# Updates in the Language of Histoplasma Biodiversity

Pierre Gladieux

► To cite this version:

Pierre Gladieux. Updates in the Language of Histoplasma Biodiversity. mBio, 2018, 9 (3), 10.1128/mBio.00181-18. hal-02619830

HAL Id: hal-02619830

https://hal.inrae.fr/hal-02619830

Submitted on 25 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Check for
updates

# Updates in the Language of *Histoplasma* Biodiversity

Pierre Gladieuxª

ªBGPI, Université de Montpellier, INRA, CIRAD, Montpellier SupAgro, Montpellier, France

**ABSTRACT** In a recent article, Sepúlveda et al. (mBio 8:e01339-17, 2017, https://doi.org/10.1128/mBio.01339-17) investigated the genetic structure and evolutionary history of the human pathogen *Histoplasma*. Using whole-genome resequencing data, Sepúlveda et al. found that the *Histoplasma* genus is composed of at least four strongly differentiated lineages. Their tour de force is to use a smart combination of population genomic approaches to show that the advanced stage of intraspecific divergence observed within *Histoplasma* does not simply reflect population structure, but instead results from previously unidentified speciation events. The four independently evolving *Histoplasma* lineages are elevated to the species status and assigned names. The newly described species exhibit medically important differences in phenotype, and these findings, therefore, have important epidemiological implications. This work provides a blueprint for phylogenomic species recognition in fungi, opening the way for a new age of enlightenment in which fungal species are diagnosed using highly discriminatory tools within a hypothesis-testing framework.

**KEYWORDS** fungi, gene flow, genomics, pathogens, speciation, systematics, taxonomy

## FROM PHYLOGENETIC TO PHYLOGENOMIC FUNGAL SPECIES RECOGNITION

The last three decades have witnessed a sustained and concerted attempt to modernize fungal taxonomy, the Latin gibberish used as the language of fungal biodiversity. This modernization effort culminated in the abolishment of the dual nomenclature that permitted the use of different names for the asexual and sexual forms of the same species (1) and translates into a massive weeding out of name redundancy. Besides the dilemma of choosing meaningful and acceptable names, mycologists are also striving to clarify the language of fungal biodiversity by probing into the two main sources of taxonomic ambiguity, uncertainty, and error: the tremendous diversity of fungi and the difficulty of identifying discontinuities in the distribution of genotypes and phenotypes that reflect the existence of independently evolving lineages called species. A great effort is being made to fill in gaps in the fungal tree of life, via large-scale studies of the functional and genetic diversity of fungi in the environment and the sustained production of well-characterized, foundational reference data of fungal genomes (2). Attempts to characterize more finely species boundaries and the structure of fungal biodiversity have also accumulated since the beginning of the millennium. Mycologists tend to abandon methods of species delineation based on *in vitro* measurements of (mostly prezygotic) interfertility and reproductive morphology, given the notorious uncooperativeness of fungi for such experiments (3). Because large-scale crossing studies are formidably challenging in fungi, and because fungal morphology is essentially cryptic, phylogenetic species recognition based on genealogical concordance has emerged as the new "gold standard" for fungal species delineation.

The principle of phylogenetic species recognition schemes is generally to compare nucleotide polymorphisms at typically <10 regions of ca. 500 nucleotides and to identify highly supported monophyletic groups that are concordant across genealogies inferred from different regions and that reflect the existence of reproductively isolated

Published 8 May 2018

**Citation** Gladieux P. 2018. Updates in the language of *Histoplasma* biodiversity. mBio 9:e00181-18. https://doi.org/10.1128/mBio.00181-18.

**Copyright** © 2018 Gladieux. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to
pierre.gladieux@inra.fr.

For the article discussed, see https://doi.org/10.1128/mBio.01339-17.

