Taxonomic status of the nominal forms assigned to Necromys lactens (Rodentia, Cricetidae) as revealed by molecular and morphometric evidence

Necromys is a genus of sigmodontine rodent that inhabits grasslands and scrublands in South America. Eight extant species are recognized in the genus; one of these is Necromys lactens, which inhabits high-elevation grasslands in the Yungas from south-central Bolivia to northwestern Argentina. Morphological variation in N. lactens has been recognized by the description of three nominal forms. Geographically structured genetic diversity also has been observed, but a thorough revision of these nominal forms within an integrative framework has yet to be performed. We conducted a phylogeographic assessment based on an 801 base-pair fragment of the cytochrome-b gene that guided morphometric analyses (univariate and multivariate comparisons) of patterns of geographic variation in the species, and the distinction of its nominal forms. Haplotypes of N. lactens form a well-supported and geographically structured clade. Within it, there are two main clades; haplotypes from the northern range form a well-supported clade, sister and allopatric to a weakly supported southern clade, which includes variants collected at or near the type localities of three nominal forms. In turn, both main clades are composed by two allopatric subclades. Morphometric analyses indicated no differences in shape of the skull among the three nominal forms or between the recovered clades and subclades. Taking together all the available evidence, we consider N. lactens to be a monotypic species.

their limited sampling (e.g., omitting Bolivian variants), those authors called for a broader phylogeographic analysis of this and other allied grassland specialist species. Moreover, a thorough revision of the nominal forms associated to N. lactens, including representative series of specimens analyzed within the framework of an integrative approach (including molecular and morphometric data), has yet to be performed.
In the present work, additional newly collected specimens of N. lactens from southern Bolivia and northwestern Argentina allowed us to undertake a range-wide phylogenetic reconstruction and an associated analysis of morphometric variation. These analyses allowed us to test the taxonomic status of its three nominal forms in an integrative framework (Padial et al. 2010;Schlick-Steiner et al. 2010).

Materials and Methods
Materials examined.-Specimens of N. lactens used in this study were newly collected by us using Sherman folding traps of 8 × 9 × 23 cm (H. B. Sherman Co., Tallahassee, Florida) or already housed at natural history collections ( Fig. 1; Appendix I). Newly captured specimens (Appendix I) were deposited in the mammal collections of the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia" (MACN-Ma; Ciudad Autónoma, Buenos Aires, Argentina); in the Centro Nacional Patagónico (CNP; Puerto Madryn, Chubut, Argentina); and in the Instituto Miguel Lillo (CML; San Miguel de Tucumán, Tucumán, Argentina). Sampling included specimens collected near the type locality of A. lactens Thomas, 1918, A. orbus Thomas, 1919, and B. negrito Thomas, 1926. All aspects of the study involving live animals followed the guidelines of the American Society of Mammalogists (Sikes et al. 2016).
Genetic and phylogenetic analyses.-Genetic and phylogenetic analyses were based on an 801 base-pair fragment of the Cytb gene following the general protocol delineated in D' Elía et al. (2008; see also ). Our sample of N. lactens is more complete than the earlier study: we analyzed sequences of 31 specimens of N. lactens, of which 23 were newly generated in this study and eight were retrieved from GenBank (accession numbers in Appendix II). Geographic coverage was relatively broad; we included sequences from specimens of Necromys collected in 15 localities in Argentina and Bolivia, including sites located in the vicinity of the type localities of the three nominal forms allocated to N. lactens (Fig. 1A). A homologous sequence from one individual of each of the six available species of Necromys (N. amoenus, N. lasiurus, N. lenguarum, N. lilloi, N. obscurus, and N. urichi) was used to form the outgroup. Sequences gathered here were generated using primers MVZ 05 and MVZ 16 (da Silva and Patton 1993) following the protocol outlined in Cañón et al. (2010). Amplicons were purified and sequenced by Macrogen Inc., Korea, or Macrogene USA. Sequences were edited and proofread (e.g., checked for the existence of internal stop codons and reading frame shifts) with Codon Code (Codon Code Aligner, Dedham, Massachusetts). Newly generated sequences were deposited in GenBank (MN102727-MN102749). showing the geographic localities of specimens used in this study. A) Collection localities of the sequenced specimens of Necromys (white circles) and the type localities (white stars) of Akodon lactens (León), A. orbus (Otro Cerro), and Bolomys negrito (Las Pavas). B) Collection localities for specimens of Necromys used in morphometric analyses assigned to Akodon lactens (white triangles), A. orbus (white squares), and Bolomys negrito (white circles). C) Collection localities for specimens of Necromys used in morphometric analyses assigned to the two main clades: northern clade (NC, white circles) and southern clade (SC, white squares). D) Collection localities for specimens of Necromys used in morphometric analyses assigned to the four subclades: northernmost clade (NNC, white circles), central-northern clade (CNC, white circles with a midpoint), central-southern clade (CSC, white squares with a midpoint), and southernmost clade (SSC, white squares). Gray shading corresponds to areas above 2,000 m elevation.
