Molecular Phylogenetics and Evolution

The systematics of the family Dendrodorididae poses a challenge to evolutionary biologists, as their mitochondrial and nuclear markers provide contradictory phylogenetic signals. Nuclear pseudogenes or exogenous contamination are hypothesized to cause the molecular discordance. However, these hypotheses have not been tested. We used genomic data from seven Dendrodorididae species to investigate the evolution of this family. Two mitogenomes displayed a novel structural rearrangement in nudibranchs, involving the translocation of three collinear genes and five surrounding tRNAs. Additionally, we found numerous mitogenomic regions with non-synonymous mutations and multiple indels in both coding and ribosomal genes. Protein modeling resulted in similar structures, suggesting that functionality is conserved. Phylogenies using mitogenomic data confirmed a specific clade membership for the rearranged mitogenomes. The incorporation of nuclear data did not fully resolve the systematic relationships of Dendrodorididae, acknowledging the evolutionary complexity of this group. The present study provides novel evidence on sudden molecular changes in mitogenomes, and highlights the relevance of using genomic data to unveil rare evolutionary processes, which is critical for understanding evolution of neglected taxa.

We provide the first comprehensive analysis of the origin of two enigmatic Satyrinae genera of uncertain affinities. Calisto, the only Satyrinae genus from the West Indies and endemic to these islands, has resisted numerous attempts at phylogenetic placement, regardless of the data type or methods used. Llorenteana, a monotypic genus from northwestern Mexico, has never been included in a molecular phylogenetic study, and past authors have placed it in five different genera and subtribes. We used mostly published genomic data, but also newly sequenced whole genome data from museum specimens and old DNA extracts, extracted BUSCO genes and prepared several datasets. These datasets differed in the degree of heterogeneity and saturation, the number of nucleotide positions used (all positions or only the first two), and were analyzed as nucleotides or as amino acids. We employed several methods for phylogenetic reconstruction using both partitioned and mixture models, as well as ASTRAL, and we inferred divergence times and ancestral areas of origin for Calisto and Llorenteana. The phylogenetic placement of Calisto varied among datasets when we used partitioned models and ASTRAL; however, most datasets resulted in the same relationships under mixture models. Our results suggest that Calisto is part of a clade of Old World origin that colonized the New World from north to south, thus sharing ancestry with Nearctic taxa. Llorenteana constitutes one of the earliest splits within the Euptychiina, a subtribe of Neotropical origin, but descending together with the Pronophilina from Nearctic ancestors. We propose the recognition of Erebiina stat. rev. as the only subtribe comprising the former Calistina syn. nov., Callerebiina syn. nov, Maniolina syn. nov., and Ypthimina syn. nov.

It is commonly assumed that increasing the number of characters has the potential to resolving radiations. We studied photosynthetic stramenopiles (Ochrophyta) using alignments of heterogeneous size and origin (6,762 sites for mitochondrion, 21,692 sites for plastid and 209,105 sites for nucleus). While statistical support for the relationships between the six major Ochrophyta lineages increases when comparing the mitochondrion and plastid trees, it decreases in the nuclear tree. Statistical support is not simply related to the dataset size but also to the quantity of phylogenetic signal available at each position and our ability to extract it. Here, we show that proper signal extraction is difficult to attain, as demonstrated by conflicting results obtained when varying taxon sampling. Even though the use of a better fitting model improved signal extraction and reduced the observed conflicts, the plastid dataset provided higher statistical support for the ochrophyte radiation than the larger nucleus dataset. We propose that the higher support observed in the plastid tree is due to an acceleration of the evolutionary rate in one short deep internal branch, implying that more phylogenetic signal per position is available to resolve the Ochrophyta radiation in the plastid than in the nuclear dataset. Our work therefore suggests that, in order to resolve radiations, beyond the obvious use of datasets with more positions, we need to continue developing models of sequence evolution that better extract the phylogenetic signal and design methods to search for genes/characters that contain more signal specifically for short internal branches.

