3.1. Phylogenetic relationships within the genus Lophuromys
The deeper phylogenetic relations in the genus Lophuromys, including the validity of the subgeneric divisions (Lophuromys and Kivumys) and older lineages (Kivumys group and L. sikapusi group), and their respective monophyly have been relatively uncontested in recent checklists [5, 6, 22]. In contrast, species accounts in the ‘speckled pelage’ groups, the L. flavopunctatus group, combining the Ethiopian endemics (herein as Ethiopian L. flavopunctatus members – ETHFLAVO) and the non-Ethiopian ones (herein as non-Ethiopian L. flavopunctatus members – NONETHFLAVO), have changed rapidly recently. In consensus, our genus-wide phylogenetic inference based on the CYTB gene supports the deep divergence of the genus Lophuromys into three distinct deeply-diverged groups that correspond with the widely recognized species groupings; i) Kivumys group (Kivumys subgenus), ii) L. sikapusi group (Lophuromys subgenus), and iii) L. flavopunctatus group (Lophuromys subgenus). The two Lophuromys groups – L. sikapusi group and L. flavopunctatus group – are separated by a much lower mtDNA divergence (p-distance) compared to the almost equidistant p-distance separating them from the Kivumys group. Based on the CYTB divergence, the Kivumys group appear distinct enough to be classified in a distinct genus, with the L. sikapusi group and L. flavopunctatus group also distinct enough to be included in separate subgeneric groups. However, testing these conjectures hinges on broader nuclear loci sampling and morphological characterizations across the genus. Overall, these findings broadly agree with the current classification of species into the Kivumys group . However, we importantly highlight that classifying species into the L. sikapusi and L. flavopunctatus groups based on morphological characterization is essentially ambiguous unless backed up by genetic evidence.
Within the Kivumys group (subgenus Kivumys), high CYTB differentiation (13.39% p-distance) between the two species represented in our dataset – L. woosnami and L. luteogaster – clearly delimitate them as distinct lineages. Together with L. medicaudatus, whose CYTB sequences were not included in the study, all three species in the Kivumys subgenus have been recorded from overlapping ranges, i.e., in the northeastern and eastern DRC forests and bordering montane forests of the Albertine Rift, with L. woosnami extending into western Burundi, Rwanda, and Uganda [5, 6, 22, 35, 38]. A thorough investigation of niche partitioning and other ecomorphological strategies inherent in gene flow and adaptive genetic divergence within the Kivumys subgenus is necessary to clear up their evolutionary history. Such a study would also illuminate the precise nature and limits of their ranges (whether sympatric, syntopic, or parapatric).
The eight clades in the L. sikapusi group correspond to seven described species (L. angolensis, L. ansorgei, L. huttereri, L. nudicaudus, L. rahmi, L. roseveari, and L. sikapusi) and the unidentified taxon (L. sp.1 in Fig. 2). The L. sp.1 clade is separated by 10.47-13.85% p-distance from all other species in the L. sikapusi group and forms a sister relationship with L. sikapusi (separated by 11.42% p-distance), representing a potentially undescribed species. While describing rodents of western and southwestern Guinea, Denys et al.  considered their Lophuromys samples from Mankountan, Boffa prefecture, Guinea, as a tentative new species in the sikapusi group due to its unique head and body length, pending morphometric and genetic evidence for a taxonomic description. Our CYTB evidence suggests that the Lophuromys from the Conakry region of Guinea might belong to their undescribed taxa. Still, without more the morphometric evidence to confirm its morphological relationships in the sikapusi group and range limits, we retain a similar provisional species status for L. sp.1.
