The cryptic case of Otomys sloggetti (Sloggett’s vlei rat): Interpreting murid molar morphology in the fossil record

FUNDING: South African National Research Foundation, National Research Council of Norway Vlei rats (Family: Muridae; Subfamily: Otomyinae) have a widespread distribution in southern Africa. They are favoured prey of barn and spotted eagle owls, and frequently become associated with archaeological deposits when the owls roost in cave sites. The phylogeny of several Otomyinae species is enigmatic, and Otomys sloggetti (Sloggett’s vlei rat) is no exception. This species has been referred to as the ‘ice rat’ and present distribution ranges are seemingly limited to mountainous areas, at high altitude, in Lesotho, Drakensberg and the Karoo. It was thus surprising and unexpected when specimens closely resembling Otomys sloggetti (identification was based on molar morphology) were found in several archaeological sites on the south and west coasts of South Africa, and also in modern owl pellet assemblages – all extralimital to the current reported distribution. However, further examination of and comparison between these specimens, as well as extensive differences observed between comparative Otomys sloggetti specimens from museum collections, highlighted potential problems associated with the common practice of using tooth morphology to identify fossil murid species. We identified six molar morphotypes from the fossil and modern material, all of which bore a morphological resemblance to O. sloggetti. The material discussed in this paper suggests that cryptic, undescribed vlei rat species, or subspecies, have been in the past, and may yet be, co-occurring with modern populations of O. karoensis and O. irroratus. Phylogenetic studies need to be done in conjunction with morphological studies, as, currently, the relationship between the huge variation seen in interspecific morphology with genetics is little understood, different Otomys species are not always distinguishable morphologically, and considerable chromosomal polytypes have been found. Our findings highlight the need for extensive cladistic and genetic research on the Otomyinae.


Introduction
The identity, and biogeographical affiliations, of fossil rats and mice (Family: Muridae) are important as these small animals, which have short lifespans, small ranges, and in some cases particular habitat requirements, are used to provide environmental information in order to reconstruct ancient environments, and trace palaeoenvironmental change over time. The correct identification of these animals in fossil sites is thus important as errors can lead to erroneous assumptions and conclusions being made about past environments, and the effects of climate change.
Here we focus on a vlei rat species, namely Otomys sloggetti, and use the fossil record of this taxon on the south coast of South Africa to illustrate some of the issues and potential problems associated with the common practice of using tooth morphology to identify fossil murid species.
Research into the murid species present in fossil assemblages is usually done on a single site basis, and it is extremely rare for researchers to compare the same taxon across sites. The taxonomic conundrums presented in this paper indicate that such comparisons might result in the re-evaluation of identifications, as inconsistencies and errors may ensue if museum specimens have been incorrectly identified -a scenario which is particularly likely in the case of cryptic species (species which look morphologically the same, but belong to different species). Cross-site comparisons, such as those made here, may also indicate variability within a species suggestive of misidentification, and/or the presence of cryptic species. We compare the molar morphology of a variety of fossil Otomys cf. sloggetti from a number of south coast archaeological sites, and a west coast site, with each other, with modern Otomys sloggetti, and with two other Otomys species (O. irroratus and O. karoensis). These comparisons include comparative material from museum collections, and from two south coast barn owl pellet collections.

Taxonomy and phylogeny of Otomys sloggetti
Otomys sloggetti is a rat which belongs to the relatively speciose 'vlei rat' (also called 'laminate toothed rat') tribe. These herbivorous short-tailed rodents (Subfamily: Murinae) are frequently mentioned as being 'volelike' in appearance. The Otomyini are represented by some 32 recognised species (including two of which are undescribed) and are endemic and widespread in sub-Saharan Africa, with a number of species occurring in South Africa. 1 The exact number of extant Otomys species is contentious and a number of cryptic species have been identified, while others await further elucidation. [2][3][4][5] Some studies suggest that speciation has occurred along biomes, with phenotypic and genotypic divergent lineages corresponding to the Fynbos/Albany Thicket and Grassland biomes in the case of Otomys irroratus and Otomys karoensis. 2,3 Geometric morphometric analyses carried out on the crania of Otomys unisulcatus also failed to support the genetic groupings, but rather followed biome boundaries, indicating previous environmental adaptations. 6 This, together with the adaptability observed in fossil Otomys communities when faced with environmental and climatic change on the south coast of South Africa 7 , as well as the fact that up to three or more Otomys species may live sympatrically in an area 1,2,7 , indicates the flexibility and plasticity of this murid subfamily.
The phylogeny of Otomys sloggetti, and the number of lineages and species present, as is the case with many of the other otomyine species, is currently unresolved. 1,2 In a study that looked at mitochondrial and nuclear genes, as well as morphological characteristics, specimens previously identified as Otomys sloggetti were found to represent two clades, and three lineages. 8 A cladistic analysis of 45 morphological (craniodental) and 46 binary allozyme characters indicated that Otomys sloggetti was basal to a 'mesic clade' of southern and eastern African species 9 , and the idea that Otomys sloggetti represents a basal lineage has been reiterated elsewhere 10