Whole mitochondrial genomes have been helpful in estimating phylogenetic relationships in many organismal groups, including caecilian amphibians. Despite the increasing ease of obtaining mitochondrial genome sequences from high-throughput sequencing, several species of caecilian lack this important molecular resource. As part of a targeted-sequence capture project of nuclear ultraconserved elements for a small but substantially diverse radiation of caecilian amphibians found on the granitic Seychelles, we examined off-target sequences to determine if we captured enough mitochondrial fragments to reconstruct mitogenomes. We reconstructed (near-)complete mitogenomes for six of the eight species of Seychelles caecilians and completed 14 independent phylogenetic analyses (Bayesian inference and maximum likelihood) on different mitochondrial datasets assembled using different alignment techniques. As with other studies, we were unable to fully resolve internal phylogenetic relationships of the group. However, we found strong support in most analyses that a recently described miniaturized species, Hypogeophis pti, and another similarly-sized miniaturized species, H. brevis are not sister taxa. Our study suggests that miniature species of caecilians likely evolved at least twice on the Seychelles and highlights the need to revise genus-level taxonomy of Seychelles caecilians while providing further evidence that off-target sequences often contain enough mitochondrial fragments to reconstruct mitogenomes.

The Formicoxenus genus-group comprises six genera within the tribe Crematogastrini. The group is well known for repeated evolution of social parasitism among closely related taxa and cold-adapted species with large distribution ranges in the Nearctic and Palearctic regions. Previous analyses based on nuclear markers (ultraconserved elements, UCEs) and mitochondrial genes suggest close relationship between Formicoxenus Mayr, 1855, Leptothorax Mayr, 1855 and Harpagoxenus Forel, 1893. However, scant sampling has limited phylogenetic assessment of these genera. Also, previous phylogeographic analyses of L. acervorum (Fabricius, 1793) have been limited to its West-Palearctic range of distribution, which has provided a narrow view on recolonization, population structure and existing refugia of the species. Here, we inferred the phylogenenetic history of genera within the Formicoxenus genus-group and reconstructed the phylogeography of L. acervorum with more extensive sampling. We employed four datasets consisting of whole genomes and sequences of the COI. The topologies of previous nuclear and our inferences based on mitochondrial genomes were overall congruent. Further, Formicoxenus may not be monophyletic. We found several monophyletic lineages that do not correspond to the current species described within Leptothorax, especially in the Nearctic region. We identified a monophyletic L. acervorum lineage that comprises both Nearctic and Palearctic locations. The most recent expansion within L. acervorum probably occurred within the last 0.5 Ma with isolated populations predating the Last Glacial Maximum (LGM), which are localized in at least two refugial areas (Pyrenean and Northern plateau) in the Iberian Peninsula. The patterns recovered suggest a shared glacial refugium in the Iberian Peninsula with cold-adapted trees that currently share high-altitude environments in this region.

Robber flies or assassin flies (Diptera: Asilidae) are a diverse family of venomous predators. The most recent classification organizes Asilidae into 14 subfamilies based on a comprehensive morphological phylogeny, but many of these have not been supported in a subsequent molecular study using traditional molecular markers. To address questions of monophyly in Asilidae, we leveraged the recently developed Diptera-wide UCE baitset to compile seven datasets comprising 151 robber flies and 146 - 2,508 loci, varying in the extent of missing data. We also studied the behavior of different nodal support metrics, as the non-parametric bootstrap is known to perform poorly with large genomic datasets. Our ML phylogeny was fully resolved and well-supported, but partially incongruent with the coalescent phylogeny. Further examination of the datasets suggested the possibility that GC bias had influenced gene tree inference and subsequent species tree analysis. The subfamilies Brachyrhopalinae, Dasypogoninae, Dioctriinae, Stenopogoninae, Tillobromatinae, Trigonomiminae, and Willistonininae were not recovered as monophyletic in either analysis, consistent with a previous molecular study. The inter-subfamily relationships are summarized as follows: Laphriinae and Dioctriinae (in part) are successively sister to the remaining subfamilies, which form two clades; the first consists of a grade of Stenopogoninae (in part), Willistonininae (in part), Bathypogoninae+Phellinae, Stichopogoninae, Leptogastrinae, Ommatiinae, and Asilinae; the second clade consists of a thoroughly paraphyletic assemblage of genera from Dioctriinae (in part), Trigonomiminae, Stenopogoninae (in part), Tillobromatinae, Brachyrhopalinae, and Dasypogoninae. We find that nodal support does not significantly vary with missing data. Furthermore, the bootstrap appears to overestimate nodal support, as has been reported from many recent studies. Gene concordance and site concordance factors seem to perform better, but may actually underestimate support. We instead recommend quartet concordance as a more appropriate estimator of nodal support. Our comprehensive phylogeny demonstrates that the higher classification of Asilidae is far from settled, and it will provide a much-needed foundation for a thorough revision of the subfamily classification.