The utilization of pelage coloration to resolve the systematic grouping of species is rather debatable in the genus Lophuromys. For instance, the inclusion of L. dieterleni (Mt Oku, Cameroon) and L. eisentrauti (Mt Lefo, Cameroon) in the L. flavopunctatus group by Musser and Carleton  (citing Verheyen et al. ) is quite doubtful. Our CYTB tree shows all unspeckled-pelage taxa clusters in the L. sikapusi group (L. angolensis, L. ansorgei, L. huttereri, L. nudicaudus, L. rahmi, L. roseveari, L. sikapusi, and L. sp.1), well distinct from the speckled-pelage L. flavopunctatus group. From an ecomorphological outlook, the craniodental relationship between L. dieterleni and L. eisentrauti and the L. flavopunctatus group [25, 26] might simply be signals of convergent adaptive responses to local environments [40, 41], which are taxonomically uninformative without genetic evidence. Then again, the genetic and craniodental affinity of the unspeckled L. pseudosikapusi to the Ethiopian endemics  confounds further the overall phylogenetic relationships within and between the speckled-pelage (L. flavopunctatus group) and unspeckled-pelage (L. sikapusi group) species. From our findings, assigning clades to either the L. sikapusi group or the L. flavopunctatus group is unambiguous based on genetic relationships, and future genetic studies are likely to resolve the L. dieterleni and L. eisentrauti membership.
The assignment of ETHFLAVO clades to corresponding species is a nontrivial task due to the recently clarified taxonomic accounts of the Ethiopian Lophuromys [15-17]. For example, the pairs of highly divergent CYTB clades of L. simensis (L. simensis 1 and L. simensis 2) and L. melanonyx (L. melanonyx 1 and L. melanonyx 2) comprise the multiple haplogroups within the same species due to past mtDNA introgression events. Such introgressions have also been confirmed in L. brunneus, of which we only sampled one haplogroup, summing up to the 12 mtDNA Lophuromys lineages endemic to Ethiopia. Nuclear genomic data support these 12 lineages represent nine species (L. chrysopus, L. melanonyx, L. simensis, L. flavopunctatus, L. brunneus, L. pseudosikapusi, L. menageshae, L. chercherensis, and L. brevicaudus) which differ by karyotypes, morphology, and preferred elevation, i.e., types of ecosystems [13, 15, 16]. Interestingly, mitochondrial introgression, apparently common in the flavopunctatus group, was not detected in the rest of the genus Lophuromys, although it should be noted that nuclear genetic data are relatively scarce outside the Ethiopian flavopunctatus members.
Among the NONETHFLAVO, three clades, L. aquilus, L. verhageni, and L. kilonzoi, form a distinct subgroup (NONETHFLAVO1) that is phylogenetically isolated from a second subgroup, NONETHFLAVO2 (L. cf. cinereus, L. dudui, L. laticeps, L. machangui, L. makundii, L. rita, L. sabuni, L. stanleyi, and L. zena). The NONETHFLAVO1 and NONETHFLAVO2 are deeply nested within the Ethiopian endemics to form a monophyletic ‘L. flavopunctatus group’, agreeing with previous conjectures that the NONETHFLAVO colonized the current ranges following dispersals out of Ethiopia [13, 14].
3.2. Species divergence and biogeography
The nested phylogenetic relationships between the ETHFLAVO and NONETHFLAVO conform generally to evolutionary processes speculated previously [13, 14]. While our findings support the Ethiopian highlands as the cradle of the speckled-pelage Lophuromys, the precise nature of their evolutionary radiation, including processes characterizing the observed differentiation between clades remains a matter for speculation, mainly owing to the strong effect of mtDNA on the inferred phylogeny. In any case, it is currently not possible to ascertain whether long-distance dispersal and/or montane-forest bridges promoted the divergence and dispersal of non-Ethiopian flavopunctatus members out of the Ethiopian Highlands. Dispersal along a north-south axis, i.e., out of Ethiopia to southern Afromontane Highlands is relatively like that of other montane-adapted rodents [42-44] and attributed to montane forest expansion during Pliocene-Pleistocene interglacials.
