Corresponding author: Danilo E. Bustamante (
Academic editor: P. Chaverri
The genus
Bustamante DE, Oliva M, Leiva S, Mendoza JE, Bobadilla L, Angulo G, Calderon MS (2019) Phylogeny and species delimitations in the entomopathogenic genus
Around 1800, a silkworm disease called “calcine”, “real del segno” or “muscardine” was causing great trouble in Italy and France (
The genus
Based on the end of dual nomenclature for different morphs of the same fungus in 2011 (
Initially,
In the Amazonian region, a total of five species have been reported (
Given the problems with species delimitation in fungi using morphology, molecular data are becoming the standard for delimiting species and testing their traditional boundaries (
In this study, we analyzed species of the newly circumscribed genus
Fungal strains were isolated from infected coffee borers (
Collections of the 55 strains of
Fifty-five fungal strains were incubated as monosporic cultures on
Genomic DNA was extracted from semisolid
List of species used in the molecular analyses.
Species | Country | Strain |
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Colombia | HUA 179219 | – |
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Colombia | HUA 179221 | – |
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Colombia | HUA 179220 | – |
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Colombia | MCA 1181 | – |
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– | |
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Australia | ARSEF4149 |
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USA, Colorado | ARSEF7542 |
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Chile | B518a |
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Peru | ARSEF1969 |
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Brazil | ARSEF2641 |
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China | ARSEF4384 |
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China | ARSEF4474 |
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Korea | ARSEF4850 |
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Australia | ARSEF4580 |
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Australia | ARSEF4622 |
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Australia | WCN2015 |
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Japan | ARSEF1040 |
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Australia | ARSEF300 |
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Italy | ARSEF1564 |
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Japan | ARSEF7518 |
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Vietnam | ARSEF751 |
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Brazil | ARSEF1478 |
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Morocco | ARSEF1811 |
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Japan | ARSEF7516 |
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USA, Oregon | ARSEF10278 |
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Korea | ARSEF7268 |
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USA, New York | ARSEF6213 |
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Japan | ARSEF4363 |
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Japan | ARSEF4362 |
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USA, Kentucky | ARSEF2271 |
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USA, Oregon | ARSEF10277 |
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France | ARSEF979 |
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Switzerland | ARSEF1567 |
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Scotland | ARSEF2567 |
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Denmark | ARSEF8024 |
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Brazil | ARSEF2251 |
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USA, Georgia | ARSEF7117 |
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Australia | ARSEF4302 |
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Ecuador | QCNE 186272 | – |
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Ecuador | QCNE 186714 | – |
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Ecuador | MCA 1557 | – |
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Reunion | Bt116 |
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Reunion | Bt121 |
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Reunion | Bt124 |
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Reunion | Bt125 |
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Reunion | Bt128 |
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Reunion | Bt129 |
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Madagascar | Bt96 |
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Reunion | Bt99 |
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USA, Hawaii | ARSEF7032 |
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China | RCEF5500 |
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China | GZU12142 |
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China | GZU12141 |
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Australia | ARSEF4755 |
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Australia | BCC17613 |
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Malawi | ARSEF7760 |
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Peru | UTRF21 |
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Peru | UTRF24 |
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Peru | UTRF25 |
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Peru | UTRF26 |
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Peru | UTRF35 |
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Peru | UTRF37 |
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Peru | UTRF38 |
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Peru | UTRF40 |
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Peru | UTRF42 |
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Peru | UTRF58 |
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Peru | UTRP6 |
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Peru | UTRP7 |
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Peru | UTRP13 |
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Peru | UTRP17 |
