Historical biogeography of the Neotropical noctilionoid bats (Chiroptera: Noctilionoidea), revisited through a geographically explicit analysis.

Biogeographic studies have generally relied on methods that use a few, large predefined areas, which may overlook fine-scale patterns. Here we test previous hypotheses about the biogeographic history of a diverse bat clade regarding its association with major Neotropical geological formations, particularly the Antilles, the South American Dry Diagonal, the Andes and the Panamanian land bridge, by applying a recently available method that uses actual distributions instead of predefined areas. We compiled and curated spatially explicit, georeferenced data of 173 bat species (Mammalia: Chiroptera: Noctilionoidea) from the online database Global Biodiversity Information Facility. By taking a previous comprehensive phylogeny as an evolutionary framework, we performed computationally intensive analyses using the Geographically-explicit Event Model. This method uses the observed species distributions to reconstruct the ancestral areas and biogeographic events at each phylogeny node. We found that sympatric speciation was the most frequently reconstructed event, and involved mainly the Panamanian Isthmus and northern South America (SA), but all sympatry reconstructions were different and specific to each node. Allopatric events were important in the Andes; vicariance caused both west/east and north/south disjunctions that went unnoticed previously. Founder events indicated bidirectional dispersal between the mainland and the Antilles since the Miocene, and across the incomplete Panamanian bridge and the SA Dry Diagonal since the early Pliocene. Overall, we found support for previous hypotheses on the influence of major Neotropical paleogeographic events in the diversification of the group, but additionally revealed multi-scale patterns that are embedded within the mainland and were previously overlooked. Our results highlight a trans-isthmian centre of diversification in the biogeographic history of Noctilionoidea including the Panamanian Isthmus and Northern SA.

DUALCOR: a phylogenetic comparative method to evaluate phylogenetic correlation between a character and a non-evolving external variable.

A new phylogenetic comparative method called DUALCOR is presented to evaluate the evolutionary response of a character to non-evolving external factors, such as environmental variables. The method treats the character as a typical evolving feature of an organism that is reconstructed on a given tree, whereas the external factor is treated as unrelated to the phylogeny. DUALCOR first calculates the correlation/regression between the observed values of the character and the external factor; then it maps the character onto the phylogeny, shuffles the changes among branches, and re-evolves the character to yield new terminal values uncorrelated with the observed values of the external factor, allowing users to examine whether the observed degree of correlation can be attained at random. This is repeated n (say, 999) times, thereby using the dual nature of characters to construct a permutation test that is shown to satisfy requirements of Generalized Monte Carlo procedures. In addition, we provide an empirical example with a reverse test where the external variable (features determined largely by non-heritable factors) is the dependent variable.

Aerodynamic reconstruction of the primitive fossil bat Onychonycteris finneyi (Mammalia: Chiroptera).

Bats are the only mammals capable of powered flight. One of the oldest bats known from a complete skeleton is Onychonycteris finneyi from the Early Eocene (Green River Formation, Wyoming, 52.5 Ma). Estimated to weigh approximately 40 g, Onychonycteris exhibits the most primitive combination of characters thus far known for bats. Here, we reconstructed the aerofoil of the two known specimens, calculated basic aerodynamic variables and compared them with those of extant bats and gliding mammals. Onychonycteris appears in the edges of the morphospace for bats, underscoring the primitive conformation of its flight apparatus. Low aerodynamic efficiency is inferred for this extinct species as compared to any extant bat. When we estimated aerofoil variables in a model of Onychonycteris excluding the handwing, it closely approached the morphospace of extant gliding mammals. Addition of a handwing to the model lacking this structure results in a 2.3-fold increase in aspect ratio and a 28% decrease in wing loading, thus greatly enhancing aerodynamics. In the context of these models, the rapid evolution of the chiropteran handwing via genetically mediated developmental changes appears to have been a key transformation in the hypothesized transition from gliding to flapping in early bats.

Comparative cranial ontogeny of Tapirus (Mammalia: Perissodactyla: Tapiridae).

