Wing pattern diversity in Eunica butterflies (Nymphalidae: Biblidinae): phylogenetic analysis implies decoupled adaptive trends in dorsal sexual dimorphism and ventral eyespot evolution.

Butterfly eyespots are wing patterns reminiscent of vertebrate eyes, formed by concentric rings of contrastingly coloured scales. Eyespots are usually located close to the wing margin and often regarded as the single most conspicuous pattern element of butterfly wing colour displays. Recent efforts to understand the processes involved in the formation of eyespots have been driven mainly by evo-devo approaches focused on model species. However, patterns of change implied by phylogenetic relationships can also inform hypotheses about the underlying developmental mechanisms associated with the formation or disappearance of eyespots, and the limits of phenotypic diversity occurring in nature. Here we present a combined evidence phylogenetic hypothesis for the genus Eunica, a prominent member of diverse Neotropical butterfly communities, that features notable variation among species in eyespot patterns on the ventral hind wing surface. The data matrix consists of one mitochondrial gene region (COI), four nuclear gene regions (GAPDH, RPS5, EF1a and Wingless) and 68 morphological characters. A combined cladistic analysis with all the characters concatenated produced a single most parsimonious tree that, although fully resolved, includes many nodes with modest branch support. The phylogenetic hypothesis presented corroborates a previously proposed morphological trend leading to the loss of eyespots, together with an increase in the size of the conserved eyespots, relative to outgroup taxa. Furthermore, wing colour pattern dimorphism and the presence of androconia suggest that the most remarkable instances of sexual dimorphism are present in the species of Eunica with the most derived eyespot patterns, and are in most cases accompanied by autapomorphic combinations of scent scales and "hair pencils". We discuss natural and sexual selection as potential adaptive explanations for dorsal and ventral wing patterns.

Hierarchies, classifications, cladograms and phylogeny.

Figure 18 of Hennig's Phylogenetic Systematics (University of Illinois Press, Urbana, IL, 1966) shows a phylogenetic tree (a generative hierarchy) and what appear to be nested sets (an inclusive hierarchy) that he stated were two representations of the same pattern of relationships. This essay questions whether this is correct or not, explores the meanings of different hierarchical patterns, reviews various interpretations of Hennig's figure, and discusses the conceptual path from systematic evidence to phylogenetic explanation. The crux of the argument is that systematic hierarchies as we know them scientifically are nested sets that group theoretical entities based on patterns of synapomorphy. The notions of phylogeny and common ancestry reflect this hierarchical pattern.

Background knowledge: the assumptions of pattern cladistics.

This paper reviews the ontogeny of pattern cladistics from the 1970s and 1980s, rebuts criticisms by contemporary anti-cladists, and endeavours to clarify persistent misunderstandings about the philosophical foundations of the approach.

"Maximum support" = 100% BS.

Bootstrap and other percentage-based measures of nodal "support" cannot distinguish between strong support and very strong support. Clearly, if there is no upper bound to the number of potential characters, there should be no upper bound to measures of support.

Anchored phylogenomics illuminates the skipper butterfly tree of life.

BACKGROUND: Butterflies (Papilionoidea) are perhaps the most charismatic insect lineage, yet phylogenetic relationships among them remain incompletely studied and controversial. This is especially true for skippers (Hesperiidae), one of the most species-rich and poorly studied butterfly families. METHODS: To infer a robust phylogenomic hypothesis for Hesperiidae, we sequenced nearly 400 loci using Anchored Hybrid Enrichment and sampled all tribes and more than 120 genera of skippers. Molecular datasets were analyzed using maximum-likelihood, parsimony and coalescent multi-species phylogenetic methods. RESULTS: All analyses converged on a novel, robust phylogenetic hypothesis for skippers. Different optimality criteria and methodologies recovered almost identical phylogenetic trees with strong nodal support at nearly all nodes and all taxonomic levels. Our results support Coeliadinae as the sister group to the remaining skippers, the monotypic Euschemoninae as the sister group to all other subfamilies but Coeliadinae, and the monophyly of Eudaminae plus Pyrginae. Within Pyrginae, Celaenorrhinini and Tagiadini are sister groups, the Neotropical firetips, Pyrrhopygini, are sister to all other tribes but Celaenorrhinini and Tagiadini. Achlyodini is recovered as the sister group to Carcharodini, and Erynnini as sister group to Pyrgini. Within the grass skippers (Hesperiinae), there is strong support for the monophyly of Aeromachini plus remaining Hesperiinae. The giant skippers (Agathymus and Megathymus) once classified as a subfamily, are recovered as monophyletic with strong support, but are deeply nested within Hesperiinae. CONCLUSIONS: Anchored Hybrid Enrichment sequencing resulted in a large amount of data that built the foundation for a new, robust evolutionary tree of skippers. The newly inferred phylogenetic tree resolves long-standing systematic issues and changes our understanding of the skipper tree of life. These resultsenhance understanding of the evolution of one of the most species-rich butterfly families.

Going rogue.

The problem of 'rogue taxa' explored by Buenaventura et al. (2017, Cladistics 33: 109-133) is due to complementary patterns of missing data in their matrix, which are evident from perusal of their supplementary table S1.

Statistical consistency and phylogenetic inference: a brief review.

The claim that parsimony can be statistically inconsistent remains the chief criticism of the cladistic approach, and also the main justification for alternative model-based approaches such as maximum likelihood and Bayesian inference. Despite its refutation in the 1980s, this persistent myth of parsimony's Achilles' heel is entrenched in the primary literature, and has metastasized into textbooks, as well. Here, I review historical controversies, and offer three short arguments as to why statistical consistency is not only irrelevant to systematics, but to empirical science in general.

Missing data, clade support and "reticulation": the molecular systematics of Heliconius and related genera (Lepidoptera: Nymphalidae) re-examined.

Kozak et al. (2015, Syst. Biol., 64: 505) portrayed the inference of evolutionary history among Heliconius and allied butterfly genera as a particularly difficult problem for systematics due to prevalent gene conflict caused by interspecific reticulation. To control for this, Kozak et al. conducted a series of multispecies coalescent phylogenetic analyses that they claimed revealed pervasive conflict among markers, but ultimately chose as their preferred hypothesis a phylogenetic tree generated by the traditional supermatrix approach. Intrigued by this seemingly contradictory set of conclusions, we conducted further analyses focusing on two prevalent aspects of the data set: missing data and the uneven contribution of phylogenetic signal among markers. Here, we demonstrate that Kozak et al. overstated their findings of reticulation and that evidence of gene-tree conflict is largely lacking. The distribution of intrinsic homoplasy and incongruence homoplasy in their data set does not follow the pattern expected if phylogenetic history had been obscured by pervasive horizontal gene flow; in fact, noise within individual gene partitions is ten times higher than the incongruence among gene partitions. We show that the patterns explained by Kozak et al. as a result of reticulation can be accounted for by missing data and homoplasy. We also find that although the preferred topology is resilient to missing data, measures of support are sensitive to, and strongly eroded by too many empty cells in the data matrix. Perhaps more importantly, we show that when some taxa are missing almost all characters, adding more genes to the data set provides little or no increase in support for the tree.

Incompatible Ages for Clearwing Butterflies Based on Alternative Secondary Calibrations.

The recent publication of a time-tree for the plant family Solanaceae (nightshades) provides the opportunity to use independent calibrations to test divergence times previously inferred for the diverse Neotropical butterfly tribe Ithomiini. Ithomiini includes clades that are obligate herbivores of Solanaceae, with some genera feeding on only one genus. We used 8 calibrations extracted from the plant tree in a new relaxed molecular-clock analysis to produce an alternative temporal framework for the diversification of ithomiines. We compared the resulting age estimates to: (i) a time-tree obtained using 7 secondary calibrations from the Nymphalidae tree of Wahlberg et al. (2009), and (ii) Wahlberg et al.'s (2009) original age estimates for the same clades. We found that Bayesian clock estimates were rather sensitive to a variety of analytical parameters, including taxon sampling. Regardless of this sensitivity however, ithomiine divergence times calibrated with the ages of nightshades were always on average half the age of previous estimates. Younger dates for ithomiine clades appear to fit better with factors long suggested to have promoted diversification of the group such as the uplifting of the Andes, in the case of montane genera. Alternatively, if ithomiines are as old as previous estimates suggest, the recent ages inferred for the diversification of Solanaceae seem likely to be seriously underestimated. Our study exemplifies the difficulty of testing hypotheses of divergence times and of choosing between alternative dating scenarios, and shows that age estimates based on seemingly plausible calibrations may be grossly incongruent.

What is a cladogram and what is not?

The origins and meanings of "cladogram" are reviewed. Traditionally, "cladogram" has been defined as a graphical representation of an empirical hypothesis of relationships among taxa, based on evidence from synapomorphies alone. Disturbingly, numerous recent authors treat "cladogram" as synonymous with "dendrogram" and do not appreciate the particular methodological connotations of the former term. This is lamented.

Transformational and taxic homology revisited.

Usages and meanings of the terms "taxic" and "transformational homology" are reviewed from 1982 to the present. While "taxic homology" has been relatively invariant in its connotation, "transformational homology" as employed by different authors refers to at least three different concepts. This has resulted in confusion.

About nothing.

In light of recent terminological controversy, this article reviews cladistic conceptions of character states coded as absences, symplesiomorphies, and secondary losses. The first section addresses absence as a question of ontology vs. epistemology. The second and third sections address the evidentiary status of symplesiomorphy in cladistics, the fourth contrasts primitive absence with secondary loss, and the fifth clarifies the meaning of "grouping". While secondary losses (reversals) are often synapomorphies, symplesiomorphies ("absent" or otherwise) have no evidentiary import to cladistic hypotheses of relationship. Thus, we argue that identifying symplesiomorphic character states as "homologous" is conceptually vacuous, because they are either synapomorphies (homologues) of more inclusive taxa, or complementary absences that unite no group.

DNA Barcodes in Taxonomic Descriptions.

This chapter discusses methods for incorporating DNA barcode information into formal taxonomic descriptions. We first review what a formal description entails and then discuss previous attempts to incorporate barcode information into taxonomic descriptions. Several computer programs are listed that extract diagnostics from DNA barcode data. Finally, we examine a test case (Astraptes taxonomy).

Introgression of wing pattern alleles and speciation via homoploid hybridization in Heliconius butterflies: a review of evidence from the genome.

The diverse Müllerian mimetic wing patterns of neotropical Heliconius (Nymphalidae) have been proposed to be not only aposematic signals to potential predators, but also intra- and interspecific recognition signals that allow the butterflies to maintain their specific identities, and which perhaps drive the process of speciation, as well. Adaptive features under differential selection that also serve as cues for assortative mating have been referred to as 'magic traits', which can drive ecological speciation. Such traits are expected to exhibit allelic differentiation between closely related species with ongoing gene flow, whereas unlinked neutral traits are expected to be homogenized to a greater degree by introgression. However, recent evidence suggests that interspecific hybridization among Heliconius butterflies may have resulted in adaptive introgression of these very same traits across species boundaries, and in the evolution of new species by homoploid hybrid speciation. The theory and data supporting various aspects of the apparent paradox of 'magic trait' introgression are reviewed, with emphasis on population genomic comparisons of Heliconius melpomene and its close relatives.

Homology and errors.