Sequences were aligned with Clustal X (Thompson et al. 1997) as implemented in MEGA7 (Kumar et al. 2016), using the default values for all alignment parameters; after a visual inspection in search of internal stop codons and reading frame shifts, no adjustment was needed. Genetic distances (p-distances) were calculated using MEGA7. A gene tree was constructed by Bayesian inference (reviewed in Huelsenbeck et al. 2001) conducted in MrBayes 3.1 (Ronquist and Huelsenbeck 2003). The best-fit model of nucleotide substitution, HKY+G (Hasegawa et al. 1985), was determined based on the Bayesian Information Criterion (BIC) using jModeltest2 (Darriba et al. 2012). Two independent runs, each with five heated and one cold Markov chains, were performed. All model parameters were estimated in MrBayes. Uniform interval priors were assumed for all parameters except base composition and HKY parameters, which assumed a Dirichlet process prior. Runs were allowed to proceed for 20 million generations; trees were sampled every 1,000 generations for each chain. To check that each run converged on a stable log-likelihood value, we plotted the log-likelihood values against generation time for each. The first 25% of the trees were discarded as burn-in and the remaining trees (all sampled at the convergence zone) used to compute a 50% majority rule consensus tree and obtain posterior probability (PP) estimates for each clade.
Taxonomic assignment of the samples.-To conduct quantitative morphological analyses, we followed two different criteria for grouping examined specimens. First, we tested the existence of quantitative morphological differences among the three described nominal forms, grouping the individuals nearby type localities (Fig. 1B). Second, the same sets of individuals were assigned to one of the mitochondrial lineages documented in our phylogenetic analysis (Figs. 1C and 1D) following a criterion of geographic proximity among localities (with respect to those of the sequenced specimens). For both groupings, we took into account the absence of discontinuities in size and shape within groups.
Specimens were assigned to age classes based on tooth wear following the model shown in the Fig. 2. Five age classes were defined: age class 1, M3 is incompletely erupted or unworn; age class 2, M3 is fully erupted and exhibits moderate wear, M1-2 unworn; age class 3, M3 is well worn, its occlusal surface is flat or concave, M1-2 exhibit moderate wear; age class 4, M3 heavily worn, being generally concave, M1-2 have worn and flattened cusps, M2 with no trace of the paraflexus; age class 5, M1-3 are all worn and concave; most details of the occlusal topography are obliterated. Age class 1: M3 incompletely erupted or unworn; age class 2: M3 fully erupted and exhibits moderate wear, M1-2 unworn; age class 3: M3 well worn, its occlusal surface is flat or concave, M1-2 exhibit moderate wear; age class 4: M3 heavily worn, being generally concave, M1-2 have worn and flattened cusps, M2 with no trace of the paraflexus; age class 5: M1-3 are all worn and concave; most details of the occlusal topography are obliterated.

JOURNAL OF MAMMALOGY
Descriptive morphometric and univariate comparisons for samples of each of the three nominal forms (Tukey's pairwise comparison) and for the specimens recovered for the two main clades (t-tests) recovered via phylogenetic analysis (NC and SC; see below) were carried out with the software PAST (Hammer et al. 2001). Specimens of age classes 2 and 3 were pooled in tests of significant size differences (for both, P ≤ 0.05 and P ≤ 0.01).
