A new species of Eigenmannia Jordan & Evermann (Gymnotiformes: Sternopygidae) from rio Tapajós, Brazil, with discussion on its species group and the myology within Eigenmanniinae

A new species of Eigenmannia is described from the rio Mutum, tributary of upper rio Juruena, rio Tapajós basin, Comodoro, Mato Grosso, Brazil. The new species is distinguished from all congeners by coloration pattern, position of the mouth, number of scales rows above lateral line, number of premaxillary and dentary teeth, number of precaudal vertebrae, orbital diameter, mouth width, relative depth of posterodorsal expansion on infraorbitals 1+2 and relative size of coronomeckelian bone. Comments on potentially useful characters in phylogenetic studies derived from musculature, discussion on Eigenmannia species-group and the first dichotomous key for Eigenmannia are provided.


Material examined
In addition to the comparative material examined and listed in [8,9,25,45], the following species were examined: Apteronotidae:   38-45 and 37-38, respectively). It additionally differs from E. vicentespealea by having 9-12 scale rows above lateral line (vs. seven or eight) and the coronomeckelian bone corresponding to 20-25% of length of Meckel's cartilage (Fig 3B vs. 45%). The new species can be distinguished from Archolaemus species by the eye completely covered by thin membrane (vs. a free orbital rim); from Distocyclus by the rounded snout in profile (vs. conical snout); from Japigny by the absence of alternating dark bands on flanks (vs. presence); and from Rhabdolichops by the region above the lateral line on the anterior portion of the body covered by scales (vs. absence of scales above the lateral line on the anterior portion of the body).

Description
Body shape and pigmentation in Figs 1, 4 and 5. Morphometric data for examined specimens in Table 1. Largest examined specimen 127.5 mm L EA . Body elongate, distinctly compressed. Greatest body depth at vertical through distal margin of pectoral fin. Dorsal profile of body convex to straight. Ventral profile slightly convex. Caudal filament elongated, about a half of the length to end of anal-fin in adult specimens.

Relevant osteological features of Eigenmannia sirius
Mesethmoid oriented at about 45˚angle from vomer, until reaching anterior margin of frontals; anterior portion with small lateral process on each side (Fig 6A). Paired frontals convex in lateral profile, about 75% of length of skull. Anterior portion of anterior fontanelle limited by contralateral posterior processes of mesethmoid and completely surrounded by frontals. Posterior fontanelle about 80-90% length of anterior fontanelle. Anterior one third of posterior fontanelle surrounded by frontals, posterolateral portion by parietals and posterior edge by supraoccipital. Parietals form lateral margins of posterior fontanelle and contact frontals anteriorly, supraoccipital posteriorly, epioccipitals and pterotics laterally ( Fig 6B).  Vomer arrow shaped anteriorly, with small anterior processes on each side; becoming larger posteriorly and diverging in two posterolateral process, contacting anterior margin of parasphenoid. Parasphenoid larger than posterior portion of vomer, with a long crest that serves as a site of origin of adductor arcus palatini. Anteriorly, parasphenoid reaches half portion of vomer, posteriorly surrounding anteroventral margin of prootics and ventral surface of basioccipital. At its posterolateral portion, parasphenoid contacts posteroventral margin of pterosphenoid through a tapered lateral process; and dorsally contacts orbitosphenoid entirely.
Orbitosphenoid connected dorsally to neurocranium and posteriorly separated from pterosphenoid by a segment of cartilage. Entire ventral surface of orbitosphenoid contacting dorsal margin of parasphenoid. Pterosphenoid contacts orbitosphenoid anteriorly, and associates with frontal dorsally. Pterosphenoid contacts parasphenoid only posteroventrally, with its anteroventral surface not contacting dorsal margin of parasphenoid, forming a lateral fenestra. Lateral ethmoids as small elements Y-shaped that are positioned in a vertical thought contact between mesethmoid and frontals; connected ventrally to parasphenoid by a connective tissue and to frontals by two strong and short ligaments.
Base of quadrate roughly trapezoidal in shape extending into pointed triangular shape anterodorsally, articulating with preopercle and sympletic at base through posteroventral process; its condyle extending anteroventrally from base and articulating with retroarticular and anguloarticular. Metapterygoid slightly rectangular, without posterodorsal process. Preopercle crescent-shaped, with five bony arches that corresponds to laterosensory canal tubes along lateral surface. Interopercle teardrop-shaped, with posterodorsal expansion and margins rounded. Opercle roughly triangular, dorsal margin convex, with a pointed anterodorsal process that anchors dilatator operculi and a medially expansion forming a crest for levator operculi insertion. Subopercle sickle-shaped, tapering posterodorsally, forming concave dorsal profile. Subopercle and interopercle becoming membranous distally. Hyomandibula at roughly 90˚to horizontal line through long axis of head; dorsal articulating head roughly one and a half time wider than ventral margin; laminar anterior shelf from widest hyomandibula point to anteroventral margin, which serves as site of adductor arcus palatini insertion (Figs 3D and 6). Neurocranial articulatory head of hyomandibula pointed; opercular articulatory head distinct from main body of hyomandibula and directed posteroventrally. Foramen for hyomandibular trunk of facial nerve positioned anterodorsally, near base of neurocranial articulatory head. Second foramen for a branch of this nerve reduced and located near dorsal portion of preopercle; third foramen, located anteroventrally in relation of second foramen that serves for exit of ramus mandibularis trigeminus. All foraminae positioned entirely within body of hyomandibula. Sympletic elongate and triangular, located in a medial crest of preopercle and posteroventral portion of quadrate ( Fig 3D).
Posttemporal bones fused with supracleithrum, and lie at posterolateral surface of skull at epioccipitals and pterotic sutures. Scapula broad, visible laterally, and contacts mesocoracoid anterodorsally and anteroventrally, and coracoid ventrally. Scapular foramen present. Supports for pectoral fin include propterygium and three proximal radials (proximals three and four co-ossified).

