Characterization of Two Distinct Species of Arvicanthis (Rodentia

J. Zool., Lond. (1997) 241,709-723

Characterization of two distinct species of Awìcanthìs (Rodentia: Muridae)

in West Africa: cytogenetic, molecular and reproductive evidence I

J. F. DUCROZ,L.f GRANJON Laboratoire Mammvères et Oiseaux, Muséum National d'Histoire Naturelle, 55 rue Buffon, 75005 Paris, France P. CHEVRET,J. M.f DUPLANTIER Institut des Sciences de I'Evolution, URA 327 CNRS, Université Montpellier II, Case courrier 064, 34095 Montpellier, France

M. LOMBARDAND V. VOLOBOUEV Laboratoire de Cytogénétique moléculaire et Oncologie, UMR 147 CNRS, Institut Curie, 26 rue d'Ulm, 75231 Paris, France

(Accepted 3 April 199Q

(With 4 plates and 2 figures in the text)

. The unstriped grass rat, Arvicunthis Lesson 1842, is one of the most common genera of murid rodents in African savannas. However, from a systematic viewpoint, very little is known about this group. Following recent investigations which showed karyotypic variability within the species A. niloticus, the present study attempts to clarify the nature and distribution of these chromosomal variants, as well as to determine their taxonomic rank. The chromosomes of 15 individuals from different West African localities were prepared from fibroblast cultures, and R- and C-banded karyotypes were constructed. In addition, the levels of genetic divergence (DNA/DNA hybridization) and reproductive isolation (attempted crossbreeding in captivity) were examined. The results confirm the existence of two differentiated karyomorphs, differing by numerous chromosomal rearrangements such as pericentric inversions and translocations, as well as differences in the quantity of constitutive heterochromatin. These karyomorphs appear to be genetically and reproductively isolated and are parapatrically distributed; their areas of distribution correspond to the sahelian and sudano-guinean domains, respectively. The distinctness of these karyomorphs, the absence of hybrids in laboratory crosses, and the pronounced genetic divergence ' provide good evidence for the recognition of two distinct sibling species. We propose to keep the designation A. niloticus for the noxthem sahelian form and discuss the naming alternatives for the other.

Introduction

i Arviianthis Lesson 1842, the umtriped grass rat and Nile rat; is one $f the most widespread and abundajlt murid rodent genera of tropical Africa. These semi-diurng, greg4ous and herbivorous rodents (Rosev$ìr, 1969) are common in the savannas and grasslands of $ub-Sah$an Africa, their distribution i ranging from Senegal to Somalia and from Egypt to Zambia (Mussbr & Cqeton, 1993). They are major i agriculfural pests (Poulet & Poupon, 1978), as well as a reservoir for various tropical infections (Trape et al., 199 1). Well-differentiated species of Arvicanthis have been recognized and characterized (Afework Bekele et al., 1993, and reference herein) in East Africa but, in contrast, all western and central African individuals are referred to a sole species, A. niloticus. In many respects, the systematics of this genus I remains unsatisfactory and deserves a comprehensive revision (Musser & Carleton, 1993). 709 O 1997 The Zoological Society of London 710 J. F. DUCROZ ETAL. The first diploid number for the species A. doticus, namely 2N = 56 for an individual from Bangui (Central African Republic), was reported by Matthey in 1965. Following this pioneering work, Volobouev et al. (1988) described three karyotypic forms, differing from each other by important chromosomal rearrangements, such as pericentric inversions, reciprocal translocations and Robertsonian fusions. The fist karyomorph, provisionally labelled ANI-1 and represented by specimens from Egypt and northem Senegal, was characterized by a karyotype of 62 chromosomes. All the chromosomes were acrocentric except the sexual chromosomes and one or two small pairs, resulting in a low autosomal fundamental number (N.F.a = 62 or 64). Another morph, ANI-2, represented by a specimen from Central African Republic displayed a 58 chromosomes karyotype and carried two Robertsonian fusions. The individual karyotyped by Matthey (1965) appears closely related to this morph. An additional form, AM-3, found in Burkina-Faso and Mali, had a diploid number of 62, but differed from ANI-1 by a series of pericentric inversions increasing the autosomal fundamental number to 76. These findings were confirmed by Granjon et al. (1992) who also reported karyotypic variability among sampled sites: specimens from southem Senegal (Basse-Casamance) displayed karyotypes closely related to the ANI-3 form of Mali and Burkina-Faso, whereas specimens from northem Senegal and Niger were karyotypically similar to ANI-1. Moreover, Orlov, Baskevich & Bulatova (1992) showed that Awicanthis specimens from Ethiopia (Omo valley) belonged to the ANI-1 type. A striking specimen from Somalia should also be mentioned, which had a diploid number of 44, but was still referred to as A. niloticils by Capanna & Civitelli (1988). Electrophoretic studies on Arvicanthis have provided estimates of the genetic variability and degree of divergence among populations (Kaminski, Rousseau & Petter, 1984; Kaminski et aZ., 1987). - Biochemical differences mirror the clear distinction of populatiom observed in the cytogenetic data, and group animals from Egypt and northem Senegal separately from those of southern Senegal and Burkina-Faso. Surprisingly, these authors, cognizant of all this evidence, never discussed the possible taxonomic significance of these data. Further evidence can be found from laboratory breeding experiments. Crosses between individuals of the same cytotype but from distant geographical origins (northem Senegal and Egypt; northern Senegal and Ethiopia; Sudan and Egypt) yielded offspring without any noticeable reduction in fertility (Petter et al., 1969; Kaminski, Rousseau & Petter, 1984; Philippi, 1994), whereas crosses between animals from northern Senegal (ANI-1) and southem

