A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrad

Mammalian Biology Zeitschrift fuÈr SaÈugetierkunde www.elsevier.de/mammbiol

Original investigation

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado By P. R. GONCËALVES, FRANCISCA C. ALMEIDA, and CIBELE R. BONVICINO Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; The American Museum of Natural History, New York, USA; Genetics Division, Instituto Nacional de CaÃncer and IOC ± FIOCRUZ, Rio de Janeiro, Brazil Receipt of Ms. 06. 10. 2003 Acceptance of Ms. 15. 12. 2003

Abstract On the basis of integrated analyses of karyologic, molecular and morphologic data of Wiedomys samples distributed throughout north-eastern Brazil, we suggest the existence of more than one evolutionary lineage within the genus and describe a new species apparently restricted to western Bahia state, in the Cerrado morphoclimatic domain. This new taxon differs from W. pyrrhorhinus by narrower incisive foramina, smaller molar rows, presence of an alisphenoid strut, and diploid number of 60 chromosomes. Phylogenetic analyses of cytochrome b sequences of this new species and samples of W. pyrrhorhinus from the Caatinga of Bahia state, depict the two taxa as reciprocally monophyletic clades separated by 14.7±15.6% corrected genetic distance. This new species and W. pyrrhorhinus are allopatric, the former occurring in the Cerrado morphoclimatic domain and the latter endemic of the Brazilian semi arid Caatinga. Key words: Wiedomys, new species, phylogeny, karyotype

Introduction The Caatinga and Cerrado morphoclimatic domains occupy an extensive area in South America, corresponding to approximately 2 650 000 km2, which together with the Chaco is commonly termed as the open diagonal belt due to the predomination of xeric formations and grasslands dissected by semideciduous forests (Eiten 1972; Reis 1976). The knowledge of this region increases with the descriptions of new rodent species from Cerrado (Bonvicino and Weksler 1998; Bonvicino 2003; Langguth and Bonvicino 2002) and by the clarifi1616-5047/05/70/01-046 $ 30.00/0.

cation of geographic limits of endemic rodent species from Caatinga (Oliveira et al. 2003). In this latter case, one of the poorly known rodent taxa apparently restricted to the Caatinga biome in northeastern Brasil is the red nosed mouse genus Wiedomys (Oliveira et al. 2003), the unique extant representative of the tribe Wiedomyini (Reig 1980). Recent phylogenetic studies have considered this genus as an independent and basal offshoot within the subfamily Sigmodontinae with apparently no closely related exMamm. biol. 70 (2005) 1´ 46±60

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado tant tribal groups (Steppan 1995; Smith and Patton 1999). Despite the relatively wide distribution of Wiedomys throughout northeastern Brazil (Thomas 1928; Pine 1980; Oliveira et al. 2003), only W. pyrrhorhinus (Wied, 1821) has been regarded as extant species since the description of the genus (Hershkovitz 1959). Few ecologic (Mares et al. 1981; Streilen 1982 a) and karyologic (Maia and Langguth 1987) studies were carried out with W. pyrrhorhinus as a consequence of its rarity and occasional occurrences in localities of the Caatinga (Streilen 1982 b). In the same way, morphologic and genetic variability within and among populations of W. pyrrhorhinus is poorly known and the species diversity within this genus may have been neglected. Here we describe a new Wiedomys species from the Cerrado morphoclimatic domain on the basis of integrated karyologic, molecular (cytochrome b) and morphological analyses of samples from north-eastern Brazil.

Material and methods We collected and karyotyped 5 specimens of Wiedomys from 2 Brazilian localities in Bahia state (Fig. 1): Jaborandi, Faz. SertaÄo do Formoso (14°37¢ S, 45°51¢ W) (males MN 61657, MN 67023, and female MN 67022); and Caetite (13°50¢ S, 42°23¢ W) (males MN 63357 and MN 63420). All specimens were deposited in Museu Nacional (MN), Rio de Janeiro, Brazil. Chromosome preparations from these individuals were obtained from short-term bone marrow cultures. Cell suspensions were cultivated for two hours at 37° C in RPMI 1640 culture medium supplemented with 20% fetal calf serum, ethidium bromide (5 lg/ml) and colchicine (10±6 M). FNa refers to autosome fundamental number. Karyologic data were compared with previously published karyologic data of specimens from Bom Conselho and Cachoeirinha, Pernambuco state (localities 11 and 12 in Fig. 1, see Maia and Langguth 1987). DNA samples of the 3 specimens from Jaborandi and 1 from Caetite were isolated from livers preserved in ethanol following the procedures of Sambrook et al. (1989). Cytochrome b mt DNA (ca. 1140 bp) was amplified with primers MVZ 05 and MVZ 14 and sequenced with the same primers, in addition to MVZ 16 and

