Revisiting chromosomal diversity in Otomys irroratus: meiotic behaviour and mitochondrial divergence

Thesis (MSc)--Stellenbosch University, 2019.ENGLISH ABSTRACT: In this study, immunofluorescence allowed the evaluation of meiotic chromosomal behaviour of the vlei rat, Otomys irroratus, a species documented for its karyotypic variability. This species was recently reported to form a species complex, as high sequence divergence values of the mtDNA cyt b gene, revealed a cryptic sister species, O.auratus. Pericentric inversions, tandem- and Robertsonian fusions are found in O. irroratus s.l karyotypes, however these classes of rearrangements are often negatively heterotic. Immunofluorescence was used to provide insight into the persistence of these chromosomal rearrangements believed to be deleterious to the carrier. The aim was to uncover the mechanisms which override the deleterious consequences of chromosome rearrangements, and gain a better understanding of the chromosomal evolution in this species. Firstly, specimens were G- banded to identify chromosomal rearrangements. Secondly, specimens were immunostained to assess the influences of chromosomal rearrangements on meiotic configurations. Lastly specimens were sequenced for the mtDNA cyt b and COI genes to determine to which extent chromosomal rearrangements correlate with mtDNA clades. The cytogenetic screening revealed two general cytotypes. The first has karyotypes which consists of 4 – 9 bi-armed chromosomes (2n= 28 - 30/NA= 44 – 50) and an abundance of rearrangements that appears as either pericentric inversions or centromere shifts on OIR 1, 4, 6,7 and 10. From these, the rearrangement on OIR 7 was described for the first time. The immunofluorescence trials with the SYCP3 antibody, targeting the proteins of the lateral elements of the synaptonemal complex revealed no inversion loops, even in specimens indicated to carry inversions by G-banding data. Rearrangements therefore did not appear to hamper pairing of chromosomes during meiosis. In addition, one specimen was indicated to have two centromeric signals per chromosome, by the CENPC antibody, which targets centromeric proteins. This suggests that a centromeric shift took place recently with the second centromere likely being an evolutionary new centromere. The immunostaining data hence casts doubt onto pericentric inversions, and rather suggests centromeric shifts. Also these rearrangements does not seem to affect reproduction given the data gathered here. The second cytogenetic group (2n= 24, 30/FA= 32 - 37) has karyotypes with acrocentric chromosomes, while the fusion chromosome involving OIR 7, 8 and 12, as well as Rb fusion 1/6 are also present in this group. These cytotypes differ in chromosomal arms (NA), 2n, rearrangements present, and localities where they have been found and is consistent with the two main chromosomal blueprints described for O. irroratus s.l. The mtDNA sequence data revealed two large clades with 5.6% divergence (uncorrected p-distances) that corresponds to the two cytogenetic groups. These groups have the same geographical distribution as the two main cytotypes, i.e. clade 1 encompassing the Western and Eastern Cape, whereas clade 2 stretches north-eastwards from the Eastern Cape to Kwazulu-Natal. These groupings were retrieved with both the cyt b and the COI genes, indicating that the COI may be a useful marker for phylogeographies of small mammals. The sequence divergence values were compared among specimens with and without inversions and revealed that specimens without inversions are equally divergent from specimens with one or many inversions. In effect the data suggests that inversions are not a strong driver for mtDNA divergence in the species complex. The sequence and immunofluorescence data therefore substantiate the idea that these rearrangements exist as neutral