Population Genetics of Hirsutella rhossiliensis, a Dominant Parasite ofCyst Nematode Juveniles on a Continental Scale

Niuniu Wang,a,b Yongjie Zhang,c Xianzhi Jiang,a Chi Shu,a,b M. Imran Hamid,a Muzammil Hussain,a,b Senyu Chen,d Jianping Xu,e

Meichun Xiang,a Xingzhong Liua

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, Chinaa; University of Chinese Academy of Sciences, Beijing, Chinab;School of Life Sciences, Shanxi University, Taiyuan, Chinac; Southern Research and Outreach Center, University of Minnesota, Waseca, Minnesota, USAd; Department ofBiology, McMaster University, Hamilton, ON, Canadae

ABSTRACT

Hirsutella rhossiliensis is a parasite of juvenile nematodes, effective against a diversity of plant-parasitic nematodes. Its globaldistribution on various nematode hosts and its genetic variation for several geographic regions have been reported, while theglobal population genetic structure and factors underlying patterns of genetic variation of H. rhossiliensis are unclear. In thisstudy, 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 sequenceanalyses. 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 asexualfungus, recombination events were frequently detected. In addition, significant genetic isolation by geography and nematodehosts was revealed. Overall, our analyses showed that recombination, geographic isolation, and nematode host adaptation haveplayed 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 providesinteresting insights into the biology of this species.

It is well known that fungi possess diverse strategies to obtainnutrients, as saprobes, pathogens (of plant and animal hosts),

and symbionts (with other microbes, plants, or animals) (1). Oneof the important living strategies is parasitoidism, where parasi-toids sterilize and/or kill their hosts before the hosts reach repro-ductive age. Plant-parasitic nematodes are worldwide agriculturalpests and are responsible for global agricultural losses amountingto an estimated $157 billion each year (2). Endoparasitic fungi arepredominantly soil-dwelling organisms that have the ability tosterilize plant-parasitic nematodes (3). They produce characteris-tic spores that adhere to, penetrate, and consume nematode bod-ies (4, 5).

Hirsutella rhossiliensis (Ophiocordycipitaceae, Hypocreales,Ascomycota) (6) lives in the soil, and it has been mainly isolatedfrom five nematodes including Criconemella xenoplax (7), Het-erodera schachtii (8), Heterodera glycines (9), Rotylenchus robustus(10), and Globodera (11). Though a dominant parasitoid of H.glycines J2s (second-stage juveniles) in the United States, mainlyfrom Minnesota, H. rhossiliensis occurs less frequently in north-east China (12). In the agricultural fields, the plants, nematodes,and endoparasitic fungi constitute a tight tripartite system withthe plants providing food to nematodes and the nematodes serv-ing as nitrogen sources to endoparasitic fungi. This existing natu-ral interaction suggests that endoparasitic fungi could have greatpotential for use as biocontrol agents to control nematodes in asustainable manner (13–15).

To date, H. rhossiliensis has been detected in many localities inEurope, the United States, and China (11, 12). Genetic variationamong different isolates of H. rhossiliensis has been detected usingrandomly amplified polymorphic DNA (RAPD) markers (16) and

analyses of two housekeeping genes, internal transcribed spacer(ITS) of ribosomal DNA (rDNA) and the mitogen-activated pro-tein kinase (MAPK) gene (13). Because of its global distributionand wide host range, H. rhossiliensis provides an ideal model tostudy the effects of geography and host nematodes on fungal pop-ulation genetic divergence and factors driving genetic differentia-tion of fungi.

Investigating the evolutionary patterns and processes of nem-atode endoparasitic fungi over their geographical and ecologicaldistribution provides valuable information on the radiation be-tween nematode endoparasitic fungi and hosts (17). While phe-notypic plasticity and genomic heterogeneity can lead to broadgeographic and ecological distribution, low levels of gene flowoften result in microbial speciation (18, 19). Population genetic

Received 6 June 2016 Accepted 4 August 2016

Accepted manuscript posted online 19 August 2016

Citation Wang N, Zhang Y, Jiang X, Shu C, Hamid MI, Hussain M, Chen S, Xu J,Xiang M, Liu X. 2016. Population genetics of Hirsutella rhossiliensis, a dominantparasite of cyst nematode juveniles on a continental scale. Appl Environ Microbiol82:6317–6325. doi:10.1128/AEM.01708-16.

