A new method for handling missing species in diversification analysis applicable to randomly or nonrandomly sampled phylogenies

Chronograms from molecular dating are increasingly being used to infer rates of diversification and their change over time. A major limitation in such analyses is incomplete species sampling that moreover is usually nonrandom. While the widely used γ statistic with the Monte Carlo constant-rates test or the birth-death likelihood analysis with the δ AICrc test statistic are appropriate for comparing the fit of different diversification models in phylogenies with random species sampling, no objective automated method has been developed for fitting diversification models to nonrandomly sampled phylogenies. Here, we introduce a novel approach, CorSiM, which involves simulating missing splits under a constant rate birth-death model and allows the user to specify whether species sampling in the phylogeny being analyzed is random or nonrandom. The completed trees can be used in subsequent model-fitting analyses. This is fundamentally different from previous diversification rate estimation methods, which were based on null distributions derived from the incomplete trees. CorSiM is automated in an R package and can easily be applied to large data sets. We illustrate the approach in two Araceae clades, one with a random species sampling of 52% and one with a nonrandom sampling of 55%. In the latter clade, the CorSiM approach detects and quantifies an increase in diversification rate, whereas classic approaches prefer a constant rate model; in the former clade, results do not differ among methods (as indeed expected since the classic approaches are valid only for randomly sampled phylogenies). The CorSiM method greatly reduces the type I error in diversification analysis, but type II error remains a methodological problem.     

EXTINCTION RATES SHOULD NOT BE ESTIMATED FROM MOLECULAR PHYLOGENIES

Molecular phylogenies contain information about the tempo and mode of species diversification through time. Because extinction leaves a characteristic signature in the shape of molecular phylogenetic trees, many studies have used data from extant taxa only to infer extinction rates. This is a promising approach for the large number of taxa for which extinction rates cannot be estimated from the fossil record. Here, I explore the consequences of violating a common assumption made by studies of extinction from phylogenetic data. I show that when diversification rates vary among lineages, simple estimators based on the birth–death process are unable to recover true extinction rates. This is problematic for phylogenetic trees with complete taxon sampling as well as for the simpler case of clades with known age and species richness. Given the ubiquity of variation in diversification rates among lineages and clades, these results suggest that extinction rates should not be estimated in the absence of fossil data.     

COSPECIATION ANALYSIS OF AN OBLIGATE POLLINATION MUTUALISM: HAVEGLOCHIDION TREES (EUPHORBIACEAE) AND POLLINATING EPICEPHALA MOTHS(GRACILLARIIDAE) DIVERSIFIED IN PARALLEL?

Species-specific obligate pollination mutualism between Glochidion trees (Euphorbiaceae) and Epicephala moths (Gracillariidae) involves a large number of interacting species and resembles the classically known fig-fig wasp and yucca-yucca moth associations. To assess the extent of parallel cladogenesis in Glochidion-Epicephala association, we reconstruct phylogenetic relationships of 18 species of Glochidion using nuclear ribosomal DNA sequences (internal and external transcribed spacers) and those of the corresponding 18 Epicephala species using mitochondrial (the cytochrome oxidase subunit I gene) and nuclear DNA sequences (the arginine kinase and elongation factor-1alpha genes). Based on the obtained phylogenies, we determine whether Glochidion and Epicephala have undergone parallel diversification using several different methods for investigating the level of cospeciation between phylogenies. These tests indicate that there is generally a greater degree of correlation between Glochidion and Epicephala phylogenies than expected in a random association, but the results are sensitive to selection of different phylogenetic hypotheses and analytical methods for evaluating cospeciation. Perfect congruence between phylogenies is not found in this association, which likely resulted from host shift by the moths. The observed significant discrepancy between Glochidion and Epicephala phylogenies implies that the one-to-one specificity between the plants and moths has been maintained through a complex speciation process or that there is an underestimated diversity of association between Glochidion trees and Epicephala moths.     

Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.     

The evolution of generalization? Parasitoid flies and the perils of inferring host range evolution from phylogenies

It is widely assumed that high resource specificity predisposes lineages toward greater likelihood of extinction and lower likelihood of diversification than more generalized lineages. This suggests that host range evolution in parasitic organisms should proceed from generalist to specialist, and specialist lineages should be found at the 'tips' of phylogenies. To test these hypotheses, parsimony and maximum likelihood methods were used to reconstruct the evolution of host range on a phylogeny of parasitoid flies in the family Tachinidae. In contrast to predictions, most reconstructions indicated that generalists were repeatedly derived from specialist lineages and tended to occupy terminal branches of the phylogeny. These results are critically examined with respect to hypotheses concerning the evolution of specialization, the inherent difficulties in inferring host ranges, our knowledge of tachinid-host associations, and the methodological problems associated with ancestral character state reconstruction. Both parsimony and likelihood reconstructions are shown to provide misleading results and it is argued that independent evidence, in addition to phylogenetic trees, is needed to inform models of the evolution of host range and the evolutionary consequences of specialization.     

