Morphology and angiosperm systematics in the molecular era

Several ways in which morphology is used in systematic and evolutionary research in angiosperms are shown and illustrated with examples: 1) searches for special structural similarities, which can be used to find hints for hitherto unrecognized relationships in groups with unresolved phylogenetic position; 2) cladistic studies based on morphology and combined morphological and molecular analyses; 3) comparative morphological studies in new, morphologically puzzling clades derived from molecular studies; 4) studies of morphological character evolution, unusual evolutionary directions, and evolutionary lability based on molecular studies; and 5) studies of organ evolution. Conclusions: Goals of comparative morphology have shifted in the present molecular era. Morphology no longer plays the primary role in phylogenetic studies. However, new opportunities for morphology are opening up that were not present in the premolecular era: 1) phylogenetic studies with combined molecular and morphological analyses; 2) reconstruction of the evolution of morphological features based on molecularly derived cladograms; 3) refined analysis of morphological features induced by inconsistencies of previous molecular and molecular phylogenetic analyses; 4) better understanding of morphological features by judgment in a wider biological context; 5) increased potential for including fossils in morphological analyses; and 6) exploration of the evolution of morphological traits by integration of comparative structural and molecular developmental genetic aspects (Evo-Devo); this field is still in its infancy in botany; its advancement is one of the major goals of evolutionary botany.     

Circularity,evolution, systematics … and circularity

Many have argued strongly that incorporation of evolutionary theory into systematics is dangerously circular, while others have maintained that such an integrated approach increases the accuracy of phylogenetic inference. Here, it is demonstrated that such blanket statements regarding exclusion or inclusion of evolutionary principles in systematics fail to distinguish between two very different types of principles. ‘Phylogeny-neutral’ evolutionary principles are those inferred without any recourse to specific phylogenetic hypotheses (e.g. via developmental genetics, biomechanics). In contrast, ‘phylogeny-dependent’ principles are those which can only be inferred on the basis of specific phylogenetic hypotheses (e.g. character associations detected via ‘comparative methods’). Inclusion of phylogeny-neutral principles in systematic studies as a priori assumptions can be justified, since these principles have (often strong) external empirical support from other spheres of investigation. However, inclusion of phylogeny-dependent principles in systematic studies is circular, since such principles have no external empirical support but are themselves derived from systematic studies. Advocating inclusion or exclusion of all (or as many as possible) evolutionary principles from phylogenetic analysis is therefore misguided. Rather, phylogeny-neutral principles are independently supported and can be included, while phylogeny-dependent principles are unjustified assumptions and should be excluded to avoid circularity. However, integration of complex phylogeny-neutral principles in systematics can create operational problems, even though there are no methodological reasons against their inclusion.     

The series,the network,and the tree: changing metaphors of order in nature

The history of biological systematics documents a continuing tension between classifications in terms of nested hierarchies congruent with branching diagrams (the ‘Tree of Life’) versus reticulated relations. The recognition of conflicting character distribution led to the dissolution of the scala naturae into reticulated systems, which were then transformed into phylogenetic trees by the addition of a vertical axis. The cladistic revolution in systematics resulted in a representation of phylogeny as a strictly bifurcating pattern (cladogram). Due to the ubiquity of character conflict—at the genetic or morphological level, or at any level in between—some characters will necessarily have to be discarded (qua noise) in favor of others in support of a strictly bifurcating phylogenetic tree. Pattern analysts will seek maximal congruence in the distribution of characters (ultimately of any kind) relative to a branching tree-topology; process explainers will call such tree-topologies into question by reference to incompatible evolutionary processes. Pattern analysts will argue that process explanations must not be brought to bear on pattern reconstruction; process explainers will insist that the reconstructed pattern requires a process explanation to become scientifically relevant, i.e., relevant to evolutionary theory. The core question driving the current debate about the adequacy of the ‘Tree of Life’ metaphor seems to be whether the systematic dichotomization of the living world is an adequate representation of the complex evolutionary history of global biodiversity. In ‘Questioning the Tree of Life’, it seems beneficial to draw at least four conceptual distinctions: pattern reconstruction versus process explanation as different epistemological approaches to the study of phylogeny; open versus closed systems as expressions of different kinds of population (species) structures; phylogenetic trees versus cladograms as representations of evolutionary processes versus patterns of relationships; and genes versus species as expressions of different levels of causal integration and evolutionary transformation.     

