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Genetic diversity in the gypsy moth fungal pathogen Entomophaga maimaiga from founder populations in North America and source populations in Asia
Mycol. Res. 109 (8): 941–950 (August 2005). f The British Mycological Society
941
doi:10.1017/S0953756205003278 Printed in the United Kingdom.
Genetic diversity in the gypsy moth fungal pathogen Entomophaga maimaiga from founder populations in North America and source populations in Asia
Charlotte NIELSEN1*, Michael G. MILGROOM2 and Ann E. HAJEK1 1
Department of Entomology, Cornell University, Comstock Hall, Ithaca, NY 14853-0901, USA. Department of Plant Pathology, Cornell University, Ithaca, NY 14853-5904, USA. E-mail : [email protected] 2
Received 25 October 2004; accepted 21 March 2005.
Entomophaga maimaiga is a naturally occurring fungal pathogen specific to larvae of the gypsy moth, Lymantria dispar. E. maimaiga is thought to be native to Asia where its epizootics can suppress gypsy moth outbreaks. However, in the USA this beneficial fungal pathogen was not observed until 1989, although an isolate of E. maimaiga from Tokyo was released in Massachusetts to control gypsy moths as early as in 1910–1911, and another isolate from Ishikawa Prefecture in Japan was later released in 1985 and 1986 in New York and Virginia. Our objectives were to : (1) test the hypothesis that E. maimaiga populations in the USA have reduced genetic variability due to founder effects compared to the putative ancestral populations in Asia; (2) track the origin of the North American populations of this fungus; and (3) assess whether genetic differences among E. maimaiga isolates are correlated to morphological differences. We compared genetic diversity among 30 E. maimaiga isolates originating from seven states in the USA, five prefectures in Japan, one province of China and one region of far eastern Russia by AFLPs. Among 14 USA isolates, only ten polymorphic AFLP loci were found, whereas 56 polymorphic loci were found among 16 Asian isolates ; 29 loci were polymorphic among 12 isolates from Japan alone. Average gene diversity (h) for the polymorphic loci was 0.223¡0.005 for Asia (including Japan), 0.131¡0.006 for Japan only, and 0.041¡0.004 for the USA. Thus, native populations from Asia were more diverse than the USA populations. These results are consistent with the expectation of a population founded from a source population by a small number of individuals. Distance and parsimony analyses of AFLP data showed that the isolates from the USA formed one distinct clade that was most closely related to Japanese isolates collected outside the Tokyo area. No morphological variation of E. maimaiga from different geographical locations was detected.
INTRODUCTION Studies of population structures of fungi over the last 20 years have enhanced our understanding of the processes of selection, mutation, gene flow, genetic drift, and mating systems. The likelihood of drift increases as population size decreases, so drift is very likely and, therefore, a very important evolutionary mechanism in small populations, e.g. populations that pass through bottlenecks. Severe bottlenecks often occur when a population is established in a new geographical area because only a few colonists or founders typically establish the new population. The consequence for a population founded by a small number of individuals is fewer alleles per locus, reduced gene diversity and often modified allele frequencies compared to their source population (Nei, Maruyama & Chakraborty 1975). * Corresponding author.
Such so-called founder effects have been described previously in relation to source populations for several fungal or oomycete plant pathogens, including the chestnut blight fungus Cryphonectria parasitica (Milgroom, Lipari & Wang 1992, Milgroom et al. 1996), the beech bark disease fungus Neonectria coccinea (Mahoney et al. 1999) and the potato late blight pathogen Phytophthora infestans (Fry & Spielman 1991). Even though a substantial number of papers have reported the successful use of molecular techniques to study different aspects of entomopathogenic Entomophthorales for biocontrol of insects (e.g. Walsh et al. 1990, Bidochka et al. 1997, Jensen, Thomsen & Eilenberg 2001, Nielsen et al. 2001, Hajek et al. 2003, Tymon, Shah & Pell 2004), little is known about their population structure. One of the few exceptions is the use of cluster analysis of RAPD data to infer the origin of Zoophthora phytonomi in North America (Hajek et al. 1996b).
AFLP diversity of Entomophaga maimaiga Entomophaga maimaiga is a naturally occurring fungal pathogen specific to larvae of the gypsy moth, Lymantria dispar (Lepidoptera : Lymantriidae) with multinucleate spores. The life-cycle of this fungus consists of several asexual generations of airborne conidia during summer, followed by dormancy over winter as azygospores (resting spores) in the soil (Hajek 1999). No sexual stage is known in this fungus although McCabe, Humber & Soper (1984) inferred cytological evidence for nuclear fusion in azygospores for the closely related fungus E. grylli. The prevalence of this fungus in gypsy moth larvae may, in some periods, exceed 99% infection, leading to complete suppression of outbreak populations (Koyama 1954, Takamura & Sato 1973, Aoki 1974, Yanbe 1976). The fungus is thus considered to be the most important natural enemy of gypsy moth (Hajek 1999). E. maimaiga was originally described from Japan (Soper et al. 1988) and is, as the gypsy moth, thought to be native to Asia. Despite many years of intensive surveys for diseased gypsy moths by entomologists in the USA, E. maimaiga was not observed in the field until 1989 (Andreadis & Weseloh 1990, Hajek et al. 1990, Hajek, Humber & Elkinton 1995). Since then, it has spread across the distribution of gypsy moths from its epicentre in the north-eastern coastal area (Andreadis & Weseloh 1990, Hajek et al. 1995, Hajek, Elkinton & Witcosky 1996a). Nevertheless, before the sudden appearance of the fungus in 1989, a small number of introductions of E. maimaiga to USA gypsy moth populations for control purposes had been attempted. The first releases of this fungus were as early as 1910 and 1911, when two diseased gypsy moth cadavers collected in the Tokyo area provided inoculum for infection of a large number of larvae which subsequently were released near Boston, Massachusetts. However, in both 1910 and 1911, no fungal infections were recovered as a result of the field release (Speare & Colley 1912), and establishment was presumed to have failed (Soper et al. 1988, Hajek et al. 1995, Hajek 1999). The next attempts to establish E. maimaiga were in 1985 and 1986 when an isolate originating from Ishikawa Prefecture, Japan, was released in New York State and Virginia, respectively. Careful collection of gypsy moth from the release sites documented that disease transmission probably did not occur in 1985 and was extremely low in 1986. Intensive collection of gypsy moth larvae from these sites in 1987, 1989 and 1991 failed to document the presence of E. maimaiga (Hajek et al. 1995). The origin of the E. maimaiga that was found in the USA in 1989 is still unknown. Several hypotheses for its origin and establishment have been proposed and are discussed in detail in Hajek et al. (1995). In summary, one hypothesis proposes that the fungus presently in the USA originated from one of the deliberate introductions, either from 1910–1911 or 1985–1986, but that it was poorly adapted to USA conditions and persisted only at undetectable levels ; subsequently,
942 a more aggressive strain arose through natural selection, and then E. maimaiga began to spread (Hajek et al. 1995). However, based on a model including records of temperature and precipitation from 1965 to 1995, Weseloh (1998) concluded that if E. maimaiga had originated from the 1910–1911 introductions, epizootics of E. maimaiga would have been observed in 1945 and 1971. Nevertheless, since the basic appearances of E. maimaiga-killed larvae and those killed by the gypsy moth nuclear polyhedrosis virus (gmNPV) are similar, E. maimaiga-killed cadavers could potentially have been mistaken for NPV-killed larvae, especially in the absence of any indication that the fungus was operating among gypsy moths whereas the gmNPV had been well known and it effects monitored long before this time. It is unlikely that the 1985–1986 introductions gave rise to the establishment of E. maimaiga in the USA because intensive sampling especially targeting E. maimaiga from release plots failed to find this fungus (Hajek et al. 1995). Another hypothesis proposed that E. maimaiga was only recently introduced to the USA by accident (Andreadis & Weseloh 1990, Hajek et al. 1995). Multiple sources for introduction of inoculum are possible, for example imported materials, especially wooden packing materials contaminated by resting spores shipped from anywhere within the natural distribution of E. maimaiga, or contaminated footwear (Hajek et al. 1995). Additionally Andreadis & Weseloh (1990) speculated that E. maimaiga could have been inadvertently introduced along with egg parasitoids from Japan or contaminated gypsy moth egg masses, which have been shown to harbour resting spores. This, however, also implies an introduction long ago, before the use of gypsy moth quarantine laboratories in the USA in 1928 (Coulson & Soper 1989). The last hypothesis suggests that E. maimaiga has evolved from E. aulicae, a naturally occurring complex of fungal pathogens of forest Lepidoptera in the USA, which also includes E. maimaiga. Based on morphological data and transmission experiments (Soper et al. 1988), as well as more recent genetic data (Hajek et al. 1990, Walsh 1996), this last hypothesis has now been rejected. Thus several convincing lines of evidence support the hypothesis of an undocumented accidental introduction of E. maimaiga into the USA, although this has never been tested empirically. The releases of E. maimaiga isolates for biocontrol described above, as well as establishment and spread of this fungus in the USA, have been carefully recorded, providing an unique opportunity to explore changes in the genetic diversity from putative source populations in Asia and founder populations in the USA. The objectives for this study were to: (1) test the hypothesis that the E. maimaiga populations in the USA have reduced genetic variability due to founder effects compared to the putative ancestral populations in Asia; (2) track the origin of the North American populations of this fungus ; and (3) assess whether genetic differences
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among E. maimaiga isolates are correlated to morphological differences. MATERIALS AND METHODS Fungal isolates 30 Entomophaga maimaiga isolates from different geographical locations within the USA, Japan, China and far-eastern Russia were selected for study (Table 1, Fig. 1). All isolates were isolated in vitro from hemolymph of infected gypsy moth larvae as described by Papierok & Hajek (1997). The infected larvae were obtained either by collections of naturally infected larvae or from larvae infected in the laboratory upon exposure to soil containing germinating resting spores, as described by Hajek, Shimazu & Knoblauch (2000). For comparison, one isolate of E. aulicae from a brown-tail moth, Euproctis chrysorrhoea (Lepidoptera : Lymantriidae), was included. All fungal isolates were grown in liquid culture (Grace’s insect culture media, Cellgro, Herndorn, VA ; supplemented with 5% fetal
bovine serum, Gibco BRL, Grand Island, NY). Before isolation of DNA all isolates were sub-cultured at least three times to get rid of insect hemocytes. All isolates are stored and maintained at the Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF, Ithaca, NY). Amplified fragment length polymorphism (AFLP) The AFLP procedure was performed essentially as described in the protocol for plant mapping by Applied Biosystems (Foster City, CA). The protocol is based on the methods described by Vos et al. (1995) but uses fluorescent dyes to label the primers instead of radioactivity. Initially, several combinations of EcoRI and MseI primers with various numbers of selective bases were screened. We found that the best resolution was obtained with a preselective amplification followed by a combination of MseI primers with three selective bases, and EcoRI primers with two to three selective bases. Details of these methods are given below.
Table 1. Entomophaga maimaiga and E. aulicae isolates included in this study. Cornell no.a
ARSEF no.
Country of origin
State/Prefecture/ Province/nearest city
Year of collection
92NY14-1-1 96NY1-13A 00NY1-1-2 89MA1-1-1b,c,d 98MA2-4A 03MA3-2-1 03MA3-1-1b,c 98MI1-1Ab,c 96VA9-1A 97VA10-1A 03PA1-1-5b 03PA5-1-5 03PA6-1-4 96MD-B2-6 84JP1-1392 84JP1-1400b,d 86JP2-2370 98JP2-9Ab,c 98JP2-4A 98JP2-14A 98JP3-8A 98JP3-5A 01JP4-11-1b 01JP4-12-1 03JP5-1-2b 03JP5-1-4 99RU1-1-1b,c,d 02CN1-1-1b,c,d 02CN1-2-1 02CN1-2-2 03ME3-1-2b (E. aulicae)
3828 5396 6625 2779 6051 7124 7123 6053 5569 5568 7190 7352 7353 5384 1392 1400 2370 6162 6253 6255 6258 6171 7104 7114 7186 7187 7127 7139 7183 7185 7142
USA USA USA USA USA USA USA USA USA USA USA USA USA USA Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Russia China China China USA
New York New York New York Massachusetts Massachusetts Massachusetts Massachusetts Michigan Virginia Virginia Pennsylvania Pennsylvania Pennsylvania Maryland Ishikawa Ishikawa Chiba Chiba Chiba Chiba Ibaraki Ibaraki Iwate Iwate Hiroshima Hiroshima Khabarowsk, Russia Heilongjiang Heilongjiang Heilongjiang Maine
1996 1996 2000 2000 1998 2003 2003 1998 1996 1997 2003 2003 2003 1996 1984 1984 1986 1998 1998 1998 1998 1998 2001 2001 2003 2003 1999 2002 2002 2002 2003
a In the Cornell identification number, the first digits give the year for collection of soil samples containing resting spores or collection of a cadaver in the field; the next two letters indicate either State (USA) or Country of origin; numbers indicate the site number, followed the soil sample number/plot number or tree number from which soil was collected, and the last number (when applicable) indicates the isolate number in cases in which more than one isolate was isolated sample. b Length and width of primary conidia measured. c Diameter of resting spores measured. d Number of nuclei per primary conidia counted.
AFLP diversity of Entomophaga maimaiga
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(A) 500 km MA1,2 NY1,14 MA3
MI1 PA1, 5,6 MD1
VA9,10
(B) 500 km RU1 CN1
Russia
China
North JP4
Korea JP1
South Korea
JP5
Japan
JP3 JP2
Fig. 1. Sampling locations of Entomophaga maimaiga in the USA and eastern Asia. (A) Map of the northeastern states of the US showing the sampling localities (dots) and E. maimaiga release sites in 1910–1911 (star) and 1985–1986 (triangles). (B) Map of eastern Asia showing the sampling localities (dots) and approximate locations of the source populations for E. maimaiga releases in the US (grey circles). The isolate released in Massachusetts in 1910–1911 originated from near Tokyo (close to current samples from Chiba and Ibaraki Prefectures, JP2 and JP3); while the isolate (84JP1-1400) released in New York state and Virginia in 1985 and 1986, respectively, originated from Ishikawa Prefecture (JP1).
DNA extraction Protoplasts were harvested after centrifugation under sterile conditions at 300 g for 10 min and re-suspended in 200 ml phosphate-buffered saline (PBS) (pH 7.4). Total genomic fungal DNA was prepared using the DNeasy Tissue Kit from Qiagen Inc. (Valencia, CA) following the protocol given for DNA extraction from cultured animals cells. DNA was eluted in Buffer AE (10 mM Tris-Cl, 0.5 mM EDTA ; pH 9.0). RNA was degraded by adding 1: 100 Ribonuclease A (10 mg mlx1 ; Sigma, St Louis, MO) and incubating samples for 20 min at 37 xC. DNA concentrations were quantified by UV spectrophotometry using a Beckman DU 640 Spectrophotometer (Columbia, MD).
DNA digestion and ligation of adapters DNA digestion and ligation of adapters were performed in a single step. Each reaction was conducted in a 50 ml volume containing 200 ng of sample DNA, 5 U EcoRI (New England Biolabs, Beverly, MA), 5 U MseI
(New England Biolabs), 1r T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 mg mlx1 BSA (pH 7.5) ; New England Biolabs), 2 Weiss U T4 ligase (New England Biolabs), and 2.5 pmol EcoRI- and 25 pmol MseI-specific adapter pairs (Integrated DNA Technology, Coralville, IA). The reactions were incubated overnight at room temperature, and reaction mixtures were then diluted to 100 ml with TE0.1 buffer (1 mM Tris-Cl; 0.1 mM EDTA ; pH 8.0) and stored at x20 x until use. Preselective amplification Preselective amplification was performed in 0.2 ml Thermowell tubes (Corning, Corning, NY) with a total volume of 20 ml containing 1r supplied PCR buffer (New England Biolabs), 200 mM of each dNTP (New England Biolabs), 0.4 mM EcoRI-A primer (Integrated DNA Technology), 0.4 mM MseI-C primer (Integrated DNA Technology), 0.8 U Taq polymerase (New England Biolabs) and 4 ml of the diluted digestionligation solution. Amplification was performed in
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a thermal cycler (PTC-100 Peltier ; MJ Research, Boston, MA) with the following parameters : 72 x for 2 min, 25 cycles of 20 s DNA denaturation at 94 x, 30 s annealing at 56 x and 2 min extension at 72 x followed by 30 min at 60 x to avoid split peaks. The amplification product was diluted 1 :20 with TE0.1 buffer.
PowerMarker v. 3.07 (Liu & Spencer 2001) using Nei’s gene diversity estimate h=1xSp2i , where pi is the frequency of the ith reaction pattern (Nei 1973). Variance and standard errors were estimated by nonparametric bootstrapping across different loci 1000 times (Weir 1996).
Selective amplification
Cluster analysis
Four different primer combinations were used : (1) EcoRI-AAC/MseI-CTC ; (2) EcoRI-AAC/MseI-CTA ; (3) EcoRI-AC/MseI-CTC ; and (4) EcoRI-AC/MseICTA. All EcoRI primers were labelled with fluorescent dye (FAM). The reaction mixture was identical to the preselective PCR, apart from using 0.05 mM EcoRI primer (Integrated DNA Technology), 0.25 mM MseI primer (Integrated DNA Technology), and 4 ml of the diluted preselective amplification product as template. Amplification was performed in a thermal cycler with the following cycle parameters : 30 s DNA denaturation at 94 x, 30 s annealing and 1 min extension at 72 x. The annealing temperature used in the first cycle was 66 x and then subsequently reduced by 1 x at each cycle for the next 10 cycles and continued at 56 x for another 25 cycles.
Both distance and parsimony analysis of AFLP data were performed. For the distance analysis neighbourjoining was performed with the statistical software program TreeCon for Windows v. 1.3b (van de Peer & de Wachter 1994) using the Dice coefficient (Nei & Li 1979) for calculating of the distance matrix. The isolate from Russia (99RU1-1-1) was chosen as out-group because it was genetically distinct from all other isolates (see results below). Parsimony analysis was implemented using the computer software PAUP* 4.0 (Swofford 1998) using unweighted maximum parsimony analysis. In both analyses, support for internal branches was assessed by 1000 bootstrap replications.
Morphology Microscope slides
Electrophoresis Prior to electrophoresis, 1.0 ml of selective amplification product was added to a loading buffer mix (9 ml Hi-Di formamide, Applied Biosystems ; and 0.05 ml GenScan 500 LIZ, internal lane size standard, Applied Biosystems), heated to 95 x for 5 min and then quickly chilled on ice. The mixture was then loaded on an automated DNA capillary sequencer (ABI Prism1 3700 DNA Analyzer ; Applied Biosystems).
Scoring of data Computer files containing sizing data for all primer combinations for each isolate were visualized and scored using Genemapper v. 3.0 (Applied Biosystems). Each unique peak in the chromatogram was interpreted as an independent genetic locus. Only loci between 50 and 500 bp were included in the following analysis and differences in intensity were not taken into account. Furthermore, only strong and clearly distinct loci were scored. Repeatability was tested for three of the isolates included in this study by isolating DNA and running assays independently twice. Identical AFLP haplotypes were obtained.
Estimation of diversity Data for all primer combinations were pooled and transformed to a binary matrix excluding invariant characters. Gene diversity was estimated with
The isolates used for morphological studies were selected to represent groups demonstrated by analysis of AFLP data (Table 1). Measurements were based on in vivo material. Sporulating cadavers and cadavers filled with resting spores were obtained as described by Hajek, Butler & Wheeler (1995). Sporulating cadavers were placed between microscope slides under humid conditions, and conidia were collected for a maximum of 1 h to prevent production of secondary conidia. Non-sporulating cadavers were squashed and microscopically checked for the presence of resting spores. Resting spores were allowed to reach full maturation (ca 10 d after host death at room temperature) before microscope slides were prepared by placing a droplet of body fluid containing resting spores on the microscope slide.
Quantification Primary conidia and resting spores were mounted in lactic acid and sealed with nail polish until examination. The length and width of primary conidia and diameter of resting spores were measured % using an ocular micrometer under a Zeiss Axioskop light microscope with phase-contrast (400r). For each cadaver 20 conidia or resting spores were measured. The numbers of nuclei in each of 10 primary conidia were determined after staining with DAPI (4,6diamidino-2-phenylindole, 5 mg ml-1) using a Zeiss Axioskop (Filter 0.5 ; violet, excitation 395–440) at 400rmagnification.
AFLP diversity of Entomophaga maimaiga
946
Table 2. Number of DNA fragments amplified in 30 E. maimaiga isolates using four AFLP primer sets. EcoRI-AAC/ MseI-CTC
EcoRI-AAC/ MseI-CTA
EcoRI-AC/ MseI-CTC
EcoRI-AC/ MseI-CTA
Totals
Population
Na
Tb
Pc
%d
T
P
%
T
P
%
T
P
%
T
P
%
All isolates North America Asia Japan
30 14 16 12
65 54 64 60
14 1 13 6
21.5 1.9 20.3 10.0
77 71 74 70
15 2 10 4
19.4 2.8 13.5 5.7
87 76 86 82
17 2 15 9
19.5 2.6 17.4 11.0
91 81 90 86
19 5 18 10
20.9 6.2 20.0 11.6
320 282 314 298
65 10 56 29
20.3 3.5 17.8 9.7
a b c d
N, number of isolates included. T, total number of fragments. P, number of polymorphic fragments. %, percentage of polymorphic fragments out of total number of fragments.
Table 3. Number of haplotypes and gene diversitya.
Population
N
No. of haplotypes
Gene diversity¡SE
All isolates North America Asia Japan
30 14 16 12
26 11 15 12
0.227¡0.004 0.041¡0.004 0.223¡0.005 0.131¡0.006
a Calculated by Nei’s gene diversity estimate h=1xSpi2, where pi is the frequency of the ith allele at each locus using the software program PowerMarker v. 3.07.
RESULTS Diversity estimation All primer combinations successfully amplified multiple DNA fragments for all isolates included in this study. The AFLP profiles of Entomophaga aulicae were different from the E. maimaiga isolates, with the two species sharing only a few alleles (data not shown). For each primer combination, between 65 and 91 loci were scored giving rise to 19–22 % polymorphic loci, excluding E. aulicae (Table 2). AFLP haplotypes were different for all Asian isolates except for two Chinese isolates originating from the same soil sample. In contrast, identical haplotypes were obtained for US isolates collected from different states ; e.g. the same haplotype was found in New York and Maryland, and another haplotype was found in Pennsylvania and Massachusetts. For the 14 USA isolates, only tenpolymorphic AFLP loci were found, whereas 56 loci were polymorphic among the 16 Asian isolates and 29 loci were polymorphic among the 12 isolates from Japan alone (Table 2). In all, 26 unique haplotypes were identified for the 30 isolates in this study (Table 3). Average gene diversity estimates h for the polymorphic loci were 0.223¡0.005 for Asia (including Japan), 0.131¡0.006 for Japan only, and 0.041¡0.004 for the USA. Cluster analysis The neighbour-joining analysis (Fig. 2) and parsimony analysis of AFLP data produced essentially identical results, with only the hypothesized relationships among
the most similar isolates from the USA differing between the two techniques ; all other hierarchical relationships were identical in the two analyses. Both distance and parsimony analyses formed distinct clades that correlated with geographical regions of origin. Among the Japanese isolates, one clade consisted of isolates collected near Tokyo (Chiba and Ibaraki Prefectures) ; three other clades of Japanese isolates correlated to the other three prefectures sampled (Ishikawa, Iwate, and Hiroshima). On the Asian mainland, one clade was formed by the three Chinese isolates. The single isolate available from Russia was used as an out-group to root the tree because it was the most genetically different isolate in an unrooted neighbour-joining tree (not shown). However, we could not calculate bootstrap values from an unrooted tree ; we used it only to choose an out-group. All 14 isolates from the USA were included in one clade (Fig. 2). The US clade is part of the larger Japanese clade, and is in a subclade that includes isolates from Japan outside of the Tokyo area. No correlations between geographical location and clade were found among the USA isolates. Morphology Morphological dimensions of conidia and resting spores of Entomophaga maimaiga and E. aulicae and numbers of nuclei per conidia are listed in Table 4. For E. maimaiga, the length of conidia varied between 33–38 mm, the width between 25–29 mm and the lengthwidth ratio between 1.25 and 1.38. E. maimaiga contained 17–36 nuclei per conidium. The diameter of resting spores was ca 37 mm. The primary conidia of E. aulicae measured 36r25 mm. DISCUSSION The number of polymorphic loci, number of haplotypes found, and gene diversity demonstrate that populations of Entomophaga maimaiga are more genetically diverse in Asia than in the USA. The average gene diversity is approximately five times greater in the Asian sample than the USA sample, and three times greater in the Japanese sample compared to the USA sample. These results are consistent with what would be
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Table 4. Dimensions of conidia and resting spores (mm¡SE) and number of nuclei per conidium in Entomophaga maimaiga and E. aulicae. All structures were obtained from cadavers of Lymantria dispar injected with an in vitro isolate of fungus. Values given are based on 20 objects per specimen except for number of nuclei per conidium, which is only based on counts from 10 conidia per isolate. Primary conidia, mean length
Primary conidia, mean width
Mean ratio (Primary conidia)
No. of nuclei per conidium
RSa, mean diam
Isolate no. Cadaver no.