SYNDROME‐DRIVEN DIVERSIFICATION IN A MEDITERRANEAN ECOSYSTEM

Phylogenetic methods to detect lineage diversification have been traditionally used within a particular taxonomic clade, but rarely applied to detect local diversification. For understanding in situ diversification triggered by novel conditions it is necessary to focus on the time slice where such conditions occur. These new conditions may differentially affect the diversification rate of lineages with different morpho‐functional syndromes. A prominent example of these processes occurs in the Mediterranean Basin, where climate arising along the Tertiary/Quaternary transition acted as an environmental filter. In this context, lineages with different syndromes (sclerophyllous and nonsclerophyllous) are hypothesized to have different local diversification rates after the rise of the Mediterranean conditions. We used macroevolutionary methods of time‐dependent diversification on a calibrated local phylogeny accommodating topological and chronological uncertainty to test syndrome‐driven diversification in Mediterranean shrublands from the eastern Iberian Peninsula. We found phylogenetic evidence of higher speciation associated with the nonsclerophyllous syndrome, although extinction rates were similar between syndromes. Consequently a syndrome‐driven local diversification has occurred in shrublands under Mediterranean conditions. The results provide an example of how the integration of the environmental filter in a dated phylogeny may recreate the local history of lineages and help to explain assembly processes in Mediterranean ecosystems.     

Mega-phylogeny approach for comparative biology: an alternative to supertree and supermatrix approaches

Background  

Biology has increasingly recognized the necessity to build and utilize larger phylogenies to address broad evolutionary questions. Large phylogenies have facilitated the discovery of differential rates of molecular evolution between trees and herbs. They have helped us understand the diversification patterns of mammals as well as the patterns of seed evolution. In addition to these broad evolutionary questions there is increasing awareness of the importance of large phylogenies for addressing conservation issues such as biodiversity hotspots and response to global change. Two major classes of methods have been employed to accomplish the large tree-building task: supertrees and supermatrices. Although these methods are continually being developed, they have yet to be made fully accessible to comparative biologists making extremely large trees rare.     

Understanding angiosperm diversification using small and large phylogenetic trees

How will the emerging possibility of inferring ultra-large phylogenies influence our ability to identify shifts in diversification rate? For several large angiosperm clades (Angiospermae, Monocotyledonae, Orchidaceae, Poaceae, Eudicotyledonae, Fabaceae, and Asteraceae), we explore this issue by contrasting two approaches: (1) using small backbone trees with an inferred number of extant species assigned to each terminal clade and (2) using a mega-phylogeny of 55473 seed plant species represented in GenBank. The mega-phylogeny approach assumes that the sample of species in GenBank is at least roughly proportional to the actual species diversity of different lineages, as appears to be the case for many major angiosperm lineages. Using both approaches, we found that diversification rate shifts are not directly associated with the major named clades examined here, with the sole exception of Fabaceae in the GenBank mega-phylogeny. These agreements are encouraging and may support a generality about angiosperm evolution: major shifts in diversification may not be directly associated with major named clades, but rather with clades that are nested not far within these groups. An alternative explanation is that there have been increased extinction rates in early-diverging lineages within these clades. Based on our mega-phylogeny, the shifts in diversification appear to be distributed quite evenly throughout the angiosperms. Mega-phylogenetic studies of diversification hold great promise for revealing new patterns, but we will need to focus more attention on properly specifying null expectation.     

Habitat use affects morphological diversification in dragon lizards

Habitat use may lead to variation in diversity among evolutionary lineages because habitats differ in the variety of ways they allow for species to make a living. Here, we show that structural habitats contribute to differential diversification of limb and body form in dragon lizards (Agamidae). Based on phylogenetic analysis and ancestral state reconstructions for 90 species, we find that multiple lineages have independently adopted each of four habitat use types: rock‐dwelling, terrestriality, semi‐arboreality and arboreality. Given these reconstructions, we fit models of evolution to species’ morphological trait values and find that rock‐dwelling and arboreality limit diversification relative to terrestriality and semi‐arboreality. Models preferred by Akaike information criterion infer slower rates of size and shape evolution in lineages inferred to occupy rocks and trees, and model‐averaged rate estimates are slowest for these habitat types. These results suggest that ground‐dwelling facilitates ecomorphological differentiation and that use of trees or rocks impedes diversification.     

