New records of the rare little rufous mouse opossum , Marmosa lepida ( Thomas , 1888 ) ( Mammalia , Didelphidae ) in southeastern Amazonia , Brazil

Here we present new records of the mouse opossum Marmosa (Stegomamosa) lepida (Thomas, 1888) collected in the recently flooded region of the Xingu River during the construction of the Belo Monte hydropower dam in the state of Pará, Brazil. This taxon is rarely captured, and it has often been misidentified as other similar genera of arboreal marsupials, such as Gracilinanus. Our specimens were identified morphologically and genetically using partial DNA of the mitochondrial cytochrome b gene. The new records increase the known distribution by about 250 km to the southeast.


Introduction
The genus Marmosa comprises 19 recognized species allocated to 5 subgenera (Voss et al. 2014), widely distributed in the Neotropical region.Marmosa (Stegomarmosa) lepida, also known as the little rufous mouse opossum, is rarely captured by conventional sampling methods and its habits are therefore poorly known (Brito and Pozo-Zamora 2015).
Species of this genus are usually nocturnal, arboreal, solitary, and feed mainly on fruits and insects (Creighton andGardner 2008, Voss 2013).Marmosa lepida has a broad distribution in the Amazon region, although only a few specimens deposited in museums are known (Fig. 1).It occurs mainly in western Amazonia, in cis-Andean areas from sea level to 1,580 m above sea level (a.s.l.), in Colombia, Venezuela, Ecuador, Peru, Brazil, Suriname, Guyana and French Guiana (Creighton and Gardner 2008, Voss 2013, Voss et al. 2014, Astúa 2015, Brito and Pozo-Zamora 2015).
We present here new records of M. lepida, increasing its known distribution area to the state of Pará, Brazil, in eastern Amazonia, to the south of the Amazon river.

Methods
Three dead specimens were collected in 2015 at three locations (Fig. 1) on the left bank of the Xingu River in the municipality of Vitória do Xingu, state of Pará, Brazil, by a wildlife rescue company during the flooding of the Belo Monte hydropower dam reservoir.This region is known as the great curve of the Xingu River and encompasses the Xingu-Tapajós moist forest ecoregion (Fig. 1), formed by open ombrophilous forests of low-altitude close to sea level (Salomão et al. 2007).
The 3 specimens are deposited and catalogued at the Mammal Collection of the Universidade Federal do Espírito Santo (UFES-MAM).Age criteria were determined by tooth eruption and wear following Voss and Jansa (2009).
The mitochondrial cytochrome b (Cyt b) gene was sequenced for confirmation of taxonomic identification.This marker was chosen because of its efficiency in diagnosing mammals at the species level (Bradley andBaker 2001, Agrizzi et al. 2012).Polymerase chain reactions (PCR) were carried out using 2.5 μl of 10× buffer, 1.0 μl of MgCl 2 at 50 mM, 0.5 μl of deoxynucleotide triphosphate mix (10 mM for each nucleotide), 0.3 μl of each primer at 10 mM, 3 units of Taq Platinum (Invitrogen Corporation, Carlsbad, California), and 1.0 μL template DNA with the final volume of 25 µL.We used primers MVZ05 and MVZ16 (Smith and Patton 1993) to amplify the first 801 base pairs (bp) of the Cytochrome b gene (Cyt b).PCRs were performed with an initial denaturation temperature of 94 °C for 5 min followed by 39 cycles (30 s denaturation at 94 °C, 45 s annealing at 48 °C and extension for 45 s at 72 °C) and a final extension at 72 °C for 10 min.Samples were sequenced in both directions using the same PCR primers listed before.Sequences were aligned in Geneious R9.1 (Biomatters, Auckland, New Zealand) using ClustalW algoritm and deposited in GenBank (Benson et al. 2004) (under accession numbers MG586948, MG586949 and MG586949).We used pairwise p-distance calculated in Geneious to compare sequence divergence to other sequences of M. lepida deposited in GenBank (accession numbers HM106376, U34668, AJ606452 and HM106377) and previously identified by experts in the field such as James L. Patton and Robert Voss, as mentioned on the GBIF website (see Table 1 for GBIF specimens).
The nomenclature of cranial structures measured followed Voss and Jansa (2009).We took 29 cranial measurements with a digital caliper following Rossi et al. (2010) (Table 2).1).Orange circles in the amplified map correspond to collecting localities of the three new specimens of Marmosa lepida.Red solid line is the interstate highway (BR-230).
The current distribution area of the species used in the present study follows Cáceres et al. (2016).
Identification.We examined the 3 specimens and compared external and cranial morphology with descriptions available in the literature (Creighton and Gardner 2008, Voss et al. 2014, Astúa 2015) to confirm the genetic identification of all 3 individuals (pairwise identity of 98.4% and query cover of 98.33% with reference sequence under Genbank accession number HM106376 for all 3 sequences of M. lepida from this study).Significant features of the genus, which distinguish it from other similar genera (e.g.Gracilinanus and Thylamys), are the presence of a strong supra-orbital process and the absence of an anteromedial process of the alisphenoid portion of the auditory bulla and generally spiral tail scales pattern (Figs 2, 4) (Creighton and Gardner 2008).Marmosa lepida is distinguished from other congeners by its smaller size (head and body length 97-120 mm and tail length 140-150 mm) (Table 2), brighter reddish dorsal pelage and brighter chestnut fur (Fig. 3) (Voss et al. 2014).Voss et al. (2001) warned about the problems of morphological identification of young individuals, as they could be easily misidentified as Gracilinanus emilae.Therefore, our 2 younger specimens (UFES-MAM 2921 and 2948) were analyzed morphologically, with only a few measurements (Table 1).The karyotype is unknown and no sexual dimorphism has been recorded for skull size and shape (Astúa 2015).

