Integrative taxonomy reveals a new species of pacu (Characiformes: Serrasalmidae: Myloplus) from the Brazilian Amazon

Pacus of the genus Myloplus represent a formidable taxonomic challenge, and particularly so for the case of M. asterias and M. rubripinnis, two widespread and common species that harbor considerable morphological diversity. Here we apply DNA barcoding and multiple species discovery methods to find candidate species in this complex group. We report on one well-supported lineage that is also morphologically and ecologically distinct. This lineage represents a new species that can be distinguished from congeners by the presence of dark chromatophores on lateral-line scales, which gives the appearance of a black lateral line. It can be further diagnosed by having 25–29 branched dorsal-fin rays (vs. 18–24), 89–114 perforated scales from the supracleithrum to the end of hypural plate (vs. 56–89), and 98–120 total lateral line scales (vs. 59–97). The new species is widely distributed in the Amazon basin, but seems to have a preference for blackand clearwater habitats. This ecological preference and black lateral line color pattern bears a striking similarity to the recently described silver dollar Metynnis melanogrammus.


INTRODUCTION
Myloplus Gill, 1896 is currently represented by 15 valid species widely distributed in South America, being found in the Amazon, Orinoco, La Plata and São Francisco drainages, as well as the coastal rivers of the Guiana Shield in Brazil, Guyana, French Guiana, Suriname, and Venezuela Andrade et al., 2016a,b;Andrade et al., , 2019Nico et al., 2018). The species richness of the genus has been underestimated for decades, as attested by the recent descriptions of four species-M. lucienae Andrade, Ota, Bastos, Jégu, 2016, M. taphorni Andrade, López-Fernández, Liverpool, 2019, M. tumukumak Andrade, Jégu, Gama, 2018, and M. zorroi Andrade, Jégu, Giarrizzo, 2016increasing the species diversity by 25% since the revalidation of the genus (Jégu et al., 2004). Jégu et al. (2004) diagnosed the genus Myloplus by the following characters: premaxillary molariform teeth arranged in two rows, spaced by an internal gap among each other; teeth of labial row not in contact at the symphysis; narrow olfactory fossa; long lateral premaxillary process extending beyond the last tooth base; and welldeveloped prepelvic spines. In their study, Jégu et al. (2004) also redescribed M. asterias (Müller, Troschel, 1844), the type species of the genus, and Myloplus rubripinnis (Müller, Troschel, 1844). Both M. asterias and M. rubripinnis were described from Guyana and are morphologically very similar, but diagnosable from one another by the presence of 25-28 dorsal-fin rays and circular-shaped anterior fontanel in M. asterias (Jégu et al., 2004: 136, fig . 13b); vs. 25 or fewer dorsal-fin rays, and an oval-shaped fontanel in M. rubripinnis (Jégu et al., 2004: 150, fig. 17b). Machado et al. (2018) in a taxonomically broad DNA barcoding study of the Serrasalmidae comprising more than 1,000 specimens representing 69 described species and 16 genera, and using four species discovery methods, recovered Myloplus as the serrasalmid genus with the greatest intraspecific lineage diversity. Six of the 12 species included in the study were estimated to comprise multiple lineages, with up to seven lineages solely in M. asterias, highlighting the difficulties in the current diagnosis of this species, and the potentially hidden diversity within Myloplus.
The accessibility of molecular techniques to taxonomists has increased the number of species descriptions that include molecular data (e.g., Benine et al., 2009;Melo et al., 2016;Andrade et al., 2017;Escobar et al., 2019). In the "candidate species" framework (Padial et al., 2010), these genetic data can provide initial hypotheses for the existence of new species, and then assist in their subsequent description, if corroborated (e.g., Costa et al., 2014;Escobar et al., 2015Escobar et al., , 2019. Here we carried out detailed morphological analysis of the voucher specimens from one of the seven M. asterias lineages reported by Machado et al. (2018), and reveal morphological characters that unambiguously diagnose it from remaining congeners. We describe this lineage as a new species of Myloplus, following the diagnosis of the genus by Jégu et al. (2004). . Counts were taken under a stereomicroscope, and measurements with a digital caliper to the nearest 0.1 mm, taken point-to-point on the left side whenever possible. All measurements are presented as percentages of standard length (SL), except for the subunits of head, expressed as percentages of head length (HL). The ventral keel is described as being composed of prepelvic spines (including those anterior to pelvic-fin origin), and postpelvic spines (those posterior to and including the spines near pelvic-fin origin), followed by the number of paired spines around anus.

