Unveiling the identity of Kerr's Atlantic tree rat, Phyllomys kerri (Rodentia, Echimyidae)

Mammalian Biology 91 (2018) 57–70

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Original investigation

Unveiling the identity of Kerr’s Atlantic tree rat, Phyllomys kerri (Rodentia, Echimyidae) Edson Fiedler de Abreu-Júnior a , Alexandre Reis Percequillo a,b , Lena Geise c , Yuri L.R. Leite d , Ana Carolina Loss d,∗ a Laboratório de Mamíferos, Departamento de Ciências Biológicas, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Av. Pádua Dias 11, Caixa Postal 9, 13418-900, Piracicaba, São Paulo, Brazil b Department of Life Sciences, The Natural History Museum, SW7 5BD, London, United Kingdom c Laboratório de Mastozoologia, Universidade do Estado do Rio de Janeiro, Departamento de Zoologia, Instituto de Biologia, R. São Francisco Xavier, 524, Maracanã, 20550-900, Rio de Janeiro, Brazil d Laboratório de Mastozoologia e Biogeografia, Universidade Federal do Espírito Santo, Departamento de Ciências Biológicas, Av. Fernando Ferrari 514, Goiabeiras, 29075-910, Vitória, Espírito Santo, Brazil

a r t i c l e

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Article history: Received 25 January 2018 Accepted 19 March 2018 Handled by Daisuke Koyabu Available online 29 March 2018 Keywords: Atlantic Forest emended diagnosis historical DNA molecular phylogeny

a b s t r a c t Arboreal spiny rats of the genus Phyllomys are the most diverse group of echimyid rodents in the Atlantic Forest. Many species of Phyllomys have small geographic ranges and are rare in scientific collections. One of them is Phyllomys kerri known from only three specimens collected in a single locality almost 80 year s ago. The identity and the taxonomic validity of this enigmatic species has been questioned in recent decades. Field surveys at different sites along the southeastern portion of the Atlantic Forest in Brazil recovered specimens of Phyllomys that we hypothesized to be P. kerri based on external similarities to the type specimen and proximity to its type locality. Here we obtained DNA sequences from these recently collected specimens and historic DNA from a topotype of P. kerri collected in 1941. Our results supported the status of P. kerri as a valid species and showed it is phylogenetically positioned among the southern clade of Phyllomys species. We therefore provide an emended diagnosis of P. kerri, comparing it with sympatric congeners, and provide comments on its evolutionary affinities, geographic distribution, and conservation status. ¨ Saugetierkunde. ¨ © 2018 Deutsche Gesellschaft fur Published by Elsevier GmbH. All rights reserved.

Introduction Arboreal spiny rats of the genus Phyllomys Lund, 1839 are the most diverse group of echimyid rodents in the Atlantic Forest comprising 13 described species plus four undescribed lineages distributed throughout this biome (see Araújo et al., 2013; Loss and Leite, 2011). Species of Phyllomys are characterized by medium to large size, dorsal pelage with spiny to soft fur, large eyes, small and rounded ears, long vibrissae, broad and short hindfeet with strong claws, and body hairs extending onto the base of the tail, the latter varying from thickly haired to almost naked at its tip (Leite and Loss, 2015). Many species of Phyllomys have small geographic ranges and are known from few collected specimens because they are nocturnal, elusive, and usually scarce (Emmons and Feer, 1997; Leite,

∗ Corresponding author. E-mail addresses: [email protected] (E.F. de Abreu-Júnior), [email protected] (A.R. Percequillo), [email protected] (L. Geise), yuri [email protected] (Y.L.R. Leite), [email protected] (A.C. Loss).

2003). One of them is Phyllomys kerri (Moojen, 1950), known from only three specimens collected at Ubatuba, São Paulo, southeastern Brazil, between 13 June and 27 July 1941 by G. Dutra (probably Gentil Dutra, see Pacheco and Parrini, 1999) (Emmons et al., 2002; Leite, 2003). No specimen of Phyllomys has been unambiguously assigned to this species ever since, making P. kerri both rare and enigmatic. Leite (2003) raised questions about the identity and taxonomic validity of P. kerri because the diagnostic characteristics provided in the original description by Moojen (1950) fall into the range of morphological variation of Phyllomys nigrispinus (Wagner, 1842). Nevertheless, Leite (2003) provided additional morphological features distinguishing P. kerri from P. nigrispinus and provisionally maintained P. kerri as a valid species but cautioned that additional specimens of both taxa are needed to evaluate their taxonomic statuses. Our recent field surveys at different sites along the southeastern portion of the Atlantic Forest in Brazil recovered specimens of Phyllomys that we hypothesized to be P. kerri based on external similarities to the type specimen and proximity to its type locality. Their taxonomic identification, however, proved to be a very difficult task, especially considering the overlap of morphological traits

https://doi.org/10.1016/j.mambio.2018.03.008 ¨ Saugetierkunde. ¨ Published by Elsevier GmbH. All rights reserved. 1616-5047/© 2018 Deutsche Gesellschaft fur