The evolution of plant development

The last decade has witnessed a resurgence in the study of the evolution of plant development, combining investigations in systematics, developmental morphology, molecular developmental genetics, and molecular evolution. The integration of phylogenetic studies, structural analyses of fossil and extant taxa, and molecular developmental genetic information allows the formulation of explicit and testable hypotheses for the evolution of morphological characters. These comprehensive approaches provide opportunities to dissect the evolution of major developmental transitions among land plants, including those associated with apical meristems, the origins of the root/shoot dichotomy, diversification of leaves, and origin and subsequent modification of flower structure. The evolution of these major developmental innovations is discussed within both phylogenetic and molecular genetic contexts. We conclude that it is the combination of these approaches that will lead to the greatest understanding of the evolution of plant development.     

Flies and congruence

Competing phylogenetic hypotheses have become the rule in modern systematics. While the problem of incongruence between character sets has become extremely acute due to the generation of molecular data, it is by no means specific to molecular and morphological comparisons. The role of the modern systematist is to interpret incongruence between character sets and to come to some conclusion regarding a phylogenetic hypothesis of the organisms in question. Two aspects of congruence analysis are examined using the Drosophilidae as an example. The first includes the quantification of congruence and the types of phylogenetic inference that can be made from such analyses. The second aspect concerns an examination of character evolution in order to identify characters and taxa that might be contributing to incongruence in phylogenetic analysis. © 1994 Wiley-Liss, Inc.     

Detecting the historical signature of key innovations using stochastic models of character evolution and cladogenesis

Phylogenetic evidence for biological traits that increase the net diversification rate of lineages (key innovations) is most commonly drawn from comparisons of clade size. This can work well for ancient, unreversed traits and for correlating multiple trait origins with higher diversification rates, but it is less suitable for unique events, recently evolved innovations, and traits that exhibit homoplasy. Here I present a new method for detecting the phylogenetic signature of key innovations that tests whether the evolutionary history of the candidate trait is associated with shorter waiting times between cladogenesis events. The method employs stochastic models of character evolution and cladogenesis and integrates well into a Bayesian framework in which uncertainty in historical inferences (such as phylogenetic relationships) is allowed. Applied to a well-known example in plants, nectar spurs in columbines, the method gives much stronger support to the key innovation hypothesis than previous tests.     

Combined support for wholesale taxic atavism in gavialine crocodylians

Morphological and molecular data sets favor robustly supported, contradictory interpretations of crocodylian phylogeny. A longstanding perception in the field of systematics is that such significantly conflicting data sets should be analyzed separately. Here we utilize a combined approach, simultaneous analyses of all relevant character data, to summarize common support and to reconcile discrepancies among data sets. By conjoining rather than separating incongruent classes of data, secondary phylogenetic signals emerge from both molecular and morphological character sets and provide solid evidence for a unified hypothesis of crocodylian phylogeny. Simultaneous analyses of four gene sequences and paleontological data suggest that putative adaptive convergences in the jaws of gavialines (gavials) and tomistomines (false gavials) offer character support for a grouping of these taxa, making Gavialinae an atavistic taxon. Simple new methods for measuring the influence of extinct taxa on topological support indicate that in this vertebrate order fossils generally stabilize relationships and accentuate hidden phylogenetic signals. Remaining inconsistencies in minimum length trees, including concentrated hierarchical patterns of homoplasy and extensive gaps in the fossil record, indicate where future work in crocodylian systematics should be directed.     

Systematic and evolutionary implications of stamen position in Myrteae (Myrtaceae)

The taxonomy of Myrteae, the most species‐rich tribe of Myrtaceae, is notoriously difficult. Although the understanding of the phylogeny has improved, the morphological characteristics that support its cladistic configuration are still unknown. The present study evaluates stamen position and anthesis type as characters of systematic and evolutionary relevance. Sixty‐nine species from 41 genera across the tribe were checked using herbarium material and spirit collections. The results recognize three patterns of stamen position in the pre‐anthetic bud: straight, semi‐curved and strongly incurved. The three patterns of stamen position correspond to the phylogenetic structure of the tribe, supporting the topology of the clades. Incurving of stamens across the evolutionary history of Myrteae appears to be linked to hypanthium extension and leads to different anthesis types that may be related to shifts in pollination strategy. The accessibility of stamen position and its systematic consistency make this character a useful tool for field and herbarium identification, and allow the inference of relationships for taxa not yet sampled in molecular analyses. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 388–402.     

