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Diversity and evolution of African Grass Rats (Muridae: Arvicanthis) : from radiation in East Africa to repeated colonization of northwestern and southeastern savannas

Reference: Bryja Josef, Colangelo Paolo, Lavrenchenko Leonid A., Meheretu Yonas, Šumbera Radim, Bryjová Anna, Verheyen Erik K., Leirs Herw ig, Castiglia Riccardo.- Diversity and evolution of African Grass Rats (Muridae: Arvicanthis) : from radiation in East Africa to repeated colonization of northw estern and southeastern savannas Journal of zoological systematics and evolutionary research - ISSN 0947-5745 - 57:4(2019), p. 970-988 Full text (Publisher's DOI): https://doi.org/10.1111/JZS.12290 To cite this reference: https://hdl.handle.net/10067/1601280151162165141

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1 Title: Diversity and evolution of African Grass Rats (Muridae: Arvicanthis) - from radiation in East 2 Africa to repeated colonization of north-western and south-eastern savannahs 3 4 Short running title: Grass rats in sub-Saharan savannas 5 6 Josef Bryja1,2, Paolo Colangelo3, Leonid A. Lavrenchenko4, Yonas Meheretu5, Radim Šumbera6, Anna 7 Bryjová1, Erik Verheyen7,8, Herwig Leirs8, Riccardo Castiglia9 8 9 10 1 Institute of Vertebrate Biology of the Czech Academy of Sciences, Brno, Czech Republic 11 2 Department of Botany Forand Zoology, Review Faculty of Science, MasarykOnly University, Brno, Czech Republic 12 3 National Research Council, Institute of Agro-environmental and Forest Biology (CNR-IBAF), Rome, Italy 13 Italy 14 4 A.N.Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Moscow, 15 Russia 16 5 Department of Biology and Institute of Mountain Research and Development, Mekelle University, 17 Mekelle, Tigray, Ethiopia 18 6 Department of Zoology, Faculty of Science, University of South Bohemia, České Budějovice, Czech 19 Republic 20 7 Royal Belgian Institute for Natural Sciences, Operational Direction Taxonomy and Phylogeny, 21 Brussels, Belgium 22 8 Evolutionary Ecology Group, Biology Department, University of Antwerp, Antwerp, Belgium 23 9 Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy 24 Italy 25 26 Prepared for: Journal of Zoological Systematics and Evolutionary Research as Original article 27 28 *Corresponding author: Josef Bryja, Institute of Vertebrate Biology of the Czech Academy of 29 Sciences, Research Facility Studenec, Studenec 122, 675 02 Koněšín, Czech Republic; E-mail: 30 [email protected] 31 32 33 Word count: 10598 words incl. References, 7 Figures, 2 Tables 34 35 Keywords: taxonomy, Ethiopia, biogeography, reticulate evolution, tropical Africa

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36 Abstract 37 African grass rats of the genus Arvicanthis Lesson, 1842, are one of the most important groups of 38 rodents in sub-Saharan Africa. They are abundant in a variety of open habitats, they are major 39 agricultural pests and they became a popular model in physiological research because of their diurnal 40 activity. Despite this importance, information about their taxonomy and distribution is 41 unsatisfactory, especially in Eastern Africa. In this study, we collected the most comprehensive 42 multilocus DNA dataset to date across the geographic and taxonomic range of the genus (229 43 genotyped specimens from 130 localities in 16 countries belonging to all currently recognized 44 species). We reconstructed phylogenetic relationships, mapped the distribution of major genetic 45 clades, and used the combination of cytogenetic, nuclear and mitochondrial markers for species 46 delimitations and taxonomicFor suggestions. Review The genus is composed Only of two major evolutionary groups, 47 called here the ANSORGEI and NILOTICUS groups. The former contains four presumed species, while 48 the latter is more diverse and we recognized nine species. Most relationships among species are not 49 resolved, which suggests a rapid radiation (dated to early-middle Pleistocene). Further, there is an 50 indication of reticulate evolution in Ethiopia, i.e. the region of the highest Arvicanthis diversity. The 51 distribution of genetic diversity suggests diversification in Eastern Africa, followed by repeated 52 dispersals to the west (Sudano-Guinean savannahs), and to the south (Masai steppe). We propose 53 nomenclatural changes for Ethiopian taxa and provide suggestions for future steps towards solving 54 remaining taxonomic questions in the genus. 55

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56 Introduction 57 Despite their practical importance for human, e.g. as vectors of pathogens or agricultural pests, the 58 general knowledge of rodents in sub-Saharan Africa is relatively limited. Surprisingly, this is often 59 true also for abundant taxa, where even the basic information about their taxonomy and distribution 60 is missing. One of the most successful groups of African murine rodents in open habitats is the genus 61 Arvicanthis Lesson, 1842, which has a wide distribution ranging along the entire Nile Valley south to 62 the Zambezi River, and from Senegal in the west to the Horn of Africa in the east. These rodents 63 occur in a variety of ecosystems, from the lowland savannahs and dry coastal areas to the high 64 altitude grasslands of the Ethiopian highlands. They are major agricultural pests, as their 65 reproductive success (especially after annual dry-season depletion) makes them more successful 66 than other competing rodentsFor (Sicard, Review Diarra & Cooper, 1999). Only Because most species are diurnal and 67 easy to breed, Arvicanthis became a popular model also in physiological research, especially in 68 studies of circadian rhythms, vision and endocrinology (e.g. Castillo-Ruiz, Indic & Schwartz, 2018; 69 Gianesini, Clesse, Tosini, Hicks & Laurent, 2015; Langel, Smale, Esquiva & Hannibal, 2015). 70 71 The genus Arvicanthis shows a remarkable degree of diversity and constitutes a typical historical 72 example of the taxonomic issues caused by the subsequent activities of lumpers and splitters during 73 the last century. For example, Allen (1939) listed 37 taxa within the genus, while Honaki, Kinman & 74 Koeppl (1982) considered the genus monotypic with the single species A. niloticus. The genus has 75 been later on the subject of intense taxonomic, systematic and evolutionary studies. Numerous 76 papers over three last decades have summarised the information so far collected through 77 morphometric data (Baskevich & Lavrenchenko, 2000; Bekele, Capanna, Corti, Marcus & Schlitter, 78 1993; Corti & Fadda, 1996; Fadda & Corti, 2001) and karyotype analyses (Castiglia et al., 2003; 79 Castiglia, Bekele, Makundi, Oguge & Corti, 2006; Civitelli, Castiglia, Codja & Capanna, 1995; Corti, 80 Civitelli, Bekele, Castiglia & Capanna, 1995; Corti, Civitelli, Castiglia, Bekele & Capanna, 1996; Corti et 81 al., 2005; Volobouev et al., 2002). Especially karyotypes were found very useful for understanding the 82 evolution of these rodents (see review in Castiglia et al., 2006) helping significantly to delimit 83 currently recognized species (e.g. Volobouev et al., 2002). In the most recent compendium of the 84 Handbook of the Mammals of the World (Denys et al. 2017), seven species are recognized. However, 85 the review of described karyotypes suggests cryptic diversity in the genus (Castiglia et al., 2006) and 86 the high numbers of synonyms included in some taxa reflects a complex situation (Musser & 87 Carleton, 2005). The definition and distribution limits of several species clearly require further 88 investigation, and additional species may prove to be present especially in central and eastern Africa 89 (Happold, 2013). 90

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91 In contrast to cytogenetic and morphometric approaches, the use of DNA sequence data in 92 phylogenetic studies of Arvicanthis is relatively scarce (Abdel Rahman Ahmed et al., 2008; Dobigny et 93 al., 2011, 2013; Ducroz, Volobouev & Granjon, 1998, 2001). These studies are limited in number of 94 markers, based mainly on mitochondrial sequences (mostly cytochrome b, CYTB), except Dobigny et 95 al. (2013), who used one nuclear gene with very limited variability. They are also geographically 96 biased to west-central Africa and only one clade, A. niloticus sensu lato, is analysed in wider 97 geographical context (Dobigny et al., 2013). Other studies either addressed the taxonomic diversity in 98 geographically limited regions (Abdel Rahman Ahmed et al., 2008 in Sudan; Dobigny et al., 2011 in 99 northern Cameroon), or attempted to derive phylogenetic relationships for the genus with a very 100 limited number of specimens collected throughout the distribution range of this genus (Ducroz et al., 101 1998, 2001). Available resultsFor suggest Review that most genetic diversity Only is located in Eastern Africa, which is 102 in agreement with cytogenetic data (Castiglia et al., 2006). However, no detailed study based on DNA 103 sequences from Eastern Africa have been published yet. As a result of relative morphological 104 uniformity, general lack of DNA data, and especially complete absence of morphological studies of 105 genotyped individuals, the species in the genus are delimited only vaguely, there have been 106 numerous confusions and misunderstandings (e.g. misuse of species names; see Table 1), and the 107 distributional limits for most taxa are not well defined. 108 109 For this study, we collected the most comprehensive multilocus dataset of DNA sequences to date 110 across the geographic range of the genus, with special focus on undersampled region of eastern 111 Africa. With these data in hand, we had following aims. (1) We used CYTB-barcoded samples for 112 realistic assessment of the distribution of main mitochondrial genetic clades, possibly corresponding 113 to different species. (2) Using CYTB and six nuclear markers, we reconstructed phylogenetic 114 relationships in the genus. (3) In areas of sympatric occurrence of multiple genetic clades (especially 115 in Ethiopia that seems to be the region of the highest Arvicanthis diversity), the combination of 116 nuclear and mitochondrial markers together with available karyotypes was employed for species 117 delimitation, taxonomic suggestions, and definitions for future systematic research. (4) Data on pan- 118 African distribution of the genetic diversity of the genus were used to propose evolutionary 119 hypotheses for the Plio-Pleistocene changes of open habitats in sub-Saharan Africa. 120 121 Materials and methods 122 Sampling 123 This study is based on 229 specimens of Arvicanthis genotyped at CYTB, 184 of them were newly 124 genotyped and 45 sequences were downloaded from GenBank (Appendix S1). Five additional 125 individuals from Castiglia et al. (2006) were unambiguously assigned to particular clades based on