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Fig. 1. Geographic location of ($) type-locality of Wiedomys pyrrhorhinus and (*) samples analyzed in this study: Bahia state: (1) Tremedal, (2) Jaborandi, (3) CaetiteÂ, (4) Seabra, (5) Canudos, (6) Feira de Santana. Alagoas state: (7) QuebraÃngulo, (8) Santana do Ipanema. Pernambuco state: (9) Caruaru, (10) Pesqueira, (11) Bom Conselho (karyotyped by Maia and Langguth 1987) and (12) Cachoeirinha (karyotyped by Maia and Langguth 1987). The shaded area delimits the Caatinga biome as defined by IBGE (1965). MEU1 in an ABI Prismä 377 automatic DNA sequencer following Smith and Patton (1993). Sequences were manually aligned using the Sequence Navigator software (Applied Biosystems, Inc. 1994) and deposited in GenBank. We also included sequence data available in GenBank for one exemplar of Wiedomys pyrrhorhinus from Canudos (GenBank AF108685, Fig. 1, locality 5), Bahia state, and for selected species of the muroid rodents Oryzomys nitidus (GenBank AF251529), Oryzomys lamia (AF181273), Eligmodontia puerelus (AF159289), Eligmodontia morgani (AF108691), Thomasomys aureus (U03540), Thomasomys gracilis (AF108674) and Scotinomys teguina (AF109796). This last taxon was used as outgroup on the basis of previous muroid phylogeny assessments (Smith and Patton 1999). Kimura 2 parameters model (K2p) was used to calculate genetic distances between haplotypes and for constructing neighbour-joining dendrograms using the software MEGA 2.1 (Kumar et al. 2000). Maximum parsimony trees were obtained through heuristic searches using the tree-bisection-reconnection branch-swapping

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algorithm in PAUP 4.0b10 (Swofford 1999), with all sites equally weighted. Confidence intervals for neighbour-joining and maximum parsimony trees were obtained by Bootstrap analysis based on 1000 replicates. For morphologic comparisons, in addition to the karyotyped specimens from Jaborandi and CaetiteÂ, we included 61 specimens from the following Brazilian localities: Bahia state, Seabra (MN 18507, 18509±511, 18514, 18516±517, 18705, 18707±708); Feira de Santana (MN 18715±716, 18719±720, 18723, 18760±762, 18765±766); Alagoas state, Quebrangulo (MN 18549, 18776±777, 18779±781, 18784, 18786, 61603), Santana do Ipanema (MN 18597, 18686, 18690, 18767±772, 18774); Pernambuco state, Caruaru (MN 18468, 18470, 18472, 18661±663, 18666±668, 60736), Pesqueira (MN 18588, 18675, 18685, 18743±744, 18747, 52229, 60742±746). Morphological descriptions and comparisons were based on qualitative anatomical external and cranial characters, using terminology referenced by Reig (1980) and Voss (1988, 1993). For morphometric characterisation we used external measurements recorded on specimen tags, and measured 16 cranial dimensions defined by Bonvicino and Weksler (1998) with the aid of a digital calliper. Greatest skull length (GSL), condylo-incisive length (CIL), breadth of the occipital condyles (BOC), length of diastema (LD), palatal

bridge (PB), length of incisive foramen (LIF), breadth of incisive foramen (BIF), length of maxillary molar row (LM), breadth of first maxillary molar (BM1), external alveolar breadth (M1M), cranial height (CH), rostrum length (RL), rostrum breadth (BRO), least interorbital breadth (LIB), zygomatic breadth (ZB), and breadth of braincase (BB). Only adult animals (with all molar teeth erupted and functional) were included in morphometric comparisons. Principal component analyses were performed to explore patterns of size and shape variation within and among samples using the software SYSTAT and MatLab 4.0 (MathWorks, Inc.) routines available at http://www.biol.ttu.edu/Faculty/FacPages/Strauss/ Matlab/matlab.htm.

Results Karyologic analyses Karyologic analyses from Caetite specimens showed 2n = 62, FNa = 90 (Fig. 2). The autosomal complement is composed by 15 pairs of biarmed chromosomes varying in size from large to small, and 15 pairs of acrocentric chromosomes varying in size from medium to small. The X chromosome

Fig. 2. Conventional Giemsa coloration of specimens of Wiedomys from (A) Jaborandi and (B) CaetiteÂ.