On the Evolutionary and Biogeographic History of Saxifraga sect. Trachyphyllum (Gaud.) Koch (Saxifragaceae Juss.)

Arctic-alpine plants in the genus Saxifraga L. (Saxifragaceae Juss.) provide an excellent system for investigating the process of diversification in northern regions. Yet, sect. Trachyphyllum (Gaud.) Koch, which is comprised of about 8 to 26 species, has still not been explored by molecular systematists even though taxonomists concur that the section needs to be thoroughly re-examined. Our goals were to use chloroplast trnL-F and nuclear ITS DNA sequence data to circumscribe the section phylogenetically, test models of geographically-based population divergence, and assess the utility of morphological characters in estimating evolutionary relationships. To do so, we sequenced both genetic markers for 19 taxa within the section. The phylogenetic inferences of sect. Trachyphyllum using maximum likelihood and Bayesian analyses showed that the section is polyphyletic, with S. aspera L. and S bryoides L. falling outside the main clade. In addition, the analyses supported several taxonomic re-classifications to prior names. We used two approaches to test biogeographic hypotheses: i) a coalescent approach in Mesquite to test the fit of our reconstructed gene trees to geographically-based models of population divergence and ii) a maximum likelihood inference in Lagrange. These tests uncovered strong support for an origin of the clade in the Southern Rocky Mountains of North America followed by dispersal and divergence episodes across refugia. Finally we adopted a stochastic character mapping approach in SIMMAP to investigate the utility of morphological characters in estimating evolutionary relationships among taxa. We found that few morphological characters were phylogenetically informative and many were misleading. Our molecular analyses provide a foundation for the diversity and evolutionary relationships within sect. Trachyphyllum and hypotheses for better understanding the patterns and processes of divergence in this section, other saxifrages, and plants inhabiting the North Pacific Rim.     

Phylogenetic Relationships Among Treeshrews (Scandentia): A Review and Critique of the Morphological Evidence

Although the supraordinal relationships of Scandentia (treeshrews) have been studied in great detail from both morphological and molecular perspectives, the phylogenetic relationships among treeshrews have been largely ignored. Here we review several published studies of qualitative morphological variation among living treeshrews and their contribution to our understanding of intraordinal phylogenetic relationships. Reanalysis of the data from each of these studies demonstrates that none of the trees in the original publications represents the most parsimonious interpretation. In addition to performing new analyses, we argue that all such studies to date suffer from one or more fundamental shortcomings, notably the failure to include reference to nonscandentian outgroups and the a priori assumption of generic monophyly of the relatively speciose genus Tupaia. Parsimony analyses of these data sets fail to resolve either intergeneric or interspecific relationships. Finally, several inconsistencies and conflicts with respect to character coding both within and between published studies are discussed. We conclude that a more rigorous investigation of morphological character state variation is sorely needed, one that explicitly identifies voucher specimens and does not make any assumptions of generic monophyly. This is necessary not only for the purpose of resolving phylogenetic relationships, but also for inference of ancestral states in a group that continues to figure prominently in studies of placental mammal diversification.     

Systematics and evolution of the Pan‐Alcidae (Aves,Charadriiformes)

Puffins, auks and their allies in the wing‐propelled diving seabird clade Pan‐Alcidae (Charadriiformes) have been proposed to be key pelagic indicators of faunal shifts in Northern Hemisphere oceans. However, most previous phylogenetic analyses of the clade have focused only on the 23 extant alcid species. Here we undertake a combined phylogenetic analysis of all previously published molecular sequence data (～ 12 kb) and morphological data (n = 353 characters) with dense species level sampling that also includes 28 extinct taxa. We present a new estimate of the patterns of diversification in the clade based on divergence time estimates that include a previously vetted set of twelve fossil calibrations. The resultant time trees are also used in the evaluation of previously hypothesized paleoclimatic drivers of pan‐alcid evolution. Our divergence dating results estimate the split of Alcidae from its sister taxon Stercorariidae during the late Eocene (～ 35 Ma), an evolutionary hypothesis for clade origination that agrees with the fossil record and that does not require the inference of extensive ghost lineages. The extant dovekie Alle alle is identified as the sole extant member of a clade including four extinct Miocene species. Furthermore, whereas an Uria + Alle clade has been previously recovered from molecular analyses, the extinct diversity of closely related Miocepphus species yields morphological support for this clade. Our results suggest that extant alcid diversity is a function of Miocene diversification and differential extinction at the Pliocene–Pleistocene boundary. The relative timing of the Middle Miocene climatic optimum and the Pliocene–Pleistocene climatic transition and major diversification and extinction events in Pan‐Alcidae, respectively, are consistent with a potential link between major paleoclimatic events and pan‐alcid cladogenesis.     

