Optimizing an integrated biovigilance toolbox to study the spatial distribution and dynamic changes of airborne mycobiota, with a focus on cereal rust fungi in western Canada.

In the face of evolving agricultural practices and climate change, tools towards an integrated biovigilance platform to combat crop diseases, spore sampling, DNA diagnostics and predictive trajectory modelling were optimized. These tools revealed microbial dynamics and were validated by monitoring cereal rust fungal pathogens affecting wheat, oats, barley and rye across four growing seasons (2015-2018) in British Columbia and during the 2018 season in southern Alberta. ITS2 metabarcoding revealed disparity in aeromycobiota diversity and compositional structure across the Canadian Rocky Mountains, suggesting a barrier effect on air flow and pathogen dispersal. A novel bioinformatics classifier and curated cereal rust fungal ITS2 database, corroborated by real-time PCR, enhanced the precision of cereal rust fungal species identification. Random Forest modelling identified crop and land-use diversification as well as atmospheric pressure and moisture as key factors in rust distribution. As a valuable addition to explain observed differences and patterns in rust fungus distribution, trajectory HYSPLIT modelling tracked rust fungal urediniospores' northeastward dispersal from the Pacific Northwest towards southern British Columbia and Alberta, indicating multiple potential origins. Our Canadian case study exemplifies the power of an advanced biovigilance toolbox towards developing an early-warning system for farmers to detect and mitigate impending disease outbreaks.

DNA barcode sequencing discovered the aecial host of Puccinia chunjiei.

The telial stage of Puccinia chunjiei was described in 2012 from a single specimen collected in China (DAOM 240982). This species is the closest relative of P. graminis but differs in telial morphology on their grass hosts and in DNA sequences. As part of a DNA-barcoding project for rust herbarium specimens, collections of the aecial stage of P. graminis on Berberis were processed. For one specimen, BPI 1103856, the ITS sequence matched that of P. chunjiei and the aecial morphology differed from P. graminis. An expanded description of P. chunjiei is presented with photographs of the aecial stages of both species.

Genetic time traveling: sequencing old herbarium specimens, including the oldest herbarium specimen sequenced from kingdom Fungi, reveals the population structure of an agriculturally significant rust.

Sequencing herbarium specimens can be instrumental in answering ecological, evolutionary, and taxonomic inquiries. We developed a protocol for sequencing herbarium specimens of rust fungi (Pucciniales) and proceeded to sequence specimens ranging from 4 to 211 yr old from five different genera. We then obtained sequences from an economically important biological control agent, Puccinia suaveolens, to highlight the potential of sequencing herbarium specimens in an ecological sense and to evaluate the following hypotheses: (1) The population structure of a plant pathogen changes over time, and (2) introduced pathogens are more diverse in their native range. Our efforts resulted in sequences from 87 herbarium specimens that revealed a high level of diversity with a population structure that exhibited spatial-temporal patterns. The specimens sequenced from Europe showed more diversity than the ones from North America, uncovering an invasion pattern likely related to its European native host in North America. Additionally, to the best of our knowledge, the specimen from France collected in c. 1811 is the oldest herbarium specimen sequenced from kingdom Fungi. In conclusion, sequencing old herbarium specimens is an important tool that can be extrapolated to better understand plant-microbe evolution and to evaluate old type specimens to solidify the taxonomy of plant pathogenic fungi.

Towards Improved Detection and Identification of Rust Fungal Pathogens in Environmental Samples Using a Metabarcoding Approach.

The dispersion of fungal inocula such as the airborne spores of rust fungi (Pucciniales) can be monitored through metabarcoding of the internal transcribed spacer 2 (ITS2) of the rRNA gene in environmental DNAs. This method is largely dependent on a high-quality reference database (refDB) and primers with proper taxonomic coverage and specificity. For this study, a curated ITS2 reference database (named CR-ITS2-refDB) comprising representatives of the major cereal rust fungi and phylogenetically related species was compiled. Interspecific and intraspecific variation analyses suggested that the ITS2 region had reasonable discriminating power for the majority of the Puccinia species or species complexes in the database. In silico evaluation of nine forward and seven reverse ITS2 primers, including three newly designed, revealed marked variation in DNA amplification efficiency for the rusts. We validated the theoretical assessment of rust-enhanced (Rust2inv/ITS4var_H) and universal fungal (ITS9F/ITS4) ITS2 primer pairs by profiling the airborne rust fungal communities from environmental samples via a metabarcoding approach. Species- or subspecies-level identification of the rusts was improved by use of CR-ITS2-refDB and the Automated Oligonucleotide Design Pipeline (AODP), which identified all mutations distinguishing highly conserved DNA markers between close relatives. A generic bioinformatics pipeline was developed, including all steps used in this study from in silico evaluation of primers to accurate identification of short metabarcodes at the level of interest for defining phytopathogens. The results highlight the importance of primer selection, refDBs that are resolved to reflect phylogenetic relationships, and the use of AODP for improving the reliability of metabarcoding in phytopathogen biosurveillance.

Four In Silico Designed and Validated qPCR Assays to Detect and Discriminate Tilletia indica and T. walkeri, Individually or as a Complex.

Several fungi classified in the genus Tilletia are well-known to infect grass species including wheat (Triticum). Tilletia indica is a highly unwanted wheat pathogen causing Karnal bunt, subject to quarantine regulations in many countries. Historically, suspected Karnal bunt infections were identified by morphology, a labour-intensive process to rule out other tuberculate-spored species that may be found as contaminants in grain shipments, and the closely-related pathogen T. walkeri on ryegrass (Lolium). Molecular biology advances have brought numerous detection tools to discriminate Tilletia congeners (PCR, qPCR, etc.). While those tests may help to identify T. indica more rapidly, they share weaknesses of targeting insufficiently variable markers or lacking sensitivity in a zero-tolerance context. A recent approach used comparative genomics to identify unique regions within target species, and qPCR assays were designed in silico. This study validated four qPCR tests based on single-copy genomic regions and with highly sensitive limits of detection (~200 fg), two to detect T. indica and T. walkeri separately, and two newly designed, targeting both species as a complex. The assays were challenged with reference DNA of the targets, their close relatives, other crop pathogens, the wheat host, and environmental specimens, ensuring a high level of specificity for accurate discrimination.

Development and evaluation of a target enrichment bait set for phylogenetic analysis of oomycetes.

Target enrichment is a term that encompasses multiple related approaches where desired genomic regions are captured by molecular baits, leaving behind redundant or non-target regions in the genome, followed by amplification and next-generation sequencing of those captured regions. A molecular bait set was developed based on 426 single-copy, oomycete-specific orthologs and 3 barcoding genes. The bait set was tested on 27 oomycete samples (belonging to the Saprolegniales, Albuginales, and Peronosporales) derived from live and herbarium specimens, as well as control samples of true fungi and plants. Results show that (i) our method greatly enriches for the targeted orthologs on oomycete samples, but insignificantly on fungal and plant samples; (ii) an average of 263 out of 429 orthologs (61%) were recovered from oomycete live and herbarium specimens; (iii) sequencing roughly 100 000 read pairs per sample is sufficient for optimal ortholog recovery while maintaining low sequencing costs; and (iv) the expected relationships were recovered by phylogenetic analysis from the data generated. This is the first report of an oomycete-specific target enrichment method with broad potential applications for evolutionary and taxonomic studies. A key benefit of our target enrichment method is that it allows researchers to easily unlock the vast and unexplored oomycete genomic diversity stored in natural history collections.

Taxonomic revision of Blumeria based on multi-gene DNA sequences, host preferences and morphology.

