Shallow Nodes Research Articles

Cytoskeletal architecture and its evolutionary significance in amoeboid eukaryotes and their mode of locomotion.

The cytoskeleton is the hallmark of eukaryotic evolution. The molecular and architectural aspects of the cytoskeleton have been playing a prominent role in our understanding of the origin and evolution of eukaryotes. In this study, we seek to investigate the cytoskeleton architecture and its evolutionary significance in understudied amoeboid lineages belonging to Amoebozoa. These amoebae primarily use cytoplasmic extensions supported by the cytoskeleton to perform important cellular processes such as movement and feeding. Amoeboid structure has important taxonomic significance, but, owing to techniques used, its potential significance in understanding diversity of the group has been seriously compromised, leading to an under-appreciation of its value. Here, we used immunocytochemistry and confocal microscopy to study the architecture of microtubules (MTs) and F-actin in diverse groups of amoebae. Our results demonstrate that all Amoebozoa examined are characterized by a complex cytoskeletal array, unlike what has been previously thought to exist. Our results not only conclusively demonstrate that all amoebozoans possess complex cytoplasmic MTs, but also provide, for the first time, a potential synapomorphy for the molecularly defined Amoebozoa clade. Based on this evidence, the last common ancestor of amoebozoans is hypothesized to have had a complex interwoven MT architecture limited within the granular cell body. We also generate several cytoskeleton characters related to MT and F-actin, which are found to be robust for defining groups in deep and shallow nodes of Amoebozoa.

The EMSO-ERIC Pan-European Consortium: Data Benefits and Lessons Learned as the Legal Entity Forms

AbstractThe European Multidisciplinary Seafloor and water-column Observatory (EMSO) European Research Infrastructure Consortium (ERIC) provides power, communications, sensors, and data infrastructure for continuous, high-resolution, (near-)real-time, interactive ocean observations across a multidisciplinary and interdisciplinary range of research areas including biology, geology, chemistry, physics, engineering, and computer science, from polar to subtropical environments, through the water column down to the abyss. Eleven deep-sea and four shallow nodes span from the Arctic through the Atlantic and Mediterranean, to the Black Sea. Coordination among the consortium nodes is being strengthened through the EMSOdev project (H2020), which will produce the EMSO Generic Instrument Module (EGIM). Early installations are now being upgraded, for example, at the Ligurian, Ionian, Azores, and Porcupine Abyssal Plain (PAP) nodes. Significant findings have been flowing in over the years; for example, high-frequency surface and subsurface water-column measurements of the PAP node show an increase in seawater pCO2 (from 339 μatm in 2003 to 353 μatm in 2011) with little variability in the mean air-sea CO2 flux. In the Central Eastern Atlantic, the Oceanic Platform of the Canary Islands open-ocean canary node (aka ESTOC station) has a long-standing time series on water column physical, biogeochemical, and acidification processes that have contributed to the assessment efforts of the Intergovernmental Panel on Climate Change (IPCC). EMSO not only brings together countries and disciplines but also allows the pooling of resources and coordination to assemble harmonized data into a comprehensive regional ocean picture, which will then be made available to researchers and stakeholders worldwide on an open and interoperable access basis.

DBR-MAC: A Depth-Based Routing Aware MAC Protocol for Data Collection in Underwater Acoustic Sensor Networks

Due to long propagation delays, routing discovery is very expensive in underwater acoustic sensor networks. Therefore, depth-based routing (DBR) protocols are usually preferred whose routing discovery cost is almost zero. However, DBR protocols working with broadcast medium access control (MAC) bring severe collisions especially in the data collection network. Moreover, in DBR, all the packets are forwarded from deep to shallow nodes along the direction toward the sink node. The directional packet transmissions may lead to load imbalance, where some nodes undertake most loads and we call these nodes key nodes. However, the majority of existing MAC protocols cannot provide the key nodes priority of accessing channel in the collision domain. It is necessary to design a MAC protocol coupling with DBR. In this paper, we adopt a cross-layer approach to propose a DBR aware MAC protocol called DBR-MAC, which smoothly integrates a handshaking-based MAC and a DBR protocol. It improves the throughput, energy, and time efficiency at the cost of fairness. A depth-based transmission scheduling scheme is introduced based on the depth information, angle information, and overheard one-hop neighboring nodes' transmissions to make key nodes have higher priority to access the channel than other nodes. DBR-MAC is a cross-layer scheme and its directional forwarding tries best to forward packets from source to the floating sink node by the least hops. Moreover, an adaptive depth-based backoff algorithm cuts down the backoff time for the nodes and prevents the congestions at key nodes. Extensive simulations show that the throughput, energy, and time efficiency of DBR-MAC outperform existing MAC protocols when applied in the data collection network.