In order to reduce the dimensionality of morphometric data and explore the differences in the form of the skull among samples of Necromys we used a "size-free" Principal Component Analysis (sfPCA) including individuals of the five age classes. To reduce the effect of size, Mosimann shape variables were calculated through geometric mean transformation of data prior to statistical analyses (as developed by Meachen-Samuels and Van Valkenburgh 2009). The geometric mean is a size variable derived from the nth root of the product of n measurements (each species measurement is divided by the nth root of the product of values of a species vector of n variables), and the ratio of any particular measurement to the overall geometric mean is a Mosimann shape variable. Statistical significance of the PCs was evaluated following the Broken-stick test (Frontier 1976;Jackson 1993). We developed three sfPCA (using individuals of all age classes to obtain sample sizes sufficiently large): one was designed to test morphometric differences among the three nominal forms (as represented by samples coming from the environs of each of the three type localities), and two others sought to test morphometric differences among the main clades obtained in the phylogenetic analysis. All multivariate statistical analyses were performed with the software PAST (Hammer et al. 2001).

Results
Genetic and phylogenetic analyses.-Average observed p-distance of all haplotype pairs of the N. lactens sample is 2.1%. Haplotypes of N. lactens form a well-supported clade (PP = 1.0), which is geographically structured, although with variable support (Fig. 3; Table 1). Haplotypes from the northern range (NC) form a well-supported clade (PP = 0.96), which is sister and allopatric to a weakly supported (PP = 0.54) clade (SC) formed by haplotypes of the southern part of the species distributional range. The NC is divided into two allopatric subclades; a moderately supported (PP = 0.86) northernmost clade (NNC) is formed by all Bolivian haplotypes and those from northern Salta province (localities of Los Toldos and Rodeo Pampa). The NNC is sister to a highly supported (PP = 1.0) central-northern clade (CNC) formed by haplotypes from Jujuy province and one locality (Abra Ciénaga Negra) in Salta province. Clade SC also shows geographic structure. Haplotypes recovered from the southernmost localities, in Catamarca and Tucumán provinces, form a strongly supported (PP = 1.0) clade (SSC), which is sister to a weakly supported (PP = 0.52) central-southern clade (CSC) formed by haplotypes recovered from specimens collected at Jujuy and Tucumán provinces.
Internally, NC and SC clades show low levels of genetic variation (0.008 and 0.011, respectively); similarly, the four subclades are genetically homogenous (range: 0-0.006; Table  1). However, p-distances between the NC and SC clade is 0.033, while the differentiation between NNC and CNC is 0.013 and between CSC and SSC is 0.017.
Morphometric characterizations.-The univariate morphometric analysis did not reveal differences among the three nominal forms assigned to N. lactens. Two of the 25 measurements evaluated (ZP and RW2) differed significantly among samples assigned to A. lactens and B. negrito, and one (NL) differed significantly among samples of the three forms (Table 2). Four (MTRL, ZB, IOC, and MDL) of the 25 measurements differed significantly among samples assigned to the northern (NC) and southern (SC) clades ( Table 2).
The first sfPCA also failed to differentiate the three nominal forms assigned to N. lactens. Representative specimens of these forms widely overlapped on the morphospace (Fig. 4A). The first three PCs summarized 68.97% of the total variation but only PCI (36.61% of the variance) and PCII (21.43%) were assessed as statistically significant by the Broken-stick test (Table 3). Mandibular diastema length was the most influential variable along these two axes, but do not appear to separate any of the three nominal forms.
The second and third sfPCA also failed to differentiate the two main clades (NC and SC) as well as the four subclades (NNC, CNC, CSC, SSC) resulting from the phylogeographic analysis. Samples of specimens of all these clades almost completely overlap in morphospace (Figs. 4B and 4C). Both analyses included the same specimens, so the numerical results of the two sfPCAs were coincident. The first three PCs summarized 64.94% of the total variation but only PCI (30.31% of the variance) and PCII (25.64%) were judged statistically significant by the Broken-stick test (Supplementary Data SD1). The zygomatic plate depth (ZP) and the molar series (MTRL and mTRL) were the most influential variable along these two axes, but do not appear to separate any of the clades.

