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ABSTRACT
Hirsutella rhossiliensis is a parasite of juvenile nematodes, effective against a diversity of plant-parasitic nematodes. Its global
distribution on various nematode hosts and its genetic variation for several geographic regions have been reported, while the
global population genetic structure and factors underlying patterns of genetic variation of H. rhossiliensis are unclear. In this
study, 87 H. rhossiliensis strains from five nematode species (Globodera sp., Criconemella xenoplax, Rotylenchus robustus, Het-
erodera schachtii, and Heterodera glycines) in Europe, the United States, and China were investigated by multilocus sequence
analyses. A total of 280 variable sites (frequency, 0.6%) at eight loci and six clustering in high accordance with geographic popu-
lations or host nematode-associated populations were identified. Although H. rhossiliensis is currently recognized as an asexual
fungus, recombination events were frequently detected. In addition, significant genetic isolation by geography and nematode
hosts was revealed. Overall, our analyses showed that recombination, geographic isolation, and nematode host adaptation have
played significant roles in the evolutionary history of H. rhossiliensis.
IMPORTANCE
H. rhossiliensis has great potential for use as a biocontrol agent to control nematodes in a sustainable manner as an endopara-
sitic fungus. Therefore, this study has important implications for the use of H. rhossiliensis as a biocontrol agent and provides
interesting insights into the biology of this species.
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TABLE 1 Summary information for the Hirsutella rhossiliensis populations analyzed in this study
Geographic Host nematode- Host nematode No. of
populationa associated populationb Geographic location species isolates Latitude (north) Longitude (east)c
NL-P NL-GL Netherlands Globodera sp. 5 52.28 to 52.78 6.59 to 6.62
USA-P USA-CX California, USA Criconemella xenoplax 5 39.02 to 39.98 (121.18 to 121.98)
USA-P USA-CX Pennsylvania, USA Criconemella xenoplax 2 41.12 to 41.35 (78.28 to 78.82)
USA-P USA-RR California, USA Rotylenchus robusta 6 34.17 to 34.95 (115.21 to 115.39)
USA-P USA-RR Pennsylvania, USA Rotylenchus robusta 1 40.98 79.89
USA-P USA-HS California, USA Heterodera schachtii 6 36.14 to 36.89 (118.73 to 118.96)
USA-P USA/CN-HG Minnesota, USA Heterodera glycines 28 43.6 to 44.9 (92.63 to 94.93)
analyses based on genotyping and genealogical analyses can help population structure and their modes of reproduction. In this
to infer past evolutionary events and evaluate the potential pro- study, we used a multilocus sequence analysis (MLSA) scheme
cesses that generated the patterns of genetic variation (20, 21). An based on SNP markers to analyze population genetics of H.
analysis of the population genetics of Hirsutella minnesotensis in- rhossiliensis by sampling from three geographic populations
dicated that this dominant species in China had a clonal popula- and five host nematode-associated populations. We aimed to
tion structure and likely experienced a founder effect after coloni- understand (i) the level and partitioning of genetic variation
zation of China but without a significant bottleneck (22). within H. rhossiliensis, (ii) genetic structure and modes of re-
However, the population genetic structure, possible geographic production in H. rhossiliensis, and (iii) whether geographic isola-
origins, and host nematode adaptation of H. rhossiliensis are tion and host nematode adaptation lead to rapid differentiation
largely unknown. among populations of H. rhossiliensis. This study not only pro-
In recent years, single-nucleotide polymorphism (SNP) analy- vides essential insights into the population differentiation and
ses have been widely used to detect intraspecific variation in mi- host adaptation of H. rhossiliensis but also helps scientists to
croorganisms, including endoparasitic fungi (23, 24). When ana- better understand the ecological mechanism underlying the
lyzing the genetic diversities of human fungal pathogens, for natural control of H. rhossiliensis in its role as a biocontrol agent
instance, Histoplasma capsulatum (25), Candida albicans (26), against nematodes.
Aspergillus fumigatus (23), and Cryptococcus neoformans (27), it
has been demonstrated that SNP analyses were highly discrimina- MATERIALS AND METHODS
tive in these species. Based on multilocus sequence analyses (ML- Sample collection and DNA extraction. We employed all available H.
SAs), genetic diversity in plant-pathogenic fungi was also reported rhossiliensis isolates (87 in total) around the world for this study. They
for Magnaporthe grisea and Ustilaginoidea virens (28, 29). were classified into three geographic populations (Europe, the United
States, and China) and five host nematode-associated populations (Glo-
Field experiments demonstrated that H. rhossiliensis was effec-
bodera, H. schachtii, C. xenoplax, R. robusta, and H. glycines). Except for 8
tive for control of H. glycines in greenhouses (30). Current biolog- isolates from the CBS-KNAW Fungal Biodiversity Centre and 17 from the
ical control applications using H. rhossiliensis, however, have USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF),
shown relatively limited success in agricultural fields. We think the other 62 isolates were all recovered by us from parasitized H. glycines in
that an effective application of H. rhossiliensis or other fungi for Heilongjiang Province, China, or northern Minnesota, USA (Table 1 and
biological control requires a thorough understanding of their Fig. 1; see also Table S1 in the supplemental material).
