Revisiting Molossus (Mammalia: Chiroptera: Molossidae) diversity: Exploring southern limits and revealing a novel species in Argentina

Understanding species diversity and delineating their boundaries are crucial for effective management and conservation efforts. In the case of bats, species identification holds particular importance from an epidemiological standpoint. The genus Molossus (Chiroptera: Molossidae) encompasses 15 species distributed across the Neotropics, ranging from the southeastern United States to Argentina. This genus exhibits two contrasting patterns of variation: some species are cryptic, while others are morphologically dis - tinct yet genetically similar. This study explores the diversity of Molossus in Argentina through a molecular phylogenetic approach. We analyzed sequences from three molecular markers (cyt b , COI, and FGB) along with morphology data obtained from a sample of 64 individuals. Uni-and multivariate analyses of external and cranial measurements were conducted, alongside comparisons of external and cranial characteristics among species. Based on molecular and morphological differences, we describe a new species within the Molossus genus. This newly discovered species exhibits a broad distribution spanning the Paraná River basin across three distinct ecoregions. It is noteworthy that this species is pseudo-cryptic with respect to similar-sized species such as M. molossus and M. melini . Additionally, it is important to mention that all species in Argentina have overlapping distribution ranges. In summary, this study provides valuable insights into the diversity and distribution of Molossus bats in Argentina, employing molecular and morphological analyses. The discovery of a new species underscores the ongoing importance of comprehensive research efforts in understanding and conserving bat populations in the Neotropics


Introduction
Accurate species identification is of paramount importance for various reasons.Firstly, it serves as the foundation for understanding and documenting biodiversity, allowing the recognition and appreciation of the variety of life on Earth (Mora et al. 2011).Precise species identification is crucial for effective conservation efforts, as it enables the assessment of population sizes, monitoring distribution patterns, and evaluation of threats and vulnerabilities specific species face (Gaston and Spicer 2004;Pimm et al. 2014).Additionally, accurate species identification is essential for studying ecological interactions, unraveling evolutionary processes, as well as for the control, prevention, and elimination of pathogens (Heywood et al. 1995;De Benedictis et al. 2022).The advent of molecular techniques and modern taxonomic tools revealed differentiation within previously recognized species uncovering the existence of cryptic species in most types of organisms and habitats (Pfenninger and Schwenk 2007).
With over 1400 species comprising 20% of mammal diversity, bats (Order Chiroptera) hold a prominent position in the Tree of Life (Wilson and Mittermeier 2019;Simmons and Cirranello 2024).Genetic studies and molecular techniques have uncovered hidden diversity of cryptic species in a wide variety of bat families (see Tsang et al. 2016 and references therein), usually discovered within the boundaries of what has been previously considered a single species (Jones et al. 2009).In this respect, one particularly challenging group is the genus Molossus É. Geoffroy, 1805.
Bats of genus Molossus are distributed throughout the continental and insular Neotropics, from southeastern United States to central Argentina (Eger 2008), making them an ideal group to study speciation processes, diversity, and biogeographic patterns (Loureiro et al. 2019).The alpha taxonomy of this group has been historically unstable due to the occurrence of two opposing patterns that tend to generate uncertainty in the systematics of the genus.On one hand, there are morphologically similar species that exhibit higher levels of genetic differentiation than expected (e.g., M. molossus, M. fentoni, M. milleri, and M. verrilli) (Loureiro et al. 2018a).Many of these cryptic species have geographically restricted ranges in areas of high anthropogenic pressure, requiring high priority for conservation.The second pattern corresponds to morphologically divergent species with very low levels of genetic differentiation (e.g., M. aztecus, M. pretiosus, M. currentium, and M. sinaloae) (Lim and Arcila Hernandez 2016; Lindsey and Ammerman 2016; Lim 2017; Loureiro et al. 2019).The occurrence of these contrasting patterns can be ex-plained by some typical attributes of the group such as low levels of morphological differentiation at the species level, significant differences in sexual characteristics, and a tendency towards genetic conservatism (Eger 2008;González-Ruiz et al. 2011;Loureiro et al. 2018aLoureiro et al. , 2019)).
In a recent study, Loureiro et al. (2020) applied the genotyping-by-sequencing methodology (GBS) analyzing tens of thousands of single nucleotide polymorphisms (SNPs) covering the entire distribution of the genus providing the first high-resolution phylogeny for Molossus.This study offered new insights into species delimitation and taxon relationships, increasing the number of species from 11 to 14 (Fig. S1).
Recently, a fifteenth species, M. melini, was described in Argentina (Montani et al. 2021).Besides the new species, three other Molossus species are known to occur in Argentina (Barquez and Díaz 2020;Loureiro et al. 2020).Molossus currentium has a restricted distribution and few records in the country, while M. molossus and M. fluminensis have a broader distribution, principally in central and northern provinces of the country (Fig. S1).
It should be noted that except for two specimens of M. melini (Montani et al. 2021), other populations of Molossus in Argentina have not been studied from a multi-locus perspective.This is a necessary approach since the use of a single molecular marker (cyt b) has proved to be insufficient for species identification in Argentinian mastiff bats (Caraballo et al. 2020).
Due to the peculiarity of the natural history of this bat genus, the identification of Molossus at the species level poses a common problem in epidemiological surveillance laboratories, fieldwork, and biological collections.Most specimens are cataloged as Molossus sp.or, what is worse, assigned an incorrect species name (typically M. molossus).Therefore, it is necessary to evaluate genes that can provide sufficient information for the identification of Molossus species without employing highly complex and costly methodologies such as GBS.
In this study, we conducted an extensive examination of the Molossus species diversity in Argentina, assessing the taxonomic differentiation at the species level using one nuclear and two mitochondrial markers.To facilitate further research, we compiled a meticulously curated dataset of genetic sequences, enabling the comprehensive assessment of the phylogenetic classification of any Molossus specimen across its entire geographic range.The discovery of a previously unrecognized lineage was confirmed as a distinct species through multivariate analysis of cranial morphology as well as comparisons of cranial and external characteristics with other known species present in Argentina.In summary, our study significantly contributes to the understanding of the diversity and geographical distribution of mastiff bats in the southernmost extents of their range, shedding new light on the biology of this group.