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and geographic ranges of Phyllomys species in this region, where four taxa might occur (Leite and Loss, 2015): P. kerri, P. nigrispinus, Phyllomys medius (Thomas, 1909), and Phyllomys sulinus Leite et al., 2008. Here we obtained DNA sequences from these recently collected specimens and historic DNA from a topotype of P. kerri collected more than 75 years ago, to infer their phylogenetic affinities. We also evaluated the morphological variation of these samples, in comparison to sympatric and allopatric samples of other congeners, aiming to elucidate the specific status and taxonomic identity of P. kerri. Our results supported the status of P. kerri as a valid species, and we therefore provide an emended diagnosis of this species and comments on its evolutionary affinities, geographic distribution, and conservation status. Material and methods Samples We analyzed three recently collected specimens of P. kerri: one from Parque Estadual da Serra do Mar, Núcleo Picinguaba (hereafter Picinguaba), Ubatuba, São Paulo, Brazil, housed at Museu Nacional da Universidade Federal do Rio de Janeiro (MN), and two from Estac¸ão Ecológica de Bananal (hereafter Bananal), Bananal, São Paulo, Brazil, housed at Museu de Zoologia da Universidade de São Paulo (MZUSP) (see details in Specimens examined section). We also examined all other known specimens of P. kerri, the holotype and the two topotypes, housed at MN (see details in Specimens examined section), as well as comparative specimens of P. nigrispinus (MN 83181 and 83187, and EBB 16 and 28 housed at Colec¸ão do Laboratório de Mamíferos da Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Brazil – LMUSP) and P. sulinus (MN 81062, EEB 798 housed at MZUSP, and VK 19 housed at LMUSP). Molecular analysis Molecular analyses were conducted using two different datasets. The first included 47 mitochondrial cytochrome b (CytB) DNA sequences from Phyllomys species used to confirm the taxonomic identification of specimens in this study. Then we added mitochondrial cytochrome oxidase I (COI) and nuclear exon 28 of the von Willebrand factor gene (vWF) sequences to a subset of 26 specimens from the first dataset, in order to infer the phylogenetic position of these specimens assigned to P. kerri within Phyllomys species. Sequences of Makalata didelphoides (Desmarest, 1817) and Echimys chrysurus (Zimmermann, 1780) were used as outgroups. Details on sequences and GenBank accession numbers are given in Appendix A. We sequenced 801 base pairs (bp) of CytB from the three recently collected P. kerri specimens (MN 84019, EEB 734 and EEB 751) and from one topotype collected in 1941 (MN 5463). We also sequenced CytB from specimens of P. nigrispinus (EBB 16 and EBB 28) and P. sulinus (EEB 798 and VK 19) collected closely (in Estac¸ão Biológica de Boracéia and Picinguaba) or in sympatry (in Bananal) to P. kerri. Additionally, 657 bp of COI and 1164 bp of vWF were sequenced for one specimen of P. kerri (EEB 751). Total genomic DNA was extracted from muscle samples preserved in 95% ethanol or from dried skin sampled from museum specimens. DNA from muscle was extracted using salt and proteinase K (Bruford et al., 1992) protocol. Polymerase chain reaction (PCR) conditions followed Loss and Leite (2011) using primers MVZ05 and MVZ16 (Smith and Patton, 1993) for CytB; primers LCO1490 and HCO2198 (Hebert et al., 2003) for COI; and primers V10, W13 (Galewski et al., 2005), V2 and W1 (Huchon et al., 1999) for vWF. DNA from museum specimens (MN 5463, MN 84019, VK

19, EEB 798) was extracted in a separate DNA clean room dedicated to historical samples using the Qiagen DNAeasy protocol and the steps prior to the extraction following Patterson and Velazco (2008). For skin samples we only amplified and sequenced CytB. As historical DNA is usually degraded in small fragments, CytB amplification was performed using 7 overlapping fragments of ca. 200 bp each. We designed 11 primers using Primer3 (Koressaar and Remm, 2007; Untergasser et al., 2012) implemented in Geneious 6.1.8 (Biomatters Ltd.), based on an alignment of 53 CytB Phyllomys sequences. To check primer specificity and avoid chimeric sequences, designed primers were tested in PCR reactions using DNA extracted from ethanol-preserved tissues of other Phyllomys species as template. All specimens used for this test had known CytB sequences generated using primers MVZ05 and MVZ16, which span larger regions (∼800 pb). After the amplification, the small fragments were sequenced and then compared to reference sequences. Only primers that produced sequences identical to the reference were used to amplify historical DNA. PCR of historical DNA was performed in a final volume of 25 ␮L with 2.5 ␮L of 10× buffer, 1.0 ␮L of MgCl2 (50 mM), 1.0 ␮L of bovine serum albumin (10 m g/mL), 1.0 ␮L of deoxynucleoside triphosphate mix (10 mM for each nucleotide), 0.5 ␮L of each primer (10 ␮M), 1 unit of Taq Platinum (Invitrogen Corporation, Carlsbad, California), and 4 ␮L of DNA template. PCR profiles included an initial denaturation at 94 ◦ C for 2 minute s, followed by 60 cycles and a final extension at 72 ◦ C for 7 minute s. Cycles began with a denaturation at 94 ◦ C for 30 seconds, annealing at 51–54 ◦ C for 30 seconds and extension at 72 ◦ C for 20 seconds. Details of primer sequences, primer combinations and PCR annealing temperatures are given as supplementary material S1. PCR products were purified using ExoSAP enzymes (GE Healthcare Life Sciences). Cycle-sequencing reactions were performed using BigDye Terminator 3.1 (Applied Biosystems, Inc.) and with the same primers used in PCR. Sequences were read in both directions on an ABI-3500 capillary automated sequencer (Applied Biosystems, Inc.) and aligned using Geneious 6.1.8. Pairwise uncorrected genetic p-distances between sequences were calculated in MEGA version 7 (Kumar et al., 2016). PartitionFinder2 (Lanfear et al., 2017) was used to establish the best nucleotide substitution model and partition scheme for the data using the corrected Akaike Information Criterion (AICc) and all models implemented in MrBayes, excluding those with proportion of invariant sites (I) due its dependence on site rate variation (G) (Mayrose et al., 2005; Stamatakis, 2006). The dataset was partitioned by gene and codon position allowing each partition to evolve under an independent model. Nexus files for the alignments including MrBayes block are available as supplementary material S2-3. Bayesian inference was performed in MrBayes version 3.2 (Ronquist et al., 2012) with default settings for the Markov chain Monte Carlo (MCMC) analysis, running for 106 generations, sampling one tree every 5 × 102 generations, resulting in 2 × 103 trees. We discarded the first 25% samples as burn-in and estimated a 50% majority-rule consensus from the remaining trees. Only nodes with Bayesian posterior probability (BPP) equal to or greater than 95% were considered robust. Maximum likelihood and bootstrap analyses were performed using RAxML-HPC2 on XSEDE available at CIPRES Science Gateway (Miller et al., 2010, available at https://www.phylo.org/portal2) with default settings. Only nodes with bootstrap values equal to or greater than 70% were considered robust. Morphological analysis We analyzed qualitative traits of external and craniodental morphology following Emmons et al. (2002), Leite (2003) and Leite et al. (2008). We classified the specimens on age categories based on toothwear pattern following Leite (2003). Craniodental measure-

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Fig. 1. Bayesian inference phylogeny of Phyllomys based on the CytB molecular marker. Statistical support for clades are shown along branches (Bayesian Posterior Probability ¨ ¨indicates clades not recovered in likelihood phylogeny. ≥ 0.95 / Likelihood bootstrap ≥70). -¨ ¨indicates support below cutoff values. NA

ments were performed using a digital caliper (0.01 mm precision) based on Leite (2003): greatest skull length (GSL), nasal length (NL), rostral length (RL), orbital length (OL), rostral breadth (RB), interorbital constriction (IOC), mastoid breadth (MB), zygomatic breadth (ZB), condyloincisive length (CIL), basilar length (BaL), diastema length (D), maxillary toothrow length (MTRL), palatal length a (PLa), palatal length b (PLb), incisive foramina length (IFL), bullar length (BuL), postpalatal length (PPL), maxillary breadth (MaxB), occipital condyle width (OccW), rostral depth (RD), cranial depth (CD), and cranial depth at M1 (CDM1). External measurements were obtained from collector’s field notes: body mass (BM), head and body length (HB), tail length (TL), external ear length (EE), internal ear length (IE), hindfoot length with claws (HC), and hindfoot length without claws (HW).