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado is a medium sized acrocentric, and the Y chromosome a small sized metacentric. Karyologic analysis from Jaborandi specimens showed 2n = 60, FNa = 88 (Fig. 2). The autosome complement is composed by 15 pairs of biarmed chromosomes one large, the largest of the chromosome complement, and 14 decreasing in size from medium to small, and 14 acrocentric pairs varying in size from medium to small. The X chromosome is a medium sized acrocentric, and the Y chromosome a small acrocentric. Molecular analyses The genetic distance estimates revealed relatively low divergence levels between Caetite and Canudos haplotypes (0.025 K2p) and between Jaborandi haplotypes (0.013± 0.015 K2p) (Tab. 1). Conversely, higher values of genetic divergence were found in comparisons between Jaborandi haplotypes and either Caetite or Canudos haplotypes (0.147±0.156 K2p). These values are higher than those found between selected species of Oryzomys (0.079 K2p), Thomasomys (0.143 K2p) and Eligmodontia (0.123 K2p) (Tab. 1). The neighbour-joining and maximum parsimony trees placed Wiedomys specimens in a well-supported monophyletic clade (100% bootstrap value), divided in two major lineages, one constituted by Jaborandi specimens and another by Caetite and

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Canudos samples (Fig. 3). In both analyses Oryzomys, Eligmodontia and Thomasomys were depicted as monophyletic genera, although their intergeneric relationships are not fully resolved. The only exception is the clustering of Thomasomys with Wiedomys in both NJ and MP analyses, although there is no bootstrap support in the last one. Morphological variation Considering the karyologic and molecular distinctiveness of the Jaborandi sample (2n = 60, FNa = 88), we evaluated the hypothesis that this population is morphologically distinct from the Caetite sample (2n = 62, FNa = 90) including 6 additional larger samples from Bahia, Alagoas and Pernambuco states. In addition to the principal components analysis we evaluated the craniometric diagnosis of the sample from Jaborandi by inspecting bivariate plots portraying combinations between the different craniometric characters. The principal component analysis summarized the majority (62.9%) of the total craniometric variance in the first component (PC1) and only 7.5% in the second component (Fig. 4 a). The PC1 rather represents the cranial variability related to variation in size due to the positive correlation of all craniometric variables with this axis (GSL0.2348; CH-0.1218; CIL-0.2489; ZB-0.2601; BB-0.1318; LIB-0.1239; RL-0.3164; BRO-

Table 1. Pairwise Kimura 2-parameters genetic distances among cytochrome b haplotypes used in molecular analyses. Taxa

1

2

3

4

5

6

7

10

11

12

1. W. pyrrhorhinus ± Caetite 2. W. pyrrhorhinus ± Canudos 3. Wiedomys sp.n. ± Jaborandi 4. Wiedomys sp.n. ± Jaborandi 5. Wiedomys sp.n. ± Jaborandi 6. Oryzomys lamia 7. Oryzomys nitidus 10. Eligmodontia puerelus 11. Eligmodontia morgani 12. Thomasomys aureus 13. Thomasomys gracilis

0.025 0.156 0.152 0.154 0.202 0.201 0.218 0.206 0.223 0.209

0.147 0.150 0.148 0.203 0.205 0.216 0.206 0.207 0.190

0.014 0.015 0.205 0.211 0.242 0.227 0.215 0.221

0.013 0.214 0.218 0.248 0.225 0.217 0.218

0.214 0.222 0.248 0.226 0.214 0.218

0.079 0.182 0.192 0.197 0.179

0.185 0.204 0.123 0.200 0.242 0.232 0.187 0.235 0.219 0.143

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Fig. 3. Phylogenetic relationships among Wiedomys haplotypes and selected muroid species based on 1 140 bp of the cytochrome b gene. (A) Neighbour-joining dendrogram and (B) single Maximum Parsimony tree obtained (tree statistics: 944 steps, C. I. = 0.66, R. I. = 0.63). Numbers above nodes are bootstrap support values estimated on 1000 replications.

0.2294; BOC-0.1262; BL-0.2380; BM1± 0.1672; LM-0.1203; M1M-0.1824; PB-0.1998; LIF-0.2767; LD-0.3293; BIF-0.4344), while PC2 depicts shape variation. Most samples overlap along PC1 and PC2 suggesting extensive intrapopulational size and shape variation across samples. Specimens from Caetite generally have larger skulls while the specimen from Jaborandi presents a relatively smaller skull. The remaining samples are intermediate in size and display some specimens with scores near Caetite or Jaborandi samples. The inspection of bivariate plots revealed that the specimen from Jaborandi segregates best from the other samples in comparisons considering both length of maxillary tooth row and breadth of incisive

foramina (Fig. 4 b; Tab. 2). In this combination, the Jaborandi specimen diverges from most samples displaying relatively shorter maxillary molar row and narrower incisive foramina. One specimen from Caetite presents the widest incisive foramina whereas the other specimen is superimposed within most samples presenting intermediate values for the two characters. The qualitative character variation related to the alisphenoid foramina further contributed for the discrimination of the specimens from Jaborandi in relation to the other samples. The Jaborandi specimens present the buccinator-masticatory foramen separated from the foramen ovale accessorius by a distinct bony alisphenoid strut whereas specimens from the remaining samples lack this struc-