~ Burkina-Faso (ANI-3) remained sterile (Kaminski & Petter, 1984). Unfortunately, most studies to date have sampled too few individuals (a total of fewer than 15 specimens were karyotyped) and from too distant geographical sites, thus providing a fragmentary outline of the situation. The present study, using several different technical approaches, attempts to characterize more precisely these different karyomorphs and to determine whether they represent true biological species or merely geographical variants. This analysis includes a larger sample size and uses a finer spatial scale (sahelo-sudanian West African belt) to provide a better understanding of the nature and distribution of these morphs. Chromosomal characterization (R- and C-banding), global genomic comparisons (DNADNA hybridization), and level of reproduetive isolation (breeding experiments in captivity) are examined. !i IL.. ?f I’ :i Materials and methodk

Cytogenetic experiments

A total of 15 individuals (12 males and 3 females) from 5 different West African countries were karyotyped. The origin of the specimens studied is indicated in Table I and the localities in Fig. 1. A more intensive collecting effort Y .

SYSTEMATICS OF ARVICANTHIS IN WEST AFRICA 711 TABLEI List of locality names, sample size und sex (in = male, f =female), diploid number, findamental autosomal number und name of collector of the specimens involved in the cytogenetic analysis i

Locality Geographic coordinates Sample 2n NFa Collector

Velingara, Senegal 12"2S'N 12'04' W Im 62 74 J.-M. Duplantier Kédougou, Senegal 12"33'N 12'11'W Im 62 74 J.-M. Duplantier Pondala, Senegal 12'40" 12'02'W lm 62 74 J.-M. Duplantier Saraya, Senegal 12'50" 1I"45'W 2m 62 74, 76 J.-M. Duplantier Fadiga, Senegal 12"31'N 12'15'W If 62 76 J.-M. Duplantier I Bankoumana, Mali 12"lS'N 08"lS'W 2m 62 76 B. Sicard Samaya, Mali 12'20" 08"IO'W If . 62 76 B. Sicard Ougadougou, Burkina-Faso 12"22'N Ol"31'W Im 62 76 A. Ouedraogo Chott Boul, Mauritania 16"35'N 16"25'W lm 62 64 L. Granjon Richard-Toll, Senegal 16'28'N 15'45'W Im 62 64 J.-M. Duplantier Oursi, Burkina-Faso 14'41'N OO"27'W lm 62 64 B. Sicard Kolo, Niger 13"14'N 02'20'E lm, If 62 64 B. Sidiki