Species delimitation Leite (2003) presents a discussion on species concepts applied to species delimitation of Phyllomys, advocating that the Phylogenetic Species Concept (PSC; e.g. Cracraft, 1983; Queiroz and Donoghue, 1988) – including its two fundamental elements (monophyly and diagnosability) – is an operational criterion that can support accurate taxonomic decisions for this group. Although there are some pros and cons (see Gutiérrez and Garbino 2018; Zachos et al., 2013; Zachos, 2015, 2016), we follow Leite (2003) on the application of the PSC as an operational and philosophical approach to establish the validity of P. kerri. We are aware of the limitations and problems associated to the concept (Zachos et al., 2013; Zachos, 2015, 2016) and minimized them by generating a phylogeny through molecular analyses based on mitochondrial and nuclear markers, and subse-

quently examining morphological diagnosability, considering both qualitative and quantitative traits, and employing the best available samples of sympatric and closely related species. We also considered the spatial distribution of samples to investigate whether monophyly and morphological differences could be interpreted as a result of geographic isolation only.

Results Phylogenetic analyses recovered 14 main lineages associated with 10 currently valid species of Phyllomys (P. blainvilii, P. brasiliensis, P. dasythrix, P. kerri, P. lamarum, P. lundi, P. mantiqueirensis, P. nigrispinus, P. pattoni, P. sulinus) and 4 possibly undescribed species within the genus (Figs. 1 and 2) already reported in the literature (Araújo et al., 2013; Loss and Leite, 2011). The CytB phylogeny (Fig. 1) confirmed the identification of specimens EEB 734, EEB 751 and MN 84019 as P. kerri, as they clustered with the P. kerri topotype MN 5463 in a monophyletic group with high statistical support (BPP = 1; bootstrap = 94) and low genetic distances among them (0.5–1.3%). The CytB genetic distance between P. kerri and other Phyllomys lineages varies from 12.2% from the farthest Phyllomys sp. 4 to 7.7% from the nearest P. dasythrix. CytB genetic distances within and between Phyllomys species are given in supplementary file S4. The multilocus phylogeny (Fig. 2) showed P. kerri as sister to the southern clade of Phyllomys, formed by P. dasythrix (P. sulinus + P. nigrispinus) with high statistical support (BPP = 1; bootstrap = 94). These four species clustered in a polytomy with 7 other Phyllomys lineages: P. lundi, Phyllomys sp. 1, Phyllomys sp. 2, Phyllomys sp. 3, P. blainvilii (P. lamarum + P. brasiliensis).

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Fig. 2. Bayesian inference phylogeny for Phyllomys species based on CytB, COI and vWF molecular markers. Statistical support for clades are shown along branches (Bayesian ¨ ¨indicates clades not recovered in likelihood phylogeny. Posterior Probability ≥ 0.95 / Likelihood bootstrap ≥70). -¨ ¨indicates support below cutoff values. NA

Therefore, these molecular results confirmed our hypothesis that these recently collected specimens in the southeastern portion of the Atlantic Forest represent new individuals of P. kerri. The molecular data combined with sympatric occurrence of other lineages from the southern clade support P. kerri as an independent monophyletic lineage. These specimens, in addition to the holotype and topotypes, present a unique combination of morphological features that deserve specific status. We present emended diagnosis, distribution, habitat information and field observations of P. kerri, and comparisons with similar congeners, as follows: Phyllomys kerri (Moojen, 1950) Specimens examined BRAZIL: SÃO PAULO: Ubatuba: Estac¸ão Experimental de Ubatuba: MN 6241 (holotype; female; age 8; skin and skull preserved; collected by G. Dutra under field number M 13108 on 3 June 1941), MN 5463 (topotype; male; age 9; skin, skull, and limb bones preserved; collected by G. Dutra under field number M 13191 on 26 June 1941), MN 5464 (topotype; female; age 6; skin and skull preserved; collected by G. Dutra under field number M 13202 on 27 July 1941); Parque Estadual da Serra do Mar, Núcleo Picinguaba: MN 84019 (female; age 8; skin, skull, skeleton, and tissue preserved; collected by P. S. Pinheiro under field number PSP 51 in April 2002); Bananal: Estac¸ão Ecológica de Bananal: EEB 734 (MZUSP uncatalogued; male; age 5; skin, skull, skeleton, and tissue preserved; collected by MZUSP field expedition team on 17 December 2003), EEB 751 (MZUSP uncatalogued; female; age 3; skin, skull, and tissue preserved; collected by MZUSP field expedition team on 18 December 2003).

Emended diagnosis Medium-sized for the genus, head and body length varying from 212 to 253 mm, tail length varying from 215 to 237 mm, hindfoot length varying from 38 to 44 mm, ear length varying from 14 to 18 mm, and body mass varying from 200 to 352 g, based on measurements of adult specimens (MN 5463, MN 6241–holotype, and MN 84019). Dorsal pelage spiny, orangish brown streaked with black (Fig. 3). Aristiforms on rump long (ca. 24 mm), wider in the middle (ca. 0.85 mm), ending in a thin, whip-like tip, pale from the base to the middle portion, gradually darkening toward the tip; some hairs with an orange subterminal band. Lateral pelage orangish brown, paler than dorsum. Ventral color yellowish gray with hairs usually white or pale gray at base (Fig. 4). Mystacial vibrissae numerous and long, surpassing the top of the pinnae when laid back. Pinnae small and covered by dark hairs, with a long tuft of dark hairs at the base. Dorsal surface of manus covered by grayish hairs; digits with medium sized claws. Pes dorsally covered by grayish hairs, whitish digits with long claws and long ungual tufts, extending beyond the claws. Tail covered by long brownish hairs, paler ventrally, giving a slightly bicolored appearance; tail tip tufted. Skull long and narrow (Figs. 5 and 6; Table 1); rostrum short and wide. Nasals not extending beyond the maxillary-frontal-lacrimal suture. Frontal-premaxillary suture collinear with frontal-nasal suture. Supraorbital ridges developed, interorbital region diverging posteriorly, with inconspicuous or absent postorbital process. Parietal largely expanded to the lateral surface of braincase. Upper incisors slightly opisthodont. Zygomatic arch robust with maximum height approximately one-third of jugal length. Postorbital process of zygoma spinose and formed only or mainly by jugal.