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado

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Fig. 4. Bivariate plots of (A) individual scores of the first and second principal components, (B) measurements of breadth of incisive foramina and maxillary tooth row length of specimens. Samples: (*) Feira de Santana, Bahia state; (*) Seabra, Bahia state; (^) Pesqueira, Pernambuco state; (´) Caruaru, Pernambuco state; (&) Santana do Ipanema, Alagoas state; (^) Jaborandi, Bahia state; (~) QuebraÃngulo, Alagoas state; (~) CaetiteÂ, Bahia state. See Material and methods for character abbreviations.

ture (Fig. 5). Samples from Pesqueira, Santana do Ipanema and Seabra were the only polymorphic samples for this character, presenting one out of 10 individuals bearing the alisphenoid strut. Nevertheless, these samples lie well apart from Jaborandi specimen in comparisons of breadth of incisive foramina and length of maxillary tooth row mentioned above. Therefore, the combination of these two craniometric characters with the conditions of the alisphenoid strut consistently discriminates the Jaborandi specimen from other samples of Wiedomys. Wiedomys cerradensis sp. nov. Holotype: An adult female (MN 67022) represented by skin, skull, mandible, partial skeleton and ethanol-preserved liver tissues, field number CRB 1878 (Fig. 6). Collected at Fazenda SertaÄo do Formoso (14°37¢888² S and 45°51¢293² W), Jaborandi, state of Bahia, October 29. 1999, by C. R. Bonvicino and J. F. Vilela in a semi deciduous forest in Brazilian Cerrado. Paratypes: Two males, collected at the same locality and date of the holotype. The young

(MN 61657) is represented by skin and complete skull, M2 and M3 are not fully erupted. The adult male (MN 67023) is represented only by skin. Etymology: Wiedomys pyrrhorhinus has been suggested as a Caatinga endemic and the new form is apparently found outside this biome, in a semi deciduous forest in western Bahia, an area enclosed by the Cerrado morphoclimatic domain. Therefore, the epithet cerradensis refers to the morphoclimatic domain where this species was found. Diagnosis: A small sigmodontine rodent with tail longer than head and body, pelage of muzzle, ears, eye-ring and rump bright orange, narrow incisive foramina and small molar rows, buccinator-masticatory and accessory oval foramina separated by a distinct alisphenoid strut, and diploid number of 60 chromosomes. Measurements: See table 2. Distribution: Known only from the type locality. External characters: Dorsal pelage soft and dense with overall color grizzled-brown, but with the rump, ears and muzzle dis-

Wiedomys pyrrhorhinus

Char

type

CaetiteÂ

Seabra

Feira de Santana

QuebraÃngulo

Santana do Ipanema

Caruaru

Pesqueira

HB

107

116.5 107±126

111 102±120

116 100±125

105.7 95±120

110 100±130

109 100±115

110.3 100±120

T

142

188.5 183±194

184 172±205

173 149±188

158 140±180

162 145±180

149.6 110±164

168.8 150±185

HF

25.5

25.5 25±26

26 24±28

27 26±30

25 24±27

24 22±25

24.4 22±26

25.8 25±29

E

19

21.5 20±23.5

21 20±23

21 19±23

19 18±20

21.5 19±24

19.6 17±21

19.7 18±20

GSL

27.85

32.26 32.05±32.47

29.93 28.22±32.1

29.82 27.39±30.99

29.21 26.87±30.79

28.38 26.6±30.61

27.88 25.7±29.29

28.92 27.05±30.58

CH

8.54

9.55 9.53±9.57

8.87 8.14±9.4

8.96 8.66±9.32

8.80 8.24±9.44

8.72 7.91±9.19

8.55 8.23±8.82

8.81 8.4±9.29

CIL

25.15

29.85 29.11±30.6

27.12 25.29±28.91

27.22 24.72±28.26

26.48 24.17±27.95

25.79 24.22±27.84

25.37 22.95±26.95

26.52 24.61±28.62

ZB

13.89

16.78 16.48±17.08

15.25 14.41±16.14

15.19 14.03±16.06

14.50 13.07±15.56

14.51 13.84±15.45

14.14 13.5±14.83

15.03 14.06±16.35

BB

11.61

13.1 13±13.2

11.99 11.5±12.33

11.95 11.59±12.2

11.86 11.22±12.29

11.77 11.05±12.27

11.39 11.28±11.74

12.04 11.38±13.14

LIB

4.44

4.65 4.6±4.69

4.54 4.26±4.79

4.6 4.28±4.95

4.37 3.97±4.61

4.34 4.22±4.75

4.40 4.06±4.62

4.41 4.22±4.7

RL

9.55

12.13 12.12±12.14

10.85 9.63±11.73

10.75 9.36±11.52

10.73 9.86±11.71

10.40 9.46±11.48

9.95 8.13±10.46

10.47 9.38±11.68

P. R. GONCËALVES et al.

n. sp.