When fossils are sparse and the lineages studied are very divergent morphologically, analyses based exclusively on morphology may lead to conflicting and unexpected hypotheses. Through integration of data from conservative genes/gene regions the terminals including these data can anchor or constrain the search, thereby practically circumscribing the search space of the combined analyses. In this study, we revisit the phylogeny of a highly divergent group of mosses, class Polytrichopsida. We supplemented the morphological matrix by adding sequence data of the nuclear gene 18S, chloroplast genes rbcL and rps4, plus the mitochondrial gene nad5. For the phylogenetic analyses we used parsimony as the optimality criterion. Analyses that included all the terminals resulted in one most parsimonious tree with a clade comprised of Alophosia azorica and the fossil Meantoinea alophosioides representing the basal-most lineage. Analyses with different outgroup sampling produced the same topology for most ingroup relationships. An analysis excluding morphological characters and the four terminals for which only morphological characters were scored (the two fossil and two extant terminals) resulted in one optimal tree with identical topology to the one obtained when including all terminals. These results are largely congruent with those obtained in the recent analyses based exclusively on sequence level data of a larger number of terminals. Our results indicate that large size and complexity of the gametophyte have evolved independently in several lineages. Notably, the nodes of the backbone of the most parsimonious tree have very low support values, thus these inferred relationships could change if new additional information conflicts with the current data. Future studies should be aimed at incorporating all terminals into phylogenetic analyses, which is not an unrealistic goal for a group with less than 200 species. Also, additional fossils, some of which await detailed examination and description, need to be included. Whether these will affect the overall pattern of phylogeny presented here remains to be seen. In a group that is obviously very ancient, we cannot assume, a priori, that currently known fossil taxa, which go back in time less than 140 Ma, represent the oldest lineages of the group.

The use of molecular data to study evolutionary history of different organisms, revolutionized the field of systematics. Now with the appearance of high throughput sequencing (HTS) technologies more and more genetic sequence data is available. One of the important sources of genetic data for phylogenetic analyses has been mitochondrial DNA. The limitations of mitochondrial DNA for the study of phylogenetic relationships have been thoroughly explored in the age of single locus phylogenies. Now with the appearance of genomic scale data, more and more mitochondrial genomes are available. Here we assemble 47 mitochondrial genomes using whole genome Illumina short reads of representatives of the family Erebidae (Lepidoptera), in order to evaluate the accuracy of mitochondrial genome application in resolving deep phylogenetic relationships. We find that mitogenomes are inadequate for resolving subfamily level relationships in Erebidae, but given good taxon sampling, we see its potential in resolving lower level phylogenetic relationships.