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Fig. 3. Dorsal views of skins of Phyllomys kerri. From left to right: MN 84019 (female; age 8), MN 5463 (male; age 9), MN 6241 (female; age 8), MN 5464 (female; age 6), EEB 751 (female; age 3).

Fig. 4. Ventral views of skins of Phyllomys kerri. From left to right: MN 84019 (female; age 8), MN 5463 (male; age 9), MN 6241 (female; age 8), MN 5464 (female; age 6), EEB 751 (female; age 3).

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Fig. 5. Dorsal and ventral views of skulls of Phyllomys kerri. From left to right: MN 84019 (female; age 8; GSL = 53.34 mm), MN 5463 (male; age 9; GSL = 52.06 mm), MN 6241 (female; age 8; GSL = 51.69 mm), MN 5464 (female; age 6; GSL = 46.07 mm), EEB 734 (male; age 5; GSL = 44.27 mm).

Lateral process of supraoccipital short, reaching horizontal midline of external auditory meatus, or long, extending below midline of external auditory meatus. Incisive foramina ovate or tear drop in shape. Palatine width equal to or greater than tooth width at M1. Upper toothrows nearly parallel or slightly divergent posteriorly. Mesopterygoid fossa wide, forming an angle of nearly 60◦ , not surpassing M3 or reaching last laminae in M2. Sphenopalatine vacuities absent. Alisphenoid strut present, buccinator and masticatory foramina not confluent. Foramen ovale large and ovate or nearly rounded in shape. Posterior opening of alisphenoid canal varying from completely visible to almost invisible on ventral surface of skull. Auditory bullae large. Angular process of mandible long, surpassing the condylar process. Ventral root of the angular process not deflected or slightly deflected laterally. Ventral mandibular spine absent. Two specimens (MN 84019 and EEB 734) have complete skeleton preserved. The axial skeleton includes 7 cervical, 14 thoracic, 8 (EEB 734) or 7 (MN 84019) lumbar, 3 sacral, and 33 caudal vertebrae. EEB 734 has an incomplete tail with 29 caudal vertebrae. In MN 84019 only sacral vertebrae s1 and s2 are fused, whereas EEB

734 has all three sacral vertebrae fused. Both specimens have 14 ribs, but EEB 734 has only vestiges of the 14th rib. Distribution Phyllomys kerri is known from the northeastern region of the state of São Paulo (Fig. 7). Its geographic distribution includes two sites in Ubatuba municipality, namely Estac¸ão Experimental de Ubatuba (the type locality, currently known as Unidade de Pesquisa e Desenvolvimento de Ubatuba) and Parque Estadual da Serra do Mar, Núcleo Picinguaba, both near sea level, and one site at Serra da Bocaina, Estac¸ão Ecológica de Bananal, at around 1,200 m of elevation. Habitat and field observations In this section we present habitat information and field observations for the three localities where P. kerri was collected: Unidade de Pesquisa e Desenvolvimento de Ubatuba (UPDU, known as Estac¸ão Experimental de Ubatuba in 1941, when P. kerri was described, and also popularly known as Horto Florestal): This area (ca. 1,100 ha) is contiguous with the Parque Estadual da Serra

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Fig. 6. Lateral view of skull and mandible of Phyllomys kerri. From top to bottom: MN 84019 (female; age 8; GSL = 53.34 mm), MN 5463 (male; age 9; GSL = 52.06 mm), MN 6241 (female; age 8; GSL = 51.69 mm), MN 5464 (female; age 6; GSL = 46.07 mm), EEB 734 (male; age 5; GSL = 44.27 mm).

do Mar, Núcleo Picinguaba (see below), and is on the outskirts of ¨ Ubatuba, ca. 6 km NW of downtown (23◦ 25’00S¨ and 45◦ 06’47W), along the road to Taubaté (Rodovia Oswaldo Cruz, SP–125). This was an agricultural experimental station during most of the 20th century, but today it seems to be largely abandoned, with no signs of research activity, and only a few families of state employees still living in the area. In addition to cultivated areas, UPDU also has large patches of rainforest, especially on the slopes, where Myrtaceae and Lauraceae are the two most diverse families in number of species, while Euphorbiaceae, Palmae, and Rubiaceae are the most frequently encountered families (Silva and Leitao Filho, 1982). Canopy height ranges from 10 to 30 m, and some emergent trees reach up to 40 m (Goerck, 1999). There is a weather station at UPDU, and a 30-year record (1966–1995) showed average precipitation

of 2,100 mm/year and average temperature of 22 ◦ C, with a super humid season from October to April (Bencke and Morellato, 2002). Parque Estadual da Serra do Mar, Núcleo Picinguaba (PESM-P): The PESM-P has about 8,000 ha in the municipality of Ubatuba, in the northern coast of São Paulo, close to the border with Rio de Janeiro, around 23◦ 22’ S and 44◦ 48’ W (Hartmann, 2005; Sanchez et al., 1999). The elevation at PESM-P ranges from sea level to 100 m (Hartmann, 2005). The Núcleo Picinguaba connects the Parque Nacional da Serra do Mar to the Parque Nacional da Serra da Bocaina, making a large (about 118,000 ha) and continuous protected area (Sanchez et al., 1999). The climate is categorized as tropical humid, with rainfall year-round (Pinheiro and Geise, 2008; Sanchez et al., 1999). The mean annual temperature ranges from 20 to 24 ◦ C and the annual precipitation ranges from 1,500 to 4,000 mm (Pinheiro and Geise, 2008). The vegetation includes

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Table 1 External body and craniodental measurements (mm) and body mass (g) of Phyllomys kerri specimens and comparative specimens of P. nigrispinus and P. sulinus. Only adults were used. “NA” indicates missing data due absence of information on catalogue notes or skull damage. Specimens acronyms are described in Appendix A and measurements’ in morphological analysis subsection. P. kerri Specimen Source Sex Age BM HB TL EE IE HC HW GSL NL RL OL RB IOC MB ZB CIL BaL D MTRL PLa PLb IFL BuL PPL MaxB OccW RD CD CDM1

