Diogo N. Silva

Application of the Apn2/MAT locus to improve the systematics of the Colletotrichum gloeosporioides complex: an example from coffee (Coffea spp.) hosts

To improve phylogenetic resolution of the Colletotrichum gloeosporioides species complex we developed and tested the performance of a new set of primers for the Apn2/MAT locus with a case study of 22 isolates. These were isolated mainly from coffee plants and represent six divergent and well characterized species within the C. gloeosporioides complex. Following previous studies on this locus, we have generated sequence data from an expanded region (> 4600 bp), revealing increased phylogenetic informativeness when compared to other commonly used markers such as ITS, β-tub2 and GS. Within the Apn2/MAT locus the ApMAT marker alone was almost as informative in terms of phylogenetic resolution as a seven-gene concatenated dataset. Our results further revealed that gene-tree discordance may come to be a common issue in resolving evolutionary relationships in the C. gloeosporioides complex, highlighting the importance of multilocus approaches. The use of state-of-the-art data analysis techniques and a highly informative dataset as employed here may abate this issue and hopefully assist in disentangling the C. gloeosporioides complex.

Host‐jump drives rapid and recent ecological speciation of the emergent fungal pathogen Colletotrichum kahawae

Ecological speciation through host‐shift has been proposed as a major route for the appearance of novel fungal pathogens. The growing awareness of their negative impact on global economies and public health created an enormous interest in identifying the factors that are most likely to promote their emergence in nature. In this work, a combination of pathological, molecular and geographical data was used to investigate the recent emergence of the fungus Colletotrichum kahawae. C. kahawae emerged as a specialist pathogen causing coffee berry disease in Coffea arabica, owing to its unparalleled adaptation of infecting green coffee berries. Contrary to current hypotheses, our results suggest that a recent host‐jump underlay the speciation of C. kahawae from a generalist group of fungi seemingly harmless to coffee berries. We posit that immigrant inviability and a predominantly asexual behaviour could have been instrumental in driving speciation by creating pleiotropic interactions between local adaptation and reproductive patterns. Moreover, we estimate that C. kahawae began its diversification at <2200 bp leaving a very short time frame since the divergence from its sibling lineage (c. 5600 bp), during which a severe drop in C. kahawae’s effective population size occurred. This further supports a scenario of recent introduction and subsequent adaptation to C. arabica. Phylogeographical data revealed low levels of genetic polymorphism but provided the first geographically consistent population structure of C. kahawae, inferring the Angolan population as the most ancestral and the East African populations as the most recently derived. Altogether, these results highlight the significant role of host specialization and asexuality in the emergence of fungal pathogens through ecological speciation.

Evolutionary and Biogeographic Insights on the Macaronesian Beta-Patellifolia Species (Amaranthaceae) from a Time-Scaled Molecular Phylogeny.

The Western Mediterranean Region and Macaronesian Islands are one of the top biodiversity hotspots of Europe, containing a significant native genetic diversity of global value among the Crop Wild Relatives (CWR). Sugar beet is the primary crop of the genus Beta (subfamily Betoideae, Amaranthaceae) and despite the great economic importance of this genus, and of the close relative Patellifolia species, a reconstruction of their evolutionary history is still lacking. We analyzed nrDNA (ITS) and cpDNA gene (matK, trnH-psbA, trnL intron, rbcL) sequences to: (i) investigate the phylogenetic relationships within the Betoideae subfamily, and (ii) elucidate the historical biogeography of wild beet species in the Western Mediterranean Region, including the Macaronesian Islands. The results support the Betoideae as a monophyletic group (excluding the Acroglochin genus) and provide a detailed inference of relationships within this subfamily, revealing: (i) a deep genetic differentiation between Beta and Patellifolia species, which may have occurred in Late Oligocene; and (ii) the occurrence of a West-East genetic divergence within Beta, indicating that the Mediterranean species probably differentiated by the end of the Miocene. This was interpreted as a signature of species radiation induced by dramatic habitat changes during the Messinian Salinity Crisis (MSC, 5.96–5.33 Mya). Moreover, colonization events during the Pleistocene also played a role in shaping the current diversity patterns among and within the Macaronesian Islands. The origin and number of these events could not be revealed due to insufficient phylogenetic resolution, suggesting that the diversification was quite recent in these archipelagos, and unravelling potential complex biogeographic patterns with hybridization and gene flow playing an important role. Finally, three evolutionary lineages were identified corresponding to major gene pools of sugar beet wild relatives, which provide useful information for establishing in situ and ex situ conservation priorities in the hotspot area of the Macaronesian Islands.