Editor: A. A. Brakhage, HKI and University of Jena

Address correspondence to Meichun Xiang, xiangmc@im.ac.cn.

N.W. and Y.Z. contributed equally to this work.

C.S. met authorship criteria but was unreachable for final approval of the bylineand article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01708-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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analyses based on genotyping and genealogical analyses can helpto infer past evolutionary events and evaluate the potential pro-cesses that generated the patterns of genetic variation (20, 21). Ananalysis of the population genetics of Hirsutella minnesotensis in-dicated that this dominant species in China had a clonal popula-tion structure and likely experienced a founder effect after coloni-zation of China but without a significant bottleneck (22).However, the population genetic structure, possible geographicorigins, and host nematode adaptation of H. rhossiliensis arelargely unknown.

In recent years, single-nucleotide polymorphism (SNP) analy-ses have been widely used to detect intraspecific variation in mi-croorganisms, including endoparasitic fungi (23, 24). When ana-lyzing the genetic diversities of human fungal pathogens, forinstance, Histoplasma capsulatum (25), Candida albicans (26),Aspergillus fumigatus (23), and Cryptococcus neoformans (27), ithas been demonstrated that SNP analyses were highly discrimina-tive in these species. Based on multilocus sequence analyses (ML-SAs), genetic diversity in plant-pathogenic fungi was also reportedfor Magnaporthe grisea and Ustilaginoidea virens (28, 29).

Field experiments demonstrated that H. rhossiliensis was effec-tive for control of H. glycines in greenhouses (30). Current biolog-ical control applications using H. rhossiliensis, however, haveshown relatively limited success in agricultural fields. We thinkthat an effective application of H. rhossiliensis or other fungi forbiological control requires a thorough understanding of their

population structure and their modes of reproduction. In thisstudy, we used a multilocus sequence analysis (MLSA) schemebased on SNP markers to analyze population genetics of H.rhossiliensis by sampling from three geographic populationsand five host nematode-associated populations. We aimed tounderstand (i) the level and partitioning of genetic variationwithin H. rhossiliensis, (ii) genetic structure and modes of re-production in H. rhossiliensis, and (iii) whether geographic isola-tion and host nematode adaptation lead to rapid differentiationamong populations of H. rhossiliensis. This study not only pro-vides essential insights into the population differentiation andhost adaptation of H. rhossiliensis but also helps scientists tobetter understand the ecological mechanism underlying thenatural control of H. rhossiliensis in its role as a biocontrol agentagainst nematodes.

MATERIALS AND METHODSSample collection and DNA extraction. We employed all available H.rhossiliensis isolates (87 in total) around the world for this study. Theywere classified into three geographic populations (Europe, the UnitedStates, and China) and five host nematode-associated populations (Glo-bodera, H. schachtii, C. xenoplax, R. robusta, and H. glycines). Except for 8isolates from the CBS-KNAW Fungal Biodiversity Centre and 17 from theUSDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF),the other 62 isolates were all recovered by us from parasitized H. glycines inHeilongjiang Province, China, or northern Minnesota, USA (Table 1 andFig. 1; see also Table S1 in the supplemental material).

TABLE 1 Summary information for the Hirsutella rhossiliensis populations analyzed in this study

Geographicpopulationa

Host nematode-associated populationb Geographic location

Host nematodespecies

No. ofisolates Latitude (north) Longitude (east)c

NL-P NL-GL Netherlands Globodera sp. 5 52.28 to 52.78 6.59 to 6.62USA-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 �79.89USA-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)CN-P USA/CN-HG Heilongjiang, Heihe, China Heterodera glycines 8 49.13 to 50.13 127.16 to 127.25CN-P USA/CN-HG Heilongjiang, Jixi, China Heterodera glycines 11 45.18 to 45.38 131.11 to 131.25CN-P USA/CN-HG Heilongjiang, Jiamusi, China Heterodera glycines 6 46.5 130.26 to 130.28CN-P USA/CN-HG Heilongjiang, Suihua, China Heterodera glycines 7 46.13 to 46.48 125.28 to 126.30CN-P USA/CN-HG Jilin, China Heterodera glycines 1 43.63 122.46CN-P USA/CN-HG Heilongjiang, Nehe, China Heterodera glycines 1 48.49 124.89Total samples 87 36.14 to 52.78 6.59 to 131.25;

�(78.28 to 121.98)a NL-P, population from Netherlands; USA-P, population from USA; CN-P, population from China.b NL-GL, strains isolated from Globodera sp.; USA-CX, strains isolated from C. xenoplax; USA-RR, strains isolated from R. robusta; USA-HS, strains isolated from H. schachtii; USA/CN-HG, strains isolated from H. glycines.c Negative values (with parentheses) represent west longitude.