was made in south-eastem Senegal, a region likely to be a contact zone with intermediate forms between ANI-1 and ANI-3. Chromosome preparations were obtained from fibroblast cell cultures established from tail biopsy, or from sternal muscle tissues when killing an animal was unavoidable. After disruption, the explants were grown at 37 "C-in a CO2 incubator as monolayer cultures in Falcon flasks (25 cm2) containing McCoy nutritive medium supplemented with 20% foetal calf serum, Fibroblast Growth Factor (FGF) and antibiotks (Ampicillin, Gentamicin). Once the cultures were established, explants were removed and cells were grown in McCoy nutritive medium supplemented with 10% foetal calf serum. Mitotic chromosomes were studied using RBG- banding (R-bands by BrdU using Giemsa) and CBG-banding (C-bands by Barium hydroxide using Giemsa) techniques following the procedures of Viegas-Péquignot & Dutrillaux (1978) and Sumner (1972), respectively. Cultures were synchronized by adding FudR (5-fluorodeoxyuridine) in order to increase the yield of metaphase and early metaphase cells. To induce RBG-banding, 5-bromo-2-deoxyuridine (BrdU) was added at a final concentration of 10-20 pg/ml. Colchicine was added 45 minutes (R-banding) to 2 hours (C-banding) before harvesting. The cells were then treated with a hypotonic solution of potassium chloride (KCI) for 5 minutes at 37 OC, fixed with Camoy's solution and spread on previously cooled slides. To obtain RBG-bands, a Fluorochrome-Photolysis-Giemsa (FPG) staining was performed as described by Viegas-Péquignot & Dutrillaux (1978). At least 15 good quality metaphase plates were analysed for each specimen. The best metaphases were photographed and karyotypes were then prepared, using one metaphase per karyotype. Chromosomes were al., ordered according to their relative size and shape, as was previously described (Volobouev et 1988). Voucher specimens (skin & skull) are deposited in the collection of the Muséum National d'HiStoire Naturelle, - Paris (Laboratoire de Zoologie, Mammifères et Oiseaux). The cells and explants'of studied specimens are kept in liquid ;nitrogen in the Cell and Tissues Collection of the Laboratoire de Cytogénetique Moléculaire et Oncologie, I Instit4 Curie, paris. 'i : i i i DNNDNA hybridization experiments 1 The DNA/DNA hybridization survey was performed on 5 individuals (7 DNA samples were. extracted from al. these 5 tissue samples), which had been previously karyotyped by Volobouev et (1988). Their geographical location and collectors' names are listed in Table II. DNA samples were extracted and purified from 95% ethanol preserved tissues housed in the Collection of Preserved Mammalian Tissues of the Institut des Sciences de I'Evolution, Montpellier (Catzeflis, 1991). km O 100 200 300 I . ,..,.."._..*.,.. .-."..(... '\ I.

FIG. I.Map of West Africa showing the locality of origin of the karyotyped individuals. Circles represent ANI-I specimens and triangles ANI-3 specimens. Closed symbols represent localities sampled in the present study, open symbols show localities previously studied. The insert in the lower comer is an enlarged map of south-eastem Senegal. Localities are: 1=Chott Boul, 2 = Richard-Toll, 3 =Dakar, 4 =Nema Nding, 5 =BranSan, 6 =Basse-Casamance National Park, 7=Kédougou, S=Velingara, 9=Pondala, 10=Fadiga, 11 =Saraya, 12=Bankoumana, 13=Samaya, 14=Bamako, 15=Ouagadougou, 16=Oursi, 17 =Niamey, 18 = Kolo, 19= Lokossa, 20 =Toff0 and 21 = Attogon. > _. . .--.----... --. I ~ , -- .-..-___.__-______,-."--- I.~_I_~_-__---- .-- . .,. SYSTEMATICS OF ARVICANTHIS IN WEST AFRICA 713 TABLEII List of taxa (with DNA sample numbers) used in the DNA/DNA hybridization experiments with their geographic origins and name of collectors