Model‐based approach to test hard polytomies in the Eulaemus clade of the most diverse South American lizard genus Liolaemus (Liolaemini,Squamata)

Lack of resolution in a phylogenetic tree is usually represented as a polytomy, and often adding more data (loci and taxa) resolves the species tree. These are the ‘soft’ polytomies, but in other cases additional data fail to resolve relationships; these are the ‘hard’ polytomies. This latter case is often interpreted as a simultaneous radiation of lineages in the history of a clade. Although hard polytomies are difficult to address, model‐based approaches provide new tools to test these hypotheses. Here, we used a clade of 144 species of the South American lizard clade Eulaemus to estimate phylogenies using a traditional concatenated matrix and three species tree methods: *BEAST, BEST, and minimizing deep coalescences (MDC). The different species tree methods recovered largely discordant results, but all resolved the same polytomy (e.g. very short internodes amongst lineages and low nodal support in Bayesian methods). We simulated data sets under eight explicit evolutionary models (including hard polytomies), tested these against empirical data (a total of 14 loci), and found support for two polytomies as the most plausible hypothesis for diversification of this clade. We discuss the performance of these methods and their limitations under the challenging scenario of hard polytomies. © 2015 The Linnean Society of London     

Molecular phylogeny and biogeography of Linanthus (Polemoniaceae)

To better understand the evolutionary history of Linanthus (Polemoniaceae) and its relatives, molecular phylogenies based on DNA sequence data from the internal transcribed spacer (ITS) region of nrDNA and the chloroplast gene matK were estimated using several methods. Our data suggest two separate and well-supported lineages of Linanthus in close association with two other genera-Leptodactylon and Phlox. These results agree with previous molecular systematic work on the Polemoniaceae, but do not support the traditional classification of the genus as a natural group, nor do they support the sectional classification within the genus. With a distribution centered primarily in western North America and a high degree of endemism in the California Floristic Province, it has been suggested by Raven and Axelrod that the origin and diversification of Linanthus and its relatives were tied to the development of a summer-dry climate in western North America, which began around 13-15 million years ago (mya). Increased drying during the Pliocene (1.2-5 mya) has also been hypothesized by Axelrod to have led to an increase in plant speciation in California and adjacent areas. Divergence times within the Linanthus lineages were estimated from the ITS and matK gene trees. A log-likelihood ratio test could not reject clock-like evolution for the matK data; however, the clock was strongly rejected for the ITS data set. Although ITS molecular evolution was not clock-like, the estimated times of divergence were similar to those of the matK data set. Within both lineages of Linanthus there seems to have been considerable diversification that has occurred since the Pliocene.     

Phylogenetic estimates of diversification rate are affected by molecular rate variation

Molecular phylogenies are increasingly being used to investigate the patterns and mechanisms of macroevolution. In particular, node heights in a phylogeny can be used to detect changes in rates of diversification over time. Such analyses rest on the assumption that node heights in a phylogeny represent the timing of diversification events, which in turn rests on the assumption that evolutionary time can be accurately predicted from DNA sequence divergence. But there are many influences on the rate of molecular evolution, which might also influence node heights in molecular phylogenies, and thus affect estimates of diversification rate. In particular, a growing number of studies have revealed an association between the net diversification rate estimated from phylogenies and the rate of molecular evolution. Such an association might, by influencing the relative position of node heights, systematically bias estimates of diversification time. We simulated the evolution of DNA sequences under several scenarios where rates of diversification and molecular evolution vary through time, including models where diversification and molecular evolutionary rates are linked. We show that commonly used methods, including metric‐based, likelihood and Bayesian approaches, can have a low power to identify changes in diversification rate when molecular substitution rates vary. Furthermore, the association between the rates of speciation and molecular evolution rate can cause the signature of a slowdown or speedup in speciation rates to be lost or misidentified. These results suggest that the multiple sources of variation in molecular evolutionary rates need to be considered when inferring macroevolutionary processes from phylogenies.     

