the genus Myotis ( Chiroptera , Vespertilionidae ) in Brazil : phylogeny , distribution , and cytogenetics

Myotis is the largest genus of the Vespertilionidae, showing a cosmopolitan geographical distribution and is considered an example of adaptive radiation. Nine species occurs in Brazil and this study synthesized aspects of the geographic distribution, karyotype, and phylogeny. A search in bibliographic databases was carried out using keywords. The phylogeny study was based on the sequencing of a specimen of Myotis ruber collected in a fragment of the Altantic Forest of Minas Gerais; this specimen was deposited at the Newton Baião de Azevedo Museum of Zoology. The genus showed to be widely distributed in the Brazilian territory, with Myotis nigricans being the most widespread. In addition, high karyotypic conservatism was observed in all species of the genus. The phylogenetic analyses using the mt-Cytb gene corroborated the monophyletic aspect of the genus and the Myotis ruber species.


Introduction
Myotis Kaup, 1829 is the most representative genus among the Vespertilionidae and has a wide distribution (LaVal, 1973;Koop-man, 1993;Ruedi & Mayer, 2001;Simmons, 2005;Tavares et al., 2008).Its species exhibit great similarities in forms and seldom display specialized features (LaVal, 1973;Ruedi et al., the genus Myotis (Chiroptera, Vespertilionidae) in Brazil: phylogeny, distribution, and cytogenetics 1990; Gannon et al., 2001;López-González et al., 2001;Ruedi & Mayer, 2001;Stadelmann et al., 2004), hindering the process of identification and the systematic organization of the group.Tate (1941) divided the genus into seven subgenera by grouping species within each subgenus according to their morphological similarity: Selysius, Isotus, Paramyotis, Myotis, Chrysopteron, Leuconoe, and Rickettia.Findley (1972) analyzed 48 external and cranial characters grouping the species only in the three subgenera Selysius, Myotis, and Leuconoe, also according to the specimens' morphological affinities related to different flight and feeding modes.A fourth subgenus, Cistugo, was described by Koopman (1993), and as containing species restricted to South Africa.Cistugo is now recognized as a distinct genus from Myotis (Lack et al., 2010).The other subgenera are currently considered an ecomorphological group, that is, all Myotis of the New World are grouped into one single subgenus yet without a formally known name (Wilson, 2008).Despite being a broad and diverse genus, Myotis is karyotypically one of the most conserved (Baker & Jordan, 1970); in general, phylogenetic studies classify the genus as being monophyletic (Stadelmann et al., 2007).
Regarding the genus taxonomic complex of species that occur in Brazil, Myotis dinellii was described as a subspecies of Myotis levis by LaVal (1973) based on the allopatric distribution of organisms and their morphological similarities.Subsequently, because sympatric traits were recorded in the spatial distribution of M. levis levis and M. l. dinellii (see Passos et al., 2010) in Southern Brazil, they could no longer be described as species and subspecies.Miranda et al (2013) performed morphological and morphometric studies on these organisms and detected differences in the sexual dimorphism of both species, with M. levis showing separation between males and females, and differences in color and size.Myotis levis showed more similarity with M. ruber while M. dinellii presented similarity to M. albescens and M. nigricans.
Myotis lavali and M. izecksohni were described from the M. nigricans complex based on morphological and morphometric traits for species differentiation among themselves, and for the differentiation of M. nigricans species, during a study about qualitative evaluations of this species (Moratelli et al., 2011b).
Although there are a volume considerable work describing the ecology of individuals of the genus, systematic and phylogenetic aspects remain poorly understood.Therefore, through bibliographic review and molecular study of Myotis ruber, this work brought together, briefly, data on the geographic distribution of .163 the genus Myotis in Brazil and to find out the cytogenetic and phylogenetic status, pointing out the main problems involved in these studies.

