A review of Euryoryzomys legatus (Rodentia, Sigmodontinae): morphological redescription, cytogenetics, and molecular phylogeny

Studied Sample

We studied the specimens of E. legatus based on three different approaches as follow: (i) 145 individuals were used for morphological descriptions and morphometric analyses (Fig. 1A, Table S1); (ii) two specimens out of the 145 were analyzed cytogenetically; and (iii) phylogenetic analyses were carried out using six out of those 145 individuals, in addition to the other Euryoryzomys species (Fig. 1B, Table S1). Tissues and chromosomal preparations were shipped under transportation permit CITES #19BR032169/DF.

We collected two specimens of E. legatus (CML13250 [field number JPJ2681] and CML13251 [field number JPJ2682]) with Sherman traps in Arroyo Yuto, 13 km SW Yuto, Jujuy, Argentina (Fig. 1A locality #11; Table S1), and deposited them in the Fundación Miguel Lillo collection, Tucumán, Argentina (CML). Collecting and shipping permits for these two specimens were issued by the Dirección de Recursos Genéticos y Protección de la Biodiversidad del Ministerio de Ambiente of Jujuy (#1102-122-2020/SByDS and #178/2020-SByDS). The capture and handling of the specimens followed the guidelines of the American Society of Mammalogists (Sikes et al., 2016) and with permission of the Instituto Butantan Ethics Committee (CEUAIB #1260/14).

We studied 134 additional specimens of E. legatus from Jujuy and Salta provinces, Argentina, and nine specimens from Chuquisaca and Tarija departments, Bolivia (Table S1). These specimens were housed in the following institutional collections: Colección Elio Massoia, Ciudad Autónoma de Buenos Aires, Argentina (CEM); Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Ciudad Autónoma de Buenos Aires, Argentina (MACN-Ma); Fundación Miguel Lillo, Tucumán, Argentina (CML); Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja, La Rioja, Argentina (CRILAR-Ma); Museum of Southwestern Biology, University of New Mexico, New Mexico, USA (MSB); Natural History Museum, London, United Kingdom (BMNH). Analyzed material includes the holotype of E. legatus (BMNH 25.2.1.24).

Furthermore, we examined 1,215 representative specimens of other five species of Euryoryzomys, including 772 specimens of E. russatus, 24 specimens of E. lamia, 285 specimens of E. macconnelli, 9 specimens of E. emmonsae, and 125 specimens of E. nitidus (Table S1). These specimens were studied in the following collections: American Museum of Natural History, New York, USA (AMNH); Colección Elio Massoia, Ciudad Autónoma de Buenos Aires, Argentina (CEM); Colección Julio Contreras, Corrientes, Argentina (CJC); Fundación Miguel Lillo, Tucumán, Argentina (CML), Louisiana State University Museum of Zoology, Baton Rouge, Louisiana, USA (LSUMZ); Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Ciudad Autónoma de Buenos Aires, Argentina (MACN-Ma); Museu de Biologia Mello Leitão, Santa Teresa, Brazil (MBML), Museu de História Natural Capão da Imbuia, Curitiba, Brazil (MHNCI); Museu Nacional do Rio de Janeiro, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (MN); Museu Paraense Emílio Goeldi, Belém, Brazil (MPEG); Museum of Southwestern Biology, University of New Mexico, New Mexico, USA (MSB); Museum of Vertebrate Zoology, University of California, Berkeley, CA, USA (MVZ); Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil (MZUSP); The Museum, Texas Tech University, Lubbock, TX, USA (TTU); Laboratório de Mastozoologia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (UFMG); Laboratório de Mastozoologia da Universidade Federal da Paraíba, João Pessoa, Brazil (UFPB); Laboratório de Citogenética de Mamíferos da Universidade Federal do Paraná, Curitiba, Brazil (UFPR); Laboratório de Mastozoologia da Universidade Federal de Santa Catarina, Florianópolis, Brazil (UFSC).