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126 their karyotype and geographic distribution. Collected material sufficiently covers the known 127 distribution of the genus (130 localities from 16 countries; Appendix S1) and it represents the most 128 comprehensive available material of all currently recognized Arvicanthis species (sensu Denys et al., 129 2017). The tissue samples were stored in 96% ethanol or DMSO until DNA extraction. All fieldwork 130 complied with legal regulations in the respective African countries and sampling was carried out in 131 accordance with local legislation (see Acknowledgements). 132 133 Genotyping 134 DNA was extracted by commercial kits and all samples were genotyped at mitochondrial gene for 135 cytochrome b (CYTB) using the protocol from Bryja et al. (2014). Preliminary phylogenetic analyses of 136 CYTB showed division ofFor the genus Reviewinto two main clades (seeOnly also Dobigny et al., 2013). Hereafter we 137 will call these two major groups as the NILOTICUS and ANSORGEI groups according to two 138 characteristic species within them (Dobigny et al., 2013; Volobouev et al. 2002). Because several 139 mitochondrial lineages in the NILOTICUS group seem to have overlapping distribution in eastern 140 Africa, we employed nuclear markers to assess whether they might represent separate gene pools 141 (i.e. reproductively isolated species) and to reconstruct their nuclear phylogeny. We selected 142 individuals representing all but one mitochondrial lineages from the NILOTICUS group (we were not 143 able to get nuclear sequences of one genetic lineage; in total 32 individuals + three outgroups from 144 the ANSORGEI group) and sequenced them at two nuclear exons (IRBP, RAG) and four nuclear introns 145 (WLS, DHCR, TRPV, SMO). For more details on used markers, including primer sequences and PCR 146 protocols, see Appendix S2. 147 148 Mitochondrial phylogeny and sequence divergence 149 In the first step, we reconstructed mitochondrial phylogeny by Bayesian inference (BI) and maximum 150 likelihood (ML) approaches. Sequences of CYTB were edited and aligned in Geneious 9.0.5 151 (Biomatters, Ltd.) producing a final alignment of 1140 bp. The Findmodel web application 152 (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html) was used to identify the 153 most appropriate substitution model. The Akaike information criterion (AIC), compared among 12 154 biologically relevant substitution models, revealed that the model best fitting the ingroup data was 155 GTR + G. As outgroups, we used seven sequences of other murid rodents, including three from the 156 tribe Arvicanthini, one from Otomyini, one from Murini, one from Rattini and one Acomys sequence 157 from the sister subfamily Deomyinae (see Appendix S2). BI analysis (partitioned by codon position) 158 was performed in MrBayes 3.2.6 (Ronquist & Huelsenbeck, 2003). Three heated and one cold chain 159 were employed, and runs were initiated from random trees. Two independent runs were conducted 160 with 10 million generations each and trees and parameters were sampled every 1000 generations.

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161 Convergence was checked using Tracer 1.7 (Rambaut, Drummond, Xie, Baele & Suchard, 2014). For 162 each run, the first 25% of sampled trees were discarded as burn-in. Bayesian posterior probabilities 163 were used to assess branch support of the MCMC tree. ML analysis was performed using RAxML 164 8.2.8 (Stamatakis, 2014). We used GTRCAT model as suggested by authors of the programme. The 165 robustness of the nodes was evaluated by the default bootstrap procedure with 1,000 replications. 166 All phylogenetic analyses were run on CIPRES Science Gateway (Miller, Pfeiffer & Schwartz, 2010) 167 and trees were edited in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). 168 169 Genetic p-distances among mitochondrial clades (i.e. the number of base differences per CYTB site 170 from averaging over all sequence pairs between groups) were calculated in MEGA 10.0.4 (Kumar, 171 Stecher, Li, Knyaz & Tamura,For 2018) Reviewusing the same dataset Only as for mitochondrial phylogeny, i.e. 121 172 sequences, and pairwise deletion option. Standard error estimate(s) were obtained by a bootstrap 173 procedure (1000 replicates). 174 175 Phylogeny reconstruction using nuclear markers 176 The number of genetic partitions in the concatenated alignment of six nuclear loci and the most 177 suitable nucleotide substitution models were simultaneously estimated in PartitionFinder 2 (Lanfear, 178 Frandsen, Wright, Senfeld & Calcott, 2016). Single gene trees were calculated by BI in MrBayes as 179 described above using the best substitution models. Partitioned analyses of concatenated nuclear 180 dataset (3926 bp) were performed in MrBayes (BI analysis specifying the best models for all four 181 partitions identified by PartitionFinder; see Appendix S2) and RAxML (ML analysis with GTRCAT 182 model for all partitions) using the specifications described above. In MrBayes, the fifth partition was 183 defined for six parsimony informative indels encoded as standard morphological data (i.e. 1 or 0) and 184 with substitution model specified by Nst=1 rates=gamma coding=variable. 185 186 The species tree based on sequences of nuclear markers was calculated under the fully Bayesian 187 framework using the multi-species coalescent model implemented in StarBEAST 2 (Ogilvie, Bouckaert 188 & Drummond, 2017), an extension of BEAST 2.5.0 (Bouckaert et al., 2014). Alignments for each of six 189 genes were imported into BEAUti 2.5.0, where separate and unlinked substitution and tree models, 190 but the same strict clock rate for all partitions, were defined. We set up the lognormal priors for 191 birthRate.t:Species (with M=4.0 and S=1.25) and popMean (M=-5.0 and S=1.2), the Yule speciation 192 model and linear Pop function with constant root. Two independent runs were carried out for 20 x 193 106 generations with sampling every 2000 generations in BEAST. The resulting parameter and tree 194 files from the two runs were combined using LogCombiner 2.5.0 (Bouckaert et al., 2014) and 195 examined for convergence and effective sample sizes (ESS) in Tracer v1.7 (Rambaut et al., 2018; all

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196 parameters having ESS > 200) and a maximum credible tree was found in TreeAnnotator 2.5.0 197 (Bouckaert et al., 2014). 198 199 Incongruent topologies of mitochondrial and nuclear genes (see Results) suggest that past 200 hybridization likely played a role in the evolution of Arvicanthis. We therefore reconstructed the 201 Bayesian species network (reflecting both incomplete lineage sorting and admixture) using the newly 202 developed approach SpeciesNetworks (Zhang, Ogilvie, Drummond & Stadler, 2017), implemented in 203 BEAST 2.5.0 (Bouckaert et al., 2014). We used the same dataset, substitution and tree models as well 204 as set up for clock rate as in StarBEAST analysis described above. In general, we followed the tutorial 205 for SpeciesNetworks for defining parameters of the analysis in BEAUTI, however, we modified some 206 priors as follows. We usedFor exponential Review prior for netDivRate.t:Species Only (i.e. speciation rate minus 207 hybridization rate) with mean 1.0. The exponential prior was used for the origin time of the species 208 network (originTime.t:Species) with mean 0.001. The prior for popMean.t:Species was set up as 209 gamma with shape (alpha) 2.0 and scale (beta) 0.005, which corresponds to distribution with a mean 210 of 0.01. The analysis in BEAST was run with 20 million iterations, sampled every 2000th. All ESS were 211 > 200 after checking in Tracer. The individual networks were explored in IcyTree, which is a web 212 application for visualizing phylogenies, including phylogenetic networks (Vaughan, 2017). 213 214 Divergence dating 215 The temporal frame of divergences in the genus was analysed in StarBEAST 2. For this analysis, we 216 used 35 individuals sequenced for both nuclear and mitochondrial loci (Appendix S1). The species 217 tree shape was modelled by the birth-death process using an uninformative prior for the net 218 diversification rate and a weakly informative prior, Beta(2,2), for the fraction of extinct species. 219 Because we do not expect differences in substitution rates among lineages within the complex, we 220 assumed a strict molecular clock but partition-specific clock rates (it is reasonable to assume e.g. 221 much higher substitution rate in the mitochondrial marker). Because the variability of nuclear 222 markers was relatively low, we defined more informative exponential primer (with mean = 0.002) for 223 the clock rate in all nuclear genes, based on genetic distances between the two major groups in the 224 genus. The fossil record of Arvicanthis does not allow the placing of fossils on the particular sites of 225 the phylogenetic tree of the genus, so it is not possible to calibrate the molecular clock by ingroup 226 fossils. We therefore performed a secondary calibration using the time to most recent common 227 ancestor (TMRCA) of the NILOTICUS and ANSORGEI clades estimated by Aghová et al. (2018). In their 228 study, the authors used the refined set of nine controlled fossil calibrations for the family Muridae 229 and reconstructed a dated phylogeny of the family using a comprehensive multilocus dataset. They 230 included also three species from the NILOTICUS group and two from the ANSORGEI group and

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231 estimated TMRCA of all taxa at 4.45 Mya (with 95% HPD 3.65-5.23) . We therefore specified a normal 232 prior for TMRCA of the two major Arvicanthis groups with mean 4.45 and standard deviation = 0.5, 233 providing the distribution corresponding to primary divergence estimates (i.e. median 4.45 Mya, 95% 234 HPD 3.63-5.27). The MCMC analysis in StarBEAST and analysis of outputs were performed as 235 described above (but with 50 million iterations per run, sampled every 5000th, and 25% burnin) and 236 the final figure was edited using R package 'strap' (Bell & Lloyd, 2014). 237 238 Results 239 Mitochondrial phylogeny and karyotypic variability 240 Both BI and ML analyses provided very similar topology of mitochondrial tree, with the genus 241 Arvicanthis highly supportedFor and split Review into two major clades, Only the ANSORGEI and NILOTICUS groups 242 (Fig. 1). These two groups have partially overlapping distributions across the range of the genus in 243 sub-Saharan Africa (see Fig. 2 for central-east Africa and Volobouev et al., 2002, for west-central 244 Africa), but significantly differ by DNA sequences as well as by karyotypes (with the former having 245 NFa = 72-78, while the latter NFa = 60-64; Fig. 1). Within each group we defined several usually highly 246 supported clades that we will consider below as separate "species" (but see Discussion for taxonomic 247 issues). The ANSORGEI group contains four species, while the NILOTICUS group is more diverse and 248 we recognized nine species (Fig. 1). 249 250 In the ANSORGEI group, there are two species in eastern Africa: (a) A. nairobae is the only species in 251 the group, whose monophyly at mtDNA is not significantly supported. It is highly structured with 252 three distinct geographically separated clades (with unresolved relationships) distributed along the 253 Albertine rift from Uganda to southern Tanzania, between Nairobi and north-eastern Tanzania, and 254 in southern coastal Kenya, respectively (Fig. 2B). (b) A. sp. "ANI-6" was found only in the Great Rift 255 Valley (GRV) in Ethiopia and is separated into two distinct mitochondrial clades in north-south 256 direction. In addition, there is one species in west-central Africa (A. rufinus, see Fig. 2B for central 257 African localities) and one limited to western Africa, west of the Niger River (A. ansorgei, not shown). 258 The former one is sister to all other taxa in the ANSORGEI group, while the latter is closely related to 259 Ethiopian endemic A. sp. "ANI-6". 260 261 All nine species in the NILOTICUS group can be found in eastern Africa (Fig. 2A), while only one (A. 262 niloticus "C2-C4"; named according to Dobigny et al., 2013) is also widespread in Sudanian savannas 263 up to western Senegal. Except Ethiopia, the distribution of most species is roughly parapatric. Eastern 264 border of the distribution of A. niloticus "C2-C4", represented in eastern Africa only by the clade C2, 265 seems to be limited by GRV in southern Ethiopia and Kenya. On the other hand, A. somalicus is the

