Congruence and indifference between two molecular markers for understanding oral evolution in the Marynidae sensu lato (Ciliophora, Colpodea)

Our understanding of the evolution of oral structures within the Colpodida is confounded by the low number of morphological characters that can be used in constructing hypotheses, and by the low taxon and character sampling in molecular phylogenetic analyses designed to assess these hypotheses. Here we increase character sampling by sequencing the mitochondrial SSU-rDNA locus for three isolates of the Marynidae sensu lato. We show that the inferred mitochondrial and nuclear SSU-rDNA trees, as well as concatenated and constrained analyses, are congruent in not recovering a monophyletic Marynidae. However, due to low node support, the trees are indifferent to whether the morphological characters used to unite the Marynidae are the result of retention of ancestral states or convergence. In light of this indifference and an increased amount of nuclear and mitochondrial SSU-rDNA data, alternative hypotheses of oral evolution in the Colpodida are presented.     

Corroboration versus "Strongest Evidence"

Background knowledge comprises accepted (well-corroborated) theories and results. Such theories are taken to be true for the purpose of interpreting evidence when assessing the corroboration of a hypothesis currently in question. Accordingly, background knowledge does not properly include rejected theories, false assumptions, or null models. In particular, regarding a model of random character distribution as "background knowledge" would rule out corroboration of phylogenetic hypotheses, since it would make character data irrelevant to inferring phylogeny. The presence of homoplasy is not grounds for treating characters as if they were randomly distributed, since characters can show strong phylogenetic structure even when they show homoplasy. This means that clique (compatibility) analysis is unjustified, since that method depends crucially on the assumption that characters showing any homoplasy at all are unrelated to phylogeny. Although likelihood does not measure corroboration, corroboration is closely connected to likelihood: for given evidence and background, the most likely trees are also best corroborated. Most parsimonious trees are best corroborated; the apparent clash between parsimony and likelihood is an artifact of the use of unrealistic models in most "maximum likelihood" methods.     

Homoplasy, homology, and the perceived special status of behavior in evolution

Evolutionary biologists tend to tread cautiously when considering how behavioral data might be incorporated into phylogenetic analyses, largely because of the preconception that behavior somehow constitutes a "special" set of characters that may be inherently more prone to homoplasy or subject to different selection regimes than those that operate on the morphological or genetic traits traditionally used in phylogenetic reconstruction. In this review, we first consider how the evolution of behavior has been treated historically, paying particular attention to why phylogenetic reconstruction has often failed to include behavioral traits. We then discuss, from a theoretical perspective, what reasons there are--if any--for assuming that behavioral traits should be more prone to homoplasy than other types of traits. In doing so, we review several empirical studies that tackle this issue head-on. Finally, we examine how behavioral features have been used to good effect in phylogenetic reconstruction. Our conclusion is that there seems to be little justification on theoretical grounds for assuming that behavior is in any way "special"--either particularly labile or particularly prone to exhibit high levels of homoplasy. Additionally, in reviewing historical perceptions of behavior and their links to conceptions of homology, we conclude that there is no compelling reason why behavior cannot be homologized or therefore why it should not prove phylogenetically informative. In subsequently considering several factors related to selection that influence the likelihood of homoplasy occurring in any trait system, we also found no clear trend predicting homoplasy disproportionately in behavioral systems. In fact, where studied, the degree of homoplasy seen in behavioral traits is comparable to that seen in other trait systems. Ultimately, there appear to be no grounds for dismissing behavior a priori from the class of phylogenetically informative characters.     

Evolution of the Vertebrate Central Nervous System: Patterns and Processes

AS brains do not fossilize, most proposed phylogenetic sequencesfor central nervous system characters must be based on the patternsof variation of those characters in living organisms. Similarly,hypotheses regarding how brains change through time, and theevolutionary processes that produce these changes, are ultimatelybased on the character patterns recognized. It is critical inthese analyses to distinguish between homologous and homoplasouscharacters if errors in the reconstruction and interpretationof phylogenies are to be minimized. Definitions of homologyand homoplasy are reviewed, as are the concepts that bear ontheir application. Cladistic definitions are adopted, and criteriafor distinguishing homologous from homoplasous characters arediscussed. Analysis of a number of CNS characters that are usuallyassumed to be homologous reveals that homoplasous charactersappear among them. As in other organ systems, homoplasous charactersare actually common. A number of previous hypotheses regardingCNS evolution are reviewed in the context of new data on neuralconnections and their cladistic analysis. Some of these hypothesesmay be falsified by a cladistic treatment of CNS characters,whereas sufficient data do not exist to evaluate others.     