Sampling
Genomic DNA was extracted from a set of 56 specimens belonging to the genus Molossus, sourced from various locations within Argentina (Table S1), and obtained through three distinct methods.A group of specimens was captured by fieldwork specialists during ecological studies of rabies virus, who followed a protocol involving the capture and subsequent release of the specimens.Wing membrane tissue samples were obtained using a biopsy punch with a diameter of 3 mm.Captures were carried out under the approval of the Ethics and Safety Committee of the Universidad Nacional del Litoral (Expte.FCV-0869428-17) and the Ministerio de Medio Ambiente de la Provincia de Santa Fe (Expte No. 02,101-00, 181, 129-9, Resolution No. 093/2018).Guidelines of the American Society of Mammalogists (Sikes and the Animal Care and Use Committee of the American Society of Mammalogists 2016) were followed.The second group consisted of ethanol-preserved tissue samples, including muscle and patagium, obtained from museum vouchers deposited in the Colección de Vertebrados of the Instituto Nacional de Limnología (INALI-A), Colección de Mamíferos of the Museo Provincial de Ciencias Naturales "Florentino Ameghino" (MFA-ZV-M), Colección de Mamíferos of the Museo Provincial de Ciencias Naturales "Dr.Ángel Gallardo" (MG-ZV-M), and Colección de Mamíferos of the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia" (MACN-Ma).The remaining samples including intestine, patagium, and muscle tissue were collected from bat specimens from the "Instituto de Zoonosis Luis Pasteur" (Buenos Aires city, Argentina) and personal collections.

Sequencing
One nuclear gene, the intron 7 and short partial sequences of exons 7 and 8 of the β-fibrinogen (FGB) and two mitochondrial genes, cytochrome c oxidase I (COI) and cytochrome b (cyt b), were sequenced totaling 2567 aligned base pairs (COI -657 bp; cyt b -1140 bp; FGB -770 bp).Primers and PCR conditions for each gene are given in Table S2.Sequences were obtained with both primers for each locus.Samples that failed to produce sequences were discarded, remaining 47 successfully sequenced specimens (Table S1).Coding sequences were checked for the presence of premature stop codons to discard the amplification of pseudogenes.

Data acquisition and curation
To test the resolution of each marker we downloaded all available sequences annotated as Molossus in GenBank.Although it is immensely valuable, this public database is susceptible to having lots of errors in the taxonomy of the sequences available in it, especially in taxa with cryptic species and taxonomic instability such as Molossus.Morphological species misidentification and/or lack of taxonomic update are the two sources of error that abound in Molossus sequences in GenBank.
To overcome this issue we designed a strategy consisting in updating all previous names according to the current taxonomy, taking into account species distributions (Loureiro et al. 2020;Montani et al. 2021Montani et al. , 2023;;IUCN 2022;Pavé et al. 2023;Olímpio et al. 2024).For example, M. rufus was recently subdivided into M. nigricans, M. rufus, and M. fluminensis, with non-overlapping distributions in Central America, northern South America, and southern South America, respectively (Fig. S1).Another example is that of M. milleri, which is distributed in the Cayman Islands and Cuba, and sequences uploaded previously to its description were annotated as M. molossus.A second part of this strategy was the recognition of misidentified specimens.This was done by scrutinizing majority clusters (composed of more than 50% sequences) of a given species in each single-locus phylogeny and revising those sequences that behaved as outliers in those clusters.Again, this revision was done by contemplating the updated taxonomy, identifying also which species have low levels of genetic differentiation, and also checking if the putative wrong assignment was done to a specimen captured within the distribution range of the majority cluster.Even after the correction of erroneously annotated sequences, we noted that several species failed to produce monophyletic clades, so we included sequences representing all these lineages in the multi-locus analysis.
In addition to the analysis conducted for each marker separately, phylogenies were inferred from the concatenated mitochondrial loci on one hand, and from the three combined loci on the other.The combined analysis of cyt b and COI genes allows the reconstruction of the same evolutionary history (Hurst and Jiggins 2005).Different phylogenetic patterns have been reported between mitochondrial and nuclear DNA (Spinks and Shaffer 2009), which highlights the need to compare both datasets independently (Loureiro et al. 2019).For the full dataset, we concatenated the three genes including, whenever possible, specimens with sequences available for the three loci.In cases when this was not achievable, we constructed chimeras ensuring these were formed by individuals of the same species, country, and lineage in the single-locus trees (Table S3).At least two individuals/chimeras per species and lineage were included to test their monophyly.Since the FGB analysis resulted in a less informative topology, we gave priority to specimens with sequences from both mitochondrial loci.If a specimen lacked FGB data, we completed the chimera with another specimen, following the previously mentioned criteria.The sequences of specimens/chimeras that lacked a specific locus were coded as missing data.