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266 typical species of arid Somali-Masai savanna in Ethiopia and Kenya, east of GRV. Arvicanthis 267 neumanni was recorded in the so-called Masai steppe in Tanzania, while its mitochondrial sister 268 clade A. sp. "Masai Mara" only in two localities in GRV in Tanzania. Three distinct haplogroups of A. 269 niloticus s. str. are distributed along the Nile River in Egypt and Sudan, in northern Ethiopia, and it is 270 the only Arvicanthis species confirmed from Yemen in the Arabian Peninsula. Besides these five 271 species, we distinguished four additional taxa; all of them are endemic to Ethiopia (Fig. 3). There are 272 two species living in Afroalpine grasslands in the highest mountains of Ethiopia: A. blicki in Bale and 273 Arsi Mts. (and Debre Sina, see below) and A. abyssinicus in Semien Mts and three additional highest 274 mountain ranges in north-eastern part of Ethiopian highlands. Monophyly of A. blicki is not 275 supported at CYTB, but the two species clearly differ at nuclear markers (see below). Two additional 276 Ethiopian clades are A. sp.For "Menangesha" Review (incorrectly called Only "abyssinicus" in previous genetic papers; 277 see Table 1 and Discussion) from four localities around Addis Ababa, and A. sp. "Metahara" from 278 lowland savannahs in the northern part of GRV (Fig. 3). 279 280 Mean uncorrected genetic distances at CYTB among the putative species (genetic clades) were > 8% 281 and in most cases even > 10% (Table 2). There were two exceptions for the pairs A. abyssinicus - A. 282 blicki (5.1±0.5%) and A. sp. "ANI-6" - A. ansorgei (6.9±0.6%). We also calculated p-distances in three 283 species with pronounced intraspecific structure, i.e. between three clades of A. niloticus "C2-C4" 284 (4.1-4.7%), three clades of A. nairobae (5.8-7.4%), and three clades of A. rufinus (5.1-5.8%). 285 286 Phylogeny based on nuclear markers 287 Bayesian analysis based on six concatenated nuclear loci provided relatively well-resolved tree, 288 clearly separating ANSORGEI and NILOTICUS groups (Fig. 4). All species identified by mtDNA within 289 the NILOTICUS group were supported by PP > 0.97 also at nuclear markers (and bootstrap support of 290 ML analysis > 86 in all but one species), but their mutual relationships were different from the ones 291 inferred from the mtDNA tree (compare topologies at Figs. 1 and 4). The most important difference 292 was in splitting the Afroalpine mitochondrial clade "blicki/abyssinicus" into two very distinct groups; 293 one of them (A. blicki) clustering individuals from Bale + Arsi Mts. (east of GRV) with those from 294 Debre Sina (west of GRV; Fig. 3), while the second included A. abyssinicus from the highest 295 mountains in northwestern part of Ethiopian highlands. Diagnostic indels in introns grouped together 296 (1) A. niloticus "C2-C4" with A sp. "Menangesha" and A. blicki, and (2) A. niloticus s. str. with A. sp. 297 "Metahara". It is also interesting that Tanzanian A. neumanni was sister to all seven remaining 298 species, which all were found in Ethiopia. 299

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300 However, the individual nuclear genes were not very informative, when analysed separately, and 301 individual gene trees provided slightly different topologies (Appendix S3), which can be the result of 302 incomplete lineage sorting and/or past admixture. We therefore calculated Bayesian species tree in 303 *BEAST (taking into account incomplete lineage sorting) and Bayesian species network (reflecting 304 both incomplete lineage sorting and admixture) (Fig. 5). The resolution of the output was rather 305 weak (reflecting low number of markers and their low variability), but some relationships were 306 consistently supported in both analyses. In contrast to the concatenated tree (Fig. 4), both species 307 tree and species network clustered A. blicki and A. abyssinicus, with the population from Debre Sina 308 being closer to A. abyssinicus. Their closest sister is A. sp. "Menangesha"; all three species have the 309 same NFa=64 (Fig. 1). Further, the sister relationship of A. niloticus s.str. and A. sp. "Metahara" was 310 repeatedly confirmed. OnFor the contrary, Review the phylogenetic positionOnly of A. niloticus "C2-C4", A. somalicus 311 and A. neumanni strongly depended on the used method of analysis and the nuclear markers were 312 unable to identify their sister clades. The sister relationship of A. neumanni and A. somalicus 313 (considered to be synonyms by some authors, e.g. Musser & Carleton, 2005) was not confirmed by 314 any of the used analyses and markers. 315 316 Divergence dating 317 The results of divergence dating are summarized at Fig. 6. The posterior median of TMRCA of all 318 species of Arvicanthis (4.2 Mya) is slightly younger than the prior is (4.45 Mya). The species tree 319 based on the combination of nuclear and mitochondrial data again supports two major Arvicanthis 320 group, but the topology of the NILOTICUS group is not well resolved. Most divergences leading to 321 current species diversity occurred simultaneously in early Pleistocene (1.5-2 Mya). The only 322 exception is the clade of Ethiopian Afroalpine endemics (0.4-0.7 Mya), but these estimates should be 323 taken with caution, as the evolution of Ethiopian taxa was likely affected by reticulation (see 324 Discussion), which is not assumed by multi-species coalescent model in StarBEAST. 325 326 Discussion 327 Multilocus phylogenetic analysis performed in this study identified two major genetic groups within 328 the genus (the NILOTICUS and ANSORGEI groups), thus confirming previous studies based on DNA 329 sequences (Dobigny et al., 2013) and karyotypes (Castiglia et al., 2006). Both groups are widely 330 distributed across sub-Saharan Africa and the species from these groups might occur in sympatry, 331 e.g. in the inner delta of the Niger River (Volobouev et al., 2002), northern Cameroon (Dobigny et al., 332 2011), or southern Ethiopia (this study). The delimitation of species within the two main groups, 333 however, is not yet satisfactorily done, especially in eastern Africa. 334

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335 Relatively low diversity and well-resolved taxonomy of grass rats in West Africa 336 The taxonomic situation of West African taxa is relatively clear thanks to intensive research in last 337 decades (e.g. Dobigny et al., 2011, 2013; Fadda & Corti, 2001; Volobouev et al., 2002; see Fig. 7). 338 Based on combined evidence from cytogenetics, mitochondrial sequences and geometric 339 morphometrics it was proposed that three species occupy open habitats of west-central Africa. There 340 is one species from the NILOTICUS group (A. niloticus "C2-C4" sensu Dobigny et al., 2013; ANI-1a and 341 ANI-1b cytotypes sensu Volobouev et al., 2002), widespread in dry savannahs of Sahelian and 342 northern Sudanian domains (Fig. 7A), and two species from the ANSORGEI group (Fig. 7B). The first 343 species A. ansorgei (= ANI-3 cytotype), with the holotype from Gunnal in Guinea Bissau (Volobouev 344 et al., 2002), occupies bush and woodland savannas of the Sudanian domain in southern Senegal, 345 Gambia, Guinea, Mali, Burkina-Faso,For Review northern Benin and south-westernOnly Niger (Fig. 7B based on 346 Denys et al., 2009; Dobigny, Nomao & Gautun, 2002; Fadda & Corti, 2001; Konečný, Koubek & Bryja, 347 2010; Volobouev et al., 2002). This species is parapatric with A. niloticus "C2-C4", with overlapping 348 populations in the inner delta of the Niger River in Mali (Volobouev et al., 2002). The only record east 349 of the Niger River was reported by Fadda & Corti (2001) from one locality in southern Chad, but it 350 was based on two specimens identified by a discriminant function from geometric morphometric 351 data and it should be taken with caution, awaiting genetic confirmation (not shown in Fig. 7B). The 352 second species is A. rufinus (with the karyotype ANI-4 and likely also ANI-2; see Castiglia et al., 2006; 353 Ducroz et al., 1998; and Dobigny et al., 2011 for taxonomic discussion), distributed in the most humid 354 zones of the Sudanian-Guinean region and confirmed (cyto-)genetically from Guinea (C. Denys et al., 355 pers. comm) through Benin and northern Cameroon to CAR (Dobigny et al., 2011; Volobouev et al., 356 2002). Based on the discriminant function derived from geometric morphometry of skulls, the 357 species was also reported from Sierra Leone, Ghana (where is also the type locality) and western 358 Nigeria (Fadda & Corti, 2001). This species exhibits intraspecific variation with two mitochondrial 359 lineages (both with ANI-2 cytotype) east of the Niger River and one lineage (ANI-4 cytotype) west of 360 the Niger River (Fig. 1; Dobigny et al., 2011). The study of possible reproductive barriers between the 361 two cytotypes (i.e. ANI-2 and ANI-4 sensu Volobouev et al., 2002) would require new sampling, 362 especially from Nigeria. 363 364 Species limits in the ANSORGEI group in East Africa 365 The taxonomic situation in eastern Africa is much more complicated and, unfortunately, the new 366 multilocus DNA data analysed in this paper sheds light on some groups only. The less speciose clade 367 ANSORGEI is represented in eastern Africa by two major groups that we propose to represent two 368 distinct species. The first one is A. nairobae, the taxon described from Nairobi in Kenya. Castiglia et 369 al. (2006) reported a very distinct karyotype (2n = 62, NFa = 78) from the type locality and they

