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Mortierellomycotina subphyl. nov., based on multi-gene genealogies
Abstract and Figures
The Mucoromycotina unifies two heterogenous orders of the sporangiferous, soilinhabiting fungi. The Mucorales comprise saprobic, occasionally facultatively mycoparasitic, taxa bearing a columella, whereas the Mortierellales encompass mainly saprobic fungi lacking a columella. Multi-locus phylogenetic analyses based on eight nuclear genes encoding 18S and 28S rRNA, actin, alpha and beta tubulin, translation elongation factor 1alpha, and RNA polymerase II subunits 1 and 2 provide strong support for separation of the Mortierellales from the Mucoromycotina. The existence of a columella is shown to serve as a synapomorphic morphological trait unique to Mucorales, supporting the taxonomic separation of the acolumellate Mortierellales from the columellate Mucoromycotina. Furthermore, irregular hyphal septation and development of subbasally vesiculate sporangiophores bearing single terminal sporangia strongly correlate with the phylogenetic delimitation of Mortierellales, supporting a new subphylum, Mortierellomycotina.
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... The PCR was conducted in 50 µl reaction mixtures, consisting of 1µL Direct DNA Polymerase, a small number of mycelia as a template, 2× ApexDirect Buffer 25µL, 2µL of each primer, and 20 µL sterile water. DNA fragments of ITS, LSU, and SSU were amplified using primer pairs ITS4/ITS5 (White et al. 1990), NS1/NS8 (Hoffmann et al. 2011), and NL1/NL4 (Hoffmann et al. 2011), respectively. The PCR procedure was as follows: pre-denaturation was performed at 94 °C for 4 min; then, 35 cycles were performed at 94 °C for 30 s, at 50 °C for 30 s, and at 72 °C for 30 s; finally, the cycle was extended at 72 °C for 7 min. ...
... The PCR was conducted in 50 µl reaction mixtures, consisting of 1µL Direct DNA Polymerase, a small number of mycelia as a template, 2× ApexDirect Buffer 25µL, 2µL of each primer, and 20 µL sterile water. DNA fragments of ITS, LSU, and SSU were amplified using primer pairs ITS4/ITS5 (White et al. 1990), NS1/NS8 (Hoffmann et al. 2011), and NL1/NL4 (Hoffmann et al. 2011), respectively. The PCR procedure was as follows: pre-denaturation was performed at 94 °C for 4 min; then, 35 cycles were performed at 94 °C for 30 s, at 50 °C for 30 s, and at 72 °C for 30 s; finally, the cycle was extended at 72 °C for 7 min. ...
Potassium-solubilizing microorganisms are capable of secreting acidic chemicals that dissolve and release potassium from soil minerals, thus facilitating potassium uptake by plants. In this study, three potassium-dissolving filamentous fungi were isolated from the rhizosphere soil of a poplar plantation in Jiangsu Province, China. Phylogenetic analyses based on ITS, 18 S, and 28 S showed that these three isolates were most similar to Mortierella. These strains also possessed spherical or ellipsoidal spores, produced sporangia at the hyphal tip, and formed petal-like colonies on PDA media resembling those of Mortierella species. These findings, along with further phenotypic observations, suggest that these isolates were Mortierella species. In addition, the potassium-dissolution experiment showed that strain 2K4 had a relatively high potassium-solubilizing capacity among these isolated fungi. By investigating the influences of different nutrient conditions (carbon source, nitrogen source, and inorganic salt) and initial pH values on the potassium-dissolving ability, the optimal potassium-solubilization conditions of the isolate were determined. When potassium feldspar powder was used as an insoluble potassium source, isolate 2K4 exhibited a significantly better polysaccharide aggregation ability on the formed mycelium–potassium feldspar complex. The composition and content of organic acids secreted by strain 2K4 were further detected, and the potassium-dissolution mechanism of the Mortierella species and its growth promotion effect were discussed, using maize as an example.
... 2A-C;Nguyen et al. 2016: fig. 2L-O), which is a sterile swelling of the sporangiophore apex that protrudes into the sporangium (Zycha et al. 1969;Hoffmann et al. 2011;Walther et al. 2019). No evidence of a columella was found inside any one of the morphotype-3 inflations, rendering unlikely that they are columellate mucoromycotan sporangia. ...
... However, there are also acolumellate (i.e., lacking a columella) sporangia, for instance in species of Mortierella Coem. and other taxa of the Mortierellales (Hoffmann et al. 2011;Wagner et al. 2013;Vandepol et al. 2020). ...
