Relaxed Molecular Clock Model Research Articles

Plastid phylogenomics clarifies broad-level relationships in Bulbophyllum (Orchidaceae) and provides insights into range evolution of Australasian section Adelopetalum

The hyperdiverse orchid genus Bulbophyllum is the second largest genus of flowering plants and exhibits a pantropical distribution with a center of diversity in tropical Asia. The only Bulbophyllum section with a center of diversity in Australasia is sect. Adelopetalum. However, the phylogenetic placement, interspecific relationships, and spatio-temporal evolution of this section remain largely unclear. To infer broad-level relationships within Bulbophyllum, and interspecific relationships within sect. Adelopetalum, a genome skimming dataset was generated for 89 samples, which yielded 70 plastid coding regions and a nuclear ribosomal DNA cistron. For 18 additional samples, Sanger data from two plastid loci (matK and ycf1) and nuclear ITS were added using a supermatrix approach. The study provided new insights into broad-level relationships in Bulbophyllum, including phylogenetic evidence for the non-monophyly of sections Beccariana, Brachyantha, Brachypus, Cirrhopetaloides, Cirrhopetalum, Desmosanthes, Minutissima, Oxysepala, Polymeres, and Sestochilos. Section Adelopetalum and sect. Minutissima s.s. formed a highly supported clade that was resolved as a sister group to the remainder of the genus. Divergence time estimations based on a relaxed molecular clock model placed the origin of Bulbophyllum in the Early Oligocene (ca. 33.2 Ma) and sect. Adelopetalum in the Late Oligocene (ca. 23.6 Ma). Ancestral range estimations based on a BAYAREALIKE model identified the Australian continent as the ancestral area of the sect. Adelopetalum. The section underwent crown diversification from the mid-Miocene to the late Pleistocene, predominantly in continental Australia. At least two independent long-distance dispersal events were inferred eastward from the Australian continent to New Zealand and to New Caledonia from the early Pliocene onwards, likely mediated by predominantly westerly winds of the Southern hemisphere. Retraction and fragmentation of the eastern Australian rainforests from the early Miocene onwards are likely drivers of lineage divergence within sect. Adelopetalum facilitating allopatric speciation.

Phylodynamic analysis of foot-and-mouth disease virus evolution in Mar Chiquita, Argentina.

Foot-and-mouth disease is a highly contagious disease affecting cloven-hoofed animals, resulting in considerable economic losses. Its causal agent is foot-and-mouth disease virus (FMDV), a picornavirus. Due to its error-prone replication and rapid evolution, the transmission and evolutionary dynamics of FMDV can be studied using genomic epidemiological approaches. To analyze FMDV evolution and identify possible transmission routes in an Argentinean region, field samples that tested positive for FMDV by PCR were obtained from 21 farms located in the Mar Chiquita district. Whole FMDV genome sequences were obtained by PCR amplification in seven fragments and sequencing using the Sanger technique. The genome sequences obtained from these samples were then analyzed using phylogenetic, phylogeographic, and evolutionary approaches. Three local transmission clusters were detected among the sampled viruses. The dataset was analyzed using Bayesian phylodynamic methods with appropriate coalescent and relaxed molecular clock models. The estimated mean viral evolutionary rate was 1.17 × 10- 2 substitutions/site/year. No significant differences in the rate of viral evolution were observed between farms with vaccinated animals and those with unvaccinated animals. The most recent common ancestor of the sampled sequences was dated to approximately one month before the first reported case in the outbreak. Virus transmission started in the south of the district and later dispersed to the west, and finally arrived in the east. Different transmission routes among the studied herds, such as non-replicating vectors and close contact contagion (i.e., aerosols), may be responsible for viral spread.