Skull morphology in tapirs is particularly interesting due to the presence of a proboscis with important trophic, sensory and behavioral functions. Several studies have dealt with tapir skull osteology but chiefly in a comparative framework between fossil and recent species of tapirs. Only one study examined an aspect of cranial ontogeny, development of the sagittal crest (Holbrook. J Zool Soc Lond 2002; 256; 215). Our goal is to describe in detail the morphological changes that occur during the postnatal ontogeny of the skull in two representative tapir species, Tapirus terrestris and Tapirus indicus, and to explore possible functional consequences of their developmental trajectories. We compared qualitative features of the skull on a growth series of 46 specimens of T. terrestris ordered on the basis of the sequence of eruption and tooth wear, dividing the sample into three age classes: class Y (very young juvenile), class J (from young juvenile to young adult) and class A (full and old adult). The qualitative morphological analysis consisted of describing changes in the series in each skull bone and major skull structure, including the type and degree of transformation (e.g. appearance, fusion) of cranial features (e.g. processes, foramina) and articulations (sutures, synchondroses, and synovial joints). We then measured 23 cranial variables in 46 specimens of T. terrestris that included the entire ontogenetic series from newborn to old adults. We applied statistical multivariate techniques to describe allometric growth, and compared the results with the allometric trends calculated for a sample of 25 specimens of T. indicus. Results show that the skull structure was largely conserved throughout the postnatal ontogeny in T. terrestris, so class Y was remarkably similar to class A in overall shape, with the most significant changes localized in the masticatory apparatus, specifically the maxillary tuber as a support of the large-sized permanent postcanine dentition, and correlated changes in diastemata, mandibular body, and sagittal and nuchal crests. In the nasal region, ontogenetic remodeling affected the space for the meatal diverticulum and the surfaces for the origin of the proboscis musculature. Overall, ontogenetic trajectories exhibited more negative allometric components in T. indicus than in T. terrestris, and they shared 47.83% of allometric trends. Tapirus indicus differed most significantly from T. terrestris in the allometry of postcanine toothrows, diastemata and mandibular body. Thus, some allometric trends seem to be highly conserved among the species studied, and the changes observed showed a strong functional and likely adaptive basis in this lineage of ungulates.

Each flying fox on its own branch: a phylogenetic tree for Pteropus and related genera (Chiroptera: Pteropodidae).

Pteropodidae is a diverse Old World family of non-echolocating, frugivorous and nectarivorous bats that includes the flying foxes (genus Pteropus) and allied genera. The subfamily Pteropodinae includes the largest living bats and is distributed across an immense geographic range from islands in East Africa to the Cook Islands of Polynesia. These bats are keystone species in their ecosystems and some carry zoonotic diseases that are increasingly a focus of interest in biomedical research. Here we present a comprehensive phylogeny for pteropodines focused on Pteropus. The analyses included 50 of the ∼63 species of Pteropus and 11 species from 7 related genera. We obtained sequences of the cytochrome b and the 12S rRNA mitochondrial genes for all species and sequences of the nuclear RAG1, vWF, and BRCA1 genes for a subsample of taxa. Some of the sequences of Pteropus were obtained from skin biopsies of museum specimens including that of an extinct species, P. tokudae. The resulting trees recovered Pteropus as monophyletic, although further work is needed to determine whether P. personatus belongs in the genus. Monophyly of the majority of traditionally-recognized Pteropus species groups was rejected, but statistical support was strong for several clades on which we based a new classification of the Pteropus species into 13 species groups. Other noteworthy results emerged regarding species status of several problematic taxa, including recognition of P. capistratus and P. ennisae as distinct species, paraphyly of the P. hypomelanus complex, and conspecific status of P. pelewensis pelewensis and P. p. yapensis. Relationships among the pteropodine genera were not completely resolved with the current dataset. Divergence time analysis suggests that Pteropus originated in the Miocene and that two independent bursts of diversification occurred in the Pleistocene in different regions of the Indo-Pacific realm.

Palaeoatmosphere facilitates a gliding transition to powered flight in the Eocene bat, Onychonycteris finneyi.

The evolutionary transition to powered flight remains controversial in bats, the only flying mammals. We applied aerodynamic modeling to reconstruct flight in the oldest complete fossil bat, the archaic Onychonycteris finneyi from the early Eocene of North America. Results indicate that Onychonycteris was capable of both gliding and powered flight either in a standard normodense aerial medium or in the hyperdense atmosphere that we estimate for the Eocene from two independent palaeogeochemical proxies. Aerodynamic continuity across a morphological gradient is further demonstrated by modeled intermediate forms with increasing aspect ratio (AR) produced by digital elongation based on chiropteran developmental data. Here a gliding performance gradient emerged of decreasing sink rate with increasing AR that eventually allowed applying available muscle power to achieve level flight using flapping, which is greatly facilitated in hyperdense air. This gradient strongly supports a gliding (trees-down) transition to powered flight in bats.