FIG 1 Map of the geographic distribution of H. rhossiliensis: United States, Europe, and China.
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H. rhossiliensis Population Differentiation
To isolate H. rhossiliensis from parasitized H. glycines, we collected simple stepwise-addition heuristic searches with 1,000 replicates. A P
rhizosphere soil samples from soybean fields in the main soybean-pro- value lower than 0.001 indicated statistically significant differences (35).
ducing regions across northeast China from 2007 to 2008 and across Min- After identification of loci with no significant conflict, the phylogenic
nesota (USA) from 2010 to 2011. The sucrose flotation and centrifugation relationship among 87 strains was constructed using two methods: Bayes-
method (31) was used to extract the second-stage juveniles (J2s) that ian inference (BI) and maximum likelihood (ML). To conduct phyloge-
naturally occur in the soil, and these J2s were then transferred to wells of a netic analyses, we used the program PartitionFinder 1.1.1 (36) to evaluate
6-well tissue culture plate. The extracted J2s were examined with an in- the best partitioning scheme and determine suitable substitution models
verted microscope (Olympus, Japan) at 40 magnification, washed twice under the Bayesian information criterion (see Table S5 in the supplemen-
with sterilized water, and then placed on Difco potato dextrose agar tal material). For partitioned data sets, model parameters across different
(PDA) plates at 25C for 2 weeks. All of the filamentous fungi isolated partitions were unlinked, and the overall rate of evolution among parti-
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Wang et al.
TABLE 2 Patterns of genetic variation at the individual loci and combined-locus data sets
No. of sites
Aligned Nucleotide Haplotype
Gene length Parsimony Singleton diversity, diversity No. of Significance of
Fragment name identifier (bp) Variableb informativec variable Indel (102) (Hd) allelesd Tajimas D
nrDNA ITS 476 3 3 0 0 0.124 0.503 4 P 0.1
Beta-tubulin (tub) HIR_02075 284 8 3 0 5 0.072 0.194 4 P 0.1
Translation elongation HIR_06488 803 2 2 0 0 0.024 0.191 3 P 0.1
factor (tef)
Glycoside hydrolase HIR_00714 715 67 15 0 52 0.419 0.469 7 0.1 P 0.05
both the three geographic populations and the five host nematode-asso- Phylogenetic and population structure analyses of H. rho-
ciated populations. ssiliensis. Bayesian (BI) and maximum likelihood (ML) phyloge-
Mantel tests were performed in GenAlEx 6.5 for three groups of cor- netic trees were constructed based on the 8-locus concatenated
relations: between the fungal genetic distance and longitudinal-latitudinal data set. H. rhossiliensis strains tend to cluster by geography and
coordinates of geographic distance, between the fungal genetic distance
nematode hosts, and each of the two phylogenetic approaches
and longitudinal distance, and between the fungal genetic distance and
latitudinal distance. For the genetic distances, we used the pairwise pop- identified six clusters: European strains from Globodera sp. (NL-
ulation PhiPT values, which represent genetic distances among all pairs of GL), American strains from C. xenoplax (USA-CX), American
populations as a tri-matrix, and these distances were then compared with strains from R. robusta (USA-RR), American strains from H.
the geographic distances between populations. schachtii (USA-HS), American strains from H. glycines (USA-
Accession number(s). All of the nucleotide sequences obtained in this HG), and Chinese (northeastern) strains from H. glycines (CN-
study have been submitted to the GenBank database, and the accession HG). However, there were subtle topological differences between
numbers are KP885720 to KP886014 (see also Table S2 in the supplemen- the two approaches. For example, two American strains from H.
tal material).
schachtii (CBS567.92 and ARSEF3761) did not group into the
RESULTS USA-HS subclade, and the positions of two Chinese strains from
H. glycines (CN-30-2-JX and CN-3-10-JL) and one American
Nucleotide variations. To determine appropriate markers, we
strain from C. xenoplax (ARSEF3746) had inconsistent place-
successfully amplified the 42 candidate fragments from each of
ments between the BI and ML trees (Fig. 2; see also Fig. S2 in the
the 12 test isolates. Six fragments (HIR_00714, HIR_06686,
HIR_08121, HIR_08679, HIR_01569, and HIR_09423) showed a supplemental material).