Phylogenetic analyses
A Bayesian phylogenetic analysis was conducted for each locus and the concatenated dataset including representatives of all known species of Molossus.We used sequences of Promops centralis and Eumops auripendulus as outgroups.Nucleotide substitution models were estimated using MrModeltest2 (Nylander 2004) under the Akaike Information Criterion (correcting by the number of taxa), being HKY+G for FGB and COI, and HKY+I+G for cyt b.In the multi-locus analyses, we inferred a substitution model separating each codon position being K80+I, JC and HKY+G for 1 st , 2 nd , and 3 rd positions of COI, and SYM+G, F81, and GTR+I+G for cyt b.
The phylogenetic analysis was performed in MrBayes version 3.2.7 (Ronquist et al. 2012), using computational resources from CCAD -Universidad Nacional de Córdoba (https://ccad.unc.edu.ar), which are part of SNCAD -MinCyT, República Argentina.For each single-locus analysis and for the concatenated mitochondrial (mtDNA) dataset, two independent runs for 1×10 7 MCMC (Markov chain Monte Carlo) generations, sampling every 1,000 generations, were carried out.The multi-locus analysis including nuclear and mitochondrial genes (FGB, COI, cyt b) was run for 2×10 7 MCMC generations, sampling every 1,000 generations.Convergence was assessed by analyzing the potential scale reduction factor (PSRF), and the average standard deviation of split frequencies (ASDSF).The burnin phase was set up in the generation that fulfilled PSRF values of 1.00-1.02for all estimated parameters and standard deviations lower than 0.01.We also checked that the estimated sample sizes were >200.Trees were visualized with Figtree (http://tree.bio.ed.ac.uk/software/figtree).For the comparison of phylogenetic resolution, we considered a posterior probability (bpp) of 0.95, 0.75 and <0.95, and <0.75 as high, moderate, and low node support.

Network analysis
A median-joining network was constructed with PopART v1.7 (Leigh and Bryant 2015).The software necessitates that sequences have few or no undefined states.Since the nuclear marker (FGB) was absent in several individuals/ chimeras, we opted to conduct the analysis solely considering the mtDNA loci.Additionally, individuals lacking sequences for either of the two mtDNA loci were excluded.Following this procedure, all lineages were represented, except for Molossus sp. 2. For this lineage, we created a chimera using a cyt b sequence and a COI sequence from two different individuals (Molossus sp. 2 Ecuador-A1 and Molossus sp. 2 Bolivia-A7, respectively).

Genetic distances
To compare intra-and interspecific distances we generated a p distances table with the concatenated matrix including the three loci.Pairwise genetic distances were calculated with MEGA X (Tamura et al. 2021).Of the two sequences of M. currentium, one was considered as M. fluminensis (MH185138), while the other was treated as M. currentium (MH185139), based on the results of the phylogenies obtained in this study (see below).

Comparative morphometry analyses
sis (GPA) and PCA.Detailed listings of the datasets employed for univariate, multivariate, and 2D GMM morphometry, as well as the statistical methods applied and the resulting test outcomes, are provided in File S1.

Single-locus and mtDNA phylogenies
The trees obtained for all nuclear and mitochondrial loci confirmed the monophyly of the genus Molossus.However, significant discrepancies were observed in internal nodes, particularly between FGB and both mitochondrial genes (Figs S2-S4; File S2).While the nuclear gene fails to recover most species as monophyletic, it can enhance the resolution of another dataset, such as the mtDNA genes (Fisher-Reid and Wiens 2011).The mtDNA dataset produced a similar but better-resolved phylogeny compared to cyt b, which is the better-resolved single-locus phylogeny (Fig. S5; Files S2, S3).