A taxonomic revision of the hitherto monotypic genus Blumeria was conducted incorporating multi-gene sequence analyses, host preference data and morphological criteria. The sequenced loci included rDNA ITS, partial chitin synthase gene (CHS1), as well as fragments of two unnamed orthologous genes (Bgt-1929, Bgt-4572). The combined evidence led to a reassessment and a new neotypification of B. graminiss. str. (emend.), and the description of seven additional species, viz. B. americana sp. nov. (mainly on hosts of the Triticeae), B. avenae sp. nov. (on Avena spp.), B. bromi-cathartici sp. nov. (on Bromus catharticus), B. bulbigera comb. nov. (on Bromus spp.), B. dactylidis sp. nov. (on Dactylis glomerata as the main host, but also on various other hosts), B. graminicola sp. nov. (on Poa spp. as principal hosts, but also on various other hosts), and B. hordei sp. nov. (on Hordeum spp.). Synonyms were assessed, some were lectotypified, and questionable names previously associated with powdery mildew on monocots were discussed although their identities remained unresolved. Keys to the described species were developed.

Genome sequencing and comparison of five Tilletia species to identify candidate genes for the detection of regulated species infecting wheat.

Tilletia species cause diseases on grass hosts with some causing bunt diseases on wheat (Triticum). Two of the four species infecting wheat have restricted distributions globally and are subject to quarantine regulations to prevent their spread to new areas. Tilletia indica causes Karnal bunt and is regulated by many countries while the non-regulated T. walkeri is morphologically similar and very closely related phylogenetically, but infects ryegrass (Lolium) and not wheat. Tilletia controversa causes dwarf bunt of wheat (DB) and is also regulated by some countries, while the closely related but non-regulated species, T. caries and T. laevis, both cause common bunt of wheat (CB). Historically, diagnostic methods have relied on cryptic morphology to differentiate these species in subsamples from grain shipments. Of the DNA-based methods published so far, most have focused on sequence variation among tested strains at a single gene locus. To facilitate the development of additional molecular assays for diagnostics, we generated whole genome data for multiple strains of the two regulated wheat pathogens and their closest relatives. Depending on the species, the genomes were assembled into 907 to 4633 scaffolds ranging from 24 Mb to 30 Mb with 7842 to 9952 gene models predicted. Phylogenomic analyses confirmed the placement of Tilletia in the Exobasidiomycetes and showed that T. indica and T. walkeri were in one clade whereas T. controversa, T. caries and T. laevis grouped in a separate clade. Single copy and species-specific genes were identified by orthologous group analysis. Unique species-specific genes were identified and evaluated as suitable markers to differentiate the quarantine and non-quarantine species. After further analyses and manual inspection, primers and probes for the optimum candidate genes were designed and tested in silico, for validation in future wet-lab studies.

Novel multi-target compounds in the quest for new chemotherapies against Alzheimer's disease: An experimental and theoretical study.

The lack of any effective therapy along with the aging world population anticipates a growth of the worldwide incidence of Alzheimer's disease (AD) to more than 100 million cases by 2050. Accumulation of extracellular amyloid-ß (Aß) plaques, intracellular tangles in the brain, and formation of reactive oxygen species (ROS) are the major hallmarks of the disease. In the amyloidogenic process, a ß-secretase, known as BACE 1, plays a fundamental role in the production of Aß fragments, and therefore, inhibition of such enzymes represents a major strategy for the rational design of anti-AD drugs. In this work, a series of four multi-target compounds (1-4), inspired by previously described ionophoric polyphenols, have been synthesized and studied. These compounds have been designed to target important aspects of AD, including BACE 1 enzymatic activity, Aß aggregation, toxic concentrations of Cu2+ metal ions and/or ROS production. Two other compounds (5 and 6), previously reported by some of us as antimalarial agents, have also been studied because of their potential as multi-target species against AD. Interestingly, compounds 3 and 5 showed moderate to good ability to inhibit BACE 1 enzymatic activity in a FRET assay, with IC50's in the low micromolar range (4.4 ±â€¯0.3 and 1.7 ±â€¯0.3 µM, respectively), comparable to other multi-target species, and showing that the observed activity was in part due to a competitive binding of the compounds at the active site of the enzyme. Theoretical docking calculations overall agreed with FRET assay results, displaying the strongest binding affinities for 3 and 5 at the active site of the enzyme. In addition, all compounds selectively interacted with Cu2+ metal ions forming 2:1 complexes, inhibited the production of Aß-Cu2+ catalyzed hydroxyl radicals up to a ∼100% extent, and scavenged AAPH-induced peroxyl radical species comparably to resveratrol, a compound used as reference in this work. Our results also show good anti-amyloidogenic ability: compounds 1-6 inhibited both the Cu2+-induced and self-induced Aß(1-40) fibril aggregation to an extent that ranged from 31% to 77%, while they disaggregated pre-formed Aß(1-40) mature fibrils up to a 37% and a 69% extent in absence and presence of Cu2+, respectively. Cytotoxicity was additionally studied in Tetrahymena thermophila and HEK293 cells, and compared to that of resveratrol, showing that compounds 1-6 display lower toxicity than that of resveratrol, a well-known non-toxic polyphenol.