Transformation Series as an Ideographic Character Concept

An ideographic concept of character is indispensable to phylogenetic inference. Hennig proposed that characters be conceptualized as “transformation series”, a proposal that is firmly grounded in evolutionary theory and consistent with the method of inferring transformation events as evidence of phylogenetic propinquity. Nevertheless, that concept is usually overlooked or rejected in favor of others based on similarity. Here we explicate Hennig's definition of character as an ideographic concept in the science of phylogenetic systematics. As transformation series, characters are historical individuals akin to species and clades. As such, the related concept of homology refers to a historical identity relation and is not equivalent to or synonymous with synapomorphy. The distinction between primary and secondary homology is dismissed on the grounds that it conflates the concept of homology with the discovery operations used to detect instances of that concept. Although concern for character dependence is generally valid, it is often misplaced, focusing on functional or developmental correlation (both of which are irrelevant in phylogenetic systematics but may be valid in other fields) instead of the historical/transformational independence relevant to phylogenetic inference. As an ideographic science concerned with concrete objects and events (i.e. individuals), intensionally and extensionally defined properties are inconsistent with the individuation of characters for phylogenetic analysis, the utility of properties being limited to communicating results and facilitating future rounds of testing.     

A Primer to Molecular Phylogenetic Analysis in Plants

Reconstructing a tree of life by inferring evolutionary history is an important focus of evolutionary biology. Phylogenetic reconstructions also provide useful information for a range of scientific disciplines such as botany, zoology, phylogeography, archaeology and biological anthropology. Until the development of protein and DNA sequencing techniques in the 1960s and 1970s, phylogenetic reconstructions were based on fossil records and comparative morphological/physiological analyses. Since then, progress in molecular phylogenetics has compensated for some of the shortcomings of phenotype-based comparisons. Comparisons at the molecular level increase the accuracy of phylogenetic inference because there is no environmental influence on DNA/peptide sequences and evaluation of sequence similarity is not subjective. While the number of morphological/physiological characters that are sufficiently conserved for phylogenetic inference is limited, molecular data provide a large number of datapoints and enable comparisons from diverse taxa. Over the last 20 years, developments in molecular phylogenetics have greatly contributed to our understanding of plant evolutionary relationships. Regions in the plant nuclear and organellar genomes that are optimal for phylogenetic inference have been determined and recent advances in DNA sequencing techniques have enabled comparisons at the whole genome level. Sequences from the nuclear and organellar genomes of thousands of plant species are readily available in public databases, enabling researchers without access to molecular biology tools to investigate phylogenetic relationships by sequence comparisons using the appropriate nucleotide substitution models and tree building algorithms. In the present review, the statistical models and algorithms used to reconstruct phylogenetic trees are introduced and advances in the exploration and utilization of plant genomes for molecular phylogenetic analyses are discussed.     

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.     

Bias and conflict in phylogenetic inference of myco-heterotrophic plants: a case study in Thismiaceae

Due to morphological reduction and absence of amplifiable plastid genes, the identification of photosynthetic relatives of heterotrophic plants is problematic. Although nuclear and mitochondrial gene sequences may offer a welcome alternative source of phylogenetic markers, the presence of rate heterogeneity in these genes may introduce bias/systematic error in phylogenetic analyses. We examine the phylogenetic position of Thismiaceae based on nuclear 18S rDNA and mitochondrial atpA DNA sequence data, as well as using parsimony, likelihood and Bayesian inference methods. Significant differences in evolutionary rates of these genes between closely related taxa lead to conflicting results: while parsimony analyses of 18S rDNA and combined data strongly support the monophyly of Thismiaceae, Bayesian inference, with and without a relaxed molecular clock, as well as the Swofford–Olsen–Waddell–Hillis (SOWH) test confidently reject this hypothesis. We show that rate heterogeneity in our data leads to long-branch attraction artifacts in parsimony analysis. However, using model-based inference methods the question of whether Thismiaceae are monophyletic remains elusive. On the one hand maximum likelihood nonparametric bootstrapping and parametric hypothesis tests fail to support a paraphyletic Thismiaceae, on the other hand Bayesian inference methods (both without and with a relaxed clock) significantly reject a monophyletic Thismiaceae. These results show that an adequate sampling, the use of rate homogeneous data, and the application of different inference methods are important factors for developing phylogenetic hypotheses of myco-heterotrophic plants. © The Willi Hennig Society 2009.     

