Molecular genetics of Rhabdomys subspecies boundaries : phylogeography of mitochondrial lineages and chromosomal fluorescence in situ hybridization

Rambau, Ramugondo Victor (2003-03)

Thesis (PhD)--University of Stellenbosch, 2003.

Thesis

ENGLISH ABSTRACT: The geographic genetic population structure and evolutionary history of the African four-striped mouse, Rhabdomys pumilio, was investigated using mitochondrial (mtDNA) cytochrome b gene (1140 bp) and control region (994 bp) sequences and a combination of cytogenetic banding techniques (G- and C-banding), and fluorescence in situ hybridization. Two cytotypes (2n = 46 and 2n = 48) were identified by cytogenetic analysis. No evidence of diploid number variation within populations was found nor were there differences in gross chromosome morphology, or subtle interchromosomal rearrangements at levels detected by ZOO-FISH. The comparative painting data (using the complete suite, N = 20, of Mus musculus chromosome specific painting probes) show that 10 mouse chromosomes have been retained as chromosomal arms, or intact chromosome blocks within the R. pumilio genome, six produced double signals, while the remaining four hybridized to three or more R. pumilio chromosomes. In total, the 20 mouse chromosome paints detected 40 segments of conserved synteny. Their analysis revealed eight R. pumilio specific contiguous segment associations, a further two that were shared by R. pumilio and other rodents for which comparable data are available, the Black (Rattus rattus) and Norwegian (Rattus nONegicus) rats, but not by the Chinese hamster, Cricetulus grise us. The results suggest that mouse chromosomes 1, 10, and 17 have undergone extensive rearrangements during genome evolution in the murids and may be useful markers for enhancing our understanding of the mode and tempo of chromosome evolution in rodents. Following initial studies using control region sequences, the phylogeographic appraisal of R. pumilio was done using cytochrome b gene sequences. Analyses based on a variety of analytical procedures resulted in the detection of two major mtDNA lineages that correspond roughly to the xeric and mesic biotic zones of southern Africa. One clade comprises specimens with 2n = 48, and the other representatives of two cytotypes (2n = 48 and 2n = 46). The mean sequence divergence (12.0%, range 8.3% -15.6%) separating the two mtDNA clades is comparable to among-species variation within murid genera suggesting their recognition as distinct species, the prior names for which would be R. dilecfus and R. pumilio. Low sequence divergences and the diploid number dichotomy within the mesic lineage support the recognition of two subspecies corresponding to R. d. dilecfus (2n = 46) and R. d. chakae (2n = 48). The data do not support subspecific division within the nominate, R. pumilio. Molecular dating places cladogenesis of the two putative species at less than 5 million years, a period characterised by extensive climatic oscillations which are thought to have resulted in habitat fragmentation throughout much of the species' range.

AFRIKAANSE OPSOMMING: Die geografiesebevolkingsstruktuur en evolusionêre verwantskappe binne die Afrika streepmuis, Rhabdoys pumilio, is ondersoek deur middel van mitochondriale ONS volgordebepaling van die geenfragment sitochroom b (1140 basispare) en die reguleerstreek (994 bp) in kombinasie met sitogenetiese tegnieke (G- en Cbandkleuring en f1uoreseerende in situ hibridisasie). Twee sitotipes (2n = 46 en 2n = 48) is geidentifiseer deur sitogenetiese analasie. Geen bewys van variasie in die 2n chromosoomgetal binne bevolkings is gevind nie. Verder is daar ook geen verskil in die morfologies struktuur van chromosome aanwesig binne bevolkings nie. Vergelykende data (verkry met behulp van die N = 20 Mus musculus chromosoomspesifiekepeilers) dui daarop dat 10 muis chromosome behoud gebly het as chromosoomarms of chromosoomblokke binne die R. pumilio genoom. Ses peilers het dubbel seine gelewer terwyl die oorblywende vier peilers gehibridiseer het aan drie of meer R. pumilio chromosome. In totaal het die 20 muischromosoomverwe 40 konserwatiewe segmente geidentifiseer. Die analise dui agt R. pumilio spesifieke aaneenlopende segmentassosiasies aan, met 'n addisionele twee wat deur R. pumilio en ander muisagtiges vir wie vergelykende data beskikbaar is, byvoorbeeld die swart (Rattus rattus) en Noorweegse (R. norvegicus) rot maar nie die Chinese hamster, Cricetulus grise us, gedeel word. Die resultate stel voor dat muischromosoom 1, 10 en 17 ekstensiewe herrangskikkings ondergaan het gedurende die genoom evolusie binne die Muridae en dat hulle waarskynlik waardevolle merkers kan wees om beide die patroon en tempo van chromosome evolusie in muisagtiges verder te kan verstaan. Die filogeografiese verwantskappe binne R. pumilio is ondersoek deur middel van ONS volgordebepalings van die reguleerstreek asook sitochroom b. Die resultate van hierdie studie het twee divergente mitochondriale ONS eenhede ontdek wat gekorreleer kan word met xeriese en mesiese klimaatsones binne suidelike Afrika. Een groep bestaan uit diere met 2n = 48, terwyl die ander genetiese groep twee sitotipes (2n = 46 en 2n= 48) insluit. 'n Gemiddelde genetiese divergensie van 12.0% (varieer tussen 8.3% - 15.5%) verdeel die twee mtDNS-groepe en is vergelykbaar met tussenspesievariasie binne ander muisagtige genera, wat moontlik daarop dui dat twee verskillende spesies teenwoordig is; die voorgestelde name is R. di/ectus en R. pumilio. Lae genetiese divergensie binne die mesiese groep versterk die moontlike teenwoordigheid van twee subspesies, R. d. di/ectus (2n = 46) en R. d. chakae (2n = 48). Die data verleen egter nie steun aan die divisie binne R. pumilio nie. Molekulêre datering van die twee spesies dui daarop dat die divergensie ten minste 5 miljoen jaar gelede plaasgevind het. Die periode was gekarakteriseer deur ekstensiewe klimaatsossilasies, wat gely het tot habitat fragmentasie in die spesie se verspreidingsgebied.

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