Cytogenetic analyses

We obtained chromosomal preparations from one female (CML13250) and one male (CML13251) of E. legatus. Preparations were obtained in vivo from bone marrow and spleen, following the protocols of Ford & Hamerton (1956) and Yonenaga (1972), with modifications. Slides were Giemsa stained, and CBG and GTG-banding patterns were obtained according to Sumner (1972) and Seabright (1971), respectively, after modifications. Fluorescence in situ hybridization (FISH) with telomeric probes labeled with FITC was carried out following the recommended protocol (Telomere PNA FISH Kit/FITC, Code No. K5326, DAKO). Slides were counterstained with 4′,6-Diamidino-2′-phenylindole dihydrochloride (DAPI) in antifade mounting medium (Vectashield with DAPI, Vector).

We analyzed 72 metaphases from the male and 56 from the female specimen to establish both diploid (2n) and fundamental numbers (FN = number of arms of the autosomes). Metaphases were digitally captured in an Axioskop 40 epifluorescence microscope (Carl Zeiss) equipped with an Axiocam camera and AxionVision software. Adobe Photoshop CS5.1 was used for assembling the karyotypes.

Phylogenetic and genetic distance analyses

Molecular data consisted of partial sequences from two mitochondrial genes, 801 bp of the cytochrome-b (cyt-b) and 667 bp of the cytochrome oxidase I (coxI; Table S1). In addition to newly generated sequences for 40 specimens, we also acquired sequences from seven individuals from GenBank (Table S1). DNA was extracted from liver or muscle using Chelex 5% (Walsh, Metzger & Higuchi, 1991). Cytochrome-b was amplified with primers MVZ05 and MVZ16 (Smith & Patton, 1993), and cytochrome oxidase I was amplified with primers LCO1490 and HCO2198 (Folmer et al., 1994). Polymerase chain reactions of 15 µL or 25 µL consisted of 30 ng of DNA, 10 mM of each primer, 0.2 mM of dNTP, reaction buffer (50 mM KCl, 2.5 mM MgCl2 and 10 mM Tris-HCl; ph 8.8) and 0.2 units of Platinum Taq DNA polymerase (Invitrogen). Amplification cycles were performed with an initial denaturation at 94 °C for 5 min; 30 cycles for cyt-b and 35 for coxI composed of: denaturation at 94 °C for 30 s, 45 s of annealing at 48 °C, and extension at 72 °C for 45 s; and a final cycle of extension at 72 °C for 5 min. Sequencing was performed at Laboratório de Bacteriologia II, Instituto Butantan, São Paulo, Brazil. We were unable to obtain sequences for both markers for some individuals (Table S1). The sequences were edited using Geneious R7 (Kearse et al., 2012) and deposited in GenBank (Table S1).

We used sequences from Wiedomys cerradensis, Handleyomys, Nephelomys devius, Hylaeamys megacephalus, Oecomys auyantepui, and O. roberti as outgroups in phylogenetic analyses (Table S2). Alignments using single genes and the two concatenate genes for all specimens were performed with MUSCLE (Edgar, 2004) using Geneious R7 (Kearse et al., 2012). The best evolutionary model for each analysis (cyt-b, coxI, and combined markers - cyt-b and coxI) was obtained using PartitionFinder (Lanfear et al., 2012). Bayesian inference (BI) was performed on MrBayes 3.2 (Ronquist & Huelsenbeck, 2003) and Maximum-Likelihood (ML) with bootstrap of 1000 replicates on RAxML-NG (Kozlov et al., 2019). The phylogenetic trees were edited with FigTree v1.4 (Rambaut & Drummond, 2009) and Adobe Illustrator CS4.

We employed MEGA7 (Kumar, Stecher & Tamura, 2015) to estimate cyt-b genetic distances (Kimura-2-parameter; Kimura, 1980) among all clades recovered in the phylogenetic analyses and among pairwise individuals of Euryoryzomys.