Evolution of the assassin's arms: insights from a phylogeny of combined transcriptomic and ribosomal DNA data (Heteroptera: Reduvioidea).

Assassin bugs (Reduvioidea) are one of the most diverse (>7,000 spp.) lineages of predatory animals and have evolved an astounding diversity of raptorial leg modifications for handling prey. The evolution of these modifications is not well understood due to the lack of a robust phylogeny, especially at deeper nodes. We here utilize refined data from transcriptomes (370 loci) to stabilize the backbone phylogeny of Reduvioidea, revealing the position of major clades (e.g., the Chagas disease vectors Triatominae). Analyses combining transcriptomic and Sanger-sequencing datasets result in the first well-resolved phylogeny of Reduvioidea. Despite amounts of missing data, the transcriptomic loci resolve deeper nodes while the targeted ribosomal genes anchor taxa at shallower nodes, both with high support. This phylogeny reveals patterns of raptorial leg evolution across major leg types. Hairy attachment structures (fossula spongiosa), present in the ancestor of Reduvioidea, were lost multiple times within the clade. In contrast to prior hypotheses, this loss is not directly correlated with the evolution of alternative raptorial leg types. Our results suggest that prey type, predatory behavior, salivary toxicity, and morphological adaptations pose intricate and interrelated factors influencing the evolution of this diverse group of predators.

Consequences of Secondary Calibrations on Divergence Time Estimates.

Secondary calibrations (calibrations based on the results of previous molecular dating studies) are commonly applied in divergence time analyses in groups that lack fossil data; however, the consequences of applying secondary calibrations in a relaxed-clock approach are not fully understood. I tested whether applying the posterior estimate from a primary study as a prior distribution in a secondary study results in consistent age and uncertainty estimates. I compared age estimates from simulations with 100 randomly replicated secondary trees. On average, the 95% credible intervals of node ages for secondary estimates were significantly younger and narrower than primary estimates. The primary and secondary age estimates were significantly different in 97% of the replicates after Bonferroni corrections. Greater error in magnitude was associated with deeper than shallower nodes, but the opposite was found when standardized by median node age, and a significant positive relationship was determined between the number of tips/age of secondary trees and the total amount of error. When two secondary calibrated nodes were analyzed, estimates remained significantly different, and although the minimum and median estimates were associated with less error, maximum age estimates and credible interval widths had greater error. The shape of the prior also influenced error, in which applying a normal, rather than uniform, prior distribution resulted in greater error. Secondary calibrations, in summary, lead to a false impression of precision and the distribution of age estimates shift away from those that would be inferred by the primary analysis. These results suggest that secondary calibrations should not be applied as the only source of calibration in divergence time analyses that test time-dependent hypotheses until the additional error associated with secondary calibrations is more properly modeled to take into account increased uncertainty in age estimates.

Avian Diversification Patterns across the K-Pg Boundary: Influence of Calibrations, Datasets, and Model Misspecification1

Birds represent the most diverse extant tetrapod clade, with ca. 10,000 extant species, and the timing of the crown avian radiation remains hotly debated. The fossil record supports a primarily Cenozoic radiation of crown birds, whereas molecular divergence dating analyses generally imply that this radiation was well underway during the Cretaceous. Furthermore, substantial differences have been noted between published divergence estimates. These have been variously attributed to clock model, calibration regime, and gene type. One underappreciated phenomenon is that disparity between fossil ages and molecular dates tends to be proportionally greater for shallower nodes in the avian Tree of Life. Here, we explore potential drivers of disparity in avian divergence dates through a set of analyses applying various calibration strategies and coding methods to a mitochondrial genome dataset and an 18-gene nuclear dataset, both sampled across 72 taxa. Our analyses support the occurrence of two deep divergences (i.e., the Palaeognathae/Neognathae split and the Galloanserae/Neoaves split) well within the Cretaceous, followed by a rapid radiation of Neoaves near the K-Pg boundary. However, 95% highest posterior density intervals for most basal divergences in Neoaves cross the boundary, and we emphasize that, barring unreasonably strict prior distributions, distinguishing between a rapid Early Paleocene radiation and a Late Cretaceous radiation may be beyond the resolving power of currently favored divergence dating methods. In contrast to recent observations for placental mammals, constraining all divergences within Neoaves to occur in the Cenozoic does not result in unreasonably high inferred substitution rates. Comparisons of nuclear DNA (nDNA) versus mitochondrial DNA (mtDNA) datasets and NT- versus RY-coded mitochondrial data reveal patterns of disparity that are consistent with substitution model misspecifications that result in tree compression/tree extension artifacts, which may explain some discordance between previous divergence estimates based on different sequence types. Comparisons of fully calibrated and nominally calibrated trees support a correlation between body mass and apparent dating error. Overall, our results are consistent with (but do not require) a Paleogene radiation for most major clades of crown birds.