Multi-locus phylogeny
The phylogeny inferred from the concatenated dataset depicts higher levels of phylogenetic resolution, with the most robustly supported internal nodes (609 parsimony informative sites of a total of 2567) (Fig. 1).While there are minor discrepancies between the mtDNA tree and the multilocus tree, these are slight differences.The only significant disparity involves the positioning of sequences of M. aztecus, indicating potential introgression events (discussed in File S2).Consistent with the single-locus and mtDNA phylogenies, M. fentoni, M. verrilli, M. milleri, and M. alvarezi form highly-supported basally-diverging groups.In addition, M. rufus (with high support) and M. bondae (with low support) are also monophyletic groups.
The clade of M. fluminensis has moderate support (bpp = 0.84) but is still recovered as monophyletic, and splits into two well-supported subclades.Within this group, there are two previously annotated M. rufus that were updated according to their distribution to M. fluminensis (Fig. S1) and a sequence annotated as M. currentium from Paraguay, a region where the distribution of both M. currentium and M. fluminensis overlap.Notably, the other sequence of M. currentium (also from Paraguay) clusters with M. nigricans, M. sinaloae, and M. rufus.These three species do not overlap with M. currentium, so the most reasonable explanation is that this second haplotype represents the valid M. currentium and the former is, in fact, M. fluminensis.
The species M. pretiosus and M. aztecus from Mexico form a highly supported clade with M. bondae, but fail to be reciprocally monophyletic, indicating that these species are genetically close.Interestingly, the three representatives of M. aztecus from Brazil form a monophyletic clade, sister to the former, although there are two distinct lineages (A and B), as evidenced by the long branch within this group.In contrast, in the mtDNA phylogeny, M. aztecus from Brazil is divided in two non-related clades, one related to M. fluminensis, and the other related to M. melini (Fig. S5; File S2).
The clade of M. melini includes several specimens previously identified as M. molossus, not only from the originally described localities but extending also to the Buenos Aires province.Interestingly, several specimens that fall in the clade of M. melini, do not have the typical ochraceous to orange fur coloration, but instead are chocolate to grayish brown with the venter paler, and that could be the reason for their initial identification as M. molossus (Fig. S7).Sister to this clade is the M. molossus clade which includes the subspecies M. molossus daulensis.This clade groups two of the three M. molossus lineages found with COI: the canonical lineage, and a lineage composed of sequences from Panama and Brazil (Fig. S3; File S2).We conclude that these two lineages are indeed M. molossus.However, as occurs with the cyt b-and COI-based phylogenies, there is another clade of specimens identified as M. molossus with moderate support (bpp = 0.81), that splits into well/moderately supported clades, one (bpp = 0.81) including specimens from Ecuador, Peru, Guyana, Suriname, and Bolivia, while the other is exclusive of Argentinian bats (bpp = 0.97).This last group should be treated as Molossus spp., consisting of Molossus sp.nov.(Argentinian clade) and Molossus sp. 2 (sister clade), since it is reciprocally monophyletic to the canonical lineage of M. molossus (which has a wider distribution and includes the subspecies M. molossus daulensis).It is noteworthy that, except for one sample, Molossus sp. 2 formed part of the M. molossus clade in the mtDNA analysis (Fig. S5) and in the full dataset it groups with Molossus sp.nov.

Genetic distances
The genetic distance analysis illustrates the general panorama of Molossus (Fig. 1; Tables S4, S5).Intraspecific distances depict low values, ranging from 0 to 1.7% (mean 0.85%), the highest values belonging to M. molossus, M. pretiosus, M. coibensis, and M. fluminensis.The only exception is M. aztecus, depicting 2.5%, a remarkably high value for intraspecific variation (but see an extended discussion in File S2).As expected for this genus, there is a wide variation of interspecific distances, with a mean of 4.0%, but including extremely low values such as those found between M. pretiosus, M. nigricans, M. bondae, M. rufus, M. sinaloae, and M. currentium, all of which are <1.0%.The distance between M. molossus and Molossus sp.nov. is 2.8% which is indicative of interspecific variation.In contrast, the distances between M. molossus and Molossus sp. 2, and between Molossus sp.nov.and Molossus sp. 2 are 1.1 and 1.4, respectively, and will be discussed below.As a whole, Molossus appears to be a genetically stable group.The average genetic distance among morphologically recognized species using mtDNA markers was estimated to be approximately 3.3% (Loureiro et al., 2019), a finding consistent with the results of this study.These low levels of genetic variation are comparable to certain molossid bats from the Old World but are notably lower compared to others such as Cynomops, Mops, and Eumops, where cyt b average distances range from 8.6% to 13.4% (Ratrimomanarivo et Baker et al. 2009a;Moras et al. 2018).While interspecific distances in our study are notably lower compared to the >5% threshold suggested by Baker and Bradley (2006) for species delimitation, it is crucial to recognize that such thresholds serve as guidelines and can vary across different organisms (Baker et al. 2009b).These thresholds are not universally applicable and should be determined empirically for each taxonomic group, taking into account specific characteristics such as generation time, dispersal rates, and geographic range (Hebert et al. 2003).Finally, we believe that genetic distances should prompt taxonomic reassessments but should not be considered the sole criterion for species delimitation.

Network analysis
The mtDNA median-joining network illustrates significant differences among M. melini, M. molossus, and Molossus sp.nov.(Fig. S6).Molossus molossus exhibits more than 30 substitutions distinguishing it from the other two species, while M. melini and Molossus sp.nov.display twice this distance, indicative of their classification as distinct species.It is worth noting that certain taxa, such as M. fluminensis, M. coibensis, or the subspecies M. molossus daulensis, exhibit high levels of intraspecific divergence at the mitochondrial level.The chimera constructed using two sequences representing Molossus sp. 2 is closely associated with Molossus sp.nov.However, as will be discussed further, the fragmentary availability of sequences representing Molossus sp. 2 dictates a cautious approach in its treatment.