Taxa DNA samples Geographic origin Collector

Arvicanthis sp. ANI-I 4429 Bransan, Senegal J.-M. Duplantier 4003,4419,4434 Bandia, Senegal J.-M. Duplantier 4023 Bandia, Senegal J.-M. Duplantier Arvicanthis sp. ANI-3 4092* Bamako, Mali F. Petter 4414 Ouagadougou, Burkina-Faso F. Petter Aethomys hìndei 4404 Centrafrican Republic F, Petter Aethomys namaquensis 4401 South African Republic P. Woodall Mus caroli 4108 Thailand' F. Bonhomme Mus musculus 57 Austria F. Bonhomme

*Sample used as tracer in the DNA/DNA hybridization experiments

The procedures for single-copy nuclear DNA (scnDNA) hybridization experiments used here are similar to those of Sibley & Ahlquist (1991) and Werman, Springer & Britten (1990). DNAs were sheared to fragments with an average size of 500bp (range, 300-1000bp). The scnDNA fractions were isolated by removing, on hydroxyapatite (Bio-Gel HTP, Bio-Rad) columns, the highly repeated sequences that reassociate by a Cot value of 100M.s (Cot is the product of the DNA concentration and the time of reassociation) in 0.48 M sodium phosphate buffer (pH 8.0) at 55.0"C. Tracer scnDNAs were chemically labelled with '''1, and their average c. fragment length measured on sizing gels ranged from 300 to c. 700 bp. D"NA hybrids were permitted to reassociate after heat denaturation to a Cot of 16 000M.s at 60.0"C in 0.48M sodium phosphate buffer (pH 8.0). This severe criterion value allowed the formation of stable hybrids for molecules which were at least 70% complementary, and ensured that homologous duplexes could form. Thermal elutions were begun at 55 "C in increments of 2.5 "C up to 95 OC, and the raw data are the radioactive cpm eluted at each fractionation temperature. Several statistics can be calculated to estimate the differences between the thermal elution curves of homoduplex (tracer and driver of the same species) and heteroduplex (tracer and driver of different species) hybrids. Among these statistics, we chose Mode which is less variable than other statistics for murid rodents as shown by Catzeflis (1990). Mode is the temperature at which the maximum of hybrid DNA is dissociated. One DNA sample has been radioactively labelled (tracer): Anticanthis niloticus, DNA sample number 4092. Specimens of the genera Aethomys and Mus were used as Outgroups.

Crossbreeding experiments

Laboratory crosses were simultaneously conducted in Paris (Laboratoire de Zoologie, Mammifères et biseaux) and Dakar (ORSTOM). Wild specimens were used in all crosses except the interform ones which involved laboratory-born south Senegal animals. Two types of crosses were performed: (1); Intraform crosses, between individuals idisplaying a similar karyotype; to confirm levels of fertility and fecpndity : t &orth Senegal (ANI-1)xNorth Senegal (&M-l): 56 pairs South Senegal (ANI-3) x South Senegal (?NI-3): 8 pairs I Mali (ANI-3) x Mali (ANI-3): 1 pair i (2)'Interform crosses to check for the fertili& potential between the chromosomally distinct forms: North Senegal (ANI-1) x South Senegal (ANI-3): 6 pairs Interform crosses involved bidirectional pairings (North Senegal male with South Senegal female, and South Senegal male with North Senegal female). Each pair was placed in a separate plastic cage with a metal mesh top, J. 714 F. DUCROZ ET AL. under partially controlled conditions (natural photoperiod and humidity, to = 25 OC). Pellets and apples or water were supplied ad libitum. Only the results from individuals paired for a minimum of 90 days were considered. Date of birth, size and sex ratio were recorded for each litter. The progeny were allowed to remain with their parents until weaning, at which time they were transferred to holding cages. In unproductive matings, a post- mortem examination of the genital tracts of the females was performed to detect traces of abortive development. Differences in litter size were compared using a Wilcoxon-Mann-Whitney test.