Potential for bias and low precision in molecular divergence time estimation of the Canopy of Life: an example from aquatic bird families.

Uncertainty in divergence time estimation is frequently studied from many angles but rarely from the perspective of phylogenetic node age. If appropriate molecular models and fossil priors are used, a multi-locus, partitioned analysis is expected to equally minimize error in accuracy and precision across all nodes of a given phylogeny. In contrast, if available models fail to completely account for rate heterogeneity, substitution saturation and incompleteness of the fossil record, uncertainty in divergence time estimation may increase with node age. While many studies have stressed this concern with regard to deep nodes in the Tree of Life, the inference that molecular divergence time estimation of shallow nodes is less sensitive to erroneous model choice has not been tested explicitly in a Bayesian framework. Because of available divergence time estimation methods that permit fossil priors across any phylogenetic node and the present increase in efficient, cheap collection of species-level genomic data, insight is needed into the performance of divergence time estimation of shallow (<10 MY) nodes. Here, we performed multiple sensitivity analyses in a multi-locus data set of aquatic birds with six fossil constraints. Comparison across divergence time analyses that varied taxon and locus sampling, number and position of fossil constraint and shape of prior distribution showed various insights. Deviation from node ages obtained from a reference analysis was generally highest for the shallowest nodes but determined more by temporal placement than number of fossil constraints. Calibration with only the shallowest nodes significantly underestimated the aquatic bird fossil record, indicating the presence of saturation. Although joint calibration with all six priors yielded ages most consistent with the fossil record, ages of shallow nodes were overestimated. This bias was found in both mtDNA and nDNA regions. Thus, divergence time estimation of shallow nodes may suffer from bias and low precision, even when appropriate fossil priors and best available substitution models are chosen. Much care must be taken to address the possible ramifications of substitution saturation across the entire Tree of Life.

Species limits and relationships within Otidea inferred from multiple gene phylogenies.