Patterns of ontogenetic evolution across extant marsupials reflect different allometric pathways to ecomorphological diversity.

The relatively high level of morphological diversity in Australasian marsupials compared to that observed among American marsupials remains poorly understood. We undertake a comprehensive macroevolutionary analysis of ontogenetic allometry of American and Australasian marsupials to examine whether the contrasting levels of morphological diversity in these groups are reflected in their patterns of allometric evolution. We collate ontogenetic series for 62 species and 18 families of marsupials (n = 2091 specimens), spanning across extant marsupial diversity. Our results demonstrate significant lability of ontogenetic allometric trajectories among American and Australasian marsupials, yet a phylogenetically structured pattern of allometric evolution is preserved. Here we show that species diverging more than 65 million years ago converge in their patterns of ontogenetic allometry under animalivorous and herbivorous diets, and that Australasian marsupials do not show significantly greater variation in patterns of ontogenetic allometry than their American counterparts, despite displaying greater magnitudes of extant ecomorphological diversity.

Evolutionary relationships of the old world fruit bats (Chiroptera, Pteropodidae): another star phylogeny?

BACKGROUND: The family Pteropodidae comprises bats commonly known as megabats or Old World fruit bats. Molecular phylogenetic studies of pteropodids have provided considerable insight into intrafamilial relationships, but these studies have included only a fraction of the extant diversity (a maximum of 26 out of the 46 currently recognized genera) and have failed to resolve deep relationships among internal clades. Here we readdress the systematics of pteropodids by applying a strategy to try to resolve ancient relationships within Pteropodidae, while providing further insight into subgroup membership, by 1) increasing the taxonomic sample to 42 genera; 2) increasing the number of characters (to >8,000 bp) and nuclear genomic representation; 3) minimizing missing data; 4) controlling for sequence bias; and 5) using appropriate data partitioning and models of sequence evolution. RESULTS: Our analyses recovered six principal clades and one additional independent lineage (consisting of a single genus) within Pteropodidae. Reciprocal monophyly of these groups was highly supported and generally congruent among the different methods and datasets used. Likewise, most relationships within these principal clades were well resolved and statistically supported. Relationships among the 7 principal groups, however, were poorly supported in all analyses. This result could not be explained by any detectable systematic bias in the data or incongruence among loci. The SOWH test confirmed that basal branches' lengths were not different from zero, which points to closely-spaced cladogenesis as the most likely explanation for the poor resolution of the deep pteropodid relationships. Simulations suggest that an increase in the amount of sequence data is likely to solve this problem. CONCLUSIONS: The phylogenetic hypothesis generated here provides a robust framework for a revised cladistic classification of Pteropodidae into subfamilies and tribes and will greatly contribute to the understanding of character evolution and biogeography of pteropodids. The inability of our data to resolve the deepest relationships of the major pteropodid lineages suggests an explosive diversification soon after origin of the crown pteropodids. Several characteristics of pteropodids are consistent with this conclusion, including high species diversity, great morphological diversity, and presence of key innovations in relation to their sister group.

Phylogenetic morphometrics (I): the use of landmark data in a phylogenetic framework.

A method for the direct use of aligned landmark data (2D or 3D coordinates of comparable points) in phylogenetic analysis is described. The approach is based on finding, for each of the landmark points, the ancestral positions that minimize the distance between the ancestor/descendant points along the tree. Doing so amounts to maximizing the degree to which similar positions of the landmarks in different taxa can be accounted for by common ancestry, i.e. parsimony. This method requires no transformation of the aligned data or the results: the data themselves are the x, y, z coordinates of the landmarks, and the output of mapping a character onto a given tree is the x, y, z coordinates for the hypothetical ancestors. In the special case of collinear points, the results are identical to those of optimization of (continuous) additive characters.

The phylogenetic relationships of cynopterine fruit bats (Chiroptera: Pteropodidae: Cynopterinae).

