First record of genus Cryptonanus ( Didelphimorphia ) in the state of Rio de Janeiro , Brazil

Here we report the first record of genus Cryptonanus in the state of Rio de Janeiro, Brazil. One specimen (MZUSP35409) was captured in an Atlantic Forest fragment and was identified by morphological characters and molecular analysis using cytochrome‐b (MT-CYB) and von Willebrand Factor (VWF). Phylo‐ genetic reconstructions based on VWF sequences positioned our specimen into the genus Cryptonanus, and the shortest genetic distance of MT-CYB was estimated between our specimen and an undescribed specimen from Piauí. Our record increases the genus’ geographic range approximately 350 km from Cotia and Ibiúna mu‐ nicipalities, São Paulo. The genus Cryptonanus remains taxonomically poorly understood.

DNA was isolated from the liver of one specimen with the phenol-chlorophorm protocol (Sambrook et al. 1989).Cytochrome b (MT-CYB) DNA was analyzed for inferring relationships between con-generic species and a partial sequence data of exon 28 of the von Willebrand Factor (e28-VWF) for inferring relationships between different genera.Gene nomenclature followed HUGO Gene Nomenclature Committee (http://www.genenames.org).
MT-CYB DNA was PCR amplified with primers L14724 (Irwin et al. 1991) and citb REV (Casado et al. 2010), with a pre-denaturation step at 94°C for 2 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 1 min 30 s, and final extension of 72°C for 5 min.VWF was PCR amplified with e28-vWF F104 and e28-vWF R1141 (Voss and Jansa 2009), with a pre-denaturation step at 94°C for 2 min; 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 30 s, extension at 72°C for 1 min 30 s, and final extension of 72°C for 10 min.PCR products were purified with GFX PCR DNA and Gel Band Purification kit (GE Healthcare, Brazil).Sequence reactions were performed with PCR primers, plus CB-in1 and CB-in2 (Cassens et al. 2000), MVZ16 (Silva and Patton 1993) and MEU1 (Gonçalves et al. 2005) for MT-CYB, e28-vWF F120, e28-vWF R655 and e28-vWF R743 for VWF, labeled with XL and BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and loaded to an ABI Prism™ 3130 platform.
Sequences were edited, assembled with Chromas (McCarthy 1998) and Bioedit (Hall 1999), aligned with MEGA v. 6 (Tamura et al. 2011) 1).Analyses of VWF data were carried out following 2012).Recent taxonomic studies showed that specimens previously identified as Gracilinanus may actually belong to Cryptonanus, and that three likely species of this genus may occur in the state of São Paulo (Vivo et al. 2011).Here we report the first record of Cryptonanus in the state of Rio de Janeiro (Southeastern Brazil).
The study was carried out in Serra das Araras, Piraí municipality, state of Rio de Janeiro, Brazil.Five sites of ombrophilous dense forest located in the center of Tinguá-Bocaina Biodiversity Corridor were sampled in February 2013.Non-volant small mammals were sampled with livetraps (Sherman®) and pitfall traps (IBAMA/ MMA process no.02001.007478/2008-41, authorization no. 196/2012).
At each sampling site, one 90 m transect was established, with 10 trap stations.Each station had one livetrap placed on the ground and one placed in the understory between 1.5 and 2.5 m above ground, with a total sampling effort of 500 traps-night.Pitfall traps consisted of 60 L plastic buckets arranged in transects and placed 10 m apart.Buckets were connected by a plastic-sheet drift fence 0.5 m high, buried 0.1 m below and extended perpendicularly to the ground in order to induce the capture of wandering individuals.Each of the five sampling site had eleven buckets, and the total sampling effort was 275 bucket-nights.
All specimens trapped were identified at the species level whenever possible, weighed using spring scales, sexed, measured (heady-body and tail lengths), and marked with a numbered ear-tag on first capture (Ear Tags, National Band & Tag Co., Newport, Kentucky, USA).Unidentified specimens were collected, euthanized, prepared, and deposited in Museu de Zoologia da Universidade de São Paulo (MZUSP).Liver samples were collected and preserved in 100% ethanol and deposited in Laboratório de Vertebrados da Universidade Federal do Rio de Janeiro.For marsupials, teeth eruption and functionality pattern permitted accurate age estimation (Macedo et al. 2006).Following Macedo et al. (2006), specimens were classified as young (dentition dPxMx), Genetic distance estimates were carried out with complete deletion using Kimura's two parameters, with MEGA v. 6.For phylogenetic reconstructions, the best fit model of evolution was inferred with ModelGenerator v. 0.85 (Keane et al. 2006), according to Akaike Information Criterion 2 (AIC; Burnham and Anderson 2002;Keane et al. 2006).For MT-CYB, the best fit model was the generalized time reversible model, with proportion of invariable sites estimated and gamma distributed rate (GTR+I+G).For VWF, the best fit model was the transversion model with equal base frequencies and gamma distributed rate (TVMef+G).
Phylogenetic reconstructions were performed with PHYML 3.0 (Guindon et al. 2010) for maximum likelihood (ML) with bootstrap estimates based on 1,000 replicates.Branch support was also calculated using the approximate likelihood ratio test (aLRT) with SH-like interpretation.Bayesian analyses (BA) were carried out with MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003).Posterior probabilities values were estimated sampling every 100th generation over a total of 1,000,000 with a burn-in of 25% generations (2500 trees).Effective sample size (ESS) and convergence diagnostic values were considered when above 100 and 1, respectively.Topologies were generated and edited with FigTree v1.4.0 (Rambaut 2012).DNAsp 5 was used for haplotype estimates and nucleotide diversity (Librado and Rozas 2009).Following Voss and Jansa (2009) we assumed the monophyly of tribe Thylamyini considering Marmosa murina and Marmosops paulensis as the most basal offshoots of the topology.
This specimen was morphologically identified to belong to Cryptonanus by the following anatomical features: presence of prehensile tail, tail longer than head-body length, lack of maxillary fenestrae, rostral process in premaxillae and secondary foramen ovale (Table 2; Figures 1 and 3).Apparently, there were no accessory cusps in the upper canines.
Sequence data accounted for 1,146 bp of MT-CYB and 865 bp of VWF but phylogenetic reconstructions were limited to a 713 bp and 836 bp of each gene, respectively, in view of the dearth of data available in GenBank.Genetic distance estimated between MT-CYB of Cryptonanus species ranged from 0.008 to 0.129 (Table 3) and between the two Cryptonanus sp. was  Cryptonanus sp. from Serra das Araras showed diagnostic cranial characters of this genus like lack of maxillary fenestrae, secondary foramen ovale and rostral process in premaxillae (Voss et al. 2005;Voss and Jansa 2009;Garcia et al. 2010).Apparently, accessory cusps in the upper canines were absent, as is the case of other specimens of Cryptonanus (Voss et al. 2005).The precise identification of this specimen still requires further analysis.A preliminary comparison of other anatomical features between this young and not fully developed specimen and the three Brazilian Cryptonanus species has been inconclusive.
Molecular analyses corroborated the phylogenetic position of this specimen in Cryptonanus.Measures of genetic distances are an important tool in the assessment   The genus Cryptonanus is still poorly understood, requiring new studies to describe the new species.In view that this genus is presently distributed in southeastern Brazil, specimens previously identified as Gracilinanus deposited in the zoological collections should be reanalyzed.