Molecular phylogenetics of Vandeae (Orchidaceae) and the evolution of leaflessness

Members of tribe Vandeae (Orchidaceae) form a large, pantropical clade of horticulturally important epiphytes. Monopodial leafless members of Vandeae have undergone extreme reduction in habit and represent a novel adaptation to the canopy environment in tropical Africa, Asia, and America. To study the evolution of monopodial leaflessness, molecular and structural evidence was used to generate phylogenetic hypotheses for Vandeae. Molecular analyses used sequence data from ITS nrDNA, trnL-F plastid DNA, and matK plastid DNA. Maximum parsimony analyses of these three DNA regions each supported two subtribes within monopodial Vandeae: Aeridinae and a combined Angraecinae + Aerangidinae. Adding structural characters to sequence data resulted in trees with more homoplasy, but gave fewer trees each with more well-supported clades than either data set alone. Two techniques for examining character evolution were compared: (1) mapping vegetative characters onto a molecular topology and (2) tracing vegetative characters onto a combined structural and molecular topology. In both cases, structural synapomorphies supporting monopodial Vandeae were nearly identical. A change in leaf morphology (usually reduced to a nonphotosynthetic scale), monopodial growth habit, and aeration complexes for gas exchange in photosynthetic roots seem to be the most important characters in making the evolutionary transition to leaflessness.     

The problem of characters susceptible to parallel evolution in phylogenetic analysis: a reply to Marquès and Gnaspini (2001) with emphasis on cave life phenotypic evolution

Marquès and Gnaspini [Cladistics 17 (2001) 371–381] analyzed the problem of the phylogenetic treatment of characters submitted to parallel evolution. Their proposal aimed at preserving the potential phylogenetic significance of supposedly homoplastic characters and considering them for phylogeny reconstruction. As an example, they used the troglobiomorphic features of animals restricted to caves; according to their preliminary hypothesis of homoplasy, troglobitic animals would exhibit a particular phenotype, referred to as troglobiomorphic, which they would acquire under similar selective pressures from the subterranean environment. We examined Marquès and Gnaspini's approach not from the point of view of its technical flaws, but from the point of view of the authors’ basic assertions on the treatment of troglobiomorphic characters and more generally the inclusion/exclusion/transformation of characters prior to phylogenetic analysis. In the present paper, we argue that this approach is invalidated by the repeated use of ad hoc hypotheses, which are supported neither by an existing phylogenetic pattern nor by available data. We consequently contest the adequateness of this approach with a cladistic analysis of historical questions.     

Homoplasy and adaptation in the atelid postcranium

Homoplasy is a ubiquitous phenomenon in phylogenetic investigations, but it is rarely investigated on its own. As a case study in the pattern and basis of homoplasy in primates, the atelid postcranium is discussed here. Characters available from Ford's ([1986] in Erwin J, Swindler DR, eds: Comparative Primate Biology I: Systematics, Evolution, and Anatomy (New York: Alan R. Liss), p 73-135; [1994] in Fleagle JG, Kay RF, eds: Anthropoid Origins (New York: Plenum Press), p 595-674) analyses of New World monkeys are mapped onto alternative phylogenetic trees for the family Atelidae to contrast patterns of character evolution and to develop explanatory hypotheses for differences in the trees. In an unrooted phylogenetic network, pitheciines do not group together because those pitheciines that routinely adopt hind limb suspensory postures (Chiropotes, Cacajao) share traits with atelines. Ford's (1986) work on phylogeny has shown that these traits are homoplastic and also identified potential synapomorphies of a clade comprised of modern pitheciins and atelines. However, following that work, congruence between studies of craniodental and molecular data suggested a still broader definition of atelids (including Callicebus and Cebupithecia), and in this case only one trait may define atelids, and several traits arise in parallel. The homoplastic characters in this phylogeny suggest that the phylogenetic signal in this set of postcranial data is overwhelmed by parallel adaptations to the use of climbing behaviors in all of Ford's atelids and suspensory postures in a more restricted set of taxa. These parallelisms probably indicate a bias of selective pressures in the South American environment, especially given the frequent, independent evolution of suspensory mammals there. This highlights the fact that homoplasy can be a dominant source of similarity in data partitions strongly influenced by a particular behavioral regime, in this case positional behavior.     