FIG 1 Map of the geographic distribution of H. rhossiliensis: United States, Europe, and China.

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To isolate H. rhossiliensis from parasitized H. glycines, we collectedrhizosphere soil samples from soybean fields in the main soybean-pro-ducing regions across northeast China from 2007 to 2008 and across Min-nesota (USA) from 2010 to 2011. The sucrose flotation and centrifugationmethod (31) was used to extract the second-stage juveniles (J2s) thatnaturally occur in the soil, and these J2s were then transferred to wells of a6-well tissue culture plate. The extracted J2s were examined with an in-verted microscope (Olympus, Japan) at �40 magnification, washed twicewith sterilized water, and then placed on Difco potato dextrose agar(PDA) plates at 25°C for 2 weeks. All of the filamentous fungi isolatedfrom these J2s were cultured on PDA plates covered with a piece of cello-phane paper, and 0.05 to 0.1 g of mycelia was harvested and used to extractgenomic DNA with the cetyltrimethylammonium bromide (CTAB)method (32). Fungal cultures were identified based on their morphology(30) and nonribosomal DNA (nrDNA) ITS sequences (13).

Molecular marker selection. We initially chose three housekeepinggenes as molecular markers: the nrDNA ITS region, the beta-tubulin gene(tub), and the translation elongation factor 1� gene (tef). However, thenumber of phylogenetically informative sites provided by the three frag-ments was insufficient. Therefore, novel DNA markers were determinedby searching the genome data (15). Specifically, 27 fragments of genesputatively involved in the infection processes of H. rhossiliensis into nem-atodes and 15 fragments of interest as a result of a genomic comparisonbetween H. minnesotensis and H. rhossiliensis were chosen as candidatemarkers. PCR primers were designed for each of the 42 fragments usingthe online software Primer3 (http://frodo.wi.mit.edu/primer3/). From 12randomly chosen isolates of different origins (CBS104.94, CBS105.94,ARSEF2006, ARSEF3746, ARSEF2788, ARSEF2789, ARSEF3755,ARSEF3756, CN-13-1, CN-30-1, USA-87-2, and USA-92-1) (see Table S1in the supplemental material), we amplified and sequenced each of the 42DNA fragments. Fragments with a high frequency of nucleotide variationwere finally chosen as markers used in this study. A neutrality test for theselected markers was performed by DnaSP 5.10 (33).

PCR amplification and sequence determination. Nine molecularmarkers, including ITS, tub, tef, and six newly identified markers, wereamplified from each of the 87 H. rhossiliensis isolates. Each PCR mixture(50 �l) was composed of 5 �l of 10� EasyTaq buffer, 5 �l of deoxynucleo-side triphosphate (dNTP) mixture (2.5 mM), 2 �l of each primer (10�M), 1 �l of genomic DNA, 0.5 �l of EasyTaq DNA polymerase (Trans-Gen Biotech, Beijing, China), and 36.5 �l of double-distilled water(ddH2O). The PCRs were performed in a TGradient thermocycler (Bi-ometra, Germany) under the following conditions: denaturation at 94°Cfor 3 min; 35 cycles of denaturation at 94°C for 40 s, annealing at variabletemperatures for 50 s, and elongation at 72°C for 1 min; and a final 10-minelongation step at 72°C. After confirmation of the PCR products by aga-rose gel electrophoresis, the amplicons were cleaned using the 3S SpinPCR product purification kit (Biocolor Bioscience & Technology Com-pany, China). The purified PCR products were then sequenced using anABI 3730 XL DNA sequencer (Applied Biosystems, USA) with Sangerdideoxy terminator sequencing. In the case of poor sequencing qualityresulting from the direct sequencing of PCR products, the representativePCR fragments were cloned, and individually cloned fragments were thensequenced again.

