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Does the widely distributed rodent Calomys tener (Cricetidae: Sigmodontinae) constitute a single evolutionary unit?

ABSTRACT

The nominal species Calomys tener (Winge, 1887) ranges broadly in open lands of the Caatinga, Cerrado, Pantanal and Mata Atlântica of Brazil, and was recently reported from the Pampas of southern Brazil, and in the Selva Paranaense of eastern Paraguay and northeastern Argentina. This rodent can be infected with the pathogenic Araraquara hantavirus in Brazil. Given that most epidemiological studies have not taken into account updated taxonomic findings of their rodent hosts, in this study, we obtained sequence data of the Cyt-b and COI genes of specimens of C. tener from 22 different geographical localities from throughout the currently known distribution of the species (including individuals from Argentina, Paraguay, Bolivia, and Brazil) to test if it constitutes a single genetic unit or if it presents genetic discontinuities that may represent different evolutionary lineages. Phylogenetic analyses including several species of Calomys recovered several clades with strong support. Regarding C. tener, it is recovered as sister to the node that cluster C. laucha (Fischer, 1814) sensu lato, C. expulsus (Lund, 1841) and species in the C. callosus (Rengger, 1830) species complex. At the intraspecific level there are no genetic gaps among haplotypes of C. tener that could suggest more than one species. The recent captures in the Pampas of southern Brazil and in the Selva Paranaense suggest that the species may be colonizing new geographic areas.

KEY WORDS:
Cyt-b; phylogenetic relationships; South America

INTRODUCTION

Geographic ranges are dynamic properties of species, easily modified in response to biotic and abiotic drivers (Case et al. 2005Case TJ, Holt RD, McPeek MA, Keitt TH (2005) The community context of species’ borders: ecological and evolutionary perspectives. Oikos 108(1): 28-46. https://doi.org/10.1111/j.0030-1299.2005.13148.x
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, Soberon 2007Soberon J (2007) Grinnellian and Eltonian niches and geographic distribution of species. Ecology Letters 10(12): 1115-1123. https://doi.org/10.1111/j.1461-0248.2007.01107.x
https://doi.org/10.1111/j.1461-0248.2007...
, Linder et al. 2012Linder HP, Bykova O, Dyke J, Etienne RS, Hickler T, Kühn I, Marion G, Ohlemüller R, Schymanski SJ, Singer A (2012). Biotic modifiers, environmental modulation and species distribution models. Journal of Biogeography 39(12): 2179-2190. https://doi.org/10.1111/j.1365-2699.2012.02705.x
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, Simonov 2014Simonov PS (2014) Influence of natural and anthropogenic factors on the distribution of the field mouse in the Sikhote-Alin mountains. Achievements in the Life Sciences 8(1): 85-87. https://doi.org/10.1016/j.als.2014.06.001
https://doi.org/10.1016/j.als.2014.06.00...
). New locality records, new country records, or new habitat records ought to be documented as they may indicate range extensions resulting from demographic expansions and/or deficiency in species geographical samplings. Cases like these have been observed for example in Loxodontomys micropus (Waterhouse, 1837) (see Cañon et al. 2010Cañon C, D’Elia G, Pardiñas UFJ, Lessa EP (2010) Phylogeography of Loxodontomys micropus with comments on the alpha taxonomy of Loxodontomys (Cricetidae: Sigmodontinae). Journal of Mammalogy 91(6): 1449-1458. https://doi.org/10.1644/10-MAMM-A-027.1
https://doi.org/10.1644/10-MAMM-A-027.1...
), in Dromiciops gliroides Thomas, 1894 (see D’Elia et al. 2016D’Elia G, Hurtado N, D’Anatro A (2016) Alpha taxonomy of Dromiciops (Micrtobiotheriidae) with the description of 2 new species of monito de monte. Journal of Mammalogy 97(4): 1136-1152. https://doi.org/10.1093/jmammal/gyw068
https://doi.org/10.1093/jmammal/gyw068...
, but also see Suárez-Villota et al. 2018Suárez-Villota EY, Quercia C, Nuñez J, Gallardo M, Himes CM, Kenagy GJ (2018) Monotypic status of the South American relictual marsupial Dromiciops gliroides (Microbiotheria). Journal of Mammalogy 99(4): 803-812. https://doi.org/10.1093/jmammal/gyy073
https://doi.org/10.1093/jmammal/gyy073...
), among many others.

Despite a renewed interest in the taxonomy and systematics of South American sigmodontine rodents, many taxa still lack detailed analyses of morphological, geographical, genetic and ecological variation; thus, the alpha-taxonomy of these groups is only partially resolved. Detailed morphological and genetic studies provide insights into the biology and evolution of species with large anthropogenic impact, as is the case of species of zoonotic or agriculture concern (for example, Yackulic et al. 2010Yackulic CB, Sanderson EW, Uriarte M (2010). Anthropogenic and environmental drivers of modern range loss in large mammals. Proceedings of the National Academy of Sciences of the United State of America 108(10): 4024-4029. https://doi.org/10.1073/pnas.1015097108
https://doi.org/10.1073/pnas.1015097108...
, Pigot and Tobias 2013Pigot AL, Tobias JA (2013) Species interactions constrain geographic range expansion over evolutionary time. Ecology Letters 16(3): 330-338. https://doi.org/10.1111/ele.12043
https://doi.org/10.1111/ele.12043...
, Bordes et al. 2015Bordes F, Blasdell K, Morand S (2015) Transmission ecology of rodent-borne diseases: new frontiers. Integrative Zoology 10(5): 424-435. https://doi.org/10.1111/1749-4877.12149
https://doi.org/10.1111/1749-4877.12149...
). An example of this is the genus Calomys Waterhouse, 1837 where several species share similar morphological and morphometric characteristics, many of which are only recently being duly appreciated (Almeida et al. 2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
, Haag et al. 2007Haag T, Muschner VC, Freitas LB, Oliveira LFO, Langguth AR, Mattevi M (2007) Phylogenetic relationships among species of the genus Calomys with emphasis on South American lowland taxa. Journal of Mammalogy 88(3): 769-776. https://doi.org/10.1644/05-MAMM-A-319R1.1
https://doi.org/10.1644/05-MAMM-A-319R1....
, Salazar-Bravo 2015Salazar-Bravo J (2015) Genus Calomys Waterhouse, 1837. In: Patton JL, Pardiñas UFJ, D’Elia G (Eds) Mammals of South America. Chicago, The University of Chicago Press, vol. 2, 504-505. https://doi.org/10.7208/chicago/9780226169606.001.0001
https://doi.org/10.7208/chicago/97802261...
). A couple of the species in the genus have received recent interest, for example Calomys laucha (Fischer, 1814) (González-Ittig et al. 2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
) or Calomys sorellus (Thomas, 1900) (Zeballos et al. 2014Zeballos H, Palma RE, Marquet PA, Ceballos G (2014) Phylogenetic relationships of Calomys sorellus complex (Rodentia: Cricetidae), with the description of two new species. Revista Mexicana de Mastozoología (Nueva Época) 4(1): 1-23.); in both bases, cryptic species within currently recognized taxonomic units were suggested.