MN 6241 (holotype) This study female 8 200 212 223 NA 17 38 NA 51.69 16.42 19.61 13.92 8.41 12.05 19.10 23.28 46.14 39.81 12.08 11.25 20.44 8.28 4.70 9.87 20.74 8.12 8.56 11.33 19.40 14.37

P. nigrispinus MN 84019 This study female 8 352 253 237 NA 18 44 41 53.34 15.82 19.92 14.69 10.06 11.63 20.46 25.90 47.23 40.91 12.26 11.92 21.19 9.47 4.90 11.45 22.40 8.71 8.99 11.21 17.40 15.36

MN 5463 This study male 9 250 235 215 NA 14 41 NA 52.06 15.58 18.44 15.28 7.77 11.56 18.49 23.57 45.57 39.20 11.24 10.99 20.17 8.62 4.09 8.90 21.05 7.19 8.18 11.90 18.50 14.87

EBB 28 This study female 8 199 195 NA NA 19.5 NA NA 50.11 14.68 18.93 14.57 8.41 10.35 20.24 25.27 45.79 39.05 11.20 11.58 20.21 9.94 4.66 11.10 21.95 8.87 8.50 10.91 15.87 14.56

P. sulinus MN 83187 Delciellos et al. (2017) female 8 234 229 220 NA 17 42 39 51.09 16.85 19.95 14.61 8.03 10.40 19.98 23.30 45.54 38.98 11.42 12.58 19.91 9.27 5.38 10.11 21.78 8.20 8.11 11.06 15.73 14.50

MN 83181 Delciellos et al. (2017) male 9 202 214 NA 11 16 39.5 37 50.91 15.94 19.46 13.83 7.19 10.13 20.63 23.38 45.72 39.70 11.63 11.24 20.47 9.78 5.46 10.21 22.31 8.19 8.48 10.73 16.35 14.13

MN 81062 Delciellos et al. (2017) female 7 178 208 225 12 14 42 39 47.16 13.47 17.26 13.97 6.94 10.44 19.12 23.06 41.27 35.79 9.97 NA 18.84 9.02 5.01 9.81 20.79 NA 8.79 10.16 15.77 13.75

VK 19 This study NA 8 NA NA NA NA NA NA NA 46.81 14.82 17.97 13.21 7.07 9.56 19.00 23.00 42.08 36.27 10.10 12.01 19.10 9.95 4.83 9.80 20.75 7.97 8.06 10.36 17.30 13.73

Fig. 7. Map of occurrence of Phyllomys kerri. 18- Unidade de Pesquisa e Desenvolvimento de Ubatuba, Ubatuba, SP; 25- Parque Estadual da Serra do Mar, Núcleo Picinguaba, Ubatuba, SP; 17- Estac¸ão Ecológica de Bananal, Bananal, SP. Brazilian states abbreviation: Minas Gerais (MG), Rio de Janeiro (RJ) and São Paulo (SP). See detailed information of localities on the text and Appendix B.

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Table 2 Morphological comparisons between Phyllomys kerri and the sympatric species from the southern species group. Only adults were used. Specimens acronyms are described in Appendix A and measurements’ in morphological analysis subsection. Character

P. kerri (MN 6241, MN 5463, MN 84019)

P. nigrispinus (MN 83181, MN 83187, EBB 28)

P. sulinus (MN 81062, VK 19, EEB 798)

Texture of dorsal pelage Aristiforms on rump Aristiforms color Aristiforms tip Tail tip Palatine width at M1 Maxillary toothrows

spiny 25.35 × 1.02 black to ocher whip-like, black or orange tufted > tooth width slightly divergent posteriorly

Incisive foramen Mesopterygoid fossa Supraorbital ridges Interorbital region Postorbital process Zygomatic arch height Postorbital process of zygoma

ovate to tear drop not surpassing M3 or reaching M2 developed to well developed divergent posteriorly inconspicuous or absent ≤ or ≥ 1/3 jugal length spinose, formed mainly by jugal or by jugal only

soft to stiff 25.47 × 0.39 black to gray whip-like, black tufted > tooth width parallel to slightly divergent posteriorly tear drop reaching M2 developed divergent posteriorly absent ≈ 1/3 jugal length spinose, formed mainly by jugal

Mastoid process

short to long, extending to midline or bellow of EAM

Ventral root of the angular process Ventral mandibular spine Upper incisors

does not deflect laterally or slightly deflected laterally absent slightly opisthodont

stiff to spiny 23.96 × 0.52 black to gray whip-like, black or orange tufted > tooth width parallel to slightly divergent posteriorly ovate to slightly tear drop not surpassing M3 or reaching M2 developed divergent posteriorly absent ≤ or ≥ 1/3 jugal length spinose, formed mainly by jugal or by jugal only short to long, extending to midline or bellow of EAM deflected laterally absent or present slightly opisthodont

the sub-montane Dense Ombrophilous Forest, Lowland Dense Ombrophilous Forest, and Arboreal to Shrub/Herbaceous Meadows (Pinheiro and Geise, 2008). The specimen of P. kerri was an adult female, found dead in a pitfall trap on Vietnam trail at 15 m above sea level in April 2002. Estac¸ão Ecológica de Bananal (EEB): The EEB (884 ha) is in the municipality of Bananal, Serra da Bocaina, northeastern São Paulo, o o o o between 22 15’ to 22 37’ S and 44 07’ to 44 22’ W. It is a region of steep slopes – elevation ranges from 1,100 to 2,000 m (Castro, 2001). The climate at EEB is mesothermal, without dry season and with mild summer. The average annual rainfall ranges from 1,500 to 2,000 mm, and the mean annual temperature varies from 20 to 33 ◦ C (Castro, 2001; Fundac¸ão Florestal, 2013). The vegetation at EEB is typical of montane (between 1,100 m and 1,500 m of altitude) and high-montane (above 1,500 m) formations of the Dense Ombrophilous Forest (Castro, 2001). The forest is present in different successional stages, from secondary-disturbed to pristine areas, where the canopy reaches 30 m high (Percequillo et al., 2017, 2011). The specimens of P. kerri were trapped in pitfall traps in a humid second-growth forest with diverse understory, including palm trees (e.g. Euterpe edulis Martius) and lianas. These specimens were captured in December 2003 and were both juveniles, with no signs of reproductive activity: abdominal testes in the male and unperforated vagina in the female. Comparisons P. kerri may occur in sympatry with two species of the southern clade of Phyllomys: P. sulinus and P. nigrispinus. These three species each present large intraspecific morphological variation, as noted here (Table 2) and already reported by Delciellos et al. (2017). However, some morphological traits distinguish P. kerri from specimens of P. sulinus and P. nigrispinus collected near or in the geographic range of P. kerri, such as: P. kerri has wider aristiforms on rump (1.0 mm) when compared with P. sulinus (0.4 mm) and P. nigrispinus (0.5 mm); the tail hairs are mostly pale brown in P. kerri while in P. sulinus and P. nigrispinus they are dark brown; the skull of P. kerri is larger (GSL: 51.69 –53.34 mm) than the skull of P. sulinus (46.81–47.16 mm) and P. nigrispinus (50.11–51.09 mm); and the interorbital constriction is wider in P. kerri (IOC: 11.56–12.05 mm) than in P. sulinus (9.56–10.44 mm) and P. nigrispinus (10.13–10.40 mm). Besides the putative sympatric species from the southern clade, there are two other species near