The genus Otidea is one of the more conspicuous members of the Pyronemataceae, with high species diversity in hemiboreal and boreal forests. The genus is morphologically coherent and in previous higher-level multi-gene analyses it formed a highly supported monophyletic group. Species delimitation within Otidea is controversial and much confusion has prevailed in the naming of taxa. To provide a phylogenetic hypothesis of Otidea, elucidate species diversity and limits we compiled a four-gene dataset including the nuclear LSU rDNA and three nuclear protein-coding genes (RPB1, RPB2 and EF-1α) for 89 specimens (total 4 877 nucleotides). These were selected from a larger sample of material studied using morphology and 146 ITS (ITS1-5.8S-ITS2) and 168 LSU rDNA sequences to represent the full genetic diversity. Using genealogical concordance phylogenetic species recognition (GCPSR), Bayesian and maximum likelihood analyses of the individual datasets resolved 25 species of Otidea. An additional eight singletons are considered to be distinct species, because they were genetically divergent from their sisters. Sequences of multiple genes were included from 13 holotypes, one neotype and three epitypes. Otidea angusta, O. myosotis and O. papillata f. pallidefurfuracea are nested within O. nannfeldtii, O. leporina and O. tuomikoskii, respectively and are considered synonyms. Otidea cantharella var. minor is shown to be a distinct species. Five new species were discovered: O. oregonensis and O. pseudoleporina for North America; and O. borealis, O. brunneoparva and O. subformicarum for Europe. The analyses of the individual four gene datasets yielded phylogenies that were highly concordant topologically, except for the RPB1 that showed supported conflict for some nodes in Bayesian analysis. Excluding the RPB1 from the combined analyses produced an identical topology to the four-gene phylogeny, but with higher support for several basal nodes and lower support for several shallow nodes. We argue to use the three-gene dataset to retrieve the maximum support for the higher-level relationships in Otidea, but still utilise the signal from the RPB1 for the delimitation and relationships of closely related species. From the four gene regions utilised, EF-1α and RPB1 have the strongest species recognition power, and with higher amplification success EF-1α may serve as the best secondary barcoding locus for Otidea (with ITS being a primary). The phylogeny from the three- and four-gene datasets is fully resolved and strongly supported in all branches but one. Two major clades, as part of six inclusive clades A-F, are identified - and ten subclades within these: A) O. platyspora and O. alutacea subclades, and B) O. papillata, O. leporina, O. tuomikoskii, O. cantharella, O. formicarum, O. unicisa, O. bufonia-onotica and O. concinna subclades. Morphological features in Otidea appear to be fast evolving and prone to shifts, and are poor indicators of higher-level relationships. Nevertheless, a conspicuous spore ornament is a synapomorphy for the O. unicisa subclade (/Otideopsis); all other species in Otidea have smooth or verruculose (in SEM) spores. Exclusively pale to bright yellow apothecia and straight to curved, broadly clavate to distinctly capitate paraphyses are synapomorphies for a restricted O. concinna subclade (/Flavoscypha). The curved to hooked apices of the paraphyses is suggested to be a symplesiomorphic trait for the genus. The reaction of resinous exudates on the outermost excipular cells that coalesce into amber drops in Melzer's reagent is likely an ancestral state for clade B. We estimate that Otidea consists of 47 species worldwide, based on all available information (including morphology, ITS or LSU sequences, and literature descriptions). Three fifths of the species occur in Europe, with 20 species recognised as endemic. At least 14 species occur in North America and 17 in Asia, with eight and ten species considered endemic to each continent, respectively. Our knowledge about Otidea in Asia is still fragmentary and the diversity likely much higher.

Multiplexed-shotgun-genotyping data resolve phylogeny within a very recently derived insular lineage.

Endemic plants on oceanic islands have long served as model systems for studying patterns and processes of evolution. However, phylogenetic studies of island plants frequently illustrate a decoupling of molecular divergence and ecological/morphological diversity, resulting in phylogenies lacking the resolution required to interpret patterns of evolution in a phylogenetic context. The current study uses the primarily Macaronesian flowering plant genus Tolpis to illustrate the utility of multiplexed shotgun genotyping (MSG) for resolving relationships at relatively deep (among archipelagos) and very shallow (within archipelagos) nodes in this small, yet diverse insular plant lineage that had not been resolved with other molecular markers. Genomic libraries for 27 accessions of Macaronesian Tolpis were generated for genotyping individuals using MSG, a form of reduced-representation sequencing, similar to restriction-site-associated DNA markers (RADseq). The resulting data files were processed using the program pyRAD, which clusters MSG loci within and between samples. Phylogenetic analyses of the aligned data matrix were conducted using RAxML. Analysis of MSG data recovered a highly resolved phylogeny with generally strong support, including the first robust inference of relationships within the highly diverse Canary Island clade of Tolpis. The current study illustrates the utility of MSG data for resolving relationships in lineages that have undergone recent, rapid diversification resulting in extensive ecological and morphological diversity. We suggest that a similar approach may prove generally useful for other rapid plant radiations where resolving phylogeny has been difficult.

Flying rocks and flying clocks: disparity in fossil and molecular dates for birds.

Major disparities are recognized between molecular divergence dates and fossil ages for critical nodes in the Tree of Life, but broad patterns and underlying drivers remain elusive. We harvested 458 molecular age estimates for the stem and crown divergences of 67 avian clades to explore empirical patterns between these alternate sources of temporal information. These divergence estimates were, on average, over twice the age of the oldest fossil in these clades. Mitochondrial studies yielded older ages than nuclear studies for the vast majority of clades. Unexpectedly, disparity between molecular estimates and the fossil record was higher for divergences within major clades (crown divergences) than divergences between major clades (stem divergences). Comparisons of dates from studies classed by analytical methods revealed few significant differences. Because true divergence ages can never be known with certainty, our study does not answer the question of whether fossil gaps or molecular dating error account for a greater proportion of observed disparity. However, empirical patterns observed here suggest systemic overestimates for shallow nodes in existing molecular divergence dates for birds. We discuss underlying biases that may drive these patterns.