Relevant features of dorsolateral head muscles of Eigenmannia sirius
Dorsolateral head muscles illustrated in Fig 8. Three primary sections of adductor mandibulae, segmentum facialis well differentiated, except mesialmost fibers of pars malaris and pars rictalis which are partially continuous with lateroventral fibers of pars stegalis. Adductor mandibulae, pars malaris originates from preopercle and hyomandibula, and inserts primarily by a fibrous attachment to medial face of posterodorsal expansion on infraorbitals 1+2. At portion near insertion, mesialmost fibers converges into a poorly differentiated endomaxilar ligament which, in turn, inserts on posteromedial portion of maxilla. Adductor mandibulae, pars malaris located laterally to dorsal portion of pars rictalis and lateroventrally to pars stegalis. Adductor mandibulae, pars rictalis arises from quadrate, symplectic, preopercle and hyomandibula, with its posterolateral fibers restricted to anterior margin of preopercle fossa. Insertion of pars rictalis occurs mostly on the coronoid process, however, with some lateral fibers attached to buccopalatal membrane and posterior margin of anguloarticular. Adductor mandibulae, pars stegalis originates from anterior margin of hyomandibula, metapterygoid and quadrate, and converges anteriorly onto a meckelian tendon that attaches to coronomeckelian tendon, with some anterodorsal fibers associated to mandibular tendon. Adductor mandibulae, pars stegalis located laterally only to midposterior and posterior portion of adductor arcus palatini, resulting in a partial overlap. All sections of segmentum facialis are fibrous, without intermuscular bones.
Adductor mandibulae, segmentum mandibularis present as a single component, arising from mandibular tendon and attached to medial face of dentary and anguloarticular. Adductor mandibulae, segmentum mandibularis restricted to dorsal portion of the Meckel's cartilage, extending about 60% of dorsal margin of this cartilage. Nerve ramus mandibularis trigeminus lies laterally to pars stegalis and medially to pars malaris and rictalis, with an unidentified branch medial to a set of fibers of midposterior portion of pars rictalis.
Levator arcus palatini approximately triangular in shape, arising from sphenotic bone and inserting on anterodorsal margin of hyomandibula. Levator arcus palatini origin narrower than its extension at site of insertion, about half of its insertion. Levator arcus palatini in a single mass of fibers, with four discernible sets of fibers near insertion, located distinctly to pars malaris: anterolateral and anteromedial set of fibers medial to pars malaris; posterolateral and posteromedial set of fibers lie lateral to pars malaris. At dorsal portion, posterolateral levator arcus palatini fibers located parallel to anterior portion of dilatator operculi. Dilatator operculi nearly conical in shape, located posteriorly to levator arcus palatini. This muscle arises from sphenotic, pterotic and hyomandibula, and its insertion occurs invariably on dorsal process of opercle.
Levator operculi muscle, superficial and located posteriorly to dilatator operculi and differentiated in two sections. Levator operculi anterior arises from pterotic and hyomandibula; levator operculi posterior originates from supracleithrum canal with both sections inserting on opercle. R-Avn lies medial to entire levator operculi. Adductor arcus palatini originates from parasphenoid and prootic. At anterior portion, adductor arcus palatini inserts on lateral face of endopterygoid and metapterygoid; towards posterior portion, this muscle inserts on mesial face of hyomandibula.
The adductor hyomandibulae muscle relatively well differentiated from posterior portion of adductor arcus palatini, arising from prootic and pterotic, inserting on posteromedial face of hyomandibula. Adductor operculi originates from pterotic, prootic and exoccipital, and its insertion occurs on medial face of opercle (Fig 8).