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Table 2. Measurements (in mm) of the holotype of Wiedomys cerradensis (n. sp.), means and interval of variation (Max-Min) of selected samples of W. pyrrhorhinus from northeastern Brazil. Char = variables, see material and methods from abbreviations, HB = head-body length, T = tail length, HF = hind foot length without claw, E = ear length.

n. sp. Char

type

Wiedomys pyrrhorhinus CaetiteÂ

Seabra

Feira de Santana

QuebraÃngulo

Santana do Ipanema

Caruaru

Pesqueira

4.67

5.14 5±5.29

4.85 4.46±5.23

4.81 4.56±5.14

4.78 4.26±5.19

4.61 4.17±5.03

4.62 4.44±4.84

4.81 4.33±5.67

BOC

6.04

6.67 6.67

6.48 6.23±7.05

6.56 6.38±6.79

6.33 5.86±6.84

6.48 6.24±6.73

6.27 6.04±6.54

6.74 6.24±7.23

BU

5.75

6.98 6.84±7.13

6.04 5.59±6.54

5.95 5.32±6.31

6.14 5.45±6.69

5.94 5.61±6.43

5.59 5.25±6.16

6.13 5.66±6.49

BM1

1.28

1.36 1.31±1.42

1.31 1.25±1.41

1.29 1.22±1.36

1.18 1.13±1.27

1.23 1.16±1.3

1.18 1.13±1.26

1.26 1.14±1.38

LM

4.35

4.85 4.8±4.9

4.90 4.62±5.45

4.76 4.45±5

4.62 4.46±4.87

4.70 4.41±4.8

4.65 4.31±5

4.77 4.4±5.27

M1M

5.1

5.67 5.65±5.69

5.40 5.13±5.67

5.39 5.15±5.73

5.10 4.43±5.43

5.24 5.06±5.42

5.03 4.79±5.4

5.28 5.08±5.73

PB

4.16

5.18 5.15±5.21

4.67 4.25±5.05

4.53 4.36±4.79

4.34 3.79±4.58

4.40 4.23±4.6

4.32 3.79±4.44

4.46 4.22±4.81

LIF

5.34

6.68 6.47±6.89

6.32 5.74±6.68

6.28 5.8±6.91

6.11 5.52±6.52

5.90 5.43±6.37

5.86 5.4±6.48

6.15 5.46±6.61

LD

6.24

7.89 7.7±8.08

7.10 6.74±7.65

7.17 6.61±7.77

6.84 5.84±7.46

6.59 6.05±7.41

6.45 5.53±6.95

6.84 6.46±7.4

BIF

1.78

2.45 2.24±2.66

2.36 2.21±2.62

2.25 2.09±2.62

2.22 1.9±2.51

2.07 1.83±2.45

2.02 1.88±2.36

2.16 1.83±2.45

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado

BRO

53

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P. R. GONCËALVES et al.

Fig. 5. Lateral view of the alisphenoid region showing the different conditions of the alisphenoid strut between specimens from (A) Jaborandi and (B) CaetiteÂ. (ab) auditory bulla, (als) alisphenoid strut, (bmf) buccinator-masticatory foramen, (foa) foramen ovale accessorius. Scale bar = 1 mm.

tinctly brightened orange. Two types of hairs of similar length (8±9 mm) are present in dorsum, a rather homogeneously melanic type and a banded type with basal, subapical and apical bands. In the head and dorsum, banded hairs have a large plumbeous basal band (7 mm), a very short orange colored subapical band (2 mm) and a more distal clay coloured apical band (4 mm). Melanic hairs are entirely black in their distal parts and mixed with banded hairs provide the grayish-brown tone of the dorsum. In the rump, banded hairs present apical and sub apical bands apparently fused in a large (6±7 mm) rich orange layer, strengthening the brightened orange tone of the rump. Laterals of body are slightly grayer than dorsum due to the paler and wider sub apical and apical bands. Ventral pelage

is well delineated from laterals presenting entirely white hairs extending from the gular to the inguinal region, including the ventral region of forelimbs and medial region of hind limbs. Pinnae rounded and externally covered by entirely orange hairs, which originate in the proximal helix and in the region just anterior to the base of the ear. Eyes surrounded by short orange hairs forming a conspicuous orange eyering. Muzzle well furred by small orange hairs, which extend to the caudal limit of the mystacial vibrissae array; mystacial vibrissae are well developed (3±3.5 cm long), the longest extending beyond the distal limit of pinnae; two supraciliary and one genal vibrissae also present and shorter (1.8±2 cm). Manus and pes dorsally furred by entirely orange hairs; silvery ungual tufts are pre-