short, extending to midline of EAM deflected laterally absent slightly opisthodont

the range of P. kerri: P. thomasi and P. medius. The former is endemic to São Sebastião Island and it has very large body and cranial measurements (see Leite, 2003), exceeding the range of P. kerri. The latter has a stiff pelage with very long and thin (36 × 0.4 mm) aristiforms on rump (Leite, 2003). Discussion Phyllomys is a highly diverse genus of Atlantic Forest arboreal echimyid rodents. Leite (2003) employed an integrative taxonomic approach using morphological, molecular and karyotypic data to diagnose and delimit species, and described two new species. For some species, however, only a few museum specimens were then available (e.g. 3 Phyllomys kerri, 2 P. lundi, 1 P. mantiqueirensis, 1 P. unicolor), precluding analyses of morphological variation. Sampling effort for small mammals in the Atlantic Forest has increased consistently in the past two decades, and several specimens have been added to collections, especially due to the use of pitfall traps (Bovendorp et al., 2017). Therefore, today we are able to better evaluate and understand issues regarding morphological variation and species limits within this genus. Since Leite (2003), one new species was described (Phyllomys sulinus Leite et al., 2008), four others have been recognized as putative new species but have not been described yet (see Araújo et al., 2013; Loss and Leite, 2011), and studies have added new data on morphological variation of some species (see Delciellos et al., 2017). Our goal here was to answer the question raised by Leite (2003) whether P. kerri represented a distinct species or a junior-synonym of P. nigrispinus. We argue that P. kerri represents a distinct species under the corollaries of PSC, given that: (i) available DNA sequences of P. kerri specimens form a robust clade reciprocally monophyletic to another clade containing ((P. sulinus, P. nigrispinus) P. dasythrix)); and (ii) these specimens are diagnosable from other sympatric species by genetic (karyotypic or molecular) and/or phenotypic features. Moreover, P. kerri and P. nigrispinus are not sister taxa and are morphologically distinct, so we reject the earlier hypothesis of co-specificity (Leite, 2003). Furthermore, three of these monophyletic species (P. kerri, P. nigrispinus and P. sulinus) overlap their geographic distribution ranges, and their sympatry and even syntopy suggest genetic and thus reproductive isolation, reinforcing their recognition as distinct species. Zachos (2015, 2016) cautioned

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Fig. 8. Geographical distribution of southern clade species of Phyllomys. Brazilian states abbreviation: Minas Gerais (MG), Paraná (PR), Rio de Janeiro (RJ), Rio Grande do Sul (RS), Santa Catarina (SC) and São Paulo (SP). See detailed information of localities on Appendix B.

about the inappropriate application of the PSC, but we are confident that we avoided the pitfalls enlisted by this author, and present a consistent hypothesis on the delimitation of P. kerri. Echimyid rodents are well known by the overlapping of phenotypic traits among species and thus challenging diagnoses. It makes the access of genetic information crucial to evaluate species limits within the group (e.g. Emmons and Fabre, 2018; Patton et al., 2000). This is especially true for Phyllomys where genetic data revealed four putative new species, corresponding to independent evolutionary lineages (preset study Fig. 2; Araújo et al., 2013), which have been historically misidentified based solely on phenotypic features (Loss and Leite, 2011). The recognition of P. kerri as a valid species is also important in revealing hidden variation and helping elucidate the processes underlying the evolution of morphological similarity (i.e. cryptic) and sympatry, as it seems to be a recurring pattern in Phyllomys and other Echimyidae (e.g. Costa et al., 2016). Here we presented a phylogenetic hypothesis with the broadest taxonomic coverage so far for the genus Phyllomys, including 10 described species and four putative new species. We corroborated some phylogenetic relationships obtained in previous studies, such as the northeastern and southern clades (see Araújo et al., 2013; Delciellos et al., 2017; Loss and Leite, 2011), and added Phyllomys kerri to the latter. Moreover, we doubled the sample size of P. kerri and obtained molecular data from this species for the first time. Phyllomys kerri is sister to all remaining species of the southern clade and it presents the smallest geographical range among the southern taxa, occurring near the northern limit of the distribution of this group. Phyllomys dasythrix is sister to P. sulinus + P. nigrispinus, and is distributed from the states of Rio Grande do Sul to Paraná, occurring in the southern limit of the southern clade. Phyllomys sulinus and P. nigrispinus have the broadest ranges among the southern clade, and are largely sympatric in the states of Paraná and São Paulo. Moreover, there is a contact zone between P. dasythrix,