The subfamily Cynopterinae comprises ca. 24 species of pteropodid bats (Family Pteropodidae) distributed exclusively in South and Southeast Asia. Although some studies have supported monophyly of the subfamily, molecular analyses have produced contradictory results and there has been little agreement on relationships of cynopterines to other megabat groups. However, no previous studies have included a complete sampling of cynopterine genera. Here we describe a phylogenetic analysis of Cynopterinae based on more than 6000 bp from six different genes sampled in representatives of all 14 recognized genera. Our results support the monophyly of Cynopterinae but refute a close relationship of cynopterines with Nyctimeninae. Within Cynopterinae, our analyses consistently recovered two monophyletic clades, which we recommend be recognized formally as tribes: Cynopterini and Balionycterini. Biogeographic analyses indicate a Sundaland origin of the Cynopterinae and divergence date estimates suggest different timing of diversification of the two major cynopterine clades.

A multiple peak adaptive landscape based on feeding strategies and roosting ecology shaped the evolution of cranial covariance structure and morphological differentiation in phyllostomid bats.

We explored the evolution of morphological integration in the most noteworthy example of adaptive radiation in mammals, the New World leaf-nosed bats, using a massive dataset and by combining phylogenetic comparative methods and quantitative genetic approaches. We demonstrated that the phenotypic covariance structure remained conserved on a broader phylogenetic scale but also showed a substantial divergence between interclade comparisons. Most of the phylogenetic structure in the integration space can be explained by splits at the beginning of the diversification of major clades. Our results provide evidence for a multiple peak adaptive landscape in the evolution of cranial covariance structure and morphological differentiation, based upon diet and roosting ecology. In this scenario, the successful radiation of phyllostomid bats was triggered by the diversification of dietary and roosting strategies, and the invasion of these new adaptive zones lead to changes in phenotypic covariance structure and average morphology. Our results suggest that intense natural selection preceded the invasion of these new adaptive zones and played a fundamental role in shaping cranial covariance structure and morphological differentiation in this hyperdiverse clade of mammals. Finally, our study demonstrates the power of combining comparative methods and quantitative genetic approaches when investigating the evolution of complex morphologies.

Comparative myology of the ankle of Leopardus wiedii and L. geoffroyi (Carnivora: Felidae): functional consistency with osteology, locomotor habits and hunting in captivity.

Leopardus wiedii (margay) is the only arboreal Neotropical felid able to climb head-first down trees, due to its ability to rotate its tarsal joint 180°. A closely related, similar-sized species, L. geoffroyi (Geoffroy's cat) exhibits more typical terrestrial habits and lacks the arboreal capabilities of L. wiedii. There is osteological evidence that supports a mechanical specialization of L. wiedii's tarsal joint for inversion, but there have been no studies on the myology of this specialization. Based on comparative gross-anatomy dissections of zeugo- and autopodial muscles related to the ankle joint of one margay specimen and two Geoffroýs cats, we identified myological specializations of L. wiedii that support its arboreal abilities. In addition, we documented both species hunting the same prey (domestic pigeon Columba livia, Aves: Columbidae) in captivity, to complement. We report differences in the origin, insertion and belly in 8 of the 10 dissected muscles. At least 3 of these interspecific variations can be associated with strengthening of the main muscles that command inversion/eversion movements of the tarsal joint and support the body weight in the head-down climbing position typical of L. wiedii. Frame-by-frame video reconstructions depict the sequence of movements in these species while hunting and highlight the advantages of the arboreal abilities of L. wiedii.

The endemic Patagonian vespertilionid assemblage is a depauperate ecomorphological vicariant of species-rich neotropical assemblages.

Vespertilionidae is the most diverse chiropteran family, and its diversity is concentrated in warm regions of the World; however, due to physiological and behavioral adaptations, these bats also dominate bat faunas in temperate regions. Here we performed a comparative study of vespertilionid assemblages from two broad regions of the New World, the cold and harsh Patagonia, versus the remaining temperate-to-subtropical, extra-Patagonian eco-regions of the South American Southern Cone. We took an ecomorphological approach and analyzed the craniodental morphological structure of these assemblages within a phylogenetic framework. We measured 17 craniodental linear variables from 447 specimens of 22 currently recognized vespertilionid species of the study regions. We performed a multivariate analysis to define the morphofunctional space, and calculated the pattern and degree of species packing for each assemblage. We assessed the importance of phylogeny and biogeography, and their impact on depauperate (Patagonian) versus rich (extra-Patagonian) vespertilionid assemblages as determinants of morphospace structuring. We implemented a sensitivity analysis associated to small samples of rare species. The morphological patterns were determined chiefly by the evolutionary history of the family. The Patagonian assemblage can be described as a structurally similar but comparatively depauperate ecomorphological version of those assemblages from neighboring extra-Patagonian eco-regions. The Patagonian assemblage seems to have formed by successively adding populations from Northern regions that eventually speciated in the region, leaving corresponding sisters (vicariants) in extra-Patagonian eco-regions that continued to be characteristically richer. Despite being structurally akin, degree of species packing in Patagonia was comparatively very low, which may reflect the effect of limited dispersal success into a harsh region for bat survival.

