The evolving species concepts used for yeasts:from phenotypes and genomes to speciation networks

Here we review how evolving species concepts have been applied to understand yeast diversity.Initially,a phenotypic species concept was utilized taking into consideration morphological aspects of colonies and cells,and growth profiles.Later the biological species concept was added,which applied data from mating experiments.Biophysical measurements of DNA similarity between isolates were an early measure that became more broadly applied with the advent of sequencing technology,leading to a sequence-based species concept using comparisons of parts of the ribosomal DNA.At present phylogenetic species concepts that employ sequence data of rDNA and other genes are universally applied in fungal taxonomy,including yeasts,because various studies revealed a relatively good correlation between the biological species concept and sequence divergence.The application of genome information is becoming increasingly common,and we strongly recommend the use of complete,rather than draft genomes to improve our understanding of species and their genome and genetic dynamics.Complete genomes allow in-depth comparisons on the evolvability of genomes and,consequently,of the species to which they belong.Hybridization seems a relatively common phenomenon and has been observed in all major fungal lineages that contain yeasts.Note that hybrids may greatly differ in their post-hybridization development.Future in-depth studies,initially using some model species or complexes may shift the traditional species concept as isolated clusters of genetically compatible isolates to a cohesive speciation network in which such clusters are interconnected by genetic processes,such as hybridization.

Defining honeybee subspecies in an evolutionary context warrants strategized conservation

Despite the urgent need for conservation consideration,strategic action plans for the preservation of the Asian honeybee,Apis cerana Fabricius,1793,remain lacking.Both the convergent and divergent adaptations of this widespread insect have led to confusing phenotypical traits and inconsistent infraspecific taxonomy.Unclear subspecies boundaries pose a significant challenge to honeybee conservation efforts,as it is difficult to effectively prioritize conservation targets without a clear understanding of subspecies identities.Here,we investigated genome variations in 362 worker bees representing almost all populations of mainland A.cerana to understand how evolution has shaped its population structure.Whole-genome single nucleotide polymorphisms(SNPs)based on nuclear sequences revealed eight putative subspecies,with all seven peripheral subspecies exhibiting mutually exclusive monophyly and distinct genetic divergence from the widespread central subspecies.Our results demonstrated that most classic morphological traits,including body size,were related to the climatic variables of the local habitats and did not reflect the true evolutionary history of the organism.Thus,such morphological traits were not suitable for subspecific delineation.Conversely,wing vein characters showed relative independence to the environment and supported the subspecies boundaries inferred from nuclear genomes.Mitochondrial phylogeny further indicated that the present subspecies structure was a result of multiple waves of population divergence from a common ancestor.Based on our findings,we propose that criteria for subspecies delineation should be based on evolutionary independence,trait distinction,and geographic isolation.We formally defined and described eight subspecies of mainland A.cerana.Elucidation of the evolutionary history and subspecies boundaries enables a customized conservation strategy for both widespread and endemic honeybee conservation units,guiding colony introduction and breeding.

Phylogenomics and integrative taxonomy reveal two new species of Amana(Liliaceae)

Until now the genus Amana(Liliaceae),known as ’East Asian tulips’,has contained just seven species.In this study,a phylogenomic and integrative taxonomic approach was used to reveal two new species,Amana nanyueensis from Central China and A.tianmuensis from East China.A.nanyueensis resembles Amana edulis in possessing a densely villous-woolly bulb tunic and two opposite bracts,but differs in its leaves and anthers.Amana tianmuensis resembles Amana erythronioides in possessing three verticillate bracts and yellow anthers,but differs in aspects of its leaves and bulbs.These four species are clearly separated from each other in principal components analysis based on morphology.Phylogenomic analyses based on plastid CDS further support the species delimitation of A.nanyueensis and A.tianmuensis and suggests they are closely related to A.edulis.Cytological analysis shows that A.nanyueensis and A tianmuensis are both diploid(2n=2x=24),different from A edulis,which is either diploid(northern populations) or tetraploid(southern populations,2n=4x=48).The pollen morphology of A.nanyueensis is similar to other Amana species(single-groove germination aperture),but A.tianmuensis is quite different because of the presence of a sulcus membrane,which creates the illusion of double grooves.Ecological niche modelling also revealed a niche differentiation between A.edulis,A.nanyueensis and A.tianmuensis.

