This is the first study using nuclear sequences and analyzing the Nubian giraffe

Traditionally, one giraffe species and up to eleven subspecies have been recognized []; however, nine subspecies are commonly accepted []. Even after a century of research, the distinctness of each giraffe subspecies remains unclear, and the genetic variation across their distribution range has been incompletely explored. Recent genetic studies on mtDNA have shown reciprocal monophyly of the matrilines among seven of the nine assumed subspecies []. Moreover, until now, genetic analyses have not been applied to biparentally inherited sequence data and did not include data from all nine giraffe subspecies. We sampled natural giraffe populations from across their range in Africa, and for the first time individuals from the nominate subspecies, the Nubian giraffe, Giraffa camelopardalis camelopardalis Linnaeus 1758 [], were included in a genetic analysis. Coalescence-based multi-locus and population genetic analyses identify at least four separate and monophyletic clades, which should be recognized as four distinct giraffe species under the genetic isolation criterion. Analyses of 190 individuals from maternal and biparental markers support these findings and further suggest subsuming Rothschild’s giraffe into the Nubian giraffe, as well as Thornicroft’s giraffe into the Masai giraffe []. A giraffe survey genome produced valuable data from microsatellites, mobile genetic elements, and accurate divergence time estimates. Our findings provide the most inclusive analysis of giraffe relationships to date and show that their genetic complexity has been underestimated, highlighting the need for greater conservation efforts for the world’s tallest mammal.

The survey genome assembly of a Kordofan giraffe produced 5,042 scaffolds > 10,000 bp. Repeat identification identified similar occurrences and relative numbers of short interspersed elements, endogenous retroviruses, and DNA transposons as in other Ruminantia [], which may be suitable markers for conservation genetics. The genome assembly identified 2,239 protein-coding genes, of which 588 are orthologous to other mammals. After rigorous filtering, ≈540,000 bp remained for phylogenetic analyses, which places giraffe as sister group to cattle, antelope, and sheep and allows estimating the emergence of Giraffidae at 28.2 mya with high accuracy ( Figure 3 D), slightly longer ago than previously suggested []. Extracting microsatellites with >21 repeats identified 54 putatively informative loci.

Pattern and timing of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes.

A genome survey sequencing of the Java mouse deer (Tragulus javanicus) adds new aspects to the evolution of lineage specific retrotransposons in Ruminantia (Cetartiodactyla).

Bayesian multi-locus clustering analysis shows that nuclear loci support four distinct groupings ( Figure 3 A). The highest ΔK [] is observed for K = 4 clusters, with one cluster each corresponding to (1) southern giraffe (Angolan and South African), (2) the Masai giraffe (including Thornicroft’s giraffe), (3) the reticulated giraffe, and (4) the northern giraffe (West African, Kordofan, Nubian, and Rothschild’s giraffe). At K = 3, the northern cluster and reticulated giraffe are merged, while the other clusters remain distinct. Using K = 5 or higher values does not reveal additional clusters but rather shows increasing admixture. Analyzing the northern giraffe cluster separately shows that the West African giraffe is somewhat distinct but shares haplotypes with the Nubian giraffe. The southern giraffe cluster does not show further structuring ( Figures S3 A–S3C). Accordingly, principal component analyses (PCAs) of giraffe haplotypes find significant support for only four giraffe groups ( Figure 3 B). PCAs do not find support for additional groups according to mtDNA or traditional subspecies, or for a separate West African cluster ( Figures S3 D and S3E). The distinctness of these four clusters is in addition supported by significant fixation index (Fst) values and by Bayesian posterior probability (BPP) analyses of nuclear data receiving significant support for four clusters. BPP analyses that allow for additional clusters (e.g., West African giraffe being separate), clustering according to the mtDNA data, or six clusters [] lack significant support (p = 0.65, p = 0.32, p = 0.47, respectively). Thus, population genetic, phylogenetic, and network analyses of nuclear sequences demonstrate that the giraffe is genetically well structured into four distinct species. This is consistent with divergence times of 1.25 to 2 million years ago (mya) among the four clusters ( Figure 3 C).