Intense natural selection preceded the invasion of new adaptive zones during the radiation of New World leaf-nosed bats.

The family Phyllostomidae, which evolved in the New World during the last 30 million years, represents one of the largest and most morphologically diverse mammal families. Due to its uniquely diverse functional morphology, the phyllostomid skull is presumed to have evolved under strong directional selection; however, quantitative estimation of the strength of selection in this extraordinary lineage has not been reported. Here, we used comparative quantitative genetics approaches to elucidate the processes that drove cranial evolution in phyllostomids. We also quantified the strength of selection and explored its association with dietary transitions and specialization along the phyllostomid phylogeny. Our results suggest that natural selection was the evolutionary process responsible for cranial diversification in phyllostomid bats. Remarkably, the strongest selection in the phyllostomid phylogeny was associated with dietary specialization and the origination of novel feeding habits, suggesting that the adaptive diversification of phyllostomid bats was triggered by ecological opportunities. These findings are consistent with Simpson's quantum evolutionary model of transitions between adaptive zones. The multivariate analyses used in this study provides a powerful tool for understanding the role of evolutionary processes in shaping phenotypic diversity in any group on both micro- and macroevolutionary scales.

Conflict and congruence in a combined DNA-morphology analysis of megachiropteran bat relationships (Mammalia: Chiroptera: Pteropodidae).

The phylogeny of megabats (Mammalia: Chiroptera: Megachiroptera) has been addressed only on molecular grounds, as little effort has previously been made to describe the impressive morphological variation of the group in terms of phylogenetically informative characters. Here we provide a morphological matrix of 236 characters from the integument, dentition, cranial and post-cranial skeleton, digestive apparatus and urogenital system. This data set covers most characters discussed previously in more restricted taxonomic contexts, as well a large number of new characters. Our aim was to generate a phylogenetic hypothesis for megabats based on a combined analysis of morphological characters and available gene sequence data from four mitochondrial and one nuclear loci. We used direct optimization under conventional equal costs, as well as under a cost ratio that maximizes homology when inapplicables (gaps) are present. Our results contradict the allegedly high level of conflict between the molecular and morphological partitions. We found that, although morphology alone recovered trees different and to some extent incompatible with those from previous molecular analyses, the combination of the two sources of evidence easily accommodated the morphological and molecular signals, yielding a resolved and relatively well-supported phylogeny of Megachiroptera that is in reasonable agreement with the current morphology-based taxonomy of the group. Overall congruence favored the maximization of homology by a narrow margin. In addition, partial analyses showed that implied weighting of morphology performed slightly better than equal weighting with respect to the combined analyses.

A phylogeny of megachiropteran bats (Mammalia: Chiroptera: Pteropodidae) based on direct optimization analysis of one nuclear and four mitochondrial genes.

The phylogeny of megachiropteran bats (Mammalia: Chiroptera: Pteropodidae) has been investigated using several different molecular datasets. These studies differed widely in taxonomic and locus sampling, and their results tended to lack resolution of internal nodes and were themselves largely incongruent. To address this, we assembled a data set of 5 loci (up to 3.5 kbp from 12S rDNA, 16S rDNA, tDNA-valine, cytochrome b, and the nuclear gene c-mos) for 43 species of megachiropterans and 6 microchiropteran outgroups. We analyzed these data with direct optimization under equal costs for substitutions and indels. We used POY in a parallel setting, and searches consisted of replicated swapping + refinements (ratcheting, tree fusing, and iterative pass optimization). Our results indicate that Megachiroptera and all recognized genera (including Pteropus) are monophyletic, and that Melonycteris is the sister group of the clade containing all the other genera. Clades previously proposed using molecular data, as well as many new and traditional groups, were well-supported, and various sources suggest that the degree of conflict with morphological data may be considerably less marked than previously supposed. Analysis of individual loci suffer 70% loss in the number of compatible groups recovered across all analyses with respect to combined analyses. Our results indicate that, within Megachiroptera, nectarivory and cave-dwelling originated several times, but echolocation (used for obstacle detection) evolved only once. Megachiropterans likely originated in SE Asia-Melanesia, and colonized Africa at least four times.