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370 assigned it to this taxon. An identical karyotype has been found previously at two localities in north- 371 eastern Tanzania (Castiglia et al., 2003) and all these specimens form a strongly supported 372 monophyletic group at mitochondrial DNA (Fig. 1). Its sister subclade grouped individuals from 373 Uganda (Ducroz et al., 1998) and our new samples from western and southern Tanzania. The third, 374 newly reported, mitochondrial clade clusters four individuals from two localities in coastal southern 375 Kenya (Figs. 1 and 2B). The relationships of these three groups are not satisfactorily solved at 376 mitochondrial DNA (and we were not able to amplify the nuclear markers). However, considering the 377 geographic proximity and genetic divergences at CYTB, we propose them conspecific and suggest the 378 name A. nairobae for all of them. Unfortunately, the type material of A. nairobae was not included in 379 morphometric study of Fadda & Corti (2001), so it is difficult to identify, which morphogroup in their 380 study might represent thisFor species andReview what is the real extent Only of its distribution. However, the 381 distribution of their "A. sp1" significantly overlaps with genetically identified A. nairobae in our study 382 and it is the only Arvicanthis species in western Tanzania. It is also morphologically well distinct taxon 383 in East Africa, suggesting it belongs to the ANSORGEI group (Fadda & Corti, 2001). We therefore 384 assigned their "A. sp1" to A. nairobae at Fig. 7B. Awaiting further study, the species seems to be 385 distributed in miombo habitats along the Albertine rift and along eastern part of Kenya/Tanzania 386 border (with the type locality, Nairobi, laying at the margin of its distribution; Fig. 7B). 387 388 The second East African species from the ANSORGEI group is called here A. sp. "ANI-6", based on the 389 karyotype described by Corti et al. (2005) from Ethiopia (we included some of their specimens in our 390 phylogenetic analysis). This species very likely includes also the populations with the karyotype ANI- 391 6a (Orlov, Baskevich & Bulatova, 1992; see Castiglia et al., 2006). Based on mitochondrial DNA 392 analyses, it is a significantly supported sister taxon to West African A. ansorgei and its distribution is 393 limited to Ethiopian lowlands, especially in GRV (Fig. 2B). It has clear north-south intraspecific 394 structure, separating mtDNA haplotypes into two subgroups. A similar phylogeographic pattern was 395 found also in other rodents living in Ethiopian Somali-Masai savannahs, e.g. gerbils (Aghová et al., 396 2017) or spiny mice (Aghová et al., under review). Intraspecific divergence can be a result of a 397 vicariance event caused by the African rift lakes forming important barriers to gene flow, especially 398 during humid periods of Pleistocene (Trauth et al., 2010; see also discussion in Aghová et al., 2017). 399 400 Genus Arvicanthis in Ethiopian highlands 401 Ethiopian highlands are inhabited by Arvicanthis populations with two very divergent mitochondrial 402 haplogroups, called here A. sp. "Menangesha" and A. abyssinicus/blicki (Fig. 1). The concatenated 403 nuclear dataset clearly separated A. abyssinicus and A. blicki (Fig. 4), suggesting that three highland 404 species of Arvicanthis can occur in Ethiopia (Fig. 3). Arvicanthis abyssinicus was described from

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405 Semien Mts., so we assigned this name to the only genetic clade of the genus captured in these 406 mountains during several recent expeditions (Appendix S1). It is a high-elevation specialist, occupying 407 Afroalpine habitats at 3600-3900 m a.s.l. However, there are two reasons to cast doubts on the 408 assignment of this species name to these populations. First, the type locality (Entschetqab, Simien 409 Mts.) was reported from elevation about 3250 m a.s.l. ("ca. 10,000 French feet"; Rüppell, 1842), 410 which is slightly lower than known altitudinal range of this genetic clade (besides Semien Mts. the 411 species is reported from three additional highest mountain ranges west of GRV, always above 3600 412 m a.s.l.; Fig. 3). Second, the type material of A. abyssinicus is unfortunately damaged to the extent 413 that it cannot be used for any multivariate analysis of skull morphology (O. Mikula, pers. comm.; see 414 also Yalden, Largen & Kock, 1976). Arvicanthis blicki, morphologically very distinct species (Fadda & 415 Corti, 2001) was consideredFor to be distributedReview only in Bale andOnly Arsi Mts., east of GRV (Denys et al. 416 2017), but the concatenated nuclear tree suggests that it is distributed also in Debre Sina, west of 417 GRV (Figs. 3 and 4). It seems very likely that GRV was repeatedly crossed by highland specialists 418 during glacial periods, because similar biogeographic pattern, i.e. genetically very close populations 419 on both sides of GRV, was observed in other taxa of mountain rodents (Bryja et al., 2018; 420 Lavrenchenko, Verheyen, Verheyen, Hulselmans & Leirs, 2007) and frogs (Freilich, Tollis & Boissinot, 421 2014). Finally, A. sp. "Menangesha" is the species that we reported from relatively limited area in 422 central Ethiopia based on DNA genotyping and karyotypes (Fig. 3, Appendix S1). In recent literature, 423 this species was incorrectly reported under the name "A. abyssinicus" (Table 1). Most localities 424 reported by Yalden et al. (1976) and Yalden, Largen, Kock & Hillman (1996) as "A. abyssinicus" from 425 the elevation of 2000-3400 m a.s.l. from the western part of Ethiopian plateau very likely represent 426 A. sp. "Menangesha". It usually has a mid-dorsal dark stripe and rather dark agouti fur on the venter 427 (Dollman, 1911; Yalden et al., 1976; J. Bryja and R. Castiglia, pers. obs.), but the detailed phenotypic 428 analysis of genotyped individuals is required to evaluate this interpretation. 429 430 The evolutionary history of Arvicanthis in Ethiopian highlands, however, was probably complex and 431 there are indications of past reticulate processes. Species tree and species network based on nuclear 432 loci (Fig. 5) both suggest that all Ethiopian highland populations form a monophyletic group, where 433 A. abyssinicus is closely related to A. blicki from Debre Sina. Close relationship between A. blicki east 434 of GRV (Bale and Arsi Mts.) and A. sp. "Menangesha" west of GRV is documented by concatenated 435 nuclear phylogeny, shared insertion in DHCR gene (Fig. 4), allozymes (Capula, Civitelli, Corti, Bekele & 436 Capanna, 1997) and by the same inversion on chromosome 3 (Castiglia et al., 2006). On the other 437 hand, they are very distinctive for body size, pelage coloration, diploid chromosome number and 438 skull morphology (Castiglia et al., 2006; Fadda & Corti 2001; Yalden et al., 1976). Although we are 439 unable to resolve the phylogenetic relationships of Arvicanthis in Ethiopian highlands with the

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440 present dataset, we can propose an evolutionary scenario that could be tested in future work by 441 genomic approaches. This scenario must include: (1) the gene flow across GRV, probably during 442 glacial periods, and cross-breeding of ancestors of A. sp. "Menangesha" and A. blicki, which is 443 suggested by shared traits at DHCR gene and inversion on chromosome 3; (2) rapid karyotype 444 changes (Castiglia et al., 2006) and evolution of very distinct phenotype of A. blicki (Fadda & Corti, 445 2001) in Afroalpine conditions of the Bale/Arsi mountains, probably in response to strong 446 environmental selection (see similar examples e.g. in Šumbera et al., 2018; Kostin et al., 2018); (3) 447 hybridization of ancestors of A. blicki with the ancestors of A. abyssinicus, which resulted in the 448 (adaptive?) introgression of mitochondrial DNA of A. blicki into A. abyssinicus, potentially allowing A. 449 abyssinicus to colonize the highest mountains in north-western Ethiopian plateau. The population in 450 Debre Sina may representFor one of intermediate Review populations Only of hybrid origin between these two taxa, 451 and its newly reported karyotype (2n = 62, NFa = 64) suggest that the hybridization predated the 452 karyotypic changes of A. blicki in Bale and Arsi Mts. It is also possible that Ethiopian endemic 453 Arvicanthis sp. "Menangesha", able to live also at relatively low altitudes (see Yalden et al., 1976), 454 potentially hybridized with A. niloticus "C2-C4" in southwestern Ethiopia and adopted its DHCR gene 455 (Fig. 4) and mtDNA (Fig. 1), or vice versa. Albeit this scenario seems to be quite complicated, the 456 reticulate speciation processes are common in Ethiopian highlands, at least in rodents (Bryja et al., 457 2018; Kostin et al. 2018; Lavrenchenko et al., 2004) and amphibians (Freilich et al., 2014), i.e. groups, 458 where both nuclear and mitochondrial markers have been studied so far. For example, two distinct 459 Afroalpine species exist in Stenocephalemys (S. albocaudata and S. sp. "A") and their distribution is 460 the same as that of A. blicki and A. abyssinicus, respectively (compare Fig. 3 with Bryja et al., 2018). 461 Interestingly, the two Afroalpine Stenocephalemys species have very similar mtDNA, but are clearly 462 distinct at nuclear markers, which is also in agreement with the situation in Arvicanthis. 463 464 Diversity of the NILOTICUS group in Sudanian and Somali-Masai savannahs 465 There have been numerous confusions in using the name A. niloticus (see e.g. Musser & Carleton 466 2005 for a review). This species was described from Egypt and in the most comprehensive genetic 467 work of Dobigny et al. (2013) the name was used for widespread populations with ANI-1a and ANI-1b 468 cytotypes from Senegal to eastern Sudan, harbouring four distinct mitochondrial haplogroups C1-C4. 469 We revealed similar structure at mitochondrial DNA, when using more samples especially from 470 eastern Africa, but our nuclear markers show that the easternmost clade C1 is very distinct and is not 471 even a sister species to C2-C4 (in contrast, C2-C4 individuals form a well-supported monophyletic 472 group at nuclear markers despite large geographic distances among them; Fig. 4). This supports 473 previous views, e.g. based on skull morphology (Fadda & Corti, 2001), that populations living along 474 the Nile valley, in northern Ethiopia and Yemen represent the same species (see also taxonomic