The views expressed in this Commentary do not necessarily reflect the views of this journal or of ASM.
groups (4). The immense advantage of species recognition by genealogical concor-
dance is reproducibility and archivability; the main drawbacks are that reproducibility
requires the use of the same set of sequenced loci across studies and that the resolving
power of sequenced loci is generally unknown a priori. The sequence of diagnostic
markers can be readily retrieved from whole-genome sequences, but using genomic
data to characterize a few loci is clearly overkill and ignores the mass of information
available in genomic sequences. Hence, the challenge is not so much to use genomic
data to feed classical approaches to species recognition but to develop methods that
make full use of the wealth of information encoded in whole-genome resequencing
data. A great benefit of using a genomic approach to identify fungal species is that
fungi have relatively small genomes and haploid genetics and as such are highly
amenable to genome resequencing. The current flood of fungal genomic data me-
chanically raises the demand for methods of species identification that can handle such
data, but fungal eco-evolutionary genomics lacks a unified and relatively simple
framework to elucidate taxonomic subdivisions using population genomics. Working
with the human fungal pathogen *Histoplasma*, Sepúlveda et al. (5) deploy the full
arsenal of population methods to indirectly measure the presence of gene flow based
on resequencing data, and they demonstrate the presence of independently evolving
*Histoplasma* lineages that seldom exchange genes, even while coexisting in sympatry.

SPECIATION AND SEMIPERMEABLE BARRIERS TO GENE FLOW IN HISTOPLASMA

*Histoplasma* causes a respiratory infection in humans called histoplasmosis, the most common endemic mycosis in the Americas. Histoplasmosis is also a significant health issue globally, and *Histoplasma* became a genetic model system. The HIV pandemic and increasing use of immunosuppressive medications may have contributed to spread histoplasmosis globally (6), but many aspects regarding the natural and colonization history, environmental reservoirs, and genetic structure of *Histoplasma* remain poorly understood.

In the recent literature, *Histoplasma*—historically the anamorphic classification of
the fungus—appears to be preferred over the teleomorphic names *Emmonsiella* and
*Ajellomyces*, and there seems to be no debate on this matter. *Histoplasma* was deter-
mined to be closely related to other pathogenic dimorphic fungi such as *Coccidioides*,
*Blastomyces*, and *Paracoccidioides*, but notable differences in reproductive biology
warrant distinct generic names (7). Questions rather concern the cryptic genetic
diversity encompassed by the genus, as previous studies based on multilocus Sanger
sequencing revealed strong population structure across continents, raising the possi-
bility of undiscovered events of speciation (8, 9). Sepúlveda et al. (5) use a genomic
approach to investigate the presence of cryptic species within *Histoplasma*. Their data
analysis workflow starts with an unsupervised, model-free approach to analyzing
population subdivision. Having identified at least five discrete geographic clusters, the
authors set out to explore if the clusters represented distinct phylogenetic species.

One first possible approach to explore the existence of cryptic species based on
genomic data is to identify single-copy orthologs, reconstruct genealogical relation-
ships for all orthologs, and use a computational approach that scales to large sets of loci
(e.g., see reference 10) to analyze genealogical concordance. However, this approach
assumes that single-copy orthologs were correctly identified, which is not always
guaranteed. Moreover, the size of orthologous gene sets is expected to decrease with
the inclusion of more phylogenetically distant lineages, which can hinder comparisons
across studies (11). An alternative approach, used by Sepúlveda et al. (5), is to take the
supercontigs as the individual loci. Depending on the completeness of the assembly,
one can chose to include all supercontigs in the analysis, or just the largest ones, so that
it remains feasible to analyze genealogical concordance by eye. Using the latter
approach, Sepúlveda et al. (5) were able to show that the clusters encompassed by
*Histoplasma* are reciprocally monophyletic in supercontig genealogies, with genealog-
ical concordance across genealogies inferred for the 10 largest supercontigs. These
findings show that previously unidentified speciation events occurred within *Histo-
plasma and that the five genetic groups of *Histoplasma* can be referred to as phylo-
genetic species.

Species diagnostics using species recognition remains an approximate representa-
tion of biological diversity in terms of independently evolving lineages. Methods of
species recognition are designed to detect lineages that have a long-term history of
independent evolution, but none can formally reject the presence of some postdiver-
gence gene flow. Phylogenetic species recognition is based on the premise that conflict
among gene genealogies is due to recombination among conspecifics, and the tran-
sition from concordance to conflict determines the limits of species. However, the
homogenizing effect of gene flow between divergent lineages is expected to only
affect a fraction of the genome, not necessarily enough to break concordance among
chromosomal genealogies. Hence, the pattern of genealogical concordance uncovered
in *Histoplasma* by Sepúlveda et al. (5) can be seen as a proof of the existence of
independently evolved lineages called species—warranting the development of
species-specific treatments and diagnostic tools—but the finding of phylogenomic
species does not preclude more detailed analyses of interlineage gene flow. The data
analysis workflow employed by Sepúlveda et al. (5) to explore the extent of gene
exchange across newly discovered *Histoplasma* species involved the following steps: (i)
quantify the extent of concordance at the whole-genome level and infer the branching
order of phylogenetic species using species tree analyses and total evidence genome
genealogy; (ii) measure the relative depth of population subdivision by comparing
measurements of nucleotide diversity within and between lineages; (iii) assess the
plausibility of incomplete lineage sorting as a confounding factor in the analysis of
genetic exchanges across lineages; and (iv) test for postdivergence admixture and gene
flow using model-based clustering algorithms, phylogenetic tests of gene flow based
on numbers of shared derived alleles, and graph-based analyses of population mix-
tures. Hence, the authors do not merely diagnose species, but they also examine the
divergence history of species, quantify the relative depth of divergence between them,
and assess hypotheses of interspecific gene flow, and by doing so, they not only
corroborate their species delineation work, but also contribute new knowledge about
*Histoplasma's* natural history.

A BLUEPRINT FOR ASSESSING EVOLUTIONARY INDEPENDENCE IN MICROBIAL
EUKARYOTES

The study by Sepúlveda et al. (5) provides a blueprint for the diagnosis of fungal
species using whole-genome resequencing data. Their framework both builds on
classical methods of species recognition by genealogical concordance and takes full
advantage of the inferential power of speciation genomics (12). By incorporating
state-of-the-art methods to analyze divergence and gene flow, their approach is more
precise, and also more informative, than classical phylogenetic species recognition
based on multilocus sequence typing. For instance, speciation genomic analyses can
lead to biogeographical hypotheses that would be hardly accessible to nongenomic
species recognition by genealogical concordance: species trees and whole-genome
analyses of genealogical branching patterns can provide information about the role of
vicariance in speciation, and admixture analyses can identify hybrids and show that the
species have come into contact in the past. In practical terms, the analytic framework
proposed by Sepúlveda et al. (5) has the advantage of being flexible, with the possibility
of replacing tools as new approaches become available. Another important practical
aspect is that diagnostic assays for epidemiology research and clinical microbial
identification can be designed by choosing, in Sepúlveda et al.'s data, loci that
recapitulate the supercontig genealogies. One possible limitation though is that taxo-
nomic updates will have to be based on genomic data, as the identification of species
is based on the concordance among supercontig genealogies. With species identifica-
tion implemented in a hypothesis-testing framework, it is still possible that the new
species names propelled into the taxonomic space will collide with the conservative
inclinations (which generally forms objects with large inertial mass) of the scientists
<figure><img src="image_3.png" /></figure>

concerned, but the reality of the diagnosed species can hardly be refuted without the inclusion of more data and analysis. The study by Sepúlveda et al. (5) bears the promise that "a new age of enlightenment is at hand in fungal systematics" (13), with mycol- ogists emphasizing hypothesis testing within a speciation genomics framework rather than traditional taxonomic rules and approaches as they endeavor to create a language of fungal biodiversity that captures more accurately the underlying reality of biological discontinuity.

## ACKNOWLEDGMENT

I thank Tatiana Giraud for helpful discussions.

## REFERENCES

1. Hawksworth DL, Crous PW, Redhead SA, Reynolds DR, Samson RA, Seifert KA, Taylor JW, Wingfield MJ, Abaci O, Aime C, Asan A, Bai FY, de Beer ZW, Begerow D, Berikten D, Boekhout T, Buchanan PK, Burgess T, Buzina W, Cai L, Cannon PF, Crane JL, Damm U, Daniel HM, van Diepeningen AD, Druzhinina I, Dyer PS, Eberhardt U, Fell JW, Frisvad JC, Geiser DM, Geml J, Glienke C, Gräfenhan T, Groenewald JZ, Groenewald M, de Gruyter J, Guého-Kellermann E, Guo LD, Hibbett DS, Hong SB, de Hoog GS, Houbraken J, Huhndorf SM, Hyde KD, Ismail A, Johnston PR, Kadaifciler DG, Kirk PM, Köljalg U, Kurtzman CP, Lagneau PE, Lévesque CA, Liu X, Lombard L, Meyer W, Miller A, Minter DW, Najafzadeh MJ, Norvell L, Ozerskaya SM, Oziç R, Pennycook SR, Peterson SW, Pettersson OV, Quaedvlieg W, Robert VA, Ruibal C, Schnürer J, Schroers HJ, Shivas R, Slippers B, Spierenburg H, Takashima M, Taşkin E, Thines M, Thrane U, Uztan AH, van Raak M, Varga J, Vasco A, Verkley G, Videira SI, de Vries RP, Weir BS, Yilmaz N, Yurkov A, Zhang N. 2011. The Amsterdam Declaration on fungal nomenclature. IMA Fungus 2:105-112. https://doi.org/10.5598/imafungus.2011.02.01.14.