(D) Time-calibrated phylogenomic analysis based on 540,000 bp of protein coding sequences. The divergence time of Giraffidae was estimated at 28.7 mya.

(C) Divergence times among giraffe species estimated by BEAST to 1.99, 1.89, and 1.25 mya, respectively.

(B) PCA axes 1–2 for four distinct giraffe clusters (1: southern; 2; northern; 3: Masai; 4: reticulated giraffe) according to STRUCTURE clusters (K = 4). The x axis explains 12.5% and the y axis 7.15% of variation. The oval outlines represent 95% confidential intervals and are colored after STRUCTURE clusters. Non-overlapping frames denote significantly different clusters. Analyses along axes 1–3 (data not shown) produced nearly identical results.

(A) STRUCTURE analysis of seven nuclear loci for 105 individuals. Vertical bars show the membership in a cluster for each individual. Separate colors represent separate clusters. K = 4 has the highest credibility and shows well-resolved groups: blue: southern cluster (South African plus Angolan giraffe); green: Masai giraffe; orange: reticulated giraffe; yellow: northern cluster of the remaining subspecies. K = 5 or higher shows no further resolution.

Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study.

The phylogenetic analysis of mtDNA from all nine giraffe subspecies ( Figure 2 B) produced a tree that conforms to previous analyses [], including the reciprocal monophyly of the seven distinct subspecies clades (p > 0.95). The lack of sequence variation shows that the Masai and Thornicroft’s giraffe are not genetically distinct, supporting their grouping under the Masai giraffe []. Notably, mtDNA analyses also do not place the Nubian giraffe into a separate, monophyletic clade, but instead, two Nubian giraffe individuals group with the Kordofan giraffe, and three Nubian giraffe individuals group with Rothschild’s giraffe ( Figure 2 B). Placing all Nubian giraffe individuals as a monophyletic sister group to either subspecies can be significantly rejected (approximately unbiased < 0.05). Specifically, the Nubian giraffe from Gambella National Park, Ethiopia, and two individuals from Bandingilo National Park, South Sudan, east of the Nile River, are placed in Rothschild’s giraffe. In contrast, the Nubian giraffe individuals from northwest of Shambe National Park, west of the Nile River, South Sudan, group with the Kordofan giraffe ( Figure 2 B), despite varying pelage patterns ( Figures 2 C–2E).

Parsimony haplotype networks from the nuclear intron sequences revealed in general a similar pattern, reflecting the differentiation into four major clusters ( Figure S1 ). Haplotype sharing between the Angolan and South African giraffe does not allow distinguishing between them; however, southern giraffe subspecies haplotypes are somewhat distinct. Two of the analyzed loci have exclusive haplotypes for the reticulated giraffe and the Masai giraffe (including Thornicroft’s) that separate these from other giraffe subspecies. Intron 52 is characterized by a 33-bp-long insertion, a remnant of a DNA transposon, exclusive for the Masai giraffe.

Our nuclear and mitochondrial gene analyses clearly show that giraffes are not a homogeneous taxon but are deeply structured into distinct genetic groups. The degree of population genetic differentiation of seven nuclear markers from 105 individuals and concordance to mitochondrial (mt) data of 190 individuals suggests that some of the currently recognized subspecies are distinct species. Our nuclear dataset includes all currently recognized giraffe subspecies ( Table 1 Figure 1 ) and, importantly, the elusive Nubian giraffe (Giraffa c. camelopardalis). A coalescent multi-locus (ML) tree analysis based on individual ML trees distinguishes four clusters: (1) a southern cluster comprising the South African and Angolan giraffe, (2) a Masai cluster corresponding to the Masai giraffe (including Thornicroft’s giraffe []), (3) the reticulated giraffe, and (4) a northern cluster including West African, Kordofan, and Nubian giraffe ( Figure 2 A). The monophyly of these groups is supported by p > 0.95 and is consistent with ML analysis of concatenated sequences.