Asymmetries in phylogenetic diversification and character change can be untangled

The analysis of diversification and character evolution using phylogenetic data attracts increasing interest from biologists. Recent statistical developments have resulted in a variety of tools for the inference of macroevolutionary processes in a phylogenetic context. In a recent paper Maddison (2006 Evolution, 60: 1743-1746) pointed out that uncareful use of some of these tools could lead to misleading conclusions on diversification or character evolution, and thus to difficulties in distinguishing both phenomena. I here present guidelines for the analyses of macroevolutionary data that may help to avoid these problems. The proper use of recently developed statistical methods may help to untangle diversification and character change, and so will allow us to address important evolutionary questions.     

Integration of morphological data sets for phylogenetic analysis of Amniota: the importance of integumentary characters and increased taxonomic sampling

Several mutually exclusive hypotheses have been advanced to explain the phylogenetic position of turtles among amniotes. Traditional morphology-based analyses place turtles among extinct anapsids (reptiles with a solid skull roof), whereas more recent studies of both morphological and molecular data support an origin of turtles from within Diapsida (reptiles with a doubly fenestrated skull roof). Evaluation of these conflicting hypotheses has been hampered by nonoverlapping taxonomic samples and the exclusion of significant taxa from published analyses. Furthermore, although data from soft tissues and anatomical systems such as the integument may be particularly relevant to this problem, they are often excluded from large-scale analyses of morphological systematics. Here, conflicting hypotheses of turtle relationships are tested by (1) combining published data into a supermatrix of morphological characters to address issues of character conflict and missing data; (2) increasing taxonomic sampling by more than doubling the number of operational taxonomic units to test internal relationships within suprageneric ingroup taxa; and (3) increasing character sampling by approximately 25% by adding new data on the osteology and histology of the integument, an anatomical system that has been historically underrepresented in morphological systematics. The morphological data set assembled here represents the largest yet compiled for Amniota. Reevaluation of character data from prior studies of amniote phylogeny favors the hypothesis that turtles indeed have diapsid affinities. Addition of new ingroup taxa alone leads to a decrease in overall phylogenetic resolution, indicating that existing characters used for amniote phylogeny are insufficient to explain the evolution of more highly nested taxa. Incorporation of new data from the soft and osseous components of the integument, however, helps resolve relationships among both basal and highly nested amniote taxa. Analysis of a data set compiled from published sources and data original to this study supports monophyly of Amniota, Synapsida, Reptilia, Parareptilia, Eureptilia, Eosuchia, Diapsida, Neodiapsida, Sauria, Lepidosauria, and Archosauriformes, as well as several more highly nested divisions within the latter two clades. Turtles are here resolved as the sister taxon to a monophyletic Lepidosauria (squamates + Sphenodon), a novel phylogenetic position that nevertheless is consistent with recent molecular and morphological studies that have hypothesized diapsid affinities for this clade.     

A methodological investigation of hominoid craniodental morphology and phylogenetics

The evolutionary relationships of extant great apes and humans have been largely resolved by molecular studies, yet morphology-based phylogenetic analyses continue to provide conflicting results. In order to further investigate this discrepancy we present bootstrap clade support of morphological data based on two quantitative datasets, one dataset consisting of linear measurements of the whole skull from 5 hominoid genera and the second dataset consisting of 3D landmark data from the temporal bone of 5 hominoid genera, including 11 sub-species. Using similar protocols for both datasets, we were able to 1) compare distance-based phylogenetic methods to cladistic parsimony of quantitative data converted into discrete character states, 2) vary outgroup choice to observe its effect on phylogenetic inference, and 3) analyse male and female data separately to observe the effect of sexual dimorphism on phylogenies. Phylogenetic analysis was sensitive to methodological decisions, particularly outgroup selection, where designation of Pongo as an outgroup and removal of Hylobates resulted in greater congruence with the proposed molecular phylogeny. The performance of distance-based methods also justifies their use in phylogenetic analysis of morphological data. It is clear from our analyses that hominoid phylogenetics ought not to be used as an example of conflict between the morphological and molecular, but as an example of how outgroup and methodological choices can affect the outcome of phylogenetic analysis.     

