Identifying the true number of specimens of the extinct blue antelope (Hippotragus leucophaeus)

Native to southern Africa, the blue antelope (Hippotragus leucophaeus) is the only large African mammal species known to have become extinct in historical times. However, it was poorly documented prior to its extinction ~ 1800 AD, and many of the small number of museum specimens attributed to it are taxonomically contentious. This places limitations on our understanding of its morphology, ecology, and the mechanisms responsible for its demise. We retrieved genetic information from ten of the sixteen putative blue antelope museum specimens using both shotgun sequencing and mitochondrial genome target capture in an attempt to resolve the uncertainty surrounding the identification of these specimens. We found that only four of the ten investigated specimens, and not a single skull, represent the blue antelope. This indicates that the true number of historical museum specimens of the blue antelope is even smaller than previously thought, and therefore hardly any reference material is available for morphometric, comparative and genetic studies. Our study highlights how genetics can be used to identify rare species in natural history collections where other methods may fail or when records are scarce. Additionally, we present an improved mitochondrial reference genome for the blue antelope as well as one complete and two partial mitochondrial genomes. A first analysis of these mitochondrial genomes indicates low levels of maternal genetic diversity in the ‘museum population’, possibly confirming previous results that blue antelope population size was already low at the time of the European colonization of South Africa.

www.nature.com/scientificreports/ some with frontlet, and one inaccessible specimen that is either a skull or a pair of horns (Table 1, Supplementary Document S1). All institutions housing specimens were contacted and subsequently ten bone and/or skin samples were obtained. Photographs (if available, Supplementary Figs. S1-S15) and detailed information about the specimens are presented in Supplementary Document S1. Whenever documents have been inspected by one of the authors (for example copies of catalogues), no source is stated; whenever the information was provided by others, this is stated as personal communication.
Specimens analyzed. The specimen from the Swedish Museum of Natural History (Naturhistoriska riksmuseet) in Stockholm (NRM 590107) is a mounted skin of a subadult male, its young age being obvious from its short horns with few rings. The collector A. U. Grill received the specimen from C. P. Thunberg 26 . In 1829, Grill's heirs donated it to the Swedish Museum of Natural History 17,18 . It is highly likely that no skull is inside the skin (see Supplementary Document S1). The mounted skin from the Natural History Museum Vienna (NMW ST 715) is the only known female. Recent X-ray analysis (June 2017) has shown that the Vienna specimen does not contain a skull (Supplementary Fig. S2). According to the acquisition catalogue, it became part of the collection in 1806. It was the first specimen of the species from which DNA was successfully recovered 22 . Table 1. List of definitive, potential and previously assumed historical blue antelope specimens. The historical specimens which, at some point in time, have been identified as blue antelope (Hippotragus leucophaeus), are listed with current species identification. Horn sheaths are present unless stated otherwise. Sex given as in museum catalogue or literature. a Specimens previously not mentioned in the literature. www.nature.com/scientificreports/ The complete skull (without horn sheaths) from the Hunterian at the University of Glasgow (GLAHM:Z4884) was discovered there in 1949 21 , whereas the complete skull (including both horn sheaths) in the collection of Naturalis Biodiversity Center in Leiden (ZMA.MAM.18623) was formerly stored in the Zoölogisch Museum in Amsterdam before this collection was merged with Naturalis in 2011. To our knowledge, the cranium at the Muséum national d'Histoire naturelle in Paris (MNHN-ZM-AC-1896-100) has not been previously mentioned in the literature. According to the catalogue, it is from Senegal and was donated by a certain Houdelot(?). It was first mentioned in a museum catalogue at the end of the nineteenth century (pers. comm. Joséphine Lésur, in charge of the Collections Ostéologiques d' Anatomie Comparée at the Muséum national d'Histoire naturelle). Two crania and one skull in the Mammal Collection at the Museum für Naturkunde in Berlin were at some point identified as blue antelopes. Only one of these, a cranium (ZMB MAM 8859) was sampled. It is now cataloged as a roan, possibly after Mohr's 17 revision. Its date of arrival in the collection, geographic origin, and sex are unknown.