Small mammals as palaeoenvironmental indicators
As mentioned previously, archaeologists and palaeontologists frequently use rats, mice and shrews from fossil sites as indicators of past climatic and environmental conditions. [17][18][19] These animals generally become associated with fossil deposits when barn and eagle owls, which roost in the same caves occupied by people, regurgitate the bones and teeth of their prey in their pellets. Over time, the pellets disintegrate, leaving lenses of bones and teeth in the sediment. As these owls have small home ranges, and select a broad range of prey, they provide a good sample of the small mammal species living in the vicinity of the cave site. 14,17 The teeth of rodents and soricids (shrews) are generally used to identify the small mammal species present in archaeological deposits. The laminate molars of vlei rats, and the terminology used in this paper, is illustrated in Figure 1.
Vlei rats have laminate teeth and generally show a high degree of intraspecific variability in terms of molar shape, and sometimes even in the number of laminae. For example, O. karoensis is noted as sometimes having five, rather than six, laminae on the upper third molar. 1 This variability, which includes biogeographical differences in morphology and size, and the fact that the appearance of the occlusal surface of the tooth changes over time with wear (in some species wear leads to the joining of previously separate laminae), makes this a difficult group of rodents to identify. In fossil deposits, the cranial bones are frequently broken to the degree that only isolated molars are recovered. This means that the approach of identifying features used with modern comparative material, such as presence/absence of grooves on the lower incisor, and the shape of the petrotympanic foramen, cannot be used. O. sloggetti is distinguishable from all other Otomys species in having ungrooved lower incisors, and a slit-shaped petrotympanic foramen. 2,9 In the case of material from fossil deposits, however, the lower first molar (M 1 ) of the mandible, and upper third molar (M 3 ) of the maxilla (see Figure 1), are the most distinctive and useful teeth for the identification of Otomys species.  Figure 2 shows the fossil and pellet collection sites mentioned in this paper, and the current extent of O. sloggetti's distribution based on the IUCN Red List. 20 Computed tomography (CT) scans of individual fossils, and some of the modern material, were carried out at the Central Analytical Facilities of Stellenbosch University (South Africa), using their micro-and nano-CT scanning facilities. Length and breadth measurements (see Appendix 1) of all M 1 and M 3 molars were made on nano-CT scans of the specimens, using the 'measuring' and 'dimensions' functions of VGMax, version 3.3. Measurements of all the teeth presented here are given in Appendix 1.
Note that these must be considered within the context of age and wear patterns, as older individuals, with more worn teeth, will automatically yield higher measurements than younger individuals, as the occlusal surface becomes longer, and broader, with wear. Photographs of modern specimens from EPF were taken with a Leica M205A camera attached to a Leica M275 microscope. Specimens from KDS were photographed with a Leica DFC 295 camera attached to a Leica M125 C microscope.

