A Chromosome-Scale Genome Assembly of the Okapi (Okapia johnstoni).

The okapi (Okapia johnstoni), or forest giraffe, is the only species in its genus and the only extant sister group of the giraffe within the family Giraffidae. The species is one of the remaining large vertebrates surrounded by mystery because of its elusive behavior as well as the armed conflicts in the region where it occurs, making it difficult to study. Deforestation puts the okapi under constant anthropogenic pressure, and it is currently listed as "Endangered" on the IUCN Red List. Here, we present the first annotated de novo okapi genome assembly based on PacBio continuous long reads, polished with short reads, and anchored into chromosome-scale scaffolds using Hi-C proximity ligation sequencing. The final assembly (TBG_Okapi_asm_v1) has a length of 2.39 Gbp, of which 98% are represented by 28 scaffolds > 3.9 Mbp. The contig N50 of 61 Mbp and scaffold N50 of 102 Mbp, together with a BUSCO score of 94.7%, and 23 412 annotated genes, underline the high quality of the assembly. This chromosome-scale genome assembly is a valuable resource for future conservation of the species and comparative genomic studies among the giraffids and other ruminants.

Ruminants (Giraffidae and Bovidae) from Kanapoi.

We update here our recent revision of the Kanapoi ruminants and describe recently collected material. We now regard the occurrence of reduncins as doubtful, we revise the identification of a large raphicerin as being more probably Gazella, and we add Gazella cf. janenschi and the Cephalophini to the faunal list. New material of Tragelaphus kyaloi suggests that this species held its head unlike other tragelaphins, and was not an exclusive dedicated browser, but Kanapoi pre-dates the Pliocene change of Sivatherium, Aepyceros, Alcelaphini, and even Tragelaphini toward more grazing diets. Kanapoi shares several ruminant taxa with sites in Ethiopia and Tanzania, attesting to latitudinal exchanges.

Cryopreservation of Giraffe Epidydimal Spermatozoa Using Different Extenders and Cryoprotectants.

Giraffe numbers have plummeted over the last 30 years by 30-40%. Thus, their conservation status has been raised from least concern to vulnerable. Efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques (ART) such as sperm cryopreservation could help preserve the genetic diversity of giraffe subspecies and, when used for artificial inseminations, enhance genetic exchange between isolated populations. However, to date, the post-thaw motility of recovered sperm has been low and inconsistent. In this study, epididymal sperm collected from the testes of giraffes (n = 7) was frozen in three different extenders, namely, BotuCrio, Steridyl, and test egg yolk (TEY), each supplemented with one of two different cryoprotectants (5% glycerol or a mix of 1% glycerol and 4% methylformamide) and frozen over liquid nitrogen vapor. Across all three extenders, sperm showed significantly better post-thaw results when frozen with a mix of glycerol and methylformamide compared with glycerol alone. Sperm frozen with TEY and a mix of glycerol and methylformamide achieved superior post-thaw total and progressive sperm motility of 57 ± 3% and 45 ± 3%, respectively. These results show the benefit of using alternative cryoprotectants for freezing giraffe spermatozoa and could aid in the application of ARTs for giraffe subspecies or the closely related endangered Okapi.

Large giraffids (Mammalia, Ruminantia) from the new late Miocene fossiliferous locality of Kemiklitepe-E (Western Anatolia, Turkey).

Kemiklitepe is a well-known locality with four recognised fossiliferous horizons, KTA to KTD, which have yielded a plethora of mammalian remains. Previous taxonomic studies indicate the presence of three giraffid taxa: Samotherium major and Palaeotragus rouenii from the uppermost three horizons, KTA, KTB and KTC, as well as Palaeotragus rouenii and Samotherium? sp. from the lowermost KTD horizon. In this study a new locality, Kemiklitepe-E, is presented for the first time. Kemiklitepe-E is located approximately 350 m NW of the classic Kemiklitepe locality. The fossiliferous sedimentary rocks at Kemiklitepe-E occur at the same stratigraphic level as localities KTA, KTB and KTC. The preliminary faunal list includes representatives of Proboscidea, Chalicotheriidae, Equidae, Bovidae and Giraffidae. Comprehensive descriptions and comparisons of the Kemiklitepe-E Giraffidae specimens suggest the co-occurrence of two large giraffids: Samotherium major and Helladotherium duvernoyi. Samotherium major, previously documented from this region, is the most common taxon at Kemiklitepe. Helladotherium duvernoyi is rare at Kemiklitepe and here reported for the first time. The two taxa coexisted during the middle Turolian in Greece and Western Anatolia. In addition, it is suggested that specimens of Samotherium? sp described from KTD possibly belong to Samotherium neumayri. Based on the stratigraphic position of fossiliferous rocks, as well as the faunal data presented herein, the newly discovered locality is considered to be of middle Turolian (MN12) age.

Glycosylation and immunocytochemistry of binucleate cells in pronghorn (Antilocapra americana, Antilocapridae) show features of both Giraffidae and Bovidae.