high frequency of nucleotide variation and existed as single copies The network diagram showed a linear structure among the 87
as indicated by BLAST searches against the H. rhossiliensis ge- strains (Fig. 3), with Chinese strains from H. glycines, American
nome. The six fragments, together with three commonly used strains from H. glycines, European strains from Globodera sp.,
markers (nrDNA ITS, tub, and tef), were finally chosen to amplify American strains from H. schachtii, American strains from R. ro-
from each of the 87 isolates (Table 2). Direct sequencing of PCR busta, and American strains from C. xenoplax each forming an
products from any of the nine fragments indicated no heteroge- individual cluster.
neity in sequencing chromatograms. Three to 11 alleles were iden- Genetic differentiation and gene flow among geographic or
tified in our samples depending on the locus. Moreover, the result host nematode-associated populations. A high value for the ge-
of a neutrality test showed that all nine markers were neutral netic differentiation coefficient Fst (P 0.01) revealed that signif-
(Table 2). icant genetic differentiation occurred among all of the geographic
A significant conflict was detected by PHT between HIR_00714 or host nematode-associated populations except between NL-P
and the other loci (see Table S4 in the supplemental material). and USA-P, the value for which was not significant (P 0.01)
Therefore, HIR_00714 was excluded from the concatenated data (Tables 3 and 4). Consistently, the estimated gene flow was low
set in the following analyses. From the concatenation of the re- among the three geographic populations (Nm 0.14) and among
maining eight loci, a total of 280 variable sites within the com- the five host nematode-associated populations (Nm 0.28).
bined 4,658-bp sequence and 44 multilocus haplotypes were iden- Evidence for recombination. The PrCP index provided evi-
tified from the 87 isolates (Table 2). dence for recombination except for two populations, American
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FIG 2 Bayesian tree of the H. rhossiliensis strains based on the concatenated 8-locus data set. The strict consensus tree from phylogenetic analysis was constructed
using a Bayesian inference method. The numbers above the branches indicate the posterior probability (PP) support for each node according to the Bayesian
inference (nodes with PPs of 0.5 are shown).
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TABLE 3 Population genetic parameters for each of three geographic populations of H. rhossiliensis
Fst valueb
Geographic No. of Nucleotide Haplotype
population haplotypes diversity, (102) diversity (Hd) PrCPa (P value) P value (w) NL-P USA-P
NL-P 4 0.053 0.9 0.964 (0.271) 0.042
USA-P 23 0.195 0.804 0.750 (0.001) 0.192 0.007
CN-P 17 0.378 0.868 0.857 (0.933) 0.005 0.607** 0.399**
Total 44 0.393 (0.001) 0.001
a
PrCP, proportion of phylogenetically compatible pairs of loci.
b
Fst, fixation index. **, P 0.01
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H. rhossiliensis Population Differentiation
TABLE 4 Population genetic parameters for each of five host nematode-associated populations of H. rhossiliensis
Fst valuea
Host nematode-associated No. of Nucleotide diversity, Haplotype P value
population No. haplotypes (102) diversity (Hd) PrCP (P value) (w) NL-GL USA-CX USA-RR USA-HS
NL-GL 5 4 0.053 0.9 0.964 (0.271) 0.042
USA-CX 7 7 0.04 1 1 (1) 0.248 0.532**
USA-RR 7 3 0.038 0.524 1 (1) 0.776** 0.634**
USA-HS 6 6 0.125 1 0.892 (0.717) 0.24 0.173* 0.542** 0.763**
USA/CN-HG 62 24 0.445 0.849 0.785 (0.261) 0.001 0.325* 0.749** 0.746** 0.341*
a
*, 0.01 P 0.05; **, P 0.01.
TABLE 5 Summary results of the analysis of molecular variance TABLE 6 Summary results of the analysis of molecular variance
(AMOVA) within and among three geographic populations of H. (AMOVA) within and among five host nematode-associated
rhossiliensisa populations of H. rhossiliensisa
Estimated Estimated
Source df SS MS variance % Statistic Value P Source df SS MS variance % Statistic Value P
Among populations 2 48.571 24.286 0.981 0.44 PhiPT 0.436 0.001 Among populations 4 71.073 17.768 1.634 61 PhiPT 0.615 0.001
Within populations 84 106.486 1.268 1.268 0.56 PhiPT 0.505 0.001 Within populations 84 83.985 1.024 1.024 39 PhiPT 0.527 0.002
Total 86 155.057 2.248 1 Total 86 155.057 2.658 100
a a
There were 3 geographic populations and 87 isolates. Abbreviations: df, degree of There were 5 host-nematode associated populations and 87 isolates. Abbreviations: df,
freedom; SS, sum of squared observations; MS, mean of squared observations. degree of freedom; SS, sum of squared observations; MS, mean of squared observations.
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