Results

Cytogenetic analysis The karyotype analysis showed that all the individuals studied corresponded to either of the two cytotypes (ANI-1 or ANI-3) previously established (see Table I). No intermediate form was found in the tentative contact zone in south-eastem Senegal.

ANI-1(2N= 62; N.F.a = 64) This karyomorph is represented by specimens from Mauritania, northern Senegal, Niger and northern Burkina-Faso (Plate I). They appear similar in all respects to the previously karyotyped Arvicantltis from northern Senegal. All. autosomes are acrocentric except for two small metacentric pairs (28 and 30). The X chromosome, @e largest of the set, is submetacentric and the Y chromosome is a medium metacentric. The C-banding shows that constitutive heterochromatin blocks are present in the pericentromeric region of all autosomes (Plate III). The short arm of the X chromosome is heterochromatic although less intensively stained than the juxtacentromeric region. The Y chromosome is almost entirely heterochromatic with the pericentromeric region more strongly stained.

ANI-3(2N= 62; N.F.a = 74-76) The high fundamental number and the 2N = 62 diploid number of the specimens from south-eastern Senegal, Mali and south Burkina-Faso clearly relate these individuals to the ANI-3 cytotype, although displaying minor differences from specimens from Casamance described by Granjon et al. (1992) (Plate II). The first three autosomal pairs are submetacentric, as well as the medium-sized pairs 16, 17 and 21. Pairs 26, 28 and 30 are small biarmed chromosomes. The X chromosome is a large submetacentric, but its short arm is more reduced than in ANI-1. The Y chromosome is a medium meta- to submetacentric. The C-banding pattern, illustrated in Plate IV, is quite different from the one observed for ANI-1. ! The staining is very weak or even absint, notably for the larger pairs of autosomes. Likewise, the : L heterochromatic short arm of the X chry"ome is muchjmore reduced in size. ._L 1 The ANI-3 sample studied displayed; considerable karyotypic polymorphism which accounts for the variation in N.F.a (74 to 76). Pairs i6, 17, and 21 were either acrocentric or submetacentric but always in a homozygous state. Data are summarized in Table III. In south-eastem Senegal, the six individuals studied displayed three different chromosomal associations and two individuals from the same locality (Saraya) were different. On the other hand, the submetacentric form of pair 17 seems to be restricted to the southem Burkina-Faso population and may indicate a geographical differentiation. SYSTEMATICS OF ARVICANTHIS IN WEST AFRICA 715

1 4 5 io 6 10

v -t ui or 11 ~ 14 15

#!!I 16 19 20 oft*

- 25 A& XY

PLATEI. ANI-1Karyotype. A R-banded karyotype of ahale specimen from Kollo (Niger). J. 716 F. DUCROZ ETAL.

5 li 1 2 3 4

7 8 9

iI0&

11 12 13 - 14 bC 69 d& 16 17 18 19 5$" 21 22 23 24

rl w a4 - *. 25 26 27 28

!i :I?: t 30

PLATEII. ANI-3 Karyotype. A R-banded karyotype of .a male:specimen from Saraya (Senegal). !

SYSTEMATICS OF ARVICANTHIS IN WEST AFRICA 717 c

, *: . .')

a: 3 "P

b # F m b e.i' 6j PLATEIII. AÑI-I Karyotype. A C-banded metaphase of a male specimen from Kollo (Niger) with enlargement of the sexual chromosomes.