1
2
1
2
1
2
1
3
E. maimaiga 89MA1-1-1 (2779)b 03MA3-1-1 (7123) 98MI1-1A (6053) 03PA1-1-5 (7190) 84JP1-1400 (1400) 98JP2-9A (6162) 99RU1-1-1 (7127) 02CN1-1-1 (7139)
34.5¡0.5 35.5¡0.5 38¡0.5 35¡0.5 34.5¡0.5 35.5¡0.5 33.5¡0.5 37.5¡0.5
33¡0.5 37¡0.5 35¡0.5 33.5¡0.5 35.5¡0.5 36¡0.5 34.5¡0.5 37¡0.5
26.5¡0.5 27.5¡0.5 29¡0.5 26¡0.5 25¡0.5 28.5¡0.5 25¡0.5 27.5¡0.5
25¡0.5 27.5¡0.5 26¡0.5 26¡0.5 27¡0.5 28.5¡0.5 26¡0.5 27¡0.5
1.29¡0.01 1.33¡0.01 1.32¡0.01 1.34¡0.01 1.36¡0.01 1.25¡0.01 1.35¡0.02 1.37¡0.01
1.32¡0.03 1.35¡0.01 1.36¡0.01 1.30¡0.01 1.32¡0.01 1.28¡0.01 1.31¡0.01 1.38¡0.01
21.8 [19–27]c – – – 23.3 [19–36] – 19.0 [17–24] 22.6 [18–30]
37¡0.5 – 37¡0.5 – – 37¡0.5 37¡0.5 37¡0.5
1.43¡0.01
–
–
–
E. aulicae 03ME3-1-2 a b c
36¡0.5
–
25¡0.5
–
RS, Resting spores. Number given in parentheses is the ARSEF accession number. Numbers in square brackets give the range.
Distance 0.1
96VA9-1A 98MI1-1A 54 97VA10-1A 03PA6-1-4 00NY1-1-2 03PA5-1-5 03PA1-1-5 USA 03MA3-2-1 98MA2-4A 03MA3-1-1 100 89MA1-1-1 79 92NY14-1-1 96MD-B2-6 71 96NY1-13A 1 84JP1-1400 Ishikawa, Japan 84JP1-1392 01JP4-12-1 Iwate, Japan 01JP4-11-1 57
61 92 60 100
86
98
03JP5-1-4 03JP5-1-2 98JP2-9A 98JP3-5A 99 63 98JP3-8A 87 98JP2-14A 98JP2-4A 85 86JP2-2370
02CN1-1-1 100 02CN1-2-2 02CN1-2-1
99RU1-1-1
Hiroshima, Japan
Chiba & Ibaraki,, Japan
China Russia
Fig. 2. Neighbour-joining tree based on AFLP haplotypes of Entomophaga maimaiga. This tree was generated using the Dice coefficient for calculation of the distance matrix. The calculations and the drawing were performed with the software program TreeCon for Windows v. 1.3b. USA isolate numbers are shown in grey, and Asian isolates are shown in black.
expected when comparing an ancestral source population and a founder population (Nei et al. 1975) and support the hypothesis of establishment of only a small number of E. maimaiga individuals in the USA. Consequently, the founder population in the USA lost genetic variability compared to the source population
in Asia. Our data are thus similar to previous reports of plant pathogens recently established on new continents, e.g. Cryphonectria parasitica (Milgroom et al. 1992, Milgroom et al. 1996), and Phytophthora infestans (Fry & Spielman 1991) where a loss of genetic variability was found in the founder population compared to the
AFLP diversity of Entomophaga maimaiga source population. In such studies the sampling scheme must, however, be taken into account especially when interpreting results from a relatively limited number of isolates per population. Because we used roughly comparable sampling schemes in the USA and Japan we are confident that the population of E. maimaiga is more diverse in Japan than the USA population. In other words, the number of isolates included from the USA was greater than for Japan, and the distance between some of the sampling localities in the USA was greater than in Japan (Fig. 1). Among the 30 isolates in this study, both distance and parsimony analyses formed six distinct clades separated from the out-group from Russia (99RU1-1-1). The clade that included all isolates from the USA was in the same clade as all isolates from Japan, which was distinct from isolates from China. High bootstrap support for most clades in Asia may indicate restricted migration, preventing significant genetic exchange between geographically distinct populations. In contrast to the situation in Asia, no correlation between geographical location and clade was found among the US isolates. However, this fungus has been moved between locations within the USA for control purposes (Hajek & Roberts 1991, Smitley et al. 1995, Hajek, Elkington & Witcosky et al. 1996a). For example, E. maimaiga was moved from Massachusetts to Maryland, Michigan, New York, Pennsylvania, Virginia and West Virginia (Smitley et al. 1995, Hajek et al. 1996a) and from central New York to Maryland, Virginia and West Virginia (Hajek & Roberts 1991, Hajek et al. 1996a) in the early and mid-1990s. It is therefore impossible to determine whether the fungus at an individual location is the result of spread from a release or migration from naturally infected areas. Furthermore, because E. maimaiga was introduced into the USA relatively recently, genetically distinct subpopulations may not have evolved yet. Both distance and parsimony analyses of AFLP data support an introduction of E. maimaiga into the USA from a location in Japan other than the Tokyo area (Chiba and Ibaraki), although it has to be kept in mind that the isolate from the Toyo areas released in Massachusetts in 1910 and 1911 was not included in our study. The closest relative to the North American isolates was 84JP1-1400 (ARSEF 1400), the isolate released in New York state and Virginia in 1985 and 1986, respectively (Hajek et al. 1995). However, based on the following four arguments we hypothesize that the population now present in the USA came from an accidental introduction rather than the deliberate release of 84JP1-1400. (1) Isolate 84JP1-1400 is in a clade distinct from the USA clade supported by a boot-trap value of 92%. (2) The amount of variation in AFLP haplotypes seen in one locality over time is relatively small (e.g. isolates 86JP2-2370 and 98JP2-4A/8A/9A/ 14A from Chiba were collected in 1989 and 1998, respectively, but have nearly the same AFLP profile), thus a change of haplotype from isolate 84JP1-1400
948 since its release in 1985 and 1986 to the haplotypes obtained for the USA isolates does not seems likely, although comprehensive sampling within population was not done. (3) Despite intensive sampling, E. maimaiga was never found from the release sites in 1987, 1989 or 1990 (Hajek et al. 1995). And (4) during an epizootic, the maximum geographic spread of E. maimaiga is about 100 km per season (Dwyer, Elkinton & Hajek 1998), and even shorter distances are expected in endemic situations. In 1989, E. maimaiga was documented o300 km from the release sites of isolate 84JP1-1400 in 1985 and 1986, which is probably farther than it could have migrated in 3–4 yr. This study further demonstrates that AFLPs are a reliable genotyping technique for estimating intraspecific variation of entomophthoralean fungi. Previously, RAPDs have been the predominant method for fingerprinting in insect pathology (e.g. Hodge, Sawyer & Humber 1995, Rohel et al. 1997, Nielsen et al. 2001), but this method has often been criticized for lack of reproducibility, because the fragment patterns are highly sensitive to the polymerase, the primers and the cycling parameters used (Takamatsu 1998). In contrast, AFLP assays are more robust and reliable for genotyping due to the stringent reaction conditions for primer annealing (Vos & Kupier 1998). We found that the best results were obtained using a combination of MseI primers with three selective bases and EcoRI primers with two or three selective bases. The length of the primers chosen in this study is thus more similar to protocols recommended for plants and invertebrates rather than protocols used for most fungi (Vos & Kupier 1998). This is, however, not surprising because the genome size of E. aulicae previously was estimated as y8.0r106 kb (Murrin et al. 1986) which is the same order of magnitude of most plants and about 100 times larger than most fungi (Murrin et al. 1986, Vos & Kupier 1998). For each primer combination, 65–91 loci were scored and generated 14–19 polymorphic loci, enabling us to differentiate 26 out of the 30 E. maimaiga isolates included in this study. As expected, more loci were scored for AFLP assays employing EcoRI with two compared to three base extensions. However, the shorter primers did not reveal a greater number of scorable polymorphisms possibly because several loci had to be excluded due to the closeness of the peaks in the chromatogram, which made it impossible to determine whether some of the loci consisted of one or two amplification products. Morphological and cytological characters of the primary conidia are of taxonomic importance in the E. aulicae species complex, which includes E. maimaiga (Soper et al. 1988). Our measurements of conidial length and width made it possible to distinguish E. maimaiga isolates from the only E. aulicae isolate included in this study, whereas morphological measurements could not distinguish between E. maimaiga isolates from different geographical locations. Morphometric dimensions of both conidia and resting