Materials and Methods
The phylogenetic analyses were based on cytochrome-b (cyt-b) sequences of Myotis obtained from GenBank and proper sequencing (Table 1).The mitochondrial cytochrome b (cyt-b) gene was chosen for the study because it is widely used in systematic studies of mammals, alone or in combination and because it has a database of good taxonomic coverage deposited in GenBank.
A specimen of Myotis ruber was collected during a bat survey in an Atlantic Forest remnant of Minas Gerais, located in Reserva Particular do Patrimônio Natural Santuário Ecológico Mata dos Jacus (RPPN SEMJ) (42°02'51" W; 20°29'01" S; DATUM SIRGAS 2000), in municipality the Alto Jequitibá.The ranges of temperature vary from 1,150 to 1,470 mm, the climate type is Cwb, wet tropical, with dry winter and temperate summer, according to the classification of Köppen (ALVA-RES et al., 2013).The specimen was prepared and deposited at the Newton Baião de Azevedo Museum of Zoology (voucher MZNB 378; see Faria et al. 2017).Hepatic tissue DNA was extracted using the phenol/chloroform protocol according to Sambrook et al. (1989).The DNA quality was verified by 0.8% agarose gel electrophoresis and ultraviolet (UV) transilluminator visualization and quantified in a Nanodrop spectrophotometer (ND-1000™).The gene was amplified through polymerase chain reactions (PCR; Axygen model Maxygene II) standardized with specific primers.
The phylogenetic analyses were based on a prior molecular evolution model, chosen using the Akaike Information Criterion test (AIC) with modifications following Posada & Crandall (2001), and run in the ModelGenerator 0.85 software (Keane et al., 2006).The Maximum Likelihood (ML) analyses with the cyt-b gene were performed using the HKY evolution model (Hasegawa et al., 1985) with invariant sites ratio and gamma substitution distribution rate (HKY+I+G).
The ML topology was obtained with the PhyML 3.0 software (Guindon & Gascuel, 2003) using the following parameters: optimized sequence balance, proportion of estimated variable sites, estimated α, initial BioNj tree, topology of sizes of optimized branches, and the aLRT-SH support values obtained on the basis of 1,000 replicates (Anisimova & Gascuel, 2006;Guindon et al., 2010).
Bibliographic searches were conducted in databases such as Web of Science (http:// www.webofknowledge.com/)and SciElo (http:// www.scielo.br/) to obtain data on taxonomy, geographic distribution, and cytogenetics.The searches were made from July of 2016 to May of 2018 using the following keywords: 'geographic distribution', 'karyotype', 'phylogenetic', 'Myotis'.Records were obtained in both databases.The distribution reviews consider both animals deposited in collections, as well as capture and release data.

Phylogeny
The molecular analysis showed that the dataset provided 733 base pairs of the mitochondrial cytochrome b gene (cyt-b) with a TAT stop codon.The maximum likelihood topology confirmed the morphological identification of the specimen collected by us, clustering it together with the other M. ruber, with 2.05% of genetic distance between them, while the maximum value of the genetic distance (K2p) interspecific result 16.59% (Table 2), was higher than that found .165by Ruedi & Mayer (2001) (15%).The divergence intraspecific did not exceed 5%, corroborating studies already done for species of neotropical bats (Porter & Baker, 2004;Hoffmann & Bakker, 2001).Also revealed the monophyletic as-pect of the genus Myotis, divided into two clades (Figure 1    Myotis simus and M. ruber. Studies using cyt-b have recovered M. albescens as belonging to the clades formed by M. nigricans and M. levis, while M. simus, M. riparius and M. ruber form a distinct clade (Stadelmann et al., 2007;Rueddi et al., 2013;Moratelli et al., 2013;Moratelli et al., 2016;Moratelli et al., 2017).However, the topology generated in the present work, was structured to group M. albescens with M. levis, M. nigricans and M. riparius; with M. ruber and M. simus forming the second clade.This result, although little statistically supported, was similar to that found by Larsen et al (2012), which indicates the presence of lineages not yet known for the genus, exemplified by the values of genetic divergence.
The taxonomic problems found in the group are evident, which implies difficulties in carrying out consistent phylogenetic and phylogeographic studies.Besides that, in Brazil, there is a need for a more extensive geographical sampling of Myotis, because there is insufficient genetic data to allow better construction of biogeography, conservation and evolutionary history at national level.
Myotis dinelli is recorded in the States of Santa Catarina and Rio Grande do Sul (Passos et al., 2010, Miranda et al., 2013) and is distributed in Atlantic Rainforest and Pampas (Paglia et al., 2012) (Figure 2B).

Myotis izecksohni has records in the
States of Paraná and Rio de Janeiro, both in the Atlantic Rainforest domains (Moratelli et al., 2011a).Dias et al. (2015) reported new records of the species' distribution in the State of Minas Gerais through specimens collected in a region classified as an ecotone between the Atlantic Rainforest and Cerrado, emphasizing the need for more studies to characterize species habits (Figure 2C).
Myotis lavali was first recorded in endemism in the Caatinga (Moratelli et al., 2011a) occurring in the States of Pernambuco, Bahia, and Ceará.Moratelli & Wilson (2013) found records of the species in the Atlantic Rainforest domains of the Brazilian Northeast, also reporting occurrence in a part of the Cerrado belong-.167 ing to the state of Tocantins, which composes a diagonal corridor.Maas et al (2013) recorded the occurrence of the species in the State of Piauí reinforcing the idea that it may present wider distributions within the Cerrado and Caatinga domains (Figure 2D).
Myotis simus displays an endemism for South America as well as M. izecksohni and M. lavali (LaVal, 1973;Simmons, 2005;Wilson, 2008;Moratelli et al., 2011a;Moratelli, 2012).It is the least representative species of the genus in terms of occurrence with distribution recorded in the States of Amazonas, Mato Grosso do Sul, and Pará (Moratelli et al., 2011a), and in Amazon domains (Paglia et al., 2012).It is worth mentioning that individuals occurring in the State of Mato Grosso do Sul may correspond to another species know (Moratelli et al., 2015).In addition, there are records of the species present in the Atlantic Rainforest and in Pantanal (Moratelli, 2012;Paglia et al., 2012), being found near aquatic environments (Figure 2).