2. Grigoriev IV. 2014. A changing landscape of fungal genomics, p 1-20. In Martin F (ed), The ecological genomics of fungi. John Wiley & Sons, Inc, Hoboken, NJ.

3. Taylor JW, Turner E, Townsend JP, Dettman JR, Jacobson D. 2006. Eukaryotic microbes, species recognition and the geographic limits of species: examples from the kingdom Fungi. Philos Trans R Soc Lond B Biol Sci 361:1947-1963. https://doi.org/10.1098/rstb.2006.1923.

4. Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31:21-32. https://doi.org/10.1006/fgbi.2000.1228.

5. Sepúlveda VE, Márquez R, Turissini DA, Goldman WE, Matute DR. 2017. Genome sequences reveal cryptic speciation in the human pathogen Histoplasma capsulatum. mBio 8:e01339-17. https://doi.org/10.1128/mBio.01339-17.

6. Wheat LJ, Azar MM, Bahr NC, Spec A, Relich RF, Hage C. 2016. Histoplas- mosis. Infect Dis Clin North Am 30:207-227. https://doi.org/10.1016/j.idc.2015.10.009.

7. Kwon-Chung KJ. 1973. Studies on Emmonsiella capsulata. I. Heterothal- lism and development of the ascocarp. Mycologia 65:109-121.

8. Kasuga T, White TJ, Koenig G, McEwen J, Restrepo A, Castañeda E, Da Silva Lacaz C, Heins-Vaccari EM, De Freitas RS, Zancopé-Oliveira RM, Qin Z, Negroni R, Carter DA, Mikami Y, Tamura M, Taylor ML, Miller GF, Poonwan N, Taylor JW. 2003. Phylogeography of the fungal pathogen Histoplasma capsulatum. Mol Ecol 12:3383-3401. https://doi.org/10.1046/j.1365-294X.2003.01995.x.

9. Teixeira Mde M, Patané JS, Taylor ML, Gómez BL, Theodoro RC, de Hoog S, Engelthaler DM, Zancopé-Oliveira RM, Felipe MS, Barker BM. 2016. Worldwide phylogenetic distributions and population dynamics of the genus histoplasma. PLoS Neglect Trop Dis 10:e0004732. https://doi.org/10.1371/journal.pntd.0004732.

10. Brankovics B, van Dam P, Rep M, de Hoog GS, J van der Lee TAJ, Waalwijk C, van Diepeningen AD. 2017. Mitochondrial genomes reveal recombi- nation in the presumed asexual Fusarium oxysporum species complex. BMC Genomics 18:735. https://doi.org/10.1186/s12864-017-4116-5.

11. Gladieux P, Condon B, Ravel S, Soanes D, Maciel JLN, Nhani A, Jr, Chen L, Terauchi R, Lebrun M-H, Tharreau D, Mitchell T, Pedley KF, Valent B, Talbot NJ, Farman M, Fournier E. 2018. Gene flow between diver- gent cereal- and grass-specific lineages of the rice blast fungus Magnaporthe oryzae. mBio 9:e01219-17. https://doi.org/10.1128/mBio.01219-17.

12. Seehausen O, Butlin RK, Keller I, Wagner CE, Boughman JW, Hohenlohe PA, Peichel CL, Saetre GP, Bank C, Brännström A, Brelsford A, Clarkson CS, Eroukhanoff F, Feder JL, Fischer MC, Foote AD, Franchini P, Jiggins CD, Jones FC, Lindholm AK, Lucek K, Maan ME, Marques DA, Martin SH, Matthews B, Meier JI, Möst M, Nachman MW, Nonaka E, Rennison DJ, Schwarzer J, Watson ET, Westram AM, Widmer A. 2014. Genomics and the origin of species. Nat Rev Genet 15:176-192. https://doi.org/10.1038/nrg3644.

13. Hibbett DS, Taylor JW. 2013. Fungal systematics: is a new age of en- lightenment at hand? Nat Rev Microbiol 11:129-133. https://doi.org/10.1038/nrmicro2963.