polymorphisms.AFRIKAANSE OPSOMMING: In hierdie studie, was immunofluoressensie gebruik om die meiotiese chromosoom gedrag van die vlei rot, Otomys irroratus, ‘n spesie bekend vir sy chromosoom variasie, te ondersoek. Hierdie spesie was onlangs raporteer as ‘n spesies kompleks, aangesien hoë mtDNA skeidings syfers ‘n kriptiesie suster spesie, O. auratus openbaar het. Perisentromeriese inversies, tandem en Robertsoniese fusies kom voor in die kariotipes. Hierdie klasse van chromosoom afwykings word gekoppel aan negatiewe heterose, maar ongeag is hulle, veral perikentriese inversies, volop in die kariotipes van O. irroratus s.l. Immunofluoressensie kon hier die teenwoordigheid van hierdie afwykings help verstaan, en nuwe insigte van die chromosomale evolusie van die spesie skep. Die doel was om te ondersoek of daar meganismes in plek is, wat die nadelige gevolge van hierdie chromosoom afwykings verhoed. Monsters was eerstens analiseer met G-bandbepaling om strukturele chromosoom herrangskikkings te identifiseer, tweedens het immunofluoressensie die invloed van herrangskikkings op meiotiese chromosome bepaal. Laastens, het mitochondriale volgordebepaling van die sitochroom b en sitochroom oksidasie I gene, ondersoek of chromosoom afwykings met mitochondriale groepe korreleer. Die sitogenetiese analise het twee hoof chromosoom groepe opgelewer. Die eerste groep se kariotipes (2n= 28 - 30/Nfa= 44 – 50) het heterokromatiese kort arms op 4 – 9 chromosoom pare, en kan gekenmerk word deur chromosoom herrangskikkings wat as perisentromeriese inversies op outosome OIR1, 4, 6, 7 en 10 voorkom. Die inversie op OIR 7 was hier vir die eerste keer beskryf. Die immunofluoressensie experimente met die SYCP3 antigeen, wat die laterale elemente van die synaptomale kompleks uitwys, het geen inversie lusse getoon nie, ‘n onmisbare kenmerk van inversies. Chromosoom afwykings het nie meiotiese chromosoom paring beinvloed nie. Een monster wat met die CENPC antigeen bestudeer was, het twee sentromeer seine per chromosoom in een paar opgelewer. Dit dui waarskynlik op ‘n nuwe evolusionêre sentromeer wat besig is om te vorm voordat die ou een heeltemal kon verval. Die resultate skep twyfel oor die identiteit van die chromosoom afwykings wat voorheen as perikentriese inversies aangeteken is, aangesien dit beter as sentromeer verskuiwings vertoon. Die tweede chromosoom groep (2n= 24, 30/Nfa= 32 - 37) het geen inversies en bestaan uit kariotipes met akrocentriese chromosome. ‘n Tandem fusie van OIR7, 8, en 12 asook die Robertsoniese fusie van OIR 1/6 kom voor slegs in die groep. Die twee groepe verskil dus in chromosoom vorm (met betrekking tot hoeveelhied fundementele chromosoom arms-Nfa), diploïede getal (2n), afwykings teenwoordig asook geografiese verspreiding en is konsekwent met die twee hoof chromosoom groepe wat voorheen vir die spesies kompleks beskryf is. Die mitokondriale data het twee klades wat met 5.6% (onkorregeerde p- afstande) verskil opgelewer, en stem ooreen met die twee chromosoom groepe. Hierdie klades het dieselfde geografiese verspreiding as die chromosoom groepe naamlik clade 1 wat die Wes- en Oos-Kaap insluit, terwyl clade 2 verder Noord-Oos van die Oos-Kaap strek tot Kwazulu-Natal en Zimbabwe. Beide mitokondriale merkers het dieselfde groepe oopgelewer, wat dui dat die sitokroom oksidasie I merker ook nuttig in filogeografiese studies van klein soogdiere kan wees. Die mitokondriale volgorde verskille was vergelyk tussen monsters wat inversies besit en die wat nie, en daar was gevind dat monsters wat nie inversies het nie, ewe ver geskei is van monsters wat een of meer inversies besit. Die resultate dui daarop dat inversies nie ‘n sterk dryfkrag vir mitokondriale skeiding in die spesies kompleks is nie. Die resulte hier ondersteun die idee dat hierdie chromosoom herrangskikkings as neutrale polimorfismes ontstaan.Master