Failed refutations: further comments on parsimony and likelihood methods and their relationship to Popper's degree of corroboration

Kluge's (2001, Syst. Biol. 50:322-330) continued arguments that phylogenetic methods based on the statistical principle of likelihood are incompatible with the philosophy of science described by Karl Popper are based on false premises related to Kluge's misrepresentations of Popper's philosophy. Contrary to Kluge's conjectures, likelihood methods are not inherently verificationist; they do not treat every instance of a hypothesis as confirmation of that hypothesis. The historical nature of phylogeny does not preclude phylogenetic hypotheses from being evaluated using the probability of evidence. The low absolute probabilities of hypotheses are irrelevant to the correct interpretation of Popper's concept termed degree of corroboration, which is defined entirely in terms of relative probabilities. Popper did not advocate minimizing background knowledge; in any case, the background knowledge of both parsimony and likelihood methods consists of the general assumption of descent with modification and additional assumptions that are deterministic, concerning which tree is considered most highly corroborated. Although parsimony methods do not assume (in the sense of entailing) that homoplasy is rare, they do assume (in the sense of requiring to obtain a correct phylogenetic inference) certain things about patterns of homoplasy. Both parsimony and likelihood methods assume (in the sense of implying by the manner in which they operate) various things about evolutionary processes, although violation of those assumptions does not always cause the methods to yield incorrect phylogenetic inferences. Test severity is increased by sampling additional relevant characters rather than by character reanalysis, although either interpretation is compatible with the use of phylogenetic likelihood methods. Neither parsimony nor likelihood methods assess test severity (critical evidence) when used to identify a most highly corroborated tree(s) based on a single method or model and a single body of data; however, both classes of methods can be used to perform severe tests. The assumption of descent with modification is insufficient background knowledge to justify cladistic parsimony as a method for assessing degree of corroboration. Invoking equivalency between parsimony methods and likelihood models that assume no common mechanism emphasizes the necessity of additional assumptions, at least some of which are probabilistic in nature. Incongruent characters do not qualify as falsifiers of phylogenetic hypotheses except under extremely unrealistic evolutionary models; therefore, justifications of parsimony methods as falsificationist based on the idea that they minimize the ad hoc dismissal of falsifiers are questionable. Probabilistic concepts such as degree of corroboration and likelihood provide a more appropriate framework for understanding how phylogenetics conforms with Popper's philosophy of science. Likelihood ratio tests do not assume what is at issue but instead are methods for testing hypotheses according to an accepted standard of statistical significance and for incorporating considerations about test severity. These tests are fundamentally similar to Popper's degree of corroboration in being based on the relationship between the probability of the evidence e in the presence versus absence of the hypothesis h, i.e., between p(e|hb) and p(e|b), where b is the background knowledge. Both parsimony and likelihood methods are inductive in that their inferences (particular trees) contain more information than (and therefore do not follow necessarily from) the observations upon which they are based; however, both are deductive in that their conclusions (tree lengths and likelihoods) follow necessarily from their premises (particular trees, observed character state distributions, and evolutionary models). For these and other reasons, phylogenetic likelihood methods are highly compatible with Karl Popper's philosophy of science and offer several advantages over parsimony methods in this context.     

Rethinking the phylogeny of scleractinian corals: a review of morphological and molecular data

Scleractinian corals, which include the architects of coral reefs, are found throughout the world's oceans and have left a rich fossil record over their 240 million year history. Their classification has been marked by confusion but recently developed molecular and morphological tools are now leading to a better understanding of the evolutionary history of this important group. Although morphological characters have been the basis of traditional classification in the group, they are relatively few in number. In addition, our current understanding of skeletal growth and homology is limited, and homoplasy is rampant, limiting the usefulness of morphological phylogenetics. Molecular phylogenetic hypotheses for the order, which have been primarily focused on reef-building corals, differ significantly from traditional classification. They suggest that the group is represented by two major lineages and do not support the monophyly of traditional suborders and most traditional families. It appears that once a substantial number of azooxanthellate taxa are included in molecular phylogenetic analyses, basal relationships within the group will be clearly defined. Understanding of relationships at lower taxonomic levels will be best clarified by combined analyses of morphological and molecular characters. Molecular phylogenies are being used to inform our understanding of the evolution of morphological characters in the Scleractinia. Better understanding of the evolution of these characters will help to integrate the systematics of fossil and extant taxa. We demonstrate how the combined use of morphological and molecular tools holds great promise for ending confusion in scleractinian systematics.     