ShinyTempSignal: an R shiny application for exploring temporal and other phylogenetic signals

The molecular clock model is fundamental for inferring species divergence times from molecular sequences. However, its direct application may introduce significant biases due to sequencing errors, recombination events, and inaccurately labeled sampling times. Improving accuracy necessitates rigorous quality control measures to identify and remove potentially erroneous sequences. Furthermore, while not all branches of a phylogenetic tree may exhibit a clear temporal signal, specific branches may still adhere to the assumptions, with varying evolutionary rates. Supporting a relaxed molecular clock model better aligns with the complexities of evolution. The root-to-tip regression method has been widely used to analyze the temporal signal in phylogenetic studies and can be generalized for detecting other phylogenetic signals. Despite its utility, there remains a lack of corresponding software implementations for broader applications. To address this gap, we present shinyTempSignal, an interactive web application implemented with the shiny framework, available as an R package and publicly accessible at https://github.com/YuLab-SMU/shinyTempSignal. This tool facilitates the analysis of temporal and other phylogenetic signals under both strict and relaxed models. By extending the root-to-tip regression method to diverse signals, shinyTempSignal helps in the detection of evolving features or traits, thereby laying the foundation for deeper insights and subsequent analyses.

Phylogeography of hepatitis B virus: The role of Portugal in the early dissemination of HBV worldwide.

In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.

Quantitatively defining species boundaries with more efficiency and more biological realism

We introduce a widely applicable species delimitation method based on the multispecies coalescent model that is more efficient and more biologically realistic than existing methods. We extend a threshold-based method to allow the ancestral speciation rate to vary through time as a smooth piecewise function. Furthermore, we introduce the cutting-edge proposal kernels of StarBeast3 to this model, thus enabling rapid species delimitation on large molecular datasets and allowing the use of relaxed molecular clock models. We validate these methods with genomic sequence data and SNP data, and show they are more efficient than existing methods at achieving parameter convergence during Bayesian MCMC. Lastly, we apply these methods to two datasets (Hemidactylus and Galagidae) and find inconsistencies with the published literature. Our methods are powerful for rapid quantitative testing of species boundaries in large multilocus datasets and are implemented as an open source BEAST 2 package called SPEEDEMON.

Genomic assessment of the Phyllodactylus tuberculosus complex (Reptilia: Phyllodactylidae) in America

AbstractLeaf‐toed geckos (Phyllodactylus) exhibit much cryptic diversity, at least in North America. However, the origins of Central American taxa and their relationships with North American congeners remain unknown. The P. tuberculosus species complex is ideal for evolutionary and biogeographic studies because its geographic distribution extends from northern Mexico to Costa Rica. We analyse ~2,500 nuclear loci obtained by the GBS genomic technique to test hypotheses of genetic isolation among taxa and estimate times of divergence using a relaxed molecular clock model, which constitutes the first hypothesis for the diversification of North and Central American phyllodactylids. Phylogenetic analysis recovers three main clades within polyphyletic P. tuberculosus: tuberculosus, magnus and saxatilis. ABBA‐BABA tests show moderate to low levels of gene flow within the latter two clades and yet genetic isolation in Central American lineages. The relaxed molecular clock model dates the age of the MRCA between the Central American and North American lineages to the beginning of the Paleocene (65 mya), suggesting an ancestral distribution in the proto‐Mexican region and the Chortis Block.

Western Tethys origin, tropical Asia and tropical America disjunction in Berchemia and reinstatement of Phyllogeiton (Rhamneae, Rhamnaceae)

AbstractDespite the well‐known biogeographic pattern in the Northern Hemisphere of disjunct distributions in plants between eastern Asia and North America, many details regarding the evolution of the disjunction between lineages of tropical Asia and tropical America remain poorly understood. Berchemia (Rhamnaceae), comprising ca. 32 species, is distributed predominantly in tropical regions of east to southeast Asia and North America. However, the phylogeny and biogeography of this genus are still not clear. Furthermore, fossils of the genus have been found in Europe, making it an ideal group to understand the biogeographical story of tropical disjunction in the Northern Hemisphere. With two nuclear (ETS, ITS) and three plastid (petA‐psbJ, psbA‐trnH, trnL‐F) DNA regions, we first reconstructed the phylogeny of Berchemia with Bayesian inference and maximum likelihood approaches. Our results indicated that Berchemia, excluding the African species B. discolor and B. zeyheri, which were placed into the reinstated Phyllogeiton, was well‐supported as monophyletic. Divergence times and ancestral areas were inferred using a Bayesian uncorrelated lognormal relaxed molecular clock model and Bayesian binary MCMC method, respectively. The core Berchemia likely originated in the Western Tethys during the middle Eocene, then formed the tropical Asia and tropical America disjunction during the Oligocene and further diversified in Asia from the early Miocene on. The formation and diversification of the core Berchemia likely resulted from a mixture of geologic events, such as the close of the Tethys Ocean, disappearance of the North Atlantic Land Bridges, uplift of the Qinghai‐Tibetan Plateau, and climatic events, such as the establishment and enhancement of Asian monsoons.