(B) mtDNA BEAST tree for 190 individuals. Except for the Nubian giraffe (G. c. camelopardalis), all seven subspecies are well separated and form monophyletic groups. The Masai giraffe and Thornicroft’s giraffe are subsumed into one subspecies, G. c. tippelskirchi. Misplaced individuals of the reticulated, Rothschild’s, and South African giraffe are from databases and are likely to represent misidentifications []. The MTNP (Mosi-oa-Tunya National Park) and SNNP (Sioma Ngwezi National Park) giraffes are likely South African giraffes.

(A) A coalescent multi-locus tree from seven nuclear loci (4,294 bp) from okapi and 105 giraffe individuals identifies four monophyletic clades with significant support, p > 0.95: southern giraffe (G. giraffa, G. angolensis), Masai giraffe (G. tippelskirchi), reticulated giraffe (G. reticulata), and northern giraffe (G. antiquorum, G. camelopardalis, G. peralta, G. rothschildi). This grouping is consistent with STRUCTURE, PCA, and BPP analyses ( Figures 3 A and 3B). Asterisks indicate statistically significant support (p > 0.95). Arrowheads indicate Nubian giraffe individuals. Individuals without geographic ID are from databases.

(B) Enlarged view of the South Sudan region. Note that the samples of the putative Nubian giraffe were taken west and east of the Nile River.

(A) Distribution ranges (colored shading) provided by the Giraffe Conservation Foundation [], plotted on a map of Africa ( http://www.naturalearthdata.com/ ). Circles represent sampling locations; for coding, see Figure 2

Giraffe (Giraffa camelopardalis) subspecies [], common names, principal occurrences, and population sizes. Subspecies are listed in order of their approximate appearance from north to south. According to 2015 estimates by the Giraffe Conservation Foundation, the total giraffe population is ∼ 90,000 individuals. See also Table S1 . Data are from Giraffe Conservation Foundation, 2016; http://giraffeconservation.org

G. c. rothschildi and G. c. thornicrofti are now subsumed under G. c. tippelskirchi [

G. c. rothschildi and G. c. thornicrofti are now subsumed under G. c. tippelskirchi [

Discussion

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Foster J.B. The Giraffe: Its Biology, Behaviour and Ecology. 1 Lydekker R. On the subspecies of Giraffa camelopardalis. 2 Dagg A.I.

Foster J.B. The Giraffe: Its Biology, Behaviour and Ecology. 12 Lydekker R. Two undescribed giraffes. 4 Bock F.

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Janke A. Mitochondrial DNA analyses show that Zambia’s South Luangwa Valley giraffe (Giraffa camelopardalis thornicrofti) are genetically isolated. 13 Matschie P. Einige anscheinend noch nicht beschriebene Säugetiere aus Afrika. The giraffe was first described in 1758 in Linnaeus’ Systema Naturae []. As later revealed, Linnaeus based his description on the Nubian giraffe [], corresponding to the nominotypical subspecies, Giraffa camelopardalis camelopardalis. Linnaeus had never seen a living giraffe and referred to 200-year-old descriptions []. Further descriptions of additional giraffe subspecies were later based on variable and taxonomically unreliable morphological traits, such as coat markings, ossicones, and geographic distribution []. As an example, Thornicroft’s giraffe from eastern Zambia was described as a distinct subspecies [] but is morphologically similar to the Masai giraffe that occurs some 500 km to the north. However, genetic studies could not differentiate between the two subspecies, and Thornicroft’s giraffe was therefore synonymized with the Masai giraffe [], as previously described []. Although the known geographical distribution of some giraffe subspecies remains uncertain, we genetically assigned individuals from the Sioma Ngwezi and Mosi-oa-Tunya National Parks in Zambia to the South African giraffe, in contrast to the previous assumption that they were Angolan giraffe.