From Messinian crisis to Mediterranean climate: A temporal gap of diversification recovered from multiple plant phylogenies

Paleobotanical and molecular studies link diversification of plants in the Mediterranean Basin with the onset of the Mediterranean climate. Screening diversification before this period is needed in order to analyze whether the observed increase in diversification is a legitimate footprint denoting radiation or instead the biological signal of a previous mass extinction or rate stasis period. A shared post-Messinian temporal gap of cladogenesis has been previously observed in two Mediterranean sister genera. Based on this evidence we explored recently published molecular studies to recover lineages with similar diversification profiles exhibiting a cladogenesis gap. Using this criterion, we conducted a meta-analysis of 36 Mediterranean plant lineages with a post-Messinian temporal gap of cladogenesis, including a new molecular dating of Genista (Fabaceae). Whereas 39% of these lineages have not diversified since the Miocene, another 39% began to rediversify during the onset of the Mediterranean climate and the remaining 22% began diversifying again afterwards during the Quaternary. The pattern of Mediterranean diversification recovery after a temporal gap of cladogenesis was also obtained with phylogenetic tree simulations under birth and death processes when forcing one or two temporal shifts in diversification rates. The relative importance of the Mediterranean onset as a driving force promoting speciation or triggering extinction remains as an open question, since neither the mass extinction nor the rate stasis evolutionary scenarios can be rule out. The independent analysis of individual clades within phylogenies is also essential to detect clade-dependent patterns hidden by phylogeny-level ones. We disclose the importance of analyzing diversification patterns of Mediterranean lineages since the Miocene to understand the recent history of the Mediterranean biota.     

Correlated evolution in fig pollination

This is the first comparative study of correlated evolution between figs (Ficus species, Moraceae) and their pollinators (Hymenoptera: Agaoninae) based on molecular phylogenies of both lineages. Fig relationships based on the internal transcribed spacer region (ITS) of nuclear ribosomal DNA and pollinator relationships inferred from mitochondrial cytochrome oxidase I (COI) sequences enabled the study of correlated evolution based on molecular phylogenies for the largest set of interacting species ever compared. Comparative methods have been applied to tests of adaptation, but the application of these methods in tests of coadaptation, defined as reciprocal evolutionary change in interacting lineages, has received less attention. I have extended tests of correlated evolution between two traits along a phylogeny to the case of interacting lineages, where two traits may or may not share a common phylogenetic history. Independent contrasts and phylogenetic autocorrelation rejected the null hypothesis that trait correlations within lineages are stronger than trait correlations between interacting lineages. Fig style lengths and pollinator ovipositor lengths, for example, were more highly correlated than were pollinator body size and ovipositor length. Mutualistic interactions between figs and their pollinators illustrate the novel ways in which phylogenies and comparative methods can detect patterns of correlated evolution. The most outstanding evidence of correlated evolution between these obligate mutualists is that interacting trait correlations are stronger than within-lineage allometric relationships.     

The effect of paralogous lineages on the application of reconciliation analysis by cophylogeny mapping

Paralogy defines similarity caused by duplication rather than common descent and is well known in the case of paralogous gene copies within a single genome. The term is here extended to paralogous lineages of associates within a single host. The phylogenies of four genera within the Herpesviridae were reconciled with host phylogenies using cophylogenetic mapping. The observed correspondence for each pair of phylogenies was evaluated through randomization of the viral phylogeny and demonstrated to be greater than expected by chance. A simulation study was then carried out to assess the influence of paralogous lineages on the efficacy of reconciliation analysis. Combining viral taxa from different genera that infected common hosts introduced incongruence into the cophylogenies and reduced both the minimum and maximum observed number of codivergence events relative to the initial analysis of orthologous clades. However, at an average sample size this did not alter the fundamental significance of observed correspondence. With smaller sample sizes, the number of orthologous taxa selected at random from the pool of taxa was reduced. False-negative results then increased in proportion from 0.02 to 0.33. These results demonstrated that reconciliation analysis is robust under conditions of paralogy at "normal" sample sizes but is adversely affected by a combination of paralogy and low sample size. Consideration of phylogenies for Papillomavirus, Atadenovirus, and Mastadenovirus suggest that paralogous lineages may be a widespread phenomenon among DNA viruses and that duplication irrespective of host speciation is an important cause of viral diversification.     

The molecular phylogenetic signature of clades in decline

Molecular phylogenies have been used to study the diversification of many clades. However, current methods for inferring diversification dynamics from molecular phylogenies ignore the possibility that clades may be decreasing in diversity, despite the fact that the fossil record shows this to be the case for many groups. Here we investigate the molecular phylogenetic signature of decreasing diversity using the most widely used statistic for inferring diversity dynamics from molecular phylogenies, the γ statistic. We show that if a clade is in decline its molecular phylogeny may show evidence of the decrease in the diversification rate that occurred between its diversification and decline phases. The ability to detect the change in diversification rate depends largely on the ratio of the speciation rates of the diversification and decline phases, the higher the ratio the stronger the signal of the change in diversification rate. Consequently, molecular phylogenies of clades in relative rapid decline do not carry a signature of their decreasing diversification. Further, the signal of the change in diversification rate, if present, declines as the diversity drop. Unfortunately, the molecular signature of clades in decline is the same as the signature produced by diversity dependent diversification. Given this similarity, and the inability of current methods to detect declining diversity, it is likely that some of the extant clades that show a decrease in diversification rate, currently interpreted as evidence for diversity dependent diversification, are in fact in decline. Unless methods can be developed that can discriminate between the different modes of diversification, specifically diversity dependent diversification and declining diversity, we will need the fossil record, or data from some other source, to distinguish between these very different diversity trajectories.     