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado sent in the distal phalanxes of digits 2±5 in manus and digits 1±5 in pes. Volar surface of manus displays well-developed and fleshy metacarpal pads, the thenar being the largest with an oblong shape and the hypothenar about the same size of the interdigital pads. Tail larger than combined head and body length; entirely dark brown with its proximal ventral surface slightly paler; dorsal and ventral regions covered by small black and white hairs averaging 2 scales in length, which become longer towards the tail tip, but do not form a distal hair tuft. The single adult female examined (MN 67022) displays 8 mammae: 2 inguinal, 2 abdominal, 2 thoracic, 2 pectoral. The sole young specimen examined (MN 61657) differs from adult individuals externally only by presenting more brownish dorsal coloration throughout the flanks due to the richer orange colour of apical and sub apical bands of banded hairs, thus, providing a less conspicuously brightened rump. Cranial characters: Skull with relative short rostrum and laterally expanded braincase. Frontal bones with anteriorly convergent lateral outlines and sharply defined supraorbital edges, but without distinct supraorbital crests. Suture between parietals and frontals V-shaped. Interparietal broad and wide, not contacting the squamosal laterally. Nasals short, strongly depressed in the median region of the fronto-nasal suture. Zygomatic plate narrow not projected anteriorly, with roughly vertical anterior outline. Zygomatic notch moderately deep and broad. Incisive foramina extending to the line of M1 protoflexus, with 2/3 of the septum formed by premaxillary bones. Palate short, not extending posteriorly beyond the line of M3 hypoflexus, presenting a pair of posterolateral palatal pits. Mesopterygoid fossa narrow with U-shaped anterior notch and straight lateral margins. Sphenopalatine vacuities well developed occupying approximately 2/3 of the basisphenoid roof. A distinct alisphenoid strut separates the buccinator-masticatory and accessory oval foramina. Even the young specimen (MN61657) presented the alisphenoid strut although incompletely devel-

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oped in one side of the skull, represented only by a thin bony projection. Parapterygoid fossae shallow and large, wider than mesopterygoid fossa at its caudal limit and perforated by two small fontanelles. The posterior openings of the oval foramina are reduced and unequally divided by a slender strut of the aliphenoid. Hamular process slender, delimiting a small postglenoid fenestra and a large subesquamosal foramen. Ectotympanic (auditory bulla) notably inflated and globular in its ventral surface and contacted to the squamosal by the tegmen tympani and hamular process; stapedial process well developed. Petrotympanic fissure deep, large stapedial and sphenofrontal foramina present, providing evidence of the carotid circulation pattern 1 (Voss 1988). Mastoid fenestra quadrangular and relatively large, occupying approximately 1/3 of the mastoid surface. Mandible: Coronoid process small, not projecting posterodorsally beyond the level of the condyloid process and delimiting a small sigmoid notch. Angular notch deep. Condylar process well developed, extending posterodorsally to the level of the angular process. Inferior masseteric crest more pronounced than superior masseteric crests. Dental characters: Upper incisors opisthodont with rounded yellow enameled bands in anterior face. Molars brachyodont and crested with alternated cusps. Upper series (M1±M3) parallel sided. M1 with rounded anterior margin of procingulum. M2 squared and M3 triangular with reduced metacone and hypocone. Anteromedian flexus well defined in M1, unequally dividing the anterocone in a small anterolingual conule and larger anterolabial conule; anteroloph slender in M1 and obsolescent in M2. Anterior (M1) and median mures (M1, 2) diagonally oriented. Mesoloph distinct in M1 and M2, originated at the median mure and lingually fused to mesostyle. Protostyle and enterostyle present as low stylar cusps. Mesoflexus and posteroflexus reduced as enameled islands in M1 and M2. Procingulid of m1 narrower than breadth of m2, with anteroconid equally divided by a deep antermedian flexid. Mesolophid absent. Protoflexid wide in

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Fig. 6. Dorsal, ventral and lateral view of the skull, lateral view of mandible and occlusal view of upper and lower molar rows of Wiedomys cerradensis holotype (female MN 67022).

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado m1 and deep in m2. Mesostylid present in m1 and m2. Hypoflexid wide, presenting a well developed ectostylid in the lingual margins of m1 and m2. Posteroflexid deep and pronounced in m2 but reduced to a small enamel island in m3. Comparisons: Wiedomys cerradensis differs from W. pyrrhorhinus in karyotype (diploid and fundamental numbers), and by presenting an alisphenoid strut separating the buccinator-masticatory foramen from the accessory oval foramen combined with smaller cranial and body dimensions (Tab. 2), of which the most distinctive are the relative small molar row length (4.35 mm) and narrow incisive foramina (1.78 mm). Both species display the same colour pattern, with rumps, ears and eye-rings brightened orange, strongly contrasted to the grizzledbrown color of head and dorsum. W. cerradensis differs from the recently described fossil species W. marplatensis (Quintana 2002) by displaying a much more simplified m3, which lacks a distinct protoflexid and has a posteroflexid reduced to an enamel island.