Phylotranscriptomics: Saturated Third Codon Positions Radically Influence the Estimation of Trees Based on Next-Gen Data

Recent advancements in molecular sequencing techniques have led to a surge in the number of phylogenetic studies that incorporate large amounts of genetic data. We test the assumption that analyzing large number of genes will lead to improvements in tree resolution and branch support using moths in the superfamily Bombycoidea, a group with some interfamilial relationships that have been difficult to resolve. Specifically, we use a next-gen data set that included 19 taxa and 938 genes (∼1.2M bp) to examine how codon position and saturation might influence resolution and node support among three key families. Maximum likelihood, parsimony, and species tree analysis using gene tree parsimony, on different nucleotide and amino acid data sets, resulted in largely congruent topologies with high bootstrap support compared with prior studies that included fewer loci. However, for a few shallow nodes, nucleotide and amino acid data provided high support for conflicting relationships. The third codon position was saturated and phylogenetic analysis of this position alone supported a completely different, potentially misleading sister group relationship. We used the program RADICAL to assess the number of genes needed to fix some of these difficult nodes. One such node originally needed a total of 850 genes but only required 250 when synonymous signal was removed. Our study shows that, in order to effectively use next-gen data to correctly resolve difficult phylogenetic relationships, it is necessary to assess the effects of synonymous substitutions and third codon positions.

Three modes of heterochrony explain lobule diversity inRadulasubgenusCladoradula(Porellales: Jungermanniopsida), a small lineage of early land plants today

Changes in lobule morphology in Radula subgenus Cladoradula show liverworts have the capacity for dramatic, relatively rapid morphological change by heterochrony. In individuals of R. bipinnata, R. boryana and R. tenax, lobules on secondary and tertiary shoots are progenetic with respect to lobules on primary shoots, in that the slope of the relationship between growth duration and shape does not change. However, in R. campanigera, lobules on secondary and tertiary branches exhibit different slopes from primary branches, but have the same growth duration, a pattern consistent with neoteny. The trajectory of allometric growth is extended or truncated in different species compared with outgroup and ancestral nodes. Changes in duration of lobule growth explain 85% of variation in lobule shape between species. Species are related by relatively shallow nodes in the crown of the Radula subgenus Cladoradula clade, suggesting that divergence and associated heterochronic changes have occurred relatively recently. The rapid morphological diversification in the crown contrasts with the relative stasis between the ancestral node and R. brunnea, the outgroup used in this analysis. A robust primary axis may be required to hold shoots away from vertical surfaces to maximize light interception, and hypermorphosis in lobule ontogeny could be a by-product of the longer growth durations required to build axes sufficiently large to perform this structural role. Alternatively, the large auriculate lobules could function in external water transport systems by providing continuity of surfaces for solute transport via capillary action. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 153–175.

Exploring phylogenetic informativeness and nuclear copies of mitochondrial DNA (numts) in three commonly used mitochondrial genes: mitochondrial phylogeny of peppermint, cleaner, and semi-terrestrial shrimps (Caridea:Lysmata,Exhippolysmata, andMerguia)

Of paramount importance to studies that profit from molecular trees is the accuracy and robustness of the reconstructed phylogenies. Causes of systematic error that can mislead phylogenetic methods include nuclear copies of mitochondrial DNA (numts) and low phylogenetic informativeness (PI). Herein, numts and PI were explored in three mitochondrial genes commonly used for phylogenetic reconstruction: 16S, 12S, and cytochrome c oxidase I (COI). Shrimps from the genera Lysmata, Exhippolysmata, and Merguia were used as a model system. The existence of: (1) multiple bands on gels of COI and 12S polymerase chain reaction (PCR) products from various species; (2) double peaks, background noise, and ambiguity in sequence chromatograms of COI and 12S PCR products that produced a single clear band in other species; and (3) indels, stop codons, and considerable composition bias in COI-like cloned sequences of one problematic species (Lysmata seticaudata), was interpreted as evidence of pervasive non-functional nuclear copies of mitochondrial DNA (numts) of the targeted COI (and probably 12S) mtDNA fragment. The information content of the three mtDNA markers studied was investigated using PI profiling, spectral analysis, and neighbour-nets. Marker-specific PI profiles suggested that the COI marker has the highest information content and greatest power for resolving both shallow and deep nodes in trees depicting the phylogenetic relationship among the species studied. Nonetheless, spectral analysis of splits and neighbour-nets suggested that the 16S and 12S markers were equally or even more powerful than the COI marker for resolving nodes at all phylogenetic levels. Altogether, these analyses suggest that all three mtDNA markers are equally useful for resolving phylogenetic relationships in the shrimps studied, and that PI profiling is not necessarily useful to estimate overall gene utility. A ‘total-evidence’ phylogenetic analysis that included 34 species and used a concatenated data set of 1403 characters (from reliable 16S, 12S and COI sequences), demonstrated that the genus Lysmata is paraphyletic, and that the monophyletic clade comprising species of Lysmata and Exhippolysmata can be divided into four well-supported subclades (Neotropical, Cleaner, Cosmopolitan, and Morphovariable).