Although the pronghorn (Antilocapra americana) resembles an antelope, its nearest relatives are the giraffe and okapi. In this study we have examined the placentae of 6 pronghorns using lectin- and immunocytochemistry to identify giraffid and bovid features. Binucleate cells (BNC) of the placenta exhibited features intermediate between those of the giraffe and bovine; Dolichos biflorus agglutinin binding - strong in the bovine BNC and absent in the giraffe - was evident in only a subpopulation of BNC while binding to blood vessels, as in the giraffe. Binding of Phytolacca americana agglutinin resembled that of the giraffe and okapi whereas many other glycans were found in all four clades. PAG antigens were similar to bovine and okapi but not giraffe. In summary, although the pronghorn outwardly resembles an antelope, placental BNC show giraffid features. Although each clade has its own individual characteristics, there are far more similarities than differences between them, emphasizing the common ancestry of all four clades.

Fossil evidence and stages of elongation of the Giraffa camelopardalis neck.

Several evolutionary theories have been proposed to explain the adaptation of the long giraffe neck; however, few studies examine the fossil cervical vertebrae. We incorporate extinct giraffids, and the okapi and giraffe cervical vertebral specimens in a comprehensive analysis of the anatomy and elongation of the neck. We establish and evaluate 20 character states that relate to general, cranial and caudal vertebral lengthening, and calculate a length-to-width ratio to measure the relative slenderness of the vertebrae. Our sample includes cervical vertebrae (n=71) of 11 taxa representing all seven subfamilies. We also perform a computational comparison of the C3 of Samotherium and Giraffa camelopardalis, which demonstrates that cervical elongation occurs disproportionately along the cranial-caudal vertebral axis. Using the morphological characters and calculated ratios, we propose stages in cervical lengthening, which are supported by the mathematical transformations using fossil and extant specimens. We find that cervical elongation is anisometric and unexpectedly precedes Giraffidae. Within the family, cranial vertebral elongation is the first lengthening stage observed followed by caudal vertebral elongation, which accounts for the extremely long neck of the giraffe.

The cervical anatomy of Samotherium, an intermediate-necked giraffid.

Giraffidae are represented by many extinct species. The only two extant taxa possess diametrically contrasting cervical morphology, as the okapi is short-necked and the giraffe is exceptionally long-necked. Samotherium major, known from the Late Miocene of Samos in Greece and other Eurasian localities, is a key extinct giraffid; it possesses cervical vertebrae that are intermediate in the evolutionary elongation of the neck. We describe detailed anatomical features of the cervicals of S. major, and compare these characteristics with the vertebrae of the two extant giraffid taxa. Based on qualitative morphological characters and a quantitative analysis of cervical dimensions, we find that the S. major neck is intermediate between that of the okapi and the giraffe. Specifically, the more cranial (C2-C3) vertebrae of S. major represent a mosaic of features shared either with the giraffe or with the okapi. The more caudal (C5-C7) S. major vertebrae, however, appear transitional between the two extant taxa, and hence are more unique. Notably, the C6 of S. major exhibits a partially excavated ventral lamina that is strong cranially but completely absent on the caudal half of the ventral vertebral body, features between those seen in the giraffe and the okapi. Comprehensive anatomical descriptions and measurements of the almost-complete cervical column reveal that S. major is a truly intermediate-necked giraffid. Reconstructions of the neck display our findings.

On reconstructing Giraffa sivalensis, an extinct giraffid from the Siwalik Hills, India.

Giraffa sivalensis occurred during the Plio-Pleistocene period and probably represents the terminal species of the genus in Southern Asia. The holotype is an almost perfectly preserved cervical vertebra of disputed anatomical location. Although there is also uncertainty regarding this animal's size, other specimens that have been assigned to this species include fragments of two humeri, a radius, metacarpi and teeth. Here we estimate neck length, leg length and body mass using interspecific and, unusually, ontogenetic allometry of extant giraffe skeletal parameters. The appropriateness of each equation to estimate body mass was evaluated by calculating the prediction error incurred in both extant giraffes (G. camelopardalis) and okapis (Okapia johnstoni). It followed that the equations with the lowest prediction error in both species were considered robust enough to use in G. sivalensis. The size of G. sivalensis, based on the holotype, is proposed as 400 kg (range 228 kg-575 kg), with a neck length of approximately 147 cm and a height of 390 cm. The molar lengths of tooth specimens considered agree with this size estimate. The humerus was the most appropriate long bone to establish body mass, which estimates a heavier animal of ca 790 kg. The discrepancy with the vertebral body weight estimate might indicate sexual dimorphism. Radial and metacarpal specimens estimate G. sivalensis to be as heavy as extant giraffes. This may indicate that the radius and metacarpus are unsuitable for body mass predictions in Giraffa spp. Alternatively, certain long bones may have belonged to another long legged giraffid that occurred during the same period and locality as G. sivalensis. We have concluded that if sexual dimorphism was present then males would have been about twice the size of females. If sexual dimorphism was not present and all bones were correctly attributed to this species, then G. sivalensis had a slender neck with a relatively stocky body.