Nucleotide variations. Sequences of the fragments were aligned withthe Muscle algorithm of MEGA 6.0 (34) and trimmed to remove ambig-uously aligned regions. The nucleotide diversity (�), haplotype diversity,number of polymorphic sites, and allele numbers were estimated for bothindividual fragment and combined sequences using the program DnaSP5.10 (33). By employing DnaSP 5.10, alleles at each locus were assignedand combined into an allelic profile designated a haplotype (HA) for eachstrain.

Phylogenetic and network analyses. We performed a partition ho-mogeneity test (PHT) to detect potential phylogenetic conflicts betweendifferent loci using PAUP version 4.0b10 (Sinauer Associates, Sunder-land, MA, USA; D. Swofford, 2003). PHT used informative characters and

simple stepwise-addition heuristic searches with 1,000 replicates. A Pvalue lower than 0.001 indicated statistically significant differences (35).After identification of loci with no significant conflict, the phylogenicrelationship among 87 strains was constructed using two methods: Bayes-ian inference (BI) and maximum likelihood (ML). To conduct phyloge-netic analyses, we used the program PartitionFinder 1.1.1 (36) to evaluatethe best partitioning scheme and determine suitable substitution modelsunder the Bayesian information criterion (see Table S5 in the supplemen-tal material). For partitioned data sets, model parameters across differentpartitions were unlinked, and the overall rate of evolution among parti-tions was allowed to vary. The Bayesian analysis, which was performed inMrBayes 3.2.2 (37), was initiated with random starting trees, run for 100million generations with four incrementally heated chains (Metropolis-coupled Markov chain Monte Carlo [38]), and sampled at intervals of10,000 generations. To avoid entrapment in the local optima, two inde-pendent Bayesian analyses were run, and the log-likelihood scores werecompared for convergence (38). An ML analysis was performed inRAxML version 8.0.0 (39) and conducted under the GTRGAMMAmodel, and the ML support was assessed by 1,000 bootstrap replicates.Two H. minnesotensis strains, CN3608 and CBS113353, were used as theoutgroup.

A phylogenetic network was constructed using the median-joiningmethod implemented in the program SplitsTree 4.12 (40) to further assessthe relationships among worldwide H. rhossiliensis haplotypes. This model-free method uses a parsimony approach based on pairwise differences toconnect each sequence to its closest neighbor and allows for the creationof internal nodes (median vectors), which are interpreted as unsampled orextinct ancestral genotypes to link the existing genotypes in the mostparsimonious way (41).

Population genetic analyses. To determine whether the number ofloci was sufficient to represent the genotypic diversity of the populations,we plotted the relationship between the number of loci and the genotypediversity using MultiLocus1.3b (42). A population genetic analysis wasalso performed within and between the three geographic populations andwithin and between the five host nematode-associated populations. Forthe within-population genetic analysis, we analyzed the genetic variationpatterns with DnaSP 5.10, including the number of haplotypes, nucleo-tide diversity, and haplotype diversity. For the cross-population geneticanalysis, pairwise differentiation coefficients (Fst) (43) were calculatedand tested for significance against 1,000 bootstrap replicates using Arle-quin 3.1 (44), and the average gene flow (Nm) (45) was calculated usingDnaSP 5.10.

Tests for recombination. Sexual reproduction generates random as-sociations between alleles at different loci because genes from differentindividuals are mixed in a given population by sexual reproduction.Therefore, when recombination occurs among populations, random as-sociations between alleles may be observed at different loci, even if sexualreproduction has not been observed in the natural populations (46). Toidentify evidence for recombination in H. rhossiliensis, two methods wereused: the proportion of compatible pairs of loci (PrCP) (47) and the �w

test (or pairwise homoplasy index [PHI]) (48). Tests for recombinationbased on the principle of compatibility have been proven to be the mostpowerful (49, 50). Two loci are compatible (PrCP � 1) if it is possible toaccount for all the observed genotypes by mutations without inferringhomoplasy (reversals, parallelisms, or convergences) or recombination;otherwise, the loci are incompatible (PrCP 1). The �w test means thatphylogenetic incompatibility is an indicator of recombination at thepopulation level; the lack of phylogenetic incompatibility, in contrast,implies asexual reproduction. The tests for PrCP were calculated usingMultiLocus1.3b (42) with 1,000 randomizations; the �w test was calcu-lated by SplitsTree 4.12.