One species of the genus that needs a study of its taxonomic situation is Calomys tener (Winge, 1887), the Delicate Vesper Mouse, a small body-sized species, broadly distributed in open vegetative formations in the Cerrado of central Brazil and eastern Bolivia, Caatinga and Pantanal in Brazil (Salazar-Bravo 2015Salazar-Bravo J (2015) Genus Calomys Waterhouse, 1837. In: Patton JL, Pardiñas UFJ, D’Elia G (Eds) Mammals of South America. Chicago, The University of Chicago Press, vol. 2, 504-505. https://doi.org/10.7208/chicago/9780226169606.001.0001
https://doi.org/10.7208/chicago/97802261...
). Several novel reports of occurrence for C. tener have been published: for example, the species was recently reported in the Atlantic and Paranaense forests of Argentina and Paraguay and areas in southeastern Brazil (González-Ittig et al. 2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
, Quintela et al. 2014Quintela FM, da Silveira EC, Dellagnese DG, Cademartori CV (2014) Calomys tener (Winge, 1887) (Rodentia: Cricetidae:Sigmodontinae): Filling gaps. Check List 10(3): 650-654. https://doi.org/10.15560/10.3.650
https://doi.org/10.15560/10.3.650...
), all of which were considered outside of the range of the species (Salazar-Bravo 2015Salazar-Bravo J (2015) Genus Calomys Waterhouse, 1837. In: Patton JL, Pardiñas UFJ, D’Elia G (Eds) Mammals of South America. Chicago, The University of Chicago Press, vol. 2, 504-505. https://doi.org/10.7208/chicago/9780226169606.001.0001
https://doi.org/10.7208/chicago/97802261...
). The ecology of C. tener is better known in Brazil, where it occupies a mix of habitat types, including domestic and agricultural areas, and populations appear to be stable all year-long (Umetsu and Pardini 2007Umetsu F, Pardini R (2007) Small mammals in a mosaic of forest remnants and anthropogenic habitats-evaluating matrix quality in an Atlantic forest landscape. Landscape Ecology 22(4): 517-530. https://doi.org/10.1007/s10980-006-9041-y
https://doi.org/10.1007/s10980-006-9041-...
). Individuals in a handful of populations in São Paulo state are known to be infected with Araraquara hantavirus, currently considered the most pathogenic of all South American hantaviruses (Figueiredo et al. 2010Figueiredo GG, Borges AA, Campos GM, Machado AM, Saggioro FP, Sabino Junior GS, Badra SJ, Ortiz AA, Figueiredo LT (2010). Diagnosis of hantavirus infection in humans and rodents in Ribeirao Preto, state of São Paulo, Brazil. Revista da Sociedade Brasileira de Medicina Tropical 43(4): 348-354. https://doi.org/10.1590/S0037-86822010000400002
https://doi.org/10.1590/S0037-8682201000...
). It has been estimated that more than 50% of total hantavirus pulmonary syndrome (HPS) cases in Brazil are caused by Araraquara hantavirus and that the human fatality rate is over 40% for infected patients (Figueiredo et al. 2009Figueiredo LTM, Moreli ML, Sousa RLM, Borges AA, Figueiredo GG, Machado AM, Bisordi I, Nagasse-Suqahara TK, Suzuki A, Pereira LE, Souza RP, Souza LT, Braconi CT, Harsi CM, Andrade-Zanotto PM (2009). Hantavirus pulmonary syndrome, central plateau, southwestern, and southern Brazil. Emerging Infectious Disease 15(4): 561-567. https://doi.org/10.3201/eid1504.080289
https://doi.org/10.3201/eid1504.080289...
). Although Necromys lasiurus (Lund, 1840) is the natural reservoir of this viral genotype, the role of C. tener in the circulation of the virus is not completely understood; however, reports of the occurrence of this species in other areas of Brazil and elsewhere in South America should be considered an element of risk for the potential presence of this pathogenic virus in these areas. As suggested by Mills and Childs (1998Mills JN, Childs JE (1998) Ecological studies of rodent reservoirs: their relevance for human health. Emerging Infectious Disease 4(4): 529-537. https://doi.org/10.3201/eid0404.980403
https://doi.org/10.3201/eid0404.980403...
) and Mills et al. (1999Mills JN, Ksiazek TG, Peters CJ, Childs JE (1999) Long-term studies of hantavirus reservoir populations in the southwestern United States: a synthesis. Emerging Infectious Disease 5(1): 135-142. https://doi.org/10.3201/eid0501.990116
https://doi.org/10.3201/eid0501.990116...
), reservoir studies, including those related to their systematics, taxonomy, and geographic distribution, are necessary for a comprehensive understanding of the biology of emerging zoonotic diseases.