Color in alcohol
Ground coloration of body beige (Figs 1 and 4). Body densely covered by dark brown chromatophores gradually more spaced ventrally. Chromatophores more concentrated on perforated scales forming a narrow lateral line stripe. Superior midlateral stripe thick, one or one and half scales deep, tapering from vertical between base of 23 rd to 31 st anal-fin ray to posterior two-thirds of body. Second layer of pigmentation formed by multiple, small bars of dark chromatophores situated between musculature associated with anal-fin pterygiophores. Dark individual bars in combination form two stripe-like patterns. Inferior midlateral stripe approximately as wide as orbital diameter. Anal-fin base stripe approximately as wide as orbital diameter. Superior midlateral stripe and inferior midlateral stripe separated by weakly pigmented area, absent in juveniles (Fig 4). Head densely covered by dark brown chromatophores gradually more spaced ventrally. Pectoral and anal fins hyaline with scattered dark brown chromatophores overlying fin rays.

Color in life
Based on photographs taken in field (Fig 5). Overall body translucent, darker in mid-dorsal region of trunk; dorsal region of body yellowish brown in large specimen (95.9 mm L EA ) ( Fig  5A) or greyish yellow in small specimen (79.0 mm L EA ) (Fig 5B). Dorsal region of head dark brown, ventral region lighter, with diffuse chromatophores. Opercle translucent in small specimens, exposing red color of gills; opercle light brown with a concentration of black chromatophores in large specimens. Silvery abdominal region due presence of iridophores covering external portion of peritoneal membrane, more evident in small than large specimens. Midlateral stripes equally located and similar to those found in fixed specimens. Pectoral and anal fins hyaline with scattered dark brown chromatophores overlying fin rays.

Etymology
The specific epithet sirius is an allusion to the alpha star of the Canis Major constellation that represents the state of Mato Grosso in the Brazilian national flag, a reference of the state of occurrence of the new taxon. A noun in apposition.

Distribution and habitat
Eigenmannia sirius is currently known only from rio Mutum, a tributary of the upper rio Juruena, rio Tapajós basin, Comodoro, Mato Grosso, Brazil (Fig 9). The type-locality is 502 m above sea level at the Chapada dos Parecis plateau. It is a clearwater river up to c. 3-6 m wide and 0.5-2.5 m deep, preserved riparian vegetation, swift current, and sand, pebbles and dead leaves on the bottom (Fig 10). Several types of microenvironment were sampled exhaustively, but E. sirius was captured only between root and subaquatic vegetation. Other species sampled syntopically were Aequidens cf.

Conservation status
Eigenmannia sirius is currently known only from its type-locality, and it may have a very restricted distribution. However, considering that no imminent threats to the species were detected in the area of its occurrence, E. sirius would be classified as Least Concern (LC) according to the International Union for Conservation of Nature (IUCN) categories and criteria [47].