Otomys sloggetti and the south coast fossil record
Otomys sloggetti sensu lato frequently co-occurs with two other Otomys species, namely Otomys karoensis and Otomys irroratus, in fossil deposits on the south coast. 7 O. sloggetti sensu lato was identified in the fossil deposits based on the M 3 and M 1 , and is differentiated by two specific molar morphologies. Firstly, by the presence of five laminae on the upper third molar (M 3 ) as opposed to the six laminae generally found in Otomys karoensis and Otomys irroratus, and secondly, a rounded anterior first laminae (M 1 ) (which differs to the rectangular first lamina of the other two species), and in the distribution of the laminae across the occlusal surface of the molar. Another vlei rat with the same general morphology of the M 1 and M 3 , O. unisulcatus, has also made an extralimital appearance in some south coast fossil horizons, but the shape and general appearance of the molars are differentiable from that of O. sloggetti (see Figures 5 and 7) as the M 3 has generally smaller dimensions and is roughly rectangular in shape, and the anterior and second laminae of the M 1 merge to form a characteristic 'C' shape, with only slight wear.
The appearance of O. sloggetti in a number of fossil south coast South African sites (see Figure 2 for localities) was unexpected, as it has been called a high altitude and alpine endemic species, and its current distribution includes the Drakensberg, Sneeuberg and the southern Great Escarpment 2 , with a couple of isolated populations on mountains in the Karoo 8,21-23 . This species is often referred to as the 'ice rat' due to its appearance in areas which reach icy temperatures. The appearance of O. sloggetti in the south coast fossil record was surprising as it appeared to belie the categorisation of this taxon as being a 'high altitude endemic' and 'montane-adapted' 9    and O. irroratus, in general skull form and proportions: the skull cap is rounder in shape relative to the others, the orbital foramen is broader and extends less anteriorly to posteriorly, the nasal foramen is rounded in shape rather than rectangular, and the shape and size of the maxilla, zygomatic arch and the zygomatic plate are likewise clearly different.
The upper incisors of all the taxa exhibit a single groove. The DHC-10-3 skull appears very similar, although not identical, to O. karoensis in terms of the shape of the zygomatic arch, general skull shape, the infraorbital foramen, and the premaxilla. Small differences may be attributed to the fact that it belongs to a smaller and (as indicated by the wear pattern on the molars) younger, individual. The premaxilla and maxilla from the Elandspad Bulk Sample (EPF-bulk-1) specimen differed in size and morphology, not only to O. sloggetti, but also to all the other taxa illustrated in Figure 3. In order to illustrate these morphological differences, and to compare this specimen with another of a similar M 3 molar morphology, Figure 4   The area where the incisive bone meets the zygomatic process of the maxilla is curved in O. karoensis (DHC-10-3) but straight in the indeterminate Otomys from EPF. Other differences include the size and shape of the infraorbital foramen (much larger in the case of the EPF Otomys), and the zygomatic arch is different in size and shape between the taxa. The differences observed in maxillary morphology are likewise reflected in the M 3 , as illustrated in Figure 5, where the distribution of the laminae across the occlusal surface of the tooth, and the size and shape of the anterior lamina, differ. The fact that the Elandspad specimen shows morphological differences in the maxilla and the M 3 supports the identification of this as a taxon or morphotype differentiable from O. karoensis. A search was made through the Elandspad owl pellets for M 1 teeth which looked different from those of O. karoensis and O. irroratus, and which could possibly be a match for the M 3 Elandspad specimen. Two such specimens were recovered from the Elandspad bulk collection, one of which is illustrated in Figure 7. It is distinguished by a notably large, round, first lamina (EPF-bulk-12, see inset photo in Figure 7). An Otomys irroratus specimen is included in both Figures 5 and 7 to illustrate the similarity of this species in terms of molar morphology to O. karoensis. The site name and specimen reference numbers are provided in brackets in Figure 5.   Figure 5, M and N) in terms of the degree of curvature of the laminae, and general tooth shape. Both specimens come from the so-called 'Name Chamber' deposits which are mixed in that they contain deposits from both Member 4 and Member 5E, with the Oldowan (Member 5E) deposits dating to ~2-1 Ma, and/or Member 4 (~2.8-2 Ma). 26 The taxonomic relevance of the morphological differences is unclear, given the stratigraphic/age uncertainties. The Sterkfontein fossil specimens are considerably smaller than the modern comparative O. sloggetti and almost all the fossil O. sloggetti sensu lato, providing an example of the flexibility in size of this genus in both modern and fossil populations (see Appendix 1).