In the case of C. tener, different molecular systematic studies have included sequences from few localities (see Table 1). For example, Salazar-Bravo et al. (2001Salazar-Bravo J, Dragoo JW, Tinnin DS, Yates TL (2001) Phylogeny and evolution of the Neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 20(2): 173-184. https://doi.org/10.1006/mpev.2001.0965
https://doi.org/10.1006/mpev.2001.0965...
) included sequences of the mitochondrial cytochrome b (Cyt-b) gene from individuals of one locality in Bolivia and one in Brazil. Almeida et al. (2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
) used the sequences of Salazar-Bravo et al. (2001Salazar-Bravo J, Dragoo JW, Tinnin DS, Yates TL (2001) Phylogeny and evolution of the Neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 20(2): 173-184. https://doi.org/10.1006/mpev.2001.0965
https://doi.org/10.1006/mpev.2001.0965...
) and added Cyt-b data of individuals from five sites in Brazil. Haag et al. (2007Haag T, Muschner VC, Freitas LB, Oliveira LFO, Langguth AR, Mattevi M (2007) Phylogenetic relationships among species of the genus Calomys with emphasis on South American lowland taxa. Journal of Mammalogy 88(3): 769-776. https://doi.org/10.1644/05-MAMM-A-319R1.1
https://doi.org/10.1644/05-MAMM-A-319R1....
) also used the sequences obtained by Salazar-Bravo et al. (2001Salazar-Bravo J, Dragoo JW, Tinnin DS, Yates TL (2001) Phylogeny and evolution of the Neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 20(2): 173-184. https://doi.org/10.1006/mpev.2001.0965
https://doi.org/10.1006/mpev.2001.0965...
) and added molecular information of the same gene from individuals from three extra localities in Brazil. González-Ittig et al. (2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
) in a study focused on the systematics of C. laucha, identified three individuals from Paraguay and Argentina that actually corresponded to C. tener. The results of all these studies have recovered C. tener as monophyletic without any phylogenetic structure or sub-clades, however, these conclusions are partial since none have integrated the whole information available for the species. To detect possible genetic gaps among specimens of this species over a broad geographic area, in the present study we compared sequences of the mitochondrial genes Cyt-b and cytochrome oxidase subunit 1 (COI) of individuals from Argentina, Paraguay, Bolivia, and Brazil, encompassing most of the known distribution of the rodent.

MATERIAL AND METHODS

Partial sequences of the Cyt-b (788bp) of 31 individuals identified as C. tener were used for this study: from Brazil (n = 19), Bolivia (n = 1), Paraguay (n = 4), and Argentina (n = 7) and five partial sequences of the COI from Brazil, giving a total of 22 sampled localities (Fig. 1, Table 1). The five sequences of the COI gene were published by Müller et al. (2013Müller L, Gonçalves GL, Cordeiro-Estrela P, Marinho JR, Althoff SL, Testoni AF, González EM, Freitas TRO (2013) DNA barcoding of sigmodontine rodents: identifying wildlife reservoirs of zoonoses. PLoS ONE 8(11): e80282. https://doi.org/10.1371/journal.pone.0080282
https://doi.org/10.1371/journal.pone.008...
). Of the Cyt-b sequences, 19 were published in different studies listed in Table 1, whereas 12 were generated at the lab of Jorge Salazar-Bravo (Texas Tech University, Lubbock, Texas, USA) from samples obtained on loan from several institutions (see Acknowledgements).

Figure 1
Geographical locations of individuals listed in Table 1. Vegetation formations are represented by ecoregions like Cerrado, Humid Chaco, Rainforests (including the phytogeographic regions Alto Paraná Atlantic forest + Araucaria moist forest + Serra do Mar coastal forest), Dry Forests (Mato Grosso seasonal forest + Atlantic dry forest) and Savannas (Southern cone Mesopotamian savanna + Uruguayan savanna). The star indicates the type locality of Calomys tener: Lagoa Santa in the state of Minas Gerais corresponding to the Cerrado ecoregion. This map was designed using free spatial data of QGIS (Quantum GIS Development Team, 2018), available at: http://qgis.osgeo.org.

Table 1
Field identification code or voucher numbers of specimens of the Delicate Vesper Mouse (Calomys tener). The location of the sampling sites shown in Fig. 1 with provinces or departments in parentheses; geographic coordinates; Cyt-b GenBank accession numbers (there are five accession numbers corresponding to the cytochrome oxidase I gene indicated in parenthesis with COI) and articles were the sequences were obtained.

Tissues were processed for DNA extraction using DNeasy Blood and Tissue kit (Qiagen, Cat# 69505) and the manufacturer’s recommendations. DNA was quantified using Nanodrop (NanoDropTM 1000 Spectrophotometer v3.7) and 1% agarose gels. A fragment of the mitochondrial Cyt-b gene was amplified using one of two primer combinations of Mus14095-F and Mus15398-R or L14415-F and Mus15398-R (González-Ittig et al. 2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
). Thermocycler conditions were: initial denaturation at 94 °C for 3 minutes, followed by 30 cycles of denaturation at 94 °C for 45 seconds, annealing at 50 °C for 1 minute, and extension at 72 °C for 1.5 minutes, and final extension at 72 °C for 5 minutes. Reaction mixtures were set at 25μL total volume with 1 μL of DNA (20-30 ng/μL), 0.13 μL of Promega Taq (Cat# M3008, 5U/μL), 0.8 μL of dNTPs mix (Cat#U151B, 10mM), 2 μL of MgCL2 (25 mM), 2.5 μL of 5 × Go Taq Green buffer, and 0.75 μL of each primer (10 μM). Amplified products were visualized in 1% agarose gel; 1Kb ladder was used as standard DNA size. Well amplified products were sequenced in Macrogen, USA (http://www.macrogenusa.com) with both amplifying primers. Raw chromatograms were cleaned, translated to amino-acids to check for premature stop codons and other nonfunctional mutations. Contigs for individual sequences were assembled using SeqManII 5.07 (Lasergene, DNASTAR, Madison, WI, USA). New sequences of C. tener were deposited in GenBank (Table 1).