PLATEIV. ANI-3Karyotype. C-banded metaphase of a male specimen from Saraya (Senegal) with enlargement of the sexual chromosomes. 718 J. F. DUCROZ ET AL. TABLEIII Chromosomal polymorphism of the pairs 16, 17 and 21 found in the ANI-3 type (individuals designated after location). A = acrocentric; S = submetacentric

~ Individual Pair 16 Pair 17 Pair 21

Kédougou A A S Pondala S A A Vélingara A A S Fadiga S A S Saraya 1 A A S Saraya 2 S A S Bankoumana 1 S A S Bankoumana 2 S A S Samaya S A S Ouagadougou S S A

two Comparison of the chromosomal forms Although displaying the same diploid number, ANI-I and ANI-3 differ by numerous rearrangements. The difference in morphology and fundamental number between the two cytotypes can be ascribed to a series of five to six pericentric inversions affecting pairs, 1,2,3, 16, 17,21 and 26. They are also differentiated by a reciprocal translocation involving chromosomes 4 and 7. Some difficulties were experienced in matching homologous chromosomes among the smaller pairs, owing to their indistinct R-bands and similarity in size, Additional small intrachromosomal rearrangements may have occurred, but could not be resolved. ANI-1 and ANI-3 also displayed different C-banding patterns. Whereas all autosomal pairs of ANI-1 show large heterochromatic blocks in the pericentromeric region, these blocks are reduced or even lacking (larger pairs) in ANI-3. The discrepancy in size of the X chromosomes between the two cytotypes is related to additional heterochromatic material in ANI-I.

TABLEIV * Mean values of Mode vor 4092) and deltalMode of the different samples of Arvicanthis with their fundamental numbers (NFa). The values obtained with two other genera of Murinae are indicated for comparison

. Species DNA ' Modes (OC) NFa

i Arvicanthis sp. ANI-3 4092* i 86.5 76 i i Arvicanthis sp. ANI-3 4414 0.1 76 t i Arvicanthis sp. ANI-1 4429 2.5 64 i 4434 2.7 64 i 4003 i- 2.8 64 i 4419 . 2.4 64 ' 4023 2.7 64 . Aethomys hindei 4404 7.5 Aethomys namaquensis 4401 7.8 Mus carolì 4018 15.3 Mus musculus 57 16.1

*Sample used as tracer in the DNNDNA hybridization experiments ..

SYSTEMATICS OF ARVICANTHIS IN WEST AFRICA 719

65 70 75 80 85 90 95 Tsmperature ("C)

--o- ANI-3 (4092) tracer*

, -t- ANI-1 (4434, 4003, 4419) -O- ANI-1 (4429) - f -ANI-1 (4023) -ANI-3 (4414) 4- Aethomys hindei & namaquensi .-A,. Mus caroli & musculus FIG.2. Modal melting curves for the determination of the Mode using Arvicanthis sp. (4092) as the tracer. The DNA samples n used are indicated in brackets. Each curve represents the average of hybrids. DNMDNA hybridization analysis The results are presented in Table IV and Fig. 2. The different samples of Arvicanthis can be separated at least into two groups, congruent with the chromosomal data. The first one includes samples 4092 and 4414, both belonging to the ANI-3cytotype, and separated by a very low molecular distance (delta-Mode = 0.1 OC). *The three individuals from the ANI-1 cytotype (DNA samples

TABLEV Reproductive data for the infrafonn and interfonn crosses. N, =number of pairs, N2 = number of pairs having reproduced, n = number of offspring, X = mean litter size

i i L Matings i hjating Litter size f Pairings NI NZ sucqess (%) n X

Intraform North Senegal (ANI-I) - 56 23 42 325 6.02 South Senegal (ANI-3) 8 2 25 54 5.40- Mali (ANI-3) 1 O O - Integorm North SenegalxSouth Senegal 6 O O - - I

J. 720 F. DUCROZ ETAL. ! 4434 = 4003 = 4419,4023 and 4429) are well separated from the tracer 4092 with an average value ! of delta-Mode = 2.62 If: 0.4, n = 11. I i Crossbreeding analysis l The results of all crosses are summarized in Table V. A very significant proportion (50/121) of the !1 pairs had to be removed before the minimal time of 90 days because of aggressive behaviour sometimes resulting in the death of one of the animals. These were not included in the analysis. ANI-1 i and ANI-3 intraform pairs produced offspring with respective successful mating rates of 41 and 25% which represent quite low values, and produced litters of, respectively, 6.02 ? 0.37 and 5.40 2 0.28 ! young on average. Differences in reproductive success rates and mean litter size between the two forms were not statistically significant, although ANI-3 were less fertile in both parameters. No interform pairs produced offspring and examination of the genital tracts of females revealed no sign of gestation.

