal-fin origin comprise a group of species (Fig. 2). The sister group of the C. alipioi species group is comprised by the C. grajahuense species group, which is represented in our analysis by specimens of C. pterostictum, C. timbuiense and C. vidali. In our maximum likelihood analysis (Fig. 2), the branches from the common ancestor of C. cricarense and C. alipioi correspond to 4.7% and 6.5%, respectively, of corrected nucleotide divergence. These values are substantially smaller than the 8.8% and 10.0% of anagenetic change along the lineages comprising gap between the C. alipioi and C. grajahuense species groups. These values are well above the 2% cutoff value of genetic barcode distance usually accepted for species-level divergence (Pereira et al., 2013), and corroborate our hypothesis that the C. grajahuense and the C. alipioi groups constitute distinct lineages among species with unscaled isthmus in southeastern Brazil.