Myological analysis across Sternopygidae
Anatomical studies in Gymnotiformes follow the trend of efforts implemented in other Teleostei groups and focused on detailed descriptions of distinct osteological complexes (e.g., [15,32,48]), neuroanatomical structures or anatomical components associated with electrogenesis and electroreception (e.g., [37,38,[49][50][51]). Recently, efforts were employed aiming to describe structures recently discovered in Gymnotiformes, like the caudal skeleton in Electrophorus Gill [52] and pseudotympanum [53]. Thus, studies of comparative anatomy in Gymnotiformes trends to explore traditional sources of information, resulting in a hiatus on several anatomical complexes that can be crucial in understanding the evolution of the group, such as myological tissues, which have rarely been explored [34,[54][55][56]. As a result, characters derived from myology represents less than 0.2% of the entire universe explored of the morphological traits in the cladistic studies in Gymnotiformes (e.g., [18][19][20][21]42,57,58]). After a myological analysis, additional data derived from this poorly explored source of data led us to infer putative useful variations in a phylogenetic scenario.
The Sternopygidae genera presents the insertion of the adductor mandibulae, pars malaris occurring primary in the mesial face of the posterodorsal expansion of the infraorbital 1+2 (Fig 11). Towards its insertion, the mesialmost fibers of the pars malaris converge onto an endomaxilar ligament which, in turn, inserts in the mesial face of the posterodorsal portion of the maxillary bone. This configuration is an exclusive condition shared in all Sternopygidae genera, which differs from remaining Gymnotiformes families, and has been proposed as a synapomorphy for Sternopygidae, however, with superficial homology inferences on the sections of the adductor mandibulae (see Datovo & Vari,[28,29] for discussions on the homology of adductor mandibulae sections). For instance, previous studies [20,22] proposed the aforementioned condition as a homoplastic feature found in Rhamphichthys Müller & Troschel, however, in this genus, the pars malaris converge onto a wide endomaxilar ligament which, in turn, inserts onto the mesial face of the antorbital and maxillary bones (Fig 12) (per. obs.; https://doi.org/10.1371/journal.pone.0220287.g009 [34]), without association with any infraorbital bone, thus non-homologous with those condition found in Sternopygidae genera. Additionality, the ventral fibers of Rhamphichthys converge in a ventral ligament, named herein as "accessory endomaxilar ligament", which inserts only to the mesial face of the antorbital. The monophyly of Sternopygidae was questioned only by studies grounded in molecular data [18,22], with its monophyly well-corroborated in several studies from distinct source of data [15,16,[19][20][21][40][41][42][43]49], including the aforementioned myological data source.
All members of Eigenmanniinae exhibit the adductor mandibulae, pars stegalis originating from the anterior portion of hyomandibula, metapterygoid and quadrate, without attachment to the sphenotic bone. This condition differs that observed in most gymnotiform species, in which the pars stegalis originates from the sphenotic and suspensorial bones. In all Eigenmanniinae genera, except for Distocyclus, only the mid-posterior and posterior portion of the adductor arcus palatini is located medially to the pars stegalis (Fig 13A), resulting in a partial overlapping, which is distinct from the condition found in most Gymnotiformes, where the pars stegalis completely overlaps the adductor arcus palatini (Figs 13B and 14). In Distocyclus, the pars stegalis is located laterally only in the region near adductor arcus palatini insertion, without overlapping with the mid-posterior or posterior portion of the latter. Furthermore, the R-avn is located medially to the entire levator operculi (Figs 8 and 13A) in Eigenmanniinae, that contrasts with the condition found in all other Gymnotiformes analyzed herein where the R-Avn is located laterally to the levator operculi anterior and medially to the levator operculi posterior (Figs 13B and 14). Thus, grounded in the phylogenetic relationships hypothesized by [21], the following conditions are shared by Eigenmanniinae members: (1) absence of an association of pars stegalis with the sphenotic bone, (2) R-avn entirely medial to the levator  operculi, and (3) partial overlapping between pars stegalis and adductor arcus palatini, with the loss of the overlapping between the last two muscles in D. conirostris (Eigenmann & Allen 1942).
Within Eigenmanniinae, only Archolaemus, Distocyclus, Eigenmannia and Rhabdolichops exhibit the anterior margin of the levator arcus palatini insertion nearly straight in relation to the horizontal arm of the preopercle, resulting in an angle near 90˚to the longitudinal axis of the head (Figs 8 and 13A). In contrast, the anterolateral margin of levator arcus palatini is arranged obliquely in relation to the longitudinal axis of the head, forming an angle near 45ẘ ith the longitudinal axis of the head in most gymnotiforms species, including Sternopygus and Japigny (Figs 13B and 14). Therefore, the condition found in Archolaemus, Distocyclus, Eigenmannia and Rhabdolichops could indicate an useful variation for the elucidation of a close relationship among these four genera within Eigenmanniinae, and due its absence in Japigny, could provide an additional evidence for the phylogenetically position of Japigny as a sister-group of all Eigenmaniinae genera [59], and not related to Eigenmannia species (see Remarks).