Discussion i The important karyotypic and genetic differentiation observed between AM-1 and AM-3, as well as the apparent sterility between these karyomorphs, suggest that they distinguish separate species. As such, 1 they represent another example of sibling species, morphologically similar but chromosomally and i genetically distinct, a phenomenon frequently encountered in African murid rodents (Meester, 1988). The speculation that the different karyomorphs Gght represent balanced chromosomal polymorph-- I ism is not supported by our data, as no intermediate morphs were found, even in the most likely contact ti zone in south-eastem Senegal. The last observation is not surprising, as ANI-1 and ANI-3 display a E large karyotypic differentiation which is likely to give rise to strong reproductive isolation. Chromosomal rearrangements by which these forms differ are of two kinds and comprise one t reciprocal translocation and six to seven pericentric inversions. Reciprocal translocations in chromo- [ somal evolution are scarce because they result in severe disturbance of meiosis usually producing i genetically unbalanced gametes. Fixation of such a rearrangement may by itself lead to the establishment of genetic isolation. Besides, it is generally believed that reciprocal translocations i1 result in the creation of novel genes so that their establishment needs two euchromatic breaks (Wilson, 1 Sarich & Maxson, 1974). Contrary to reciprocal translocations, pericentric inversions are common in f. karyotype evolution of vertebrates and numerous findings of intrapopulational polymorphism invol- ! ving one, two, or rarely three, inversions have been made in different groups (Gum & Greenbaum; -1986; Greenbaum er aZ., 1990). In the case of ANI-1 and ANI-3, no heterozygous individual was -4 . detected. The fact that all six to seven inversions are fixed in ANI-3 implies that their progressive accumulation must have begun at least in the Pleistocene, a view supported by the DNA/DNA hybridization data. If we use a molecular time sca1e:calibrated on aMus-Rattus dichotomy esimated at 10 Ma (Jaeger, Tong & Denys, 198$ the delta-Mode value indicates that the two forms of Arvicanthii might have diverged some 1.5 myh. Natural hybripzation between ANI-1 and ANI-3, which woul4 result in heterozygosity for six td seven inversiofis, looks highly improbable. Thus, chromosomai rearrangements separating ANI-1 !and ANI-3 are undoubtedly capable of providing a very efficient genetic barrier. The failure to produce offspring in captivity does not necessarily imply reproductive isolation under ! natural conditions (Rubinoff & Rubinoff, 1971), particularly when breeding is difficult and the mating success low, as in our case. Although the numbers of intraform and interform matings . . . ..