Discussion
Taxonomy.-As for many other species of sigmodontine rodents described in the first half of the XX century, the three nominal forms associated to N. lactens were characterized and diagnosed on the basis of small series of specimens, and assessing only a limited number of morphological characters. In fact, Oldfield Thomas named and diagnosed these three nominal forms using a single specimen each; females for A. lactens and A. orbus, and a male for B. negrito. Thomas (1919) recognized the overall similarity between A. lactens and A. orbus when stating in the original description of the latter: "General characters very much as in A. lactens." He differentiated both forms by the putative absence of a white patch on the chin, the tone of the fur "slightly duller and more drabby" (although he recognized the scant difference in this character when he said "…but the difference amounts to little."), the ears slightly shorter, the skull of lighter build, with markedly narrower muzzle and more bowed in outline, and the thinner incisors, in A. orbus. In our samples of specimens from the vicinity of the type localities, most of these characters varied, but not in association with these nominal forms. In fact, we observed specimens from the environs of the type localities of A. orbus and A. lactens with poorly expressed (almost absent) white patches on the chin; other specimens from the same localities display great variation in patch size (small, medium, or large, in size). The size of the ear (E) and the midrostral width (RW2) of specimens of both nominal forms do not show significant differences ( Table 2). As such, both forms have been traditionally considered synonyms (e.g., Musser and Carleton 2005). As far as we are aware, Capllonch et al. (1997) have been the only recent authors to recognize orbus (as Bolomys orbus) as a valid species (but see also Galliari and Pardiñas 2000:226 who considered the synonymy of orbus as dubious). They referred to this species a single specimen (CML 5989) collected in the extreme north of the Sierra de San Javier in Tucumán province (northwestern Argentina). We studied this specimen and concluded that it is not morphologically similar to A. orbus or allied to N. lactens, but is more similar in external and skull characters to the lowland species of the genus-e.g., N. lenguarum (Thomas, 1898)-and the recently described N. lilloi Jayat, D'Elía, Ortiz and Teta, 2016. Thomas (1926) was even briefer in comparisons of B. negrito, highlighting only the dark color of the fur and the "fossorial characters of its feet and teeth." In the sample examined by us, we observed several specimens with very dark tones  Table 2.-External and craniodental measurements for specimens of the nominal forms assigned to Necromys lactens and for those recovered for the two main clades (NC and SC) obtained with the phylogenetic analysis for the age classes 2 and 3. X = mean; SD = standard deviation; r = range; n = sample size. Measurement abbreviations listed in "Materials and Methods." One and two asterisks indicate a significant difference at P < 0.05 and P < 0.01, respectively.  Jayat et al. 2010Jayat et al. , 2017 as well as other species from elsewhere (e.g., Kaufman 1974;Stoner et al. 2003;Bedford and Hoekstra 2015). In general, most of the specimens we examined showed comparatively well-developed claws, although we observed variation in this regard. We also observed variation in the procumbency of the incisors across the specimens examined, but the type specimen of B. negrito seems extreme in this character state (Supplementary Data SD2B). Phylogenetic studies conducted by D'Elía et al. (2008) based on Cytb gene data recovered N. lactens as representing a highly differentiated lineage within the radiation of Necromys. Analyzing a sample of seven specimens from seven Argentinean localities, those authors documented that the genetic variation was geographically structured and suggested that lactens, negrito, and orbus were conspecific. Our results, based on a larger sample of specimens, including some collected in Bolivia, corroborate the conclusion of D' Elía et al. (2008). Neither set of results supports the recognition of these three taxa as representing distinct lineages, either at the species or subspecies level. Haplotypes assigned by locality to the three nominal forms all fall into a single clade (the SC); haplotypes of the form lactens fall in the CSC, while those of orbus and negrito are all included in the SSC. Given the low level of genetic divergence between both clades (1.7%), that clade CSC lacks significant support (PP = 0.52), and specimens of clades CSC and SSC are not morphologically differentiated (a Discriminant Analysis performed to verify this result misclassified a large proportion of cases; Supplementary Data SD3), we corroborate the hypothesis advanced by D' Elía et al. (2008) and conclude that the best taxonomic decision is to consider orbus and negrito as junior synonyms of lactens.