Climate effects on amphibian distributions depend on phylogenetic resolution and the biogeographical history of taxa

AbstractAimDisentangling the effects of climate and historical factors on biodiversity distribution remains a challenge for biogeographers. Here, we provide an analytical framework to discriminate the contributions of contemporary climate and the biogeographical history of taxa to the geographical distributions of phylogenetic lineages. Furthermore, we evaluate the constraint that the biogeographical history of clades exerts on the association between climate and clade distribution, i.e. the historical legacy of climatic effects. As a case study, we analysed the distributions of amphibian lineages across the Americas.LocationThe Americas.MethodsWe tallied the number of amphibian species per genus in each of 262 ecoregions. Each ecoregion was described by the composition of phylogenetic lineages using phylogenetic fuzzy weighting. The composition of amphibian genera and phylogenetic clades represented the distributions of shallow and deep phylogenetic nodes, respectively. We characterized each ecoregion by the biogeographical history of amphibian taxa and its current climate, whose influences on shallow and deep phylogenetic nodes were analysed using variation partitioning analysis.ResultsThe association between climate and the distributions of deep phylogenetic nodes showed a strong historical legacy, although the distribution of amphibian genera was mostly associated with climate. Hyloidea were associated with a Gondwanan origin and higher annual mean temperatures, whereas other clades (e.g. Caudata) were related to a Laurasian origin and higher temperature seasonality. Microhylidae were related to occurrence in the Early Jurassic in Gondwana and recent occurrence in the Neotropics.Main conclusionsBiogeographical patterns can be thought of as the net outcome of evolutionary, historical and ecological processes. Although temperature is likely to affect the ecology of amphibians, the effects of climate on the distributions of deep phylogenetic nodes were strongly constrained by the biogeographical history of clades. Nevertheless, local, climatically driven processes are likely to influence the distributions of shallow phylogenetic nodes. The historical biogeography of clades might help to explain the interplay between evolutionary and environmental processes in determining assembly patterns found elsewhere.

Efficient Sequencing of Anuran mtDNAs and a Mitogenomic Exploration of the Phylogeny and Evolution of Frogs

Anura (frogs and toads) constitute over 88% of living amphibian diversity but many important questions about their phylogeny and evolution remain unresolved. For this study, we developed an efficient method for sequencing anuran mitochondrial DNAs (mtDNAs) by amplifying the mitochondrial genome in 12 overlapping fragments using frog-specific universal primer sets. Based on this method, we generated 47 nearly complete, new anuran mitochondrial genomes and discovered nine novel gene arrangements. By combining the new data and published anuran mitochondrial genomes, we assembled a large mitogenomic data set (11,007 nt) including 90 frog species, representing 39 of 53 recognized anuran families, to investigate their phylogenetic relationships and evolutionary history. The resulting tree strongly supported a paraphyletic arrangement of archaeobatrachian (=nonneobatrachian) frogs, with Leiopelmatoidea branching first, followed by Discoglossoidea, Pipoidea, and Pelobatoidea. Within Neobatrachia, the South African Heleophrynidae is the sister-taxon to all other neobatrachian frogs and the Seychelles-endemic Sooglossidae is recovered as the sister-taxon to Ranoidea. These phylogenetic relationships agree with many nuclear gene studies. The chronogram derived from two Bayesian relaxed clock methods (MultiDivTime and BEAST) suggests that modern frogs (Anura) originated in the early Triassic about 244 Ma and the appearance of Neobatrachia took place in the late Jurassic about 163 Ma. The initial diversifications of two species-rich superfamilies Hyloidea and Ranoidea commenced 110 and 133 Ma, respectively. These times are older than some other estimates by approximately 30-40 My. Compared with nuclear data, mtDNA produces compatible time estimates for deep nodes (>150 Ma), but apparently older estimates for more shallow nodes. Our study shows that, although it evolves relatively rapidly and behaves much as a single locus, mtDNA performs well for both phylogenetic and divergence time inferences and will provide important reference hypotheses for the phylogeny and evolution of frogs.