Tests for genetic isolation according to geography and host nema-todes. An analysis of molecular variance (AMOVA) was performed todetermine the relative contribution of genetic variation among and withinpopulations using GenAlEx 6.5 (51). The AMOVA was conducted for

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both the three geographic populations and the five host nematode-asso-ciated populations.

Mantel tests were performed in GenAlEx 6.5 for three groups of cor-relations: between the fungal genetic distance and longitudinal-latitudinalcoordinates of geographic distance, between the fungal genetic distanceand longitudinal distance, and between the fungal genetic distance andlatitudinal distance. For the genetic distances, we used the pairwise pop-ulation PhiPT values, which represent genetic distances among all pairs ofpopulations as a tri-matrix, and these distances were then compared withthe geographic distances between populations.

Accession number(s). All of the nucleotide sequences obtained in thisstudy have been submitted to the GenBank database, and the accessionnumbers are KP885720 to KP886014 (see also Table S2 in the supplemen-tal material).

RESULTSNucleotide variations. To determine appropriate markers, wesuccessfully amplified the 42 candidate fragments from each ofthe 12 test isolates. Six fragments (HIR_00714, HIR_06686,HIR_08121, HIR_08679, HIR_01569, and HIR_09423) showed ahigh frequency of nucleotide variation and existed as single copiesas indicated by BLAST searches against the H. rhossiliensis ge-nome. The six fragments, together with three commonly usedmarkers (nrDNA ITS, tub, and tef), were finally chosen to amplifyfrom each of the 87 isolates (Table 2). Direct sequencing of PCRproducts from any of the nine fragments indicated no heteroge-neity in sequencing chromatograms. Three to 11 alleles were iden-tified in our samples depending on the locus. Moreover, the resultof a neutrality test showed that all nine markers were neutral(Table 2).

A significant conflict was detected by PHT between HIR_00714and the other loci (see Table S4 in the supplemental material).Therefore, HIR_00714 was excluded from the concatenated dataset in the following analyses. From the concatenation of the re-maining eight loci, a total of 280 variable sites within the com-bined 4,658-bp sequence and 44 multilocus haplotypes were iden-tified from the 87 isolates (Table 2).

Phylogenetic and population structure analyses of H. rho-ssiliensis. Bayesian (BI) and maximum likelihood (ML) phyloge-netic trees were constructed based on the 8-locus concatenateddata set. H. rhossiliensis strains tend to cluster by geography andnematode hosts, and each of the two phylogenetic approachesidentified six clusters: European strains from Globodera sp. (NL-GL), American strains from C. xenoplax (USA-CX), Americanstrains from R. robusta (USA-RR), American strains from H.schachtii (USA-HS), American strains from H. glycines (USA-HG), and Chinese (northeastern) strains from H. glycines (CN-HG). However, there were subtle topological differences betweenthe two approaches. For example, two American strains from H.schachtii (CBS567.92 and ARSEF3761) did not group into theUSA-HS subclade, and the positions of two Chinese strains fromH. glycines (CN-30-2-JX and CN-3-10-JL) and one Americanstrain from C. xenoplax (ARSEF3746) had inconsistent place-ments between the BI and ML trees (Fig. 2; see also Fig. S2 in thesupplemental material).

The network diagram showed a linear structure among the 87strains (Fig. 3), with Chinese strains from H. glycines, Americanstrains from H. glycines, European strains from Globodera sp.,American strains from H. schachtii, American strains from R. ro-busta, and American strains from C. xenoplax each forming anindividual cluster.

Genetic differentiation and gene flow among geographic orhost nematode-associated populations. A high value for the ge-netic differentiation coefficient Fst (P 0.01) revealed that signif-icant genetic differentiation occurred among all of the geographicor host nematode-associated populations except between NL-Pand USA-P, the value for which was not significant (P 0.01)(Tables 3 and 4). Consistently, the estimated gene flow was lowamong the three geographic populations (Nm � 0.14) and amongthe five host nematode-associated populations (Nm � 0.28).