With the advent of molecular phylogenetics, the number of taxonomic studies unveiling and describing cryptic diversity has greatly increased. However, speciation between cryptic lineages is often defined without evaluating population structure or gene flow, which can lead to false claims of species status and, subsequently, taxonomic inflation. In the present study we focus on the intriguing case of the Arabian gecko Trachydactylus hajarensis (Squamata: Gekkonidae), a species for which cryptic diversity has been previously reported. We generated mitochondrial data (12S rDNA) and genome-wide SNP data (ddRADseq) for 52 specimens to determine phylogenomic relationships, population structure and gene flow within this species. Then, we applied species delimitation methods (SDMs) to evaluate several competing species hypotheses through the Multispecies Coalescent model. Results show that T. hajarensis is comprised by three well-defined lineages, two of them in the Hajar Mountains of eastern Arabia, and one in Masirah Island, in the southeastern coast of Oman. Even though high levels of past introgression and strong mitonuclear discordances were found, current gene flow is scarce with clear boundaries between populations and shallow levels of admixture in the contact zone between lineages. Surprisingly, species tree topology differed between methods and when different individuals were used in downsampled datasets. Conventional SDMs supported up to three putative new species within the group. However, after species validation with the genealogical divergence index (gdi), none of the putative species held. Overall, this study highlights the importance of sample choice, integrative analyses, and validation methods to not incur into taxonomic inflation, providing a set of already available tools to assess and validate population structure, gene flow, and SDMs before describing new species.

Phylogenomics, which uses genome-scale data for phylogenetic inference, has clarified many controversial nodes in the tree of life. Such extensive data improve tree resolution and better reflects organismal history compared to analyses based on single or a few genetic loci. However, some relationships within the tree of life remain unresolved, as increased data can yield high node support without ensuring accuracy due to systematic errors. For example, the order-level relationships among chondrichthyans are still contentious despite the use of phylogenomic data. To address systematic errors, complex models have been developed, and filtering for less erroneous data shows great promise. Current metric-based filtering methods rank loci based on overall tree statistics, but problematic signals are often local; and topology-based data filtering approaches struggle with circular assumptions. In this study, we introduced two novel metric-based data filtering methods based on the ratio of local branch length or GC content between problematic clades. We applied these methods to a dataset of 4,452 single-copy exons extracted from 98 chondrichthyan species. The results using all loci showed that the Hexanchiformes was positioned at the root of Elasmobranchii, pulling other squalomorphs to the basal position and rendering Squalomorphii paraphyletic. Contrastingly, filtering for loci with more even branch length, the branch ratio method (absRatioLen) strongly supported the monophyly of all superorders of the chondrichthyans as well as their higher classification grouping, such as Selachii and Batoidea. By concentrating on problematic nodes, our assumption-free filtering methods demonstrate significant potential in resolving contentious relationships in the tree of life.

Quantitative traits are a source of evolutionary information often difficult to handle in cladistics. Tools exist to analyze this kind of data without subjective discretization, avoiding biases in the delimitation of categorical states. Nonetheless, the ability of continuous characters to accurately infer relationships is incompletely understood, particularly under parsimony analysis. This study evaluates the accuracy of phylogenetic reconstructions from simulated matrices of continuous characters evolving under alternative evolutionary processes and analyzed by parsimony. We generated 100 trees to simulate 9,000 matrices containing 26 terminals and 100 continuous characters evolving under: Brownian-Motion (BM), Ornstein-Uhlenbeck (OU) and Early-Burst (EB) processes assuming variable parametrizations. Our comparisons of cladograms revealed that matrices analyzed by parsimony carry phylogenetic signals to infer relationships, but the accuracy is higher for matrices simulated under BM, regardless of the parameterization schemes. Implementation of equal or implied weighting with multiple penalization strengths against homoplasies did not affect cladogram inferences. Accuracy of continuous characters in resolving relationships is skewed toward apical nodes of the trees. Our simulations provide controlled tests of the usefulness of quantitative traits in phylogenetics, specifically under neutral evolution, and demonstrate their effectiveness in estimating shallower nodes among recently diverged species, regardless of parameters and weighting schemes.