Convergent evolution of a complex fruit structure in the tribe Brassiceae (Brassicaceae)

Many angiosperms have fruit morphologies that result in seeds from the same plant having different dispersal capabilities. A prime example is found in the Brassiceae (Brassicaceae), which has many members with segmented or heteroarthrocarpic fruits. Since only 40% of the genera are heteroarthrocarpic, this tribe provides an opportunity to study the evolution of an ecologically significant novelty and its variants. We analyzed nuclear (PHYA) and plastid (matK) sequences from 66 accessions using maximum parsimony, maximum likelihood, and Bayesian inference approaches. The evolution of heteroarthrocarpy and its variants was evaluated using maximum parsimony and maximum likelihood ancestral state reconstructions. Although nuclear and plastid phylogenies are incongruent with each other, the following findings are consistent: (1) Cakile, Crambe, Vella, and Zilla lineages are monophyletic; (2) the Nigra lineage is not monophyletic; and (3) within the Cakile clade, Cakile, Didesmus, and Erucaria are paraphyletic. Despite differences in the matK and PHYA topologies at both deep and shallow nodes, similar patterns of morphological evolution emerge. Heteroarthrocarpy, a complex morphological trait, has evolved multiple times across the tribe. Moreover, there are convergent transitions in dehiscence capabilities and fruit disarticulation across the tribe. We present the first explicit analysis of fruit evolution within the Brassiceae, which exemplifies evolutionary lability. The repeated loss and gain of segment dehiscence and disarticulation suggests conservation in the genetic pathway controlling abscission with differential expression across taxa. This study provides a strong foundation for future studies of mechanisms underlying variation in dispersal capabilities of Brassiceae.

Molecular phylogeny and evolution of symbiosis in a clade of Indopacific nudibranchs

Previous efforts at understanding the evolution of the genus Phyllodesmium, based on morphological analyses, have been plagued by poorly supported phylogenies (Ortiz and Gosliner, 2008; Moore and Gosliner, 2009, in press). It has been suggested (Moore and Gosliner, 2009) that a molecular phylogeny might provide more insight into this history than can be easily discovered using morphological data. In this study, 658bp of the cytochrome c oxidase subunit I gene (CO1), 441bp of the mitochondrial large ribosomal subunit (16S) gene, and 328bp of a protein-coding nuclear gene (histone 3) were sequenced for 18 species of Phyllodesmium and six outgroup species. A total of 464 parsimony informative sites were used for parsimony, maximum likelihood, and Bayesian inference of phylogeny analyses. All three analyses produced similar topologies, with the exception of a single difference within the parsimony analysis. Bootstrap values and posterior probabilities provided strong support at many shallow nodes, and the monophyly of Phyllodesmium was supported in every case. Three distinct clades of Phyllodesmium are evident in this analysis. One of these represents the majority of asymbiotic taxa. Phyllodesmium poindimiei, an asymbiotic species, is clearly a member of a symbiotic clade and appears to have secondarily lost its symbiotic relationship with zooxanthellae. There was moderate support confirming similar topological trends seen in earlier morphological phylogenies, including the hypothesis that symbiotic species associating with zooxanthellae have evolved more recently than non-symbiotic species. Despite the inclusion of a presumably conservative nuclear locus, some deep nodes are still unresolved or are not well supported. Future inclusion of additional taxa and more slowly evolving loci will likely improve resolution of these deeper nodes. The subsequent phylogeny supports previous hypotheses by Rudman (1991), Kempf (1991) and Burghardt et al. (2008b) that evolution of more complex digestive gland structures is related to increased complexity of symbiosis with zooxanthellae and greater efficiency of photosynthetic activity. Our phylogeny also demonstrates that this symbiosis has evolved only once in Phyllodesmium and that azooxanthellate species are sister, rather than basal, to zooxanthellate species.