7 Krumbiegel I. Die Giraffe. 2 Dagg A.I.

Foster J.B. The Giraffe: Its Biology, Behaviour and Ecology. 7 Krumbiegel I. Die Giraffe. 11 Harper F. The nomenclature and type localities of certain Old World mammals. 1 Lydekker R. On the subspecies of Giraffa camelopardalis. 14 ICZN

International Commission on Zoological Nomenclature: International Code of Zoological Nomenclature. 5 Linnaeus C. Systema Naturae per Regna Tria Naturae, Secundum Classes, Ordines, Genera, Species, cum Characteribus, Differentiis, Synonymis, Locis (Tomus I, Editio Decima). Further north, the identification and classification of the nominotypical Nubian giraffe was even less certain []. The giraffe samples from west of the Nile River, which were assumed to be Nubian giraffe based on geography and morphology, turned out to share haplotypes with the Kordofan giraffe, whereas samples from east of the Nile River were genetically similar to Rothschild’s giraffe. This is in agreement with the previous vague suggestion that South Sudan’s giraffe populations from west of the Nile River ( Figure 1 ) could be either Nubian, Rothschild’s, or Kordofan giraffe []. Thus, the putative Nubian giraffe samples that group with the Kordofan giraffe provide the first evidence that giraffes west of the Nile River actually belong to Kordofan giraffe. Since the type locality of the Nubian giraffe had been previously restricted to “Sudan, Sennar” [], east of the Nile River, it is clear that this name refers only to giraffe populations in this region. Yet, Rothschild’s giraffe was also described from east, and further south, of the Nile River []; however, the Nubian and Rothschild’s giraffe are genetically indistinguishable. For nomenclatorial priority reasons [], Article 23, Rothschild’s giraffe, G. c. rothschildi Lydekker, 1903 therefore needs to be synonymized with the earlier described Nubian giraffe, G. c. camelopardalis [].

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Janke A. Nuclear genomic sequences reveal that polar bears are an old and distinct bear lineage. Numerous efforts have been made to define species, but a clear-cut consensus has not yet been reached []. Common to many species concepts is that “species” represent distinct evolutionary units with limited gene flow to other, similar units, and concordance among different character sets has been suggested to support species distinctness []. In giraffe, multi-locus nuclear gene analyses, morphological data [], mtDNA sequences [], and microsatellites [] concordantly suggest genetically distinct groupings within giraffe. Concordance between maternally inherited mitochondrial and biparentally inherited nuclear markers indicates reproductive isolation for at least four giraffe groups. This lack of gene flow is unexpected, because wild giraffes are highly mobile [] and can interbreed in captivity []. However, the genetic differentiation between the four giraffe groups is strong despite their similar appearance. Their divergence times are consistent with previous estimates [] and are on the order of divergence times of other mammals [].

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Bradley R.D. Speciation in mammals and the genetic species concept. (1) southern giraffe (G. giraffa), comprising two distinct subspecies, Angolan giraffe (G. g. angolensis) and South African giraffe (G. g. giraffa);

(2) Masai giraffe (G. tippelskirchi), which includes the formerly recognized Thornicroft’s giraffe;

(3) reticulated giraffe (G. reticulata); and

(4) northern giraffe (G. camelopardalis), which includes Nubian giraffe (G. c. camelopardalis) and its new synonym, Rothschild’s giraffe (G. c. rothschildi), with Kordofan giraffe (G. c. antiquorum) and West African giraffe (G. c. peralta) as distinct subspecies. In the face of small population sizes, especially for the West African giraffe ( Table 1 ), concerted conservation efforts are necessary for preserving these genetically differentiated subspecies. Although previous microsatellite analyses suggested the distinctness of six subspecies, with West African, Angolan, and South African giraffe being separate clusters [], the statistical support is not clear. Our multi-locus coalescent-based analyses on sequence data allow for rigorous statistical testing and did not find support for such a grouping. Based on these data and analyses, and using the genealogical concordance method of phylogenetic species recognition [] and fulfilling the requirements of the genetic species concept [], we suggest recognizing four distinct giraffe species:

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et al. Giraffe genome sequence reveals clues to its unique morphology and physiology. Genome survey approaches have been successful in other species [], including giraffe []. Our assembly and analysis of a Kordofan giraffe genome produced over 500,000 bp of protein coding sequence, numerous putatively informative microsatellites, and mobile element loci. Taken together with the recently published Masai giraffe genome [], these markers are valuable for future analyses on conservation genomics. Thus, for two of the of the four giraffe species, there are now genomes available for conservation research.