HYBRIDS AND PHYLOGENETIC SYSTEMATICS I. PATTERNS OF CHARACTER EXPRESSION IN HYBRIDS AND THEIR IMPLICATIONS FOR CLADISTIC ANALYSIS

Interspecific hybridization is considered common among plants, but the methods of cladistic systematics produce only divergently branching phylogenetic hypotheses and thus cannot give the correct phylogeny if an analysis includes hybrids. Empirical studies of the impact of known hybrids on phylogenetic analysis are lacking, and are necessary to begin to understand the problems that we face if hybrids are often included in cladistic analysis. Examination of the implications of hybrids for cladistics must begin with patterns of character expression in hybrids. This study includes 17 hybrids and their nine parental taxa that are Central American species of Aphelandra (Acanthaceae), analyzed using a set of 50 morphological characters. The hybrids are overwhelmingly intermediate as quantitatively scored for phylogenetic analysis. They express maternal and paternal, and primitive and derived characters in equal frequencies, showing no evidence of predominant inheritance of derived character states as has been assumed by most cladists who have considered hybrids theoretically. Because of their known genetic constitution, hybrids were useful in homology assessment and ordering character states. The parental character set was generally robust, but some changes were made to reflect the special evidence offered by the hybrids. These hybrids suggest that the inclusion of hybrids in phylogenetic analysis will not lead to unresolved cladograms with rampant homoplasy, as has been predicted by other authors. Instead, the patterns of character inheritance in these hybrids lead to the prediction that a hybrid will be placed by phylogenetic analysis as a basal lineage to the clade that includes its most derived parent, with relatively little effect on homoplasy. These predictions will be evaluated by incorporation of the hybrids in phylogenetic analyses, to be reported in a subsequent paper.     

Character evolution in Hydrozoa (phylum Cnidaria)

The diversity of hydrozoan life cycles, as manifested in the wide range of polyp, colony, and medusa morphologies, has been appreciated for centuries. Unraveling the complex history of characters involved in this diversity is critical for understanding the processes driving hydrozoan evolution. In this study, we use a phylogenetic approach to investigate the evolution of morphological characters in Hydrozoa. A molecular phylogeny is reconstructed using ribosomal DNA sequence data. Several characters involving polyp, colony, and medusa morphology are coded in the terminal taxa. These characters are mapped onto the phylogeny and then the ancestral character states are reconstructed. This study confirms the complex evolutionary history of hydrozoan morphological characters. Many of the characters involving polyp, colony, and medusa morphology appear as synapomorphies for major hydrozoan clades, yet homoplasy is commonplace.     

A priori assumptions about characters as a cause of incongruence between molecular and morphological hypotheses of primate interrelationships

When molecules and morphology produce incongruent hypotheses of primate interrelationships, the data are typically viewed as incompatible, and molecular hypotheses are often considered to be better indicators of phylogenetic history. However, it has been demonstrated that the choice of which taxa to include in cladistic analysis as well as assumptions about character weighting, character state transformation order, and outgroup choice all influence hypotheses of relationships and may positively influence tree topology, so that relationships between extant taxa are consistent with those found using molecular data. Thus, the source of incongruence between morphological and molecular trees may lie not in the morphological data themselves but in assumptions surrounding the ways characters evolve and their impact on cladistic analysis. In this study, we investigate the role that assumptions about character polarity and transformation order play in creating incongruence between primate phylogenies based on morphological data and those supported by multiple lines of molecular data. By releasing constraints imposed on published morphological analyses of primates from disparate clades and subjecting those data to parsimony analysis, we test the hypothesis that incongruence between morphology and molecules results from inherent flaws in morphological data. To quantify the difference between incongruent trees, we introduce a new method called branch slide distance (BSD). BSD mitigates many of the limitations attributed to other tree comparison methods, thus allowing for a more accurate measure of topological similarity. We find that releasing a priori constraints on character behavior often produces trees that are consistent with molecular trees. Case studies are presented that illustrate how congruence between molecules and unconstrained morphological data may provide insight into issues of polarity, transformation order, homology, and homoplasy.     