The skull fragments at Naturalis Biodiversity Center in Leiden consist of the anterior part of a mandible and premaxillary bones (RMNH.MAM.20681.a). They may derive from the same specimen represented also by the mounted skin in this collection (RMNH.MAM.20681.b) 24 (see Specimens not analyzed). The collection catalogue from 1887 refers to this as a complete skull 24,27 , and it may be that the remainder of the skull is within the mounted skin. Therefore, the fragments bear the same object ID as the mounted skin in the collection catalogue, but with 'b' for the mounted skin and 'a' for the skull fragments. We refer to the skull fragments as Leiden 1 in this study, to distinguish them from the complete skull from Leiden, which is referred to as Leiden 2.
The horns in the Museum of Evolution at the University of Uppsala (UPSZMC 78488) still have traces of fur attached to their bases, but are not connected by a frontlet. From this specimen, we received a fur/skin as well as a bone sample. It had already been used earlier to generate a mitochondrial genome of the blue antelope 23 . According to Rookmaaker 4 it was received in 1781 by C. P. Thunberg from D. F. Immelmann.
The frontlet with horns from the Terrestrial Vertebrate Collection at the Iziko Museums of South Africa in Cape Town (SAM ZM 40759) was first reported by Ozinsky 28 . The only available information is that it was part of the private collection of Mr. J. Piek from Observatory (Cape Town) and entered the collection in 1989 (pers. comm. Denise Hamerton, curator Terrestrial Vertebrate Collection at Iziko Museums of South Africa). The frontlet with horns from the Bell Pettigrew Museum at the University of St Andrews (BPM 2234) has, to our knowledge, never been mentioned in the literature. It is labeled as 'Blaubok? Ozanna leucophaeus (Pall.) South Africa, now extinct' . It seems to lack documentation of its history as well as information on its origin.
Specimens not analyzed. The mounted skin at Leiden Naturalis Biodiversity Center (formerly Rijksmuseum van Natuurlijke Historie) (RMNH.MAM.20681.b) is an adult male and the lectotype of the species, to which later Swellendam was assigned as location 24 . The mounted skin in the Collections d' Anatomie Comparée at the Muséum national d'Histoire naturelle in Paris is an adult male (MNHN-ZM-MO-1994-1103). It was first mentioned in a museum catalogue from the end of the nineteenth century (pers. comm. Joséphine Lésur; for a recent publication see Glenn) 29 . Mohr 17 assumed that the Paris specimen did not contain its skull, on account of the shape of its forehead (where the nasal and frontal bones would be). This was confirmed by Péquignot 30 . While the Leiden and Paris mounted skins are most likely true blue antelopes (based on their morphological similarity with the genetically confirmed Stockholm and Vienna specimens), despite the comment in Skead 31 that a blue antelope could be manufactured 'using the skin of an animal resembling it' , they should be genetically analyzed in the future.
The Museum für Naturkunde in Berlin houses two more specimens at one time labeled as blue antelopes: the cranium of a female from South Africa (ZMB MAM 8855) and the skull of a male from Sudan (ZMB MAM 8860). Mohr 17 determined ZMB MAM 8855 to be a sable after receiving measurements from Berlin, which is probably the reason for its relabeling. ZMB MAM 8860 has to our knowledge never been mentioned in the literature; unfortunately, it was discovered too late to be included in our study.