Morphometric analyses

The following standard external measurements were recorded from field catalogs and tags: TL: total length; T: tail length; HF: hind foot length (including claw); E: ear length; and W: body mass. The following skull measurements were recorded with a vernier caliper to the nearest of 0.01 mm, following Hershkovitz (1962), Myers (1989) and Myers, Patton & Smith (1990): CIL: condyloincisive length; PB: palatal bridge; RL: rostral length; OL: orbital length; RW2: mid rostral width; ZP: zygomatic plate depth; IOC: interorbital constriction; ZB: zygomatic breadth; BB: braincase breadth; OCW: occipital condyle width; DL: diastema length; MTRL: maxillary toothrow length; IFL: incisive foramina length; AW1: alveolar width (across external side of both M1); BLLT: bullar length less tube; ML: mandible length. Five age classes were defined according to tooth wear following the descriptions in Jayat et al. (2020) and Fig. S1. Descriptive morphometric and univariate comparisons (Tukey’s pairwise comparison; Table 1) for samples of the species of Euryoryzomys were carried out with the software PAST (Hammer, Harper & Ryan, 2001). Specimens of age classes 2 and 3 (the largest pool of available specimens for the whole specimen sample) were pooled in tests of significant size differences (for both, P ≤ 0.05 and P ≤ 0.01).

With the aim of reducing the dimensionality of morphometric data in comparing E. legatus with other 5 species of Euryoryzomys, we conducted two Principal Components Analyses (PCA) using only skull measurements. The first PCA explored skull size variation (“size PCA” hereafter; Table 2) and we used only specimens of the age classes 2 and 3 (again taking advantage of the largest pool of available specimens). The second PCA was developed to explore skull shape differences (“size free PCA”; Table 3) and all age classes were used (after removing the size effect). This latter analysis was conducted using Mosimann shape variables (Mosimann & James, 1979) obtained as described in Meachen-Samuels and Van Valkenburgh (2009). Principal components (PC) and their statistical significance were obtained as in Jayat et al. (2020). Finally, we conducted a Discriminant Function Analysis using Mosimann shape variables and specimens of all age classes (“size free DFA”; Table 4). All multivariate statistical analyses were conducted in PAST (Hammer, Harper & Ryan, 2001) and using only the specimens without missing data.

Morphological description

We re-described the skin color pattern and the skull of E. legatus and compared them with parapatric populations assigned to E. nitidus (its putative sister species) and other species of the genus. Terminology used to describe skull anatomical features followed Hill (1935), Carleton (1980), Voss (1988), Carleton & Musser (1989), Steppan (1995), and Weksler (2006). Descriptions of the molar cusp pattern followed Reig (1977).

Cytogenetic analyses

Both karyotyped individuals presented 2n=80 and FN=86. The karyotype is composed of 35 acrocentric pairs, with pair 1 the largest of the complement and others pairs (2 to 35) decreasing gradually in size, and four small metacentric pairs (36 to 39). The sex chromosomes are readily distinguishable from the autosomes because of their different morphologies: the X is a large submetacentric and the Y is a medium-sized submetacentric (Fig. 2A).

CBG-bands revealed subtle pericentromeric constitutive heterochromatin in all autosomes. The X-chromosome is heterochromatic in the short arm, and the Y is entirely heterochromatic (Fig. 2B). GBG-bands allowed the identification of the homologues (Fig. 3). FISH showed signals exclusively at the telomeric regions of all chromosomes (Fig. 4A) and DAPI evinces the Y strongly and entirely stained (Fig. 4B).

Phylogenetic analyses and genetic distances

The phylogenetic analyses of cyt-b provided high nodal support for the monophyly of Euryoryzomys (Clade A, BI: 0.93, ML: 95%) (Fig. 5). E. macconnelli (Clade B, BI: 0.98, ML: 78%) was recovered as the sister group to the clade containing all other species (Clade C, BI: 0.71, ML: 80%). E. emmonsae (Clade G, BI: 0.99, ML: 93%) was recovered as the sister group to a major subclade (Clade H, BI: 0.73, ML: 88%) composed of E. russatus (Clade K, BI: 1, ML: 100%), and the clade containing remaining species (Clade L, BI: 1, ML: 98%). This Clade L encompassed two subclades: Clade M (BI: 0.92, ML: 80%), with E. lamia (Clade O, BI: 1, ML: 100%) + Euryoryzomys sp. (Clade P, BI: 1, ML: 100%), and its sister clade (Clade N, BI: 0.97, ML: 89%), composed of E. nitidus and E. legatus. E. nitidus was recovered paraphyletic, splitting into E. nitidus A (Clade Q, BI: 1, ML: 100%) which is the sister group (Clade R, BI: 0.99, ML: 97%) of E. nitidus B (Clade S, BI: 1, ML: 99%) and its sister group (Clade T, BI: 1, ML: 100%), composed of E. nitidus MSB70697 and E. legatus (Clade U, BI: 1, ML: 99%).

