First record of Proechimys pattoni da Silva , 1998 ( Rodentia , Echimyidae ) in northwestern Bolivia

Proechimys pattoni da Silva, 1998 is one of the 3 small-bodied species of Proechimys and its geographic range is only known in western Brazil and eastern and southern Peru. However, based on morphological and molecular analyses, we report P. pattoni from the lowland forest of Bolivia (Pando: Rio Madre de Dios, near San Rosa). This is the first report of P. pattoni in Bolivia and extends its distributional range 315 km to the southeast in the Amazon biogeographic region of Bolivia, representing the southeastern most record. Furthermore, we document the karyotype (2n = 40 / FN = 56) and morphological variation in diagnostic characters.


Introduction
Spiny rats of the genus Proechimys are distinguished from others echimyids by an elongated head and long rostrum, large and erect ears, narrow and long hind feet, and dorsal coloration which varies from reddish-brown to gray-brown, often streaked with black along the midline (Patton et al. 2000, Patton andLeite 2015).Some 22 species are now recognized in the genus and these are organized in about 10 groups based on morphological affinities (Patton 1987;Patton and Leite 2015).Only a handful of these groups have been supported by molecular analyses, including the "gardneri" group (da Silva 1998; Patton et al. 2000).
The "gardneri"group of Proechimys includes the only 3 small-bodied species (head and body length < 185 mm) of the genus: Proechimys gardneri da Silva, 1998; P. kulinae da Silva, 1998;and P. pattoni da Silva, 1998.These 3 species are parapatric in western Amazonia (Patton et al. 2000): P. gardneri is known from western Amazonian Brazil (right bank of central and lower region of the Jurua River and both sides of Madeira River) and northern Bolivia; P. kulinae occurs only on the left bank of the central region of the Jurua River, extending north to the right bank of the Amazon river in northeastern Peru; P. pattoni occurs on both sides of the headwaters of the Jurua River in Brazil and extends into eastern and southern Peruvian Amazonia (da Silva 1998, Patton et al. 2000, Schetino 2008).Whereas P. kulinae is the most divergent species in the "gardneri" group based on morphology, DNA variation, and karyotype; P. pattoni and P. gardneri share several morphological features and almost the same karyotype (2n = 40, FN = 56) because P. gardneri from the Madeira river presents 2n = 40, FN = 54 (Eler et al. 2012); however, DNA sequence divergence  between these species at the cytochrome-b locus averages about 12% (da Silva 1998, Patton et al. 2000, Patton and Leite 2015).
Currently, Proechimys gardneri is the only smallbodied species recorded in Bolivia (Salazar-Bravo et al. 2003, Patton andLeite 2015).In the course of a revision of species of Proechimys occurring in Bolivia, we discovered other small-bodied specimens that closely resembled the characteristics of P. pattoni instead of P. gardneri.Based on those specimens, we examined morphological, chromosomal, and molecular data to test whether P. pattoni occurs in Bolivia.In addition, we highlight morphological characters for the Bolivian specimens that differ slightly from those previously considered as diagnostic for P. pattoni.
For molecular analyses, DNA was isolated from Bolivian (only the tissue from MSB 57229 was available) and Peruvian specimens of small-bodied Proechimys, using IBI Scientific Genomic DNA Mini kit and following the manufacturer's instructions.Specimens are listed in the Appendix, Table A1.Amplification of a fragment of the cytochrome b gene sequence was performed by the polymerase chain reaction (PCR) using primers MVZ 05 and MVZ 16 (Smith and Patton 1993).Phylogenetic reconstruction included sequences reported by Patton et al. (2000), from GenBank and sequences we obtained in this study (Appendix, Table A1).Alignment of sequences was performed with MEGA v7.0.14 (Kumar et al. 2016) using Clustal W (Thompson et al. 1997) with a total sequence length of 801bp.Phylogenetic analyses were Table 1.External and skull measurements (in mm) of Proechimys pattoni from Pando, Bolivia and measurements of the holotype and other individuals from the Jurua River, Brazil (da Silva 1998, Patton et. al. 2000).See text for definition of measurements.(Kimura-2p, Kimura 1980) among haplotypes was estimated in MEGA v7.0.14 (Kumar et al. 2016).In addition, a non-differentially stained karyotype was prepared from field-prepared slides for one of the 2 specimens (MSB 57230) following protocols described in Anderson et al. (1987).
Identification.Two small-bodied specimens (female: MSB 57229, age-class X; male: MSB 57230, age-class IX) agree with the description of P. pattoni by da Silva (1998) and Patton et al. (2000) in the following characteristics: dorsal and lateral pelage is uniformly reddish brown and auburn in coloration although it turns slightly darker in the rump; the tail is relatively short (65% of head and body length) with conspicuous scales distributed on rows of about 10 scales per cm; ears are relatively short ( < 23 mm, Table 1); hind feet are slim and short with white dorsal surfaces and dark brown bands around the ankles; soles of hindfeet with 6 plantar pads but with weakly developed hypothenars; small but robust skulls with narrow rostra; incisive foramen ovate in shape; postorbital processes of zygoma weakly developed, and small molars (Fig. 1).A morphological variation on tail coloration, the degree of development of the septum of the incisive foramina, the depth of the mesopterygoid fossa, and other characters are evident in Bolivian specimens (Table 2; Fig. 2).Phylogenetic reconstruction (Fig. 3) including a Bolivian sequence from Pando (MSB 57229) found it to be part of a clade that contained haplotypes of P. pattoni from the Jurua River (Brazil) and Madre de Dios (Peru).The intraspecific variation considering all those popula-   tions (Jurua, Madre de Dios, Pando) is 1.6 ± 0.3.Genetic divergence between our specimen from Pando and haplotypes from populations of P. pattoni from the Jurua River is 1.9% and 1.8%, from Madre de Dios, whereas specimens from Madre de Dios diverge in 2.1% from those of the Jurua River.
The chromosomal analysis shows a karyotype of 2n = 40 and FN = 56 (Fig. 4).The autosome complement comprises 1 medium sized submetacentric chromosome, 7 pairs of medium to small-sized metacentric chromosomes, 3 large pairs of acrocentric chromosomes, 8 pairs of small acrocentric chromosomes.The sex chromosomes are composed of a medium-sized acrocentric X-chromosome.

