Molecular cytogenetics of tragelaphine and alcelaphine interspecies hybrids: hybridization, introgression and speciation in some African antelope

Hybridization can occur naturally among diverging lineages as part of the evolutionary process leading to complete reproductive isolation, or it can result from range shifts and habitat alteration through global warming and/or other anthropogenic influences. Here we report a molecular cytogenetic investigation of hybridization between taxonomically distinct species of the Alcelaphini (Alcelaphus buselaphus 2n = 40 × Damaliscus lunatus 2n = 36) and the Tragelaphini (Tragelaphus strepsiceros 2n = 31/32 × Tragelaphus angasii 2n = 55/56). Cross-species fluorescence in situ hybridization provides unequivocal evidence of the scale of karyotypic difference distinguishing parental species. The findings suggest that although hybrid meiosis of the former cross would necessitate the formation of a chain of seven, a ring of four and one trivalent, the progeny follow Haldane's rule showing F1 male sterility and female fertility. The tragelaphine F1 hybrid, a male, was similarly sterile and, given the 11 trivalents and chain of five anticipated in its meiosis, not unexpectedly so. We discuss these findings within the context of the broader evolutionary significance of hybridization in African antelope, and reflect on what these hold for our views of antelope species and their conservation.


Provenance, Cell culture, G-band and FISH analysis:
Provenance: Following reports of interspecific hybridization of Red hartebeest (Alcelaphus buselaphus) and Tsessebe (Damaliscus lunatus) at the Itala Game Reserve (27 o 30 m' 14" S 31 o 18' 31" E) in KwaZulu-Natal, South Africa (Rushworth in litt.), and the historic report of a similar situation that predated anthropogenic influence [1], a conservation intervention that included controlled access to conspecific mates was initiated in the Free State Province where both taxa are important game species. This led to the establishment of F1 Red Hartebeest x Tsessebe hybrids (1 male and 4 females) at the Rustfontein Dam Nature Reserve (29 o 16' 44" S 26 o 36' 00" E) in 2007. Once adult, the F1 male hybrid had exclusive access to a herd comprising hybrid and Tsessebe females for consecutive breading seasons without producing offspring (substantiating the reproductive and clinical evidence of infertility, see below). Following its replacement by a Red Hartebeest bull in January 2013, the female F1 hybrids and two Tsessebe produced calves (Supplementary Fig S3). See [2] for information on the Greater kudu x Nyala hybrid.
Cell culture: Fibroblast cultures were established from skin and callus biopsies of adult male hybrids taken in the field: (i) a Red hartebeest x Tsessebe hybrid (resulting from a male A. buselaphus x female D. lunatus cross) collected at the Rustfontein Dam Nature Reserve (above), and (ii) a Greater kudu x Nyala hybrid (from a male T. strepsiceros x female T. angasii cross) collected from a private game farm in the North West Province of South Africa (October 2013). Biopsy material was grown at 37 °C in Dulbecos Modified Medium (DMEM) enriched with 15% bovine fetal serum. Cell harvest followed conventional procedures; chromosome preparations were aged at 65 °C overnight before G-banding with trypsin. Karyotypes derived from cryopreserved fibroblast cells of the two parental species were similarly prepared and used to facilitate analysis of the hybrids' chromosomes. Chromosomal nomenclature follows that for cattle [3].  . ** The Greater kudu (T. strepsiceros) has 2n=31/32. *** The Nyala (T. angasii) has 2n=55/56. The difference in 2n between sexes is due to a Y;13 translocation which results in both T. strepsiceros 31,X,t(Y;13) and T. angasii males 55,X,t(Y;13) invariably having one chromosome less than females.
In addition to the Y;13 fusion, which is characteristic of all Tragelaphini, the Greater kudu and Nyala karyotypes are characterized by a complex compound chromosomes thought to have arisen from a centric fusion of the orthologous cattle equivalents BTA 22 and BTA 2 followed by a tandem fusion with BTA 11 in the Nyala, and BTA 24 in the Greater kudu [7]. Clinical and reproductive analysis of the male hybrids: (i) T. strepsiceros x T. angasii: see [1] for details.
(ii) A. buselaphus x D. lunatus F1: Several histological and clinical parameters were evaluated. (i) Fresh epididymal sperm smears were prepared in the field and examined microscopically for the presence of spermatozoa following Giemsa staining; (ii) testicular tissue was preserved in Bouin's fluid and subsequently embedded in paraffin wax and sectioned using a microtome. Photomicrographs were taken from mounted preparations stained with Ehrlich's haemotoxylin and eosin (below).
A paucity of germ cells and the absence of spermatids and spermatozoa in tubule cross-sections was noted. Although primary spermatocytes are evident in the preparation, meiotic activity was extremely limited. Additionally, the testis, epididymis, seminal vesicles and prostate were examined by pathologists who concluded that the male reproductive tract appeared normal, but there was a virtual absence of spermatogenesis (degeneration of some microtubules was also observed); the very few spermatozoa seen were considered abnormal. S3: