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Rationale: Methicillin resistant Staphylococcus aureus (MRSA) is prevalent and consequential in cystic fibrosis (CF). Whole genome sequencing (WGS) could reveal genomic differences in MRSA associated with poorer outcomes or detect MRSA transmission. Objectives: To identify MRSA genes associated with low lung function and identify potential MRSA transmission in CF. Methods: We collected 97 MRSA isolates from 74 individuals with CF from 2017 and performed short-read WGS. We determined sequence type (ST) and the phylogenetic relationship between isolates. We aligned accessory genes from 25 reference genomes to genome assemblies. We classified the MRSA by accessory gene content and correlated the accessory genome to clinical outcomes. Results: The most prevalent ST were ST5 (N=55), ST105 (N=14), and ST8 (N=14). Closely related MRSA strains were shared by family members with CF, but rarely between unrelated individuals. Three distinct clusters of MRSA were identified by accessory genome content. The first included ST5 and ST105 strains and was common among older patients with lower FEV. The second cluster included ST8, which was generally identified in younger patients. Sputum density of MRSA and Pseudomonas aeruginosa was higher in cultures from patients with ST5/ST105 compared to patients with ST8 at similar ages. Conclusions: In this CF cohort, we identified MRSA subtypes that predominate at different ages and differ by accessory gene content. ST5 and ST105 represented the most prevalent cluster of MRSA. ST8 MRSA was more common in younger patients and thus has the potential to rise in prevalence as these patients age.

The goal of this research was to determine the prevalence of Listeria monocytogenes in Iowa retail delicatessens and assess environmental aspects that mitigate L. monocytogenes. Fifty-seven small and large retail delicatessens in Iowa were selected randomly. More small operations (n = 43) were included as compared with larger stores, given the higher frequency of violations. An environmental assessment instrument was used to determine environmental factors and practices. At least five microbial samples were collected per site. We collected 286 (74.3%) of 385 microbial samples from small deli operations and 99 (25.7%) of 385 samples from large deli operations. Samples were taken from various zone 1 and 2 areas, such as the slicer, deli case, and meat scale; three (0.08%) samples were positive for L. monocytogenes. Regarding environmental aspects, not preparing, holding or storing ready-to-eat products near raw products (n = 30, 53%) was practiced by the fewest delis. The majority of establishments were observed covering, wrapping, or protecting ready-to-eat products when not in use to prevent contamination (n = 56, 98.2%). Comparisons were made to the U.S. Department of Agriculture Food Safety and Inspection Service Guidance on environmental practices, and 60% of the operations surveyed were in adherence with at least seven of the eight recommendations.

The discovery of unanticipated microbial diversity in remote, often hostile environments has led to a greater appreciation of the complexity and richness of the natural world. Yellowstone National Park (YNP) has long been a focus of work on taxa that inhabit extreme environments. Here we report the finding of microbial flora that inhabit an unexpected niche: the cavities of bone remnants from a bison carcass in Norris Geyser Basin in YNP. Although bleached white on the surface, the bone cavities are bright green due to the presence of Stichococcus-like trebouxiophyte green algae. The cavities also harbour different fungi and bacteria. Stichococcus species are common lichen photobionts and the Thelebolales fungi present in the bone cavities have previously been found in association with animal remains. Scanning electron microscope analysis suggests the fungi and algae do not form lichen-like associations in the bone. Rather these taxa and the bacteria appear to be opportunists that have colonized an isolated oasis that provides nutrients and protection from desiccation and UV radiation.

Acarospora rosulata is revised and reported from North America and Mongolia. Acarospora bullata is not verified as occurring in South and North America and may be conspecific with A. rugulosa.

The sporadic distribution of nuclear group I introns among different fungal lineages can be explained by vertical inheritance of the introns followed by successive losses, or horizontal transfers from one lineage to another through intron homing or reverse splicing. Homing is mediated by an intron-encoded homing endonuclease (HE) and recent studies suggest that the introns and their associated HE gene (HEG) follow a recurrent cyclical model of invasion, degeneration, loss, and reinvasion. The purpose of this study was to compare this model to the evolution of HEGs found in the group I intron at position S943 of the nuclear ribosomal DNA of the lichen-forming fungus Pleopsidium. Forty-eight S943 introns were found in the 64 Pleopsidium samples from a worldwide screen, 22 of which contained a full-length HEG that encodes a putative 256-amino acid HE, and 2 contained HE pseudogenes. The HEGs are divided into two closely related types (as are the introns that encode them) that differ by 22.6% in their nucleotide sequences. The evolution of the Pleopsidium intron-HEG element shows strong evidence for a cyclical model of evolution. The intron was likely acquired twice in the genus and then transmitted via two or three interspecific horizontal transfers. Close geographical proximity plays an important role in intron-HEG horizontal transfer because most of these mobile elements were found in Europe. Once acquired in a lineage, the intron-HEG element was also vertically transmitted, and occasionally degenerated or was lost.

Pezizomycotina is the largest subphylum of Ascomycota and includes the vast majority of filamentous, ascoma-producing species. Here we report the results from weighted parsimony, maximum likelihood and Bayesian phylogenetic analyses of five nuclear loci (SSU rDNA, LSU rDNA, RPB1, RPB2 and EF-lalpha) from 191 taxa. Nine of the 10 Pezizomycotina classes currently recognized were represented in the sampling. These data strongly supported the monophyly of Pezizomycotina, Arthoniomycetes, Eurotiomycetes, Orbiliomycetes and Sordariomycetes. Pezizomycetes and Dothideomycetes also were resolved as monophyletic but not strongly supported by the data. Lecanoromycetes was resolved as paraphyletic in parsimony analyses but monophyletic in maximum likelihood and Bayesian analyses. Leotiomycetes was polyphyletic due to exclusion of Geoglossaceae. The two most basal classes of Pezizomycotina were Orbiliomycetes and Pezizomycetes, both of which comprise species that produce apothecial ascomata. The seven remaining classes formed a monophyletic group that corresponds to Leotiomyceta. Within Leotiomyceta, the supraclass clades of Leotiomycetes s.s. plus Sordariomycetes and Arthoniomycetes plus Dothideomycetes were resolved with moderate support.

About one-fifth of all known extant fungal species form obligate symbiotic associations with green algae, cyanobacteria or with both photobionts. These symbioses, known as lichens, are one way for fungi to meet their requirement for carbohydrates. Lichens are widely believed to have arisen independently on several occasions, accounting for the high diversity and mixed occurrence of lichenized and non-lichenized (42 and 58%, respectively) fungal species within the Ascomycota. Depending on the taxonomic classification chosen, 15-18 orders of the Ascomycota include lichen-forming taxa, and 8-11 of these orders (representing about 60% of the Ascomycota species) contain both lichenized and non-lichenized species. Here we report a phylogenetic comparative analysis of the Ascomycota, a phylum that includes greater than 98% of known lichenized fungal species. Using a Bayesian phylogenetic tree sampling methodology combined with a statistical model of trait evolution, we take into account uncertainty about the phylogenetic tree and ancestral state reconstructions. Our results show that lichens evolved earlier than believed, and that gains of lichenization have been infrequent during Ascomycota evolution, but have been followed by multiple independent losses of the lichen symbiosis. As a consequence, major Ascomycota lineages of exclusively non-lichen-forming species are derived from lichen-forming ancestors. These species include taxa with important benefits and detriments to humans, such as Penicillium and Aspergillus.

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.

The Cyanidiales are unicellular red algae that are unique among phototrophs. They thrive in acidic, moderately high-temperature habitats typically associated with geothermally active regions, although much remains to be learned about their distribution and diversity within such extreme environments. We focused on Yellowstone National Park (YNP), using culture-dependent efforts in combination with a park-wide environmental polymerase chain reaction (PCR) survey to examine Cyanidiales diversity and distribution in aqueous (i.e. submerged), soil and endolithic environments. Phylogenetic reconstruction of Cyanidiales biodiversity demonstrated the presence of Cyanidioschyzon and Galdieria lineages exhibiting distinct habitat preferences. Cyanidioschyzon was the only phylotype detected in aqueous environments, but was also prominent in moist soil and endolithic habitats, environments where this genus was thought to be scarce. Galdieria was found in soil and endolithic samples, but absent in aqueous habitats. Interestingly, Cyanidium could not be found in the surveys, suggesting this genus may be absent or rare in YNP. Direct microscopic counts and viable counts from soil samples collected along a moisture gradient were positively correlated with moisture content, providing the first in situ evidence that gravimetric moisture is an important environmental parameter controlling distribution of these algae. Significance and Impact of the Study The Cyanidiales are an order of unicellular eukaryotic algae that thrive in acidic geothermal environments. In this study, we report several novel observations relative to Cyanidiales ecology in Yellowstone National Park, including the following: (i) the identification of two phylogenetic lineages of Cyanidiales: Cyanidioschyzon and Galdieria; (ii) the absence of Galdieria in aquatic environments; (iii) the absence of Cyanidium and Galdieria phlegrea in prime Cyanidiales habitats; (iv) the cohabitation of Cyanidioschyzon and Galdieria in nonaqueous environments; and (v) the first in situ evidence regarding the relationship between soil moisture and Cyanidiales habitat preference and viability

The genus Lecidea Ach. sensu lato (sensu Zahlbruckner) includes almost 1200 species, out of which only 100 species represent Lecidea sensu stricto (sensu Hertel). The systematic position of the remaining species is mostly unsettled but anticipated to represent several unrelated lineages within Lecanoromycetes. This study attempts to elucidate the phylogenetic placement of members of this heterogeneous group of lichen-forming fungi and to improve the classification and phylogeny of Lecanoromycetes. Twenty-five taxa of Lecidea sensu lato and 22 putatively allied species were studied in a broad selection of 268 taxa, representing 48 families of Lecanoromycetes. Six loci, including four ribosomal and two protein-coding genes for 315- and 209-OTU datasets were subjected to maximum likelihood and Bayesian analyses. The resulting well supported phylogenetic relationships within Lecanoromycetes are in agreement with published phylogenies, but the addition of new taxa revealed putative rearrangements of several families (e.g. Catillariaceae, Lecanoraceae, Lecideaceae, Megalariaceae, Pilocarpaceae and Ramalinaceae). As expected, species of Lecidea sensu lato and putatively related taxa are scattered within Lecanoromycetidae and beyond, with several species nested in Lecanoraceae and Pilocarpaceae and others placed outside currently recognized families in Lecanorales and orders in Lecanoromycetidae. The phylogenetic affiliations of Schaereria and Strangospora are outside Lecanoromycetidae, probably with Ostropomycetidae. All species referred to as Lecidea sensu stricto based on morphology (including the type species, Lecidea fuscoatra [L.] Ach.) form, with Porpidia species, a monophyletic group with high posterior probability outside Lecanorales, Peltigerales and Teloschistales, in Lecanoromycetidae, supporting the recognition of order Lecideales Vain. in this subclass. The genus name Lecidea must be redefined to apply only to Lecidea sensu stricto and to include at least some members of the genus Porpidia. Based on morphological and chemical similarities, as well as the phylogenetic relationship of Lecidea pullata sister to Frutidella caesioatra, the new combination Frutidella pullata is proposed here.

The discovery of unanticipated microbial diversity in remote, often hostile environments has led to a greater appreciation of the complexity and richness of the natural world. Yellowstone National Park (YNP) has long been a focus of work on taxa that inhabit extreme environments. Here we report the finding of microbial flora that inhabit an unexpected niche: the cavities of bone remnants from a bison carcass in Norris Geyser Basin in YNP. Although bleached white on the surface, the bone cavities are bright green due to the presence of Stichococcus-like trebouxiophyte green algae. The cavities also harbour different fungi and bacteria. Stichococcus species are common lichen photobionts and the Thelebolales fungi present in the bone cavities have previously been found in association with animal remains. Scanning electron microscope analysis suggests the fungi and algae do not form lichen-like associations in the bone. Rather these taxa and the bacteria appear to be opportunists that have colonized an isolated oasis that provides nutrients and protection from desiccation and UV radiation.

Extremophiles are organisms that thrive in environments previously thought inhospitable to life because the physicochemical characteristics fall outside the range tolerated by human cells. These types of environments are usually fatal to most eukaryotes, but have been shown to host a diverse group of prokaryotes that rely on specialized enzymes for survival. Extremophiles are permanently exposed to harsh environmental conditions and are categorized according to their ability to thrive in a specific type of niche. For example, thermophiles grow at temperatures above 50°C, psycrophiles prefer temperatures below 15°C, piezophiles are pressure-lovers, halophiles are found in high salt concentrations, whereas acidophiles and alkaliphiles thrive at an extreme pH of ≤ 3 and ≥ 10, respectively. These taxa are found in hot and cold deserts, hot springs, salt lakes, in sulfide mines, or near deep-sea vents all around the world. It has been speculated that if extraterrestrial life exists, it would be in the form of an extremophile.

The Chromalveolata "supergroup" is a massive assemblage of single-celled and multicellular protists such as ciliates and kelps that remains to be substantiated in molecular trees. Recent multigene analyses place chromalveolates into two major clades, the SAR (Stramenopiles, Alveolata, and Rhizaria) and the Cryptophyta+Haptophyta. Here we determined 69 new sequences from different chromalveolates to study the interrelationships of its constituent phyla. We included in our trees, the novel groups Telonemia and Katablepharidophyta that have previously been described as chromalvoleate allies. The best phylogenetic resolution resulted from a 6-protein (actin, alpha-tubulin, beta-tubulin, cytosolic HSP70, BIP HSP70, HSP90) and a 5-protein (lacking HSP90) alignment that validated the SAR and cryptophyte+haptophyte clades with the inclusion of telonemids in the former and katablepharids in the latter. We assessed the Plastidophila hypothesis that is based on EF2 data and suggest this grouping may be explained by horizontal gene transfers involving the EF2 gene rather than indicating host relationships.

The Lecanoromycetes includes most of the lichen-forming fungal species (13500) and is therefore one of the most diverse class of all Fungi in terms of phenotypic complexity. We report phylogenetic relationships within the Lecanoromycetes resulting from Bayesian and maximum likelihood analyses with complementary posterior probabilities and bootstrap support values based on three combined multilocus datasets using a supermatrix approach. Nine of 10 orders and 43 of 64 families currently recognized in Eriksson's classification of the Lecanoromycetes (Outline of Ascomycota--2006 Myconet 12:1-82) were represented in this sampling. Our analyses strongly support the Acarosporomycetidae and Ostropomycetidae as monophyletic, whereas the delimitation of the largest subclass, the Lecanoromycetidae, remains uncertain. Independent of future delimitation of the Lecanoromycetidae, the Rhizocarpaceae and Umbilicariaceae should be elevated to the ordinal level. This study shows that recent classifications include several nonmonophyletic taxa at different ranks that need to be recircumscribed. Our phylogenies confirm that ascus morphology cannot be applied consistently to shape the classification of lichen-forming fungi. The increasing amount of missing data associated with the progressive addition of taxa resulted in some cases in the expected loss of support, but we also observed an improvement in statistical support for many internodes. We conclude that a phylogenetic synthesis for a chosen taxonomic group should include a comprehensive assessment of phylogenetic confidence based on multiple estimates using different methods and on a progressive taxon sampling with an increasing number of taxa, even if it involves an increasing amount of missing data.