Evidence for recombination. The PrCP index provided evi-dence for recombination except for two populations, American

TABLE 2 Patterns of genetic variation at the individual loci and combined-locus data sets

Fragment nameGeneidentifier

Alignedlength(bp)

No. of sitesNucleotidediversity,� (10�2)

Haplotypediversity(Hd)

No. ofallelesd

Significance ofTajima’s DVariableb

Parsimonyinformativec

Singletonvariable Indel

nrDNA ITS 476 3 3 0 0 0.124 0.503 4 P 0.1Beta-tubulin (tub) HIR_02075 284 8 3 0 5 0.072 0.194 4 P 0.1Translation elongation

factor (tef)HIR_06488 803 2 2 0 0 0.024 0.191 3 P 0.1

Glycoside hydrolase HIR_00714 715 67 15 0 52 0.419 0.469 7 0.1 P 0.05SignalP HIR_06686 807 191 18 2 171 0.281 0.531 8 P 0.1Abhydrolase HIR_08121 754 7 6 1 0 0.363 0.487 7 P 0.1Fucose-specific lectin HIR_08679 711 11 8 3 0 0.213 0.515 11 P 0.1Nucleoside triphosphate

hydrolasesHIR_01569 350 11 6 5 0 0.405 0.598 8 P 0.1

Exo_endo_phos HIR_09423 473 47 47 0 0 3.269 0.587 10 0.1 P 0.05Combined 9-locus data set 5,373 347 108 11 228 0.497 0.921 44Combined 8-locus data set

excluding HIR_00714a

4,658 280 93 11 176 0.493 0.921 44

a The sequences of HIR_00714 are excluded due to the partition homogeneity test results (see Table S3 in the supplemental material). The fungal combined data set withoutHIR_00714 was used in all the following analyses.b The variable sites consist of substitution sites (phylogenetically informative or uninformative) and indel (insertion/deletion) sites.c At least two mutations occurred no less than twice after sequence alignment.d The number of individuals or multiple loci is represented by the most dominant allele or multilocus haplotypes.

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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 constructedusing a Bayesian inference method. The numbers above the branches indicate the posterior probability (PP) support for each node according to the Bayesianinference (nodes with PPs of 0.5 are shown).

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strains from C. xenoplax (USA-CX) and American strains from R.robusta (USA-RR). When the 87 strains were analyzed together,recombination signals were found in 3/5 locus pairs (Table 3; seealso Table S6 in the supplemental material).

Similarly, the �w test found statistically significant evidence forrecombination (P 0.001). However, there was a slight differencein the �w results for recombination among the three geographicpopulations and five host nematode-associated populations com-pared with the PrCP results. For example, the �w test did not findstatistically significant evidence for recombination in the USA-P,USA-CX, USA-HS, or USA-RR population (Table 3).

Genetic isolation by geography or host nematodes. TheAMOVA results showed that 44% of the genetic variation could beattributed to variation among the three geographic populationsand 56% could be attributed to variation among the individualisolates within populations (Table 5). For the five host nematode-associated populations, the AMOVA results showed that 61% and39% of the genetic variation could be attributed to variationamong the populations and among individual isolates within pop-ulations, respectively. Both of these sources of variation were sig-nificant (P 0.01) (Table 6).

The Mantel tests revealed significant genetic isolation by dis-tance irrespective of the horizontal geographical distances (basedon longitudinal-latitudinal coordinates, along a longitudinal gra-dient, or along a latitudinal gradient) or population divisions(three geographic populations) employed (Table 7).

DISCUSSION

We collected 87 H. rhossiliensis strains from five host nematodes inEurope, the United States, and China. Although a large number ofstrains were obtained from H. glycines, at least three strains wereincluded from each of the other nematode hosts (Table 1; see alsoTable S1 in the supplemental material). We would explain thatour samples from four nematodes (Globodera sp., H. schachtii, C.xenoplax, and R. robusta) were isolated in the 1980s and 1990s,while samples from H. glycines were isolated during 2007 to 2011.Because some nematodes did not occur in China or we did notfind this fungus from them (11, 52–54), we failed to isolate H.rhossiliensis from other host nematodes except from H. glycines. Itwas also difficult to obtain strains from the above four host nem-atodes (except H. glycines) in the United States. The cultures fromculture collection centers (CBS and ARSEF) were preserved inlyophilized forms, which largely decreased strain mutations overthe past 20 to 30 years (see Table S1 in the supplemental material).Although there might be some genetic variations of fungus innature, we believe that the variation during 20 to 30 years couldnot affect the main results obtained from this study.