Little information is available for the frontlet with horns in the Natural History Museum in London (NHMUK GERM 636e). In Lydekker's 32 catalogue of the ungulate mammals in the British Museum it is listed as 'provisionally referred to this species [blue antelope]' . It was mentioned by Sclater & Thomas 10 as having 'been long in the Museum' . The specimen in the Royal Belgian Institute of Natural Science in Brussels (RBINS 3785) has to our knowledge never been mentioned in the literature and could not be accessed for this study due to collection renovation. According to the museum catalogue, it is either a skull or a pair of horns which was donated to the museum in 1931 by the arachnologist and ichthyologist Dr L. Giltay.
More detailed information about these specimens, as well as others documented in the literature but believed to not exist anymore, is given in Supplementary Document S1 and Supplementary Table S1. Laboratory procedures. DNA preparation. DNA was extracted from bone using the Dabney et al. 33 protocol and from skin by using a combination of Rohland et al. 34 and Dabney et al. 33 protocols with digestion buffer modifications from Taron et al. 35 (5 M GuSCN,25 nM NaCl, 50 mM Tris, 20 mM EDTA, 1% Tween-20, 1% 2-Mercaptoethanol) using columns of Roche's High Pure Viral Nucleic Acid Kit for purification. DNA extracts were built into single-stranded libraries following Gansauge et al. 36 with the adjustment of an initial 15 min. incubation step with 0.5 µl USER (Uracil-Specific Excision Reagent) Enzyme for Uracil removal (modified from Meyer et al.) 37 . A maximum of 13 ng DNA was used as input for library construction. qPCR was performed to determine the optimal number of amplification cycles for the subsequently performed dual-indexing PCR. Extraction and library blanks were run alongside all samples to check for contamination. For the sample from Uppsala, a library was built from the bone as well as the skin sample. As both the Stockholm and Vienna samples contained very short reads after initial test-sequencing, both were processed on the Pippin Prep (Sage Science) with a 3% Agarose Gel Cassette for targets from 90-250 bp (Marker C) (product no. CSD3010) with selection www.nature.com/scientificreports/ parameters set to 165-300 bp to remove short reads and thereby improve the mappable fraction of sequenced reads. All pre-PCR lab work was conducted in dedicated archival DNA facilities at the University of Potsdam, Germany. The libraries were shotgun sequenced using custom primers 38,39 on an Illumina NextSeq500 at the University of Potsdam generating 75 bp single-end reads.
Mitochondrial genome enrichment. For array capture following Hodges et al. 40 and Paijmans et al. 41 , an Agilent Sure select Array was designed based on the mitochondrial genomes of Hippotragus leucophaeus (NC_035309 23 ), H. equinus (NC_020712) 42 and H. niger (NC_020713) 42 . As H. leucophaeus contained two areas in the control region, between coordinates 15,643-15,673 and 16,396-16,422, with missing/ambiguous data, these were removed from the bait design. For H. niger, one ambiguous base within CYTB was adjusted to match the corresponding position in H. leucophaeus and H. equinus (R, position 14,921). All genomes were divided into 60-mer probes with 3 bp tiling using a custom python script. To avoid underrepresentation of the region where the mitochondrial genome was cut to linearize it, 59 bp from the beginning of each of the mitochondrial genomes were copied to its end. On the same array, mitochondrial genomes for five Caprinae species were present for enrichment of samples from a different project. Prior to capture, libraries were pooled in equimolar amounts according to their mitochondrial endogenous content as determined via shotgun sequencing (Supplementary Table S3 and S4) 43 . Due to its already high endogenous content, the Stockholm sample was not captured. The capture product was sequenced using custom primers on an Illumina NextSeq500 at the University of Potsdam generating 75 bp single-end reads.