A comprehensive phylogenetic classification of the kingdom Fungi is proposed, with reference to recent molecular phylogenetic analyses, and with input from diverse members of the fungal taxonomic community. The classification includes 195 taxa, down to the level of order, of which 16 are described or validated here: Dikarya subkingdom nov.; Chytridiomycota, Neocallimastigomycota phyla nov.; Monoblepharidomycetes, Neocallimastigomycetes class. nov.; Eurotiomycetidae, Lecarioromycetidae, Mycocaliciomycetidae subclass. nov.; Acarosporales, Corticiales, Baeomycetales, Candelariales, Gloeophyllales, Melanosporales, Trechisporales, Umbilicariales ords. nov. The clade containing Ascomycota and Basidiomycota is classified as subkingdom Dikarya, reflecting the putative synapomorphy of dikaryotic hyphae. The most dramatic shifts in the classification relative to previous works concern the groups that have traditionally been included in the Chytridiomycota and Zygomycota. The Chytridiomycota is retained in a restricted sense, with Blastocladiomycota and Neocallimastigomycota representing segregate phyla of flagellated Fungi. Taxa traditionally placed in Zygomycota are distributed among Glomeromycota and several subphyla incertae sedis, including Mucoromycotina, Entomophthoromycotina, Kickxellomycotina, and Zoopagomycotiria. Microsporidia are included in the Fungi, but no further subdivision of the group is proposed. Several genera of 'basal' Fungi of uncertain position are not placed in any higher taxa, including Basidiobolus, Caulochytrium, Olpidium, and Rozella. (c) 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

Background Group I introns have spread into over 90 different sites in nuclear ribosomal DNA (rDNA) with greater than 1700 introns reported in these genes. These ribozymes generally spread through endonuclease-mediated intron homing. Another putative pathway is reverse splicing whereby a free group I intron inserts into a homologous or heterologous RNA through complementary base-pairing between the intron and exon RNA. Reverse-transcription of the RNA followed by general recombination results in intron spread. Here we used phylogenetics to test for reverse splicing spread in a taxonomically broadly sampled data set of fungal group I introns including 9 putatively ancient group I introns in the rDNA of the yeast-like symbiont Symbiotaphrina buchneri. Results Our analyses reveal a complex evolutionary history of the fungal introns with many cases of vertical inheritance (putatively for the 9 introns in S. buchneri) and intron lateral transfer. There are several examples in which introns, many of which are still present in S. buchneri, may have spread through reverse splicing into heterologous rDNA sites. If the S. buchneri introns are ancient as we postulate, then group I intron loss was widespread in fungal rDNA evolution. Conclusion On the basis of these results, we suggest that the extensive distribution of fungal group I introns is at least partially explained by the reverse splicing movement of existing introns into ectopic rDNA sites.

Summary Thriving in the hot, acidic, and metal-rich environments associated with geothermal areas is possible for only a few eukaryotes, with the Cyanidiophytina red algae ( Cyanidium , Galdieria , and Cyanidioschyzon ) being a famous example. These unicellular taxa can live in pH 0–4 and temperatures reaching up to 56°C [1,2]. Because Cyanidiophytina is sister to a vast array of mesophilic red algae (the Rhodophytina), such as the unicellular Porphyridium and the seaweed Chondrus [3], the genetic basis of their adaptation to extreme environments is of great interest from both the perspective of biotechnology and of evolution. The recently completed 13.7 Mbp genome sequence from the hot-spring dwelling Galdieria sulphuraria demonstrated that horizontal gene transfer (HGT) from prokaryotic sources provided this taxon with remarkable metabolic versatility ( e.g ., glycerol metabolism) and the ability to survive in its hostile environment ( e.g. , genes to detoxify mercury and arsenic) [4]. To explore the role of HGT in other members of this genus, we generated an 11.4 Mbp draft genome assembly from the sister taxon G. phlegrea DBV 009 [5]. In contrast to G. sulphuraria, this species is adapted to dry habitats near fumaroles such as fissures between rocks or cryptoendolithic environments [5,6]. Here, we provide evidence for extensive gene loss in the common ancestor of Cyanidiophytina that includes the eukaryote-derived loci required for urea utilization. Surprisingly, we find that G. phlegrea has regained the complete set of genes required for urea hydrolysis through HGT from eubacteria. The unlinked nature of these genes is likely explained by multiple gene transfers that resulted in assembly of the pathway in G . phlegrea . Our study demonstrates that genome reduction, a common outcome in eukaryotes for adaptation to a specialized niche, can be ameliorated by the gain of once lost, or novel functions through HGT.

Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project ‘‘Assembling the Fungal Tree of Life’’ (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.

Phylogenetic analyses ofnon-protein-coding nucleotidesequencessuch asribosomalRNA genes, internal transcribed spacers, and introns are often impeded by regions of the alignments that are ambiguously aligned. These regions are characterized by the presence of gaps and their uncer- tain positions, no matter which optimization criteria are used. This problem is particularly acute in large-scalephylogeneticstudiesandwhenaligninghighlydivergedsequences.Accommodatingthese regions, where positional homology is likely to be violated, in phylogenetic analyses has been dealt with very differently by molecular systematists and evolutionists, ranging from the total exclusion of these regions to theinclusion of every position regardless of ambiguity in the alignment. We presenta newmethod that allowsthe inclusion of ambiguously aligned regions without violating homology. In this three-step procedure, erst homologous regions of the alignment containing ambiguously aligned sequences are delimited. Second, each ambiguously aligned region is unequivocally coded as a new character, replacing its respective ambiguous region. Third, each of the coded characters is subjected to a speciec stepmatrix to account for the differential number of changes (summing substitutions and indels)neededtotransform onesequencetoanother. Theoptimalnumberof stepsincludedinthestep matrix is the one derived from the pairwise alignment with the greatest similarity and the least num- ber of steps. In addition to potentially enhancing phylogenetic resolution and support, by integrating previouslynonaccessiblecharacterswithoutviolatingpositionalhomology,thisnewapproachcanim- provebranchlengthestimationswhenusingparsimony.(Charactercoding;character-stateweighting; crocodile 12S mtrDNA; indel-rich region; insect 16S mtrDNA; intron; large-scale phylogenies; molec- ular phylogenies; multiple sequence alignment; non-protein-coding DNA sequences; ribosomalRNA genes.)

Ribosomal DNA genes in lichen algae and lichen fungi are astonishingly rich in spliceosomal and group I introns. We use phylogenetic, secondary structure, and biochemical analyses to understand the evolution of these introns. Despite the widespread distribution of spliceosomal introns in nuclear pre-mRNA genes, their general mechanism of origin remains an open question because few proven cases of recent and pervasive intron origin have been documented. The lichen introns are valuable in this respect because they are undoubtedly of a “recent” origin and limited to the Euascomycetes. Our analyses suggest that rDNA spliceosomal introns have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. We propose that the spliceosome itself (and not an external agent; e.g. transposable elements, group II introns) has given rise to the introns. The rDNA introns are found most often between the flanking sequence G (78%) - intron-G (72%), and their clustered positions on secondary structures suggest that particular rRNA regions are preferred sites (i.e., proto-splice sites) for insertion. Mapping of intron positions on the newly available tertiary structures show that they are found most often in exposed regions of the ribosomes. This again is consistent with an intron origin through reverse-splicing. Remarkably, the distribution and phylogenetic relationships of most group I introns in nuclear rDNA genes are also consistent with a reverse-splicing origin. These data underline the value of lichens as a model system for understanding intron origin and stress the importance of RNA-level processes in the spread of these sequences in nuclear coding regions.