Assessing Performance of Spore Samplers in Monitoring Aeromycobiota and Fungal Plant Pathogen Diversity in Canada.

Spore samplers are widely used in pathogen surveillance but not so much for monitoring the composition of aeromycobiota. In Canada, a nationwide spore-sampling network (AeroNet) was established as a pilot project to assess fungal community composition in air and rain samples collected using three different spore samplers in the summers of 2010 and 2011. Metabarcodes of the internal transcribed spacer (ITS) were exhaustively characterized for three of the network sites, in British Columbia (BC), Québec (QC), and Prince Edward Island (PEI), to compare performance of the samplers. Sampler type accounted for ca. 20% of the total explainable variance in aeromycobiota compositional heterogeneity, with air samplers recovering more Ascomycota and rain samplers recovering more Basidiomycota. Spore samplers showed different abilities to collect 27 fungal genera that are plant pathogens. For instance, Cladosporium spp., Drechslera spp., and Entyloma spp. were collected mainly by air samplers, while Fusarium spp., Microdochium spp., and Ustilago spp. were recovered more frequently with rain samplers. The diversity and abundance of some fungi were significantly affected by sampling location and time (e.g., Alternaria and Bipolaris) and weather conditions (e.g., Mycocentrospora and Leptosphaeria), and depended on using ITS1 or ITS2 as the barcoding region (e.g., Epicoccum and Botrytis). The observation that Canada's aeromycobiota diversity correlates with cooler, wetter conditions and northward wind requires support from more long-term data sets. Our vision of the AeroNet network, combined with high-throughput sequencing (HTS) and well-designed sampling strategies, may contribute significantly to a national biovigilance network for protecting plants of agricultural and economic importance in Canada.IMPORTANCE The current study compared the performance of spore samplers for collecting broad-spectrum air- and rain-borne fungal pathogens using a metabarcoding approach. The results provided a thorough characterization of the aeromycobiota in the coastal regions of Canada in relation to the influence of climatic factors. This study lays the methodological basis to eventually develop knowledge-based guidance on pest surveillance by assisting in the selection of appropriate spore samplers.

Rust fungi on Panicum.

Rusts are economically important diseases of switchgrass (Panicum virgatum) and other Paniceae grasses. Phylogenetic analyses based on sequences of the nuc rDNA 5.8S internal transcribed spacer 2 region (ITS2), partial 28S region, and intergenic spacer region (IGS) of nuc rDNA showed that species of rust fungi infecting switchgrass are closely related within Puccinia. Variation among rbcLa sequences for the associated hosts sampled concurred with the original identifications. Five species infecting switchgrass were recognized: Puccinia graminicola (≡ Uromyces graminicola), P. pammelii (= P. panici), and the proposed new species P. amari, P. novopanici, and P. pascua. These species were distinct from P. emaculata, the species previously considered the principal rust pathogen infecting switchgrass but that was found exclusively on witchgrass (Panicum capillare) in this study. Rust fungi on switchgrass previously identified as P. emaculata were identified as the morphologically similar species P. amari, P. novopanici, and P. pammelii. The morphological species Puccinia graminicola was found to comprise three species, P. graminicola and the proposed new species P. pascua on switchgrass and P. cumminsii on Panicum sp.

Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi.

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi. Database URL: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353.

Molecular characterization of reptile pathogens currently known as members of the chrysosporium anamorph of Nannizziopsis vriesii complex and relationship with some human-associated isolates.

In recent years, the Chrysosporium anamorph of Nannizziopsis vriesii (CANV), Chrysosporium guarroi, Chrysosporium ophiodiicola, and Chrysosporium species have been reported as the causes of dermal or deep lesions in reptiles. These infections are contagious and often fatal and affect both captive and wild animals. Forty-nine CANV isolates from reptiles and six isolates from human sources were compared with N. vriesii based on their cultural characteristics and DNA sequence data. Analyses of the sequences of the internal transcribed spacer and small subunit of the nuclear ribosomal gene revealed that the reptile pathogens and human isolates belong in well-supported clades corresponding to three lineages that are distinct from all other taxa within the family Onygenaceae of the order Onygenales. One lineage represents the genus Nannizziopsis and comprises N. vriesii, N. guarroi, and six additional species encompassing isolates from chameleons and geckos, crocodiles, agamid and iguanid lizards, and humans. Two other lineages comprise the genus Ophidiomyces, with the species Ophidiomyces ophiodiicola occurring only in snakes, and Paranannizziopsis gen. nov., with three new species infecting squamates and tuataras. The newly described species are Nannizziopsis dermatitidis, Nannizziopsis crocodili, Nannizziopsis barbata, Nannizziopsis infrequens, Nannizziopsis hominis, Nannizziopsis obscura, Paranannizziopsis australasiensis, Paranannizziopsis californiensis, and Paranannizziopsis crustacea. Chrysosporium longisporum has been reclassified as Paranannizziopsis longispora. N. guarroi causes yellow fungus disease, a common infection in bearded dragons and green iguanas, and O. ophiodiicola is an emerging pathogen of captive and wild snakes. Human-associated species were not recovered from reptiles, and reptile-associated species were recovered only from reptiles, thereby mitigating concerns related to zoonosis.

Molecular Phylogenetic Relationships of the Brown Leaf Rust Fungi on Wheat, Rye, and Other Grasses.

The classification of brown leaf rust fungi (Puccinia recondita complex and allied species) on wheat (Triticum aestivum), rye (Secale cereale), and other grasses in the family Poaceae has experienced a long history of controversy and uncertainty due to the reduced morphological characteristics available for taxonomy and difficulty of conducting interfertility experiments. However, because these are pathogens on important crops, it is important to clarify the species delimitations reflecting the natural lineages. In this study, phylogenetic analyses were conducted with DNA sequence data from the ribosomal DNA internal transcribed spacer region and elongation factor 1-α to elucidate this species complex. Three phylogenetic lineages were recovered within the complex of rye leaf rust fungi, P. recondita sensu stricto, which is congruent with existing classifications based on DNA content, sexual compatibility, and morphological studies. The brown leaf rust fungus on wheat (P. triticina) grouped with the related species P. persistens on Elymus repens and E. intermedia as a strongly supported clade. Collections on other Elymus spp. were separated into six clades. Based on the phylogenetic affinities of nine type specimens and aecial host associations, potential taxonomic names were evaluated for selected lineages.

Puccinia chunjii, a close relative of the cereal stem rusts revealed by molecular phylogeny and morphological study.

A rust specimen with macroscopic similarities to the cereal stem rusts was collected on Elymus sp. from Gansu province, China. Phylogenetic analyses of ITS and COI DNA sequences indicated the fungus was closely related but distinct as a strongly supported sister taxon to the Puccinia graminis species complex. Microscopic examination revealed diagnostic teliospore characteristics, differentiating it from P. graminis and other morphologically similar rusts. Herein, we designate a name for this new lineage, Puccinia chunjii sp. nov.