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475 discussion in Musser & Carleton, 2005), here called A. niloticus s. str., distinct from Sudano-Sahelian 476 taxon, i.e. A. sp. "C2-C4" (sensu Dobigny et al., 2013). The samples from Egypt belong to the 477 mitochondrial clade C1 and the name "niloticus" should be therefore used for the species distributed 478 along the Nile River in Egypt and Sudan, and in northern Ethiopia and Yemen (Fig. 7A), with 479 frequently used name "dembeensis" being its younger synonym. The species A. niloticus "C2-C4", 480 widely distributed in the belt of Sudanian savanna from Senegal to southern Ethiopia and western 481 Kenya (Fig. 7A), should thus receive another name. It is important to note that Fadda & Corti (1998) 482 found conspicuous variation in skull morphology in the potential contact zone of C1 (i.e. A. niloticus s. 483 str.) and C2 (called "testicularis" in their work) in Sudan and South Sudan, where future studies 484 should investigate the extent of reproductive isolation between these two taxa and their phenotypic 485 differences. For Review Only 486 487 Within the NILOTICUS group, there are three distinct mitochondrial clades that potentially co-occur 488 in the central and northern part of GRV in Ethiopia - A. sp. "Metahara", A. somalicus, and A. niloticus 489 s. str. (Figs. 1 and 3). Also based on nuclear markers, they are clearly distinct, even if we did not 490 analyse the latter one from the central rift, but only from northern Ethiopia. In Ethiopia, A. niloticus s. 491 str. prefers higher altitudes (Koka and Dera Dilfekar in GRV, cca 1700 m a.s.l.), which is also true in 492 northern Ethiopia (up to 2700 m a.s.l.; Meheretu et al., 2012, 2014; J. Bryja et al., unpubl. data). Two 493 remaining species, however, are syntopic at low elevation (≤ 1000 m a.s.l.) in the southern part of the 494 Afar triangle, e.g. in the Awash National Park (Corbet & Yalden, 1972; Demeter, 1983; this study). 495 Arvicanthis somalicus, described from northern Somaliland (Dollman, 1911) is an arid-adapted 496 species with small body size and it is probably the only species of Arvicanthis living in dry conditions 497 of the Somali region in south-eastern Ethiopia, Somalia and eastern Kenya (Fig. 7A). The species has 498 been considered as a synonym of A. neumanni, another small-bodied species living in arid habitats of 499 the Masai steppe in Tanzania. Both nuclear and mitochondrial markers suggest that the two taxa are 500 genetically very distinct (Figs. 1, 4, 5), which agrees with earlier findings that they also differ 501 significantly in the shape of the skull (Fadda & Corti, 2001) and karyotype (Castiglia et al., 2006). 502 Hence, it seems they should be considered as two different species (see also suggestions in Musser & 503 Carleton, 2005 and Denys et al., 2017; for their distribution see Fig. 7A). All genetically identified 504 individuals of A. sp. "Metahara" were collected from the central and northern part of GRV (Fig. 3) and 505 this species was recently captured also near Dire Dawa further eastward (C. Denys, unpubl. data). It is 506 likely that previous records of larger Arvicanthis from this area (Corbet & Yalden, 1972; Demeter, 507 1983; some of them reported as A. dembeensis by Yalden et al., 1976) represent this species. Its 508 precise distributional limits and phylogenetic relationships with A. niloticus s. str. should be further

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509 investigated, but it can be anticipated that they are relatively close relatives as suggested by shared 510 insertion at SMO gene (Fig. 4) and sister relationship in the species tree (Fig. 5). 511 512 The mitochondrial lineage A. sp. "Masai-Mara" is the only one, where we were unable to sequence 513 nuclear markers and its specific status thus remains obscure. This lineage is only known from two 514 localities in the so-called Serengeti ecosystem, which hosts several endemic species of small 515 mammals, e.g. Mastomys pernanus (Denys et al., 2017). They represent relatively large-bodied 516 specimens and even if they are sister with A. neumanni based on mitochondrial DNA (Fig. 1), they 517 have different karyotype (ancestral ANI-1a; Ducroz, 1998). The name A. muansae Matschie, 1911, 518 described from Mwanza on the south coast of Lake Victoria), is a possible candidate for relatively 519 large individuals from thisFor region, butReview more detailed taxonomic Only study using new material is required 520 to solve this question. Fadda & Corti (2001) identified a morphologically distinct group of individuals 521 along the Lake Victoria (called "A. sp." in their paper) that might represent this species, but 522 unfortunately, none of these individuals was sequenced. They were morphologically most similar to 523 their "A. sp2" and "A. testicularis" that we included in A. niloticus "C2-C4" based on significant 524 overlap with the distribution ranges of our genotyped individuals (Fig. 7). The genetic and phenotypic 525 analyses of populations from northern Tanzania, western Kenya, Uganda, northern DRC and South 526 Sudan is now necessary to solve the remaining taxonomic issues in the NILOTICUS group. 527 528 Taxonomic implications 529 Using the combination of nuclear and mitochondrial DNA markers with available cytogenetic and 530 morphological data, we provide the most comprehensive description of the diversity of Arvicanthis in 531 sub-Saharan Africa (Fig. 7). Most recent compendia of rodent diversity (Denys et al., 2017; 532 Monadjem, Taylor, Denys & Cotterill, 2015) recognize seven Arvicanthis species. Our results show 533 that the species diversity within the genus is much higher and at least some of the revealed genetic 534 groups should receive a species name. We do not aim to perform a comprehensive integrative 535 taxonomic revision of the genus in this paper, as our study mainly used genetic data. For example, 536 we have insufficient information to interpret the taxonomic implications of our findings for A. 537 niloticus "C2-C4". The numerous Arvicanthis descriptions originate from Kenya and Uganda (see 538 Allen, 1939), from where the substantial morphological variability was reported (Fadda & Corti, 2001) 539 and where the taxon A. sp. "Masai Mara" could occur. It will be necessary to perform combined 540 genetic and morphological analysis from this region to answer the question of how many species 541 exist there and how they are related to A. niloticus "C2-C4" in central and western Africa. Similarly, 542 there are at least four descriptions from "Anglo-Egyptian Sudan" (Allen, 1939), i.e. current Sudan and 543 South Sudan, which is a region of potential contact zone of A. niloticus s. str. and A. niloticus "C2-C4".

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544 Detailed analysis of the type material and genomic analysis of potential contact zone, especially in 545 Sudan, is required to delimit correctly these taxa. 546 547 On the other hand, our sampling and genotyping allow to revise at least the suitable names for 548 Ethiopian species. Based on Allen (1939), following taxa were named from Ethiopia: 549 Mus abyssinicus Rüppel, 1842 - described from Entschetqab, Simien province, ca. 10,000 French feets 550 (3250 m a.s.l.). We use this name for the high-elevation taxon A. abyssinicus, recorded in 551 Afroalpine habitats in four highest mountains in north-western part of Ethiopian highlands (Fig. 552 3). It is the only species of Arvicanthis captured in Semien Mts. during our recent expeditions. 553 "Mus rufidorsalis Heuglin, 1877" from "grassy plain in Simien and Wogara" was synonymized with 554 A. abyssinicus (Allen,For 1939). The Review name "abyssinicus" has Only been frequently incorrectly used for 555 other Ethiopian taxa, especially A. sp. "Menangesha" (see below). 556 Arvicanthis abyssinicus blicki Frick, 1914 was described from South Chilalo Mts., Hora Mt., 9000 ft 557 (2743 m a.s.l.) and it represents another high-elevation specialist, A. blicki, which is also 558 morphologically very distinct (Fadda & Corti, 2001). It lives in Afroalpine habitats of Bale and Arsi 559 Mts., east of GRV, but another population (affected by hybridization with A. abyssinicus?) was 560 confirmed from Debre Sina, west of GRV (Fig. 3). The understanding of possible reticulate 561 evolution of highland Ethiopian taxa would require the use of genomic approaches. 562 Arvicanthis abyssinicus fluvicinctus Osgood, 1936 was described from Bichana, Gojjam (close to 563 Debre Marcos) and it was synonymized with "abyssinicus" in Musser & Carleton (2005), 564 corresponding to A. sp. "Menangesha" in our study. 565 Arvicanthis abyssinicus mearnsi Frick, 1914 was described from "Sadi Malka, Hawash River, 2800 ft 566 (853 m a.s.l.)", close to the current Awash NP, where two Arvicanthis species co-occur (A. sp. 567 "Metahara" and A. somalicus). The name is reported as synonym of "niloticus" in Musser & 568 Carleton (2005), but it is the most suitable name for A. sp. "Metahara", i.e. bigger lowland species 569 living (at least) in northern part of GRV and at the southern margin of the Afar triangle. The 570 hindfoot length in the type series is 27-29 mm, while all individuals of genotyped A. somalicus, i.e. 571 its sympatric taxon, in our collection had it shorter than 24.5 mm. 572 Arvicanthis abyssinicus raffertyi Frick, 1914 was described from Gardula, south of Arba Minch from 573 the elevation of 4000 ft (ca 1220 m a.s.l.). This is the area of sympatry between A. sp. "ANI-6" and 574 A. niloticus "C2-C4". Our unpublished data suggest that the two species differ by the length of 575 hindfoot (albeit overlapping), being significantly longer for A. niloticus "C2-C4". The 576 measurements provided for the type series of "raffertyi" correspond to A. sp. "ANI-6" (most of 577 them 27.0-28.5 mm, while frequently above 29.0 mm in A. niloticus "C2-C4"). Based on the 578 description, "raffertyi" also differs from A. abyssinicus zaphiri Dollman, 1911 (synonymized with A.