Palaeonitella cranii is the only known charophyte from the Lower Devonian Rhynie chert. Thalli consist of a main axis with whorls of branchlets, and rhizoids with bulbils. The above-ground parts of this alga are known to have been colonized by a variety of probably parasitic fungi; however, virtually nothing is on record about fungal associations with the rhizoids and bulbils. Here, we describe four different fungal morphotypes as colonizers of bulbils of P. cranii. Morphotypes 1 and 2 are both characterized by epibiotic or interbiotic sporangia and endobiotic apophysate rhizoidal systems. Morphotype 3 occurs in the form of long stalks terminating in pinhead-like inflations, and club-shaped endobiotic axes producing rhizoids distally, whereas morphotype 4 consists of sterile, several-times forked, hypha-like elements. A three-dimensional meshwork of interwoven rhizoids of the colonizers is found in the lumen of several bulbils, indicating that the hosts were viable and crammed with starch grains at the time of colonization. The bulbil-colonizing fungi all differ from the fungi associated with the axes and branchlets of P. cranii, which suggests organ-specific host colonization. Morpho-types 1 and 2 are probably chytrids (Chytridiomycota), while morphotype 3 could be a member of the Mucoromycota; however, affinities to other lineages of fungi cannot be ruled out. This discovery expands the inventory of fungal associations with P. cranii, and provides new data that can be used in considerations on the importance of microorganisms for other aquatic life in the Rhynie ecosystem.
... Different fungal groups have different delineations for their species, which are frequently impacted by a lack of sample availability and in-depth biological understanding. This impact is evident when ITS distances are compared between phyla, subphyla, and species (Taylor et al. 2000;Hoffmann et al. 2011). The rates of secondary fungal infections were higher in positive cases PBFD virus than in healthy cases (Gugnani 2000). ...
AB S T RA C T : Accurate identification of infectious molds from clinical specimens is crucial, as evidenced by the rising incidence of invasive fungal diseases in birds and their growing frequency. Fungal DNA was found in parrot feather samples linked to Psittacine Beak and Feather Disease (PBFD) virus infection following the detection of culture isolates. Internal transcribed spacer regions (ITS) have been employed as targets for phylogenetic research as they typically exhibit sequence variation between species, with modest change within strains of the same species. Numerous sequence variations were found when comparing ITS region sequences from reference and clinical isolates of six Aspergillus species, one Rhizopus arrhizus, and two Penicillium species, The addition of 5.8S rRNA gene sequences had little effect on the species as a whole due to the presence of little diversity between species in this region. Amplification, sequencing, and comparison with non-reference strain sequences in GenBank were performed on strains and clinical isolates of aspergilli and other fungi. Aspergillus and other species amplified ITS region had a size range of 540 to 608 base pairs (bp). A bootstrap of 1000 replications of every fungal isolate was also displayed on the phylogenetic tree, along with varying percentage rates among the fungal isolates.
... Es el grupo hermano de Dikarya, con estilos de vida dominantes asociados a plantas, lo que sugiere que el Most Recent Common Ancestor (MRCA) de Mucoromycota y Dikarya corresponde al origen de las asociaciones modernas hongo-planta. Comprende el grupo más grande y mejor estudiado de hongos zigomicetos y está formado por los subfilos: Glomeromycotina, Mortierellomycotina y Mucoromycotina (74,91). ...
Resumen
Los hongos son organismos polifacéticos presentes en casi todos los ecosistemas de la tierra, donde establecen diversos tipos de simbiosis con otros seres vivos. A pesar de ser reconocidos por los humanos desde la antigüedad -y de la cantidad de trabajos que han profundizado sobre su biología y ecología-, aún falta mucho por conocer sobre estos organismos. Algunos de los criterios que clásicamente se han utilizado para su estudio, hoy resultan limitados y hasta cierto punto permiten un agrupamiento de los aislamientos según algunas características, pero generan confusión en su clasificación y, más aún, cuando se pretende comprender sus relaciones genealógicas.