Morphological analysis. Counts and measurements follow
Osteological features included in the Description section were analyzed in two skeletonized (skel) specimens (INPA 53281), and from two x-rayed specimens and INPA 46320). Dried skeletons were prepared with addition of dermestid beetle larvae and x-ray radiographs taken in Faxitron® specimen radiography system LX-60 (www.faxitron.com) 40 kV. Vertebral counts include those of the Weberian apparatus as four elements, and the fused PU1+U1 as a single centrum. Osteological terminology follows Mattox et al. (2014).
Counts are followed by their frequency in parentheses, with an asterisk indicating the count observed in the holotype. In the comparative material list, the number of specimens of each lot is given first, followed by those skeletonized or x-rayed, by tissue voucher number (CTGA), and finally by GenBank accession number in parentheses. Information on sexual dimorphism, including sex and breeding condition of the dissected material was determined according to Vazzoler (1996).
Mouth terminal to slightly upturned. Two rows of molariform premaxillary teeth, 5*(45) teeth in lingual row, and 2*(45) in labial row. First and second tooth in labial row interspaced by gap from teeth of lingual row. Premaxillary teeth 1-3 of lingual row with moderately sharp edges. Dentary with 5*(10) or 6(28) molariform teeth gradually decreasing posteriorly in size. Pair of conical symphyseal teeth posterior to main row of teeth, with cutting edge on anterior margin. Maxillary edentulous.
Ascending premaxillary process elongate, similar width from base to distal tip, with distal tip gently pointed, and moderately inclined relative to lateral premaxillary process. Premaxillary lacking interdigitations at symphysis and strongly attached on mesethmoid. Lateral premaxillary process rectangular, with dorsolateral process extending beyond last tooth, with concavity for maxillary insertion. Three replacement teeth trenches on premaxillary. Dentary rectangular, slightly arched with three rounded or oval replacement tooth-trenches, posterior more elongate. Four bony lamellae at symphysis. Anguloarticular well developed and articulated with quadrate by thin cartilage. Retroarticular contacting posteroventral portion of dentary. Coronomeckelian elongate.
Infraorbital series well developed. Antorbital robust, with base larger than tip. Six infraorbitals forming semi-circle, leaving a small naked area on cheeks, not covering lateral surface of vertical arm of preopercle. Infraorbital 6 with "Y" shaped laterossensory channel. Supraorbital somewhat oval and narrow with slightly convex anteroventral margin, not contacting infraorbital 6. Orbital region overall wide ( Fig. 2A).

Color in alcohol.
Ground coloration gray to pale-yellow, head and body darker dorsally. Lateral line scales with melanophores forming dark stripe along flanks  (Figs. 1, 3); melanophores vertically elongate, similar to stitches (Fig. 4F). Posterior half of scales well-pigmented, anterior half pale. Males with concentration of small dark spots, forming irregular blotches over the body, most dispersed above the lateral line. Light blotches scattered on flanks, predominantly concentrated above lateral line (Figs. 4E, F). Mandibular and gular regions, first four infraorbitals, and cheek gap light brown. Eye with broad dark transversal bar crossing pupil (not discernible in specimens retained for a long period in formalin) (Fig. 4). Dorsal, anal, pelvic and caudal fins overall somewhat hyaline, becoming darker towards fin margins. Pectoral fin light to dark brown. Caudal fin with dark and diffuse distal band.

