Phylogenetic relationships and population dynamics of Calonectria

Schoch, Conrad Lamoraal (1999-12)

Thesis (Ph.D.(Agric.)) -- University of Stellenbosch, 1999.

Thesis

ENGLISH SUMMARY: This dissertation is presented as a collection of separate publications and an amount of redundancy has thus been unavoidable. Although several species are newly described they are not effectively published and will thus be formally published in scientific journals. There were two main objectives: I. To investigate the variability and mating compatibility of species and populations, in order to contribute to the systematics of Calonectria. II To identify loci that would be useful for DNA sequence comparisons in this genus and to present a reliable phylogeny of Calonectria and other closely related hypocrealean taxa. In the introductory review a synopsis of the current knowledge regarding the taxonomy and life cycle of Calonectria and Cylindrocladium spp. is presented. The importance of these pathogens are noted, as well as the problems related to identifying them. Aspects regarding specific species complexes and topics are discussed in more detail in the following chapters. The morphological and phylogenetic variation was investigated for the Cy. candelabrum species complex in Part 2. DNA sequence comparisons of the ribosomal 5.8S gene and flanking ITS1 and ITS2 spacers were employed in order to determine whether mating incompatibility and general morphology was supported by molecular evidence. Although only small differences were found these proved to be consistent and resulted in the recognition of Galonectria scoparia (anamorph Cylindrocladium candelabrum), and the description of three new species, namely Calonectria pallciramosa (anamorph Cyfindrocladium pauciramosum), Calonectria insularis (anamorph Cylindrocladium insulare) and Calonectria mexicana (anamorph Cylindrocladium mexicanum). The Cyfindrocladium scoparium cultures studied in Part 3 were isolated from several hosts in the U.S.A. Isolates were mated in all combinations, and one successful mating was selected to establish whether recombination occurred. RAPD and mating type data of parental isolates and progeny confirmed Cy. scoparium to have a heterothallic mating system. Furthermore, to determine the phylogeny of Cy. scoparium with several morphologically similar Cylindrocladium spp., DNA sequences of the ribosomal 5.8S gene and the flanking internal transcribed spacers (ITS), as well as part of the high mobility group (HMG) box (forming part of the MA T- 2 mating type gene) and the β-tubulin gene, were analysed. Maximum parsimony yielded concordant trees for all three data sets. These data supported the morphological and biological species concepts proposed for Cy. scoparium and other, similar, small-spored Cylindrocladium spp. Part 4 represented an investigation into the mating compatibility and mating type distribution of populations of Cy. pauciramosum. This enabled the determination of the effective population for the different areas studied. A sample collected over a period of six years, reflecting a number of locations in South Africa were found have 1: 1 mating type ratio, as expected in a random mating population. However, the mating type ratio was found to be significantly different in single nursery populations. In the South African nursery, the MAT-1 mating type was dominant, while the MAT-2 was more common in other samples obtained from nurseries in Italy and the U.S.A.. This was consistent with one or more founder effects. The high percentage of hermaphrodites also suggested that recent introductions had occurred in nurseries in Italy and the U.S.A. In addition to this, DNA sequence comparisons of the β-tubulin gene was used to investigate variation below species level in Cy. pauciramosum. All isolates from South Africa, Australia, U.S.A. and a group from Italy had identical sequences. A second group with identical sequences were found in the Italian sample. In addition to this, variation was found between all isolates from Brazil, Colombia and Mexico. Some of these base pairs were shared between the South and Central American isolates as well as isolates of Cy. candelabrum. This points towards a speciation event in South or Central America. After investigating variation below species level, this study was also expanded to generic level. In Part 5 information obtained in the preceding chapters culminated in a phylogeny of all known species in Calonectria and Cylindrocladium based on DNA sequence comparisons of the β-tubulin gene. Many clades, containing small numbers of isolates were strongly supported by bootstrap. However, relationships between these clades were often ambiguous. A number of phylogenetic placements based on DNA data did not always agree with preconceived morphological relationships. Two large groupings were evident and both contained small-spored, one-septate species. The only morphological character that correlated with DNA based phylogenies was vesicle shape of the anamorph. Finally, in Part 6, the generic phylogeny was investigated. In order to obtain a generic phylogeny a subset of Calonectria species was selected, as well as isolates from other genera, closely related to Calonectria. All of these genera were originally described under the broad concept of Nectria sensu lato. A gene tree phylogeny, based on β-tubulin was presented for selected nectriaceous genera with anamorphs bearing cylindrical macroconidia. Based on molecular data and the distinct anamorph genera, new teleomorph genera were proposed for Gylindrocladiella (Nectricladiella), Gliocladiopsis (Glionectria) and Xenocylindrocladium (Xenocalonectria). Ca/onectria was also found to form a monophyletic lineage. Eight species of Cylindrocladiella were recognised, with two having teleomorphs in Nectricladiella, namely N. camelliae (Ce. microcylindrica) and N. infestans (Ce. infestans). This study concluded that the current morphological species concepts in Cylindrocladium and its Calonectria teleomorphs can comprise several biological as well as phylogenetic species. The use of mating testers in this study was shown to provide a powerful tool to separate morphologically similar, but genetically isolated species. The biological and morphological species also agreed with the phylogenetic concepts used, but only vesicle shape were found to define phylogenetic clades. However, phylogenetic species concepts based on DNA sequences data obtained from genomic regions such as the β-tubulin and MA T-2 genes and additional areas will become increasingly important for further taxonomic studies in Calonectria and related genera.