Species delimitation based on diagnosis and monophyly, and its importance for advancing mammalian taxonomy

A recently proposed taxonomic classification of extant ungulates sparked a series of publications that criticize the Phylogenetic Species Concept （PSC） claiming it to be a particularly poor species concept.These opinions reiteratively stated that （1） the two fundamental elements of the ＂PSC＂,i.e.,monophyly and diagnosability,do not offer objective criteria as to where the line between species should be drawn;and （2） that extirpation of populations can lead to artificial diagnosability and spurious recognitions of species.This sudden eruption of criticism against the PSC is misleading.Problems attributed to the PSC are common to most approaches and concepts that modern systematists employ to establish species boundaries.The controversial taxonomic propositions that sparked criticism against the PSC are indeed highly problematic,not because of the species concept upon which they are based,but because no evidence （whatsoever） has become public to support a substantial portion of the proposed classification.We herein discuss these topics using examples from mammals.Numerous areas of biological research rest upon taxonomic accuracy （including conservation biology and biomedical research）;hence,it is necessary to clarify what are （and what are not）the real sources of taxonomic inaccuracy.

Systematics of the Artiodactyla of China in the 21st century

In this paper, I have introduced the concept of the Evolutionary Species, and shown how it affects the taxonomy of the Artiodactyla of China. The ＂traditional＂ taxonomy of the Artiodactyla, which has remained almost unchanged for 100 years, relies on ill-formulated notions of species and subspecies, only slightly modified by the population-thinking of the 1930s. Species are populations （or metapopu- lations） differentiated by the possession of fixed heritable differences from other such populations （or metapopulations）. In the Artiodactyla, there are many more species than ＂traditionally＂ recognised; this is by no means a drawback, as it enables the units of biodiversity to be identified in a testable fashion, and brings the taxonomy of large mammals into line with that long practised for small mammals. Species are likely to differentiate where there are natural gaps in the distribution of a genus, such as mountain blocks （for example in the genus Budorcas） or otherwise dissected habitat （for example in the genus Cervus）. Natural hybridisation between distinct species is not an uncommon phenomenon, again illustrated well in the genus Cervus, where hybridisation between the elaphus and nippon groups occurs today and evidently occurred in the past, as shown by the distribution of mtDNA.

The art and science of species delimitation

DNA-based approaches to systematics have changed dramatically during the last two decades with the rise of DNA barcoding methods and newer multi-locus methods for species delimitation. During the last half-decade, partly driven by the new sequencing technologies, the focus has shifted to multi-locus sequence data and the identification of species within the frame-work of the multi-species coalescent （MSC）. In this paper, I discuss model-based Bayesian methods for species delimitation that have been developed in recent years using the MSC. Several approximate methods for species delimitation （and their limitations） are also discussed. Explicit species delimitation models have the advantage of clarifying more precisely what is being delimited and what assumptions we are making in doing so. Moreover, the methods can be very powerful when applied to large multi-locus datasets and thus take full advantage of data generated using today＇s technologies [Current Zoology 61 （5）： 846-853,2015].

What are fungal species and how to delineate them?

This is the opening paper in the special issue of Fungal Diversity,which collates the data on defining species.Defining and recognizing species has long been a controversial issue.Since Darwin’s proposed origin of species,over 30 species criteria have been brought forth and used to define species boundaries.In recent times,phylogenetic analyses based on multiple loci have been extensively used as a method to define species boundaries.However,only a few mycologists are aware that phylogenetic species criteria can mask discordances among fungal groups,leading to inaccurately defined species bounda-ries.In the current review,we discuss species recognition criteria,how and where these criteria can be applied along with their limitations and derived alternatives.In order to delimit fungal species,authors need to take into account not only the phylogenetic and phenotypic coherence,but also the timing of events that lead to fungal speciation and subsequent diversi-fications.Variations in the rate of phenotypic diversifications and convergent fungal evolution make it difficult to establish a universal species recognition criterion.The best practice can only be defined in the context of each fungal group.In this review,we provide a set of guidelines,encouraging an integrative taxonomic approach for species delimitation that can be used to define fungal species boundaries in the future.The other papers in this special issue deal with fungal speciation in Ascomycota,Dothideomycetes,Basidiomycota,basal fungi,lichen-forming fungi,plant pathogenic fungi,and yeasts.