I E

72 SYSTEMATICS OF ARVICANTHIS IN WEST AFRICA 1 involving ANI-3 (nine and six, respectively) are low, the results suggest that hybridization between ANI-1 and ANI-3 is at best highly unlikely, and support the hypothesis thai they correspond to good biological species (sensu Mayr, 1963). The DNADNA hybridization data on Awicanthis are in agreement with the latter conclusion. As we have shown, the delta-Mode values observed between I ANI-I and ANI-3 are far above the usual level of intraspecific variation, and even exceed the levels of genetic differentiation reported between congeneric murid species such as Mastomys huhcrti and M. natalensis (delta-Mode = 0.8 2 0.3), M. huberti and M. erythroleucus (delta-Mode = 0.7 2 0.2) or Mus spretus/Mus musculus/Mus spicilegus (delta-Mode = 1.9 I0.3) (Chevret, 1994). In contrast to ANI- 1, which appears karyotypically very homogeneous throughout its distribution, ANI-3 displays important chromosomal polymorphism, which suggests that this form may be involved in an extensive process of chromosomal rearrangement. This chromosomal variation raises the problem of its taxonomic and evolutionary significance. A more intensive investigation of wild populations is required to determine if the chromosomal variation corresponds to intrapopulational polymorphism or to different karyotypic races. ANI-1 and ANI-3 would have undergone dissimilar chromosomal evolutions, and it is likely that the heterochromatin content, so different in the two forms, is somehow linked to this process. The preceding body of data prompts us to propose a systematic revision for the genus in West Africa. We agree with the designationA. niloticus Desmarest 1822, made by Volobouev et al. (1988) for ANI-I, the chromosomal formula of which (2N = 62; N.F.a = 64) is shared by specimens from Egypt, the country of the species type locality. Moreover, individuals from Egypt and northern Senegal . proved to be interfertile (Kaminiski et al., 1987). For-AM-3, the situation is more problematic as no chromosomal data are available from regions neighbouring the type localities of named forms. Volobouev et al. (1988) suggested referring ANI-3 to A. n. solatus Thomas 1925 whose type locality (AoudCras, Aïr, Niger) is the nearest to the individuals from Burkina-Faso and Mali. But solatus, characterized by a pale sandy dorsal pelage, is a race probably restricted to subdesert environments. On the four remaining races recognized by Rosevear (1969) for Awicanthis in West Africa, two would be good candidates for a renaming of ANI-3. Anticanthis niloticus mordax Thomas 1911 matches in size and coat colour the specimens of ANI-3 that we have karyotyped. The type locality is Panyam, in central Nigeria, a locality situated in the sudanian belt and likely to house ANI-3 forms. The other race, Awicanthis niloticus rujnus Temminck 1853 (type locality given as Elmina, Ghana) seems to be a wet-habitat form restricted to the southern limits of the distribution of the genus in contact with the high forest zone. It is characterized by deep reddish upper parts and flanks, as well as a weaker ' dentition, features never exhibited by our sample (Ducroz et al., unpubl. results). However, this name has priority over A. n. mordax and should these two taxa belong to the same species, the name ... A. n. rujous should be adopted. Moreover, 13 individuals from southem Benin karyotyped by Civitelli et al. (1995) are clearly related to our AM-3 moqh (2N=62; N.F.a=74). This observation demonstrates that ANI-3 reaches the southem limits of the distribution of Awicanthis. It is therefore ,..I .. i not improbable that A. niloticus rujìnus is a &ìI-3 ;orpl+ though any taxonomic revision of this i i morph must await karyotypic investigation from the dpe localities of the two races. i! A synthesis on karyomorph distribution is presente$ in @g. 1, including all previously published i:. data. It is now clear that ANI-I and ANI-3 are parapatrically distributed, following a latitudinal . pattern, with ANI-1 to the north and ANI-3 to the .south. It is worth noting that their areas of distribution seem to agree quite well with biogeographical domains of western Africa: A. niloticus is present in the low tree and shrub savannas of the sahelian and north sudanian zone, while ANI-3 is distributed in the savanna woodlands of the sudano-guinean zone. These distribution patterns may be related to certain ecological adaptations, and would greatly benefit from an eco-physiological analysis. Ø

722 J. F. DUCROZ ET AL. Extension of these phylogeographical studies to East African Arvicanthis, where similar chromosomal forms seem to occur (Orlov et aL, 1992), would also be of great interest for a better understanding of this genus.

We would like to thank all the people listed in Tables I and II who collected the specimens analysed here, as well as J. C. Gautun for help in providing specimens from Burkina-Faso and Niger. We are grateful to F. Catzeflis for assistance in DNADNA hybridization analysis. Laboratory work at Montpellier was partially funded by Region Languedoc-Roussillon, by Services Communs de Biosystématique, and by a specific grant from GREG (decision 81/94 to F. Catzeflis). This is contribution 97-001 of Institut des Sciences de I’Evolution, Montpellier, France. J. Many thanks to P. A. Ritchie, E. Capanna and especially Britton-Davidian for corrections and comments on earlier drafts of this manuscript.

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