Cryptic species present major challenges for biodiversity and evolutionary research due to the morphological similarity and frequent lack of genetic and ecological data. In parasitoids wasps, this is especially true for lineages with limited sampling and unclear species boundaries. Here we reconstruct the phylogeny of Cotesia wasps parasitizing Melitaeini butterflies, including two cryptic species complexes (Cotesia acuminata agg. and C. melitaearum agg.). Using a ten-gene dataset from samples collected in Europe, Asia, and North America, we inferred relationships among 22 Cotesia species using maximum likelihood. We also included non-Melitaeini associated Cotesia to assess whether the Melitaeini parasitoids form a monophyletic group. Our analyses yielded a highly supported phylogeny, revealing four major clades, three of which included the Melitaeini associated species. This confirms that the Cotesia attacking Melitaeini butterflies are polyphyletic, resulting from independent host shifts across the genus. Each clade is further subdivided into subclades corresponding to the different (cryptic) species, clarifying previously unresolved relationships. These results provide a robust framework for future studies on the evolution, ecology, and host use dynamics of Cotesia wasps and highlight the utility of multi-locus data for resolving phylogenies in morphologically cryptic taxa.

Mitochondrial genomes are frequently used for phylogenetic inference due to their availability and cost-efficient sequencing. In most mitogenomic phylogenetic analyses, only the two ribosomal RNA and 13 protein coding genes are used. For such multi-locus datasets it has long been established that appropriate data partition models, e.g., partitioning by gene type and/or codon position, are necessary for robust phylogenetic inference. While most studies focused on the impact of partition models on the tree topology, little is known about the impact on divergence time estimation. Furthermore, although modeling among site rate variation within a partition is common practice, the extent of substitution rate variation among partitions is less explored. Here we study the impact of four partition model strategies: (i) no subdivision of the data (uniform), (ii) partitioning by gene, (iii) partitioning by codon position, and (iv) partitioning by both gene and codon position (combined). We explore the impact of partition models on divergence time estimation in fireflies (Coleoptera: Lampyridae). To this end, we sequenced mitochondrial genomes from 22 firefly species from Europe and Central America and combined these with 82 published mitochondrial genomes. Our results represent the most comprehensive time-calibrated phylogeny of fireflies to day. While the partition models had an impact on the inferred tree topology, the divergence times were almost not affected. Furthermore, we observed up to 3-fold substitution rate variation across genes and additionally more than to 10-fold substitution rate variation across codon positions. Our study gives insights into best practices of performing partitioned-data time-calibrated phylogenetic inference from mitochondrial genomes and multi-locus datasets in general.

Comprehensive phylogenies serve as foundations for taxonomic, comparative, and coevolutionary studies. Avian feather lice comprise the most diverse Parvorder (Ischnocera) of parasitic lice (Phthiraptera). Convergence and reduction of morphological features in this group have made past attempts at understanding the evolutionary relationships of feather lice challenging. Several recent phylogenomic studies have begun shedding light on the broader scale evolutionary tree of this group. However, additional taxonomic sampling is needed for a more complete understanding of the phylogeny and for higher statistical power in comparative studies. Here we investigate the higher-level relationships of feather lice based on the most comprehensive taxon sample to date. We leverage genome sequences of 260 samples of feather lice and 25 outgroup taxa to reconstruct a phylogenomic tree based on 2,395 target nuclear ortholog genes, using both concatenated and coalescent methods. These trees provide high support across nearly all branches of the topology, resolving backbone relationships as well as more detailed relationships within major groups. These phylogenies provide a framework for future comparative studies of feather lice, including comparisons with avian phylogeny and investigating patterns of diversification.