The timing of the NONETHFLAVO and NONETHFLAVO out-of-Ethiopia dispersals, albeit based on a single mitochondrial locus, coincide with the repeated expansion and contraction/isolation of montane forests and their faunal assemblages during the humid intervals of Pleistocene glacial-interglacial cycles [17, 45-48]. Within the L. flavopunctatus group, these events likely connected the southern Ethiopian Highlands with Albertine Rift montane forests, and Kenyan and Tanzanian Highlands across the currently arid Turkana depression [43, 45, 49-51]. The L. flavopunctatus group is primarily restricted to humid/wet habitats which are currently confined to montane areas in East Africa. These species could only have dispersed when the East Africa Highlands were connected with similarly suitable habitats. The first out-of-Ethiopia dispersal by the NONETHFLAVO1 ancestor and consequent range retention in the northern EAMs concur with their prolonged stability that preceded the formation of most of the Kenya Highlands, Tanzanian Highlands, and Albertine Rift montane forests. The split and dispersal of the L. aquilus + L. verhageni clade from L. kilonzoi, the consequent split of L. aquilus from L. verhageni, and the appearance of several clades in the NONETHFLAVO2 subgroup, all happened in the mid-late Pleistocene. This coincides with wet climate periods that made it possible to cross currently dry valleys such as those isolating Mt. Kilimanjaro and Mt. Meru and the Turkana depression in northern Kenya and southern Ethiopia [44, 52]. The absence of genetic evidence of this first dispersal in East African highlands such as the Kenyan Highlands, suggests these mountains served as ‘stepping-stones’. The ‘first colonizers’ – NONETHFLAVO1 – were presumably replaced by the more successful ‘second colonizers’ – NONETHFLAVO2, such as L. zena in the Kenyan highlands. Whether or not the second colonizers hybridized with the first ones remains unclear from mtDNA, and genomic analyses should be applied to investigate this possibility.
The Albertine Rift Valley is a crucial biogeographical feature in the radiation of the NONETHFLAVO and is likely an active barrier to gene flow on either side. The four distinct clades whose ranges are separated by the Albertine Rift (L. dudui, L. stanleyi, L. cf. cinereus, and L. laticeps) suggest that they are not able to cross and have not experienced gene flow since their divergence. While L. stanleyi occurs widely eastward of the Rwenzori Mountains, it does not extend west of the mountains, whereas the range of L. dudui begins in Virunga National Park, and only extends westwards. The Albertine Rift might have been a barrier to L. stanleyi’s westward dispersal and L. dudui’s eastward dispersal. This hypothesis is also consistent with the occurrence of L. laticeps on the eastern and L. cf. cinereus on the opposite western side of the Albertine Rift around Lake Kivu and Lake Tanganyika, with either presumptively unable to cross. Notably, L. sabuni, which is the only clade whose occurrence span both flanks of the Albertine Rift, appear to have dispersed between the Rukwa Rift and Lake Tanganyika and then southwards to Chishimba Falls (Northern Zambia), where it was recently recorded (Sabuni et al. . Other forest rodents have ranges that span the Albertine Rift, unlike observed here for L. dudui, L. stanleyi, L. cf. cinereus, and L. laticeps. For instance, the Malacomys longipes  and the Praomys jacksoni  occur on both sides of the Albertine Rift Valley.
3.3. Morphological variation within the non-Ethiopian flavopunctatus members
Most species in the NONETHFLAVO have overlapping craniodental characters in morphospace, making our large dataset of linear measurements and geometric landmarks unreliable as the exclusive evidence to infer species limits. For instance, the range of skull morphology of L. stanleyi and L. zena (both linear and geometric) significantly resembles the skull forms of all other clades in the NONETHFLAVO, except L. verhageni and L. aquilus, which unambiguously cluster and have the least overlap with any other species in the group. The L. stanleyi and L. zena clades exemplify a typical systematic problem in the NONETHFLAVO, where morphological evidence cannot classify samples to meaningful species units using taxonomically informative characters. Accounting for phenetic variation in the NONETHFLAVO, beyond their common ancestry, requires more comprehensive genomic analyses to disentangle the underlying ecomorphological processes among species occurring in similar habitats. Without such genomic evidence, the taxonomic accounts of several clades are best not considered reliably resolved when based on linear or geometric morphometrics only.