When molecules and morphology concur: the ‘Gondwanan’ midges (Diptera: Chironomidae)

A phylogeny of the Chironomidae subfamily Podonominae, significant in the history of phylogenetic biogeography, is estimated from an analysis of four genes. Fragments of two ribosomal genes (18S and 28S), one nuclear protein-coding gene (CAD), and one mitochondrial protein-coding gene (COI) were sequenced from specimens representing 13 of 15 genera, and analysed using mixed model Bayesian and maximum likelihood inference methods. Podonominae is monophyletic and sister to Tanypodinae – the shared development of the larval ligula is synapomorphic and diagnostic. Tribe Podonomini is monophyletic with the inclusion of Trichotanypus; tribe Boreochlini is a grade. Monophyly is confirmed for the genera Podonomus Philippi, Podonomopsis Brundin, Podochlus Brundin, Archaeochlus Brundin and Austrochlus Cranston, Edward & Cook: Parochlus Enderlein becomes monophyletic through the inclusion of Zelandochlus Brundin (n.syn.) with its type species, P. latipalpis (Brundin) n.comb. The ‘mandibulate’Archaeochlus plus Austrochlus is monophyletic with nonmandibulate Afrochlus weakly supported as a member of, or sister to, the African Archaeochlus. Subtending this group is Lasiodiamesa, although it associates in some analyses with the sister group Tanypodinae. Generic relationships coincide with those proposed based on morphology, particularly as understood via all life history stages of some problematic (autapomorphic, adult-based) taxa. Divergence time analysis (beast) allows inference of Mesozoic diversification of higher taxa in Podonominae, of appropriate timing for fragmentation of Gondwana, post-African divergence, to have caused vicariance. Shallower nodes (within genera) imply both younger vicariance involving Antarctica and some recent dispersal, including southern to northern hemisphere movement in the New World. New Zealand taxa test controversial biogeographical relationships and show proximity to southern South America without direct Australian sister taxon pairs: dating implies persistence of midges through the ‘Oligocene’ bottleneck.

High mitogenomic evolutionary rates and time dependency

Using entire modern and ancient mitochondrial genomes of Adélie penguins (Pygoscelis adeliae) that are up to 44000 years old, we show that the rates of evolution of the mitochondrial genome are two to six times greater than those estimated from phylogenetic comparisons. Although the rate of evolution at constrained sites, including nonsynonymous positions and RNAs, varies more than twofold with time (between shallow and deep nodes), the rate of evolution at synonymous sites remains the same. The time-independent neutral evolutionary rates reported here would be useful for the study of recent evolutionary events.

Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies

BackgroundThe increase in availability of genomic sequences for a wide range of organisms has revealed gene duplication to be a relatively common event. Encounters with duplicate gene copies have consequently become almost inevitable in the context of collecting gene sequences for inferring species trees. Here we examine the effect of incorporating duplicate gene copies evolving at different rates on tree reconstruction and time estimation of recent and deep divergences in butterflies.ResultsSequences from ultraviolet-sensitive (UVRh), blue-sensitive (BRh), and long-wavelength sensitive (LWRh) opsins,EF-1α and COI were obtained from 27 taxa representing the five major butterfly families (5535 bp total). Both BRh and LWRh are present in multiple copies in some butterfly lineages and the different copies evolve at different rates. Regardless of the phylogenetic reconstruction method used, we found that analyses of combined data sets using either slower or faster evolving copies of duplicate genes resulted in a single topology in agreement with our current understanding of butterfly family relationships based on morphology and molecules. Interestingly, individual analyses of BRh and LWRh sequences also recovered these family-level relationships. Two different relaxed clock methods resulted in similar divergence time estimates at the shallower nodes in the tree, regardless of whether faster or slower evolving copies were used, with larger discrepancies observed at deeper nodes in the phylogeny. The time of divergence between the monarch butterfly Danaus plexippus and the queen D. gilippus (15.3–35.6 Mya) was found to be much older than the time of divergence between monarch co-mimic Limenitis archippus and red-spotted purple L. arthemis (4.7–13.6 Mya), and overlapping with the time of divergence of the co-mimetic passionflower butterflies Heliconius erato and H. melpomene (13.5–26.1 Mya). Our family-level results are congruent with recent estimates found in the literature and indicate an age of 84–113 million years for the divergence of all butterfly families.ConclusionThese results are consistent with diversification of the butterfly families following the radiation of angiosperms and suggest that some classes of opsin genes may be usefully employed for both phylogenetic reconstruction and divergence time estimation.