Research Progress of Molecular Clock

In the paper, related research and progress of molecular clock hypothesis were summarized, including definition of molecular clock, supporting proofs, contro- versy, significance and perfection, application and accompanying challenges.

Molecular Evolutionary Rate Calculation of Hemoglobin α Chain and γ Chain at Different Stages in Vertebrate Evolution Process

Pseudogene and fibrin peptide which has rapid evolve speed and good stability are used in this study to determine the divergence date among groups.Through calculating the evolutionary rate of hemoglobin α chain,γ chain in vertebrate（including birds and mammals）,it is concluded that the evolutionary rate of hemoglobin α chain,γ chain is not invariable.It shows different evolutionary rates in different periods,that is,faster in early evolution stage and relatively slow in later stage.

Molecular phylogeny and divergence time of Trachypithecus： with implications for the taxonomy of T. phayrei

The genus Trachypithecus is the most diverse langur taxon, distributed in southwestern China, south and southeastern Asia. In this study, we include 16 recognized Trachypithecus species to reconstruct the phylogeny with particular concern to the taxonomy of the three subspecies of Z phayrei using multiple genes. Our results support a sister-relationship between Z p. phayrei and T p. shanicus. However, the mitochondrial CYT B gene supported T. p. crepuscula as a distinct species, but the nuclear PRMI gene suggested a closer relationship between T p. crepuscula and T p. phayrei. The incongruence between nuclear and mitochondrial genes suggests that hybridization may have occurred, a fact that would benefit from re-examination using multiple unlinked nuclear genes.

Molecular phylogeny and divergence time of Trachypithecus:with implications for the taxonomy of T.phayrei

The genus Trachypithecus is the most diverse langur taxon,distributed in southwestern China,south and southeastern Asia.In this study,we include 16 recognized Trachypithecus species to reconstruct the phylogeny with particular concern to the taxonomy of the three subspecies of T.phayrei using multiple genes.Our results support a sister-relationship between T.p.phayrei and T.p.shanicus.However,the mitochondrial CYT B gene supported T.p.crepuscula as a distinct species,but the nuclear PRM1 gene suggested a closer relationship between T.p.crepuscula and T.p.phayrei.The incongruence between nuclear and mitochondrial genes suggests that hybridization may have occurred,a fact that would benefit from re-examination using multiple unlinked nuclear genes.

A fossil-calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America

Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions （amphitropical range）. Great heterogeneity in the substitution rates of Gnetales （Ephedra, Gnetum, and Welwitschia） has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species （and 7736 aligned nucleotides） to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil-based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long-distance dispersal, but finer-scale studies are needed to test this hypothesis.

Angiosperm-wide analysis of fruit and ovary evolution aided by a new nuclear phylogeny supports association of the same ovary type with both dry and fleshy fruits

Fruit functions in seed protection and dispersal and belongs to many dry and fleshy types,yet their evolutionary pattern remains unclear in part due to uncertainties in the phylogenetic relationships among several orders and families.Thus we used nuclear genes of 502 angiosperm species representing 231 families to reconstruct a well supported phylogeny,with resolved relationships for orders and families with previously uncertain placements.Using this phylogeny as a framework,molecular dating supports a Triassic origin of the crown angiosperms,followed by the emergence of most orders in the Jurassic and Cretaceous and their rise to ecological dominance during the Cretaceous Terrestrial Revolution.The robust phylogeny allowed an examination of the evolutionary pattern of fruit and ovary types,revealing a trend of parallel carpel fusions during early diversifications in eudicots,monocots,and magnoliids.Moreover,taxa in the same order or family with the same ovary type can develop either dry or fleshy fruits with strong correlations between specific types of dry and fleshy fruits;such associations of ovary,dry and fleshy fruits define several ovaryfruit"modules"each found in multiple families.One of the frequent modules has an ovary containing multiple ovules,capsules and berries,and another with an ovary having one or two ovules,achenes(or other single-seeded dry fruits)and drupes.This new perspective of relationships among fruit types highlights the closeness of specific dry and fleshy fruit types,such as capsule and berry,that develop from the same ovary type and belong to the same module relative to dry and fleshy fruits of other modules(such as achenes and drupes).Further analyses of gene families containing known genes for ovary and fruit development identified phylogenetic nodes with multiple gene duplications,supporting a possible role of whole-genome duplications,in combination with climate changes and animal behaviors,in angiosperm fruit and ovary diversification.

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.