For E. macconnelli (Clade B), the individual CMNH64561 from 1.5 km W Rudi, Brokopondo–Suriname (locality #93) was recovered as the sister to a clade (Clade D, BI: 1, ML: 98%) composed of E. macconnelli A (Clade E, BI: 1, ML: 96%) + E. macconnelli B (Clade F, BI: 0.91, ML: 89%). E. macconnelli A corresponds to individuals from Nuevo San Juan and Río Gálvez, Loreto Province–Peru (RSV2025, RSV2030 and AMNH272678–localities #91 and #92)–W Tangoshiari, Cusco Province–Peru (LLW447 and LLW462–locality #90); and E. macconnelli B is composed of specimens from Juruena and Aripuanã, Mato Grosso State (PEU 960004 and M000147–localities #76 and #89, respectively), and Marabá, Pará State–Brazil (CS32–locality #87).

E. emmonsae (Clade G) was also split into two major clades, E. emmonsae A from Cláudia, Mato Grosso State - Brazil (M97018 and M97120–locality #84) (Clade I, BI:1, ML: 100%); and E. emmonsae B from Altamira and Marabá, Pará State–Brazil (MZUSP27150, USNM549552 and CS37–localities #86 and #87) and Vila Rica, Mato Grosso State - Brazil (APC312, APC318–locality #85) (Clade J, BI:1, ML: 98%).

E. nitidus split into E. nitidus A (Clade Q), composed of individuals from Apiacás and Juruena, Mato Grosso State - Brazil (M968409, M97008 and APC153–localities #75 and #76); and E. nitidus B (Clade S), composed of individuals from Igarapé Porangaba, Acre State - Brazil (MVZ190456 and INPA3193 –locality #73), Reserva Cusco Amazónico, Cusco Province - Peru (CR97 and MVZ166027–locality #83), 45 km N Yacuma, Beni Department (MSB56057–locality #52) and Palmira, Pando Department - Bolivia (MSB57117–locality #56); and the individual E. nitidus MSB70697 from 1km NE Estancia Cuevas, Santa Cruz Department - Bolivia (locality #70) was recovered as sister to E. legatus.

The phylogenetic analyses of the concatenated cyt-b and coxI markers (Fig. 6) showed Euryoryzomys as a monophyletic group (Clade A) with high support (BI: 0.99, ML: 99%), although E. lamia was not included in the analyses. The topology was similar to the cyt-b analyses, with E. macconnelli (Clade B) as the sister-group to all the other species (Clade C, BI: 1, ML: 95%), which encompasses E. emmonsae (Clade D, BI: 1, ML: 98%) and the sister group (Clade E, BI: 0.98, ML: 93%) composed of E. russatus (Clade H, BI: 1; ML: 100%), and its sister group (Clade I, BI: 1, ML: 100%) encompassing Euryoryzomys sp., and Clade J (BI: 0.99, ML: 91%) with E. nitidus (Clade K, BI: 1, ML: 99%) + E. legatus (Clade L, BI: 0.99, ML: 100%). E. emmonsae also was split into two subclades: E. emmonsae A from Claudia, Mato Grosso State - Brazil (M97018 and M97120–locality #84) (Clade F, BI:1, ML: 100%); and E. emmonsae B from Vila Rica, Mato Grosso State - Brazil (APC312 and APC318–locality #85) (Clade G, BI: 1, ML: 100%).

Genetic distances (K2p) using cyt-b sequences (Table S3) are: (i) 4.2% between E. legatus and E. nitidus B clades; (ii) 5.1% between E. legatus and E. nitidus A clades; (iii) 6.5% between E. legatus and Euryoryzomys sp. clades; (iv) 7.5% between E. legatus and E. lamia clades; (v) 16% between E. legatus and E. russatus clades; (vi) 14.2% between E. legatus and E. emmonsae B clades; (vii) 16% between E. legatus and E. emmonsae A clades; (viii) 15% between E. legatus and E. macconnelli B clades; and (ix) 14.7% between E. legatus and E. macconnelli A clades. In this analysis, we did not include the specimen E. nitidus MSB70697, since it is a single specimen and not a clade. However, the pairwise analysis of Euryoryzomys individuals presented that E. nitidus MSB70697 had lower genetic distance with E. legatus individuals (ranging from 1.7% to 1.9%) than to E. nitidus A (ranging from 4.8% to 5.7%) and E. nitidus B individuals (ranging from 3.6% to 4.4%) (Table S4).