Bioinformatic procedures and analyses. Mitochondrial genome reconstruction. For the reconstruction of the complete mitochondrial genome of the blue antelope, both the Stockholm (NRM 590107) and the Vienna (NMW ST 715) specimens were considered since they are both mounted skins and as such their attribution to the blue antelope is likely correct. However, the Stockholm specimen was chosen as it yielded the best quality DNA (highest endogenous content). For all reads, Illumina adapter sequences (1 bp overlap) and reads < 30 bp were removed with Cutadapt v1.12 44 . Next, potential PCR-duplicates were removed by sequence identity (--by-seq) with SeqKit v0.12.0 rmdup 45 and quality trimming was conducted for a quality of 30 at the 3′ end of the reads with Cutadapt v1.12 44 . For mitochondrial genome assembly, MITObim v1.9.1 46,47 , which employs the mapper Mira v4.0.2 48 , was used with the --quick option and default settings except for the mismatch value and a limit of 500 iterations. All runs stopped before 500 iterations were reached. Details of the run results are shown in Supplementary Table S6. As reference bait sequences, the complete mitochondrial genomes of the two closest relatives H. niger (NC_020713) 42 and H. equinus (NC_020712) 42 were used. For each bait, mismatch values from zero to six were tested (Supplementary Table S6). The assembled reads were filtered for a minimum mapping quality of 30, sorted using samtools view and sort v0.1.19 49  www.nature.com/scientificreports/ calling, a minimum coverage of 3 × and the 'trim to reference sequence' option. Positions where an insertion was called relative to the reference were removed if the insertion was covered by less than three reads. To improve the coverage at the edges of the sequences it was necessary to map the obtained reads again to modified versions of the same references. For this, the circular character of the mitochondrial genome was used by cutting the last 100 bp of each reference and concatenating them to the beginning of the sequence to allow reads to map in the region where the reference had been cut before. Consensus sequences were called and insertions removed as described above. Subsequently, the first 100 bp of each sequence were cut and concatenated back to the end of the sequences to match the original reference again. Then the consensus sequences of both mappings were combined using a 50% majority rule threshold for base calling. In a final step, the sequences were edited such that all started with tRNA-Phe and ended with the control region. This resulted in improved sequences for the samples from Glasgow ( An alignment applying the MAFFT algorithm v7.450 51,52 to the consensus sequences of the reference that each individual mapped the most base pairs to was built using default parameters as implemented in Geneious R10 v10.2.3 53 with the gap opening penalty altered to 1.605. In this alignment, the references of H. niger and H. equinus from GenBank were included. Since the new reference of H. leucophaeus is identical to the sample from Stockholm, the reference was not included. As outgroup the mouflon (Ovis aries musimon) was included (HM236184) 55 . The control region from each sequence was removed from the alignment because this region is challenging to align for divergent sequences. The final alignment (four taxa) was used to generate a maximumlikelihood tree with 100 bootstrap replicates using RAxML 8.2.10 with the GTR + G substitution model (Fig. 3) 56 .
To rule out the influence of reference bias, a consensus sequence for each sample for each of the three Hippotragus references was generated in Geneious R10 v10.2.3 53 , again using a 75% majority rule threshold for base calling, a minimum coverage of 3 × and the 'trim to reference sequence' option. No insertions were deleted and no improvement of the edges of the sequences was performed. The control region was removed. An alignment and a maximum-likelihood tree using RAxML 8.2.10 with GTR + G 56 were built separately for the consensus sequences of each of the three references using the parameters described above (Supplementary Figs. S16-S18). The alignments were improved by shifting gaps by hand.
Intraspecific mitochondrial diversity comparison. The consensus sequences of the four specimens identified as blue antelopes (see "Results" section) were aligned using the MAFFT algorithm v7.450 51,52 with default parameters as implemented in Geneious R10 v10.2.3 53 . All ambiguities including missing data were removed from the alignment, leaving 6,300 bp. Subsequently, from this alignment a median joining network was built and the nucleotide diversity calculated using POPART v1.7 57 with default parameters.