Defining a species in fungal plant pathology:beyond the species level

In plant pathology,the correct naming of a species is essential for determining the causal agents of disease.Species names not only serve the general purpose of concise communication,but also are critical for effective plant quarantine,prevent-ing the introduction of new pathogens into a territory.Many phytopathogenic genera have multiple species and,in several genera,disagreements between the multiple prevailing species concept definitions result in numerous cryptic species.Some of these species were previously called by various names;forma speciales(specialised forms),subspecies,or pathotypes.However,based on new molecular evidence they are being assigned into new species.The frequent name changes and lack of consistent criteria to delineate cryptic species,species,subspecies,forms,and races create increasing confusion,often making communication among biologists arduous.Furthermore,such ambiguous information can convey misleading evo-lutionary concepts and species boundaries.The aim of this paper is to review these concepts,clarify their use,and evaluate them by referring to existing examples.We specifically address the question,“Do plant pathogens require a different ranking system?”We conclude that it is necessary to identify phytopathogens to species level based on data from multiple approaches.Furthermore,this identification must go beyond species level to clearly classify hitherto known subspecies,forms and races.In addition,when naming phytopathogenic genera,plant pathologists should provide more information about geographic locations and host ranges as well as host specificities for individual species,cryptic species,forms or races.When describing a new phytopathogen,we suggest that authors provide at least three representative strains together with pathogenicity test results.If Koch’s postulates cannot be fulfilled,it is necessary to provide complementary data such as associated disease severity on the host plant.Moreover,more sequenced collections of species causing diseases should be published in order to stabilise the boundaries of cryptic species,species,subspecies,forms,and races.

Species from Darwin onward

Controversy regarding the species problem has been going on for many decades and no consensus has ever been reached about what a“species”really is and how best to define the concept.De Queiroz(1998)introduced a distinction between two aspects of this problem:on the one hand,the definition proper,and on the other,the criteria allowing biologists to recognize species in practice.This distinction is a first step on the way toward a solution of the problem.In the present paper,we show that de Queiroz’s distinction is made possible by the radical theoretical change introduced by Darwin.We emphasize that the species problem did not appear in the 20th century,but long before,and that Darwin addresses it indirectly in the Origin of Species.It might seem paradoxical to refer to Darwin’s views about species,because they are usually considered as unclear.However,we propose that an analysis of these views in the context of Darwin’s own theory of evolution might reveal how a definition of the concept of species is made possible by being anchored to the very theory of evolution.To this aim,we present a plausible reconstruction of Darwin’s implicit conception of species and show how this conception fits with the debates on species that took place in the 18th and 19th centuries.We then turn to today’s biology and show what changes Darwin’s implicit conception of species has brought about relative to the species concept and species delimitation.

Integrative approaches for species delimitation in Ascomycota

Biodiversity loss from disturbances caused by human activities means that species are disappearing at an ever increasing rate.The high number of species that have yet to be described have generated extreme crisis to the taxonomist.Therefore,more than in any other era,effective ways to discover and delimitate species are needed.This paper reviews the historically fore-most approaches used to delimit species in Ascomycota,the most speciose phylum of Fungi.These include morphological,biological,and phylogenetic species concepts.We argue that a single property to delineate species boundaries has various defects and each species concept comes with its own advantages and disadvantages.Recently the rate of species discovery has increased because of the advancement of phylogenetic approaches.However,traditional phylogenetic methods with few gene regions lack species-level resolution,and do not allow unambiguous conclusions.We detail the processes that affect gene tree heterogeneity,which acts as barriers to delimiting species boundaries in classical low-rank phylogenies.So far,limited insights were given to the DNA-based methodologies to establish well-supported boundaries among fungal species.In addition to reviewing concepts and methodologies used to delimit species,we present a case study.We applied differ-ent species delimitation methods to understand species boundaries in the plant pathogenic and cryptic genus Phyllosticta(Dothideomycetes,Botryosphaeriales).Several DNA-based methods over-split the taxa while in some methods several taxa fall into a single species.These problems can be resolved by using multiple loci and coalescence-based methods.Further,we discuss integrative approaches that are crucial for understanding species boundaries within Ascomycota and provide several examples for ideal and pragmatic approaches of species delimitation.