Post-weaning cranial ontogeny in two bandicoots (Mammalia, Peramelomorphia, Peramelidae) and comparison with carnivorous marsupials.

The ontogeny of the skull has been studied in several marsupial groups such as didelphids, microbiotheriids, and dasyurids. Here, we describe and compare the post-weaning ontogeny of the skull in two species of bandicoots, Echymipera kalubu (Echymiperinae) and Isoodon macrourus (Peramelinae), analyzing specific allometric trends in both groups, describing common (and specific) patterns, and discussing them on functional and phylogenetic grounds. Growth patterns were analyzed both qualitatively and quantitatively, including bivariate and multivariate analyses of allometry. We also evaluated character transformation and phylogenetic signals of the allometric patterns in several groups of marsupials and some placentals. We identified morphological changes between juvenile and adult stages in both species of peramelids, many related to the development of the trophic apparatus. Notable differences were detected in the patterns of growth, suggesting divergences in ontogenetic trajectories between both species. Both bivariate and multivariate methods indicate that positive allometries in E. kalubu apply to longitudinal dimensions, whereas in I. macrourus, positive allometries are restricted to vertical dimensions of the skull. The comparison of the allometric trends of two bandicoots with previously studied taxa reveals that although peramelids exhibit a particularly short gestation period and divergent morphology compared to other marsupials, their pattern does not show any particular trend. Some allometric trends seem to be highly conserved among the species studied, showing weak phylogenetic signal. Marsupials in general do not show particular patterns of post-weaning skull growth compared with placentals.

Response to comment on "The placental mammal ancestor and the post-K-Pg radiation of placentals".

Tree-building with diverse data maximizes explanatory power. Application of molecular clock models to ancient speciation events risks a bias against detection of fast radiations subsequent to the Cretaceous-Paleogene (K-Pg) event. Contrary to Springer et al., post-K-Pg placental diversification does not require "virus-like" substitution rates. Even constraining clade ages to their model, the explosive model best explains placental evolution.

The placental mammal ancestor and the post-K-Pg radiation of placentals.

To discover interordinal relationships of living and fossil placental mammals and the time of origin of placentals relative to the Cretaceous-Paleogene (K-Pg) boundary, we scored 4541 phenomic characters de novo for 86 fossil and living species. Combining these data with molecular sequences, we obtained a phylogenetic tree that, when calibrated with fossils, shows that crown clade Placentalia and placental orders originated after the K-Pg boundary. Many nodes discovered using molecular data are upheld, but phenomic signals overturn molecular signals to show Sundatheria (Dermoptera + Scandentia) as the sister taxon of Primates, a close link between Proboscidea (elephants) and Sirenia (sea cows), and the monophyly of echolocating Chiroptera (bats). Our tree suggests that Placentalia first split into Xenarthra and Epitheria; extinct New World species are the oldest members of Afrotheria.

Delayed-response phylogenetic correlation: an optimization-based method to test covariation of continuous characters.

A new phylogenetic comparative method is proposed, based on mapping two continuous characters on a tree to generate data pairs for regression or correlation analysis, which resolves problems of multiple character reconstructions, phylogenetic dependence, and asynchronous responses (evolutionary lags). Data pairs are formed in two ways (tree-down and tree-up) by matching corresponding changes, Delta x and Delta y. Delayed responses (Delta y occurring later in the tree than Delta x) are penalized by weighting pairs using nodal or branch-length distance between Delta x and Delta y; immediate (same-node) responses are given maximum weight. All combinations of character reconstructions (or a random sample thereof) are used to find the observed range of the weighted coefficient of correlation r (or weighted slope b). This range is used as test statistic, and the null distribution is generated by randomly reallocating changes (Delta x and Delta y) in the topology. Unlike randomization of terminal values, this procedure complies with Generalized Monte Carlo requirements while saving considerable computation time. Phylogenetic dependence is avoided by randomization without data transformations, yielding acceptable type-I error rates and statistical power. We show that ignoring delayed responses can lead to falsely nonsignificant results. Issues that arise from considering delayed responses based on optimization are discussed.