Interspecific mitochondrial diversity comparison. The genetic diversity of the blue antelope specimens was estimated with an average pairwise distance (k) approach, where k is the average number of substitutions in a pairwise individual comparison. For comparison, six wild ungulate species with a wide range of characteristics were chosen: one closely related species (scimitar-horned oryx, Oryx dammah), three species that are known to have low genetic diversity, including the European bison (Bison bonasus), the American bison (Bison bison) and the Przewalski's horse (Equus przewalskii), as well as two species with large geographic ranges, the African buffalo (Syncerus caffer) and the moose (Alces alces). For each of the six comparison species, all sequences considered reliable were downloaded from GenBank. If a sequence was uploaded to GenBank only once, but was found in more than one individual within a study, it was included as many times as individuals with it were present in the respective study (Supplementary Table S5). The control region of each sequence was removed. All sequences were aligned together with the four blue antelope consensus sequences (also without the control region) using the MAFFT algorithm v7.450 51,52 with default settings as implemented in Geneious R10 v10.2.3 53 . All positions containing ambiguities/missing data or gaps were excluded from the alignment to ensure that only homologous positions were compared. Subsequently, for each species four sequences were randomly drawn without replacement using seqtk v1.3 (https ://githu b.com/lh3/seqtk ), then aligned with MAFFT v7.453 51,52 using the default settings for gap opening penalty (--op 1.53) and offset value (--ep 0.123). The pairwise distance was calculated using the command line version of MEGA-CC 10 58,59 applying the 'complete deletion' option for the treatment of gaps and missing data. This was repeated 1,000 times for each of the species. Finally, the average was calculated using an R script. For the blue antelope, this was performed once with the four available sequences. The results were visualized using RStudio v1.1.423 60 (https ://www.rstud io.com/) with R v3.63 61 (https ://www.R-proje ct.org/).

Results
Processing the Stockholm and Vienna samples on the Pippin Prep resulted in 4.5 × more mappable endogenous data for the Stockholm and 13.5 × more for the Vienna sample compared to their respective original libraries. For the mitochondrial genome reconstruction, the results for mismatch values zero contained many unknown bases. Therefore, this value appeared too strict and the results were dismissed. The results using H. niger as assembly bait sequence for mismatch values one and two were identical  Table S2).

Species identity of the museum specimens.
The mitochondrial mapping success rates show that the mounted skins from Stockholm (NRM 590107) and Vienna (NMW ST 715), the skull fragments from Leiden 1 (RMNH.MAM.20681.a) and the pair of horns from Uppsala (UPSZMC 78488) are conspecific. Since Leiden 1 is probably part of the skull of the blue antelope lectotype specimen and the Stockholm and Vienna mounts are blue antelopes beyond reasonable doubt based on their morphology, these four specimens can be attributed to Hippotragus leucophaeus (Fig. 2, Supplementary Table S2).
All four blue antelope samples map second best to the sable and all sable specimens map second best to the blue antelope mitochondrial genome. On the other hand, all roan samples map almost equally well to the sable and blue antelope mitochondrial genomes, with a slightly higher value for the blue antelope (Fig. 2).
Since the identity of all tested samples could be clearly established, this allowed for a tentative estimate of the genetic diversity of the blue antelope museum specimens.
Low levels of genetic diversity in blue antelope museum specimens. The comparison of the four unambiguous blue antelope specimens using a median joining network, taking only the positions into account where all specimens had unambiguous available data and including the control region, revealed only six segregating sites (Fig. 4) and a nucleotide diversity (pi) of 0.0005. The sequences of the Stockholm and Uppsala specimens were identical in this comparison.
In the pairwise distance comparison, the blue antelope sequences from Stockholm, Vienna, Uppsala and Leiden 1 were compared. The resulting k value was compared to the k values of 1,000 comparisons of four randomly chosen sequences of homologous positions of six wild ungulate species (Fig. 5). In comparison to the other species, the blue antelope shows a rather modest k value (3.2), similar to that in the American bison.