Inferring Evolutionary Timescales without Independent Timing Information: An Assessment of "Universal" Insect Rates to Calibrate a Collembola (Hexapoda) Molecular Clock.

Previous estimates of nucleotide substitution rates are routinely applied as secondary or “universal” molecular clock calibrations for estimating evolutionary timescales in groups that lack independent timing information. A major limitation of this approach is that rates can vary considerably among taxonomic groups, but the assumption of rate constancy is rarely evaluated prior to using secondary rate calibrations. Here I evaluate whether an insect mitochondrial DNA clock is appropriate for estimating timescales in Collembola—a group of insect-like arthropods characterized by high levels of cryptic diversity. Relative rates of substitution in cytochrome oxidase subunit 1 (COI) were inferred via Bayesian analysis across a topologically constrained Hexapod phylogeny using a relaxed molecular clock model. Rates for Collembola did not differ significantly from the average rate or from the rates estimated for most other groups (25 of 30), suggesting that (1) their apparent cryptic diversity cannot be explained by accelerated rates of molecular evolution and (2) clocks calibrated using “universal” insect rates may be appropriate for estimating evolutionary timescales in this group. However, of the 31 groups investigated, 10 had rates that deviated significantly from the average (6 higher, 4 lower), underscoring the need for caution and careful consideration when applying secondary insect rate calibrations. Lastly, this study exemplifies a relatively simple approach for evaluating rate constancy within a taxonomic group to determine whether the use of secondary rates are appropriate for molecular clock calibrations.

Genotyping and Phylogenetic Position of Trichinella spiralis Isolates from Different Geographical Locations in China.

In China, the nematode Trichinella spiralis is the main aetiological agent of human trichinellosis. We performed multi-locus microsatellite typing of T. spiralis isolates to improve the current knowledge of the evolution and population diversity. First, seven polymorphic microsatellite loci were used to infer the genetic diversity of T. spiralis collected in 10 endemic regions. Then, a Bayesian model-based STRUCTURE analysis, a clustering based on the neighbor-joining method, and a principal coordinate analysis (PCA) were performed to identify the genetic structure. Finally, the phylogenetic position of Chinese isolates was explored based on six mitochondrial and nuclear genetic markers (cox1, cytb, 5S ISR, ESV, ITS1, and 18S rDNA) using the maximum likelihood and Bayesian methods. In addition, the divergence time was estimated with multiple genes using an uncorrelated log-normal relaxed molecular-clock model. A total of 16 alleles were detected in 2,310 individuals (1,650 muscle larvae and 660 adult worms) using seven loci. The STRUCTURE analysis indicated that the T. spiralis isolates could be organized and derived from the admixture of two ancestral clusters, which was also substantiated through the clustering analysis based on the allelic data. PCA separated most samples from Tiandong, Guangxi (GX-td), and Linzhi, Tibet (Tibet-lz), from the remaining isolates. However, both maximum likelihood and Bayesian inference supported the close relationship between Xiangfan, Hubei (HB-xf), and GX-td. The molecular dating analysis suggested that the Chinese isolates started to diverge during the Late Pleistocene (0.69 Mya). Generally, T. spiralis was observed to harbor low genetic variation, and further investigation with deeper sampling is needed to elucidate the population structure.

Divergence dating using mixed effects clock modelling: An application to HIV-1.