AFRIKAANSE OPSOMMING: Hierdie studie word aangebied as 'n samevoeging van 'n aantal onafhanklike publikasies en 'n sekere mate van oorvleueling sal dus voorkom. Alhoewel 'n aantal spesies nuut beskryf word in hierdie tesis, is hulle nie effektief gepubliseer nie, en sal dergelike publikasie in toepaslike wetenskaplike joernale plaasvind. Die hoofdoel van hierdie studie was tweerlei: I. Om die varieerbaarheid en paringsvermoens van spesies en bevolkings te ondersoek en by te dra tot die sistematiek van Calonectria. II. Om die lokusse te identifiseer wat bruikbaar kan wees vir DNA volgorde vergelykings in hierdie genus en om 'n betroubare filogenie van Calonectria en naby verwante spesies in die Hyporeales te genereer. In die inleidende oorsig is die huidige kennis aangaande die taksonomie en lewensiklus van Calonectria en Cylindrocladium spp. bespreek. Die belang van hierdie spesies is aangedui, sowel as die probleme waarmee hulle geassosieer word. Punte wat van toepassing is op spesifieke spesie komplekse word later in meer detail bespreek. Die morfologiese en filogenetiese variasie op spesie vlak word ondersoek in Deel 2. DNA volgorde vergelykings van die ribosomale 5.8S geen en die naasliggende ITS1 en ITS2 intergeniese areas was gebruik om die paringsonvermoe en morfologiese karakters te onderskryf. Dit het die herbeskrywing van 'n bestaande spesie, Calonectria scoparia (anamorf Cylindrocladium candelabrum), en die beskrywing van drie nuwe spesies, Calonectria pauciramosa (anamorf Cylindrocladium pauciramosum): Calonectria insularis (anamorf Cylindrocladium insulare) and Calonectria mexicana (anamorf Cylindrocladium mexicanum) tot gevolg gehad. In die daaropvolgende deel was die herkombinering van Cy. scoparium beskou met behulp van RAPD merkers. Parings is uitgevoer en die RAPD en paringstipe data het bevestig dat hierdie spesie heterotallies is. In die tweede deel van hierdie hoofstuk was DNA volgorde vergelykings gedoen op fragmente wat verkry is van drie verskillende lokusse, die 5.8S ribosomale geen en ITS areas en dele van die MA T-2 geen se HMG kas asook die β-tubulien geen. Hierdie data was aangewend om die filogenie van Cy. scoparium en ander kleinspoorvormende heterotalliese Cylindrocladium spesies te ondersoek. Dit het die drie nuut beskryfde spesies van die vorige hoofstuk ingesluit en morfologiesen biologies spesie konsepte bevestig. Deel 4 bevat 'n ondersoek na die paringsvermoens van Cy. pauciramosum op bevolkingsvlak. As gevolg hiervan kon die effektiewe bevolking in verskillende areas bepaal word. A monster wat oor 'n tydperk van sesjaar en 'n verskeidenheid van geografies gebiede versamel is het 'n paringstipe verhouding van 1:1 gehad. Dit is volgens verwagting in 'n bevolking wat vrylik paar. In spesifieke kwekerye was die geval egter anders. In die Suid-Afrikaanse kwekery was die MAT-1 paringstipe oorheersend, terwyl MA T-2 meer voorgekom het in Italie en V.S.A. Die hoe aantal hermafrodiete dui ook daarop dat die spesie onlangs ingebring is. DNA volgorde vergelykings was ookgebruik om variasie onder spesievlak te ondersoek. Aile isolate van Suid-Afrika, Australie, V.S.A. en 'n groep van Italie het indentiese volgordes gehad. 'n Tweede groep in die Italiaanse bevolking is ook gevind met identiese DNA volgordes. In die Suid en Sentraal Amerikaanse bevolkings is die meeste variasie gevind en sommige van die basis paar verskille is gedeel met Cy. candelabrum. Dit dui op 'n spesiasie in Suid Amerika. In Deel 5 is die inligting wat verkry is vantevore uitgebrei na generiese vlak toe. Dit het 'n filogenie van aile bestaande Calonectria en Cylindrocladium spesies tot gevolg gehad, gebaseer op DNA volgordes van 'n dee van die β-tubulien geen. Verskeie klades is deur statisties analise ondersteun. Verhoudinge tussen hierdie groepe was egter minder duidelik. Twee groot groepe was ook onderskei en die engiste morfologiese karakter was met die geen filogenie ooreengestem het is die vorm van die "vesicle" op die kondiofore van die anamorf. Tot slotsom, in Deel 6 is die verteenwoordigende groepe spesies van Calonectria en naby verwante genera vergelyk. Hierdie genera is alreeds voorheen bespreek onder die wye taksonomiese konsep Nectria sensu lato. 'n Geen boom gebaseer op β- tubulien was aangedui. Aan die hand van hierdie data en unieke anamorf verwantskappe is nuwe teleomorf genera voorgestel vir Cylindrocladiella (Nectricladiella) , Gliocladiopsis (Glionectria) en Xenocylindroc!adium (Xenoca/onectria). Oit is ook bevind dat Calonectria monofileties is. Ag spesies van Cylindrocladiella is aangedui, waarvan twee teleomorwe het in Nectricladiella, naamlik N. camelliae (Ce. microcylindrica) en N. infestans (Ce. infestans). Hierdie studie het dus bevind dat die huidige morfologies spesie konsepte in Cylindrocladium ook biologiese en filogeneties spesies omskryf. Die gebruik van paringstoetsers is aangedui as 'n goeie metode om morfologies eenderse spesies te onderskei. Dit wil egter voorkom asof filogenetiese spesie konspte gebaseer op DNA gebiede soos die β-tubulien en MA T-2 geen, asook ander areas meer belangrik sal word vir verder taksonomies studies in hierdie swam.

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