Divergence dates and biogeographic patterns in the NONETHFLAVO suggest that the drivers of craniodental variation fit multiple non-exclusive hypotheses associated with the correlation of ecomorphological divergence with speciation . The relatively recent divergence of most of the clades suggests ecologically-mediated adaptive evolution might not be predominant speciation drivers between congeners in sympatry [57-59]. Except for L. aquilus and L. verhageni which are restricted to single mountain ecosystems, all the NONETHFLAVO species appear to have non-specialized niches as they are not restricted to high montane habitats. They are, thus, more likely to exhibit non-specialized morphological traits that are taxonomically uninformative . The treatment of the L. flavopunctatus group by Verheyen et al.  and Verheyen et al.  highlights the use of craniodental and external morphology data to recognize populations as unique species with minimal use of genetic data. However, inter/intraspecific taxonomic delimitation among rodents often have fewer taxonomically informative stable morphological states, possibly due to nonadaptive and or rapid adaptive radiations . These influences might hinder a replicable definition of taxon-specific phenotypic traits [62-64], leading to the subjective interpretation of valid species. While our geometric morphometry appears generally more sensitive at detecting variabilities between clades compared to linear measurements, just like in other cases , over-all, both datasets produced virtually similar results.
3.4. Taxonomic assessment of the non-Ethiopian flavopunctatus members
While most of the OTUs recovered in the L. flavopunctatus group represent species currently named, the between-clade genetic distances and CYTB incongruence with morphometric and IRBP gene results raise more taxonomic questions than resolution. For instance, only a few mutations at CYTB separate L. aquilus from L. verhageni (2.8% p-distance) and L. stanleyi from L. zena (2.9% p-distance), which is among the closest between-species CYTB divergences in the L. flavopunctatus group. While such low sequence divergence between these sister clades indicates a recent separation of gene pools (at least at mtDNA), it nonetheless, raises concerns about the species’ taxonomic validity, suggesting the need to synonymize them in future taxonomic revisions, without more genetic support, especially since no clear diagnostic morphological differences delimit them. Moreover, the IRBP failure to delimitate several distinct mtDNA clades in the NONETHFLAVO might relate to its slow mutation rate which makes it unable to resolve deeper and or short branches among rodents [66, 67]. Nevertheless, future taxonomic reassessments of the genus Lophuromys should utilize more comprehensive genomic analysis (such as multiple nuclear loci) which are likely to be more informative in delimitating phylogenetic relationships. Such genomic evidence would also quantify the level of distinctiveness between close relatives that are allopatric such as L. aquilus and L. verhageni and the level/absence of gene flow between parapatric ones such as L. stanleyi and L. zena as is prevalent in the ETHFLAVO .
The genetic diversity within lineages such as L. sikapusi and L. chrysopus, for instance, showed the delimited OTUs within them were similarly distinguishable based on CYTB comparably to several clades in the NONETHFLAVO. It appears that taxonomic classifications of Lophuromys species that is not based on extensive nuclear evidence, should yet be regarded inconclusive (i.e., within the Kivumys group, L. sikapusi group, and NONETHFLAVO).
The NONETHFLAVO1 subgroup – L. aquilus, L. verhageni, and L. kilonzoi
The samples from Mt. Kilimanjaro, Mt. Meru, and northeastern Tanzanian Eastern Arc Mountains form distinct monophyletic lineages, representing species currently recognized as valid. L. aquilus was described by True  from Mt. Kilimanjaro and confirmed by Verheyen et al.  to be the only Lophuromys along the entire elevation gradient. Lophuromys verhageni was described by Verheyen et al.  as an endemic of Mt Meru, while L. kilonzoi was described by Verheyen et al.  from the Magamba, East Usambara. Perhaps because of fewer informative sites in shorter sequences, L. aquilus, L. verhageni, and L. kilonzoi had a different phylogenetic topology in Verheyen et al. . Our expanded CYTB sampling supports the three species are minimally differentiated, forming a sister clade to one of the haplogroups of L. simensis. The current CYTB phylogeny, therefore, provides a clearer picture of the phylogenetic relationship between L. aquilus, L. verhageni, and L. kilonzoi and their position in the genus. The historical biogeographical reconstruction suggested the colonization and divergence of L. aquilus and L. verhageni resulted from vicariance events that coincide with the Pleistocene climatic oscillations which fragmented humid montane forests in East Africa [45, 54, 55, 68, 69]. The savannas separating their current ranges were substantially stable even across glacial cycles in the late Pleistocene . The occurrence of L. aquilus and L. verhageni is also consistent with the endemism of Crocidura newmarki on Mt. Meru  and Myosorex zinki on Mt Kilimanjaro . The divergence and dispersal of the NONETHFLAVO1 ancestor coincided with temporary biogeographical contacts between the Ethiopian Highlands and other Afromontane forests in the early Pleistocene . After initial colonization, montane forests were again fragmented by climatic oscillations, in the process facilitating allopatric speciation. Similarly, the patchy distribution of Praomys delectorum across the Eastern Arc Mountains and Southern Montane Forests was probably driven by comparable vicariance events . The higher genetic diversity within L. kilonzoi suggests that it remained in the ancestral range after diverging from the ancestor of L. aquilus + L. verhageni. Similar divergence and diversity patterns were observed for the forest-dependent Praomys delectorum , where the MRCA of populations from the Eastern Arc Mountains predated those from Mt. Kilimanjaro and Mt. Meru and correlated with genetic diversity.