Color in life.
Overall body coloration varying from silvery to plumbeus, with orange blotches scattered on flanks. Dorsal profile of head and inferior half of opercle with high concentration of melanophores. Fins uniformly hyaline or light yellow with scattered dark chromatophores along interradial membranes. Males during breeding period with anterior lobe of anal fin orange to intense red; with irregular-shaped orange to blood-red  blotch on posterior half of head to supracleithrum region, situated in opercular area, and near pectoral-fin origin. Males during breeding period also possessing dark, irregularly shaped and distributed blotches, more concentrated along dorsal half of flanks (Figs. 4E, F). Females during breeding period with orange and dark gray irregularly shaped blotches scattered on flanks (Fig. 4B).
Sexual dimorphism. Myloplus nigrolineatus presents the typical sexual dimorphism pattern of other Myloplus species, with males possessing an additional anal-fin lobe centered on 14th or 15th branched ray (Figs. 3A; 4C-F), the presence of stiff hooks laterally curved on distal-most lepidotrichia segment of anal-fin branched rays, and by the presence of ossified protuberances on anal-fin branched rays forming additional lobe (absent in females). Male specimens additionally present thin, long filaments extending branched dorsal-fin rays (absent in females; Figs. 4E, F), and display darker background coloration and more intense colors (Figs. 3, 4), with scattered dark and orange blotches more conspicuous than in females (see Color in life). Females have very rigid unbranched anal-fin rays, with first eight to ten rays extremely elongate forming a falcate margin (Figs. 3B; 4A, B). Representatives of Myloplus nigrolineatus in the breeding season show remarkable coloration with intense grayish-orange to blackish-red colors (Figs. 4E, F) Geographical distribution. Myloplus nigrolineatus is widespread in the Amazon basin, occurring in the Negro, Branco, Nhamundá, Purus, Madeira, Tapajós, Tocantins, and Uatumã rivers (Fig. 5). In whitewater rivers with high sediment loads such as the Madeira, Branco, and Purus, Myloplus nigrolineatus was only captured in tributaries with black-or clearwater (see Ecological notes). Ecological notes. The species inhabits slow-flowing habitats such as backwaters and lakes, feeding mainly on aquatic and terrestrial plants (Goulding, 1980). Of the collection localities, the Aripuanã, rio Água Boa do Univini (rio Branco), Nhamundá, Tapajós, Tocantins, and Uatumã rivers are clearwater rivers chemically characterized by a low concentration of suspended sediments, dissolved minerals and humic compounds (Crampton, 2011), while the rio Negro basin, rio Jutaí, Pauapixuna lake (rio Juruá), Tapauá lake (rio Purus), Serpa lake (rio Amazonas), and rio Maués-Açu are extremely acidic oligotrophic blackwaters (Sioli, 1984;Goulding et al., 1988). The Tracajá lake (holotype locality) is also a blackwater system that has its headwaters in the Purus-Madeira interfluvium and like other blackwater rivers, is of low sediment load and low pH. These blackwater environments are inhospitable to certain species of fish (Wallace, 1889;Roberts, 1972;Goulding et al., 1988;Araújo-Lima, Goulding, 1997;Saint-Paul et al., 2000;Crampton, 2011;Lima, Ribeiro, 2011).
Etymology. The epithet nigrolineatus, from the Latin nigro meaning black, and lineatus meaning lined, an allusion to the black, pigmented lateral line. An adjective.

Conservation status.
Myloplus nigrolineatus is a common and naturally abundant species, widely distributed within black-and clearwater Amazonian rivers in Brazil. As no specific threats were detected, M. nigrolineatus can be tentatively categorized as Least Concern (LC) according to the International Union for Conservation of Nature (IUCN) categories and criteria (IUCN Standards and Petitions Subcommittee, 2017).