C. Nielsen, M. G. Milgroom and A. E. Hajek spores were slightly larger in our study than those given in the original description of E. maimaiga in Soper et al. (1988), even for isolate 84JP1-1400 (ARSEF 1400) which was included in both studies; our results were similar to dimensions given by Andreadis & Weseloh (1990) for in vivo isolates collected in Connecticut. However, it has previously been shown, for the closely related entomophthoralean fungus Entomophthora schizophorae, that in vitro growth history can significantly affect the dimensions of conidia and number of nuclei per conidium, at least until these fungi have been cycled in the host twice (Eilenberg, Bresciani & Latge´ 1990). It is therefore likely that previous in vitro growth history could have affected both our morphometric measurements as well as those given in the literature. Unfortunately, neither our study nor that published by Soper et al. (1988) have recorded the growth history of the isolates included in their studies. In summary the most significant finding in this study, that the overall genetic diversity of E. maimaiga is considerably greater in Japan than the USA, is consistent with what would be expected when comparing an ancestral source population and a founder population. Furthermore, E. maimaiga appears to have been introduced to the USA from Japan, probably from a location outside the Tokyo area.
ACKNOWLEDGEMENTS We are grateful to Stephen Kresovich, Institute of Genomic Diversity at Cornell University, who placed his laboratory at our disposal. We also thank Sharon E. Mitchell, Rebecca S. Bennet and Italo Delalibera jr for their kind help, technical advice and constructive discussions throughout this project. We thank Joshua E. Izzo, Hong Sun, Michael M. Wheeler and Joshua J. Hannan for technical laboratory support. For collection of soil we are indebted to the following people: George Boettner, Joseph S. Elkinton, Shota Jikumaru, Beth Knoblauch, Gary Laudermilch, Zengzhi Li, Timothy Marasco, Galina Markova, Max McFadden, Mitsuaki Shimazu and Nathan Siegert. We thank Alison E. Burke, Michael M. Wheeler, and Richard S. Soper for isolation of several of the isolates included in this study. Finally, we thank The Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF) for providing and maintaining all the fungal isolates included in this study. This project was principally funded by USDA NRICGP grant no. 2002-01968 with additional funding from The Danish Research Agency grant no. 23-03-0009.
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AFLP diversity of Entomophaga maimaiga germination of entomophthoralean resting spores. Mycologia 76: 1104–1107. Milgroom, M. G., Lipari, S. E. & Wang, K. (1992) Comparison of genetic dversity in the chestnut bligth fungus, Cryphonectria (Endothia) parasitica, from China and the U.S. Mycological Research 96: 1114–1120. Milgroom, M. G., Wang, K., Zhou, Y., Lipari, S. E. & Kaneko, S. (1996) Intercontinental population structure of the chestnut blight fungus, Cryphonectria parasitica. Mycologia 88 : 179–190. Murrin, F., Holtby, J., Nolan, R. A. & Davidson, W. S. (1986) The genome of Entomophaga aulicae (Entomophthorales, Zygomycetes) : Base composition and size. Experimental Mycology 10, 67–75. Nei, M. (1973) Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences, USA 70: 3321–3323. Nei, M. & Li, W. H. (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences USA 76: 5269–5273. Nei, M., Maruyama, T. & Chakraborty, R. (1975) The bottleneck effect and genetic variability in populations. Evolution 29: 1–10. Nielsen, C., Sommer, C., Hansen, K. S., Eilenberg, J. & Humber, R. A. (2001) Characterization of aphid pathogenic species in the genus Pandora using PCR techniques and digital image analysis. Mycologia 93 : 864–874. Papierok, B. & Hajek, A. E. (1997) Entomophthorales. In Manual of Techniques in Invertebrate Pathology (L. A. Lacey, ed.): 187–212. Academic Press, London. Rohel, E., Couteadier, Y, Papierok, B., Cavelier, N. & Dedryver, C. A. (1997) Ribosomal internal transcribed spacer size variation correlated with RAPD-PCR pattern polymorphisms in the entomopathogenic fungus Erynia neoaphidis and some closely related species. Mycological Research 101: 573–579. Smitley, D. R., Bauer, L. S., Hajek, A. E., Sapio, F. J. & Humber, R. A. (1995) Introduction and establishment of Entomophga maimaiga, a fungal pathogen of gypsy moth (Lepidoptera: Lymantriidae) in Michigan. Environmental Entomology 24: 1685–1695. Soper, R. S., Shimazu, M., Humber, R. A., Ramos, M. E. & Hajek, A. E. (1988) Isolation and characterization of Entomophaga maimaiga sp. nov., a fungal pathogen of gypsy moth, Lymantria dispar, from Japan. Journal of Invertebrate Pathology 51: 229–241. Speare, A. T. & Colley, R. H. (1912) The Artificial Use of the BrownTail Fungus in Massachusetts, with Practical Suggestions for Private
950 Experiment, and a Brief Note on a Fungus Disease of the Gypsy Caterpillar. Wright & Potter, Boston. Swofford, D. L. (1998) PAUP: phylogenetic analysis using parasimony. Sinauer Associates, Sunderland, MA. Takamatsu, S. (1998) PCR applications in fungal phylogeny. In Applications of PCR in Mycology (P. D. Bridge, D. K. Arora, C. A. Reddy & R. P. Elander, eds): 125–152. CAB International, Wallingford. Takamura, N. & Sato, H. (1973) [Observation on the epizootic diseases in the outbreak population of the gypsy moth.] Transaction of the 84th Meeting of the Japanese Forest Society: 353–357. [In Japanese] Tymon, A. M., Shah, P. A. & Pell J. K. (2004) PCR based molecular descrimination of Pandora neoaphidis isolates from related entomopathogenic fungi and development of species-specific diagnostic primers. Mycological Research 108: 419–433. van de Peer, Y. & de Wachter, J. J. (1994) TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Computer and Applied Bioscience 10: 569–570. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23 : 4407–4414. Vos, P. & Kupier, M. (1998) AFLP analysis. In DNA markers, protocols, applications, and overviews (G. Caetano-Anolle´s & P. M. Gresshoff, eds): 115–131. Wiley-VCH, New York. Walsh, S. R. A. (1996) Development of molecular markers for the detection and differentiation of Entomophaga strains pathogen for insects. PhD dissertation, University of Toronto. Walsh, S. R. A., Tyrrell, D., Humber, R. A. & Silver, J. C. (1990) DNA restriction fragment length polymorphisms in the rDNA repeat unit of Entomophaga. Experimental Mycology 14: 381–392. Weir, B. S. (1996) Genetic Data Analysis II. Sinauer Associates, Sunderland, MA. Weseloh, R. M. (1998) Possibility for recent origin of the gypsy moth (Lepidoptera : Lymantriidae) fungal pathogen Entomophaga maimaiga (Zygomycetes : Entomophthorales) in North America. Environmental Entomology 27 : 171–177. Yanbe, T. (1976) [Influences of the population density of the gypsy moth on the thickening growth of larch tree in the forest.] Newsletter Tohoku Branch Government Forest Experimental Station. 175 : 1–4. [In Japanese]
Corresponding Editor: R. A. Humber
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doi:10.1017/S0953756205003278 Printed in the United Kingdom.