Teaching Tree-Thinking to Undergraduate Biology Students

Evolution is the unifying principle of all biology, and understanding how evolutionary relationships are represented is critical for a complete understanding of evolution. Phylogenetic trees are the most conventional tool for displaying evolutionary relationships, and “tree-thinking” has been coined as a term to describe the ability to conceptualize evolutionary relationships. Students often lack tree-thinking skills, and developing those skills should be a priority of biology curricula. Many common student misconceptions have been described, and a successful instructor needs a suite of tools for correcting those misconceptions. I review the literature on teaching tree-thinking to undergraduate students and suggest how this material can be presented within an inquiry-based framework.     

Inferring the accumulation of morphological disparity in epiphyllous liverworts

Phylogenetic studies of lineages growing in extreme environments have frequently recovered evidence not only of high level of homoplasy but also of discordance of morphological disparity and species diversity. It has been suggested that this divergence may be caused by developmental constraints and/or natural selection. Here we explored these hypotheses by inferring the phenotypic evolution of the derived liverwort genus Cololejeunea. These liverworts occur preferentially on the surface of leaves or other aerial parts of vascular plants growing in wet forests. The evolution of 12 morphological characters was studied using a phylogenetic framework comprising 70 species of Cololejeunea. The phylogeny was reconstructed using DNA sequences of one nuclear and two plastid regions and enabled the inference of the evolution of the studied morphological characters by determining the frequency of homoplasy. Mantel tests were used to test for correlations of morphological disparity?×?species diversity and morphological disparity?×?epiphytism. The phylogenetic informativeness of each binary character was estimated by the D metric of the Fritz and Purvis test, and the relationship between each character and epiphytism was inferred by Pearson’s coefficient. We evaluated the morphospace occupation using principal coordinate analyses. Our results not only recovered high levels of homoplasy but also weak correlations of morphological disparity and species diversity. Morphological disparity was not linked to epiphytism, although positive or negative relationships between some characters and epiphytism were found. The Brownian model of character evolution was not rejected for the studied morphological disparity in Cololejeunea with the exception of asexual propagules. The observations support the prediction that iterative evolution in a well-defined morphospace may result in rampant homoplasy and the observed divergence of disparity and diversity.     

Reconstructing the evolution of umbonal sculptures in the Unionida

‘Umbonal sculptures’ of freshwater mussels (Unionida), which ornament the early ontogenetic shell, have long been used for species identification. Specificity of these sculptures to higher taxonomic levels and their value for phylogenetic reconstruction are still under considerable scientific debate. In particular, the distribution of beak sculpture morphotypes across the unionoid phylogeny and, consequently, evolution of this character remain poorly understood. Based on an examination of 187 taxa, covering five of the six extant unionoid families, this study presents a new model of character evolution of umbonal sculptures in the order. Ten morphotypes were recognized and conceptualized into the cladistic characters sculpture presence and category. Optimization of sculpture presence on two recent hypotheses of palaeoheterodont phylogenetic relationships using the program Mesquite indicates a sculptured common ancestor of the extant Unionida, with multiple losses of the umbonal ornament occurring subsequently within the clade. Reconstruction of changes in sculpture category is ambiguous and demonstrates the need for further research into the evolutionary relationships of freshwater mussels in general and of their early ontogenetic sculptures in particular. Ambiguity is reduced in analyses applying a model with unequal costs of transformation between character states, which was derived from observations on intermediate forms and polymorphisms. These analyses suggest ‘V‐shaped’ or ‘nodulous’ sculpture as the plesiomorphic sculptural category for Unionida. The relatively low levels of homoplasy inferred for V‐shaped, pseudo‐radial and double‐looped sculptures suggest that these types may comprise useful guides to relationships within Unionida. The high degree of homoplasy of W‐shaped, pseudo‐concentric, wrinkled and single‐looped sculptures, on the other hand, renders these sculpture types less fit for such purposes.     