Comparison between extant and fossil Otomys taxa
As mentioned previously, O. karoensis has been noted to sometimes have five laminae on the M 3 . The DHC specimens in Figure 5 ('B' and 'D') illustrate such a case. These specimens differ slightly in that DHC 10-2 ('B') has a tiny auxiliary cusplet situated proximally on the anterior laminae, which indicates a variation occurring in the phenotype. Such additional cusplets (note this feature also appears in the fossils 'G' and J' in Figure 5) would disappear with wear and would not be discernible in older individuals. This kind of intraspecific variation is not uncommon among murids, and small additional cusplets occur infrequently on murid teeth (T.H.N. and T.M. personal observation).
The EPF M 3 (EPF-bulk-1, see 'D' in Figure 5), whose maxilla and premaxilla show differences to O. karoensis as noted above, is different from the fossil and modern O. karoensis in that it has a larger and more rounded anterior cusp, which is embedded in the second lamina to a greater degree than seen in O. karoensis. It also differs in that the spacing between the anterior to the posterior lamina of the occlusal surface is wider, and the laminae are more curved and orientated centrally towards the middle of the tooth, whereas O. karoensis laminae slant towards the labial side of the tooth.
The south coast fossil specimen from PP30 ( Figure 5  When viewed laterally, the five, or six, laminae present on the various Otomys taxa, as well as the auxillary cusplets, are clearly discernible ( Figure 6). Note that the specimens, and the order of specimen line-up, are identical in Figures 5 and 6. In Figure 7, the top row of specimens from B to E are considered to represent O. karoensis, although E from PP9C was originally differentiated from other O. karoensis at the site on the basis of size and the relative roundness of the first lamina. However, it appears very similar to the modern specimen from DHC 10-2 (Figure 7, D), and given this likeness, is most probably an O. karoensis. Fossil O. unisulcatus specimens are represented in Figure 7 (see S, T, U and V), while W represents a modern specimen (from Diepkloof Rock Shelter).  Notably, morphological differences between the fossil specimens and the one modern O. sloggetti specimen (TM-22669) are less than that observed between the two modern museum specimens in Figure 7.
As observed in the M 3 , the two museum specimens differed in the size and distribution of the cusps varying across the occlusal surface, and the extent of curvature of the laminae (see Figure 7, H and I). The two modern O. sloggetti (TM-22669, TM-16519) were broader than all the fossil specimens measured (O. unisulcatus excluded), although there was some overlap with the fossils in terms of length.
The modern specimen from EPF (EPF-bulk-12, Figure 7, 'X') (hereafter referred to as Morphotype 6) is differentiated from Morphotypes 3, 4 and 5 taxa by a larger (relative to the other laminae) and more rounded anterior cusp -the former have a smaller and more rectangular-rounded shape. This M 1 (two molars with this morphology were recovered from the bulk samples) may match the unidentified Otomys maxilla and M 3 from the EPF bulk pellet samples, or it may represent a variation of cf. O. karoensis. The similarity observed in the morphology of the M 3 of the EPF specimen with some of the fossils (i.e. Morphotype 1), is not as obvious when it comes to the M 1 , and it is not possible to match the EPF M 1 and M 3 specimens with any certainty.

Discussion and conclusions
An intrasite examination of fossil specimens identified as O. cf. sloggetti during previous research indicates that, although there is homogeneity observed between some specimens, there is a degree of variation, which, together with the differences observed between modern comparative O. sloggetti material, is very hard to interpret. There are, however, two morphotypes distinguishable in the fossil O. cf. sloggetti M 3 , and four in the M 1 , material, as described above. We suggest that the differentiation of these morphotypes, at least in the case of Morphotypes The genetic relevance of the observed similarities and differences will remain obscure until they can be interpreted within a framework which clarifies the link between skull and molar morphology, and the genotype of modern taxa.
The presence of O. cf. sloggetti in surface deposits in some of the Pinnacle Point sites was puzzling, and it was suggested that this might have occurred due to mixing with fossil deposits. 7 This review of the purported O. sloggetti material from the owl pellets and fossil sites suggests that it is probably not O. sloggetti, and, as noted above, the jury remains out as to the exact phylogeny of the different morphotypes. The observed differences between the two O. sloggetti museum specimens illustrate the fact that museum collections may not provide a reliable taxonomic reference where cryptic species are involved, and are likely to be related to the fact that the specimens were collected from different mountain ranges, and probably represent different species. 2 It has been noted that specimens identified as O. sloggetti in the field were found (in an investigation utilising nuclear and mitochondrial gene regions, as well as morphological characteristics 8 ) to represent two clades. One was consistent with the description of O. sloggetti, but the other was distinct not only from O. sloggetti, but from all other Otomys species, and the authors concluded that this could be a new, and novel, species. Subsequent to this, a new Otomys species was described from the Sneeuberg Centre of Floristic Endemism in the southern Drakensberg Mountains (Otomys willani sp. nov.), and the same publication notes the presence of two specimens from the Mountain Zebra National Park in the Eastern Cape which showed a well-supported and relatively deeply divergent lineage that has no supported relationship with any other Otomyini species. 2 The fossil and modern material discussed in this paper suggests that such cryptic, undescribed species, or subspecies, have been, and may be, co-occurring with modern populations of O. karoensis and O. irroratus. Phylogenetic studies need to be done in conjunction with morphological studies of skulls and molars, as, currently, the relationship between the huge variation seen in interspecific morphology with genetics is little understood, different Otomys species are not always distinguishable morphologically, and considerable chromosomal polytypes have been found. 2 The otomyines are an interesting family of murids and assessing the degree of morphological variation in modern O. sloggetti, O. irroratus/O. auratus, and O. karoensis populations will provide essential information on morphological variation, and how it relates to biogeography and adaptation, and will in turn provide interesting ecological and evolutionary information on a family of small mammals which exhibit great adaptability and phenotypic plasticity. Clearly, further genetic and cladistic research is needed to unravel the cryptic and complicated taxonomy of Otomys taxa. Given the issues raised by this research into the south and west coast fossil record, we recommend that future research into the Otomyini focus on modern collections (including owl pellet collections) from the south and west coasts and the Eastern Cape, using an integrated molecular, karyotypic and morphological approach.