Taxonomic study of stripe rust, Puccinia striiformis sensu lato, based on molecular and morphological evidence.

Phylogenetic analyses of ITS and ß-tubulin DNA sequences for 30 Puccinia striiformis specimens from wide geographic and host ranges revealed four strongly supported monophyletic lineages. Based on comparisons of morphological characteristics, and after consideration of previous classifications, the lineages were recognized as P. striiformis sensu stricto, Puccinia pseudostriiformis, Puccinia striiformoides and one new species, Puccinia gansensis. A new series (Puccinia Series Striiformis) was erected to accommodate this strongly supported monophyletic group of four species within Puccinia. Expanded descriptions, illustrations and a tabular identification key are provided. The morphological differences among species are subtle but host association and combinations of other characters can be used successfully for identification. Puccinia striiformis s.str. has the widest host range, within the tribe Triticeae (Aegilops, Elymus, Hordeum and Triticum) but it is less diverse than previously documented. The other three species are restricted to single genera in Poeae (P. pseudostriiformis on Poa; P. striiformoides on Dactylis) or Stipeae (P. gansensis on Achnatherum). Urediniospore size and surface echinulation, especially as seen by SEM, germ pore number, shape of uredinial paraphyses, and teliospore hilum width were the main features used to differentiate species. Relying solely on the symptom of linearly aligned uredinia on chlorotic stripes on leaves, from which the common name "stripe rust" was derived, is not recommended because sori of other leaf rust species may also form in a linear series.

Reconstructing the early evolution of Fungi using a six-gene phylogeny.

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.

User satisfaction with a constipation service: a comparative audit.

Chronic constipation in children affects their quality of life and can have far reaching consequences, causing low self-esteem in children and family conflict (McGinley 2001). A review of outcomes following the establishment of a new constipation service showed significant improvements in outcomes and in the level of support provided to families. To obtain a more in depth picture of effectiveness, quantitative findings were supplemented with the views of 16 children and families who use the service. Families generally understood the condition and were attempting to make lifestyle changes. They appreciated the education and support offered by the service: for many it was the first time that they had been given effective treatment for what was often a long term problem. Although the existing service is effective, further development could help prevent constipation, improve early intervention and prevent the need for secondary referrals.

A multigene phylogeny of the Dothideomycetes using four nuclear loci.

We present an expanded multigene phylogeny of the Dothideomycetes. The final data matrix consisted of four loci (nuc SSU rDNA, nuc LSU rDNA, TEF1, RPB2) for 96 taxa, representing five of the seven orders in the current classification of Dothideomycetes and several outgroup taxa representative of the major clades in the Pezizomycotina. The resulting phylogeny differentiated two main dothideomycete lineages comprising the pseudoparaphysate Pleosporales and aparaphysate Dothideales. We propose the subclasses Pleosporomycetidae (order Pleosporales) and Dothideomycetidae (orders Dothideales, Capnodiales and Myriangiales). Furthermore we provide strong molecular support for the placement of Mycosphaerellaceae and Piedraiaceae within the Capnodiales and introduce Davidiellaceae as a new family to accommodate species of Davidiella with Cladosporium anamorphs. Some taxa could not be placed with certainty (e.g. Hysteriales), but there was strong support for new groupings. The clade containing members of the genera Botryosphaeria and Guignardia resolved well but without support for any relationship to any other described orders and we hereby propose the new order Botryosphaeriales. These data also are consistent with the removal of Chaetothyriales and Coryneliales from the Dothideomycetes and strongly support their placement in the Eurotiomycetes.

False-positive Histoplasma capsulatum Gen-Probe chemiluminescent test result caused by a Chrysosporium species.

We describe a case in which the Histoplasma capsulatum AccuProbe test displayed cross-reactivity with a respiratory isolate thought to be Histoplasma but not morphologically consistent with H. capsulatum. The isolate was later identified as the Chrysosporium anamorph of Nannizziopsis vriesii by sequence analysis and phenotypic data.