Comparative morphometric analysis of genetically (re)assigned specimens
In order to contrast the above inferred phylogeny with morphological data, we undertook morphometric comparisons using a subset of genetically identified specimens from which we have enough confident linear metric external and cranial measurements.Molossus sp.nov.clearly differentiates from M. fluminensis by consistently exhibiting lower linear measurement values across all external and cranial variables evaluated (one way ANOVA, Fig. 2).However, Molossus sp.nov.tends to overlap with the other small species, being M. molossus the more distinguishable specially when considering the skull mea-surements (Fig. 2; File S1 [Table SM2] for the analysis with genetically defined specimens plus misidentified genetically specimens).Furthermore, between Molossus sp.nov.and the recently described M. melini only three out of fourteen linear metric cranial variables evaluated were statistically different (p<0.05):LMxT, PC, and LMdT (Fig. 2).
Global skull size variation between species, analyzed by PCA using a subset of cranial measurements of genetically confirmed specimens of M. fluminensis, M. melini, M. molossus, and Molossus sp.nov.(see File S1 for specimen details), showed that the convex hull enclosing the specimens of Molossus sp.nov.maps separated from M. molossus (p<0.001) and M. fluminensis (p<0.003)clusters and in a lesser extent with M. melini (p<0.05) (Fig. 3; File S1 [Table SM4]).In this analysis, the first two components summarize 93% of the variation.PC2 explained only 5.3% of the variation, being clearly dominated by PC and GLS wo.i variables (Fig. 3B).The PC1 explained the largest amount of variation because most cranial vari-  M3).Dot sizes of each specimen is proportional to its genetic information availability score (GIS, see File S1 for score definition details).B Percentages of each cranial variable's contribution to the first two principal components (Dim1 and Dim2).
ables contribute to it with almost equal strength (Fig. 3B).Accordingly, the centroids of species groups are distributed along PC1 from the smallest (M.molossus, M. melini, and Molossus sp.nov.) on the left to the largest (M.fluminensis) on the right side (Fig. 3A).
Based on the genetic and morphometric evidence shown above, we describe the members of the lineage Molossus sp.nov.as a new species, as follows: Holotype.MFA-ZV-M 1494, adult male, preserved as skin, skull, and postcranial skeleton (Figs 4, 5), collected on 20 January 2018 by M. E. Montani (MEM) and V. Colombo (field number MEM 237).External and craniodental measurements for the type series are presented in Table 1.-Type locality.Sociedad Rural "Las Colonias", Esperanza, Santa Fe province,44 m).This is a rural area in the Espinal ecoregion (sensu Olson et al. 2001) where the natural vegetation has been highly transformed in farmlands and there is very little tree vegetation.At the site, there are native tree species such as Tipuana tipu (Fabaceae) as well as exotic species like Eucalyptus sp.(Myrtales) and Fraxinus sp.(Oleaceae).Nine kilometers northeast of this site lies the Martín de la Peña Natural Reserve, characterized by its native vegetation.Other specimens.Five individuals, three males and two females, were captured using mist nets at "Sociedad Rural", Santa Fe city, Santa Fe province, Argentina (lat.
Distribution and habitat.This species is known from ten localities in four provinces of eastern Argentina (Buenos Aires, Corrientes, Entre Ríos, and Santa Fe) in the Espinal, Humid Chaco, Humid Pampas, and Paraná Flooded Savanna ecoregions (sensu Olson et al. 2001).Molossus paranaensis inhabits natural environments in pampas grasslands, dry shrublands and wetlands, and anthropic environments such as human constructions in cities and agricultural fields.
It is conceivable that this newly discovered species may have a wider distribution, particularly within the region influenced by the Paraná River.
Etymology.The name paranaensis is bestowed in reference to the extended distribution of the new species along the Paraná River basin, one of the largest rivers in South America.Paraná is a word from the Mbyá people who speak Tupí (one of the native languages in Argentina), pará = "sea" and nã = "similar to" or "like", which means "that looks like the sea" or "similar to the sea".This river shelters a great biodiversity and natural beauty.

Diagnosis. Molossus paranaensis is distinguished from all other
Molossus species by the following combination of characters: medium size ; dorsal coloration medium brown (cinnamon to grayish brown sensu Ridgway 1912) with individual hairs bicolored, with a large and pale basal band reaching 1/4 to 1/2 to of total length of the hair, the venter is paler than dorsum (Fig. 4); in frontal view the rostrum is triangular (Fig. 5B), and the lambdoidal crests are moderately developed (Fig. 5).
Description.The new species is a medium-sized Molossus (ToL: 90.31-115 mm; FA: 39.4-42.84mm, n = 13 specimens, 6 males and 7 females).Sexual dimorphism is observed in certain variables (body mass, ToL, CBL, MB, PL, C-C; see Table 1).Rostrum with a well-developed and straight keel, and the upper lip with a fringe of medium brown hard hairs on the border forming an upwardly projected mustache.Triangular and medium-sized ears (10-14.5 mm total length) with an oval-shaped antitragus that has an anterobasal constriction.Dorsal hairs measuring 5 mm long, markedly bicolored with a large pale band on the base from white to cream and the tips are variable from cinnamon to grayish brown (the holotype is cinnamon).Ventral hairs measuring 5 mm long, paler than the dorsal hairs and bicolored with grayish brown tips (buffy olive or citrine drab sensu Ridgway 1912; Fig. 4) and white or cream bases; ventrally, the pelage extends onto the plagiopatagium along the side of the body from the elbow to the knee.Wing membranes and uropatagium are pale brown.Feet have thickened first and fifth toes with short and curved hairs and all the toes are bordered with long, curved, and light hairs.The calcar is well-developed, longer than the hindfoot, and occupying ⅔ of the uropatagium edge.
The skull is elongated with a short rostrum (compared to the braincase); the infra-orbital foramen is frontally directed; the nasal cavity is taller than wide (in dorsal view the posterior edge of the nasal cavity is triangular); basisphenoid and basioccipitals pits moderately deep although the first more than the second; the occipital is triangular in posterior view; the sagittal crest has low development and the lambdoidal crest is moderately developed and has a quadrangular shape in posterior view as in M. fluminensis, both crests are more pronounced in males.The post-tympanic process of the squamosal is well developed and this is more visible dorsally (Fig. 5D).The dental formula is I 1/1, C 1/1, P 1/2, M 3/3, total = 26.The upper incisors are elongated, pincer-like, they are projected beyond the canines, and with parallel tips (Fig. 5A, B).