Discussion Since its original description as Mus pyrrhorinos by Wied (1821) on the basis of a specimen collected in southern Bahia, W. pyrrhorhinus had been at times included or related to a number of divergent sigmodontine genera such as Hesperomys (Burmeister 1854), Oryzomys (Thomas 1886), Rhipidomys (Trouessart 1898) and Thomasomys (Thomas 1928; Gyldenstolpe 1932), owing to its divergent set of cranial and external features. Based on the uniqueness of character-states of this taxon, Hershkovitz (1959) described the monotypic genus Wiedomys and expressed his doubts regarding its suprageneric and tribal affinities. Finally, Reig (1980) formalized the uniqueness of Wiedomys as a very distinct lineage within the Sigmodontinae, coining the tribe Wiedomyini that included the fossil Cholomys pearsoni as the sole species related to W. pyrrhorhinus. More recently,

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with the description of the fossil Wiedomys marplatensis from the Upper Pliocene deposits of Argentina (Quintana 2002), the paleontological evidence accumulated so far suggests that W. pyrrhorhinus may be an extant representative of an ancient group of sigmodontine rodents that was distributed throughout South America during PlioPleistocene times. Likewise, the molecular phylogenetic studies have confirmed the status of Wiedomys as an independent and very divergent lineage within the Sigmodontinae (Smith and Patton 1999). Despite the number of discussions concerning the systematics and evolution of W. pyrrhorhinus, most of them have been centered on its intergeneric relationships and no attempt has been made to understand its intraspecific variability. The results obtained in this study on the basis of integrated karyologic, mitochondrial sequence and morphologic data are indicative of a higher diversity and complexity within the genus. The karyotypes here described differed from those previously reported from specimens collected in Cachoeirinha and Bom Conselho, Pernambuco state (2n = 62, FNa = 86; Maia and Langguth 1987). The Jaborandi karyotype (2n = 60, FNa = 88) departs to a greater extent from the previously published 2n = 62, FNa = 86 karyotype by differing in both diploid and fundamental number, whereas the karyotype from Caetite (2n = 62, FNa = 90) differs only in fundamental number, having two additional pairs of biarmed chromosomes. Maia and Langguth (1987) reported heteromorphism of pericentric inversion affecting pair number 16 in one karyotyped specimen from Pernambuco State. The distinctive morphology of this autosome pair (a submetacentric with very short arm) made it easily recognizable in the two karyotypes here described, which apparently present no heteromorphism for this pair. Both 2n = 60, FNa = 88 and 2n = 62, FNa = 90 karyotypes described here lack the large acrocentric pair 14, the largest of the chromosomal complement reported by Maia and Langguth (1987), which in the case of the 2n = 62, FNa = 90 karyotype

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may have evolved to one of its additional biarmed chromosomes. The existence of three different karyotypes within the genus raises the possibility of the existence of at least three previously unrecognised evolutionary units. We evaluated the significance of this variation to species limits within Wiedomys by considering the additional molecular and morphological evidence. Morphological data show that despite the variation in chromosomal morphology between the 2n = 62, FNa = 86 (Maia and Langguth 1987) and 2n = 62, FNa = 90 (CaetiteÂ) karyotypes, the samples from Pernambuco state (Pesqueira and Caruaru) herein analysed overlapped extensively in craniometric characters with samples from central Bahia and Alagoas states. Moreover, these samples share the absence of alisphenoid strut with the sole exception of one individual in both Caruaru and Santana do Ipanema samples that present this structure. The absence of any remarkable morphologic divergence among samples suggests that the 2n = 62, FNa = 86 karyotype from Pernambuco state and the 2n = 62, FNa = 90 karyotype from Bahia state may represent a single species widely distributed along the semiarid Caatinga. Unfortunately, we do not have sequence data available for samples from Pernambuco state in order to provide more comprehensive comparisons. The specimens from Caetite are the geographically nearest relatives to the type specimen of W. pyrrhorhinus collected by the Prince Maximilian Wied-Neuwied in the caatingas along Riacho da Ressaca, at Tremedal municipality in Bahia state, thus, this name may be used to refer to samples from the semiarid zone of Bahia, Alagoas and Pernambuco states analysed here. The molecular analyses of the specimen from Canudos and the 2n = 62, FNa = 90 sample from Caetite revealed low genetic distances among their haplotypes (2.5% K2p), which were grouped in a highly supported monophyletic clade within the genus. These results confirm the genetic cohesion of samples along central Bahia state. Karyologic, molecular and morphologic comparisons were congruent in portraying