All sequences, including those available online, were aligned using default parameters with ClustalW in MEGA5.0 (Tamura et al. 2011Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Molecular Biology and Evolution 28(10): 2731-2739. https://doi.org/10.1093/molbev/msr121
https://doi.org/10.1093/molbev/msr121...
). Identical sequences of C. tener were identified and collapsed into haplotypes using PopART v1.7 (Leigh and Bryant 2015Leigh JW, Bryant D (2015) PopART: full-feature software for haplotype network construction. Methods in Ecology and Evolution 6(9): 1110-1116. https://doi.org/10.1111/2041-210X.12410
https://doi.org/10.1111/2041-210X.12410...
). Haplotypes were used for all further phylogenetic analyses; in the matrix we included sequences published by Almeida et al. (2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
), Haag et al. (2007Haag T, Muschner VC, Freitas LB, Oliveira LFO, Langguth AR, Mattevi M (2007) Phylogenetic relationships among species of the genus Calomys with emphasis on South American lowland taxa. Journal of Mammalogy 88(3): 769-776. https://doi.org/10.1644/05-MAMM-A-319R1.1
https://doi.org/10.1644/05-MAMM-A-319R1....
) and González-Ittig et al. (2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
) corresponding to the following species: Calomys expulsus (Lund, 1841), C. fecundus (Thomas, 1926), C. callosus (Rengger, 1830), C. hummelincki (Husson, 1960), C. lepidus (Thomas, 1884), C. venustus (Thomas, 1894), C. musculinus (Thomas, 1913), C. tocantinsiBonvicino, Lima & Almeida, 2003Bonvicino CR, Lima JFS, Almeida FC (2003). A new species of Calomys Waterhouse (Rodentia, Sigmodontinae) from the Cerrado of Central Brazil. Revista Brasileira de Zoologia 20(2): 301-307. https://doi.org/10.1590/S0101-81752003000200021
https://doi.org/10.1590/S0101-8175200300...
, C. sorellus and C. laucha (both clades A and B). Sequences of Eligmodontia typus Cuvier, 1837, Auliscomys sublimis (Thomas, 1900), Phyllotis xanthopygus (Waterhouse, 1837), Andalgalomys pearsoni (Myers, 1977) and Salinomys delicatus Braun & Mares, 1995 were used as outgroups. Maximum Parsimony (MP) analysis was performed with PAUP 4.0.b10 (Swofford 1998Swofford DL (1998) PAUP*: phylogenetic analysis using parsimony (* and other methods). Sunderland, Sinauer Associates Publishers, v. 4.0b10.), where characters were unordered and equally weighted. We performed a heuristic search of 1000 iterations of random taxon addition using the TBR (tree bisection-reconnection) branch swapping algorithm. Maximum Parsimony analyses resulting in more than one most parsimonious tree were summarized in a strict consensus tree. Nonparametric bootstrap support values were calculated using 1000 replicate searches. The best-fitting model of sequence evolution was selected using jModeltest 2 (Darriba et al. 2012Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9(8): 772. https://doi.org/10.1038/nmeth.2109
https://doi.org/10.1038/nmeth.2109...
) in which likelihood scores for 88 different models were computed. The HKY+I+G model was selected using the Bayesian information criterion and the following starting parameters were used subsequently in Bayesian inferences (BI) and Maximum Likelihood (ML) analyses: a base frequency of A = 0.3030, C = 0.3096, G = 0.0877, T = 0.2997; a transition/transversion = 4.233; a proportion of invariable sites of 0.4860; a gamma distribution with alpha = 1.0360. The BI analyses were performed using MrBayes 3.2 (Ronquist et al. 2012Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029
https://doi.org/10.1093/sysbio/sys029...
) with two independent Markov chain-Monte Carlo (MCMC) runs, with one cold and three heated chains each. Runs were performed for five million generations and trees were sampled every 1000 generations. We discarded the first 25% of the samples as “burn in” and the two runs converged on similar posterior estimates with an average standard deviation of split frequencies of 0.006. We assessed convergence and mixing visually using Tracer v1.5 to plot likelihood scores for all parameters by generation time and by calculating effective sample sizes (Rambaut and Drummond 2007Rambaut A, Drummond AJ (2007) Tracer. Oxford, University of Oxford, v. 1.5 [computer program]. Available online at Available online at http://tree.bio.ed.ac.uk/software/tracer [Accessed: 08/08/2018]
http://tree.bio.ed.ac.uk/software/tracer...
). ML trees were constructed using the online version of program PhyML ver 3.0 (http://www.atgc-montpellier.fr/phyml) (Guindon et al. 2010Guindon S, Dufayard J, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate Maximum-Likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology 59(3): 307-321. https://doi.org/10.1093/sysbio/syq010
https://doi.org/10.1093/sysbio/syq010...
). In PhyML we used the same substitution model and the parameters described above; 1000 bootstrap replicates were performed.

In addition, a median-joining network was constructed using PopART v1.7 with 1,000 permutations: the method estimates the relative abundance of each haplotype and the genealogical relationships among them. Kimura-2 parameter genetic distances (K2p) among haplotypes of C. tener were calculated with MEGA5.0.

RESULTS

A total of 28 distinct haplotypes were identified from the 31 C. tener individuals analyzed. In the alignment with other species of Calomys and outgroups of other genera, from the 794 sites, 342 characters were variable of which 275 were parsimony-informative. In the phylogenetic trees obtained with different phylogenetic methods (ML, BI and MP) for species of Calomys, several clades are identified with strong support. For example, the, one composed by the species C. lepidus/C. sorellus/C. musculinus. The phylogenetic position of C. hummelincki is not resolved, since the node has low support values. Another clade strongly supported is the one composed by the species C. expulsus and species of the C. callosus complex (C. tocantinsi/C. callosus/C. fecundus/C. venustus) (Fig. 2). Sister to this clade is the one composed by clades A and B of C. laucha (sensuGonzález-Ittig et al. 2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
). The species C. tener presents a sister position in relation to the node that clusters C. laucha sensu lato, C. expulsus and C. callosus sensu lato.

Figure 2
Phylogram of the Bayesian consensus tree obtained from Cyt-b data set for species of Calomys with emphasis in Calomys tener. The order of the support values in the nodes is as follows: Bayesian posterior probabilities/maximum likelihood bootstrap/maximum parsimony bootstrap. Sequences from GenBank are indicated with their accession number and the species names. In the clade corresponding to C. tener the haplotype number, the specimen having each haplotype and the sampling locality is indicated. The vertical bars represent associations also observed in the network of Fig. 3, although with no statistical support. The intraspecific relationships recovered with the different phylogenetic methods are indicated.