Discussion
Our results show that only four of the ten analyzed specimens are in fact blue antelopes, while the remaining six belong to either roan or sable. The mapping success per reference species (Fig. 2) together with the well-resolved phylogeny (Fig. 3) yield unequivocal evidence as to the species identity of each analyzed specimen. The pattern that all sable map second best to the blue antelope mitochondrial genome and all blue antelopes map second best to the sable mitochondrial genome (Fig. 2) is in accordance with our phylogeny (Fig. 3) and previous results, indicating that the blue and sable antelope are sister species to the exclusion of the roan 23 .
Our results suggest that historical specimens of the blue antelope in museum collections are even rarer than previously thought (although fossil remains are more abundant, see below). Further work should examine the six remaining potential blue antelope specimens not analyzed here, although two of these are currently listed as sable (ZMB MAM 8855) and roan (ZMB MAM 8860), respectively. The blue antelope is therefore among the rarest historically extinct large mammal species in museums. By comparison, the other recently extinct South  Since our study only analyzed mitochondrial DNA, we cannot exclude the possibility that some of our sampled specimens are hybrids. The roan had overlapping distributions with the blue antelope since at least the Last Glacial Maximum 5,65 , and potentially longer. In historical times Du Plessis 66 found no evidence of roan antelope as far south as the Cape Floristic Region, but Mohr 17 considered a sketch by R. J. Gordon from 1778 near Plettenberg Bay to be more likely of a roan than a blue antelope. This specimen is considered to be the holotype of the roan by Grubb 67 . This would make this the last roan to be documented in the southern Cape before its extirpation in that region (yet undescribed as a species) 17,65 . However, population level nuclear DNA data from the blue antelope, which are not currently available, and from species it might have hybridized with, would be required to investigate whether any of the blue antelope museum specimens represent hybrids.
Osteological material, especially in the form of skulls and teeth, is extraordinarily important for comparative morphological, morphometric and taxonomic studies. It is widely used as reference material for museum specimens and archaeological and paleontological material to study and delineate species. For the blue antelope Groves & Westwood 20 stated that 'The authentication of even one skull of this extinct antelope would be a significant event. ' With the only historical osteological remains of the blue antelope represented by the skull fragments in Leiden, the horns in Uppsala, and some phalanges within the Vienna mounted skin, our results suggest that there is currently not a single skull that could be used as a reference against which to evaluate historical or fossil specimens. This creates the problem whereby archaeological and paleontological specimens (mostly teeth) identified as blue antelope through morphological characters (namely distinguished from roan by smaller molars and premolars, and from sable by larger premolars) 5,65 cannot be directly compared with the historical skins, horns and bone fragments identified as blue antelope through DNA analyses. This difficulty is further demonstrated by the many misidentified skulls and frontlets presented in our study, which is a known issue in museum collections 68 . This means that, while the fossil and historical specimens assigned to the blue antelope are very likely one and the same species, this cannot be directly demonstrated. Non-invasive imaging technologies like X-ray or µCT should be applied to investigate whether the Leiden mounted skin possesses a complete (or partial) skull. Ultimately, if a skull is present, it could be scanned, and a cast could be produced by means of a 3D printer. The same could be done for the Stockholm specimen to be certain that it really does not contain a skull.
With only four to maximally ten blue antelope specimens existing, they should be highly valued in museum collections. Since misidentification can go both ways, it is also possible that further specimens of the blue antelope exist, but are mislabeled as sable or roan. This is especially true in light of the fact that their status as separate species was not settled for quite some time 10,69 and that due to its early extinction, the blue antelope was never well known. Further blue antelope specimens might be privately owned in South Africa or elsewhere. For now, the genetic analysis of known potential blue antelope specimens in collections should be prioritized to perhaps increase the number of unequivocal individuals of this iconic extinct mammal species. www.nature.com/scientificreports/ Our sample size is too small to draw well-founded conclusions about the genetic diversity of the blue antelope population in the seventeenth and eighteenth centuries. However, our results suggest that the blue antelope exhibited low genetic diversity in historical times, as previously suspected 16,22 , similar to the low levels seen in the European 70 and American bison 71 . Low genetic diversity itself does not necessarily lead to extinction 72 , but it can render species more susceptible to diseases [73][74][75][76] . However, there is no evidence that the blue antelope was affected in this way. Rather, low genetic diversity itself is likely to be an indication of low population size, and this would certainly have made the blue antelope much more susceptible to extinction in the face of hunting or environmental disturbance, as suggested by Kerley et al. 16 and Faith & Thompson 6 .