The need to estimate divergence times in evolutionary histories in the presence of various sources of substitution rate variation has stimulated a rich development of relaxed molecular clock models. Viral evolutionary studies frequently adopt an uncorrelated clock model as a generic relaxed molecular clock process, but this may impose considerable estimation bias if discrete rate variation exists among clades or lineages. For HIV-1 group M, rate variation among subtypes has been shown to result in inconsistencies in time to the most recent common ancestor estimation. Although this calls into question the adequacy of available molecular dating methods, no solution to this problem has been offered so far. Here, we investigate the use of mixed effects molecular clock models, which combine both fixed and random effects in the evolutionary rate, to estimate divergence times. Using simulation, we demonstrate that this model outperforms existing molecular clock models in a Bayesian framework for estimating time-measured phylogenies in the presence of mixed sources of rate variation, while also maintaining good performance in simpler scenarios. By analysing a comprehensive HIV-1 group M complete genome data set we confirm considerable rate variation among subtypes that is not adequately modelled by uncorrelated relaxed clock models. The mixed effects clock model can accommodate this rate variation and results in a time to the most recent common ancestor of HIV-1 group M of 1920 (1915–25), which is only slightly earlier than the uncorrelated relaxed clock estimate for the same data set. The use of complete genome data appears to have a more profound impact than the molecular clock model because it reduces the credible intervals by 50 per cent relative to similar estimates based on short envelope gene sequences.

Tropical Asian Origin, boreotropical migration and long-distance dispersal in Nettles (Urticeae, Urticaceae)

The tribe Urticeae (Urticaceae), popularly known as Nettles, include 12 genera and ca. 200 species, constituting a diverse and cosmopolitan plant clade centered in tropical Asia, Africa, and South America. The global distribution of this clade makes it an excellent group to test hypotheses regarding the processes underlying tropical intercontinental disjunctions. More specifically, it allows us to test whether current distribution patterns resulted from recent transoceanic long-distance dispersal or ancient vicariance after boreotropical migration. We reconstructed the phylogeny of Nettles with the nuclear ITS and four plastid DNA regions (rbcL, trnL-F, matK and rpl14-rpl36) using Bayesian inference and maximum parsimony approaches. We inferred divergence times using a Bayesian uncorrelated lognormal relaxed molecular clock model and ancestral areas using the divergence-extinction-cladogenesis (DEC) model. Our results indicate a tropical Asian origin for the tribe during the late Paleocene. Migration events to Eurasia, South America and Africa occurred mainly during the Oligocene and Miocene. However, several long-distance dispersal events, including dispersals from Asia to Hawaii or Australasia, were inferred to have occurred from the Miocene onwards. The fleshy fruits and winged diaspores of several taxa are suited for long-distance dispersal.

Bayesian phylogenetic analysis of the influenza-A virus genomes isolated in Tunisia, and determination of potential recombination events

The influenza-A virus (IAV) causes seasonal epidemics and presents a pandemic risk with the possibility of genetic re-assortment, allowing the emergence of new strains. The evolution of IAVes is done most often by relatively frequent re-assortment between gene segments, but the hypothesis of their evolution by recombination between RNA segments has not been justified to this date. Here, we examine this hypothesis by Bayesian phylogenetic analysis, to test if recombination events have occurred between genomic RNA segments. Different IAV subtypes are observed in co-circulation in Tunisia, which increases the probability of occurrence of double infections. Mixed infections are a prerequisite for recombination between co-infecting of viral strains.The aim of this work, and since understanding the evolutionary dynamics of IAV is essential for controlling human and avian influenza, phylogenetic analyzes (Bayesian approach) have been carried out for IAV strains isolated in Tunisia, to study their co-evolutionary history, trends, and possible recombination models.A set of IAV nucleic sequences, isolated in Tunisia from 2009 to 2013 (n = 102) were used in this study. These genomic segments encode various influenza A proteins. These viral strains studied were isolated following the 2009 H1N1 pandemic.The analyzes identified two large distinct groups of viral sequences and different subgroups. Assuming a relaxed molecular clock model (uncorrelated exponential (uced)) in a Bayesian coalescence approach and a constant effective time demographic history model (Coalescent: constant size), the substitution rate was estimated at 1.356 × 10−3 substitutions/site/year for segment 4 (haemagglutinin HA gene). Consistent estimates of the age of the most recent Common Ancestor (MRCA) were obtained for the different subgroups, the MRCA ages of the two viral populations corresponding to segment 4 and segment 6 (neuraminidase gene NA) of the genome are estimated at 443.737 years and 501.159 years respectively. A detailed phylogenetic study of the HA gene was performed. The incongruous phylogenetic models deduced for the three genomic subgroups studied corresponding to this gene were indicative of recombination events between the different subpopulations. The detection of these relative signals indicating the presence of recombination events can be considered as proof that recombination seems to play a role, even a small one, in the evolution of (IAV). Reliable recombination sites have been located with statistical significance between H3, H1 and H9 subtypes. MRCA age estimates of recombinants phylogenetic clades indicate directional gene transfers from the H1 and H9 populations to the H3 population, and from H1 and H3 to the H9 population, and their co-divergences during the study period.

