Phylogenetic relationships and taxonomy of species in ceratocystis sensu lato

Abstract

Ceratocystis is a genus of Ascomycete fungi residing in the family Ceratocystidaceae (Order: Microascales; Class: Sordariomycetes). Species of Ceratocystis are best-known as woundinfecting fungi, which are mostly carried by insects. Little is known regarding the basis of the relationship between insects and Ceratocystis spp., but in some cases it might extend to levels of mutualism. The taxonomic history of the genus is complex and has, during the course of more than 100 years, been intertwined with several other genera. However, in recent years, the resolution arising from DNA sequence comparisons has resulted in the recognition of at least four major groups within Ceratocystis. major groups within Ceratocystis. Ceratocystis species in the broad sense (including Ambrosiella) represent ecologically diverse assemblages, including four very clearly defined groups. One of these groups, which I refer to as the C. coerulescens sensu lato (s.l.) group, which mainly cause blue-stain in timber, is the only group that is known to have a very close association with conifer-infesting bark beetles. Perhaps the best-known group is the C. fimbriata s.l. assemblage of species, which includes a large number of serious pathogens of angiosperms, mostly trees. This group also includes nonpathogenic wound-infecting species. There are no specific insect vectors of these species but they produce fruity aromas, which attract a wide range of insects such as flies, ants, mites and nitidulid beetles that aid in their dissemination to fresh wounds. The majority of the species in the C. fimbriata s.l. group are primary pathogens causing cankers that can girdle and eventually kill the affected areas. The third group of fungi in the broadly defined Ceratocystis s.l. is the C. moniliformis s.l. group. This group includes only saprophytes that, similar to species in C. fimbriata s.l., produce fruity odours and they are thus vectored by opportunistic insects including flies and nitidulid beetles. Ambrosiella spp. are broadly included in the assemblage accommodating Ceratocystis spp. These fungi have no known sexual state and they live in obligate symbioses with ambrosia beetles (Scolytinae). Ceratocystis and its relatives have had a complex taxonomic history ever since the genus was first described by Halstead in 1890. Much of the confusion regarding their taxonomy has arisen from a dependence on morphology for classification and the fact that these and other fungi have undergone convergent evolution related to their association with insects. Until the late 1990s, genera and species in this group were described based only on morphology, although the importance of various morphological features was strongly debated. However, between 1950- 1999, a turning point arose in the way that these fungi were recognised. This change emerged first from the inclusion of various chemical approaches and then later the application of molecular techniques to identify them. The most dramatic changes have come with the widespread application of DNA sequence comparisons, which were first applied to the group in the early 1990’s. This made it possible to define higher-order relationships and brought the first clear evidence that species in Ceratocystis and Ophiostoma were phylogenetically unrelated. A complete taxonomic history of this group is provided in the timeline presented in Table 1. This thesis deals primarily with two groups in the broadly defined Ceratocystis. These are species in the C. moniliformis s.l. and the C. fimbriata s.l. complexes, but with a considerably greater focus on the C. fimbriata s.l. complex. Morphologically, species in the two complexes are easy to distinguish from each other even though they share some common characteristics. Species in both complexes have hat-shaped ascospores and both have the anamorphic characteristics typical of species in the genus Thielaviopsis. The ascomatal bases are mostly globose to sub-globose with elongated necks. There are two main features that species in the C. moniliformis s.l. complex have that are not observed in the C. fimbriata s.l. complex. One is a very distinct “collar-like” structure at the base of the ascomatal necks of species in the C. moniliformis s.l. complex. These “collar-like” structures result in the necks being fragile and they dislodge from the ascomatal bases very easily. The other distinct characteristic is that these fungi have conical spines on their ascomatal bases, giving the structures a textured appearance. This is in contrast to the ascomatal bases in the C. fimbriata complex that are smooth. Species in the C. moniliformis s.l. complex are non-pathogenic and cause only some sap-stain in the host tissues. The C. fimbriata s.l. complex includes a large number of primary pathogens, mainly of trees but also including root crops. A phylogenetic tree including all species in the C. fimbriata s.l and C. moniliformis s.l. complexes is presented in Figure 1-3. In generating this tree, three gene regions i.e. the Internal Transcribed Spacer Region including the 5.8S rRNA operon (ITS), part of the the Beta-Tubulin 1 (BT) gene and part of the Transcription Elongation Factor 1 alpha (TEF) gene have been combined to represent species in the two complexes. Both Bootstrap as well as Bayesian analyses were run to obtain confidence intervals. More detailed methods are presented in the legends to the trees. Chapter one of this thesis presents a review of the groups in Ceratocystis. With the aid of DNA sequence comparisons, three phylogenetically distinct groups are identified. These groups are characterised by three well-known species each now defined as a species complex i.e. the Ceratocystis coerulescens complex, the C. moniliformis s.l. complex and the C. fimbriata s.l. complex. Species of Ambrosiella are also recognised as representing a discrete and related group. These three species complexes are not only differentiated based on DNA sequence comparison but have distinct morphological features that distinguish them from each other. They are also defined by having very distinct ecological roles in nature. Chapter 2 specifically reviews the key literature dealing with the taxonomy of species in the C. fimbriata complex. This group is defined by Ceratocystis fimbriata s.s., which was first described as a pathogen causing black rot on sweet potato. Subsequent to the emergence of DNA sequencing techniques to define species, many new species in the C. fimbriata s.l. complex have been defined and described. Chapters three and four of this thesis deal with a species of Ceratocystis collected from dying Mango trees in Oman. A very serious disease known as Mango Sudden Decline disease was first observed in Oman and has recently been subjected to intensive investigation. This led to a recognition that a species related to C. fimbriata might be responsible for the disease of Mangifera indica. As part of the process of identifying the causal agent of Mango Sudden Decline, two Ceratocystis spp. were described. One is the previously described and nonpathogenic C. omanensis residing in the C. moniliformis s.l. complex and the other is C. manginecans, a virulent pathogen residing in the C. fimbriata s.l. complex (Chapter 4). Chapters five through eleven of this thesis include the descriptions of new species in the C. fimbriata complex that have emerged over a seven year period. These descriptions have all relied strongly on DNA sequence based phylogenetic inference as well as morphology. It is important to recognise, however, that the morphological features are very similar in most species and that differences rely on the size ranges of key features in this group. The genus Ceratocystis represents a complex of many species and is set to be sub-divided into at least three discrete genera, not including Ambrosiella, which is also closely related and already well-defined. Studies in this thesis began at a time when it became possible to define species of Ceratocystis based strongly on a phylogenetic concept. Thus, while morphological and ecological features were taken into account, there has been a heavy reliance on DNA sequence comparisons. It is likely that in coming years, new gene regions will be recognised that will allow a refinement of genus and species boundaries. Furthermore, having access to whole-genome sequencing will add deeply to our understanding of taxonomic relationships between these important and fascinating fungi. It is my hope that the foundation provided by the studies incorporated in this thesis will be useful as this new wave of study emerges.