Morphological comparison of E. legatus

Externally, E. legatus specimens show all the character states reported by Thomas (1925) in the original description for the species. These specimens are similar to those of other species of Euryoryzomys in having a strong countershading between the dorsal and ventral pelage coloration. Nevertheless, individuals of E. legatus generally have dorsal fur with a strong yellowish brown to orange brown tinge, with clearer and brighter flanks and cheeks, forming an orange lateral line in most of the specimens examined; and ventral color grayish-white, with pure white hairs present only in small area on the chin. This more intense “ochraceous highlights” of the pelage, mentioned by Musser et al. (1998) as a difference between specimens of E. legatus and those of E. nitidus, may be also a useful character in separating specimens of E. legatus from those of E. lamia, which have a lighter dorsal coloration. This latter species also differs from E. legatus in ventral coloration, being more grayish cream than whitish. Individuals of E. nitidus possess unicolored tails (Musser et al., 1998; Percequillo, 2015), while in almost all the specimens of E. legatus the tail is certainly bicolored (dark brown above and grayish-white below). The presence of a thin blackish eye-ring in E. legatus was not described for other species of Euryoryzomys, so this character may be also useful in separating this form.

The skull of specimens of E. legatus follows the general form for the genus, with a long rostrum, narrow interorbital region with lateral margins divergent posteriorly, and the zygomatic arches widest at their squamosal roots. The mandible is deep and robust, with coronoid and condylar processes somewhat equal in height (separated by a shallow superior notch), and the angular process not extending posteriorly behind the condyloid process, also resembling the condition in other species of the genus. Qualitative characters of the skulls of E. legatus and other species the genus are very similar (see Table 13 in Weksler, 1996). Nevertheless, an alisphenoid strut, present in all specimens of E. legatus (including the holotype), is found in about half of previously examined specimens of E. nitidus (see Musser et al., 1998), and it is present in most, but not all, specimens of E. lamia and E. russatus, and completely absent in specimens of E. macconnelli. Additionally, we observed well-developed temporal and lambdoid crests in E. legatus, which were described as not well developed in E. nitidus (Weksler, 1996). Specimens of E. legatus with unworn molars always show a labial cingulum on M3, which is well developed in most of the examined specimens; in contrast, a labial cingulum is absent or vestigial in E. lamia and E. nitidus (Weksler, 1996).

Morphometric analyses

Descriptive statistics for each of the species of Euryoryzomys are summarized in Table 1. Euryoryzomys legatus differed in several of the recorded metric characters from all other species of the genus. Euryoryzomys lamia, the most similar to E. legatus following the univariate comparison, showed significant differences in seven of the 21 morphometric characters studied. On the other extreme, E. nitidus was the most different species when compared to E. legatus (these species significatively differed in 18 of the 21 analysed measurements).

The first 3 principal components of the “size” PCA (Table 2) accounted for 50.48%, 13.70%, and 8.39% of the total variance, respectively, but only the first was judged statistically significant by the Broken-stick test. PC I was a size component, as all the variables had equal sign and loaded heavily on this axis (we confirm this by regressing PC I scores against several total length measure, e.g., CIL [slope = 17.957; intercept = 30.63; r² = 0.877, t = 64.342, p = 9.3868E−267], and DL [slope = 5.238; intercept = 8.783; r² = 0.515, t = 24.877, p = 1.3258E−93]). On bivariate plots of PC I and PC II, E. legatus mostly separated well from E. macconnelli and E. emmonsae, partially overlapped with E. nitidus and E. lamia, and widely overlapped with E. russatus in multivariate space (Fig. 7A). Specimens of E. emmonsae occupy negative values on PC I (being characterized by narrower zygomatic plates and smaller skulls) and those of E. lamia occupy positive values (broader zygomatic plates and larger skulls). The other four species widely spread over the PC I, but E. macconnelli and E. nitidus were mostly on the negative side. PC II mostly separated specimens of E. macconnelli from the other species, mostly by metric characters as the palatal bridge (longer in E. macconnelli) and the incisive foramina length (shorter in E. macconnelli).