At the intraspecific level there is no phylogenetic structure among haplotypes of C. tener that would suggest more than one species, even when samples have been collected throughout a large part of its geographical distribution (encompassing individuals from Argentina, Paraguay, Bolivia, and Brazil). In Fig. 2, haplotypes from localities 1, 4, 5, 12 and 13 situated in Brazil are the first to split off, while those from localities 15, 16 and 17 located in Argentina are among the last. The median-joining network (Fig. 3) showing the frequencies and the relationships of the 28 haplotypes does not corroborate these ancestral-derived positions, since none of the haplotypes 11, 17, 18, 20, 21, 22, 23, 24 or 25 are in the center of the network. No obvious pattern was observed between haplotype identity and geographic locations, except for a tendency that haplotypes unique to Argentina (haplotypes 1, 2, 3, 4, 5 and 6) group together on one side of the network and the Brazilian haplotypes mentioned above are in the other side of the network (Fig. 3). Although with low support, some relationships among haplotypes in Fig. 3 are also recovered with the three or with two different phylogenetic methods (indicated in Fig. 2 with a vertical bar). The K2p genetic distances among C. tener haplotypes averaged 1.29% ± 1.05, also compatible with intraspecific genetic variation (Bradley and Baker 2001Bradley RD, Baker RJ (2001) A test of the Genetic Species Concept: cytochrome-b sequences and mammals. Journal of Mammalogy 82(4): 960-973. https://doi.org/10.1644/1545-1542(2001)082<0960:ATOTGS>2.0.CO;2). In the network, haplotypes 8 and 9 from locality 11 are separated from the remaining haplotypes by more than 12 mutations, moving away from the general trend of diversification within C. tener.

Figure 3
Median-joining network showing the relationships and relative abundance of the 28 haplotypes (indicated by a number) detected in Calomys tener. The different colors indicate the country where each haplotype was detected. Each bar through the solid line represents one nucleotide difference between haplotypes. The black circle (mv1) represents a median vector.

Four CO1 haplotypes were identified among the five individuals of C. tener sequences. In the alignment with other species of Calomys and outgroups, from the 640 sites, 173 characters were variable of which 100 were parsimony-informative. The phylogenetic trees obtained ML, BI and MP have the same topology with varying levels of support for each node. The clade of C. tener is strongly supported and has a sister relationship with a clade formed by Calomys cerqueiraiBonvicino, Oliveira & Gentile, 2010Bonvicino CR, Oliveira JA, Gentile R (2010) A new species of Calomys (Rodentia: Sigmodontinae) from Eastern Brazil. Zootaxa 2336: 19-35. https://doi.org/10.11646/zootaxa.2336.1.2
https://doi.org/10.11646/zootaxa.2336.1....
and C. expulsus (Fig. 4). As in the Cyt-b dataset, there are no large genetic gaps among haplotypes of C. tener that could suggest more than one species, even though samples were collected in distantly locations from Brazil (locations 4, 10 and 14 in Fig. 1). In addition, the K2p genetic distances among C. tener haplotypes averaged 1.31 ± 1.01%.

Figure 4
Phylogram of the Bayesian consensus tree obtained from COI data set for species of Calomys with emphasis in Calomys tener. The order of the support values in the nodes is as follows: Bayesian posterior probabilities/maximum likelihood bootstrap/maximum parsimony bootstrap. Sequences from GenBank are indicated with their accession number and the species names. In the clade corresponding to C. tener the haplotype letter, the specimen having each haplotype and the sampling locality is indicated.

DISCUSSION

In this study, we used the Cyt-b gene from 31 individuals and the COI gene from five individuals collected throughout the range of the Delicate Vesper Mouse. Our sampling was broad and includes the known distribution range of the species as currently understood (Salazar-Bravo 2015Salazar-Bravo J (2015) Genus Calomys Waterhouse, 1837. In: Patton JL, Pardiñas UFJ, D’Elia G (Eds) Mammals of South America. Chicago, The University of Chicago Press, vol. 2, 504-505. https://doi.org/10.7208/chicago/9780226169606.001.0001
https://doi.org/10.7208/chicago/97802261...
). The mean genetic distance detected was 1.29% and 1.31% with the Cyt-b and COI genes, respectively, which are in line with the estimations for intraspecific comparisons according to Bradley and Baker (2001Bradley RD, Baker RJ (2001) A test of the Genetic Species Concept: cytochrome-b sequences and mammals. Journal of Mammalogy 82(4): 960-973. https://doi.org/10.1644/1545-1542(2001)082<0960:ATOTGS>2.0.CO;2). These estimates are also in accordance with intraspecific variation reported for other species of Calomys (e.g., Salazar-Bravo et al. 2001Salazar-Bravo J, Dragoo JW, Tinnin DS, Yates TL (2001) Phylogeny and evolution of the Neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 20(2): 173-184. https://doi.org/10.1006/mpev.2001.0965
https://doi.org/10.1006/mpev.2001.0965...
, Almeida et al. 2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
, González-Ittig et al. 2007González-Ittig RE, Patton JL, Gardenal CL (2007). Analysis of Cytochrome-b nucleotide diversity confirms a recent range expansion in Calomys musculinus (Rodentia, Muridae). Journal of Mammalogy 88(3): 777-783. https://doi.org/10.1644/06-MAMM-A-091R1.1
https://doi.org/10.1644/06-MAMM-A-091R1....
, Bonvicino et al. 2010Bonvicino CR, Oliveira JA, Gentile R (2010) A new species of Calomys (Rodentia: Sigmodontinae) from Eastern Brazil. Zootaxa 2336: 19-35. https://doi.org/10.11646/zootaxa.2336.1.2
https://doi.org/10.11646/zootaxa.2336.1....
, Nascimento et al. 2011Nascimento FF, Pereira LG, Geise L, Bezerra AMR, D’Andrea PS, Bonvicino CR (2011) Colonization process of the Brazilian common vesper mouse, Calomys expulsus (Cricetidae, Sigmodontinae): A Biogeographic hypothesis. Journal of Heredity 102(3): 260-268. https://doi.org/10.1093/jhered/esr012
https://doi.org/10.1093/jhered/esr012...
). It is important to highlight that the monophyletic nature of C. tener was supported with two markers (Figs 2, 4). Notwithstanding, this study was performed only with mitochondrial DNA which does not undergo recombination, has uniparental inheritance, and reflects only the female evolutionary history. The shortcomings of this approach are evident when males and females have different dispersal patterns or when closely related species hybridize, so the resulting genetic structure could potentially be different for both sexes. Therefore, to confirm that C. tener has low levels of intraspecific divergence and to reflect the diversification processes of both sexes, nuclear genes should be included in further studies (Dupuis et al. 2012Dupuis JR, Roe AD, Sperling FAH (2012) Multi-locus species delimitation in closely related animals and fungi: one marker is not enough. Molecular Ecology 21(18): 4422-4436. https://doi.org/10.1111/j.1365-294X.2012.05642.x
https://doi.org/10.1111/j.1365-294X.2012...
).