Figure 2 .
Figure 2. Record of a Cryptonanus specimen (red circle; MZUSP35409) in the state of Rio de Janeiro, Brazil.

0. 032 .
Distance between C. chacoensis and C. agricolai was 0.008 while distances between the three specimens of C. guahybae ranged from 0.002 to 0.003 (Table3).Phylogenetic reconstructions based on MT-CYB showed two main lineages splitting from a strongly supported node; the one leading to C. unduaviensis and the second one to the clade (((C.chacoensis, C. agricolai) (Cryptonanus sp. 1, Cryptonanus sp.2)) C. guahybae) (Figure 4).Effective sample size (ESS) values were above 100, indicating that the parameter was not undersampled.The convergence diagnostic (PSRF, potential scale reduction factor) showed values above 1.Reconstructions based on VWF positioned our specimen in Cryptonanus.The specimen herein identified is the first record of the genus Cryptonanus in the state of Rio de Janeiro, in addition to others in southeastern Brazil (Umetsu and Pardini 2007; Martin et al. 2012), a region previously considered outside the range of this genus.Species of Cryptonanus are frequently described as inhabitants of open areas (Voss and Jansa 2009), but the establishment of large plantations with open understory, and the Delciellos et al. | Cryptonanus, first record in Rio de Janeiro stateof genetic diversity.For the marsupials analyzed here, the genetic distance of MT-CYB ranged from 0.008 to 0.129 between species.The genetic distance between the two Cryptonanus sp. was estimated in 0.032 while the distance of 0.008 between the recognized species C. chacoensis and C. agricolai was the shortest one.In the phylogenetic topology, our specimen from Rio de Janeiro state grouped with the Cryptonanus sp.captured in Uruçui-Una Ecological Station, municipality of Uruçui, state of Piauí, Brazil, 1,600 km from the municipality of Piraí, state of Rio de Janeiro.The nearest Cryptonanus records corresponded to specimens captured in São Paulo (Umetsu and Pardini 2007: ca.350 km; Martin et al. 2012: ca.470 km) and Paraná (Dias et al. 2015: ca.745 km) states.For the specimens from São Paulo there are no molecular data available in the literature or in GenBank.The specimens of Angatuba municipality were identified as C. agricolai (Martin et al. 2012), and the recent record in Paraná state was attributed to C. guahybae (Dias et al. 2015).Cryptonanus guahybae specimens from southern Brazil grouped in a separate clade with support of 100% in the molecular analyses, ruling out any possibility of being identical with the specimens herein studied.Moreover, this specimen has been found to be different from C. unduaviensis, C. guahybae, and the poorly understood taxonomic group that includes C. chacoensis and C. agricolai.

Figure 3 .
Figure 3. Dorsal, ventral, and lateral views of the skull, and lateral view of the mandible of the Cryptonanus specimen (MZUSP35409) from Serra das Araras, municipally of Piraí, state of Rio de Janeiro, Brazil.

Table 1 .
Mitochondrial Cytochrome b sequences utilized in this study, with Museum or field number, GenBank accession number, locality and reference of published study.BR = Brazil; BOL = Bolivia; PAR = Paraguay.
Delciellos et al. | Cryptonanus, first record in Rio de Janeiro state

Table 2 .
Comparison of diagnostic morphological and cranial features between described Brazilian Cryptonanus species and the specimen of Cryptonanus captured in this study (MZUSP 35409).

Table 3 .
Genetic distance estimates carried out with complete deletion using Kimura's two parameters among Cytochrome b sequences of Cryptonanus species.