New mitochondrial and nuclear evidences support recent demographic expansion and an atypical phylogeographic pattern in the spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae)

Philaenus spumarius is a widespread insect species in the Holarctic region. Here, by focusing on the mtDNA gene COI but also using the COII and Cyt b genes and the nuclear gene EF-1α, we tried to explain how and when its current biogeographic pattern evolved by providing time estimates of the main demographic and evolutionary events and investigating its colonization patterns in and out of Eurasia. Evidence of recent divergence and expansion events at less than 0.5 Ma ago indicate that climate fluctuations in the Mid-Late Pleistocene were important in shaping the current phylogeographic pattern of the species. Data support a first split and differentiation of P. spumarius into two main mitochondrial lineages: the “western”, in the Mediterranean region and the “eastern”, in Anatolia/Caucasus. It also supports a following differentiation of the “western” lineage into two sub-lineages: the “western-Mediterranean”, in Iberia and the “eastern-Mediterranean” in the Balkans. The recent pattern seems to result from postglacial range expansion from Iberia and Caucasus/Anatolia, thus not following one of the four common paradigms. Unexpected patterns of recent gene-flow events between Mediterranean peninsulas, a close relationship between Iberia and North Africa, as well as high levels of genetic diversity being maintained in northern Europe were found. The mitochondrial pattern does not exactly match to the nuclear pattern suggesting that the current biogeographic pattern of P. spumarius may be the result of both secondary admixture and incomplete lineage sorting. The hypothesis of recent colonization of North America from both western and northern Europe is corroborated by our data and probably resulted from accidental human translocations. A probable British origin for the populations of the Azores and New Zealand was revealed, however, for the Azores the distribution of populations in high altitude native forests is somewhat puzzling and may imply a natural colonization of the archipelago.

Genomic Patterns of Positive Selection at the Origin of Rust Fungi

Understanding the origin and evolution of pathogenicity and biotrophic life-style of rust fungi has remained a conundrum for decades. Research on the molecular mechanisms responsible for rust fungi evolution has been hampered by their biotrophic life-style until the sequencing of some rust fungi genomes. With the availability of multiple whole genomes and EST data for this group, it is now possible to employ genome-wide surveys and investigate how natural selection shaped their evolution. In this work, we employed a phylogenomics approach to search for positive selection and genes undergoing accelerated evolution at the origin of rust fungi on an assembly of single copy genes conserved across a broad range of basidiomycetes. Up to 985 genes were screened for positive selection on the phylogenetic branch leading to rusts, revealing a pervasive signal of positive selection throughout the data set with the proportion of positively selected genes ranging between 19.6–33.3%. Additionally, 30 genes were found to be under accelerated evolution at the origin of rust fungi, probably due to a mixture of positive selection and relaxation of purifying selection. Functional annotation of the positively selected genes revealed an enrichment in genes involved in the biosynthesis of secondary metabolites and several metabolism and transporter classes.

Age estimates of Frullania (Frullaniaceae, Porellales) main lineages: another example of rapid and recent diversification in liverwort evolution

Frullania Raddi is an extant genus of liverworts (Bryophytes) widespread around the world. It belongs to the family Frullaniaceae Lorch., with a large number of species distributed into several subgenera, sections and subsections according with different morphological classifications. As shown in previous studies, most Frullania sub-generic classifications are supported by molecular data, indicating that morphological characters appear well suitable to discriminate species. However, deep among-clade relationships remain unclear, probably due to the rapid diversification of main clades, paralleling other plant lineages. In this study, we reconstruct Frullania phylogeny based on available molecular data used in previous studies, and we present time estimates for the origin of its main branches. Results supported the monophyly of most subgenera as demonstrated in previous studies and supported a rapid diversification of these main lineages. Time estimates under a relaxed molecular clock and with integrated fossil evidence and nucleotide mutation rates further suggested a Jurassic origin of the genus and a rapid diversification during Palaeogene and Neogene. This may have been influenced by geographical and climate changes during these periods as predicted for most leafy liverworts.