Species in lichen-forming fungi:balancing between conceptual and practical considerations,and between phenotype and phylogenomics

Lichens are symbiotic associations resulting from interactions among fungi(primary and secondary mycobionts),algae and/or cyanobacteria(primary and secondary photobionts),and specific elements of the bacterial microbiome associated with the lichen thallus.The question of what is a species,both concerning the lichen as a whole and its main fungal component,the primary mycobiont,has faced many challenges throughout history and has reached new dimensions with the advent of molecular phylogenetics and phylogenomics.In this paper,we briefly revise the definition of lichens and the scientific and vernacular naming conventions,concluding that the scientific,Latinized name usually associated with lichens invariably refers to the primary mycobiont,whereas the vernacular name encompasses the entire lichen.Although the same lichen mycobiont may produce different phenotypes when associating with different photobionts or growing in axenic culture,this discrete variation does not warrant the application of different scientific names,but must follow the principle"one fungus=one name".Instead,broadly agreed informal designations should be used for such discrete morphologies,such as chloromorph and cyanomorph for lichens formed by the same mycobiont but with either green algae or cyanobacteria.The taxonomic recognition of species in lichen-forming fungi is not different from other fungi and conceptual and nomenclatural approaches follow the same principles.We identify a number of current challenges and provide recommendations to address these.Species delimitation in lichen-forming fungi should not be tailored to particular species concepts but instead be derived from empirical evidence,applying one or several of the following principles in what we call the LPR approach:lineage(L)coherence vs.divergence(phylogenetic component),phenotype(P)coherence vs.divergence(morphological component),and/or reproductive(R)compatibility vs.isolation(biological component).Species hypotheses can be established based on either L or P,then using either P or L(plus R)to corroborate them.The reliability of species hypotheses depends not only on the nature and number of characters but also on the context:the closer the relationship and/or similarity between species,the higher the number of characters and/or specimens that should be analyzed to provide reliable delimitations.Alpha taxonomy should follow scientific evidence and an evolutionary framework but should also offer alternative practical solutions,as long as these are scientifically defendable.Taxa that are delimited phylogenetically but not readily identifiable in the field,or are genuinely cryptic,should not be rejected due to the inaccessibility of proper tools.Instead,they can be provisionally treated as undifferentiated complexes for purposes that do not require precise determinations.The application of infraspecific(gamma)taxonomy should be restricted to cases where there is a biological rationale,i.e.,lineages of a species complex that show limited phylogenetic divergence but no evidence of reproductive isolation.Gamma taxonomy should not be used to denote discrete phenotypical variation or ecotypes not warranting the distinction at species level.We revise the species pair concept in lichen-forming fungi,which recognizes sexually and asexually reproducing morphs with the same underlying phenotype as different species.We conclude that in most cases this concept does not hold,but the actual situation is complex and not necessarily correlated with reproductive strategy.In cases where no molecular data are available or where single or multi-marker approaches do not provide resolution,we recommend maintaining species pairs until molecular or phylogenomic data are available.This recommendation is based on the example of the species pair Usnea aurantiacoatra vs.U.antarctica,which can only be resolved with phylogenomic approaches,such as microsatellites or RADseq.Overall,we consider that species delimitation in lichen-forming fungi has advanced dramatically over the past three decades,resulting in a solid framework,but that empirical evidence is still missing for many taxa.Therefore,while phylogenomic approaches focusing on particular examples will be increasingly employed to resolve difficult species complexes,broad screening using single barcoding markers will aid in placing as many taxa as possible into a molecular matrix.We provide a practical pro-tocol how to assess and formally treat taxonomic novelties.While this paper focuses on lichen fungi,many of the aspects discussed herein apply generally to fungal taxonomy.The new combination Arthonia minor(Lücking)Lücking comb.et stat.nov.(Bas.:Arthonia cyanea f.minor Lücking)is proposed.