Los caracteres fenotípicos no son suficientes para identificar una especie de hongos y, menos aún, para construir una filogenia amplia o de un grupo particular. Hay grandes vacíos que hacen que los árboles generados sean inestables y fácilmente debatidos. Para los profesionales de la salud, parece que la identificación de los hongos hasta niveles inferiores como género y especie es suficiente para elegir el tratamiento más adecuado para su control, comprender la epidemiología de los cuadros clínicos asociados y reconocer los brotes y los factores determinantes de la resistencia a los antimicrobianos. No obstante, la ubicación taxonómica dentro del reino permitiría establecer relaciones filogenéticas entre los taxones fúngicos, facilitando la comprensión de su biología, su distribución en la naturaleza y la evolución de su potencial patogénico. Los avances de las técnicas de biología molecular y las ciencias de la computación en los últimos 30 años han permitido cambios importantes dirigidos a establecer los criterios para definir una especie fúngica y alcanzar una construcción filogenética más o menos estable. Sin embargo, el camino por recorrer aún es largo, y supone un trabajo mancomunado de la comunidad científica a nivel global y el apoyo a la investigación básica.
... In our study, soil depth was associated with large declines in fungal taxa (i.e. Mucoromycota, Rozellomycota, Mortierellomycota, and Glomeromycota) previously reported in the literature as saprophytic and rhizospheric fungi (Bonfante and Venice, 2020;Hoffmann et al., 2011;Wagner et al., 2013;Yurkov et al., 2016). ...
Forest soils are fundamental in regulating the global carbon (C) cycle; their capacity to accumulate large stores of C means they form a vital role in mitigating the effects of climate change. Understanding the processes that regulate forest soil C dynamics and stabilisation is important to maximise the capacity and longevity of C sequestration. Compared with surface soil layers, little is known about soil C dynamics in subsoil layers, sensu those below 30 cm depth. This knowledge gap creates large uncertainties when estimating the distribution of global soil C stocks and assessing the vulnerability of soil C reserves to climate change. This study aimed to dive deep into the subsoils of Puruki Experimental Forest (New Zealand) and characterise the changes in soil C dynamics and the soil microbiome down to 1 m soil depth. ITS and 16S rRNA sequencing and quantitative real-time PCR were used to measure changes in soil microbial diversity, composition, and abundance. Stable (δ13C) and radioactive (14C) C analyses were performed to assess depth-driven changes in the stability and age of soil C. Our research identified large declines in microbial diversity and abundance with soil depth, alongside significant structural shifts in community membership. Importantly, we conservatively estimate that more than 35 % of soil C stocks are present in subsoil layers below 30 cm. Although the age of soil C steadily increased with depth, reaching a mean radiocarbon age of 1571 yr BP (years before present) in the deepest soil layers, the stability of soil C varied between different subsoil depth increments. These research findings highlight the importance of quantifying subsoil C stocks for accurate C accounting. By performing a broad range of analytical measures, this research has comprehensively characterised the abiotic and biotic properties of a subsoil environment – a frequently understudied but significant component of forest ecosystems.
... Soil depth was associated with large declines in fungal taxa (i.e. Mucoromycota, Rozellomycota, Mortierellomycota, and Glomeromycota) previously reported in the literature as saprophytic and rhizospheric fungi (Bonfante and Venice, 2020;Hoffmann et al., 2011;Wagner et al., 2013;Yurkov et al., 2016). Understandably, the abundance of saprophytic fungi would 405 decline with increasing physical distance from plant litter inputs on surface soil layers. ...
Forest soils are fundamental in regulating the global carbon (C) cycle; their capacity to accumulate large stores of C means they are vital in mitigating the effects of climate change. Understanding the processes that regulate forest soil organic C (SOC) dynamics and stabilisation is important to maximise the capacity and longevity of C sequestration. Compared to surface soil layers, little is known about the SOC dynamics in subsoil layers, sensu those below 30 cm depth. This knowledge gap creates large uncertainties when estimating the global distribution and vulnerability of SOC reserves to climate change. This study aimed to dive deep into the subsoils of Puruki Experimental Forest (New Zealand) and characterise the incremental changes in SOC dynamics and the soil microbiome down to 1 metre soil depth. ITS and 16S rRNA sequencing and quantitative real-time PCR were used to measure changes in soil microbial diversity, composition, and abundance. Stable (δ13C) and radioactive (14C) C analyses were performed to assess depth-driven changes in SOC stability and age. We conservatively estimate more than 35 % of total C stocks are present in subsoil layers below 30 cm. Although C age steadily increased with depth, reaching a mean radiocarbon age of 1571 yBP (years before present) in the deepest soil layers, the stability of SOC varied between different subsoil depth increments. Declines in soil carbon were associated with lower microbial diversity, abundance, and significant shifts in community membership. These research findings highlight the importance of quantifying subsoil C stocks for accurate systems-level global and local C budgets and modeling. Furthermore, performing a broad range of analytical measures (i.e. 13C & 14C natural abundance, and microbiome analysis) is vital to assess the vulnerability of subsoil C to climate change.