A recent review of the homology concept in cladistics is critiqued in light of the historical literature. Homology as a notion relevant to the recognition of clades remains equivalent to synapomorphy. Some symplesiomorphies are "homologies" inasmuch as they represent synapomorphies of more inclusive taxa; others are complementary character states that do not imply any shared evolutionary history among the taxa that exhibit the state. Undirected character-state change (as characters optimized on an unrooted tree) is a necessary but not sufficient test of homology, because the addition of a root may alter parsimonious reconstructions. Primary and secondary homology are defended as realistic representations of discovery procedures in comparative biology, recognizable even in Direct Optimization. The epistemological relationship between homology as evidence and common ancestry as explanation is again emphasized. An alternative definition of homology is proposed. © The Willi Hennig Society 2012.

Hybrid speciation in Heliconius butterflies? A review and critique of the evidence.

The evidence supporting the recent hypothesis of a homoploid hybrid origin for the butterfly species Heliconius heurippa is evaluated. Data from selective breeding experiments, mate-choice studies, and a wide variety of DNA markers are reviewed, and an alternative hypothesis for the origin of the species and its close relatives is proposed. A scenario of occasional red wing-pattern mutations in peripheral populations of Heliconius cydno with subsequent adaptive convergence towards sympatric mimicry rings involving H. melpomene and H. erato is offered as an alternative to the HHS hypothesis. Recent twists of this tale are addressed in a postscript.

Do model-based phylogenetic analyses perform better than parsimony? A test with empirical data.

The use of model-based methods to infer a phylogenetic tree from a given data set is frequently motivated by the truism that under certain circumstances the parsimony approach (MP) may produce incorrect topologies, while explicit model-based approaches are believed to avoid this problem. In the realm of empirical data from actual taxa, it is not known (or knowable) how commonly MP, maximum-likelihood or Bayesian inference are inaccurate. To test the perceived need for "sophisticated" model-based approaches, we assessed the degree of congruence between empirical phylogenetic hypotheses generated by alternative methods applied to DNA sequence data in a sample of 1000 recently published articles. Of 504 articles that employed multiple methods, only two exhibited strongly supported incongruence among alternative methods. This result suggests that the MP approach does not produce deviant hypotheses of relationship due to convergent evolution in long branches. Our finding therefore indicates that the use of multiple analytical methods is largely superfluous. We encourage the use of analytical approaches unencumbered by ad hoc assumptions that sap the explanatory power of the evidence. © The Willi Hennig Society 2010.

Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary.

The butterfly family Nymphalidae contains some of the most important non-drosophilid insect model systems for evolutionary and ecological studies, yet the evolutionary history of the group has remained shrouded in mystery. We have inferred a robust phylogenetic hypothesis based on sequences of 10 genes and 235 morphological characters for exemplars of 400 of the 540 valid nymphalid genera representing all major lineages of the family. By dating the branching events, we infer that Nymphalidae originated in the Cretaceous at 90 Ma, but that the ancestors of 10-12 lineages survived the end-Cretaceous catastrophe in the Neotropical and Oriental regions. Patterns of diversification suggest extinction of lineages at the Cretaceous/Tertiary boundary (65 Ma) and subsequent elevated speciation rates in the Tertiary.

Alternative facts: a reconsideration of putatively natural interspecific hybrid specimens in the genus Heliconius (Lepidoptera: Nymphalidae).

Mallet et al. (2007 BMC Evolutionary Biology, 7, 28) employed a database of putative interspecific hybrid specimens of the genus Heliconius to advance a hypothesis of "the species boundary as a continuum." Here, each of those specimens, as well as subsequently documented specimens, is individually reassessed regarding its phenotype, potential parentage and chain of custody in collections. Using a quantified scale of reliability, most of the specimens are interpreted differently than Mallet et al.'s identifications, and the actual number of interspecific hybrids is estimated to be much smaller than they proposed. To be specific, of 163 putative hybrid specimens examined, 11% suffered from ambiguous identity, 5% from confounding issues with their data labels, 50% were arguably intraspecific (depending upon alternative species concepts), and 22% were almost certainly reared, commercial specimens. Only eleven of the specimens meet the criteria established here to be legitimate and reliable interspecific hybrids, and all of those are between closely-related species. This result has potentially important implications for current hypotheses of frequent genomic introgression of wing pattern alleles among Heliconius clades.