Genetic diversity in the gypsy moth fungal pathogen Entomophaga maimaiga from founder populations in North America and source populations in Asia
Charlotte NIELSEN1*, Michael G. MILGROOM2 and Ann E. HAJEK1 1
Department of Entomology, Cornell University, Comstock Hall, Ithaca, NY 14853-0901, USA. Department of Plant Pathology, Cornell University, Ithaca, NY 14853-5904, USA. E-mail : [email protected] 2
Received 25 October 2004; accepted 21 March 2005.
Entomophaga maimaiga is a naturally occurring fungal pathogen specific to larvae of the gypsy moth, Lymantria dispar. E. maimaiga is thought to be native to Asia where its epizootics can suppress gypsy moth outbreaks. However, in the USA this beneficial fungal pathogen was not observed until 1989, although an isolate of E. maimaiga from Tokyo was released in Massachusetts to control gypsy moths as early as in 1910–1911, and another isolate from Ishikawa Prefecture in Japan was later released in 1985 and 1986 in New York and Virginia. Our objectives were to : (1) test the hypothesis that E. maimaiga populations in the USA have reduced genetic variability due to founder effects compared to the putative ancestral populations in Asia; (2) track the origin of the North American populations of this fungus; and (3) assess whether genetic differences among E. maimaiga isolates are correlated to morphological differences. We compared genetic diversity among 30 E. maimaiga isolates originating from seven states in the USA, five prefectures in Japan, one province of China and one region of far eastern Russia by AFLPs. Among 14 USA isolates, only ten polymorphic AFLP loci were found, whereas 56 polymorphic loci were found among 16 Asian isolates ; 29 loci were polymorphic among 12 isolates from Japan alone. Average gene diversity (h) for the polymorphic loci was 0.223¡0.005 for Asia (including Japan), 0.131¡0.006 for Japan only, and 0.041¡0.004 for the USA. Thus, native populations from Asia were more diverse than the USA populations. These results are consistent with the expectation of a population founded from a source population by a small number of individuals. Distance and parsimony analyses of AFLP data showed that the isolates from the USA formed one distinct clade that was most closely related to Japanese isolates collected outside the Tokyo area. No morphological variation of E. maimaiga from different geographical locations was detected.
INTRODUCTION Studies of population structures of fungi over the last 20 years have enhanced our understanding of the processes of selection, mutation, gene flow, genetic drift, and mating systems. The likelihood of drift increases as population size decreases, so drift is very likely and, therefore, a very important evolutionary mechanism in small populations, e.g. populations that pass through bottlenecks. Severe bottlenecks often occur when a population is established in a new geographical area because only a few colonists or founders typically establish the new population. The consequence for a population founded by a small number of individuals is fewer alleles per locus, reduced gene diversity and often modified allele frequencies compared to their source population (Nei, Maruyama & Chakraborty 1975). * Corresponding author.
Such so-called founder effects have been described previously in relation to source populations for several fungal or oomycete plant pathogens, including the chestnut blight fungus Cryphonectria parasitica (Milgroom, Lipari & Wang 1992, Milgroom et al. 1996), the beech bark disease fungus Neonectria coccinea (Mahoney et al. 1999) and the potato late blight pathogen Phytophthora infestans (Fry & Spielman 1991). Even though a substantial number of papers have reported the successful use of molecular techniques to study different aspects of entomopathogenic Entomophthorales for biocontrol of insects (e.g. Walsh et al. 1990, Bidochka et al. 1997, Jensen, Thomsen & Eilenberg 2001, Nielsen et al. 2001, Hajek et al. 2003, Tymon, Shah & Pell 2004), little is known about their population structure. One of the few exceptions is the use of cluster analysis of RAPD data to infer the origin of Zoophthora phytonomi in North America (Hajek et al. 1996b).
AFLP diversity of Entomophaga maimaiga Entomophaga maimaiga is a naturally occurring fungal pathogen specific to larvae of the gypsy moth, Lymantria dispar (Lepidoptera : Lymantriidae) with multinucleate spores. The life-cycle of this fungus consists of several asexual generations of airborne conidia during summer, followed by dormancy over winter as azygospores (resting spores) in the soil (Hajek 1999). No sexual stage is known in this fungus although McCabe, Humber & Soper (1984) inferred cytological evidence for nuclear fusion in azygospores for the closely related fungus E. grylli. The prevalence of this fungus in gypsy moth larvae may, in some periods, exceed 99% infection, leading to complete suppression of outbreak populations (Koyama 1954, Takamura & Sato 1973, Aoki 1974, Yanbe 1976). The fungus is thus considered to be the most important natural enemy of gypsy moth (Hajek 1999). E. maimaiga was originally described from Japan (Soper et al. 1988) and is, as the gypsy moth, thought to be native to Asia. Despite many years of intensive surveys for diseased gypsy moths by entomologists in the USA, E. maimaiga was not observed in the field until 1989 (Andreadis & Weseloh 1990, Hajek et al. 1990, Hajek, Humber & Elkinton 1995). Since then, it has spread across the distribution of gypsy moths from its epicentre in the north-eastern coastal area (Andreadis & Weseloh 1990, Hajek et al. 1995, Hajek, Elkinton & Witcosky 1996a). Nevertheless, before the sudden appearance of the fungus in 1989, a small number of introductions of E. maimaiga to USA gypsy moth populations for control purposes had been attempted. The first releases of this fungus were as early as 1910 and 1911, when two diseased gypsy moth cadavers collected in the Tokyo area provided inoculum for infection of a large number of larvae which subsequently were released near Boston, Massachusetts. However, in both 1910 and 1911, no fungal infections were recovered as a result of the field release (Speare & Colley 1912), and establishment was presumed to have failed (Soper et al. 1988, Hajek et al. 1995, Hajek 1999). The next attempts to establish E. maimaiga were in 1985 and 1986 when an isolate originating from Ishikawa Prefecture, Japan, was released in New York State and Virginia, respectively. Careful collection of gypsy moth from the release sites documented that disease transmission probably did not occur in 1985 and was extremely low in 1986. Intensive collection of gypsy moth larvae from these sites in 1987, 1989 and 1991 failed to document the presence of E. maimaiga (Hajek et al. 1995). The origin of the E. maimaiga that was found in the USA in 1989 is still unknown. Several hypotheses for its origin and establishment have been proposed and are discussed in detail in Hajek et al. (1995). In summary, one hypothesis proposes that the fungus presently in the USA originated from one of the deliberate introductions, either from 1910–1911 or 1985–1986, but that it was poorly adapted to USA conditions and persisted only at undetectable levels ; subsequently,