Molecular results:
The 29 COI barcode sequences generated in this study were deposited in GenBank under the accession codes MN702885-MN702913. Combined with the 377 sequences from Machado et al. (2018) the alignment comprised 406 individuals from 32 morphologically assigned species (https://github.com/legalLab/ publications). After alignment and trimming, the matrix was 621 bp with a median sequence length of 621 bp of non-gap characters (min. = 477 bp; mean = 605 bp), and comprised 161 unique haplotypes. Myloplus nigrolineatus was represented by 68 sequences (20 unique haplotypes) from 18 unique sampling localities.

DISCUSSION
The primary diagnostic feature of Myloplus nigrolineatus, its dark pigmented lateral line, is also shared with another serrasalmid, Metynnis melanogrammus Ota, Rapp Py-Daniel & Jégu, 2016. However, unlike Metynnis melanogrammus, the new species described herein presents variation in the degree of intensity of melanin concentration along the lateral line (Fig. 4), with specimens from the Branco and Tapajós rivers presenting a lateral line with very few melanophores (Figs. 4A-D). Another feature shared between Myloplus nigrolineatus and Metynnis melanogrammus is that both species are widely distributed in black-and clearwater environments of the Amazon basin. Few fish species have such broad spatial distributions extending across multiple Amazonian river basins, but at the same time being restricted to a narrow range of ecological conditions (e.g., Metynnis melanogrammus). Such a pattern may indicate the extreme hydrogeographic dynamism of the whitewater rivers that isolated widespread populations (Ruokolainen et al., 2018), as well as potentially strong intrinsic dispersal ability. Based on data presented here and in Ota et al. (2016), Myloplus nigrolineatus and Metynnis melanogrammus appear to have a contemporary ecological preference for habitats with low pH and low concentration of suspended sediments and dissolved minerals, with sediment-loaded waters acting as ecological barrier to contemporary dispersal and survival of these black-and clearwater species. However, inferences into the past are unclear at this moment. Studies on fishes with similar distributions spanning such barriers have suggested periods of reduced precipitation and riverine sediment load in the late Pleistocene that may have permitted historical dispersal (Collins et al., 2018). The DNA barcode data analysed recovered Myloplus nigrolineatus as sister taxon to a lineage of Myloplus from the Tapajós River (Fig. 6), although with low posterior probability. In Machado et al. (2018) this clade was identified as Myloplus rubripinnis. However,  restricted M. rubripinnis to the Guiana Shield, including the drainages of the Essequibo and Maroni rivers in Guyana and Suriname, the Oyapock River separating French Guiana and Brazil, and the Araguari and Jari rivers in Brazil. Thus, the new species is not sympatric with M. rubripinnis, and its possible sister taxon in the Tapajós may represent a new species that needs further investigation. We refer to this taxon here as M. aff. rubripinnis because it has 23-25 branched dorsal-fin rays, 80-89 total lateral line scales, and an oval anterior fontanel (vs. a circular anterior fontanel, Fig. 2b). A circular anterior fontanel has only been reported in M. asterias, by Jégu et al. (2004: 136, fig. 13b), who proposed the character as a diagnostic feature of that species. However, the specimen illustrated by Jégu et al. (2004) was from the Uatumã River basin, where M. asterias and M. nigrolineatus are sympatric, and we were not yet able to confirm this character state in the type material of M. asterias.
We found that DNA barcodes were highly effective in highlighting and partitioning unrecognized diversity of lineages. This is especially the case within the genus Myloplus, and serrasalmids more generally, where high taxonomic diversity is combined with body shape and color pattern changes during ontogeny and as a result of sexual dimorphism, make them a difficult taxonomic challenge. DNA barcodes can triage large collections and provide starting points for further investigation with more focused methods. Machado et al. (2018) estimated that the epithets M. asterias and M. rubripinnis contained between 11 and 13 COI lineages, while the analysis presented here estimated between 12 and 15 (including M. nigrolineatus). Therefore, Myloplus remains a priority for taxonomic revision, and we strongly recommend the use of integrative taxonomic techniques in future studies.