Comparisons. Molossus paranaensis is a medium-sized
Molossus similar to M. bondae, M. currentium, M. molossus, M. melini, and M. verrilli (Loureiro et al. 2018a).The new species can be easily distinguished from M. bondae and M. verrilli by dorsal fur coloration and secondarily by geographical distribution.From the species occurring in Argentina, M. paranaensis differs from M. currentium in dorsal coloration and in some cranial characters such as the shape of upper incisors (see Table 2); from M. molossus in the length of forearm (in general < 40 mm in M. molossus and 39.4-42.8mm in M. paranaensis) and LMTx (5.7-6.4 mm in M. molossus and 6.3-7.4 mm in M. paranaensis), the format of the rostrum in frontal view  2); from M. melini, in addition to the ochraceous or orange individuals of this last species, in the color of membranes (pale brown in M. paranaensis and dark brown in M. melini), in the direction of the infra-orbital foramen (frontal in M. paranaensis and lateral in M. melini), in the development of sagittal crest (higher in M. melini), and in the length of LMxT (6.0-7.0 mm in M. melini and 6.3-7.4 mm in M. paranaensis), PC (3.7-4.2 mm in M. melini and 3.8-4.6mm in M. paranaensis), and LMdT (6.4-7.9 mm in M. melini and 7.3-8.0mm in M. paranaensis) (Figs 2, 3; Table 2).
Molossus paranaensis can be readily differentiated from larger-sized Molossus, such as M. alvarezi, M. fluminensis, M. nigricans, M. pretiosus, M. rufus, and M. sinaloae, all of which have forearm longer than 42.8 mm and dorsal color in general dark brown.Furthermore, among large-sized species, M. paranaensis occurs in sympatry only with M. fluminensis.Finally, M. paranaensis differs from M. aztecus, M. coibensis, M. fentoni, and M. milleri due to its larger forearm (see Table 2).Additionally, M. aztecus and M. coibensis exhibit unicolored dorsal hairs, spatulated upper incisors, and a quadrangular occipital region, while M. fentoni and M. milleri are characterized by their dark fur.The external and cranial differences distinguishing M. paranaensis from the other Molossus species are presented in Table 2.