The “size free” PCA (Table 3) shows a similar general pattern to that observed in the “size” PCA, but E. legatus better separated from E. lamia and E. macconnelli (Fig. 8B). The first 3 principal components (all judged statistically significant according to the Broken Stick test) summarized 68.3% (PC I 34.3%, PC II 19.3%, and PC III 14.8%) of the explained variance. E. lamia occupied negative values on PC I (Fig. 7B), being the most specimens characterized by zygomatic plates (ZP) relatively broad and short molar series (MTRL). E. legatus mostly occupied the positive side of this PC (comparatively small ZP and large MTRL). PC II once again separated specimens of E. macconnelli from other species of Euryoryzomys, mostly by the palatal bridge (PB) and the incisive foramina length (IFL).

The “size free” DFA (Table 4) almost completely separated E. legatus from E. macconnelli and E. lamia, slightly overlapped E. legatus with E. emmonsae and E. nitidus, and partially overlapped E. legatus with E. russatus (Fig. 7C). DF I mostly segregated E. macconnelli from all other species on the basis of palatal bridge (PB) and diastema length (DL). DF II separated E. legatus from other species of Euryoryzomys except E. russatus. Incisive foramina length (IFL), interorbital constriction (IOC), and braincase breadth (BB) were important metric characters in this separation. The percentage of correct classifications following the jackknifed confusion matrix in this analysis was high (Table 5). Only 9.6% of the specimens of E. legatus were misclassified.

Given all the evidence, we consider E. legatus as a valid taxon and provide a taxonomic account and an emended diagnosis.

Taxonomic Account

Holotype: adult male, BMNH 25. 2. 1. 24 (Original number 1777), collected 6th August, 1924.

Type locality: “Carapari [= Caraparí], 1000 m., about 35 kilometers north of Yacuiba, on the way towards Tarija,” Tarija, Bolivia.

Geographic Distribution: eastern Andean slope from southern Bolivia (Chuquisaca, Santa Cruz, and Tarija departments) to northernmost Argentina (Jujuy and Salta provinces), at elevations from 500 to 2,100 m.

Habitat: found in Subtropical mountain forest or Yungas, from the foothills in transition areas with chacoan habitats at 500 m, to the cloud forests in areas of contact with high altitude grasslands, at 2,100 m.

Emended diagnosis: E. legatus can be differentiated from the rest of the species of the genus by the following combinations of characters: intermediate size (external and skull measurements for specimens of age class 3: total length 205–315 mm, tail 118–159 mm, maximum skull length 32.45–37.36 mm, maxillary toothrow length 4.87–5.44 mm); skin with dorsum yellowish brown to orange brown; flanks and cheeks bright orange; belly predominantly whitish, with a small white spot (all white hairs) on the chin; eyes surrounded by a thin blackish ring; ears darker than dorsum; tail bicolored; rostrum long and robust; zygomatic arches slightly convergent anteriorly; alisphenoid strut present; capsular projection of the lower incisor forming a conspicuous expansion; presence of a labial cingulum on M3.

Morphological description: External measurements for adults (age classes 4 and 5) are: total length 281–340 mm; tail 132–161 mm; hindfoot 31–37 mm; ears 23–26 mm; body weight 57–102 g (Table 1).

Skin with dorsum yellowish brown to orange brown (Fig. S2); individual hairs plumbeous at base and orange at tip (underfur), or plumbeous at base and black at the tip (guard hairs). Flanks and cheeks clearer than dorsum, bright orange in most of the specimens examined. Belly strongly contrasting with the rest of the body, predominantly whitish, with hairs gray based and tipped whitish; most examined specimens possess a small white spot (all white hairs) on the chin. Eyes surrounded by a thin blackish ring. Ears darker than dorsum, internally and externally covered with brown hairs. Tail bicolored, dark brown above and grayish-white below, with scales evident without magnification. Hand and feet covered dorsally by whitish hairs, with a tuft of white hairs over the claws.