BackgroundA robust and stable phylogenetic framework is a fundamental goal of evolutionary biology. As the third largest insect order following by Diptera and Coleoptera in the world, lepidoptera (butterflies and moths) play a central role in almost every terrestrial ecosystem as the indicators of environmental change and serve as important models for biologists exploring questions related to ecology and evolutionary biology. However, for such charismatic insect group, the higher-level phylogenetic relationships among its superfamilies are still poorly unresolved. Resultswe increased taxon sampling among Lepidoptera (40 superfamilies and 76 families contained 286 taxa) and filtered the unqualified samples, then acquired a series of large amino-acid datasets from 69,680 to 400,330 for phylogenomic reconstructions. Using these datasets, we explored the effect of different taxon sampling on tree topology by considering a series of systematic errors using ML and BI methods. Moreover, we also tested the effectiveness in topology robustness among the three ML-based models. The results showed that taxon sampling is an important determinant in tree robustness of accurate lepidopteran phylogenetic estimation. Long-branch attraction (LBA) caused by site-wise heterogeneity is a significant source of bias given rise to topologies divergence of ditrysia in phylogenomic reconstruction. Phylogenetic inference showed a most comprehensive framework by far to reveal the relationships among lepidopteran superfamilies, but limited by taxon sampling, it could only represent the current understanding of the lepidopteran tree of life. The relationships within the species-rich and relatively rapid radiation Ditrysia and especially Apoditrysia remain poorly unresolved, which need to increase taxon sampling and adopt lineage-specific genes for further phylogenomic reconstruction. ConclusionsThe present study further expands the taxon sampling of lepidopteran phylogeny and provides a potential phylogenomic foundation for further understanding its current higher-level relationships.

Most of the worlds biodiversity is described primarily or exclusively using morphological traits that may not always reflect the true evolutionary units. Accurate taxonomy is critical for conservation efforts and re-evaluation of traditional taxonomy may often be warranted since species and subspecies are frequently the focus of conservation and faunistic studies. Here, we test comprehensive taxonomic hypotheses of morphologically defined subspecies in the tiger beetle, Eunota togata (LaFerte-Senectere, 1841). The four recognized subspecies were delineated based mainly on the dorsal coloration and extent of white markings termed maculations. We combine inferences from mtDNA genealogies and genome-wide multilocus data to elucidate the evolutionary relationships within the group and assess the taxonomic implications. Three of the four subspecific taxa delineated by morphology were not supported by the genomic or mtDNA data. In fact, the species-level diversity in this group was underestimated, as E. togata was found to represent three well-supported distinct species in all genetic analyses. Emerging from these analyses, we also document an intriguing example of convergent evolution in lighter colored E. togata adapting to similar white sand backgrounds. Our collective work underscores the importance of integrating molecular methods with morphology for species and subspecies delimitation and conservation.

With the advent of genomics, sequencing thousands of loci from hundreds of individuals now appears feasible at reasonable costs, allowing complex phylogenies to be resolved. This is particularly relevant for cnidarians, for which insufficient data due to the small number of currently available markers, coupled with difficulties in inferring gene trees and morphological incongruences, encrypts species boundaries, thereby blurring the study and conservation of these organisms. Yet, can genomics alone be used to delimit species in an integrative taxonomic context? Here, focusing on the coral genus Pocillopora, which plays key roles in Indo-Pacific reef ecosystems but has challenged taxonomists for decades, we explored and discussed the usefulness of multiple criteria (genetics, morphology, biogeography and symbiosis ecology) to delimit species of this genus. Phylogenetic inferences, clustering approaches and species delimitation methods based on genome-wide single-nucleotide polymorphisms (SNPs) were first used to resolve Pocillopora phylogeny and propose genomic species hypotheses from 356 colonies sampled across the Indo-Pacific (western Indian Ocean, tropical southwestern Pacific and south-east Polynesia). These species hypotheses were then compared to previous genetic evidences, as well as to evidences based on morphology, biogeography and symbiosis. Genomics allowed to delimit 21 species hypotheses where only seven are currently recognised based on current taxonomy. Moreover, 13 species were strongly supported by all approaches, either confirming their currently recognised species status, or supporting the presence of new species that need to be formally described. Some of the other genomic species hypotheses were supported by biogeographic or symbiosis evidences, but additional investigations are needed to state on their species status. Altogether, our results support (1) the obsolescence of macromorphology (i.e., overall colony and branches shape) but the relevance of micromorphology (i.e., corallite structures) to refine Pocillopora species limits, (2) the need to identify molecularly species prior to their study, as morphology can blur species identification on the field, (3) the relevance of the mtORF (coupled with other markers in some cases) as a diagnostic marker of most species, and (4) the need for a taxonomical revision in the Pocillopora genus. These results give new insights into the usefulness of multiple criteria for resolving Pocillopora species limits and will ultimately provide helpful insights for the conservation of the species from this scleractinian genus. [biogeography; cryptic species delimitation; Indo-Pacific; microsatellites; morphology; phylogenetics; single-nucleotide polymorphism (SNP); Symbiodiniaceae]