942 a more aggressive strain arose through natural selection, and then E. maimaiga began to spread (Hajek et al. 1995). However, based on a model including records of temperature and precipitation from 1965 to 1995, Weseloh (1998) concluded that if E. maimaiga had originated from the 1910–1911 introductions, epizootics of E. maimaiga would have been observed in 1945 and 1971. Nevertheless, since the basic appearances of E. maimaiga-killed larvae and those killed by the gypsy moth nuclear polyhedrosis virus (gmNPV) are similar, E. maimaiga-killed cadavers could potentially have been mistaken for NPV-killed larvae, especially in the absence of any indication that the fungus was operating among gypsy moths whereas the gmNPV had been well known and it effects monitored long before this time. It is unlikely that the 1985–1986 introductions gave rise to the establishment of E. maimaiga in the USA because intensive sampling especially targeting E. maimaiga from release plots failed to find this fungus (Hajek et al. 1995). Another hypothesis proposed that E. maimaiga was only recently introduced to the USA by accident (Andreadis & Weseloh 1990, Hajek et al. 1995). Multiple sources for introduction of inoculum are possible, for example imported materials, especially wooden packing materials contaminated by resting spores shipped from anywhere within the natural distribution of E. maimaiga, or contaminated footwear (Hajek et al. 1995). Additionally Andreadis & Weseloh (1990) speculated that E. maimaiga could have been inadvertently introduced along with egg parasitoids from Japan or contaminated gypsy moth egg masses, which have been shown to harbour resting spores. This, however, also implies an introduction long ago, before the use of gypsy moth quarantine laboratories in the USA in 1928 (Coulson & Soper 1989). The last hypothesis suggests that E. maimaiga has evolved from E. aulicae, a naturally occurring complex of fungal pathogens of forest Lepidoptera in the USA, which also includes E. maimaiga. Based on morphological data and transmission experiments (Soper et al. 1988), as well as more recent genetic data (Hajek et al. 1990, Walsh 1996), this last hypothesis has now been rejected. Thus several convincing lines of evidence support the hypothesis of an undocumented accidental introduction of E. maimaiga into the USA, although this has never been tested empirically. The releases of E. maimaiga isolates for biocontrol described above, as well as establishment and spread of this fungus in the USA, have been carefully recorded, providing an unique opportunity to explore changes in the genetic diversity from putative source populations in Asia and founder populations in the USA. The objectives for this study were to: (1) test the hypothesis that the E. maimaiga populations in the USA have reduced genetic variability due to founder effects compared to the putative ancestral populations in Asia; (2) track the origin of the North American populations of this fungus ; and (3) assess whether genetic differences
C. Nielsen, M. G. Milgroom and A. E. Hajek
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among E. maimaiga isolates are correlated to morphological differences. MATERIALS AND METHODS Fungal isolates 30 Entomophaga maimaiga isolates from different geographical locations within the USA, Japan, China and far-eastern Russia were selected for study (Table 1, Fig. 1). All isolates were isolated in vitro from hemolymph of infected gypsy moth larvae as described by Papierok & Hajek (1997). The infected larvae were obtained either by collections of naturally infected larvae or from larvae infected in the laboratory upon exposure to soil containing germinating resting spores, as described by Hajek, Shimazu & Knoblauch (2000). For comparison, one isolate of E. aulicae from a brown-tail moth, Euproctis chrysorrhoea (Lepidoptera : Lymantriidae), was included. All fungal isolates were grown in liquid culture (Grace’s insect culture media, Cellgro, Herndorn, VA ; supplemented with 5% fetal
bovine serum, Gibco BRL, Grand Island, NY). Before isolation of DNA all isolates were sub-cultured at least three times to get rid of insect hemocytes. All isolates are stored and maintained at the Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF, Ithaca, NY). Amplified fragment length polymorphism (AFLP) The AFLP procedure was performed essentially as described in the protocol for plant mapping by Applied Biosystems (Foster City, CA). The protocol is based on the methods described by Vos et al. (1995) but uses fluorescent dyes to label the primers instead of radioactivity. Initially, several combinations of EcoRI and MseI primers with various numbers of selective bases were screened. We found that the best resolution was obtained with a preselective amplification followed by a combination of MseI primers with three selective bases, and EcoRI primers with two to three selective bases. Details of these methods are given below.
Table 1. Entomophaga maimaiga and E. aulicae isolates included in this study. Cornell no.a
ARSEF no.
Country of origin
State/Prefecture/ Province/nearest city
Year of collection
92NY14-1-1 96NY1-13A 00NY1-1-2 89MA1-1-1b,c,d 98MA2-4A 03MA3-2-1 03MA3-1-1b,c 98MI1-1Ab,c 96VA9-1A 97VA10-1A 03PA1-1-5b 03PA5-1-5 03PA6-1-4 96MD-B2-6 84JP1-1392 84JP1-1400b,d 86JP2-2370 98JP2-9Ab,c 98JP2-4A 98JP2-14A 98JP3-8A 98JP3-5A 01JP4-11-1b 01JP4-12-1 03JP5-1-2b 03JP5-1-4 99RU1-1-1b,c,d 02CN1-1-1b,c,d 02CN1-2-1 02CN1-2-2 03ME3-1-2b (E. aulicae)
3828 5396 6625 2779 6051 7124 7123 6053 5569 5568 7190 7352 7353 5384 1392 1400 2370 6162 6253 6255 6258 6171 7104 7114 7186 7187 7127 7139 7183 7185 7142
USA USA USA USA USA USA USA USA USA USA USA USA USA USA Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Russia China China China USA
New York New York New York Massachusetts Massachusetts Massachusetts Massachusetts Michigan Virginia Virginia Pennsylvania Pennsylvania Pennsylvania Maryland Ishikawa Ishikawa Chiba Chiba Chiba Chiba Ibaraki Ibaraki Iwate Iwate Hiroshima Hiroshima Khabarowsk, Russia Heilongjiang Heilongjiang Heilongjiang Maine
1996 1996 2000 2000 1998 2003 2003 1998 1996 1997 2003 2003 2003 1996 1984 1984 1986 1998 1998 1998 1998 1998 2001 2001 2003 2003 1999 2002 2002 2002 2003
a In the Cornell identification number, the first digits give the year for collection of soil samples containing resting spores or collection of a cadaver in the field; the next two letters indicate either State (USA) or Country of origin; numbers indicate the site number, followed the soil sample number/plot number or tree number from which soil was collected, and the last number (when applicable) indicates the isolate number in cases in which more than one isolate was isolated sample. b Length and width of primary conidia measured. c Diameter of resting spores measured. d Number of nuclei per primary conidia counted.
AFLP diversity of Entomophaga maimaiga
944
(A) 500 km MA1,2 NY1,14 MA3
MI1 PA1, 5,6 MD1
VA9,10
(B) 500 km RU1 CN1
Russia
China
North JP4
Korea JP1
South Korea
JP5
Japan
JP3 JP2
Fig. 1. Sampling locations of Entomophaga maimaiga in the USA and eastern Asia. (A) Map of the northeastern states of the US showing the sampling localities (dots) and E. maimaiga release sites in 1910–1911 (star) and 1985–1986 (triangles). (B) Map of eastern Asia showing the sampling localities (dots) and approximate locations of the source populations for E. maimaiga releases in the US (grey circles). The isolate released in Massachusetts in 1910–1911 originated from near Tokyo (close to current samples from Chiba and Ibaraki Prefectures, JP2 and JP3); while the isolate (84JP1-1400) released in New York state and Virginia in 1985 and 1986, respectively, originated from Ishikawa Prefecture (JP1).
DNA extraction Protoplasts were harvested after centrifugation under sterile conditions at 300 g for 10 min and re-suspended in 200 ml phosphate-buffered saline (PBS) (pH 7.4). Total genomic fungal DNA was prepared using the DNeasy Tissue Kit from Qiagen Inc. (Valencia, CA) following the protocol given for DNA extraction from cultured animals cells. DNA was eluted in Buffer AE (10 mM Tris-Cl, 0.5 mM EDTA ; pH 9.0). RNA was degraded by adding 1: 100 Ribonuclease A (10 mg mlx1 ; Sigma, St Louis, MO) and incubating samples for 20 min at 37 xC. DNA concentrations were quantified by UV spectrophotometry using a Beckman DU 640 Spectrophotometer (Columbia, MD).