DOES IMBALANCE IN PHYLOGENIES REFLECT ONLY BIAS?

Phylogenetic tree imbalance was originally believed to indicate differences in evolutionary rates within trees, but other sources of imbalance have been identified, such as tree incompleteness and low quality of the data. To examine the effect of data quality, I calculated Colless's index for 69 recent complete phylogenies. On average, these phylogenies were more unbalanced than phylogenies generated by the equal rates Markov (ERM) model. I tried Mooers's (1995) method to correct for tree size, but his measure appeared to become dependent on tree size when there are large trees (i.e., > 14 tips) in a collection. Instead I corrected for tree size by taking the difference between Colless's index of observed trees and the ERM model expectation for a tree of the same size. The balance measure thus obtained did not correlate significantly to consistency and retention indices as indicators of data quality. It was also independent of the factors kingdom (plants and animals) and taxon level at the tips and type of data (molecular, morphological, and combined).     

Prolonging the past counteracts the pull of the present: protracted speciation can explain observed slowdowns in diversification

Phylogenetic trees show a remarkable slowdown in the increase of number of lineages towards the present, a phenomenon which cannot be explained by the standard birth-death model of diversification with constant speciation and extinction rates. The birth-death model instead predicts a constant or accelerating increase in the number of lineages, which has been called the pull of the present. The observed slowdown has been attributed to nonconstancy of the speciation and extinction rates due to some form of diversity dependence (i.e., species-level density dependence), but the mechanisms underlying this are still unclear. Here, we propose an alternative explanation based on the simple concept that speciation takes time to complete. We show that this idea of "protracted" speciation can be incorporated in the standard birth-death model of diversification. The protracted birth-death model predicts a realistic slowdown in the rate of increase of number of lineages in the phylogeny and provides a compelling fit to four bird phylogenies with realistic parameter values. Thus, the effect of recognizing the generally accepted fact that speciation is not an instantaneous event is significant; even if it cannot account for all the observed patterns, it certainly contributes substantially and should therefore be incorporated into future studies.     

Molecular systematics,biogeography and taxonomy of forest‐falcons in the Micrastur ruficollis species complex (Aves: Falconidae)

We gathered molecular data to assess phylogenetic and phylogeographic patterns for widespread lineages of Neotropical forest falcons in the genus Micrastur to: 1) investigate the comparative phylogeography of four species from the M. ruficollis complex (M. ruficollis, M. gilvicollis, M. plumbeus and M. mintoni), to identify the temporal and spatial context of the group's diversification; and 2) to reevaluate, based on molecular characters, the taxonomic status and interspecific boundaries within this complex. Molecular phylogenies were based on sequences of the mitochondrial genes ND2 and Cyt b and the nuclear genes FIB5 and MUSK from 119 specimens, including M. mirandollei and M. semitorquatus as outgroups. The phylogenetic trees obtained by BI and a Species Tree analysis recovered the monophyly of currently accepted species belonging to the M. ruficollis complex. The dates in our tree indicate that the separation of species within the complex occurred 2–4 million yr ago, initiating during the Neogene (Pliocene). However, when compared to most such widely distributed Neotropical lineages, the diversification within the M. ruficollis complex appears more recent (i.e. centered in the Late Pleistocene). Our results demonstrate the existence of eleven geographic lineages (subclades) in M. ruficollis, M. gilvicollis and M. mintoni, which differ genetically from each other and therefore can be interpreted as distinct evolutionary lineages and possibly separate species under lineage‐based species concepts. However, BPP results failed to recognize with strong statistical support any of these subclades as distinct species. Distinct subclades in the M. ruficollis complex are limited by the principal tributaries of the Amazon River and the Andes, suggesting that these modern barriers limit gene flow and thereby could have promoted differentiation mostly during the Pleistocene. However, our results indicate widely disparate responses to individual barriers across subclades, supporting lineage‐specific histories throughout the Neotropics.