Taxonomic considerations
Although there is no agreement about the relationships within Eigenmanniinae, all genera have been demonstrated as monophyletic assemblages, except for Eigenmannia. In an effort to define Eigenmannia, Mago-Leccia [16] distinguished the genus among the remaining Sternopygidae by the presence of intermuscular bones at precaudal vertebrae 7 to 9 having a high branched structure, and presence of five branchiostegal rays, the two anteriormost narrow and the three remaining triangular in shape. However, despite their occurrence in E. sirius, the Mago-Leccia's definition for Eigenmannia has not been recoveredin a phylogenetic scenario based on morphologial (e.g., [19,20]), molecular ([22]; in part) or model-based total evidence [21] data. Due to the doubtful status of Eigenmannia as a monophyletic genus, the inclusion of the E. sirius in Eigenmannia is provisionally justified by the presence in the former of the synapomorphies of Eigenmanniinae [15,[19][20][21], and by the absence of those synapomorphies of the remaining Eigenmanniinae genera as discussed by previous studies [9,10,23,25,45]. In addition to our conservative position regarding the proposal of a new genus, we detail the differences between the species described herein and the current definition of all eigenmanniin genera (for external features see Diagnosis). The new species does not fit in Japigny by the absence of single row teeth at the base of the upper oral valve (vs. presence); the presence of the contact of the lateral process of the second vertebrae with the parapophysis of the fourth vertebrae (vs. absence); and the retroarticular is included in the socket of the lower jaw with the quadrate (vs. retroarticular not included in the socket of the lower jaw with the quadrate) [59]. Eigenmannia sirius does not fit in the Archolaemus definition by the teeth of the first row immobile and firmly attached on the ventral surface of the premaxilla margins (vs. anterobasal margins of the teeth of the first tooth row attached to the dentigerous surface of the premaxilla) [59]. Eigenmannia sirius can be distinguished from the Distocyclus by the presence of endopterygoid teeth (vs. absence); dentary with 15-33 teeth arranged in two or three complete rows (vs. a single tooth row limited to the anterior portion of dentary) [7]. Further, the new species is distinguished from Rhabdolichops by the premaxilla approximately rectangular (vs. premaxilla trapezoidal and elongate); one prootic foramen (vs. two); the extrascapular independent from the neurocranium (vs. extrascapular fused with the neurocranium); and gill rakers short and not ossified (vs. gill rakers long and bony) [20,31].
The historical status of Eigenmannia as a non-monophyletic genus resulted in a classification based on species groups. The first author who advocates the classification grounded in species group was Alves-Gomes [22], which proposed the clade "Eigenmannia virescens species-group" composed by five undescribed species (Eigenmannia cf. virescens 1, 2, 3, 4 and 5; Alves-Gomes [22]). Few years later, Albert [20] did also not recover the Eigenmannia monophyly and proposed its classification in two species group: "Eigenmannia microstoma species-group" and "Eigenmannia virescens species-group". In the same study, Albert [20] listed "Eigenmannia gr. macrops", composed by E. macrops and an undescribed species (designated as "Eigenmannia sp. B"), however, without discussion about its relationships or classification.
According to Albert [20], the "Eigenmannia microstoma species-group" (proposed to allocate E. humboldtii, E. limbata, E. microstoma, E. nigra and an undescribed species from rio Paraiba) can be distinguished by the body depth in mature specimens with more than 11% T L and total length over 350 mm in sexually mature individuals. The Eigenmannia virescens species-group sensu [20] (composed by E. virescens, E. trilineata and an undescribed species from Río Salí, Argentina) was proposed to distinguish species that share the presence of two or three longitudinal stripes on flanks, lateral valvula of cerebellum large, and anterior intermuscular bones highly branched. Despite the valuable contribution to the classification in Gymnotiformes in the early 2000's, the classification of Eigenmannia presented in [20] failed to establish reliable data. Exemplifying, measurement taken as a proportion of total