Primate phylogeny:molecular evidence for a pongid clade excluding humans and a prosimian clade containing tarsiers

Unbiased readings of fossils are well known to contradict some of the popular molecular groupings among primates,particularly with regard to great apes and tarsiers.The molecular methodologies today are however flawed as they are based on a mistaken theoretical interpretation of the genetic equidistance phenomenon that originally started the field.An improved molecular method the 'slow clock' was here developed based on the Maximum Genetic Diversity hypothesis,a more complete account of the unified changes in genotypes and phenotypes.The method makes use of only slow evolving sequences and requires no uncertain assumptions or mathematical corrections and hence is able to give definitive results.The findings indicate that humans are genetically more distant to orangutans than African apes are and separated from the pongid clade ～17.6 million years ago.Also,tarsiers are genetically closer to lorises than simian primates are.Finally,the fossil times for the radiation of mammals at the K/T boundary and for the Eutheria-Metatheria split in the Early Cretaceous were independently confirmed from molecular dating calibrated using the fossil split times of gorilla-orangutan,mouse-rat,and opossum-kangaroo.Therefore,the re-established primate phylogeny indicates a remarkable unity between molecules and fossils.

The genetic equidistance result:misreading by the molecular clock and neutral theory and reinterpretation nearly half of a century later

In 1963,Margoliash discovered the unexpected genetic equidistance result after comparing cytochrome c sequences from different species.This finding,together with the hemoglobin analyses of Zuckerkandl and Pauling in 1962,directly inspired the ad hoc molecular clock hypothesis.Unfortunately,however,many biologists have since mistakenly viewed the molecular clock as a genuine reality,which in turn inspired Kimura,King,and Jukes to propose the neutral theory of molecular evolution.Many years of studies have found numerous contradictions to the theory,and few today believe in a universal constant clock.What is being neglected,however,is that the failure of the molecular clock hypothesis has left the original equidistance result an unsolved mystery.In recent years,we fortuitously rediscovered the equidistance result,which remains unknown to nearly all researchers.Incorporating the proven virtues of existing evolutionary theories and introducing the novel concept of maximum genetic diversity,we proposed a more complete hypothesis of evolutionary genetics and reinterpreted the equidistance result and other major evolutionary phenomena.The hypothesis may rewrite molecular phylogeny and population genetics and solve major biomedical problems that challenge the existing framework of evolutionary biology.

Taxonomy,phylogeny and evolution of freshwater Hypocreomycetidae(Sordariomycetes)

Hypocreomycetidae is a highly diverse group with species from various habitats. This subclass has been reported as pathogenic,endophytic, parasitic, saprobic, fungicolous, lichenicolous, algicolous, coprophilous and insect fungi from aquatic andterrestrial habitats. In this study, we focused on freshwater fungi of Hypocreomycetidae which resulted 41 fresh collectionsfrom China and Thailand. Based on morphological and phylogenetic analyses, we identified 26 species that belong to twoorders (Hypocreales and Microascales) and six families (Bionectriaceae, Halosphaeriaceae, Microascaceae, Nectriaceae,Sarocladiaceae and Stachybotryaceae). Ten new species are introduced and 13 new habitats and geographic records arereported. Mariannaea superimposita, Stachybotrys chartarum and S. chlorohalonatus are recollected from freshwater habitatsin China. Based on phylogenetic analysis of combined LSU, ITS, SSU, rpb2 and tef1-α sequences data, Emericellopsisis transferred to Hypocreales genera incertae sedis;Pseudoacremonium is transferred to Bionectriaceae;Sedecimiella isplaced in Nectriaceae;Nautosphaeria and Tubakiella are excluded from Halosphaeriaceae and placed in Microascalesgenera incertae sedis;and Faurelina is excluded from Hypocreomycetidae. Varicosporella is placed under Atractium as asynonym of Atractium. In addition, phylogenetic analysis and divergence time estimates showed that Ascocodina, Campylospora,Cornuvesica and Xenodactylariaceae form distinct lineages in Hypocreomycetidae and they evolved in the family/order time frame. Hence, a new order (Xenodactylariales) and three new families (Ascocodinaceae, Campylosporaceae andCornuvesicaceae) are introduced based on phylogenetic analysis, divergence time estimations and morphological characters.Ancestral character state analysis is performed for different habitats of Hypocreomycetidae including freshwater, marineand terrestrial taxa. The result indicates that marine and freshwater fungi evolved independently from terrestrial ancestors.The results further support those early diverging clades of this subclass, mostly comprising terrestrial taxa and freshwaterand marine taxa have been secondarily derived, while the crown clade (Nectriaceae) is represented in all three habitats. Theevolution of various morphological adaptations towards their habitual changes are also discussed.