Genetic divergence between the two main clades is appreciable, reaching 3.3%, a value distinguishing some other akodontine species (e.g., Coyner et al. 2013). By this measure, the recognition of two (lactens and one new form for the NC) or three (lactens and two new forms for NNC and CNC) taxa would be an option. Notwithstanding, the southern clade lacks significant support (PP = 0.54) and the morphometric analyses of skull shape failed to separate any of the groups emerging from these arrangements, with samples representing each of the groups highly overlapped in the morphospace (a Discriminant Analyses applied to these groups also performed poorly in classification; Supplementary Data SD3). Examined specimens, although numerous enough to allow testing for shape differences  among groups, were insufficient to evaluate size differences for any of the five age classes (no age class has adequate series across all groups). Nevertheless, our quantitative morphometric analysis of specimens of age classes 2 and 3 fails to show the existence of distinctive size-based classes, either among populations of the three recognized nominal forms or between the two main clades (NC and SC). As such, taking together all the available evidence, we treat all studied specimens as belonging to a single and monotypic species, N. lactens. Biogeography.-Beyond the taxonomic assignments of the clades of N. lactens as here understood, the pattern of variation of the mitochondrial genome, showing a deep, geographically structured genealogy, suggests a history of population differentiation in isolation. Necromys lactens occur in high-altitude grasslands that develop on pre-Andean mountain ranges (Jayat and Pacheco 2006). These mountain systems are a complex of parallel ridges of different heights and extensions that replace themselves from east to west and that mostly run north to south, parallel to the Andes. Their mountaintop grasslands are separated from each other by relatively large river valleys covered by arid or semiarid habitats (e.g., Chaco, Monte, and Prepuna) and the humid Yungas forest. Our results suggest that the phylogeography of N. lactens was shaped by the history of these high-altitude grasslands in the central pre-Andean and associated mountain systems. Today, glaciers occupy some of the mountain ridges in the area (e.g., Nevados del Aconquija, Nevados del Chañi, and Nevados de Cachi) above 5,000 m; however, during the ice ages, glaciers in these areas extended far lower (Martini et al. 2017), reaching between 4,200 m and even to 4,000 m in some places (Halloy 1978;Haselton et al. 2002). This would have lowered the vegetation belts on mountain slopes and caused some montane vegetative zones to extend beyond the mountains. Under these conditions, patches of highaltitude grasslands inhabited by N. lactens may have come into  Table 3 and Supplementary Data SD1. contact and allowed dispersal of the species among the different mountain ranges. This hypothesis is supported by the similar patterns of genetic variation observed in other taxa strikingly associated to high-altitude grasslands (e.g., Jayat et al. , 2017 and by the discovery of several late Pleistocene to lower Holocene small mammal assemblages, in which several rodent species that currently live in high-altitude areas occurred at lower altitude (e.g., Ortiz and Pardiñas 2001;Ortiz et al. 2011aOrtiz et al. , 2011bOrtiz et al. , 2019. Future phylogeographic studies, based on larger series of specimens and on more loci (e.g., Lessa et al. 2014), could potentially answer the question of whether the differentiation scenario suggested here is corroborated or falsified.

Acknowledgments
We thank R. González, D. Miotti, and G. Namen, for helping us during fieldwork. We are indebted to the curators of all mammal collections visited, especially to P. Teta of the MACN and M. Díaz of the CML. Special thanks to R. Harbord who kindly provided the photographs of the holotypes of A. lactens, A. orbus, and B. negrito. Two anonymous reviewers made valuable suggestions on an earlier version of this contribution. Financial support was provided by Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT) Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT) (to PEO), Fondo para la Investigación Científica y Tecnológica (FONCYT) (to JPJ), and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1180366 (to GD). This work was also made possible thanks to the institutional support of the Instituto de Ambiente de Montaña y Regiones Áridas (IAMRA), Universidad Nacional de Chilecito (UNdeC), the Instituto de Ecología Regional-Laboratorio de Investigaciones Ecológicas de las Yungas (IER UNT-CONICET), the Instituto Superior de Correlación Geológica (INSUGEO), and Cátedra de Paleontología, Universidad Nacional de Tucumán.

Supplementary Data
Supplementary data are available at Journal of Mammalogy online. Supplementary Data S1.-Size-free Principal Component Analysis of the two main clades and the four subclades.
Supplementary Data S3.-Classification matrix and percentage of correct classifications of the Discriminant Analysis for the three nominal forms, the two main clades, and the four subclades.