The northern part of NONETHFLAVO: L. stanleyi and L. zena
The sister clades from the Kenyan and Ugandan highlands, L. zena and L. stanleyi, comprise the northern part of the non-Ethiopian flavopunctatus members. Our sample coverage of these two clades was the most comprehensive to date and substantially extend their known ranges. Lophuromys zena , thought to be endemic to the higher elevations of central Kenya [12, 14], occurs in all the stably humid ecosystems in Kenya, including Loita Hills forests in the southeast of their range to western (Mt. Elgon, Cherangani Hills, and Kakamega Forest) and southwestern (Victoria Basin – Yala Swamp) Kenya. The distribution of L. zena overlaps with L. stanleyi and L. ansorgei in the Kakamega Forest and with L. ansorgei in Yala Swamp. This distribution supports the conclusions of Onditi et al.  that L. zena could have been much more widespread in Kenya than previously known [12, 14]. The range of L. stanleyi is also much more extensive than previously described. Sabuni et al.  extended the range of L. stanleyi (delimited by mtDNA) into northwestern Tanzania beyond its Mt Rwenzori type locality , where it was thought to be restricted. Here we provide evidence that L. stanleyi occurs through much of Uganda, spanning southeastern South Sudan and northeastern Uganda forests eastwards to the Kakamega Forest in Kenya (its eastern limit) and south into northern Rwanda. The ‘Karamoja/Uganda gap’ [47, 73] was not a barrier to the dispersal of L. stanleyi through Uganda to connect the Kenya Highlands and Albertine Rift montane forests, as was the case for the forest-dependent Hylomyscus [47, 74]. Generally, the sister relationship of L. zena and L. stanleyi (minimal CYTB divergence) reinforce biogeographic affinities between the Albertine Rift montane forests and the Kenya Highlands [47-49]. Furthermore, the occurrence of L. zena and L. stanleyi in both lowland and highland forests suggest a phylogeographic pattern shaped also by an opportunistic ecological strategy, unlike true forest-specialists such as the Hylomyscus denniae and Sylvisorex granti groups that are restricted to high-elevation forests [47, 48]. The biogeographies of L. zena and L. stanleyi mirror similar patterns as the more widespread Praomys jacksoni which colonized both montane and lowland forests. However, the absence of a taxonomic structure based on IRBP further reinforces the need to apply genomic analyses, especially in zones of secondary contacts, such as in Kakamega, to shed light on the level of their reproductive isolation and the taxonomic validity.
The southern part of NONETHFLAVO: L. machangui and L. sabuni
These two significantly supported sister clades correspond to L. machangui and L. sabuni, both described by Verheyen et al.  from Mount Rungwe and the Mbizi Mountains (Ufipa Plateau), respectively. Their sister relationship and late Pleistocene divergence coincide with the split of L. verhageni and L. aquilus, attributable to the late Pleistocene expansion of moist forests that likely enabled them to disperse to the current ranges, whose suitability was later restricted to highland forests. Overall, the distribution of L. machangui and L. sabuni reveal biogeographical trends that both coincide and contrast with other small mammals in the region, suggesting that other taxon-specific functional traits, such as dispersal ability and habitat specificity versus generality also influenced their evolutionary radiation. For instance, the distribution of L. machangui suggests the Makambako Gap has not barred its dispersal, similar for other small mammals including Myosorex kihaulei , but has barred the dispersal of Praomys delectorum  and Otomys lacustris . Within the range of L. sabuni, Kerbis Peterhans et al.  recently described two species in the genus Hylomyscus, Hylomyscus stanleyi from Mbizi Forest Reserve and Hylomyscus mpungamachagorum from Mahale National Park, suggesting that the so-called Karema Gap was a barrier to the dispersal of these Hylomyscus species but not to the dispersal of L. sabuni. Overall, the close craniodental and genetic affinity between L. sabuni and L. machangui to each other in comparison with members from other clades in the NONETHFLAVO2 subgroup suggests they have experienced somewhat similar ecological selection resulting in similar ecomorphological characteristics . The craniodental and genetic affinities between L. sabuni and L. machangui also concurs with the floral and faunal affinity between the Southern highlands of the northern end of Lake Malawi and the Mbizi Forest, attributed mostly to the absence of a substantial biogeographical barrier between them. More studies are needed to delineate genetic differentiation across the range of L. machangui and L. sabuni, and detail how isolation by distance and geographical features have impacted dispersal.