Genome skimming provides well resolved plastid and nuclear phylogenies, showing patterns of deep reticulate evolution in the tropical carnivorous plant genus Nepenthes (Caryophyllales)

Nepenthes is a genus of carnivorous plants consisting of ~160 species that are distributed in the paleotropics. Molecular systematics has so far not been able to resolve evolutionary relationships of most species because of the limited genetic divergence in previous studies. In the present study, we used a genome-skimming approach to infer phylogenetic relationships on the basis of 81 plastid genes and the highly repetitive rRNA (external transcribed spacer (ETS)–26S) for 39 accessions representing 34 species from eight sections. Maximum-likelihood analysis and Bayesian inference were performed separately for the nuclear and the plastid datasets. Divergence-time estimations were conducted on the basis of a relaxed molecular-clock model, using secondary calibration points. The phylogenetic analyses of the nuclear and plastid datasets yielded well resolved and supported phylogenies. Incongruences between the two datasets were detected, suggesting multiple hybridisation events or incomplete lineage sorting in the deeper and more recent evolutionary history of the genus. The inclusion of several known and suspected hybrids in the phylogenetic analysis provided insights into their parentage. Divergence-time estimations placed the crown diversification of Nepenthes in the early Miocene, c. 20 million years ago. This study showed that genome skimming provides well resolved nuclear and plastid phylogenies that provide valuable insights into the complex evolutionary relationships of Nepenthes.

Comprehensive evolutionary and phylogenetic analysis of Hepacivirus N (HNV).

Hepaciviruses (HVs) have been detected in several domestic and wild animals and present high genetic diversity. The actual classification divides the genus Hepacivirus into 14 species (A-N), according to their phylogenetic relationships, including the bovine hepacivirus [Hepacivirus N (HNV)]. In this study, we confirmed HNV circulation in Brazil and sequenced the whole genome of two strains. Based on the current classification of HCV, which is divided into genotypes and subtypes, we analysed all available bovine hepacivirus sequences in the GenBank database and proposed an HNV classification. All of the sequences were grouped into a single genotype, putatively named 'genotype 1'. This genotype can be clearly divided into four subtypes: A and D containing sequences from Germany and Brazil, respectively, and B and C containing Ghanaian sequences. In addition, the NS3-coding region was used to estimate the time to the most recent common ancestor (TMRCA) of each subtype, using a Bayesian approach and a relaxed molecular clock model. The analyses indicated a common origin of the virus circulating in Germany and Brazil. Ghanaian sequences seemed to have an older TMRCA, indicating a long time of circulation of these viruses in the African continent.

Molecular phylogenetics of the genus Costularia (Schoeneae, Cyperaceae) reveals multiple distinct evolutionary lineages

We investigated the monophyly of Costularia (25 species), a genus of tribe Schoeneae (Cyperaceae) that illustrates a remarkable distribution pattern from southeastern Africa, over Madagascar, the Mascarenes and Seychelles, to Malesia and New Caledonia. A further species, Tetraria borneensis, has been suggested to belong to Costularia. Relationships and divergence times were inferred using an existing four marker phylogeny of Cyperaceae tribe Schoeneae expanded with newly generated sequence data mainly for Costularia s.l. species. Phylogenetic reconstruction was executed using Bayesian inference and maximum likelihood approaches. Divergence times were estimated using a relaxed molecular clock model, calibrated with fossil data. Based on our results, Tetraria borneensis is not related to the species of Costularia. Costularia s.l. is composed of four distinct evolutionary lineages. Two lineages, one including the type species, are part of the Oreobolus clade, i.e. a much reduced genus Costularia restricted to southeastern Africa, Madagascar, the Mascarenes and Seychelles, and a small endemic genus from New Caledonia for which a new genus Chamaedendron is erected based on Costularia subgenus Chamaedendron. The other two lineages are part of the Tricostularia clade, i.e. a separate single-species lineage from the Seychelles for which a new genus (Xyroschoenus) is described, and Costularia subgenus Lophoschoenus. For the latter, more research is needed to test whether they are congeneric with the species placed in the reticulate-sheathed Tetraria clade.