Discussion
As species are essential units of analysis in biology, their delimitation is the most fundamental aspect of systematics.However, there are many distinct, and partially incompatible, epistemological views of the species concept that emerge from considering different biological features (reviewed by Mayden 1997;de Queiroz 2005).In this study, we adopt two of the most commonly used species concepts to analyze the diversity of Molossus in Argentina.The evolutionary species concept, as a general principle, posits that a species constitutes a lineage resulting from populations or metapopulations evolving independently from any other lineage (Wiley and Mayden 2000;Zachos 2016).In a similar vein, the phylogenetic species concept proposes that a species represents a lineage that is evolutionarily distinct from any other, with its constituent populations forming a monophyletic group discernible through the pattern of character distribution, be it morphological or molecular (Wheeler 1999;Zachos 2016).In this study, we integrated molecular phylogenetic and morphological approaches to revise the diversity of Molossus at the southernmost limits of their distribution.The complexity of this genus, where both cases of morphological and genetic conservatism have been report-ed, makes the delimitation of species a difficult, at least non-straightforward, task.We consider that a species can be defined as a monophyletic group in the species-level phylogeny, that has enough genetic divergence from other species, and that can (or not) be morphologically distinguishable from other species of the genus.In this sense, we allow for the recognition of cryptic and non-cryptic species.
In this study, we created a multi-locus reference dataset for precise Molossus species-level identification.The first obstacle that we found in this way was to distinguish erroneously annotated sequences from possible biologically admissible conflicts (see a detailed discussion of this aspect in File S2).As mentioned above, the presence of cryptic taxa may contribute to this problem.In addition to morphological species misidentification, the lack of taxonomic updating in public databases also contributes to conflict in sequence annotation.To address these concerns, we implemented a strategy involving a thorough review of the current taxonomy and distribution of all species within the genus (see File S2).
Three groups of cryptic taxa are currently recognized in Molossus: 1) M. molossus, M. fentoni, M. milleri, and M. verrilli (Loureiro et al. 2018b); 2) M. rufus, M. nigricans, and M. fluminensis (Loureiro et al. 2020); 3) M. bondae and M. currentium (Loureiro et al. 2020).Regarding this aspect, it is important to distinguish between true cryptic species and pseudo-cryptic species.The latter can be discerned through fine-scale morphological analyses, although they may appear cryptic when using traditional morphological techniques (Lajus et al. 2015).One example of pseudo-cryptic species among bats is that of Chiroderma gorgasi and C. trinitatum, which are genetically distinguishable but only differ in the presence/absence of an accessory cusp on the second lower premolar (Lim et al. 2020).
In addition to recognized species, we could successfully identify two cryptic/pseudo-cryptic lineages that were previously classified as M. molossus.Molossus spp.comprises two lineages, one distributed in Argentina (M.paranaensis), and the other in northern countries of South America (Molossus sp.2).The separation between M. paranaensis and M. molossus, is supported by both the phylogeny and genetic distances which positions this value within the range of interspecific distances (Fig. 1).Interestingly, Molossus sp. 2 groups with M. molossus in the mtDNA phylogeny, but is allied to M. paranaensis in the full dataset, and, depicts similar levels of genetic differentiation with both, within the range of intraspecific variation (Fig. 1).A possible scenario is that M. paranaensis and M. molossus are different species, and that Molossus sp. 2 is the product of hybridization between either species or other unsampled taxa, given the geographical separation between M. paranaensis and Molossus sp. 2. However, the fragmentary availability of sequences representing Molossus sp. 2 (there is no single individual with both mtDNA loci sequenced) requires further analyses to corroborate this hypothesis.
The morphological analyses mirror the results of the molecular analysis.The PCA of 12 cranial morphomet-ric measurements allowed to distinguish M. paranaensis from all other genetically defined species occurring in Argentina.However, taking into account that the distinction from M. melini is only slightly statistically significant, and that all M. paranaensis specimens analyzed were previously misclassified as M. molossus, according to their external morphology, we can consider these species as pseudo-cryptic (based on differences in morphology and morphometry showed above), as occurs with many other species groups in the genus.It is noteworthy that all genetically corroborated specimens of M. molossus are more differentiated from M. paranaensis, compared to those that were not sequenced (File S1 [Fig.SM1]).Although there could be some degree of misidentification among those samples, we decided to keep them in the analysis shown in File S1 [Figure SM1] because PCA aims to find principal components that capture the maximum variance, and to accurately estimate the variance and covariance of a dataset, a representative sample of data points is needed.It is notable that M. currentium, the only species that lacks genetically confirmed specimens in the PCA dataset (File S1 [Fig.SM1, Table SM3]), is sufficiently distant from M. paranaensis, corroborating that these are two different entities.Furthermore, in the exploratory 2D geometric morphometric analysis conducted for nasal cavity shape comparisons (File S1 [Fig.SM3]), it was observed that the species M. melini, which exhibits significant size overlap with M. paranaensis and M. molossus, distinctly separates from them.This observation suggests that subtle shape variations may not be captured when only size-based comparisons (classic morphometry) are considered.
The integration of morphological and molecular findings leads to the conclusion that all Molossus lineages present in Argentina represent non-cryptic species.Among them, M. fluminensis stands out as the most distinct, notably larger than other species.Despite similar sizes and external morphologies, M. molossus, M. melini, M. currentium, and M. paranaensis can be reliably distinguished by cranial characteristics, categorizing them as pseudo-cryptic species.While field observers may struggle to differentiate these taxa, detailed examination of cranial characters or molecular diagnostic techniques can definitively identify each species.Fur color has been suggested as a marker for species identification in these bats; however, fur coloration can vary across habitats, leading to uncertain identifications.For instance, M. melini was previously distinguished from M. molossus by its characteristic ochraceous to orange fur coloration, but instances of chocolate to grayish brown specimens have been reported recently in Brazil and Argentina, indicating exceptions to this previously stated rule (Olímpio et al. 2024; this study) (Fig. S7).
The approach implemented in this study facilitated a revision of the diversity and geographic range of Molossus in Argentina, marking the southernmost limits of the genus' distribution.We confirmed the presence of three of the four species previously reported for the country, as well as reported the presence of the new species M. paranaensis (Fig. 6).Molossus molossus is the species with the broadest distribution, being the only species sampled in the northwestern region (Salta and Jujuy provinces) but also overlapping with the distribution of all remaining lineages, in agreement with previous assessments (Barquez and Díaz 2020).Molossus fluminensis was found in the northeastern region (Corrientes and center/north of Santa Fe), hence, according to our results, the distribution of the species (Barquez and Díaz 2020;Pavé et al. 2021) should be extended, being more likely that the two disjunct distributions are indeed connected (Fig. 6).Molossus melini was found in the south and center of Santa Fe and Entre Ríos, but also in Buenos Aires, expanding the known distributional boundaries of this species, which is characterized by an extremely limited distribution, with only four previous localities documented so far (Montani et al. 2021(Montani et al. , 2023;;Pavé et al. 2023;Olímpio et al. 2024).Molossus paranaensis is also distributed along the Paraná River, overlapping with M. molossus, M. fluminensis, and M. melini.Despite sampling mostly within the distribution range of M. currentium, no living samples compatible with this species were identified in our study.This result is particularly intriguing as the majority of our samples were collected from areas expected to harbor M. currentium, specifically the area of influence of the Paraná River (Barquez and Díaz 2020).One possibility that we considered is that M. paranaensis could indeed be M. currentium, but this was discarded by both molecular-phylogenetic and morphometric evidence.Molossus currentium appears to possess an elusive nature, as evidenced by the significant scarcity of available sequences and limited understanding regarding its distributional range and conservation status (Montani et al. 2019).Therefore, more research is needed to clarify its situation.