In this study, recombination was found in most of the H. rho-ssiliensis populations and when the whole samples were consid-ered. It can be inferred and supported by two lines of evidence, thePrCP values and the �w test (Tables 3 and 4). The PrCP test mea-sures whether two loci are compatible, and the �w test examines

FIG 3 Median joining of phylogenetic network inferred with H. rhossiliensis strain concatenated 8-locus data set. This network includes all of the mostparsimonious trees linking the isolates. Each unique haplotype is represented by a block, with the block’s size indicating the haplotype’s frequency in the data set.Red blocks denote haplotypes parasitizing H. glycines in China, blue blocks denote haplotypes from H. glycines in the United States, green blocks denotehaplotypes parasitizing Globodera, yellow blocks denote haplotypes parasitizing R. robusta, black blocks denote haplotypes parasitizing H. schachtii, and pinkblocks denote haplotypes parasitizing C. xenoplax.

TABLE 3 Population genetic parameters for each of three geographic populations of H. rhossiliensis

Geographicpopulation

No. ofhaplotypes

Nucleotidediversity, � (10�2)

Haplotypediversity (Hd) PrCPa (P value) P value (�w)

Fst valueb

NL-P USA-P

NL-P 4 0.053 0.9 0.964 (0.271) 0.042USA-P 23 0.195 0.804 0.750 (0.001) 0.192 0.007CN-P 17 0.378 0.868 0.857 (0.933) 0.005 0.607** 0.399**Total 44 0.393 (0.001) 0.001a PrCP, proportion of phylogenetically compatible pairs of loci.b Fst, fixation index. **, P 0.01

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phylogenetic incompatibility. So, it is reasonable that there is con-flict between the two results (49). Indeed, the sexual cycles of mostfungi are difficult to observe in nature (55), and inferences of thepotential for a sexual cycle to occur have largely relied on analysesof gene and genotype frequencies in natural populations. The sex-ual stage of H. rhossiliensis has not been discovered in nature or inthe laboratory, although we detected mating type genes in its ge-nome (unpublished data). In plant fungal pathogens, a high levelof recombination provides an advantage in rapidly generatingmany new combinations of virulence genes to counterbalancecorresponding resistance genes in the host (56, 57). In arbuscularmycorrhizal fungi, recombination or recombination-like eventsin addition to clonality have greatly contributed to genetic diver-sity (58). This study has demonstrated evidence of recombinationamong H. rhossiliensis strains, and recombination appears to beone of the factors shaping the evolution of H. rhossiliensis. Similarresults were also observed in Candida albicans, which showed bothclonality and recombination, even though a complete sexualitystage is not known to exist in this fungus (59). However, sexualrecombination may not be the sole factor because we cannot ex-clude the possibility that parasexual recombination has contrib-uted to the observed linkage equilibrium and phylogenetic incom-patibility (60).

Our population genetics analyses of H. rhossiliensis were basedon two types of population divisions: three geographic popula-tions (NL-P, USA-P, and CN-P) and five host nematode-associ-ated populations (NL-GL, USA-CX, USA-RR, USA-HS, and USA/CN-HG) (Table 1). The significance of geography and the hostnematodes in shaping the structure of H. rhossiliensis populationson a large scale was supported by the strong divergent signals inthe phylogenetic and haplotype network analyses (Fig. 2 and 3). Inthis study, a significant positive correlation was observed betweenH. rhossiliensis genetic distance and latitudinal and longitudinalgeographic distance, thus indicating that geography is an im-portant barrier to gene flow and has promoted the genetic di-vergence within fungal species (Table 7). Moreover, differenti-ation among the three geographic populations was also

observed from high Fst values and low Nm values. We suggestthat the North Pacific Ocean may be a boundary separating theChinese population and the American population of H. rhossil-iensis because of limited opportunities to disperse via wind orsoil. A similar geography-based separation has been reported inColletotrichum truncatum from chili peppers in China that weregenetically differentiated into southern and northern popula-tions (61). Previously, we found significant differences amonglocal populations of H. minnesotensis from China and the UnitedStates, where isolation by geography plays a key role in geneticdifferentiation (22). Based on these results, we conclude that geo-graphic distance contributes to driving population genetic differ-entiation of H. rhossiliensis.