Phylogenetic relationships of spatangoid sea urchins (Echinoidea): taxon sampling density and congruence between morphological and molecular estimates

A phylogeny for 21 species of spatangoid sea urchins is constructed using data from three genes and results compared with morphology-based phylogenies derived for the same taxa and for a much larger sample of 88 Recent and fossil taxa. Different data sets and methods of analysis generate different phylogenetic hypotheses, although congruence tests show that all molecular approaches produce trees that are congruent with each other. By contrast, the trees generated from morphological data differ significantly according to taxon sampling density and only those with dense sampling (after a posteriori weighting) are congruent with molecular estimates. With limited taxon sampling, secondary reversals in deep-water taxa are interpreted as plesiomorphies, pulling them to a basal position. The addition of fossil taxa with their unique character combinations reveals hidden homoplasy and generates a phylogeny that is compatible with molecular estimates. As homoplasy levels were found to be broadly similar across different anatomical structures in the echinoid test, no one suite of morphological characters can be considered to provide more reliable phylogenetic information. Some traditional groupings are supported, including the grouping of Loveniidae, Brissidae and Spatangidae within the Micrasterina, but the Asterostomatidae is shown to be polyphyletic with members scattered amongst at least five different clades. As these are mostly deep-sea taxa, this finding implies multiple independent invasions into the deep sea.     

ESTIMATING CHARACTER WEIGHTS DURING TREE SEARCH

Abstract— A new method for weighting characters according to their homoplasy is proposed; the method is non-iterative and does not require independent estimations of weights. It is based on searching trees with maximum total fit, with character fits defined as a concave function of homoplasy. Then, when comparing trees, differences in steps occurring in characters which show more homoplasy on the trees are less influential. The reliability of the characters is estimated, during the analysis, as a logical implication of the trees being compared. The "fittest" trees imply that the characters are maximally reliable and, given character conflict, have fewer steps for the characters which fit the tree better. If other trees save steps in some characters, it will be at the expense of gaining them in characters with less homoplasy.     

THE EFFECT OF ORDERED CHARACTERS ON PHYLOGENETIC RECONSTRUCTION

Abstract Morphological structures are likely to undergo more than a single change during the course of evolution. As a result, multistate characters are common in systematic studies and must be dealt with. Particularly interesting is the question of whether or not multistate characters should be treated as ordered (additive) or unordered (non-additive). In accepting a particular hypothesis of order, numerous others are necessarily rejected. We review some of the criteria often used to order character states and the underlying assumptions inherent in these criteria.
The effects that ordered multistate characters can have on phylogenetic reconstruction are examined using 27 data sets. It has been suggested that hypotheses of character state order are more informative then hypotheses of unorder and may restrict the number of equally parsimonious trees as well as increase tree resolution. Our results indicate that ordered characters can produce more, equal or less equally parsimonious trees and can increase, decrease or have no effect on tree resolution. The effect on tree resolution can be a simple gain in resolution or a dramatic change in sister-taxa relationships. In cases where several outgroups are included in the data matrix, hypotheses of order can change character polarities by altering outgroup topology. Ordered characters result in a different topology from unordered characters only when the hierarchy of the cladogram disagrees with the investigator's a priori hypothesis of order. If the best criterion for assessing character evolution is congruence with other characters, the practice of ordering multistate characters is inappropriate.     

Revisiting the phylogeny of microsporidia

Canonical microsporidians are a group of obligate intracellular parasites of a wide range of hosts comprising ~1,300 species of >220 genera. Microsporidians are related to fungi, and many characterised and uncharacterized groups closely related to them have been discovered recently, filling the knowledge gaps between them. These groups assigned to the superphylum Opisthosporidia have provided several important insights into the evolution of diverse intracellular parasitic lineages within the tree of eukaryotes. The most studied among opisthosporidians, canonical microsporidians, were known to science more than 160 years ago, however, the classification of canonical Microsporidia has been challenging due to common morphological homoplasy, and accelerated evolutionary rates. Instead of morphological characters, ssrRNA sequences have been used as the primary data for the classification of canonical microsporidians. Previous studies have produced a useful backbone of the microsporidian phylogeny, but provided only some nodal support, causing some confusion. Here, we reconstructed phylogenetic trees of canonical microsporidians using Bayesian and Maximum Likelihood inferences. We included rRNA sequences of 126 described/named genera, by far the broadest taxon coverage to date. Overall, our trees show similar topology and recovered four of the five main clades demonstrated in previous studies (Clades 1, 3, 4 and 5). Family level clades were well resolved within each major clade, but many were discordant with the recently revised classification. Therefore, revision and some reshuffling, especially within and between Clades 1 and 3 are required. We also reconstructed phylogenetic trees of Opisthosporidia to better integrate the evolutionary history of canonical microsporidians in a broader context. We discuss several traits shared only by canonical microsporidians that may have contributed to their striking ecological success in diverse metazoans. More targeted studies on the neglected host groups will be of value for a better understanding of the evolutionary history of these interesting intracellular parasites.