Notes,outline and divergence times of Basidiomycota

The Basidiomycota constitutes a major phylum of the kingdom Fungi and is second in species numbers to the Ascomycota.The present work provides an overview of all validly published,currently used basidiomycete genera to date in a single document.An outline of all genera of Basidiomycota is provided,which includes 1928 currently used genera names,with 1263 synonyms,which are distributed in 241 families,68 orders,18 classes and four subphyla.We provide brief notes for each accepted genus including information on classification,number of accepted species,type species,life mode,habitat,distribution,and sequence information.Furthermore,three phylogenetic analyses with combined LSU,SSU,5.8s,rpb1,rpb2,and ef1 datasets for the subphyla Agaricomycotina,Pucciniomycotina and Ustilaginomycotina are conducted,respectively.Divergence time estimates are provided to the family level with 632 species from 62 orders,168 families and 605 genera.Our study indicates that the divergence times of the subphyla in Basidiomycota are 406-430 Mya,classes are 211-383 Mya,and orders are 99-323 Mya,which are largely consistent with previous studies.In this study,all phylogenetically supported families were dated,with the families of Agaricomycotina diverging from 27-178 Mya,Pucciniomycotina from 85-222 Mya,and Ustilaginomycotina from 79-177 Mya.Divergence times as additional criterion in ranking provide additional evidence to resolve taxonomic problems in the Basidiomycota taxonomic system,and also provide a better understanding of their phylogeny and evolution.

A well-supported nuclear phylogeny of Poaceae and implications for the evolution of C4 photosynthesis

Poaceae(the grasses)includes rice,maize,wheat,and other crops,and is the most economically important angiosperm family.Poaceae is also one of the largest plant families,consisting of over 11000 species with a global distribution that contributes to diverse ecosystems.Poaceae species are classified into 12 subfamilies,with generally strong phylogenetic support for their monophyly.However,many relationships within subfamilies,among tribes and/or subtribes,remain uncertain.To better resolve the Poaceae phylogeny,we generated 342 transcriptomic and seven genomic datasets;these were combined with other genomic and transcriptomic datasets to provide sequences for 357 Poaceae species in 231 genera,representing 45 tribes and all 12 subfamilies.Over 1200 low-copy nuclear genes were retrieved from these datasets,with several subsets obtained using additional criteria,and used for coalescent analyses to reconstruct a Poaceae phylogeny.Our results strongly support the monophyly of 11 subfamilies;however,the subfamily Puelioideae was separated into two non-sister clades,one for each of the two previously defined tribes,supporting a hypothesis that places each tribe in a separate subfamily.Molecular clock analyses estimated the crown age of Poaceae to be101 million years old.Ancestral character reconstruction of C3/C4 photosynthesis supports the hypothesis of multiple independent origins of C4 photosynthesis.These origins are further supported by phylogenetic analysis of the ppc gene family that encodes the phosphoenolpyruvate carboxylase,which suggests that members of three paralogous subclades(ppc-aL1a,ppc-aL1b,and ppcB2)were recruited as functional C4 ppc genes.This study provides valuable resources and a robust phylogenetic framework for evolutionary analyses of the grass family.

Lack of evolutionary changes identified in SARS-CoV-2 for the re-emerging outbreak of COVID-19 in Beijing,China

Although significant achievements have shown that the coronavirus disease 2019(COVID‐19)resurgence in Beijing,China,was initiated by contaminated frozen products and transported via cold chain transportation,international travelers with asymptomatic symptoms or false‐negative nucleic acid may have another possible transmission mode that spread the virus to Beijing.One of the key differences between these two assumptions was whether the virus actively replicated since,so far,no reports showed viruses could stop evolution in alive hosts.We studied severe acute respiratory syndrome coronavirus 2(SARS‐CoV‐2)sequences in this outbreak by a modified leaf‐dating method with the Bayes factor.The numbers of single nucleotide variants(SNVs)found in SARS‐CoV‐2 sequences were significantly lower than those called from B.1.1 records collected at the matching time worldwide(P=0.047).In addition,results of the leaf‐dating method showed ages of viruses sampled from this outbreak were earlier than their recorded dates of collection(Bayes factors>10),while control sequences(selected randomly with ten replicates)showed no differences in their collection dates(Bayes factors<10).Our results which indicated that the re‐emergence of SARS‐CoV‐2 in Beijing in June 2020 was caused by a virus that exhibited a lack of evolutionary changes compared to viruses collected at the corresponding time,provided evolutionary evidence to the contaminated imported frozen food should be responsible for the reappearance of COVID‐19 cases in Beijing.The method developed here might also be helpful to provide the very first clues for potential sources of COVID‐19 cases in the future.