Jaborandi sample as a more divergent unit within Wiedomys. In addition to karyologic divergence of Jaborandi specimens to the 2n=62, FNa=90 and 2n=62, FNa=86 karyotypes, sequence divergence is high (14.7± 15.6%) in comparisons with either Caetite or Canudos haplotypes. These values are higher than those found between species of other sigmodontine genera included in the molecular analyses such as Oryzomys, Thomasomys and Eligmodontia. Moreover, the maximum parsimony and neighbour-joining analyses depicted the two karyotypes as two reciprocally monophyletic clades suggesting the existence of two divergent and relatively old evolutionary lineages within the genus. The morphological analyses revealed that the Jaborandi specimen displays a unique combination of traits related to the alisphenoid, molar series length and incisive foramina width, which are useful for the taxonomic distinction of this new lineage within Wiedomys. Since the name Wiedomys pyrrhorhinus may be applied to the lineage represented by the central Bahia samples, the western sample from Jaborandi represents an unnamed form, which we described in the preceding sections as W. cerradensis. Wiedomys pyrrhorhinus has been considered as one of the few endemic mammal species of the Caatinga in a recent revision of the geographic records of this species (Oliveira et al. 2003). The recognition of two evolutionary lineages within the genus Wiedomys, one presumably restricted to the Caatinga and the other apparently outer to this biome, raises an important evidence for the zoogeographic differentiation of the Caatinga fauna in relation to more mesic surrounding biomes. Oliveira et al. (2003) argued that the set of Caatinga endemic mammals, although reduced in species number, comprises autochthonous and strikingly differentiated rodent genera favouring a hypothesis of an ancient origin of the biome. In order to further evaluate the antiquity of the Caatinga we assessed divergence dates between W. cerradensis and the Caatinga endemic W. pyrrhorhinus, correlating the amount of sequence diver-

A new species of Wiedomys (Rodentia: Sigmodontinae) from Brazilian Cerrado gence between these two lineages with time considering the calibrated evolutionary rate of 3rd position transversions of the cytochrome b gene of sigmodontine rodents (2.3% sequence difference per million years Smith and Patton 1993, 1999). Applying the calibrated rate, the two lineages split at approximately 3.4 millions of years before present, at the Upper Pliocene, corroborating the hypothesis of an ancient origin of the Caatinga rodent fauna proposed by Oliveira et al. (2003). The time estimates generated by the molecular analyses are also congruent with the fossil record of Wiedomys, which documents the first appearance of the genus in the Upper Pliocene of the Buenos Aires province, Argentina, on the basis of the discovery of W. marplatensis, the sole fossil species of

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the genus (Quintana 2002). Therefore, the molecular and fossil evidence available suggest that diversification within the genus may have occurred during Pliocene times.

Acknowledgements We are grateful to the owners of Fazenda SertaÄo do Formoso for permission to collect animals at their land, to S. Lindbergh, J. F. Vilela for their collaboration in fieldwork. To P. S. D'Andrea for specimens from Caetite and J. A. Oliveira for granting access to the Museu Nacional mammal collection and for revising earlier versions of this manuscript. To A. Langguth for the Zusamenfassung. License to collect the specimens was granted by Brazilian Institute of Environment and Natural Resources (IBAMA).

Zusammenfassung Eine neue Art von Wiedomys (Rodentia: Sigmodontinae) aus dem Cerrado Brasiliens Auf Grund von karyologischen, molekularen und morphologischen Daten beschreiben wir eine neue Art von Wiedomys aus West Bahia Staat, in der Cerrado DomaÈne von Nord-Ost-Brasilien. Diese neue Art unterscheidet sich von W. pyrrhorhinus durch schmaÈlere Foramina incisiva, kleinere Backenzahnreihe, einen Alisphenoid-Fortsatz und einen diploiden-Satz von 60 Chromosomen. Phylogenetische Analysen von Cytochrom b Sequenzen der neuen Art und von W. pyrrhorhinus aus dem Caatinga und aus Bahia weisen die beiden Taxa als Schwestergruppen aus, die durch eine korrigierte genetische Distanz von 14,7±15,6% getrennt sind. Die Wiedomys-Arten sind allopatrisch, eine kommt in Cerrado und die andere in Caatinga vor.

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Authors' addresses: Pablo R. GoncËalves, Museu Nacional, Universidade Federal do Rio de Janeiro, Departamento de Vertebrados, Quinta da Boa Vista s/n, 20940-040, Rio de Janeiro, RJ, Brazil (E-mail: [email protected]); Francisca C. Almeida, The American Museum of Natural History, Central Park West, USA; Cibele R. Bonvicino, Genetics Division, Diretoria de Pesquisa, Instituto Nacional de CaÃncer, 20231-050, Rio de Janeiro, Brazil and Laboratory of Biology and Control of Schistosomiasis, Tropical Medicine Department, IOC ± FIOCRUZ, Av. Brasil, 4365, 23045-900, Rio de Janeiro, Brazil