The taxonomic uniformity of the species was already suggested by cytogenetic studies performed by other authors over the last 30 years. For example, Mattevi et al. (2005Mattevi MS, Haag T, Oliveira LFB, Langguth AR (2005) Chromosome characterization of Brazilian species of Calomys Waterhouse, 1837 from Amazon, Cerrado and Pampa domains (Rodentia, Sigmodontinae). Arquivos do Museu Nacional 63(1): 175-181.) reported 2n = 66 (FN = 66-68) for specimens from Minaçú (Goiás). A 2n = 66 (FN = 66) was reported by Bonvicino et al. (2010Bonvicino CR, Oliveira JA, Gentile R (2010) A new species of Calomys (Rodentia: Sigmodontinae) from Eastern Brazil. Zootaxa 2336: 19-35. https://doi.org/10.11646/zootaxa.2336.1.2
https://doi.org/10.11646/zootaxa.2336.1....
) for individuals from Santa Tereza (Espírito Santo), Jaborandi (Bahia), Mimoso de Goiás (Goiás) (specifically the Cyt-b of individual MN36437, karyotyped in that study, was included in the present study, Table 1). This chromosome number was also reported for specimens from Distrito Federal, Cocos (Bahia), Campinas, Itirapina, Itapetininga, Pedreira and Rio Claro, all in São Paulo state and Gaúcha do Norte (Mato Grosso) (Yonenaga 1975Yonenaga Y (1975) Karyotypes and chromosome polymorphisms in Brazilian rodents. Caryologia (Florence) 28(2): 202-210. https://doi.org/10.1080/00087114.1975.10796617
https://doi.org/10.1080/00087114.1975.10...
, Bonvicino and Almeida 2000Bonvicino CR, Almeida FC (2000) Karyotype, morphology and taxonomic status of Calomys expulsus (Rodentia: Sigmodontinae). Mammalia 64(3): 339-351. https://doi.org/10.1515/mamm.2000.64.3.339
https://doi.org/10.1515/mamm.2000.64.3.3...
, Fagundes et al. 2001Fagundes V, Sato Y, Silva MJJ, Rodrigues F, Yonenaga-Yassuda Y (2001) A new species of Calomys (Rodentia: Sigmodontinae) from Central Brazil identified by its karyotype. Hereditas 133(3): 195-200. https://doi.org/10.1111/j.1601-5223.2000.00195.x
https://doi.org/10.1111/j.1601-5223.2000...
). In addition, Haag et al. (2007Haag T, Muschner VC, Freitas LB, Oliveira LFO, Langguth AR, Mattevi M (2007) Phylogenetic relationships among species of the genus Calomys with emphasis on South American lowland taxa. Journal of Mammalogy 88(3): 769-776. https://doi.org/10.1644/05-MAMM-A-319R1.1
https://doi.org/10.1644/05-MAMM-A-319R1....
) detected the chromosomal number 2n = 66 (FN = 66-70) for specimens from Serra da Mesa (Goiás), Aliança do Tocantins (Tocantins), Quintão (Rio Grande do Sul), Tupi Paulista (São Paulo) all in Brazil and one specimen from Santa Rosa de la Roca (Santa Cruz) in Bolivia. Haag et al. (2007Haag T, Muschner VC, Freitas LB, Oliveira LFO, Langguth AR, Mattevi M (2007) Phylogenetic relationships among species of the genus Calomys with emphasis on South American lowland taxa. Journal of Mammalogy 88(3): 769-776. https://doi.org/10.1644/05-MAMM-A-319R1.1
https://doi.org/10.1644/05-MAMM-A-319R1....
) also sequenced the Cyt-b of all the karyotyped specimens which were also included in the present study (Table 1).

In this study, the only discordant result that alters genetic continuity, is that involving haplotypes 8 and 9 from locality 11, which are very divergent. Given the central geographic position of this locality (Fig. 1) and the general level of genetic differentiation within the species, these haplotypes could be the result of sequencing errors as was already advocated by Almeida et al. (2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
). Therefore, the molecular results obtained here and the substantial cytogenetic evidence, support the inference that C. tener constitutes only one taxonomic unit. The type locality of the species is Lagoa Santa in the state of Minas Gerais in Central Brazil corresponding to the Cerrado ecoregion. Notwithstanding, it should be noted that Mattevi et al. (2005Mattevi MS, Haag T, Oliveira LFB, Langguth AR (2005) Chromosome characterization of Brazilian species of Calomys Waterhouse, 1837 from Amazon, Cerrado and Pampa domains (Rodentia, Sigmodontinae). Arquivos do Museu Nacional 63(1): 175-181.) reported the chromosomal complement for two specimens (LF4974 and LF5020) from Rondônia state, Brazil, with 2n = 64 (FN = 64) and suggested they could correspond to C. tener. However, the authors, with caution, denominated these individuals as Calomys sp. Given the chromosomal uniformity detected by many authors for C. tener, it is unlikely that the karyotype from Rondônia corresponds to this species because it lacks one autosomal pair and presents differences in the size and shape of the X chromosome. Unfortunately, no molecular data is available for the two specimens from Rondônia, therefore we cannot explicitly test the hypothesis that either of these correspond to C. tener.