Of the four confirmed blue antelope specimens, all lack verified, detailed location information. Hence, we cannot draw conclusions concerning their geographic origin. However, the two specimens from Swedish museums-Stockholm and Uppsala-have most likely identical mitochondrial genomes, suggesting that they might have originated from the same herd. This seems likely considering that both were acquired by C. P. Thunberg, who was in charge of the collection in Uppsala at the time.
For a better estimate of the genetic diversity of the blue antelope in the seventeenth and eighteenth centuries, larger sample sizes would be needed. For now, this is not feasible since there is an insufficient number of potential blue antelope specimens that remain to be tested. However, there are large numbers of archaeological and www.nature.com/scientificreports/ paleontological specimens attributed to this species from late Pleistocene and Holocene sites in the southern and western Cape region 7 , with which ancient DNA analyses can be carried out to address questions of past population size changes. There is an interesting parallel to the bontebok (Damaliscus pygargus pygargus) that had a similar historical distribution and was also endemic to the Cape Floristic Region 15,66,77 . Like the blue antelope, the bontebok is thought to have been severely impacted by hunting and pastoralism, resulting in fragmented populations, bringing the species close to extinction at the beginning of the nineteenth century 1,78 . Its genetic diversity is considered to be low today 79 , and its survival is only thanks to the efforts of private landowners who enclosed some of the remaining animals 1 . Unfortunately, the blue antelope went extinct before similar measures could be taken.
The blue antelope was likely one of the inhabitants of the Palaeo-Agulhas plain, the coastal platform off the southern Cape that was exposed during marine regressions (glacials) and provided a nutrient-rich ecosystem that supported diverse ungulate communities. However, during high sea levels (interglacials), the inhabitants would have been restricted to the nutrient-poor and therefore suboptimal fynbos habitats [80][81][82][83][84] where they might have undergone population fragmentation 6,84 . On the other hand, the blue antelope (like other species) clearly survived such Pleistocene climatic fluctuations without going extinct 6,84,85 . Kerley et al. 16 , based on historical sightings, estimated a total population size of as few as 370 individuals in only a single population at the time of the arrival of the European colonists, which would have rendered the species already highly threatened 6,84,85 . Therefore, it seems likely that other factors must have contributed to its demise in historical times. Resource competition with livestock, habitat deterioration in addition to a suboptimal and probably fragmented habitat likely put the remaining blue antelope population under extreme pressure at the time of European colonization 6,84 . Unsustainable hunting and further habitat transformation at the time would have been the last contributing piece that led to the extinction of the species 84 .

Conclusions
We show that the blue antelope is not only blue but also lonesome, with only four unequivocal historical specimens in collections today: Stockholm and Vienna (mounted skins), Leiden (skull fragments) and Uppsala (pair of horns). The mounted skins from Leiden and Paris (although the former is probably from the same individual as the skull fragments) can be considered blue antelopes as well. In addition, there are four more potential specimens (in Berlin, Brussels and London). This makes it one of the scarcest historical mammal species in museums, with currently no skull identified that could serve as a comparative morphological reference. Our study is yet another example that misidentifications can be an issue in museum collections, which may seriously impact morphological studies. It highlights the promising potential of the application of archival DNA to resolve existing uncertainties of museum specimens. Furthermore, our preliminary assessment hints at a rather low level of mitochondrial diversity within the blue antelope 'museum population' . www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.