Discussion
Our results confirm the presence of Proechimys pattoni in Bolivia based on morphological, molecular, and karyotype data.This new record corresponds to the Santa Rosa locality (Pando, Bolivia) and extends the current distribution range of P. pattoni about 315 km to the southeast from Pakitza (Madre de Dios, Peru).
Although resembling typical P. pattoni, these Bolivian specimens also highlight geographic variation in the species.For example, populations assigned to P. pattoni from the Jurua River and Ucayali have a deep mesopterygoid fossa that reaches the midpoint of M2 (Patton and Gardner 1972: fig. 3F;Patton et al. 2000: fig. 140); our specimens from Bolivia, on the other hand, show a moderately deep mesopterygoid fossa, reaching almost to the midpoint of M3.In addition, populations from the Jurua River usually have an incomplete septum in the incisive foramen (Pat-ton et al. 2000: fig. 141); on the contrary, specimens from Bolivia and Ucayali (Patton and Gardner 1972: figs. 3F, 7, 8) have incisive foramen with complete septa.
We agree with Patton et al. (2000) that the dark band around the ankle and the distinct postorbital processes on the zygomatic arch in P. pattoni are the best features to distinguish it from P. gardneri.Our analysis of the morphological variation in P. pattoni (including the Bolivian samples) suggests, in addition, that the supraorbital ridge in this species is well developed and level with the interorbital region, whereas in P. gardneri, the supraorbital ridge is well-developed but lies on a plane or rises above the interorbital region.
Incorporation of haplotypes from Pando raises the percentage of corrected sequence divergence from 1.3% (Patton et al. 2000) to 1.6% (present work) despite adding considerable new geographic range to known populations (Fig. 5).Moreover, in a group where morphological and karyotype variation appears to be the norm, our preliminary work suggests chromosomal stability: all populations of P. pattoni present the same karyotype (2n = 40 and FN = 56) as reported by Patton and Gardner (1972), da Silva (1998), and Patton et al. (2000).
Along its distribution, including the new locality reported herein, P. pattoni is sympatric with P. brevicauda (Günther, 1876), P. simonsi Thomas, 1900, and P. steerei Goldman, 1911(Patton and Gardner 1972, Patton et al. 2000: fig. 133), while only in Brazil is P. pattoni in sympatry with P. cuvieri Petter, 1978.Proechimys pattoni is easily recognized from those species principally by its small body size, but also because it has slim, short, and dorsally mostly white hind feet, with 6 plantar pads.Dif-  ferences in the skull include narrower molars, the shape of the incisive foramina, and the depth of the mesopterygoid fossa (see other details in Patton et al. 2000).
The new geographic record is about 244 km distant from the nearest, southernmost record of P. gardneri (Fig 5), and because there are no significant geographic barriers between these localities, both species may occur in sympatry in northern Bolivia.A meticulous examination of newly collected specimens is needed to avoid any misidentification.
PSV obtained sequences and conducted the molecular analyses.

Figure 3 .
Figure 3. Genealogical relationships of 17 cytochrome-b gene haplotypes from specimens assigned to the "gardneri" group of Proechimys.Numbers above branches represent bootstrap support, values below are intraspecific and interspecific variation.Terminal designations are the specimen catalog and locality information (Bo = Bolivia, Pe = Peru, Br = Brazil).The blue tag indicates our P. pattoni specimen from Bolivia.