P. sulinus, and P. nigrispinus in the state of Paraná, and a region of sympatry of P. sulinus, P. nigrispinus, and P. kerri in northeastern São Paulo. In three localities, we found sympatry between two species of Phyllomys: at Estac¸ão Ecológica de Boracéia and Parque Nacional da Serra da Bocaina we recorded P. nigrispinus and P. sulinus; and at Estac¸ão Ecológica de Bananal we collected P. sulinus and P. kerri. The altitudinal variation present in southeastern Atlantic Forest also contributes to its diversity, since distinct species occur in different habitats along the altitudinal gradient (Leite, 2003; Mustrangi and Patton, 1997). The southeastern portion of the Atlantic Forest has been recognized as the region of greatest mammal richness along the biome, especially considering rodent species (see Amori et al., 2013; Costa et al., 2000; Prado and Percequillo, 2013; Prado et al., 2015). This remarkable diversity might be a result of its geological history that shaped a complex relief with large mountain ranges (e.g. Serra do Mar, Serra da Mantiqueira) separated by extensive valleys (e.g. Vale do Rio Paraíba do Sul). These mountains and valleys could have acted as geographical barriers, driving the speciation process (see Costa et al., 2012). Climatic fluctuations can be highlighted as a cause of this great diversity as well, including forest retractions and expansions and sea level fluctuations that strongly influenced the genetic diversity of taxa and occurred in this region along the Pliocene and Pleistocene (Carnaval and Moritz, 2008; Leite et al., 2016). Loss and Leite (2011) suggested a parapatric diversification within Phyllomys through ecological latitudinal gradients in the Atlantic Forest based on the first multilocus phylogeny of this genus. For the southern clade, there is some level of latitudinal structure that could be interpreted as a response to latitudinal gradients. Nevertheless, we can also correlate the southern clade diversification with altitudinal and vegetation gradients (see Fig. 8). Phyllomys kerri is sister to all other species from the southern

E.F. de Abreu-Júnior et al. / Mammalian Biology 91 (2018) 57–70

clade and its restrict geographic range is associated to the lowlands and slopes of Serra do Mar that represent one of the most stable areas of southern Atlantic Forest throughout past climatic changes (Carnaval and Moritz, 2008; Leite et al. 2016). The sister lineages from southern clade, on the other hand, are associated to interior and Araucaria forests, that are mostly distributed on the highlands and possibly represent less stable and more recent areas. This suggests an earlier parapatric diversification event driven by environmental gradient, originating the linage that led to P. kerri, followed by more recent allopatric diversification events, driven by climatic changes, originating the remaining species from southern clade. Regarding conservation status, Kerr’s Atlantic tree rat is listed as “Data Deficient” on the IUCN Red List of Threatened Species (Leite and Loss, 2016), and there is much to be learned about its biology, ecology, and conservation. Here we provided important information about its taxonomic status and phylogenetic affinities, and we also found two new localities of occurrence of P. kerri, extending its geographical and altitudinal range. Since its discovery, in the early 1940s, only six specimens of P. kerri have been collected, leading us to believe that this species is rare. It is important to note, however, that all collected specimens of P. kerri came from protected areas, indicating that the known populations are already under protection. Field efforts on surveying species of Phyllomys are crucial and must continue in the Atlantic Forest. Moreover, field biologists and ecologists who find individuals of Phyllomys should integrate with taxonomists to correctly identify the specimens at species level due to their morphological complexity. By doing so, valuable ecological data will be associated to a correct taxonomic entity, and this is the only way to further promote conservation of Phyllomys species. Declarations of interest None. Acknowledgements We are thankful to the curators that granted us access to the specimens under their care: J. A. de Oliveira (MN) and M. de Vivo Phyllomys species and outgroups

Catalog number

Makalata didelphoides Echimys chrysurus

UFMG 3012 V8M5 (at MZUSP) USNM 549594 UFMG 3014 UFMG 3017 UFMG 2376 MCNU 828 MCNU 844 MN 5463 MN 84019 EEB 751 EEB 734 UFES 284 UFMG 3016 MCNM 2275 MN 62392 MN 62393 B 723 NSV 160599 MN 83181 MN 83183 MN 83187 MN 83196 EBB 16 EBB 28 AB 740 AB 527

Phyllomys blainvilii Phyllomys brasiliensis Phyllomys dasythrix Phyllomys kerri

Phyllomys lamarum

Phyllomys lundi Phyllomys mantiqueirensis Phyllomys nigrispinus

67

(MZUSP). We thank J. Justino (UFES) for valuable help with laboratory work, especially on the implementation of the DNA clean room dedicated to historical samples. Financial support was provided to E. F. de Abreu-Júnior by Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo – FAPESP (2010/14633-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (147145/20163); to A. C. Loss by Fundac¸ão de Amparo à Pesquisa e Inovac¸ão do Estado do Espírito Santo – FAPES/CAPES (68854315/14); to L. Geise by UERJ/Prociência and CNPq; to Y. L. R. Leite by FAPES and CNPq; to A. R. Percequillo by FAPESP (2009/16009-1 and 2015/20055-2) and CNPq. We also thank Paula Soares Pinheiro who carried on the field work in Picinguaba, with grants provided by the World Wide Fund for Nature (WWF). Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.mambio.2018. 03.008. Appendix A. Catalog number and GenBank accession code of specimens analyzed. GenBank accession number of the sequences generated for this study are given in bold. Abbreviation for scientific collection: MCNM = Museu de Ciências Naturais, Pontifícia Universidade Católica de Minas Gerais, Brazil; MCNU = Museu de Ciências Naturais, Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil; MN = Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; UFES = Colec¸ão de Mamíferos, Universidade Federal do Espírito Santo, Espírito Santo, Brazil; MZUSP = Museu de Zoologia, Universidade de São Paulo, São Paulo, Brazil; UFMG = Colec¸ão de Mamíferos do Departamento de Zoologia, Universidade Federal de Minas Gerais, Minas Gerais, Brazil; USNM = National Museum of Natural History, Smithsonian Institution, Washington, DC, USA. Collector responsible for field number listed: A. Christoff (AC); A. R. Percequillo (EEB, EBB); J. C. Voltolini (NSV); P. H. Asfora (PHA); R. Pardini (AB, B); Y. Yonenaga-Yassuda and V. Fagundes (CIT), Vanessa Kuhnen (VK). GenBank accession codes CytB

COI

vWF

EU313232 KF874584 EF608180 EU313239 EF608182 EF608185 JF297832 MH068845 MH068846 MH068847 MH068848 JF297816 EF608181 KF874585 EF608183 EF608179 JF297809 EU313243 KX618646 KX618647 KX618648 KX618649 MH068849 MH068850 JF297811 JF297807

JF297658 KF874588 JF297686 JF297684 JF297680 JF297660 JF297659 MH068843 JF297682 JF297681 JF297672 JF297671 JF297667 JF297669 -

JF297707 AJ251141 JF297735 JF297733 JF297729 JF297709 JF297708 MH068844 JF297731 JF297730 JF297721 JF297720 JF297716 JF297718 -