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579 niloticus, see below) by the lack of the broad brain-case (Frick, 1914). To sum up, we propose the 580 name A. raffertyi for A. sp. "ANI-6", which is genetically the most distinct among Ethiopian 581 Arvicanthis, but has been overlooked in previous studies (Fadda & Corti reported it as "A. sp.3", 582 morphologically similar to A. ansorgei, which correspond to genetics as well). It should be noted 583 that Yalden et al. (1976) mentioned that the altitude of the type locality (Gardula) has an 584 approximate elevation of 2500 m a.s.l. (and not 1200 m as reported by Frick, 1914), but at the 585 same time it is possible that the type material was collected in the lowlands "near Gardula". 586 Arvicanthis abyssinicus saturatus Dollman, 1911 from Didessa River near Guma is the oldest available 587 name for A. sp. "Menangesha", reported in most previous works (except the original description) 588 as A. abyssinicus. The description by Dollman (1911) perfectly fits this taxon, i.e. general colour 589 dark blackish, backs ofFor feet dark Review brown, toes black and Onlydorsal stripe (usually) well marked. In 590 addition, the type locality is not far from our genotyped material of this taxon (Fig. 3) and the 591 holotype was assigned to this taxon in morphometric analysis of skulls (Fadda & Corti, 2001). 592 Based on the analysis of 3D geometric morphometry (Fadda & Corti, 2001) the species is reported 593 from additional localities at 1600-2880 m a.s.l., mostly west of GRV. Occurrence at two localities 594 east of GRV should be confirmed genetically as the species identification is based only on 595 discriminant function. The name "fluvicinctus" is the younger synonym of this taxon (see above). 596 Arvicanthis abyssinicus zaphiri Dollman, 1911 was described from Guma, 2200 ft (650 m a.s.l.) and 597 was synonymized with A. niloticus by Musser & Carleton (2005). Based on the geographic location 598 of the type locality, it will rather belong to A. niloticus "C2-C4", even if the hindfoot (31 mm) is 599 relatively long and rather similar to A. niloticus s. str. (our unpubl. data). 600 Meriones lacernatus Rüppel, 1842 was described from grassy plains around Lake Dembea (= Lake 601 Tana). Allen (1939) mentioned "dembeensis Rüppel, 1842" and "pelliceus Thomas, 1928" 602 described from the same area as its synonyms and both "dembeensis" and "pelliceus" are 603 considered synonyms to A. niloticus by Musser & Carleton (2005) (even if they do not list 604 "lacernatus"). The genotyped material from around Lake Tana confirms that all these taxa very 605 likely belong to A. niloticus s. str. 606 Mus ochropus Heuglin, 1877 was described from the Bogos country, Abyssinia, which in fact now 607 belong to Eritrea (=Keren). The species is listed as synonym of A. niloticus by Musser and Carleton 608 (2005), and it geographically corresponds to A. niloticus s. str. 609 610 Based on this information, we propose that Ethiopia is inhabited by one species from the ANSORGEI 611 group (A. raffertyi = A. sp. "ANI-6") and seven species from the NILOTICUS group. Four of them were 612 described from Ethiopia and represent the endemic species for the country: A. abyssinicus, A. blicki, 613 A. saturatus (= A. sp. "Menangesha") and A. mearnsi (= A. sp. "Metahara"). Three additional

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614 Ethiopian species are more widespread (Fig. 7): A. niloticus (= A. niloticus s. str. described from 615 Egypt), A. niloticus "C2-C4" (the naming of this taxon requires additional taxonomic wok, see above) 616 and A. somalicus (described from Somaliland and widely distributed in Somali-Masai savannah). 617 618 Further cryptic diversity? 619 Even if we have provided a significant amount of new data illuminating the taxonomic and 620 evolutionary diversity of Arvicanthis, especially in Eastern Africa, there are still unanswered questions 621 concerning the systematics of the genus. Besides those highlighted in previous text (e.g. reticulate 622 evolution in Ethiopia or species limits of A. niloticus "C2-C4"), the previous cytogenetic research 623 suggested that diversity of Arvicanthis can be even higher than revealed by the DNA markers in our 624 study (see review in CastigliaFor et al., Review2006). Some populations Only with very distinct karyotypes were 625 included in our study. For example, the ANI-5 (sensu Castiglia et al., 2006) clearly represents an 626 example of chromosomal fusions within A. niloticus "C2-C4" (Fig. 1). The ANI-5 population was 627 described from the margin of the distribution range of this species, hence it is possible that peripatric 628 evolution in marginal populations has facilitated the fixation of the chromosomal mutations; see 629 similar examples e.g. in the rodent genera Rhabdomys (Castiglia et al., 2012) or Taterillus (Dobigny et 630 al., 2015). Two other reported karyotypes (ANI-7 and ANI-8 sensu Castiglia et al., 2006), are worth of 631 further work, which might reveal new (species) diversity in the ANSORGEI group. The ANI-7 (2n=56, 632 NFa=78 described from the Gambela area by Bulatova et al., 2002) originates from the westernmost 633 karyotyped locality in Ethiopia and may represent a missing link between A. sp. "ANI-6" and non- 634 Ethiopian taxa of the ANSORGEI group. Whether it represents just intraspecific variation within A. sp. 635 "ANI-6" (as suggested by morphological study of Fadda & Corti, 2006, who identified part of 636 specimens from Gambela as A. sp.3 = A. sp. "ANI-6"; see Table 1 and Fig. 7B) or it is a separate taxon 637 should be further researched. Similarly, the karyotype ANI-8 (2n=44, NF=72, NFa not known) 638 described by Capanna & Civitelli (1988) from Afgoi in Somalia stands that the genus is prone to 639 karyotypic divergence. In this case, the divergence seems relatively huge and may result in 640 reproductive isolation between the Afgoi population and geographically closest taxa A. sp. "ANI-6" or 641 A. nairobae. Considering the cradle of the genus in East Africa, the occurrence of yet undetected 642 species in poorly sampled areas is definitely possible. 643 644 Acknowledgements 645 This study was supported by the Czech Science Foundation (project no. 18-17398S) and the Russian 646 Foundation for Basic Research (project no. 18-04-00563-a). Samples utilised in the study have been 647 lawfully acquired and were collected prior to The Nagoya Protocol on Access to Genetic Resources 648 and the Fair and Equitable Sharing of Benefits Arising from Their Utilization to the Convention on

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649 Biological Diversity has been in effect. We are indebted to many local authorities for providing 650 permits to carry out the research, especially the Ethiopian Wildlife Conservation Authority (EWCA), 651 Government of Ethiopia and the Oromia Forest and Wildlife Enterprise (OFWE) in Ethiopia, the 652 Sokoine University of Agriculture in Morogoro (Tanzania), the Kenyan Forest Service and the Kenyan 653 Wildlife Service (Kenya). For help during the field work and providing additional samples we 654 acknowledge E. Capanna, A. Bekele, N Oguge, R. Makundi, C. Denys, P. Benda, S. Gryssels, J. Šklíba, T. 655 Vlasatá, E. Hrouzková, S. Šafarčíková, M. Lövy, O. Mikula, A. Konečný, A. Hánová, L. Cuypers, A. Ribas, 656 J. Krásová, J. Sádlová, D. Frynta, J. Votýpka, W. T. Stanley, E. C. Craig, C. Sabuni, A. Katakweba, A. 657 Massawe, K. Welegerima, A.A. Warshavsky, Yu. F. Ivlev, D. Yu. Alexandrov, D. S. Kostin, A. A. 658 Martynov, A. R. Gromov, Z. Tomass and all local collaborators. For help with genotyping we 659 acknowledge T. Aghová Forand A. Hánová. Review Constructive comments Only during the data analysis and writing 660 were provided by O. Mikula and L. Granjon. 661 662 Authors' Contributions 663 JB, RS, PC, EV, HL, RC conceived and designed the study; JB, LAL, YM, RS, RC collected important part 664 of samples; AB, YM performed laboratory analysis; JB analysed data; JB wrote the first version of the 665 manuscript. All authors contributed to the final version of the paper. All authors read and approved 666 the final manuscript. 667 668

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For Review Only

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882 Figure legends 883 Fig. 1 Mitochondrial BI tree of Arvicanthis based on the alignment of 1140 bp of CYTB. The colour of 884 squares indicate posterior probability from MrBayes, circles indicate statistical support from 1000 885 bootstraps in ML analysis (see legend for range of values represented by each colour). Only one 886 outgroup is shown. The colour bars indicate separate species discussed in the paper. Information 887 about karyotypes, where available, was placed on the tree. 888 889 Fig. 2 Geographical distribution of genetic lineages within the two major Arvicanthis groups. (A) 890 NILOTICUS group; (B) ANSORGEI group. Different species are marked by different symbols, whose 891 colours and names correspond to Fig. 1. The symbols grouped by dashed lines belong to separate 892 subclades within the species.For The distributionReview of western AfricanOnly taxa is not shown, i.e. for A. 893 niloticus "C2-C4" only the easternmost C2 clade is shown. 894 895 Fig. 3 Genetic diversity of the NILOTICUS group in Ethiopia. Different species are marked by different 896 symbols, whose colours and names correspond to Fig. 1. The dashed lines indicate three separate 897 subclades of A. niloticus s. str. The type localities of four taxa described from Ethiopia and proposed 898 as Ethiopian endemic species are shown by arrows and corresponding names (see Taxonomic 899 implications). 900 901 Fig. 4 Nuclear BI tree based on partitioned analysis of concatenated dataset of DNA sequences of two 902 exons and four introns. Parsimony informative indels were also included and encoded as 903 morphological characters. The numbers above branches indicate posterior probabilities estimated in 904 MrBayes/bootstrap support in ML tree produced by partitioned analysis of the same dataset in 905 RAxML (without indels). "-" means that topology was different in ML tree. Coloured squares indicate 906 informative indels in particular introns: orange = 1 bp insertion in DHCR, blue = 3 bp insertion in 907 SMO, light green = 3 bp insertion in WLS, pink = 2 bp deletion in DHCR, yellow = 8 bp insertion in 908 DHCR + 38 bp deletion in TRPV, dark green = 22 bp deletion in TRPV + 1 bp insertion in SMO. 909 910 Fig. 5 (A) Species tree of the NILOTICUS group reconstructed by multi-species coalescent approach in 911 *BEAST (Helled and Drummond 2010). The numbers above branches indicate posterior probabilities 912 of particular clades. (B) The most probable Bayesian species network calculated in BEAST2 from 913 nuclear multilocus data using the approach of Zhang et al. (2018). It is one of the 444 networks and 914 its posterior probability is 0.02. We therefore summarized the topologies of 10 networks with highest 915 posterior probabilities (in sum they had PP = 0.128) and the numbers above branches indicate in how