Delimiting species in Basidiomycota:a review

Species delimitation is one of the most fundamental processes in biology.Biodiversity undertakings,for instance,require explicit species concepts and criteria for species delimitation in order to be relevant and translatable.However,a perfect species concept does not exist for Fungi.Here,we review the species concepts commonly used in Basidiomycota,the second largest phylum of Fungi that contains some of the best known species of mushrooms,rusts,smuts,and jelly fungi.In general,best practice is to delimitate species,publish new taxa,and conduct taxonomic revisions based on as many independent lines of evidence as possible,that is,by applying a so-called unifying(or integrative)conceptual framework.However,the types of data used vary considerably from group to group.For this reason we discuss the different classes of Basidiomycota,and for each provide:(i)a general introduction with difficulties faced in species recognition,(ii)species concepts and methods for species delimitation,and(iii)community recommendations and conclusions.

Phylogeny and taxonomic revision of Thelonectria discophora(Ascomycota,Hypocreales,Nectriaceae)species complex

Specimens regarded as Thelonectria discophora(Thelonectria,Nectriaceae,Hypocreales)constitute a conspicuous group of saprobic fungi on decaying plant material,characterized by red perithecia each with a broad mammiform(nipple-like)apex.The asexual state is characterized by a cylindrocarpon-like morphology,with 3–5 septate macroconidia,unicellular microconidia and chlamydospores that are rarely produced in culture.In the past,T.discophora was regarded as one species with a wide geographic distribution.However,a recent study rejected the monophyly and cosmopolitan distribution of this species,and showed the existence of at least 16 cryptic species distributed in three main groups.By combining the results of phylogenetic analyses of six nuclear loci and morphological studies,we revised the taxonomy of the T.discophora species complex,resulting in the description of 12 new species and four new combinations based on historic names.Even though molecular phylogenetic analyses strongly support the segregation of these species,and are in agreement with previous studies,individual diagnostic morphological characters for each species could not be identified.However,discrete morphological traits corresponding to each of the three main groups of species were discovered.Lineages could be differentiated based on the average values of morphological traits as well as the presence/absence of characteristic asexual propagules and colony growth at 30C.Descriptions,illustrations are provided for the recognized species.

Predicting global numbers of teleomorphic ascomycetes

Sexual reproduction is the basic way to form high genetic diversity and it is beneficial in evolution and speciation of fungi.The global diversity of teleomorphic species in Ascomycota has not been estimated.This paper estimates the species number for sexual ascomycetes based on five different estimation approaches,viz.by numbers of described fungi,by fungus:substrate ratio,by ecological distribution,by meta-DNA barcoding or culture-independent studies and by previous estimates of species in Ascomycota.The assumptions were made with the currently most accepted,“2.2–3.8 million”species estimate and results of previous studies concluding that 90%of the described ascomycetes reproduce sexually.The Catalogue of Life,Species Fungorum and published research were used for data procurement.The average value of teleomorphic species in Ascomycota from all methods is 1.86 million,ranging from 1.37 to 2.56 million.However,only around 83,000 teleomorphic species have been described in Ascomycota and deposited in data repositories.The ratio between described teleomorphic ascomycetes to predicted teleomorphic ascomycetes is 1:22.Therefore,where are the undiscovered teleomorphic ascomycetes?The undescribed species are no doubt to be found in biodiversity hot spots,poorly-studied areas and species complexes.Other poorly studied niches include extremophiles,lichenicolous fungi,human pathogens,marine fungi,and fungicolous fungi.Undescribed species are present in unexamined collections in specimen repositories or incompletely described earlier spe-cies.Nomenclatural issues,such as the use of separate names for teleomorph and anamorphs,synonyms,conspecific names,illegitimate and invalid names also affect the number of described species.Interspecies introgression results in new species,while species numbers are reduced by extinctions.