Evolutionary rates of mitochondrial sequences and gene orders in Spirurina(Nematoda)are episodic but synchronised

In contrast to highly conserved mitogenomic architecture in most metazoan lineages,which indicates that rearrangement events are generally strongly selected against,a limited number of often unrelated lineages exhibit highly elevated architectural evolution rates.The underlying reasons for this discontinuity in the mitogenomic evolution remain unknown.Previously we sequenced the mitochondrial genome of the first Camallanoidea species,Camallanus cotti(Nematoda:Chromadorea:Spirurina:Camallanidae),and found that it exhibited a highly disrupted architecture.We hypothesised that disrupted architecture might be a synapomorphic feature of the sister-clades Camallanoidea and Dracunculoidea.In this study,we sequenced mitogenomes of three freshwater fish-parasitic nematodes:Camallanus lacustris(Camallanidae),and two Philometridae(Dracunculoidea)species,Clavinema parasiluri,and Philometra sp.In partial agreement with the working hypothesis,both Camallanoidea species had exceptionally large mitogenomes of 18–19 Kbp,albeit the underlying reasons differed:in C.lacustris it was the existence of a single enlarged noncoding region of
5.5 Kbp.A segment of this region exhibited an inverted base composition skew,which is indicative of a sequence inversion or recombination event.Camallanidae is the second identified chromadorean(first for Spirurina)family that exhibits within-family protein-coding gene rearrangements,and the absence of trnL1 and trnF may be a synapomorphy for Camallanoidea.The underlying reason for the disrupted architecture of Camallanidae does not appear to be a particular event shared by their common ancestor,but rather an underlying mechanism that makes disruptive events more likely in this lineage.In disagreement with the working hypothesis,Spiruromorpha and Oxyuridomorpha exhibited even more highly rearranged gene orders and greater overall branch lengths than Camallanomorpha.However,withininfraorder architecture was highly conserved and leaf nodes very short.This indicates that common ancestors of Spiruromorpha and Oxyuridomorpha clades underwent a period of rapid mitochondrial evolution(both sequence and architecture),followed by a stabilisation after the taxonomic radiation.In contrast to this,Camallanomorpha,and particularly Camallanidae,appear to have entered a period of elevated evolutionary rates after the initial radiations of these two taxa.As a result of this evolutionary discontinuity,there was a strong correlation between the gene order rearrangement rate(GORR)and the overall branch length(0.81),but there was no correlation between the strength of purifying selection(ω?dN/dS)and the overall branch lengths(0.05)and GORR(0.04).These findings have important repercussions for future phylogenetic and other evolutionary studies of Spirurina.

Tertiary montane origin of the Central Asian flora,evidence inferred from cpDNA sequences of Atraphaxis(Polygonaceae)

Atraphaxis has approximately 25 species and a distribution center in Central Asia. It has been previously used to hypothesize an origin from montane forest. We sampled 18 species covering three sections within the genus and sequenced five cpDNA spacers, atpB -rbcL, psbK -psbI, psbA -trnH, rbcL, and trnL -trnF. BEAST was used to reconstruct phylogenetic relationship and time divergences, and S-DIVA and Lagrange were used, based on distribution area and ecotype data, for reconstruction of ancestral areas and events. Our results appear compatible with designation of three taxonomic sections within the genus. The generic stem and crown ages were Eocene, approximately 47 Ma, and Oligocene 27 Ma,respectively. The origin of Atraphaxis is confirmed as montane,with an ancestral area consisting of the Junggar Basin and uplands of the Pamir-Tianshan-Alatau-Altai mountain chains,and ancestral ecotype of montane forest. Two remarkable paleogeographic events, shrinkage of the inland Paratethys Sea at the boundary of the late Oligocene and early Miocene, and the time intervals of cooling and drying of global climate from24（22） Ma onward likely facilitated early diversification of Atraphaxis, while rapid uplift of the Tianshan Mountains during the late Miocene may have promoted later diversification.

Reconstructing evolutionary timescales using phylogenomics

Reconstructing the timescale of the Tree of Life is one of the principal aims of evolutionary biology. This has been greatly aided by the development of the molecular clock, which enables evolutionary timescales to be estimated from genetic data. In recent years, high-throughput sequencing technology has led to an increase in the feasibility and availability ofgenome-scale data sets. These represent a rich source of biological information, but they also bring a set of analytical challenges. In this review, we provide an overview of phylogenomic dating and describe the challenges associated with analysing genome-scale data. We also report on recent phylogenomic estimates of the evolutionary timescales of mammals, birds, and insects.