Conclusion
In this study, we conducted a review of Molossus bat diversity in Argentina and provided a curated dataset of genetic sequences.This dataset can serve as a valuable resource for testing the phylogenetic assignment of any Molossus specimen across its entire range.Additionally, we compiled a dataset of linear metric variables for five species within the genus, which inhabit central-east Ar-gentina and south-east Brazil.This dataset can facilitate future morphometric comparisons.In the future, other lines of evidence such as echolocation data, or genomic approaches may complement the information provided in this study to have a deeper insight into the differences between the species of Molossus, especially those found at the southern limits of its distribution.
Based on molecular and morphometric evidence, we propose the existence of a novel species within the genus.We believe that this study can aid various stakeholders, including the research community, conservationists, policymakers, and zoonotic surveillance laboratories, to name some, in the challenging task of identifying unknown specimens.The systematics of this genus still has something to say.
to expand the biological collections.We would like to thank María Eugenia Montani of the vertebrate collection from the Museo Gallardo for her generosity in sample sharing and for a critical discussion of the main results of this study.We would also like to acknowledge Pablo Teta and Alejandro Giraudo, for their expert recommendations in the description of the species.We thank Alejandra Gavazza for some illustrations.To Leonardo Leiva, curator of the vertebrate collection from the Museo Florentino Ameghino and Germán Saigo, of the vertebrate collection from the Museo Gallardo, for providing access to and lending us specimens.

Figure 1 .A
Figure 1.A Bayesian phylogeny obtained from the concatenated dataset (FGB, COI, and cyt b).Branch colors indicate lineage/ species membership.The letters accompanying each terminal indicate the lineage based on the analysis of individual genes, while the numbers refer to the number of representatives of each lineage.Node sizes are proportional to their posterior probability (red: 1-0.95; purple-cyan: 0.94-0.75;green-yellow: <0.75).Node support above 0.8 is shown for main lineages.The scale is expressed in substitutions per site.B Uncorrected p distances.Histogram showing intra-and interspecific distances based on the concatenated dataset (FGB, COI, and cyt b).Different colors indicate intraspecific distances (pink), interspecific distances (dark gray), intraspecific distance of M. aztecus (green), distance between Molossus sp.nov.and M. molossus (yellow), distance within Molossus sp.nov.and Molossus sp. 2 (light blue), distance between Molossus sp.nov.and M. melini (red), and distance between M. molossus and Molossus sp. 2 (purple).

Figure 2 .
Figure 2. A Genetic confidence score (GIS) was defined for each species group by weighted consideration of known sequences (see File S1 for equation details).Univariate pairwise comparisons of external (B) and cranial measurements (C) of Molossus species present in Argentina (and Brazil) based on genetically confirmed specimens (M.fluminensis, M. melini, M. molossus, and Molossus sp.nov.).Box-and-whisker plots show data distributed across quartiles, the vertical lines indicate the ranges, and outliers are shown as separate data points.*, **, and *** represent significant pairwise differences among species.

Figure 3 .
Figure 3. Principal component analysis (PCA) of selected cranial metric variables (GLS wo.i, CBL, PC, BB, ZB, MB, LMxT, PL, C-C, M2-M2, LM and LMdT) of genetically confirmed specimens of Molossus distributed in Argentina.A PCA results are presented in a biplot chart with individuals (dots) and variables (blue arrows) plotted together in the PC1 vs PC2 space.Individuals of the same species are enclosed by a convex hull.The mass centroid of each group is indicated by an unlabeled plus sign.Data labels are referenced in File S1 (TableM3).Dot sizes of each specimen is proportional to its genetic information availability score (GIS, see File S1 for score definition details).B Percentages of each cranial variable's contribution to the first two principal components (Dim1 and Dim2).

Finally
, we would like to thank Priscila Medeiros Olímpio for kindly sharing measurements of Brazilian samples.This study was funded by the grants PIP-0587 awarded by the National Scientific and Technical Research Council of Argentina to Diego Caraballo, PICT-2019-03347 awarded by the National Agency for the Promotion of Science and Technology (Ministry of Science) from Argentina to Valeria Colombo, the Instituto Nacional de Enfermedades Infecciosas ( INEI-ANLIS) Dr. Carlos G. Malbrán to Daniel Cisterna, CAI + D Orientado 2016 (Res.C.S. N° 632/17), PIBAA (28720210100082CO) awarded by the National Scientific and Technical Research Council of Argentina (CONICET) to Romina Pavé, Universidad Nacional del Litoral and the Agencia Santafesina de Ciencia, Tecnología e Innovación (ASACTEI), Gobierno de la Provincia de Santa Fe (Código IO-2017-00068).

Table 1 .
External and cranial measurements of the type series of Molossus paranaensis sp.nov.For descriptions of the abbreviations of measurements see "Methods".