... The Zygomycota is considered to depict the most basal terrestrial phylum to have evolved from flagellate, aquatic ancestors. Molecular phylogenetic analyses revealed dispersal into five subphyla containing one to four orders (Hibbett et al. 2007;Hoffmann et al. 2011). ...
The increasing number of new fungal species described from all over the world along with the use of genetics to define taxa, has dramatically changed the classification system of early-diverging fungi over the past several decades. The number of phyla established for non-Dikarya fungi has increased from 2 to 17. However, to date, both the classification and phylogeny of the basal fungi are still unresolved. In this article, we review the recent taxonomy of the basal fungi and re-evaluate the relationships among early-diverging lineages of fungal phyla. We also provide information on the ecology and distribution in Mucoromycota and highlight the impact of chytrids on amphibian populations. Species concepts in Chytridiomycota , Aphelidiomycota , Rozellomycota , Neocallimastigomycota are discussed in this paper. To preserve the current application of the genus Nephridiophaga ( Chytridiomycota : Nephridiophagales ) , a new type species, Nephridiophaga blattellae , is proposed.
Zygomycetes are phylogenetically early diverging, ecologically diverse, industrially valuable, agriculturally beneficial, and clinically pathogenic fungi. Although new phyla and subphyla have been constantly established to accommodate spe- cific members and a subkingdom Mucoromyceta, comprising Calcarisporiellomycota, Glomeromycota, Mortierellomycota and Mucoromycota, was erected to unite core zygomycetous fungi, phylogenetic relationships within phyla have not been well resolved. Taking account of the information of monophyly and divergence time estimated from ITS and LSU rDNA sequences, the present study updates the classification framework of the phylum Mucoromycota from the class down to the generic rank: three classes, three orders, 20 families (including five new families Circinellaceae, Protomycocladaceae, Rhizomucoraceae, Syzygitaceae and Thermomucoraceae) and 64 genera. The taxonomic hierarchy was calibrated with estimated divergence times: phylum earlier than 617 Mya, classes and orders earlier than 547 Mya, families earlier than 199 Mya, and genera earlier than 12 Mya. Along with this outline, all genera of Mucoromycota are annotated and 58 new species are described. In addition, three new combinations are proposed. In this study, we update the taxonomic backbone of the phylum Mucoromycota and reinforce its phylogeny. We also contribute numerous new taxa and enrich the diversity of Mucoromycota.
Research into freshwater fungi has generated a wealth of information over the past decades with various published articles, i.e., reviews, books, and monographs. With the advancement of
methodologies used in freshwater fungal research, and numerous mycologists working on this ecological group, our knowledge progress and understanding of freshwater fungi, including novel
discoveries and new insights in the ecology of freshwater fungi, has advanced. With this enormous progress, it is timely that an updated account of freshwater fungi be compiled in one volume. Thus, this account is published to give a comprehensive overview of the different facets of freshwater fungal biology. It includes an updated classification scheme based on the latest taxonomic and phylogenetic analysis of freshwater fungal taxa, including their evolutionary history. The biology, diversity, and geographical distribution of higher and basal freshwater fungi are also discussed in the entries. A section on dispersal and adaptation of filamentous freshwater fungi is included in the present work. The ecological importance and role of fungi in the breakdown of wood in freshwater habitats, including their physiology, are discussed in detail. The biotechnological potential of freshwater fungi as producers of bioactive metabolites are reviewed, with methodologies in antimicrobial drug discovery. The present volume also provides an overview of different high throughput sequencing (HTS) platforms for freshwater fungal research highlighting their advantages and challenges, including recent studies of HTS in identification and quantification of fungal communities in freshwater habitats. The present volume also identifies the knowledge gaps and direction of future research in freshwater fungi.