A biogeographic and ecological perspective to the evolution of reproductive behaviour in the family Salamandridae

Amphibians have a complex reproductive behaviour, which shows the highest diversity among tetrapodes. The family Salamandridae, distributed across the entire Holarctic, is one of the most diverse groups of extant salamanders comprising 114 species in 21 genera. The family has a remarkable diversity of courtship modes, amplexus and sperm transfer. It is often hypothesised that this diversity has evolved in adaptation to a specific mating and/or breeding habitat. We test this hypothesis based upon a phylogenetic reconstruction using the complete mitochondrial genome sequences of 45 Salamandridae species, representing all existing genera. We used ancestral character state reconstruction methods and geographic range models and applied relaxed Bayesian molecular clock models to discuss the results in a temporal framework of Salamandridae evolution. Our results show that the family Salamandridae started to diversify in the Late Cretaceous (ca. 87 mya) and is of Western Palearctic origin. Ancestral character state reconstruction predicts that its common ancestor was oviparous, mated on land without amplexus and probably showed a pin wheel spermatophore transfer, which is still found in the Italian endemic Salamandrina terdigidata. Our results suggest that several colonization of continents with subsequent radiations took place, once to the Nearctic and twice into Eastern Asian realms. However, these events were only in one case associated with a change in mating behaviour (dorsal amplexus in Nearctic newts). Around the Cretaceous-Paleogene boundary (K-Pg boundary) several Salamandridae lineages further diverged, again with no obvious changes in mating behaviour. Overall, there is no significant signal for mating character evolution being caused by changes in habitat type, with only a slight tendency that changes in mating habitat might have led to changes in the type of sperm transfer which in turn was associated with changes in the presence or absence of amplexus.

A Post-pleistocene Calibrated Mutation Rate from Insect Museum Specimens.

Quantifying the age of recent species divergence events can be challenging in the absence of calibration points within many groups. The katydid species Neoconocephalus lyristes provides the opportunity to calibrate a post-Pleistocene, taxa specific mutation rate using a known biogeographic event, the Mohawk-Hudson Divide. DNA was extracted from pinned museum specimens of N. lyristes from both Midwest and Atlantic populations and the mitochondrial gene COI sequenced using primers designed from extant specimens. Coalescent analyses using both strict and relaxed molecular clock models were performed in BEAST v1.8.2. The assumption of a strict molecular clock could not be rejected in favor of the relaxed clock model as the distribution of the standard deviation of the clock rate strongly abutted zero. The strict molecular clock model resulted in an intraspecific calculated mutation rate of 14.4-17.3 %/myr, a rate substantially higher than the common rates of sequence evolution observed for insect mitochondrial DNA sequences. The rate, however, aligns closely with mutation rates estimated from other taxa with similarly recent lineage divergence times.

Australasian orchid diversification in time and space: molecular phylogenetic insights from the beard orchids (Calochilus, Diurideae)

Phylogenetic relationships in Calochilus (~30 species) were inferred based on a supermatrix of 81 loci including 22 species. To examine the spatio-temporal evolution of Calochilus, divergence-time estimations were conducted within a Bayesian framework using an uncorrelated relaxed molecular-clock model, followed by maximum-likelihood ancestral-range reconstructions comparing four biogeographic models. To trace the evolution of key floral and vegetative characters, maximum-likelihood ancestral-character reconstructions were carried out. The stem age of Calochilus was dated to ~12.0 million years ago in the mid-Miocene. Divergence of Calochilus into a tropical and a temperate clade was inferred to have occurred ~7.6 million years ago in the late Miocene. Northern Australia was reconstructed as the ancestral area of the tropical clade and the Euronotian region for the temperate clade. Range expansions from Australia to other Australasian regions, such as New Zealand and New Guinea, were inferred to have occurred only in recent geological times, commencing in the Pleistocene. The infrageneric classification for Calochilus was revised, erecting two subgenera, subgenus Calochilus and subgenus Tropichilus subgen. nov. Section Calochilus Szlach. was recircumscribed, and sect. Abrochilus sect. nov., and section Placochilus sect. nov. were erected. Identification keys to subgenera and sections and a taxonomic synopsis of the genus are provided.

