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.

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.

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. 2021, 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.

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 1×10⁷ 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 2×10⁷ 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 1.00–1.02 for 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.

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).

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).

We recorded the following data of each specimen analyzed: locality, age class (i.e., subadult or adult), sex, external and cranial measurements, and body mass, following Barquez et al. (1999). External and cranial measurements were obtained for 64 adult specimens of Molossus species from Argentina, including six M. currentium, 12 M. fluminensis, 13 M. melini, 20 M. molossus, and 13 specimens of the novel Molossus species (Molossus sp. nov.). Measurements were taken using a digital caliper, supplemented in some cases by data obtained from the existing literature (Thomas 1901; Cláudio et al. 2020; Montani et al. 2021; 2023; Pavé et al. 2023; Olímpio et al. 2024). We included five external [total length (ToL), tail length (TL), hindfoot length (HFL), ear length (EL), forearm length (FA)] and 14 cranial measurements [greatest length of skull with incisors (GLS w.i), greatest length of skull excluding incisors (GLS wo.i), condylobasal length (CBL), postorbital constriction (PC), braincase breadth (BB), zygomatic breadth (ZB), mastoideal breadth (MB), maxillary toothrow length (LMxT), palatal length (PL), width across canines (C-C), width across second molars (M2-M2), mandible length (LM), mandibular toothrow length (LMdT), height of the sagittal crest (SAR)] for each species. In some cases, information was recorded directly from labels of museum specimens (File S1).

Univariate (using one-way ANOVA or Kruskal–Wallis test with corresponding pairwise post hoc tests) and multivariate statistical analyses (employing principal component analysis—PCA—coupled with PERMANOVA and its corresponding pairwise post hoc tests) of cranial and external linear measurements were conducted in R Studio (RStudio Team 2020) utilizing R version 4.2.1 (R Core Team 2021). The following R packages were utilized: FactoMineR version 2.8 (Lê et al. 2008), ggplot2 version 3.4.3 (Wickham 2016), Vegan version 2.6–4 (Oksanen et al. 2008) and pairwiseAdonis (downloaded from the author’s github site: pmartinezarbizu/pairwiseAdonis/pairwiseAdonis). For shape based comparisons, an exploratory two dimensional geometric morphometric (2D GMM) study of nasal cavity perimeter of the three species that showed the lesser morphological differentiation (M. molossus, M. melini, and Molossus sp. nov.) was performed. The tpsDig2 version 2.32 (https://sbmorphometrics.org) was employed for manual definition of 13 landmarks and one semi-landmark on picture files (*.jpg) of rostral views. The R based geomorph package version 4.0.6 (https://cran.r-project.org/web/packages/geomorph/index.html) was used for Generalized Procrustes Analysis (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.

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).

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.

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).

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.

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 al. 2007; 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.

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.

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 measurements (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).

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.

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 variables 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).

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 (Table 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).

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:

Family Molossidae Gervais, 1856

Genus Molossus É. Geoffroy, 1805

 Molossus paranaensis Caraballo, Pavé, Argoitia, Schierloh & Chambi Velasquez, sp. nov.

https://zoobank.org/8608EDEB-5643-4482-9E73-C7699832742E

Chresonymy

Molossus molossus – Pavé et al. (2017: 158)

Molossus molossus – Caraballo et al. (2020: 4)

Molossus molossus – Montani et al. (2021: 17)

Molossus molossus – Pavé et al. (2021: 17)

Molossus molossus – Pavé et al. (2023: 423)

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, Argentina (lat. –31.4257, long. –60.9912, 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.

Table 1.

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External and cranial measurements of the type series of Molossus paranaensis sp. nov. For descriptions of the abbreviations of measurements see “Methods”.