Zygomycetes are phylogenetically early diverged, ecologically diverse, industrially valuable, agriculturally beneficial, and clinically pathogenic fungi. Although new phyla and subphyla have been constantly established to accommodate specific members and a subkingdom, Mucoromyceta, was erected to unite core zygomycetous fungi, their phylogenetic relationships have not been well resolved. Taking account of the information of monophyly and divergence time estimated from ITS and LSU rDNA sequences, the present study updates the classification framework of the subkingdom Mucoromyceta from the phylum down to the generic rank: six phyla (including two new phyla Endogonomycota and Umbelopsidomycota), eight classes, 15 orders (including five new orders Claroideoglomerales, Cunninghamellales, Lentamycetales, Phycomycetales and Syncephalastrales), 41 families (including six new families Circinellaceae, Gongronellaceae, Protomycocladaceae, Rhizomucoraceae, Syzygitaceae and Thermomucoraceae), and 121 genera. The taxonomic hierarchy was calibrated with estimated divergence times: phyla 810–639 Mya, classes 651–585 Mya, orders 570–400 Mya, and families 488–107 Mya. Along with this outline, 71 genera are annotated and 73 new species are described. In addition, three new combinations are proposed. In this paper, we update the taxonomic backbone of the subkingdom Mucoromyceta and reinforce its phylogeny. We also contribute numerous new taxa and enrich the diversity of Mucoromyceta.
A maximum likelihood method for inferring evolutionary trees from DNA sequence data was developed by Felsenstein (1981). In evaluating the extent to which the maximum likelihood tree is a significantly better representation of the true tree, it is important to estimate the variance of the difference between log likelihood of different tree topologies. Bootstrap resampling can be used for this purpose (Hasegawa et al. 1988; Hasegawa and Kishino 1989), but it imposes a great computation burden. To overcome this difficulty, we developed a new method for estimating the variance by expressing it explicitly.The method was applied to DNA sequence data from primates in order to evaluate the maximum likelihood branching order among Hominoidea. It was shown that, although the orangutan is convincingly placed as an outgroup of a human and African apes clade, the branching order among human, chimpanzee, and gorilla cannot be determined confidently from the DNA sequence data presently available when the evolutionary rate constancy is not assumed.
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, Lecanoromycetidae, 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 Zoopagomycotina. 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. © 2007 The British Mycological Society.
Zygomycetes encompass microscopic fungi forming zygospores in sexual interactions. The most prominent and largest order is the Mucorales comprising saprotrophic and facultatively parasitic species. The traditional family system of the Mucorales deviates substantially from molecular phylogenies. Multi-gene genealogies (btub) and translation elongation factor 1 alpha (tef), the nuclear genes for the small (18S) and large (28S) subunit ribosomal RNA (comprising 811, 1168, 1112, 871, 402 characters, respectively) of twenty-seven genera of the Mucorales shows that the traditional family structure is highly unnatural at the level of groups with monophyletic origin. Four families can be considered to be monophyletic; the Umbelopsidaceae, the Phycomycetaceae, the Absidiaceae and the Choanephoraceae including Gilbertella. The establishment of a natural system suggests Spinellus as the closest phylogenetic relative to Phycomyces, which converts the former monogeneric family Phycomycetaceae to becoming bigeneric. The Mucoraceae and the Thamnidiacaeae, both representing the largest families, are polyphyletic and therefore highly unnatural. Special emphasis was given to the determination of micromorphological and ultrastructural apomorphies derived by light and scanning electron microscopy. Furthermore, phylogenetic reconstructions based on a combined profile distance analysis of small and large subunit rDNA (1431 and 387 characters, respectively) of seventy-two taxa suggest a close phylogenetic relationship between the Mucorales and the Entomophthorales. The Mortierellales, which has been recently classified to the Mucormycotina, appear as the basal group of the Dikarya.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
The estimation procedure utilizes a compatibility analysis between enzyme data sets of the most parsimonious trees constructed from the restriction enzyme. Next, a non-parametric test is given for comparing alternative phylogenies. A 2nd non-parametric test is developed for testing the molecular clock hypothesis. To illustrate the power of these procedures, data derived from the mitochondrial DNA and globin DNA of man and the apes are analyzed. Although previous analyses of these data led to the speculation that 10 times more information would be required to resolve the evolutionary relationships between man with chimps and gorillas, this algorithm resolved these relationships at the 5% level of significance. The molecular clock hypothesis was rejected at the 1% level. The implications of this phylogenetic inference when coupled with other types of data lead to the conclusion that knuckle-walking - not bipedalism - is the evolutionary novelty in mode of locomotion in the primates and that many other hominid features are primitive whereas their African ape counterparts are derived.-from Author
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.