Structure_threader: an improved method for automation and parallelization of programs structure, fastStructure and MavericK on multicore CPU systems

Structure_threader is a program to parallelize multiple runs of genetic clustering software that does not make use of multithreading technology (structure, fastStructure and MavericK) on multicore computers. Our approach was benchmarked across multiple systems and displayed great speed improvements relative to the single‐threaded implementation, scaling very close to linearly with the number of physical cores used. Structure_threader was compared to previous software written for the same task—ParallelStructure and StrAuto and was proven to be the faster (up to 25% faster) wrapper under all tested scenarios. Furthermore, Structure_threader can perform several automatic and convenient operations, assisting the user in assessing the most biologically likely value of ‘K’ via implementations such as the “Evanno,” or “Thermodynamic Integration” tests and automatically draw the “meanQ” plots (static or interactive) for each value of K (or even combined plots). Structure_threader is written in python 3 and licensed under the GPLv3. It can be downloaded free of charge at https://github.com/StuntsPT/Structure_threader.

Legitimacy and Implications of Reducing Colletotrichum kahawae to Subspecies in Plant Pathology

Colletotrichum kahawae Waller and Bridge is a highly aggressive and specialized fungal pathogen of coffee, causing the devastating Coffee Berry Disease (CBD), particularly at high altitudes. The disease arises from the unique ability of the pathogen to infect green developing coffee berries. This pathogen is currently confined to the African continent in all countries that grow Arabica coffee (Coffea arabica L.), leading to up to 80% yield losses, if no control measures are applied (Silva et al., 2006; Vossen and Walyaro, 2009; Hindorf and Omondi, 2011). For such huge economic impact, it is ranked as a quarantine pathogen and even as a biological weapon (Australia Group, 2014). Consequently, the pathogens potential dispersal to other Arabica coffee cultivation regions is greatly feared, particularly to those at high altitude also found in Latin America and Asia. Recently, this recognized species was brought down to a subspecific level (C. kahawae subsp. kahawae) based on molecular data (Weir et al., 2012), clustering together with a generalist and cosmopolitan group of Colletotrichum isolates unable to cause CBD (C. kahawae subsp. ciggaro). Since then a growing number of studies have reported the identification of C. kahawae in various hosts and regions of the world (Liu et al., 2013; Afanador-Kafuri et al., 2014; Mosca et al., 2014; Schena et al., 2014; Ismail et al., 2015; Garibaldi et al., 2016a,b; Perrone et al., 2016). Although these reports are referring to C. kahawae subsp. ciggaro, some of them could not distinguish the pathogen at the subspecific level, and this is leading to a wave of confusion of whether the long accepted species C. kahawae, the CBD pathogen, has escaped from Africa and extended its host range. Given the extreme impact that this situation may trigger and the subsequent biosecurity implications, there is a practical need to completely distinguish these pathogens taxonomically as to avoid the risk of misidentification, and caution should be taken on assigning/reassigning taxonomic ranking and nomenclature. Here we consider the evidences sustaining and contradicting the classification proposed by Weir et al. (2012), and discuss the risks and practical implications of changing the CBD pathogens species status in a plant pathology context.

Novel insights on colonization routes and evolutionary potential of Colletotrichum kahawae, a severe pathogen of Coffea arabica: Demographic history of Ck: a genomic perspective

Pathogenic fungi are emerging at an increasing rate on a wide range of host plants, leading to tremendous threats to the global economy and food safety. Several plant pathogens have been considered to be invasive species, rendering large-scale population genomic analyses crucial to better understand their demographic history and evolutionary potential. Colletotrichum kahawae (Ck) is a highly aggressive and specialized pathogen, causing coffee berry disease in Arabica coffee in Africa. This pathogen leads to severe production losses and its dissemination out of Africa is greatly feared. To address this issue, a population genomic approach using thousands of single nucleotide polymorphisms (SNPs) spaced throughout the genome was used to unveil its demographic history and evolutionary potential. The current study confirms that Ck is a true clonal pathogen, perfectly adapted to green coffee berries, with three completely differentiated populations (Angolan, Cameroonian and East African). Two independent clonal lineages were found within the Angolan population as opposed to the remaining single clonal populations. The most probable colonization scenario suggests that this pathogen emerged in Angola and immediately dispersed to East Africa, where these two populations began to differentiate, followed by the introduction in Cameroon from an Angolan population. However, the differentiation between the two Angolan clonal lineages masks the mechanism for the emergence of the Cameroonian population. Our results suggest that Ck is completely differentiated from the ancestral lineage, has a low evolutionary potential and a low dispersion ability, with human transport the most likely scenario for its potential dispersion, which makes the fulfilment of the quarantine measures and management practices implemented crucial.