The taxonomic status of C. tener and C. laucha are historically intertwined; originally described in the late 19th century, C. tener maintained its specific status until Philip Hershkovitz included it in his polytypic concept of C. laucha (Hershkovitz 1962Hershkovitz P (1962) Evolution of Neotropical cricetine rodents (Muridae) with special reference to the phyllotine group. Fieldiana, Zoology 46:1-524. https://doi.org/10.5962/bhl.title.2781
https://doi.org/10.5962/bhl.title.2781...
). The status of C. tener, with respect to C. expulsus and C. callosus was forcefully established by Bonvicino and Almeida (2000Bonvicino CR, Almeida FC (2000) Karyotype, morphology and taxonomic status of Calomys expulsus (Rodentia: Sigmodontinae). Mammalia 64(3): 339-351. https://doi.org/10.1515/mamm.2000.64.3.339
https://doi.org/10.1515/mamm.2000.64.3.3...
), but it was not until Salazar-Bravo et al. (2001Salazar-Bravo J, Dragoo JW, Tinnin DS, Yates TL (2001) Phylogeny and evolution of the Neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 20(2): 173-184. https://doi.org/10.1006/mpev.2001.0965
https://doi.org/10.1006/mpev.2001.0965...
) that a C. laucha and C. tener were included in a phylogenetic analysis of the genus. In this work, Salazar-Bravo et al. (2001Salazar-Bravo J, Dragoo JW, Tinnin DS, Yates TL (2001) Phylogeny and evolution of the Neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 20(2): 173-184. https://doi.org/10.1006/mpev.2001.0965
https://doi.org/10.1006/mpev.2001.0965...
) resolved these species as sister taxa, with little or no support. Further work by Almeida et al. (2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
) showed -with a larger sampling of taxa and individuals - that C. tener was in fact the sister taxon to all lowland forms of Calomys. Similar results were reported by Bonvicino et al. (2010Bonvicino CR, Oliveira JA, Gentile R (2010) A new species of Calomys (Rodentia: Sigmodontinae) from Eastern Brazil. Zootaxa 2336: 19-35. https://doi.org/10.11646/zootaxa.2336.1.2
https://doi.org/10.11646/zootaxa.2336.1....
). A great part of the current taxonomic confusion stems from the similar morphological and morphometric characteristics of C. tener and C. laucha. In fact, they are so similar morphologically that even the last reviser of the genus confused individuals of C. tener from Mato Grosso for C. laucha (Olds 1988Olds N (1988) A revision of the genus Calomys (Rodentia: Muridae). PhD thesis, New York, University of New York.). Although C. tener is more or less uniform across its geographic range (upper parts of body yellowish to dark brown, with a reddish hue in most specimens; ventral region grayish to whitish with base of hairs gray) the geographical variation in C. laucha is extensive (as described in Salazar-Bravo 2015Salazar-Bravo J (2015) Genus Calomys Waterhouse, 1837. In: Patton JL, Pardiñas UFJ, D’Elia G (Eds) Mammals of South America. Chicago, The University of Chicago Press, vol. 2, 504-505. https://doi.org/10.7208/chicago/9780226169606.001.0001
https://doi.org/10.7208/chicago/97802261...
). In fact, in areas of potential geographical overlap between these species (Misiones region of Argentina or Paraguay) morphological patterns of geographic variation would make it difficult to separate these forms on body coloration alone. Morphometrically, these forms also overlap in multivariate space (Olds 1988Olds N (1988) A revision of the genus Calomys (Rodentia: Muridae). PhD thesis, New York, University of New York., Bonvicino et al. 2003Bonvicino CR, Lima JFS, Almeida FC (2003). A new species of Calomys Waterhouse (Rodentia, Sigmodontinae) from the Cerrado of Central Brazil. Revista Brasileira de Zoologia 20(2): 301-307. https://doi.org/10.1590/S0101-81752003000200021
https://doi.org/10.1590/S0101-8175200300...
, Teta et al. 2017Teta P, González-Ittig RE, González EM, Pardiñas UFJ, Salazar-Bravo J (2017) Notes on the taxonomy of Calomys laucha (Rodentia, Cricetidae), with the designation of a neotype. Mastozoología Neotropical 24(2): 419-429.). Calomys tener is however, on average, larger (TL = 143.18 vs 132.4) and has a relatively longer tail than C. laucha (T/HB = 80.4 in C. tener, but 72.7 in C. laucha). Calomys tener has a longer maxillary toothrow than C. laucha (3.45 vs 3.2 mm); finally, C. tener can be distinguished from C. laucha by the presence of ridges along the sides of the supraorbital region (not present in C. laucha).

In a recent study, González-Ittig et al. (2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
) also confounded the two species; three specimens corresponding to C. tener (one from Argentina and two from Paraguay) were wrongly classified as C. laucha according to external characters. In the present study, the same occurred with specimens from localities 16- Estancia Santa Inés (Misiones) and 17- RN 12 y Arroyo Itaembé Miní (Misiones) both in Argentina. Considering body size and cranial measurements, Salazar-Bravo et al. (2001Salazar-Bravo J, Dragoo JW, Tinnin DS, Yates TL (2001) Phylogeny and evolution of the Neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 20(2): 173-184. https://doi.org/10.1006/mpev.2001.0965
https://doi.org/10.1006/mpev.2001.0965...
) and Bonvicino et al. (2010Bonvicino CR, Oliveira JA, Gentile R (2010) A new species of Calomys (Rodentia: Sigmodontinae) from Eastern Brazil. Zootaxa 2336: 19-35. https://doi.org/10.11646/zootaxa.2336.1.2
https://doi.org/10.11646/zootaxa.2336.1....
) separated two groups of Calomys, a larger-bodied species group, including C. callosus, C. expulsus, C. tocantinsi, C. callidus and C. cerqueirai, and a smaller-bodied species group, including C. tener and C. laucha. However, in the phylogenetic tree obtained here (Fig. 2) and in those obtained by Almeida et al. (2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
) and by Haag et al. (2007Haag T, Muschner VC, Freitas LB, Oliveira LFO, Langguth AR, Mattevi M (2007) Phylogenetic relationships among species of the genus Calomys with emphasis on South American lowland taxa. Journal of Mammalogy 88(3): 769-776. https://doi.org/10.1644/05-MAMM-A-319R1.1
https://doi.org/10.1644/05-MAMM-A-319R1....
), C. tener is not the sister species of C. laucha. It is important to highlight that phylogenetic trees reported by those authors and that of Fig. 2 show the same global topology with the following relationships: (C. tener (C. laucha and the big clade including all the large-bodied species of Calomys)).