Journal of Zoological Systematics and Evolutionary Research Page 30 of 40

916 many of them the same group of species was clustered. The taxon blicki "DS" represent the high 917 elevation population from Debre Sina, west of GRV (see Fig. 3). 918 919 Fig. 6 Divergence dating of the species tree inferred using a multi-species coalescent approach and 920 the combination of both mitochondrial and nuclear markers in StarBEAST 2. The numbers in circles 921 are medians of TMRCAs of particular clades (with 95% HPD indicated by red bars). PP = posterior 922 probability for each clade; note that the topology of the NILOTICUS group is not resolved, indicating 923 rapid radiation around 1.5-2 Mya. 924 925 Fig. 7 Distribution of Arvicanthis taxa in sub-Saharan Africa based on DNA sequences (Ducroz et al. 926 1998, Abdel Rahman AhmedFor et al. 2008,Review Dobigny et al. 2011, Only 2013, this study), cytogenetics 927 (Volobouev et al. 2002, Castiglia et al. 2006) and geometric morphometrics (Fadda and Corti 2001). 928 Particular species include localities of taxa as specified in Table 1. The proposed names for Ethiopian 929 species are shown in the legend. See text for more details. 930 931 932

Page 31 of 40 Journal of Zoological Systematics and Evolutionary Research

933 List of Supporting Information 934 935 Appendix S1 List of used specimens with details on their karyotypes, DNA sequences and localities. 936 937 Appendix S2 Additional details about the used methods (primers and PCR conditions, list of outgroup 938 sequences and used substitution models. 939 940 Appendix S3 Bayesian gene trees of six nuclear markers. 941 For Review Only

Page 32 of 40

Type locality Gunnal, Gunnal, Gardula, Entschetqab, Mts., Chilalo Egypt - Guinea- Bissau Ethiopia Mts., Simien Ethiopia Ethiopia Elmina, Elmina, Ghana Nairobi, Nairobi, Kenya

Burungo, É. (

Frick,

Proposed Proposed name ansorgei raffertyi abyssinicus blicki niloticus unresolved Thomas, Thomas, 1910 1914 Frick, (Rüppel, 1842) 1914 Geoffroy, 1803) rufinus (Temminck, 1853) nairobae J.A.Allen, 1909 neumanni

Dobigny et (2013) al. ansorgei - - - C1 niloticus niloticus cf. ANI-2, cf. ANI-2, rufinus - -

Dobigny et (2011) al. - - - - niloticus - rufinus rufinus - -

rufinus rufinus ANI-4, ANI-2, Abdel Abdel Rahman etAhmed (2008) al. ansorgei - - - dembeensi, - niloticus niloticus -

6, ANI 6, -

Castiglia et (2006) al. - ANI - blicki niloticus niloticus 6a 6a nairobae nairobae - neumanni neumanni neumanni somalicus neumanni 31

3 -

Volobouev Volobouev et al. (2002) ANI ------

3 -

Fadda & Corti (2001) ANI sp3 - blicki niloticus sp.(?)

Journal of Zoological Systematics and Evolutionary Research

Ducroz et et Ducroz (2001) al. sp 3 sp - - - niloticus - For Review 2 sp sp1 Only

7 - used in previous studies and their correspondence with the names used in this study. inthis used names the with correspondence their and studies in previous used

sp1 - - - dembeensis, - Ducroz etDucroz (1998) al. sp nairobae, 11 sp8-10 1 sp ANI-4 ANI-2, ANI-4 ANI-2, - niloticus somalicus neumanni sp4

Arvicanthis

6" str. s.

Species Species ansorgei nairobae "ANI sp. rufinus abyssinicus blicki niloticus Mara" "Masai sp. neumanni (this study) study) (this

Species names of of names Species

Group ANSORGEI NILOTICUS

Tables 1 Table 942 943 944

Shuk, Didessa Malka, Sadi - - Northern Northern Somaliland River, Ethiopia Ethiopia Irangi, Irangi, Tanzania

somalicus saturatus mearnsi unresolved unresolved (Matschie, (Matschie, 1894) Thomas, 1903 Dollman, 1911 1914 Frick, unresolved unresolved -

C3, C4 C3,C4 - abyssinicus - - -

- - - - -

- abyssinicus - - - niloticus niloticus niloticus C2, niloticus

7 8 - - somalicus, cf. somalicus, abyssinicus niloticus ANI ANI somalicus somalicus (Gambella, Ethiopia) (Somalia) niloticus, niloticus, ANI-5 32

ANI-1a, ANI- ANI-1a, 1b - - - - -

sp2, testicularis somalicus abyssinicus - - - ANI-1, sp. (?), sp.(?), ANI-1,

us c Journal of Zoological Systematics and Evolutionary Research

- abyssini For - Review- - Only

- abyssinicus - - - niloticus niloticus -

C4" -

"C2

7 8 - - niloticus niloticus somalicus sp. "Metahara" sp. ANI ANI "Menangesha" "Menangesha"

not sequenced

945 946 947 Page 33 of 40 Page 34 of 40 0.113 0.113 - 0.094 0.094 0.125 - 0.0074 ansorgei ansorgei nairobae rufinus sp. sp. 0.069 0.069 0.081 - 0.117 0.0064 0.0079 "ANI-6" "ANI-6" 0.137 0.137 0.141 - 0.141 0.131 0.0060 0.0061 0.0081 0.051 0.051 - 0.0082 0.0087 0.0082 0.0082 abyssinicus abyssinicus blicki sp. sp. 0.105 0.105 0.104 - 0.0050 0.0089 0.0095 0.0085 0.0086 Mara" Mara" "Masai "Masai 33 0.091 0.091 0.105 - 0.105 0.0082 0.0075 0.0088 0.0090 0.0079 0.0081 -distances in MEGA using the same dataset as for mitochondrial phylogeny, i.e. phylogeny, formitochondrial as dataset same the using inMEGA -distances p 0.105 0.105 0.097 0.100 - 0.103 0.0074 0.0084 0.0074 0.0089 0.0089 0.0086 0.0080 somalicus somalicus neumanni - 0.0082 0.0081 0.0084 0.0088 0.0082 0.0091 0.0088 0.0085 0.0083 sp. sp. 0.100 0.100 0.108 0.103 - 0.111 0.116 0.0077 0.0076 0.0085 0.0080 0.0094 0.0094 0.0086 0.0090 0.0083 0.0083 0.0079 0.0077 0.0076 0.0085 0.0075 0.0090 0.0092 0.0085 0.0087 "Metahara" "Metahara" Journal of Zoological Systematics and Evolutionary Research For Review Only sp. sp. 0.109 0.109 0.101 0.099 0.102 0.116 0.115 "Menangesha" "Menangesha"

s. str. s. str. 0.086 0.086 0.114 0.107 - 0.105 0.103 0.106 0.111 niloticus

0.132 0.132 0.131 0.139 0.137 0.137 0.133 0.123 0.132 0.132 0.132 0.135 0.144 0.137 0.142 0.147 0.135 0.143 0.140 0.140 0.133 0.147 0.143 0.135 0.141 0.129 0.140 0.085 0.085 0.096 - 0.101 0.101 0.106 0.0073 0.095 0.097 0.0088 0.101 0.0088 0.0078 0.0079 0.0081 0.0075 0.0080 0.0082 0.0084 0.0087 niloticus "C2-C4" "C2-C4" Mean genetic distances between groups calculated as uncorrected uncorrected as calculated groups between distances genetic Mean "C2-C4" "C2-C4" str. s. - 0.0066 0.0071 0.0080 0.0075 0.0076 0.0074 0.0082 0.0074 0.0085 0.0088 0.0081 0.0080 sp. "ANI-6" "ANI-6" sp. 0.137 0.127 0.142 0.144 0.141 0.145 0.133 0.136 rufinus rufinus ansorgei ansorgei abyssinicus abyssinicus sp. "Menangesha" "Menangesha" sp. neumanni neumanni nairobae nairobae blicki blicki somalicus somalicus sp. "Masai Mara" Mara" "Masai sp. Species Species niloticus niloticus sp. "Metahara" "Metahara" sp. 951 Table 2 Table 121 sequences. Standard error estimates are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). Shaded cells indicate indicate cells Shaded (1000replicates). procedure bootstrap aby obtained were and the diagonal above shown are estimates error Standard sequences. 121 genetic distances within the NILOTICUS and ANSORGEI groups, respectively. respectively. groups, ANSORGEI and NILOTICUS the within distances genetic 952 950 948 949 niloticusC3_KF478412_CBGP_KB2816_DarouWolof_SEN Page 35 of 40 Journal of ZoologicalniloticusC3_KF478350_CBGP_M5639_San_MAL Systematics and Evolutionary Research ● niloticusC3_KF478357_CBGP_M4934_SareMama_MAL niloticusC3_KF478396_CBGP_JMD441_Gouniang_SEN RAxML niloticusC3_KF478345_CBGP_M4065_Makana_MAL ● niloticusC4_KF478386_CBGP_N4251_Boumba_NIG niloticusC4_KF478366_CBGP_M5295_Tiloa_NIG