	Holotype MFA-ZV-M 1494		Paratypes
		INALI-A 389	INALI-A 390	INALI-A 457	INALI-A 588	INALI-A 589	MFA-ZV-M 1414	MG-ZV-M 176	MG-ZV-M 208	MACN-Ma 30420	MACN-Ma 30878
Sex	male	female	female	female	male	male	male	female	female	male	male
ToL	107.5	—	99	98	115	100	92	109.14	103	90.3	112.2
TL	40.5	—	39	—	38	33	35	35.4	37	29.5	37.4
HFL	8	—	8.5	8.5	8.5	9.2	7.2	10.73	9	6.7	6.2
EL	14.5	—	12	—	11	11.8	8.3	12	12	10	11
FA	42.84	39.4	41	42.7	40.7	40.6	41.8	40.68	41.3	39.4	41.4
W	22	—	—	17.3	23.5	22	—	22	19	—	18.8
CBL	18.8	16.3	16.5	16.7	18.2	17.2	—	16	16.7	—	16.9
ZB	12	11.4	11.1	11.5	12.2	12.3	—	11.4	11.5	—	11.2
GLS w.i	19	18.2	18.6	18.7	19.2	18.9	—	18.7	18.3	—	17.3
GLS wo.i	18.6	17.7	17.4	16.2	16.8	16.5	—	17.7	17.6	—	16.6
PC	4.3	4.3	4.2	4.5	4.6	4.6	—	4.3	4.2	—	3.8
BB	9.2	10.1	9.6	9.6	9.9	9.9	—	9.2	9.2	—	8.8
LMxT	7.4	6.7	6.5	6.9	7.2	7.2	—	6.8	6.9	—	6.3
PL	6.6	5.5	5.6	5.8	6.1	6.5	—	5.7	5.4	—	6.1
MB	10.4	11	10.1	11.2	11.5	11.6	—	10	10.2	—	10.8
LM	13.5	12.5	12.3	12.7	13.3	13.2	—	12.7	12.6	—	12.3
LMdT	7.9	7.8	7.3	7.4	7.6	8	—	7.5	7.7	—	7.5
C-C	5	4.9	4.6	5.3	5.5	5.4	—	4.8	5	—	4.5
M2-M2	8.6	8.6	8.2	8.4	9.1	8.2	—	8	8.1	—	7.9
SAR	1.2	1	1	0.9	1	1.2	—	1.2	0.9	—	—

Figure 4. 

Dorsal (A) and ventral (B) views of the skin of the holotype of Molossus paranaensis (MFA-ZV-M 1494), adult male. Scale bar = 10 mm.

Figure 5. 

Lateral view of skull and mandible (A), and frontal (B), ventral (C) and dorsal (E) views of the skull of the holotype of Molossus paranaensis (MF-ZV-M 1494). Scale bar = 10 mm.

Paratypes

INALI-A 389 and 390 adult females, with INALI-A 389 preserved as a skull specimen only, while both specimens are preserved in 70% alcohol, collected on October 2017 at “Desvío Arijón”, San Jerónimo, Santa Fe province, Argentina (lat. –31.88, long. –60.89, 17 m); MG-ZV-M 176 adult female preserved as skin, skull and postcranial skeleton, collected on 16 May 2015 at “Reserva Hídrica Río Carcarañá”, Area Natural Protegida, Pueblo Andino, Iriondo, Santa Fe province, Argentina (lat. –32.67, long. –60.87, 25 m); MG-ZV-M 208 adult female preserved as skin, skull, and postcranial skeleton, collected on 7 May 2016 at “Parque Villarino”, Zavalla, Rosario, Santa Fe province, Argentina (lat. –33.03, long. –60.89, 57 m); MG-ZV-M 296 and MFA-ZV-M 1414 subadult males preserved in 70% alcohol from Rosario, Santa Fe province, Argentina (lat. –32.69, long. –60.72, 30 m); INALI-A 457 adult female preserved as skull and in 70% alcohol, collected on 7 March 2018 at “Estancia Las Gamas”, Vera, Santa Fe province, Argentina (lat. –29.42, long. –60.38, 62 m); INALI-A 588 and 589 adult males with scrotal testes, preserved as skin, skull and postcranial skeleton the first and in 70% alcohol the second, collected on 8 December 2018 at “Establecimiento Inchala”, La Picada, Paraná, Entre Ríos province, Argentina (lat. –31.74, long. –60.26, 29 m); MACN-Ma 30878 an adult male preserved as skull and in 70% alcohol, collected on 7 April 2017 at “Campus Universitario ­F­­a­­C­E­N­A­-UNNE”, Corrientes city, Corrientes province, Argentina (lat. –27.47, long. –58.78, 61 m); MACN-Ma 30420 adult male preserved in 70% alcohol from Tigre, Buenos Aires province, Argentina (lat. –34.42, long. –58.57, 4 m).

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. –31.63, long. –60.71, 20 m), obtained tissue samples, marked with a haircut and then released (collector Valeria Colombo number: MR 192, 193, 194, 197, 198).

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 (FA 39.4–42.8 mm; GLS w.i 17.3–19.2 mm; PC 3.8–4.6 mm; LMxT 6.3–7.4 mm); 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: 39.4–42.84 mm, 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 39.4–42.8 mm 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 (triangular in M. paranaensis and square in M. molossus; Loureiro et al. 2018a), the development of lambdoidal crests (moderately developed in M. paranaensis and underdeveloped in M. molossus), and in several cranial measurements (see Figs 2, 3; Table 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 3.8–4.6 mm in M. paranaensis), and LMdT (6.4–7.9 mm in M. melini and 7.3–8.0 mm in M. paranaensis) (Figs 2, 3; Table 2).