Quintela et al. (2014Quintela FM, da Silveira EC, Dellagnese DG, Cademartori CV (2014) Calomys tener (Winge, 1887) (Rodentia: Cricetidae:Sigmodontinae): Filling gaps. Check List 10(3): 650-654. https://doi.org/10.15560/10.3.650
https://doi.org/10.15560/10.3.650...
) recorded for the first time the sympatry between C. tener and C. laucha and warned that the distribution limits in the Rio Grande do Sul state in Brazil should be investigated. Considering the two clades found for C. laucha by González-Ittig et al. (2014González-Ittig RE, Kandel N, Levis S, Calderon G, Salazar-Bravo J, Gardenal CN (2014). Molecular systematic of the South American rodent Calomys laucha (Cricetidae: Sigmodontinae), a reservoir of the Laguna Negra hantavirus. Canadian Journal of Zoology 92(12): 1093-1098. https://doi.org/10.1139/cjz-2014-0133
https://doi.org/10.1139/cjz-2014-0133...
), it is highly probable that what Quintela et al. (2014Quintela FM, da Silveira EC, Dellagnese DG, Cademartori CV (2014) Calomys tener (Winge, 1887) (Rodentia: Cricetidae:Sigmodontinae): Filling gaps. Check List 10(3): 650-654. https://doi.org/10.15560/10.3.650
https://doi.org/10.15560/10.3.650...
) found corresponds to our clade B. Thus, there is no evidence of sympatry with C. laucha sensu stricto (or clade A) (see a discussion on the geographic distribution of the species in Teta et al. 2017Teta P, González-Ittig RE, González EM, Pardiñas UFJ, Salazar-Bravo J (2017) Notes on the taxonomy of Calomys laucha (Rodentia, Cricetidae), with the designation of a neotype. Mastozoología Neotropical 24(2): 419-429.). In Rio Grande do Sul both clade B of C. laucha and C. tener could be getting in contact occasionally; it is known the two species have very low densities, because dozens of studies (and thousands of trap-nights) have been conducted throughout the state and until recently none reported captures of the two species (Cademartori et al. 2004Cademartori CV, Fábian ME, Menegheti JO (2004) Variações na abundância de roedores (Rodentia, Sigmodontinae) em duas áreas de floresta ombrófila mista, Rio Grande do Sul, Brasil. Revista Brasileira de Zoociências 6(2): 147-167., Badzinski et al. 2012Badzinski C, Galiano D, Marinho JR (2012) Mammalia, Rodentia, Cricetidae, Calomys laucha (Fischer, 1814): distribution extension in southern Brazil. Check List 8(2): 264-266. https://doi.org/10.15560/8.2.264
https://doi.org/10.15560/8.2.264...
, Quintela et al. 2012Quintela FM, Santos MB, Christoff AU, Gava A (2012) Pequenos mamíferos não-voadores (Didelphimorphia, Rodentia) em dois fragmentos de matas de restinga de Rio Grande, Planície Costeira do Rio Grande do Sul. Biota Neotropica 12(1): 261-266. https://doi.org/10.1590/S1676-06032012000100021
https://doi.org/10.1590/S1676-0603201200...
, 2014Quintela FM, da Silveira EC, Dellagnese DG, Cademartori CV (2014) Calomys tener (Winge, 1887) (Rodentia: Cricetidae:Sigmodontinae): Filling gaps. Check List 10(3): 650-654. https://doi.org/10.15560/10.3.650
https://doi.org/10.15560/10.3.650...
). González-Ittig et al. (2014Quintela FM, da Silveira EC, Dellagnese DG, Cademartori CV (2014) Calomys tener (Winge, 1887) (Rodentia: Cricetidae:Sigmodontinae): Filling gaps. Check List 10(3): 650-654. https://doi.org/10.15560/10.3.650
https://doi.org/10.15560/10.3.650...
) suggested that clade B of C. laucha may be experiencing a process of population expansion and dispersal into this region. The same could be occurring in C. tener. In the present study, many of the basal haplotypes of the Cyt-b gene are from localities of north-central Brazil (localities 1, 4 and 5), however, the two most basal haplotypes, H_11 and H_25, are from localities 12 and 13 in Rio Grande do Sul. In the COI gene, the most basal haplotype is H_D from locality 14 also in Rio Grande do Sul; thus the results of both genes are challenging the hypothesis of a north-south colonization of C. tener (Figs 2, 4). Contradicting the phylogenetic trees, in the network of the Cyt-b gene, both H_11 and H_25 are in a peripheral position which is more compatible with new haplotypes (Fig. 3). Almeida et al. (2007Almeida FC, Bonvicino CR, Cordeiro-Estrela P (2007) Phylogeny and Temporal diversification of Calomys (Rodentia, Sigmodontinae): Implications for the biogeography of an endemic genus of open/dry biomes of South America. Molecular Phylogenetics and Evolution 42(2): 449-466. https://doi.org/10.1016/j.ympev.2006.07.005
https://doi.org/10.1016/j.ympev.2006.07....
) described an intraspecific geographic structure given that they found a clade containing samples from São Paulo state, which was clearly separated from the remaining specimens. In the present study we did not find this pattern. We found instead that some haplotype-relationships in Fig. 2 were also detected in the network (Fig. 3), but without a clear geographic pattern. Nevertheless, it is important to consider that the sample size is low and it is not possible to make too many interpretations about the demographic history of the species, what should be performed in a future phylogeographic study.

In summary, C. tener constitutes only one evolutionary unit corroborated by the molecular data presented here using individuals encompassing most of the known geographical distribution of the species. In the present study we tried to survey the overall genetic differentiation of the species not deepening in the genetic structure itself. Because C. tener is a potential human health risk by Araraquara hantavirus in South America, additional surveys in areas not represented in our sampling, coupled with a broader phylogeographic study is needed to expand our understanding of the ecological and biogeographical processes experienced by the species.

ACKNOWLEDGEMENTS

We thank the Natural Sciences Research Laboratory of the Museum of Texas Tech University, Lubbock, Texas, USA for providing tissue samples for the specimens of C. tener. We are very grateful to Ulyses F.J. Pardiñas (CENPAT, Puerto Madryn, Argentina), for providing most of the samples from Misiones (Argentina) used in this study. We also thank to Juan Diego Pinotti (IDEA, UNC-CONICET, Córdoba, Argentina) for his assistance to make the map. This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica, Argentina (PICT 2016 #1328), by the Secretaría de Ciencia y Tecnología (SECyT-UNC), Universidad Nacional de Córdoba, Argentina. We acknowledge the contribution of the Graduate School, Texas Tech University (Doctoral Dissertation Completion Fellowship to NPK) and Texas Tech University Proposal Stimulus Program (to JSB).

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Publication Notes

  • Available online:

    May 28, 2019
  • Zoobank Register:

    http://zoobank.org/27DD8BCE-FE46-4234-9202-619A11DB3582
  • Publisher:

    © 2019 Sociedade Brasileira de Zoologia. Published by Pensoft Publishers at https://zoologia.pensoft.net

Edited by

Editorial responsibility:

Diego Astúa de Moraes

Publication Dates

  • Publication in this collection
    06 June 2019
  • Date of issue
    2019

History

  • Received
    06 Oct 2018
  • Accepted
    30 Jan 2019
  • Published
    28 May 2019
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