In honor of John Bissett:authoritative guidelines on molecular identification of Trichoderma

Modern taxonomy has developed towards the establishment of global authoritative lists of species that assume the standard-ized principles of species recognition,at least in a given taxonomic group.However,in fungi,species delimitation is fre-quently subjective because it depends on the choice of a species concept and the criteria selected by a taxonomist.Contrary to it,identification of fungal species is expected to be accurate and precise because it should predict the properties that are required for applications or that are relevant in pathology.The industrial and plant-beneficial fungi from the genus Tricho-derma(Hypocreales)offer a suitable model to address this collision between species delimitation and species identification.A few decades ago,Trichoderma diversity was limited to a few dozen species.The introduction of molecular evolutionary methods resulted in the exponential expansion of Trichoderma taxonomy,with up to 50 new species recognized per year.Here,we have reviewed the genus-wide taxonomy of Trichoderma and compiled a complete inventory of all Trichoderma species and DNA barcoding material deposited in public databases(the inventory is available at the website of the Interna-tional Subcommission on Taxonomy of Trichoderma www.trich oderm a.info).Among the 375 species with valid names as of July 2020,361(96%)have been cultivated in vitro and DNA barcoded.Thus,we have developed a protocol for molecular identification of Trichoderma that requires analysis of the three DNA barcodes(ITS,tef1,and rpb2),and it is supported by online tools that are available on www.trich okey.info.We then used all the whole-genome sequenced(WGS)Trichoderma strains that are available in public databases to provide versatile practical examples of molecular identification,reveal short-comings,and discuss possible ambiguities.Based on the Trichoderma example,this study shows why the identification of a fungal species is an intricate and laborious task that requires a background in mycology,molecular biological skills,training in molecular evolutionary analysis,and knowledge of taxonomic literature.We provide an in-depth discussion of species concepts that are applied in Trichoderma taxonomy,and conclude that these fungi are particularly suitable for the implementa-tion of a polyphasic approach that was first introduced in Trichoderma taxonomy by John Bissett(1948-2020),whose work inspired the current study.We also propose a regulatory and unifying role of international commissions on the taxonomy of particular fungal groups.An important outcome of this work is the demonstration of an urgent need for cooperation between Trichoderma researchers to get prepared to the efficient use of the upcoming wave of Trichoderma genomic data.

Early divergence and differential population histories of the Indo-Pacific humpback dolphin in the Pacific and Indian Oceans

The currently recognized Indo-Pacific humpback dolphin occurs in estuaries and surrounding shallow waters from the South China Sea to the Asian coast of the Indian Ocean.However,a recent study suggested that the humpback dolphin from the Bay of Bengal may represent a distinct phylogenetic species.In this study,we sequenced 915-bp mtDNA segments from five geographic populations in both Chinese and Thai waters;together with previously published sequences,these data revealed that the ancestral Indo-Pacific humpback dolphin might have split during the transition from the Oligocene to Miocene(23.45 Mya,95%HPD:16.65–26.55 Mya),and then dispersed along the Pacific and Indian Ocean coasts of Asia.Genetic differentiation was detected between most of the examined populations,except for only a few pairwise populations in the northern South China Sea.Genetic differentiation/distance between the humpback dolphins from the northern and southern South China Sea met the sub-species threshold value proposed for marine mammals,whereas that between the humpback dolphins in the Pacific and the Indian Ocean was above the species threshold.Bayesian inference of historic gene flow indicated low but constant northward gene flow along the Indian Ocean coast;however,there was a recent abrupt increase in gene flow in the Pacific region,likely due to the shortening coastline at the low stand of sea level.Our results revealed that the current taxonomic classification of Indo-Pacific humpback dolphins may not reflect their phylogeography.

Molecular phylogeny of the genus Sticta (lichenized Ascomycota: Lobariaceae) in Colombia

We present a molecular phylogenetic study of the lichen genus Sticta focusing on Colombia,using the ITS fungal barcoding gene for a total of 370 ingroup OTUs,with 322 newly generated sequences.The topology resulting from a maximum likelihood approach does not support current species concepts in Sticta,which use a morphological concept,but in contrast shows that similar morphodemes evolved multiple times independently within the genus.As a consequence,currently applied names such as S.fuliginosa and S.weigelii comprise numerous(up to more than 20)unrelated species-level lineages,which can be distinguished also phenotypically using previously unrecognized characters such as lobe configuration,lobe surface structure,tomentum type,and anatomy of the basal membrane of the cyphellae.We conclude that the genus Sticta contains about four to five times the number of species currently recognized.In Colombia alone,approximately 150 species of Sticta are present.