Among the five host nematode-associated populations studiedhere, we identified positive correlations between H. rhossiliensisgenetic distance at different latitudinal and longitudinal distances.We observed 61% of the molecular genetic variance among thosenematode-associated populations. This might result from host-related selection driving population genetic differentiation at neu-tral and selected loci (62). Another explanation was that host shiftsmight occur frequently between neighboring nematodes, whichincreased the mating probability of H. rhossiliensis across thenearby hosts and also increased the genetic variation within andamong populations.

Our analyses revealed that both nematode hosts and geographycontributed to genetic differentiation among H. rhossiliensis pop-ulations (Tables 5 to 7). We, however, could not perform a two-way AMOVA to further look at their relative effects because pop-ulation assignments of our samples did not meet the requirementfor performing such an analysis. In this study, we confirmed thatdifference in host nematode was a key factor in regulating geneticdivergence of nematode parasitic fungus from the phylogeneticapproaches and the haplotype network (Fig. 2 and 3). Similarly,the strong association of genetic groups with different geographiclocations suggested the important role of geographic isolation.Previous studies on microorganisms such as Bacillus simplex con-cluded that genetic differentiation was strongly attributed to geo-

TABLE 4 Population genetic parameters for each of five host nematode-associated populations of H. rhossiliensis

Host nematode-associatedpopulation No.

No. ofhaplotypes

Nucleotide diversity,� (10�2)

Haplotypediversity (Hd) PrCP (P value)

P value(�w)

Fst valuea

NL-GL USA-CX USA-RR USA-HS

NL-GL 5 4 0.053 0.9 0.964 (0.271) 0.042USA-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(AMOVA) within and among three geographic populations of H.rhossiliensisa

Source df SS MSEstimatedvariance % Statistic Value P

Among populations 2 48.571 24.286 0.981 0.44 PhiPT 0.436 0.001Within populations 84 106.486 1.268 1.268 0.56 PhiPT 0.505 0.001Total 86 155.057 2.248 1a There were 3 geographic populations and 87 isolates. Abbreviations: df, degree offreedom; SS, sum of squared observations; MS, mean of squared observations.

TABLE 6 Summary results of the analysis of molecular variance(AMOVA) within and among five host nematode-associatedpopulations of H. rhossiliensisa

Source df SS MSEstimatedvariance % Statistic Value P

Among populations 4 71.073 17.768 1.634 61 PhiPT 0.615 0.001Within populations 84 83.985 1.024 1.024 39 PhiPT 0.527 0.002Total 86 155.057 2.658 100a There were 5 host-nematode associated populations and 87 isolates. Abbreviations: df,degree of freedom; SS, sum of squared observations; MS, mean of squared observations.

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graphic locations (63). Numerous studies found that geographicregion and host species contribute equally to the population ge-netic differentiation, for instance, Grosmannia clavigera (64),Claviceps purpurea (65), Venturia inaequalis (66), Arthrobotrys oli-gospora (67), Colletotrichum gloeosporioides (68), and Metarhiz-ium anisopliae (69).

In conclusion, our study identified the population genetic struc-ture of the nematode endoparasitic fungus H. rhossiliensis and founda high level of genetic variation among its populations worldwide.Using multiple sources of evidence, we demonstrated that recombi-nation among strains, isolation by geography, and isolation based onthe host nematode were factors driving the genetic differentiation ofH. rhossiliensis. Therefore, the findings of this study on the popula-tion structure of H. rhossiliensis and the associated multiple drivingfactors affecting its genetic diversity provide an essential understand-ing of the mechanisms of evolution and differentiation of the funguson a global scale. These findings are expected to provide importantinsights into nematode control by this fungus.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation ofChina (grant no. 31430071) and the National Basic Research Program ofChina (973 program, grant no. 2013CB127506).

We thank Richard A. Humber, who kindly provided 15 strains fromthe USDA-ARS Collection of Entomopathogenic Fungal Cultures(ARSEF) database at the USDA-ARS Biological Integrated Pest Manage-ment Research Unit, Cornell University, New York, USA.

FUNDING INFORMATIONThis work, including the efforts of Xingzhong Liu, was funded by NationalBasic Research Program of China (2013CB127506). This work, includingthe efforts of Xingzhong Liu, was funded by National Natural ScienceFoundation of China (NSFC) (31430071).

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