Cytogenetic
Although considered a specious and successful genre in adaptive issues, Myotis presents, karyotypically, high conservatism.Baker & Jordan (1970) determined 2n = 44 e FN = 50, based on cytogenetic studies of M. auriculus, M. nigricans, M. simus and M. riparius.They described the autosomal complement of the species as three large pairs and a small pair of metacentric chromosomes; and 17 acrocentric pairs ranging in size from medium to small.The sex chromosomes were characterized as an average submetacentric chromosome (X) and a small chromosome acrocentric (Y).Such karyotype was described for another eighteen species of the genus.However, some studies FN = 52 (Strelkov & Volobluev, 1969;Bickham & Hafner, 1978). .169 Banding techniques proved to be important for studies of systematic and evolutionary aspects (Bickham & Baker, 1976;Bickman & Hafner, 1978).Bickham & Hafner (1978) used G and C band standards for Myotis myotis and M. oxynathus, where both were identical and showed remarkable similarity with the Miniopterus, suggesting that both genders reflect the karyotype composition of the ancestor from which they evolved.Bickham & Baker (1976) examined patterns of New World species bands and found small differences in the size of the heterochromatic pair and the smaller autosomes.Bickham (1979) determined 218 bands for M. nigricans, which presented 25 autosomes with exclusive pattern.Another 11 species presented the same pattern, and three differed minimally.Bickham et al (1986) found chromosomal variations only in chromosome size Y and in the presence or absence of short heterochromatic arms in the small autosomes.Some authors indicate that the numbering of each arm is efficient, Robertsonian mergers and translocations are forms that, commonly, cause chromosome change within the family Vespertilionidae (Capanna & Civitelli, 1970;Bickham & Baker, 1976).Some authors attribute this conservatism to the fact that the genre is older, and, as well, intraspecific and interspecific chromosome variation is uncommon among Vespertilionidae (Baker & Patton, 1967;Baker, 1970;Bickham & Hafner, 1978).Sotero-Caio et al (2017) found variation in the number and location of heterochromatic segments and nucleoli organizing regions for the genus, but the diploid number remains unchanged.Evidence suggests that repetitive DNA may play an important role in promoting events of chromosomal rearrangements.Therefore, the authors point out that the exploration of the repetitive region should be considered for a better understanding of the role of non-coding DNA in the chromosome structure.
Cytogenetic studies of the genus Myotis in Brazil are uncommon, a factor that is perhaps related to the conservatism presented by the genre, that ends up not arousing interest in these works.However, Ao et al (2006), in study of bands, showed chromosomal differences among species of Old World Myotis.But, due to the scarcity of resources, it is not possible to apply cytogenetic methods to understand the karyotype evolution of the species, which does not allow this tool to be applicable to the taxonomic diagnosis.

Conclusion
The distribution of the genus varies according to the species, which, for the most part, are distributed over a large part of the Brazilian territory with some coexisting in the same location; M. nigricans is the most widespread.A high karyotypic conservatism is observed in all species of the genus, but, studies point to the importance of the use of banding techniques for karyotype delineation.The results obtained through the molecular data using the cyt-b gene corroborate the monophyletic aspect of the genus Myotis and the Myotis ruber species.Howev- ); one with support value (aLRT = 46) and formed by Myotis levis, M. nigricans, M. albescens e M. riparius; and the other with support value (aLRT = 100), divided into two strains, with

Figure 1 .
Figure 1.Maximum likelihood topology for cyt-b showing the phylogenetic relationships of Myotis.

Figure 2 .
Figure 2. Occurrence of the genus Myotis in Brazil based on bibliographic data.

Table 1 .
List of species analyzed with name, GenBank or feld number (number), locality and reference.
The genus Myotis in Brazil Revista Brasileira de Zoociências 19(3): 161-175.2018 er, the number of individuals of the genus Myotis sequenced in Brazil is incipient, and with little data of geographical scope, suggesting the need for additional genetic investigations to be able to test any phylogenetic hypotheses.