The western part of NONETHFLAVO: L. dudui and L. rita
The L. dudui clade comprised samples from the northeastern DRC montane highlands of the Albertine Rift –Rwenzori Mountains, westwards to the Kisangani – Bomane – Yaenero areas. This distribution leaves a ca. 480 km sample gap between the eastern limits (Epulu – Tshiabirimu – Ituri) and western limits near Baliko, Boende on the left bank of Congo River. The inclusion of samples from both sides of the Congo River in the L. dudui clade modifies the original description as well as consequent accounts of L. dudui, where it has been thought to be restricted between the right bank of the Congo River and the western foothills of the Albertine Rift mountains [12, 14]. The current range of L. dudui resembles that of Praomys mutoni and Praomys jacksoni  both of which occupy lowland forests on both banks of Congo River in the Kisangani region [55, 78]. Morphologically, L. dudui is easily diagnosable from the nearby NONETH2 members due to its distinctly small skull (Fig. 6, Additional file 2 Table S3), consistent with previous findings [12, 14]. The L. dudui range overlaps with that of L. rita, which was assigned to samples spread over an expansive area in the Congo Basin, spanning southwestern DRC (Kinshasa) to the northeast (Kisangani, left bank of Congo River) and southwards to northwestern Zambia. Although we are unable to make skull comparisons with the holotype, this clade forms a well-defined mtDNA lineage, probably representing the L. rita described by Dollman  from south of Lake Tanganyika in NE Zambia (Mporokoso) and Lufupa River, Katanga, DRC. Despite its expansive range, L. rita appears bound to the central Congo basin by the Congo and Lualaba Rivers, which have likely limited its dispersal, like Praomys minor in the central Congo Basin . Our geographic sampling of L. rita is notably sparse relative to its distribution and more surveys are necessary to resolve the full range and genetic diversity within the L. rita clade. Importantly, a formal taxonomic reassessment is required to validate the morphological relationship of the L. rita clade with the holotype and topotypes.
Specimens attributed to the monophyletic L. makundii derive from the Mount Hanang (type locality) northwards over the Lake Manyara and Ngorongoro crater to Mt Kitumbeine. Several ‘unsuitable’ dry corridors which currently isolate L. makundii from Eastern Arc Montane forests, Albertine Rift Mountains, Kenyan Highlands, and even the nearby Mount Kilimanjaro and Mount Meru seem to have impacted its dispersal after the initial colonization event. However, the occurrence of Crocidura montis, Crocidura hildergadea, Otomys angoniensis, Grammomys dolichurus/macmillani, Graphiurus murinus, and Praomys taitae in similar habitats as L. makundii in the north-central Tanzania region [53, 79] suggest that its biogeographical affiliation to other Eastern Afromontane forests in the region is recent. The relatively isolated range of L. makundii likely imposed a more rigid barrier to genetic exchange with other lineages after divergence , and might explain why it is the only other clade in the NONETHFLAVO, besides L. kilonzoi, that retains monophyly in the IRBP tree. Still, the minimum divergence time and possible sister relationship to either L. dudui or L. laticeps show that it is more closely affiliated to the Albertine Rift than the NONETHFLAVO1 range. As such, L. makundii probably colonized its current range when moist forests connected the currently isolated volcanic mountains during the late Pleistocene climate fluctuations.