length in Gymnotiformes is not truthful given that fishes often suffer damages in caudal filament by predations, and posteriorly regeneration [60]. Additionally, even when considering only undamaged specimens, the body depth taken as proportion of L EA are useful only in interspecific levels within Eigenmannia, being irrelevant to supra-specific taxa. Further, E. microstoma shows four dark longitudinal stripes in flanks [8,61] and anterior intermuscular bones highly branched (MCP 45216). The presence of stripes on flanks in E. microstoma is mentioned by Albert [20], however, the author did not explain the positioning of E. microstoma in its group, since the presence of stripes on flanks and the anterior intermuscular bones highly branched are synapomorphies for the Eigenmannia virescens species-group. In addition, E. virescens is characterized by a uniform color pattern, without midlateral stripes (see Peixoto et al. [8] for taxonomic discussion of E. virescens). Peixoto et al. [8] did not follow the classification proposed by Albert [20] because of these difficulties in recognizing the characters or the taxonomic compositions and proposed the so called "Eigenmannia trilineata species-group", in which all species share the superior midlateral stripe.
Moreover, Waltz & Albert [26] classified Eigenmannia into two groups, besides the E. trilineata species-group. The so called "Eigenmannia humboldtii species-group" sensu [26] was proposed as grouping species with: (1) larger body size (>45 cm T L ); (2) deep body at maturity (body depth >11% T L ); and a (3) darker body coloration in some specimens. Subsequently, Waltz & Albert [10] redefined the Eigenmannia humboldtii species-group by: (1) large adult body sizes-(>300 mm total length), (2) deep body shape-body depth greater than 18% of the length to the end of anal fin (L EA ), (3) broad and opaque body in life, and (4) absence of longitudinal stripes. In the latter contribution, the authors indicated E. humboldtii, E. limbata and E. nigra as members of the Eigenmannia humboldtii species-group. Herein, we corroborate the taxonomic composition of the Eigenmannia humboldtii species-group. However, through the discrepancies in the definition of this group, and in addition to the problematic reference to the measurements taken as proportion of T L in Gymnotiformes, we redefine the Eigenmannia humboldtii species-group to accomplish species that share: (1) the presence of anal fin margin distinctly darkened and (2) absence of longitudinal stripes. The second group proposed by Waltz & Albert [26] was the"Eigenmannia macrops speciesgroup", including only E. macrops, and according to authors, distinguished by: (1) body fairly laterally compressed, (2) translucent white/yellow coloration in life, (3) longitudinal stripes absent, (4) eye large (greater than ore qual to snout length), (5) long caudal filament (half of body length without head). Posteriorly, Waltz & Albert [10] exclude the character "body fairly laterally compressed" of its definition. Previously, the "Eigenmannia macrops species-group" had already been proposed by [20], and, not only it is composed by only one species, therefore lacking evidence for the proposition of a group of species, but the characters described are generalized features of the remaining Eigenmannia species, except for "eye large (greater than or equal to snout length)" and "(5) long caudal filament (half of body length without head)" which are putative diagnostic for E. macrops. Therefore, there is no evidence for the recognition of a species group designated as E. macrops until the detection of additional species putatively related to this species.
Consequently, the best resolution is a classification of Eigenmannia into two species groups. The striped species compose the E. trilineata species-group, in which all species share the superior midlateral stripe, and the species with anal-fin margin darkened and without longitudinal stripes on flanks constitute the Eigenmannia humboldtii species-group. In addition, we suggest the remaining species on hold for a formal taxonomic review or for having their phylogenetic relationships clarified (e.g. "Eigenmannia" goajira and E. meeki; see Table 2). Aiming the summarization of the taxonomic considerations synthetized herein, the first dichotomous key is provided for all Eigenmannia species ("Eigenmannia" goajira not included).