68

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Phyllomys pattoni

Phyllomys sulinus

Phyllomys sp. 1 Phyllomys sp. 2 Phyllomys sp. 3 Phyllomys sp. 4

B 374 B 733 MN 84017 MN 84018 MN 62391 UFES 620 B 304 MCNU 833 MCNU 826 MCNU 837 AC 632 MN 81062 EEB 798 VK 19 AB 490 UFMG 2487 UFMG 2489 PHA 355 PHA 358 MN 84015 MN 84016 MCNM 2027 MCNM 2709

JF297808 JF297810 JF297826 JF297825 EF608187 JF297827 JF297806 JF297833 KX618650 JF297834 KX618644 KX618645 MH068851 MH068852 JF297805 JF297813 JF297815 JF297819 JF297822 JF297835 EF608184 KF874586 KF874587

JF297700 JF297699 JF297695 JF297701 JF297662 JF297663 JF297677 JF297679 JF297689 JF297692 JF297673 JF297675 KF874590 KF874591

JF297749 JF297748 JF297744 JF297750 JF297711 JF297712 JF297726 JF297728 JF297738 JF297741 JF297722 JF297724 KX852280 KX852281

Appendix B. Localities of occurrence of southern clade species of Phyllomys presented in the map of Fig. 8. Number

Locality

Decimal latitude

Decimal longitude

Source

1 2 3 4

Brazil: Rio Grande do Sul: Bairro Agronomia, Porto Alegre Brazil: Rio Grande do Sul: Itapuã, Viamão Brazil: Rio Grande do Sul: Parque Estadual de Itapuã, Viamão Brazil: Rio Grande do Sul: Parque Nacional dos Aparados da Serra, Cambará do Sul Brazil: Rio Grande do Sul: Pinheiros, Candelária Brazil: Rio Grande do Sul: Porto Alegre Brazil: Rio Grande do Sul: São Francisco de Paula Brazil: Rio Grande do Sul: Usina Hidrelétrica de Itá, Aratiba Brazil: Santa Catarina: Ilha de Santa Catarina Brazil: Santa Catarina: Serra do Tabuleiro Brazil: Paraná: Guajuvira Brazil: Paraná: Palmira Brazil: Paraná: Parque Barigüi, Bairro Mercês, Curitiba Brazil: São Paulo: Barra de Icapara Brazil: São Paulo: Barra do Rio Juquiá Brazil: São Paulo: Estac¸ão Biológica de Boracéia, Salesópolis Brazil: São Paulo: Estac¸ão Ecológica de Bananal, Bananal Brazil: São Paulo: Estac¸ão Experimental, Ubatuba Brazil: São Paulo: Floresta Nacional de Ipanema, 20 km NW Sorocaba Brazil: São Paulo: Ilha do Cardoso, Cananéia Brazil: São Paulo: Interlagos, São Paulo Brazil: São Paulo: Itapetininga Brazil: São Paulo: Itatiba Brazil: São Paulo: near São Paulo Brazil: São Paulo: Núcleo Picinguaba, Trilha do Vietnã, Ubatuba Brazil: São Paulo: Parque Estadual da Serra do Mar, Núcleo Santa Virgínia, 10 km NW Ubatuba Brazil: São Paulo: Primeiro Morro Brazil: São Paulo: Ribeirão Fundo Brazil: São Paulo: Rio Guaratuba, Bertioga Brazil: São Paulo: São Paulo Brazil: São Paulo: Sítio Baleia, Piedade Brazil: São Paulo: Sítio Cac¸ador, Reserva Florestal do Morro Grande, Cotia Brazil: São Paulo: Sítio Jupará, Piedade Brazil: São Paulo: Sítio Palmito, Reserva Florestal do Morro Grande, Cotia Brazil: São Paulo: Sítio Pexe, Reserva Florestal do Morro Grande, Cotia Brazil: São Paulo: Sítio Pseudópode, Reserva Florestal do Morro Grande, Cotia Brazil: São Paulo: Taboão da Serra Brazil: São Paulo: Teodoro Sampaio Brazil: São Paulo: Vanuire Brazil: Rio de Janeiro: Fazenda Alpina, Teresópolis Brazil: Rio de Janeiro: Parque Nacional da Serra da Bocaina - RJ-165, Paraty, Rio de Janeiro, Area 1 Brazil: Rio de Janeiro: Parque Nacional da Serra da Bocaina - RJ-165, Paraty, Rio de Janeiro, Area 2 Brazil: Rio de Janeiro: Teresópolis

-30.079167 -30.266666 -30.400000 -29.250000

-51.124722 -51.016666 -50.966667 -49.833332

Leite (2003) Leite (2003) Leite (2003) Leite (2003)

-29.783333 -30.079167 -29.450001 -27.266666 -27.600000 -27.833332 -25.600000 -25.700001 -25.415556 -24.683332 -24.366667 -23.650000 -22.806667 -23.433332 -23.435278 -25.133333 -23.716667 -23.583333 -23.000000 -23.533333 -23.333333 -23.358333

-52.733334 -51.125000 -50.583332 -52.316666 -48.500000 -48.783333 -49.533333 -50.150002 -49.300833 -47.466667 -47.816667 -45.900000 -44.367778 -45.066666 -47.628056 -47.966667 -46.700000 -48.050000 -46.849998 -46.616669 -44.850000 -45.125000

Leite (2003) Leite (2003) Leite (2003) Leite et al. (2008) Leite et al. (2008) Leite et al. (2008) Leite (2003) Leite (2003) Leite et al. (2008) Leite (2003) Leite (2003) Present study Present study Leite (2003) Leite (2003) Leite (2003) Leite (2003) Leite (2003) Leite (2003) Leite (2003) Present study Loss and Leite (2011)

-24.300000 -24.333333 -23.740833 -23.533333 -23.888996 -23.743800

-47.733333 -47.750000 -45.891111 -46.616669 -47.459761 -47.001385

Leite (2003) Leite (2003) Leite (2003) Leite (2003) Loss and Leite (2011) Loss and Leite (2012)

-23.960370 -23.771107

-47.382619 -47.001077

Loss and Leite (2013) Loss and Leite (2014)

-23.790704 -23.747132

-47.006283 -47.002741

Loss and Leite (2015) Leite et al. (2016)

-23.600000 -22.516666 -21.783333 -22.416667 -23.189722

-46.766666 -52.166668 -50.383333 -42.833333 -44.837222

Leite (2003) Leite (2003) Leite (2003) Leite (2003) Delciellos et al. (2017)

-23.198611

-44.839722

Delciellos et al. (2017)

-22.433332

-42.983334

Leite (2003)

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 21 32 33 34 35 36 37 38 39 40 41 42 43

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