Skull elongated and slightly compressed laterally (Figs. S3 and S4). Rostrum long and robust, nasals extending anteriorly well ahead of the anterior face of upper incisors and the gnathic process, and posteriorly not extending beyond the level of the lacrimals (Figs. 8A and 8B). Nasolacrimal capsules well developed, except in very young specimens. Zygomatic notches excavated, usually as wide as deep, but deeper than wider in some individuals. Lacrimals well visible, generally large and pointy laterally, except in very young and some adult specimens examined (Fig. 8A). Interorbital region posteriorly divergent, with dorsolateral margins beaded showing an overhanging shelf in most specimens examined (even vertically raised in some individuals; Fig. 8C). Frontoparietal suture predominantly U-shaped (Fig. 8C). Interparietal large, approximately 2.5 to 3 times wider than long (Fig. 8D). Zygomatic arches slightly convergent anteriorly and not well expanded laterally. Zygomatic plates comparatively broad and with straight or slightly concave anterior margins (Figs. 8B and 8E). Braincase relatively small, with temporal and lambdoidal crests well visible in most adult specimens (age classes 3 to 5; Fig. 8D). Interpremaxillary foramen is small and rounded (Fig. 8E). Incisive foramina proportionally short, posteriorly not extending beyond the anterior face of the procingulum of M1 (Fig. 8E). Bony palate extending behind the level of the alveolus of M3, with large posteropalatal pits - located slightly ahead, or at the same line, with the anterior margin of the mesopterygoid fossa - and with small palatine excrescences (Fig. 8F). Mesopterygoid fossa with the roof completely ossified, approximately of the same width of the parapterygoid fossae, with a rounded anterior margin, and without a well-developed median spine (Fig. 8F), except for a few specimens examined which shows a small median spine. Parapterygoid fossae somewhat excavated, with straight or divergent backwards lateral margins (Fig. 8F). Tympanic bullae small and with large and flattened Eustachian tubes (Fig. 8G). Alisphenoid strut present in all the specimens examined. Hamular process of the squamosal large, slender in most of the specimens, and distally pointy (Fig. 8H). Postglenoid foramen larger than subsquamosal foramen (Fig. 8H). Carotid arterial supply follows the Patterns 1 of Voss (1988), with the common carotid artery bifurcated behind the auditory bulla to form the external and internal carotid arteries, and the stapedial artery split into infraorbital and supraorbital branches.

Coronoid process of the mandible approximately at the same level as the condyloid process (Fig. 8I), but slightly above or below in some specimens examined. Angular process generally do not extend backward beyond the condylar process. Capsular projection of the lower incisor forming a conspicuous expansion located generally just behind the base of the coronoid process. Sigmoid notch comparatively shallow and lunar notch not well excavated (Fig. 8I). Upper and lower rami of the masseteric crest converging anteriorly, the lower ramus more developed than the upper one, extended approximately to the same level of the anterior border of the m1 (or slightly behind), and ending at level of the mental foramen (or just above) (Fig. 8J).

Upper incisors opisthodont (angular index lower than 90° sensu Thomas, 1919) and faced with orange enamel (Fig. 8B). Molars bunodont and pentalophodont (Fig. 9). M1 with procingulum without anteromedian flexus but with a depression on the base of its anterior face and with an enamel island at the level of the anteroloph; anteroloph, parastyle, and mesoloph well-developed, and posteroloph small (only visible in newly erupted molars) (Fig. 9A). M2 with a reduced procingulum but with mesoloph and posteroloph visible, and with an enamel island between protocone and paracone (just ahead of the median mure). M3 comparatively small, with the same general pattern (although with vestigial structures) as the M2 (Fig. 9A). Procingulum of the m1 without anteromedian flexid but with an enamel island; anterolabial cingulid, mesolophid, and posterolophid well developed (Fig. 9B). The m2 with a reduced procingulum, but mesolophid, and posterolophid distinct, and with a penetrating hypoflexid on the labial side. Although with less developed structures, the m3 shows the same general pattern that m2 (Fig. 9B).