Evidence of Biparental Mitochondrial Inheritance from Self-Fertile Crosses between Closely Related Species of Ceratocystis

Abstract

Hybridization is recognized as a notable driver of evolution and adaptation, which closely related species may exploit in the form of incomplete reproductive barriers. Three closely related species of Ceratocystis (i.e., C. fimbriata, C. manginecans and C. eucalypticola) have previously been shown to hybridize. In such studies, naturally occurring self-sterile strains were mated with an unusual laboratory-generated sterile isolate type, which could have impacted conclusions regarding the prevalence of hybridization and inheritance of mitochondria. In the current study, we investigated whether interspecific crosses between fertile isolates of these three species are possible and, if so, how mitochondria are inherited by the progeny. For this purpose, a PCR-RFLP method and a mitochondrial DNA-specific PCR technique were custom-made. These were applied in a novel approach of typing complete ascospore drops collected from the fruiting bodies in each cross to distinguish between self-fertilizations and potential hybridization. These markers showed hybridization between C. fimbriata and C. eucalypticola and between C. fimbriata and C. manginecans, while no hybridization was detected in the crosses involving C. manginecans and C. eucalypticola. In both sets of hybrid progeny, we detected biparental inheritance of mitochondria. This study was the first to successfully produce hybrids from a cross involving self-fertile isolates of Ceratocystis and also provided the first direct evidence of biparental mitochondrial inheritance in the Ceratocystidaceae. This work lays the foundation for further research focused on investigating the role of hybridization in the speciation of Ceratocystis species and if mitochondrial conflict could have influenced the process.

Keywords: Ceratocystis; PCR RFLP; hybridization.

Figure 1

Ceratocystis growing on MEA media, showing the sexual fruiting bodies (ascomata). Ascomata have round, dark bases with long necks, at the top out of which slimy masses of ascospores exude.

Figure 2

A cross of C. fimbriata and C. eucalypticola following incubation on MEA-TS medium. (a) Parallel blue lines show the zone of interaction. (b) Close-up of the interaction zone.

Figure 3

In silico PCR-RFLP analysis of the three nuclear genomic regions containing HindIII and PstI restriction enzyme cut site variation in the examined Ceratocystis species. (a) Marker region 1 contains a PstI cut site that is absent only in C. manginecans. (b) Marker region 2 contains a PstI cut site present only in C. eucalypticola. (c) Marker region 3 has PstI cut site unique to C. fimbriata. (d) Predicted profiles following digestion of the three marker regions with HindIII and PstI (molecular marker in base pairs).

Figure 4

An example the PCR-RFLP profiles produced for nuclear marker 2 applied to a C. fimbriata × C. eucalypticola mating. The amplicon of 836 bp of marker 2 (primers marker2_R and marker2_F) is double digested using restriction enzymes HindIII and PstI. In both species, the presence of a band at approximately 100 bp indicates successful digestion. A 750 bp band is unique to C. fimbriata, while in C. eucalypticola the presence of a unique cut produces a band at 400 bp. Therefore, only lanes that clearly contain all three bands (100 bp, 400 bp and 750 bp) following digestion are considered representative of a hybrid.

Figure 5

An agarose gel showing biparental mitochondrial inheritance in an interspecific cross of C. fimbriata × C. manginecans. The amplicons shown were produced using mitochondrial primer set 1 (Primer F and EucMangR), which can effectively distinguish C. manginecans from the other two species. Amplification in C. fimbriata yields a large band of around 1800 bp, while in C. manginecans a smaller band of around 560 bp is produced. In a spore drop from a hybrid cross between these two species, two distinct bands of approximately 1800 bp and 560 bp are seen, confirming the presence of mitochondria from both parents.