Discussion
These new records of M. lepida increase the known distribution of the species by about 250 km to the southeast, to the south of Amazon (Fig. 1).This species is currently known from ca 20 localities (Table 1), and they are difficult to capture by conventional methods (Ochoa et al. 2009).Despite a small number of museum specimens of M. lepida (Creighton and Gardner 2008), its conservation status in the IUCN red list is Least Concern (Cáceres et al. 2016) because of its wide geographic range (Fig. 1).
This arboreal genus occurs in moist forest areas throughout the neotropical region, being replaced by Gracilinanus, another genus of arboreal marsupial, in more open and dry areas (e.g.Cerrado of central Brazil).Although Gracilinanus also occurs in moist forests, the 2 genera do not appear to co-occur (Creighton andGardner 2008, Voss et al. 2014).However, we have found one individual of Gracilinanus emilae in the same area (BM 63456 = UFES-MAM 2937, Genbank accession number of Cyt-b sequence MG586951), showing that these 2 genera do occur in sympatry and reinforcing the use of reliable taxonomic methods to diagnose them.
The sampling area of the specimens reported here corresponds to the flooded area to impound water for the operation of the Belo Monte hydropower dam.The total flooded area reaches 516 km 2 , spreading across 3 municipalities in the state of Pará, Brazil: Altamira, Vitória do Xingu, and Brasil Novo (Ministério das Minas e energia 2011, Ministério do Meio Ambiente 2017) (Fig. 1).This project was very controversial, with low social acceptance, including the scientific community, which  argued strongly against the construction of the dam in this region (Nazareno and Lovejoy 2011).The environmental impacts generated by flooding the forest cause not only the loss of habitat, but also changes in flood and droughts dynamics (characteristic of the Amazon region), which directly affect the cycle of the fluvial fauna and the exploitation of the river by local communities and other predators (Winemiller et al. 2016).

Figure 1 .
Figure 1.Map showing known records occurrence for Marmosa lepida.Hatched area represents occurrence range from IUCN red list.Gray area corresponds to Amazonia biome.Black circles correspond to locations of specimens found at on line databases (GBIF and GenBank) and/or literature (numbers correspond to localities listed in Table1).Orange circles in the amplified map correspond to collecting localities of the three new specimens of Marmosa lepida.Red solid line is the interstate highway (BR-230).

Table 1 .
Records of Marmosa lepida sorted according to° Numbered localities mapped in Figure 1, and corresponding reference and/ or voucher° Number and Genbank accession° Number when available.Global Biodiversity Information Facility = GBIF; Field Museum of° Natural History = FMNH; Museum of Vertebrate Zoology = MVZ; Royal Ontario Museum = ROM; Muséum d'histoire° Naturelle de la Ville de Genève = MHNG; Kansas University Museum = KU; Colección de Mamíferos del Instituto de Ciencias° Naturales = ICN-MHN-Ma; University of Michigan Museum of Zoology = UMMZ; Museo de la Escuela Politécnica° Nacional = MEPN.