L. cf. cinereus and L. laticeps
The L. cf. cinereus samples overlap the Kahuzi-Biega National Park locality from where Dieterlen and Gelmroth  described L. cinereus. Following the initial proposal by Dieterlen  that the external and craniodental distinctness used by Dieterlen and Gelmroth  to describe L. cinereus were, in fact, morphotypes of L. laticeps, there has since been no formal taxonomic reassessment of its validity, with the foundational references [6, 12] maintaining its synonymy to L. laticeps. Our mtDNA, nuclear (IRBP), and craniodental tests showed similar differences between the L. cf. cinereus and L. laticeps clades, comparable to the distances within and between other NONETHFLAVO2 clades, including the sister clade, L. rita. The L. cf. cinereus skulls overlapped most with L. laticeps, L. dudui, and L. stanleyi, consistent with the earlier rationale for its synonymy [6, 12]. A formal taxonomic revision of L. cf. cinereus, is needed to validate and update its distribution, and genetic and phenetic relationship to other non-Ethiopian L. flavopunctatus members. Such a revision would importantly update the occurrence of L. cinereus (herein as L. cf. cinereus), which was perceived restricted to the type locality [28, 81], to extend from Kahuzi-Biega NP to the Itombwe Massif and southwards ca. 300 kilometers to Mt. Kabobo – on the western shore of Lake Tanganyika. Thomas and Wroughton  considered L. laticeps as a morphologically unique lineage among its close relatives allied to L. aquilus  due to a broader lower braincase and shorter palatal foramina. In agreement, our L. Laticeps skulls had the broadest BBC and one of the shortest PPL in NONETHFLAVO2 dataset (Additional file 2 Table S3). The L. laticeps clade is also genetically well-differentiated, comparably to close relatives – L. cf. cinereus, L. stanleyi, and L. laticeps. There is a need to formally reassess the taxonomy of L. cf. cinereus and L. laticeps, to clarify and update their distinctness from other lineages in the NONETHFLAVO, not to be synonymized under L. aquilus.
L. margarettae, L. rubecula, and L. major
No genetic OTUs could be matched to L. margarettae, L. rubecula, or L. major, despite sampling from their respective ranges – Mathews Range, Mount Elgon, and proximity of Ubangi River. L. margarettae was described by Heller  from the Mount Gargues (Mathews range), north-central Kenya, with Verheyen et al. , Verheyen et al.  confirming its presence on the lower elevations of the Kenya highlands. However, Onditi et al.  did not record L. margarettae in the entire elevation gradient of Mount Kenya (ca. 1,700 – 4,000 meters). In the current study, the samples from Kaptagat that Verheyen et al.  assigned to L. margarettae are completely nested within the L. zena clade, including those from the nearby Mau Forest fragments. During this study, despite ~500 trap nights (standard trapping protocol using Sherman live traps) at intermediate elevations (1,210-1,930 meters) of the Mathews Range, not a single Lophuromys was captured. Although we cannot challenge the taxonomic validity of L. margarettae in the Mathews Range yet, it is absent from all the localities where L. zena was sampled – virtually all the wet highlands of Kenya. It may be that ongoing forest degradation and changing climates might have led L. margarettae to shift range and thus become rarer. More surveys of the higher, more intact forest of the Mathews Range are required to resolve with certainty whether L. margarettae is still resident in the area or is simply an L. zena variant.
Similarly, L. rubecula described by Dollman  is another species we are unable to confirm without new material. Our Mt. Elgon samples cluster genetically and craniodentally with L. zena. However, we lacked samples from other parts of the Mt Elgon ecosystem, without which we cannot dismiss L. rubecula’s occurrence or its validity. Future surveys of Mt Elgon should employ elevational stratified sampling transects on the Kenya and Uganda sides to substantiate the occurrence limits (or the absence thereof) of L. rubecula.
Finally, we were also unable to verify the validity of L. major, which was described by Thomas and Wroughton  from the Bwanda area, Ubangi River, DRC. The ranges of the presupposed nearest congeners – L. dudui and L. rita are considerably south of its type locality and without new material from the area, we cannot verify the validity of L. major or approximate its relationship to other species in the Lophuromys genus.