<50 niloticusC4_KF478376_CBGP_N4147_Gaya_NIG niloticus "C2-C4" 50 − 75 75 − 90 90 − 100 ● niloticusC4_KF478367_CBGP_N4078_Chetimari_NIG ANI-1a: 2n=62, NFa=62 (C2,C4) <0.80 ● ● ● niloticusC4_KF478336_MNHN_M_VV1999_049_Tarabarat_MAL ANI-1b: 2n=62, NFa=64 (C3,C4) ● ● ● ● niloticusC2_KF478326_CBGP_N3085_ZakoumaNP_CHA 0.80−0.90 ● niloticusC2_KF478329_MNHN_M_2000_061_GozDjerat_CHA 2n=62, NFa=60 (Gambela, ETH; Bulatova et al. 2002) 0.90−0.95 ● ● ● ● niloticusC2_KF478323_CBGP_C353_Maga_CAM ANI-5: 2n=56, NFa=62 (C2-Kenya) niloticusC2_KF478330_MNHN_M_VV1998_061_Farcha_CHA MrBayes 0.95−1.00 ● ● ● ● niloticusC2_5309_Gisenyi_RWA niloticusC2_KE119_Kitale_KEN niloticusC2_NKR28632_Mumias_KEN ● niloticusC2_KE147_Rongai_KEN niloticusC2_KE907_Nanyuki_KEN ANI-5 niloticusC2_ETH291_MagoNP_ETH niloticusC2_LAV2026_ChamoLake_ETH niloticusC2_ETH245_NechisarNP_ETH niloticusC2_ETH877_Yabelo_ETH ● dembeensis_ETH515_Desea_ETH dembeensis_ETH1223_AlamataAbergelle_ETH ● dembeensis_AF004568_ET37_Koka_ETH ● dembeensis_LAV2213_DeraDilfekar_ETH dembeensis_ETH1234_AlamataLate_ETH dembeensis_pb3657_AshShukayrah_YEM dembeensis_KF478332_MNHN_M_VV1995_073_BreedingColony_EGY dembeensis_EF128062_Dongola_SUD niloticus s. str. ● dembeensis_LAV1797_AlatishNP_ETH dembeensis_EF128065_Shandi_SUD ANI-1a: 2n=62, NFa=62 (Koka, ETH) dembeensis_LAV1153_Vanzaye_ETH dembeensis_LAV1270_BahrDar_ETH dembeensis_KF478426_CBGP_DM8993_ElSabagola_SUD ● ● dembeensis_EF128081_Medani_SUD dembeensis_KF478424_CBGP_DM9103_ElSuki_SUD dembeensis_EF128074_Khartoum_SUD abyssinicus_AF004566_Sululta_ETH sp. "Menangesha" ● abyssinicus_AF004567_Menangesha_ETH 2n=62, NFa=64 (as "abyssinicus") abyssinicus_ETH0027_Menangesha_ETH metahara_ET110_Zeway metahara_ET117_Zeway_ETH sp. "Metahara" ● metahara_LAV2092_AdamiTulu_ETH metahara_Et182_Abaura_ETH ANI-1a: 2n=62, NFa=62 metahara_Et195_Qobo_ETH (Zeway, ETH, and Awash NP, ETH) somalicus_EU349737_Arvicanthis_somalicus_voucher_H894_Rowe2008_noLocality somalicus_KE115_MarsabitNP_KEN somalicus_ETH1068_GeralleNP_ETH somalicus_KE879_MeruNP_KEN somalicus_ETH1046_ElKere_ETH ● somalicus ● somalicus_ETH1055_Gode_ETHFor Review Only 2n=62, NFa=62-63 (Awash NP, ETH) somalicus_ETH1060_Imi_ETH somalicus_ETH0032_AwashNP_ETH somalicus_ETH059_DireDawa_ETH neumanni_TZ27309_Berega_TAN neumanni_AF004573_Arvicanthis_somalicus_Ducroz1998_Berega_TAN neumanni_TZ27338_Chakwale_TAN neumanni_AF004574_Arvicanthis_somalicus_Ducroz1998_Berega_TAN neumanni_CTZ979_Ihanda_TAN neumanni_50145b_Ndale_TAN neumanni_TZ57_Matongolo_TAN neumanni neumanni_TZ25_Zoissa_TAN 2n=53-54, NFa=62 neumanni_9290b_Msembe_TAN ● ● neumanni_2992b_Tagam_TAN neumanni_9325b_Mwagusi_TAN 1 neumanni_9349b_Mweyembe_TAN ● neumanni_220c_Itigi_TAN neumanni_TZ34_Singida_TAN neumanni_2999b_Mbarali_TAN muansae_7282_Gerodom_TAN ● sp. "Masai Mara" muansae_KF478314_MNHN_M_VV1996_009_MasaiMaraNP_KEN 2n=62, NFa=62 (Ducroz 1998, PhD thesis) blicki_LAV1112_MtGuna_ETH abyssinicus ● blicki_ETH1140_AboyeGara_ETH 2n=62, NFa=64 (Mt. Guna) ● blicki_ETH0620_MtChoqa_ETH blicki_LAV1920_DebreSina_ETH ● blicki_LAV1921_DebreSina_ETH ● blicki_ET11_BaleSinburru_ETH blicki blicki_LAV2402_BaleSanneti_ETH 2n=48, NFa=64 (Bale Mts.) ● blicki_ET25_BaleSinburru_ETH 2n=62, NFa=64 (Debre Sina, this study) NILOTICUS group blicki_ETH0951_BaleSodota_ETH blicki_ET10_BaleSinburru_ETH ANI6_ETH0049_Babile_ETH ANSORGEI group ● ANI6_Et200_Ile_Harar_ETH ANI6_Getachew2616_Alamata_ETH sp. "ANI-6" ● ● ANI6_ET129_Zeway_ETH ANI6_ETH0272_NechisarNP_ETH ANI-6: 2n=60, NFa=72 ● ANI6_ETH0290_MagoNP_ETH ANI-6a: 2n=60, NFa=76 ANI6_ET147_Derito_Yabello_ETH ● ANI6_310Y_NechisarNP_ETH ansorgei_AF004581_Ouagadougou_BUR ansorgei_KF478311_CBGP_M5619_Seniena_MAL ● ansorgei_AF004580_Samaya_MAL ansorgei ● ansorgei_AF004579_Bankoumana_MAL ANI-3: 2n=62, NFa=74-76 ansorgei_AF004577_Kedougou_SEN ● ansorgei_AF004578_Saraya_SEN nairobae_AF004585_QueenMiles_UGA ● nairobae_2998b_Mbarali_TAN nairobae_TA267_Kibondo_TAN ● nairobae_6072_Manolo_TAN nairobae_KE145_Nairobi_KEN ● nairobae_7631_Viti_TAN ● nairobae_8205b_Emau_TAN only this clade karyotyped nairobae_7633_Viti_TAN nairobae nairobae_281_Manolo_TAN 2n=62, NFa=78 nairobae_50233_Lwami_TAN nairobae_AN3_TiwiBeach_KEN ● nairobae_AN2_TiwiBeach_KEN ● nairobae_AN5_MidaCreek_KEN nairobae_AN4_TiwiBeach_KEN rufinus_AF004584_CAR rufinus_HM635841_C243_Zera_CAM ● ANI-2 ● rufinus_HM635840_C239_Zera_CAM rufinus_HM635839_C332_Gamnaga_CAM rufinus_AF004582_Lokossa_BEN ● rufinus rufinus_B57_Lokossa_BEN ANI-2: 2n=58, NFa=72 rufinus_B47_Setto_BEN rufinus_B69_Atcheribe_BEN ANI-4 ANI-4: 2n=62, NFa=74 ● rufinus_AF004583_Tanougou_BEN rufinus_JX292886_Schwan2012_LZP_2009_221_NTessoni_MAL AF533116_Rhabdomys_pumilio

0.05 Journal of Zoological Systematics and Evolutionary Research Page 36 of 40

(A) NILOTICUS group (B) ANSORGEI group For Review Only

niloticus „C2-C4“ niloticus s. str. sp. „Menangesha“ sp. „Metahara“ ANI-5 somalicus neumanni nairobae sp. „Masai Mara“ sp. „ANI-6“ abyssinicus rufinus blicki

Fig. 2 Page 37 of 40 Journal of Zoological Systematics and Evolutionary Research Semien Mts. (abyssinicus)

niloticus „C2-C4“ niloticus s. str. sp. „Menangesha“ sp. „Metahara“ For ReviewSadi Malka Onlysomalicus abyssinicus (mearnsi) blicki Debre Sina Guma (saturatus) Arsi Mts. (blicki)

Fig. 3 ETH0245_Nechisar NP_ETH Journal of Zoological Systematics and Evolutionary Research Page 38 of 40 1.00/63 ETH0291_Mago NP_ETH KE907_Nanyuki_KEN niloticus "C2-C4" 0.57/- M4065_Makana_MAL M5295_Tiloa_NIG 0.99/- 1.00/100 ETH0027_Menangesha_ETH sp. "Menangesha" ETH0028_Menangesha_ETH ETH0951_BaleSodota_ETH

0.97/38 LAV2401_BaleSanetti_ETH LAV2402_BaleSanetti_ETH blicki 0.99/95 LAV1920_DebreSina_ETH LAV1921_DebreSina_ETH For Review OnlyETH0515_Desea_ETH ETH1223_Alamata_ETH 1.00/- 1.00/98 ETH0516_Desea_ETH niloticus s.str. 1.00/- LAV2213_DeraDilfekar_ETH LAV1797_AlatishNP_ETH 1.00/100 Et182_Abaura_ETH sp. "Metahara" 0.93/62 LAV2092_AdamiTulu_ETH ETH0032_AwashNP_ETH

1.00/96 KE115_MarsabitNP_KEN ETH0059_DireDawa_ETH somalicus 1.00/100 ETH1068_GeralleNP_ETH 1.00/49 ETH1055_Gode_ETH KE879_MeruNP_KEN ETH0620_MtChoqa_ETH 1.00/86 ETH0623_MtChoqa ETH0624_MtChoqa abyssinicus LAV1112_MtGuna_ETH ETH1140_AboyeGara_ETH 0.99/100 TZ27309_Berega_TAN neumanni TZ27338_Chakwale_TAN 1.00/85 ETH0272_NechisarNP_ETH sp. "ANI-6" 1.00/100 ETH0049_Babile_ETH TA267_Kibondo_TAN nairobae

0.002 Page 39 of 40 Journal of Zoological Systematics and Evolutionary Research (A) Species tree from *BEAST (B) Species network

niloticus "C2-C4"

2 of 10

0.262 sp. "Metahara"

0.667 8 of 10

0.413 niloticus s. str. For Review Only somalicus

1 of 10 neumanni

0.497 6 of 10 sp. "Menangesha"

0.488 8 of 10 blicki

0.858 10 of 10

abyssinicus 0.884 0.8848 of 10

blicki "DS"

3.0E-4 Journal of Zoological Systematics and Evolutionary Research nairobaePage 40 of 40 1.2 sp. "ANI-6"

somalicus

1.8 neumanni 4.2 PP: For Review Only 1.5 abyssinicus < 0.50 0.4 ≥ 0.50 blicki "Debre Sina" ≥ 0.80 0.7 ≥ 0.90 2.0 blicki ≥ 0.95

niloticus "C2-C4"

1.7 sp."Metahara" 1.4 1.8 niloticus s.str.

sp. "Menangesha" Upper Middle Gelasian Zanclean Calabrian Piacenzian

Pliocene Pleistocene Holocene Neogene Quat. 5 4 3 2 1 0 Mya Page 41 of 40 Journal of Zoological Systematics and Evolutionary Research (A) NILOTICUS group

sp. (= niloticus „C2-C4“) niloticus (= niloticus s. str.) saturatus (sp. „Menangesha“) mearnsi (= sp. „Metahara“) somalicus neumanni For Review Only sp. (= sp. „Masai Mara“) abyssinicus blicki

(B) ANSORGEI group

nairobae raffertyi (= sp. „ANI-6“) rufinus ansorgei

Fig. 7