Genealogical and niche modeling analyses reveal recent expansion and limited genetic divergence in the Formicivora serrana complex (Passeriformes: Thamnophilidae)

The principal aim of this study was to infer the phylogeographic pattern of the antwrens of the Formicivora serrana complex, an endemic faunal component of the semi-deciduous forests and restinga formations of the Atlantic Forest of southeast Brazil. We combined niche-based modeling and phylogeographic approaches in describing the genetic spatial structure of the taxon, inferring the phylogenetic hypothesis of lineage diversification, and providing estimates of population growth, timing of coalescence, and gene flow. We also modeled the species potential distribution to examine the putative consequences of the Last Glacial Maximum (LGM) (21,000 years ago) and Holocene climate variability. To perform this, we analyzed a dataset composed of two mitochondrial (cytochrome oxidase I and NADH dehydrogenase subunit 2) and four nuclear (anonymous loci) DNA regions from 35 specimens. Genealogical analyses were performed using phylogenetic maximum likelihood and coalescent Bayesian approaches; genetic diversity was assessed using analysis of molecular variance and network analysis; ages of coalescent events were estimated using a relaxed molecular clock model. Ecological niche modeling of current and LGM ranges were performed using six algorithms (Bioclim, general additive model, general linear model, Maxent, support vector machine and Random forest) based on 178 occurrence localities and 19 bioclimatic variables. Limited genetic divergence among recognized forms of the F. serrana complex was evidenced in the low mitochondrial nucleotide divergence among populations (Jukes–Cantor distance between subspecies varied from 0.17 to 0.21%), unresolved phylogenetic trees, star-shaped haplotype networks, and overall lack of population genetic structure. Fixation index analyses, however, suggested an incipient geographic structure. Ecological niche modeling suggested expansion of the distribution range since the LGM. Our results provide the first evidence that bird taxa of xerophytic and semi-deciduous habitats of the AF were also affected by Quaternary climatic fluctuations. The recovered genealogical pattern indicates that F. serrana should be regarded as a single evolutionary unit, corroborating current taxonomic hypotheses.

Adaptive evolution during the establishment of European avian-like H1N1 influenza A virus in swine.

An H1N1 subtype influenza A virus with all eight gene segments derived from wild birds (including mallards), ducks and chickens, caused severe disease outbreaks in swine populations in Europe beginning in 1979 and successfully adapted to form the European avian‐like swine (EA‐swine) influenza lineage. Genes of the EA‐swine lineage that are clearly segregated from its closest avian relatives continue to circulate in swine populations globally and represent a unique opportunity to study the adaptive process of an avian‐to‐mammalian cross‐species transmission. Here, we used a relaxed molecular clock model to test whether the EA‐swine virus originated through the introduction of a single avian ancestor as an entire genome, followed by an analysis of host‐specific selection pressures among different gene segments. Our data indicated independent introduction of gene segments via transmission of avian viruses into swine followed by reassortment events that occurred at least 1–4 years prior to the EA‐swine outbreak. All EA‐swine gene segments exhibit greater selection pressure than avian viruses, reflecting both adaptive pressures and relaxed selective constraints that are associated with host switching. Notably, we identified key amino acid mutations in the viral surface proteins (H1 and N1) that play a role in adaptation to new hosts. Following the establishment of EA‐swine lineage, we observed an increased frequency of intrasubtype reassortment of segments compared to the earlier strains that has been associated with adaptive amino acid replacements, disease severity and vaccine escape. Taken together, our study provides key insights into the adaptive changes in viral genomes following the transmission of avian influenza viruses to swine and the early establishment of the EA‐swine lineage.