The phylogenetic information contained in sequence data is partly determined by the overall rate of nucleotide substitution in the genomic region in question. However, phylogenetic signal is affected by various other factors, such as heterogeneity in substitution rates across lineages. These factors might be able to predict the phylogenetic accuracy of any given gene in a data set. We examined the association between the accuracy of phylogenetic inference across genes and several characteristics of branch lengths in phylogenomic data. In a large number of published data sets, we found that the accuracy of phylogenetic inference from genes was consistently associated with their mean statistical branch support and variation in their gene tree root-to-tip distances, but not with tree length and stemminess. Therefore, a signal of constant evolutionary rates across lineages appears to be beneficial for phylogenetic inference. Identifying the causes of variation in root-to-tip lengths in gene trees also offers a potential way forward to increase congruence in the signal across genes and improve estimates of species trees from phylogenomic data sets.

Even after decades of research on diversification in the Neotropics, our understanding of the evolutionary processes shaping Neotropical clades is still incomplete. In the current study, we used different divergence times and likelihood-based methods to investigate the influence of biogeography and host associations on the diversification of the most species-rich Neotropical psyllid genus Melanastera (Liviidae) using molecular phylogenetic data from seven gene fragments (four mitochondrial and three nuclear). The putatively monophyletic group of Neotropical Melanastera species has an estimated crown node age of 20.2 Ma (ML, CI 20.2-30.6) or 23.2 Ma (BI, 95% HPD 16.6-32.6), with diversification occurring mainly in the Upper Miocene, although some species groups diversified in the Pliocene and Pleistocene. Biogeographic analysis suggests that the Neotropical Melanastera originated from the Pacific region of South and Central America. We detected a shift in diversification rates that likely occurred either at the time of origin of Melanastera or during the main colonisation of the Atlantic and Amazon Forests, followed by a subsequent slowdown in speciation rates. State-dependent speciation and extinction models revealed a significant relationship between this diversification shift and the shift of Melanastera to the plant families Melastomataceae and Annonaceae, reflecting the impact of host switching on speciation rates in this group. This period also coincides with several independent dispersal events from the Atlantic and Amazon Forests to other parts of the Neotropics. Taken together, the results of the current study suggest that diversification of Melanastera was facilitated by major shifts to new host families, which may have promoted the dispersal of Melanastera into new adaptive zones with subsequent processes of local speciation.

Despite increased recent attention towards Bayesian phylogenetics and its applications in understanding macroevolutionary processes, it remains unclear how many discrete characters are needed to accurately estimate tree topologies in a Bayesian framework. This could be particularly relevant for morphological datasets used in phylogenetics, as they usually consist of few dozens to few hundreds of characters--orders of magnitude smaller than most molecular datasets. I designed a simulation study in the software RevBayes to explore how the number of sampled discrete characters affects accuracy and precision of Bayesian phylogenetic estimates, under various setups differing in number of taxa, average number of state changes per character (i.e., tree length), and number of states per character. Results indicate that between 100 and 500 variable characters are necessary to reach sufficient accuracy and precision of phylogenetic estimates for as low as 20 tips. All other parameters being equal, multistate characters produce slightly more accurate estimates than binary characters, and more labile characters produce more accurate estimates for trees above 50 tips. The results of this study highlight the continuous need for global research efforts geared towards the characterization and digitization of interspecific morphological diversity in both extant and extinct taxa.