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Peru | UTRP19 |
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Portugal | ARSEF3220 |
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USA, Kentucky | ARSEF3405 |
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USA, Wisconsin | ARSEF3216 |
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USA, Maryland | ARSEF3529 |
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France | ARSEF4933 |
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Canada | ARSEF1855 |
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Canada | ARSEF2997 |
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China | ARSEF6229 |
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Korea | ARSEF7242 |
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Korea | ARSEF5689 | – |
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Japan | ARSEF1685 |
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Korea | ARSEF5689 |
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Korea | ARSEF7043 |
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Korea | ARSEF7044 |
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Korea | ARSEF7279 |
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Korea | ARSEF7280 |
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Korea | ARSEF7281 |
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China | RCEF3903 | – |
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Korea | ARSEF5718 | – |
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France | ARSEF8259 |
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Switzerland | ARSEF2694 |
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France | ARSEF8257 |
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Chile | ARSEF2922 |
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Korea | ARSEF7260 |
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USA | OSC93610 | – |
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– | PC546 | – |
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The phylogeny was based on concatenated data combining
Although 26 species have been molecularly confirmed in
We explored five different DNA-based delimitation methods using
To perform the
To validate the outcomes of single locus species delimitation, a multilocus
In the phylogeny of
Phylogenetic tree based on maximum likelihood inference of combined
Genetic distance (p-distances) in percentage for species of
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1.3 | 0.4 | 0.2 | |
3.1 | 0.5 | 0.2 | |
3.5–4.1 | 0.3–0.5 | 0.2–0.4 | |
4.1–4.7 | 0.7–1.1 | 0.2 |
The species-delimitation methods based on genetic distance (
Species number in
Taxa | Genetic distance | Coalescence | Genealogical concordance | Phylogeny | ||||||||
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– | 1 | 1 | – | x | x | – | 1 | 1 | – | 1 | 1 |
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1 | 4 | 3 | 5 | 2 | x | 5 | 3 | 2 | 1 | 1 | |
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2 | x | 1 | 2 | 1 | x | 2 | 1 | 1 | 1 | 1 | |
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x | 1 | 1 | 3 | 1 | x | 2 | 1 | 1 | 1 | 1 | |
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6 | x | 5 | 6 | x | x | 6 | 3 | 3 | 1 | 1 | |
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– | x | 1 | – | x | x | – | 1 | 1 | – | 1 | |
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x | 1 | 2 | 2 | 1 | x | 8 | 1 | 1 | 1 | 1 | |
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1 | 1 | 2 | 1 | 1 | x | 6 | 1 | 1 | 1 | 1 | |
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– | 2 | 2 | – | x | x | – | 2 | 2 | – | 1 | |
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1 | 1 | 1 | 8 | 1 | 1 | 8 | 1 | 1 | 1 | 1 | |
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1 | 1 | 1 | 1 | 1 | x | 1 | 1 | 1 | 1 | 1 | |
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1 | 1 | 1 | 1 | 1 | x | 1 | 1 | 1 | 1 | 1 | |
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– | 1 | 1 | – | x | x | – | 1 | 1 | – | 1 | |
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1 | x | x | x | 1 | x | 1 | 1 | 1 | 1 | 1 | |
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1 | 1 | 1 | 2 | 1 | 1 | 2 | 1 | 1 | 1 | 1 | |
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1 | 3 | 1 | 2 | 1 | 1 | 9 | 3 | 2 | 1 | 1 | |
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1 | 1 | 1 | x | 1 | x | 6 | 1 | 1 | 1 | 1 | |
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– | 1 | 1 | – | 1 | x | 1 | 1 | 1 | – | 1 | |
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– | x | x | – | x | x | – | x | x | – | 1 | |
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1 | 1 | 1 | 1 | 1 | x | 2 | 1 | 1 | 1 | 1 | |
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1 | 1 | 1 | x | 1 | x | 1 | 1 | 1 | 1 | 1 | |
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1 | x | x | 1 | x | x | 2 | 1 | 1 | 1 | 1 | |
Total | 20 | 22 | 28 | 35 | 16 | 3 | 63 | 28*** | 26*** | 16* | 1 | 22 |
x = non recognized as species, - = not evaluated, * = posterior probabilities higher or equal than 0.53, *** =highly significant
Species very similar morphologically to
Morphology of
PERU. Amazonas: Prov. Rodríguez de Mendoza, Dist. Huambo, latitude -6.469, longitude -77.376, elev. 1642 m, entomopathogenic, 08 Nov. 2017, G. Ángulo, UTRP19 (holotype: UFV5609; isotype: ARSEF14196).
Colony growth on
This species is widely spread on coffee plantations in the middle altitudes of the Amazon region in northeastern Peru.
The specific epithet ‘
PERU. Amazonas: Prov. Rodríguez de Mendoza, Dist. Chirimoto, Achamal,
Accurate species identification within the entomopathogenic fungi
The use of multi-locus sequence data is essential to establish robust species boundaries (Lumbsch and Levitt 2011), and our results for
Regarding the genetic distance methods, the
In the coalescence methods, although 6 species were not included in the
Regarding
Recently, polyphasic approaches have been used to reflect the natural classification of species within many important fungal genera (
This study was supported by the National Institute of Agrarian Innovation of Peru (Project number: 002-2016-INIA-PNIA-UPMSI/IE).
Tables S1, S2, Figures S1–S4
molecular data