Table 2.

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Diagnostic external and cranial characters for each Molossus species following Loureiro et al. (2018b, 2019, 2020) and Montani et al. (2021). References: NA, not available; *: data from this study.

Species	Forearm (mm)	Dorsal hair color	Dorsal hair basal band	GLS with incisors (mm)	Upper incisors	Occipital region	Infra-orbital foramen direction	Format of rostrum
M. alvarezi	> 42	Chocolate (cocoa) brown	Bicolor. Large band (1/2)	19.3 (19.0–20.1)	Pincer-like	Triangular	Lateral	NA
M. aztecus	< 42	Dark brown	Unicolor	17.2 (16.5–18.3)	Spatulated	Quadrangular	Lateral	Triangular
M. bondae	< 44	Dark to reddish brown (coffee brown to blackish)	Unicolor	NA (17.3–19.4)	Spatulated	Quadrangular	Lateral	NA
M. coibensis	< 38	Medium to dark brown (cocoa brown to blackish)	Unicolor	16.0 (14.9–16.9)	Spatulated	Quadrangular	Frontal	Square
M. currentium	< 45	Dark brown (coffee brown to blackish)	Bicolor	18.3 (17.9–19.4)	Spatulated	Quadrangular	Lateral	Square
M. fentoni	< 36	Medium to dark brown	Bicolor. Short band (1/4)	15.3 (15.2–16.8)	Pincer-like	Triangular	Lateral	NA
M. fluminensis	> 46	Dark brown to blackish	Uni or bicolor. Short band (1/4)	NA (19.0–23.2)	Pincer-like	Quadrangular	Lateral	Triangular
M. melini	> 41	Orange to dark brown (yellow ocher, clay, cinnamon and grayish brown)	Bicolor. Short band (1/4)	18.7 (17.8–19.5)	Pincer-like	Triangular	Lateral	Triangular*
M. milleri	< 40	Medium to dark brown	Bicolor. Variable band (1/4–1/2)	16.1 (15.8–16.4)	Pincer-like	Triangular	Frontal	NA
M. molossus	< 40	Light to dark brown (cinnamon to cocoa and grayish brown)	Bicolor. Variable band (1/4–1/2)	17.4* (17.0–17.9)*	Pincer-like	Triangular	Frontal	Square
M. nigricans	> 47	Dark brown to blackish	Uni or bicolor. Short band (1/4)	20.1–24.1	Pincer-like	Quadrangular	Lateral	Triangular
M. paranaensis	> 39*	Medium brown (cinnamon to grayish brown)*	Bicolor. Variable band (1/4–1/2)*	18.5 (17.3–19.2)*	Pincer-like*	Triangular*	Frontal*	Triangular*
M. pretiosus	> 44	Dark brown to blackish or reddish (burnt umber to tawny)	Uni or Bbicolor. Short band (1/3)	20.5 (18.9–22.4)	Spatulated	Quadrangular	Lateral	Square
M. rufus	> 46	Dark brown to blackish	Uni or bicolor. Short band (1/4)	22.1 (19.9–23.8)	Pincer-like	Quadrangular	Lateral	Triangular
M. sinaloae	> 46	Dull, dark brown	Bicolor. Large band (2/3)	21.0 (19.4–22.4)	Pincer-like	Triangular	Lateral	Triangular
M. verrilli	< 41	Medium to dark brown	Bicolor. Large band (1/2)	17.2 (17.0–17.4)	Pincer-like	Triangular	Frontal	NA

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.

For each specimen, the localities are listed alphabetically by province/state, department, and specific site, and between parentheses: Geographical coordinates, collection acronym and number. The biological collections and their specimens acronyms are: Natural History Museum (formerly British Museum of Natural History, London, United Kingdom (BMNH); Universidade Federal de São Carlos, Campus Sorocaba, Sorocaba, Brazil (ZSP); Colección de Mamíferos Lillo, San Miguel de Tucumán, Tucumán, Argentina (CML); Colección de Vertebrados of the Instituto Nacional de Limnología, La Capital, Santa Fe, Argentina (INALI-A); Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Ciudad Autónoma de Buenos Aires, Argentina (MACN-Ma); Colección de Mamíferos of the Museo Provincial de Ciencias Naturales "Florentino Ameghino”, Santa Fe, Santa Fe, Argentina (MFA-ZV-M); Colección de Mamíferos of the Museo Provincial de Ciencias Naturales “Dr. Ángel Gallardo”, Rosario, Santa Fe, Argentina (MG-ZV-M). Reference: * taken from bibliography.