Paraphyletic species no more – genomic data resolve a Pleistocene radiation and validate morphological species of the Melanoplus scudderi complex (Insecta: Orthoptera)

Rapid speciation events, with taxa generated over a short time period, are among the most investigated biological phenomena. However, molecular systematics often reveals contradictory results compared with morphological/phenotypical diagnoses of species under scenarios of recent and rapid diversification. In this study, we used molecular data from an average of over 29 000 loci per sample from RADseq to reconstruct the diversification history and delimit the species boundary in a short-winged grasshopper species complex (Melanoplus scudderi group), where Pleistocene diversification has been hypothesized to generate more than 20 putative species with distinct male genitalic shapes. We found that, based on a maximum likelihood molecular phylogeny, each morphological species indeed forms a monophyletic group, contrary to the result from a previous mitochondrial DNA sequence study. By dating the diversification events, the species complex is estimated to have diversified during the Late Pleistocene, supporting the recent radiation hypothesis. Furthermore, coalescent-based species delimitation analyses provide quantitative support for independent genetic lineages, which corresponds to the morphologically defined species. Our results also showed that male genitalic shape may not be predicted by evolutionary distance among species, not only indicating that this trait is labile, but also implying that selection may play a role in character divergence. Additionally, our findings suggest that the rapid speciation events in this flightless grasshopper complex might be primarily associated with the fragmentation of their grassland habitats during the Late Pleistocene. Collectively, our study highlights the importance of integrating multiple sources of information to delineate species, especially for a species complex that diversified rapidly, and whose divergence may be linked to ecological processes that create geographic isolation (i.e. fragmented habitats), as well as selection acting on characters with direct consequences for reproductive isolation (i.e. genitalic divergence).     

No speed dating please! Patterns of social preference in male and female house mice

Glass sponges (Class Hexactinellida) are important components of deep-sea ecosystems and are of interest from geological and materials science perspectives. The reconstruction of their phylogeny with molecular data has only recently begun and shows a better agreement with morphology-based systematics than is typical for other sponge groups, likely because of a greater number of informative morphological characters. However, inconsistencies remain that have far-reaching implications for hypotheses about the evolution of their major skeletal construction types (body plans). Furthermore, less than half of all described extant genera have been sampled for molecular systematics, and several taxa important for understanding skeletal evolution are still missing. Increased taxon sampling for molecular phylogenetics of this group is therefore urgently needed. However, due to their remote habitat and often poorly preserved museum material, sequencing all 126 currently recognized extant genera will be difficult to achieve. Utilizing morphological data to incorporate unsequenced taxa into an integrative systematics framework therefore holds great promise, but it is unclear which methodological approach best suits this task. Here, we increase the taxon sampling of four previously established molecular markers (18S, 28S, and 16S ribosomal DNA, as well as cytochrome oxidase subunit I) by 12 genera, for the first time including representatives of the order Aulocalycoida and the type genus of Dactylocalycidae, taxa that are key to understanding hexactinellid body plan evolution. Phylogenetic analyses suggest that Aulocalycoida is diphyletic and provide further support for the paraphyly of order Hexactinosida; hence these orders are abolished from the Linnean classification. We further assembled morphological character matrices to integrate so far unsequenced genera into phylogenetic analyses in maximum parsimony (MP), maximum likelihood (ML), Bayesian, and morphology-based binning frameworks. We find that of these four approaches, total-evidence analysis using MP gave the most plausible results concerning congruence with existing phylogenetic and taxonomic hypotheses, whereas the other methods, especially ML and binning, performed more poorly. We use our total-evidence phylogeny of all extant glass sponge genera for ancestral state reconstruction of morphological characters in MP and ML frameworks, gaining new insights into the evolution of major hexactinellid body plans and other characters such as different spicule types. Our study demonstrates how a comprehensive, albeit in some parts provisional, phylogeny of a larger taxon can be achieved with an integrative approach utilizing molecular and morphological data, and how this can be used as a basis for understanding phenotypic evolution. The datasets and associated trees presented here are intended as a resource and starting point for future work on glass sponge evolution.