DNA digestion and ligation of adapters DNA digestion and ligation of adapters were performed in a single step. Each reaction was conducted in a 50 ml volume containing 200 ng of sample DNA, 5 U EcoRI (New England Biolabs, Beverly, MA), 5 U MseI
(New England Biolabs), 1r T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 mg mlx1 BSA (pH 7.5) ; New England Biolabs), 2 Weiss U T4 ligase (New England Biolabs), and 2.5 pmol EcoRI- and 25 pmol MseI-specific adapter pairs (Integrated DNA Technology, Coralville, IA). The reactions were incubated overnight at room temperature, and reaction mixtures were then diluted to 100 ml with TE0.1 buffer (1 mM Tris-Cl; 0.1 mM EDTA ; pH 8.0) and stored at x20 x until use. Preselective amplification Preselective amplification was performed in 0.2 ml Thermowell tubes (Corning, Corning, NY) with a total volume of 20 ml containing 1r supplied PCR buffer (New England Biolabs), 200 mM of each dNTP (New England Biolabs), 0.4 mM EcoRI-A primer (Integrated DNA Technology), 0.4 mM MseI-C primer (Integrated DNA Technology), 0.8 U Taq polymerase (New England Biolabs) and 4 ml of the diluted digestionligation solution. Amplification was performed in
C. Nielsen, M. G. Milgroom and A. E. Hajek
945
a thermal cycler (PTC-100 Peltier ; MJ Research, Boston, MA) with the following parameters : 72 x for 2 min, 25 cycles of 20 s DNA denaturation at 94 x, 30 s annealing at 56 x and 2 min extension at 72 x followed by 30 min at 60 x to avoid split peaks. The amplification product was diluted 1 :20 with TE0.1 buffer.
PowerMarker v. 3.07 (Liu & Spencer 2001) using Nei’s gene diversity estimate h=1xSp2i , where pi is the frequency of the ith reaction pattern (Nei 1973). Variance and standard errors were estimated by nonparametric bootstrapping across different loci 1000 times (Weir 1996).
Selective amplification
Cluster analysis
Four different primer combinations were used : (1) EcoRI-AAC/MseI-CTC ; (2) EcoRI-AAC/MseI-CTA ; (3) EcoRI-AC/MseI-CTC ; and (4) EcoRI-AC/MseICTA. All EcoRI primers were labelled with fluorescent dye (FAM). The reaction mixture was identical to the preselective PCR, apart from using 0.05 mM EcoRI primer (Integrated DNA Technology), 0.25 mM MseI primer (Integrated DNA Technology), and 4 ml of the diluted preselective amplification product as template. Amplification was performed in a thermal cycler with the following cycle parameters : 30 s DNA denaturation at 94 x, 30 s annealing and 1 min extension at 72 x. The annealing temperature used in the first cycle was 66 x and then subsequently reduced by 1 x at each cycle for the next 10 cycles and continued at 56 x for another 25 cycles.
Both distance and parsimony analysis of AFLP data were performed. For the distance analysis neighbourjoining was performed with the statistical software program TreeCon for Windows v. 1.3b (van de Peer & de Wachter 1994) using the Dice coefficient (Nei & Li 1979) for calculating of the distance matrix. The isolate from Russia (99RU1-1-1) was chosen as out-group because it was genetically distinct from all other isolates (see results below). Parsimony analysis was implemented using the computer software PAUP* 4.0 (Swofford 1998) using unweighted maximum parsimony analysis. In both analyses, support for internal branches was assessed by 1000 bootstrap replications.
Morphology Microscope slides
Electrophoresis Prior to electrophoresis, 1.0 ml of selective amplification product was added to a loading buffer mix (9 ml Hi-Di formamide, Applied Biosystems ; and 0.05 ml GenScan 500 LIZ, internal lane size standard, Applied Biosystems), heated to 95 x for 5 min and then quickly chilled on ice. The mixture was then loaded on an automated DNA capillary sequencer (ABI Prism1 3700 DNA Analyzer ; Applied Biosystems).
Scoring of data Computer files containing sizing data for all primer combinations for each isolate were visualized and scored using Genemapper v. 3.0 (Applied Biosystems). Each unique peak in the chromatogram was interpreted as an independent genetic locus. Only loci between 50 and 500 bp were included in the following analysis and differences in intensity were not taken into account. Furthermore, only strong and clearly distinct loci were scored. Repeatability was tested for three of the isolates included in this study by isolating DNA and running assays independently twice. Identical AFLP haplotypes were obtained.
Estimation of diversity Data for all primer combinations were pooled and transformed to a binary matrix excluding invariant characters. Gene diversity was estimated with
The isolates used for morphological studies were selected to represent groups demonstrated by analysis of AFLP data (Table 1). Measurements were based on in vivo material. Sporulating cadavers and cadavers filled with resting spores were obtained as described by Hajek, Butler & Wheeler (1995). Sporulating cadavers were placed between microscope slides under humid conditions, and conidia were collected for a maximum of 1 h to prevent production of secondary conidia. Non-sporulating cadavers were squashed and microscopically checked for the presence of resting spores. Resting spores were allowed to reach full maturation (ca 10 d after host death at room temperature) before microscope slides were prepared by placing a droplet of body fluid containing resting spores on the microscope slide.
Quantification Primary conidia and resting spores were mounted in lactic acid and sealed with nail polish until examination. The length and width of primary conidia and diameter of resting spores were measured % using an ocular micrometer under a Zeiss Axioskop light microscope with phase-contrast (400r). For each cadaver 20 conidia or resting spores were measured. The numbers of nuclei in each of 10 primary conidia were determined after staining with DAPI (4,6diamidino-2-phenylindole, 5 mg ml-1) using a Zeiss Axioskop (Filter 0.5 ; violet, excitation 395–440) at 400rmagnification.
AFLP diversity of Entomophaga maimaiga
946
Table 2. Number of DNA fragments amplified in 30 E. maimaiga isolates using four AFLP primer sets. EcoRI-AAC/ MseI-CTC
EcoRI-AAC/ MseI-CTA
EcoRI-AC/ MseI-CTC
EcoRI-AC/ MseI-CTA
Totals
Population
Na
Tb
Pc
%d
T
P
%
T
P
%
T
P
%
T
P
%
All isolates North America Asia Japan
30 14 16 12
65 54 64 60
14 1 13 6
21.5 1.9 20.3 10.0
77 71 74 70
15 2 10 4
19.4 2.8 13.5 5.7
87 76 86 82
17 2 15 9
19.5 2.6 17.4 11.0
91 81 90 86
19 5 18 10
20.9 6.2 20.0 11.6
320 282 314 298
65 10 56 29
20.3 3.5 17.8 9.7
a b c d
N, number of isolates included. T, total number of fragments. P, number of polymorphic fragments. %, percentage of polymorphic fragments out of total number of fragments.
Table 3. Number of haplotypes and gene diversitya.
Population
N
No. of haplotypes
Gene diversity¡SE
All isolates North America Asia Japan
30 14 16 12
26 11 15 12
0.227¡0.004 0.041¡0.004 0.223¡0.005 0.131¡0.006
a Calculated by Nei’s gene diversity estimate h=1xSpi2, where pi is the frequency of the ith allele at each locus using the software program PowerMarker v. 3.07.
RESULTS Diversity estimation All primer combinations successfully amplified multiple DNA fragments for all isolates included in this study. The AFLP profiles of Entomophaga aulicae were different from the E. maimaiga isolates, with the two species sharing only a few alleles (data not shown). For each primer combination, between 65 and 91 loci were scored giving rise to 19–22 % polymorphic loci, excluding E. aulicae (Table 2). AFLP haplotypes were different for all Asian isolates except for two Chinese isolates originating from the same soil sample. In contrast, identical haplotypes were obtained for US isolates collected from different states ; e.g. the same haplotype was found in New York and Maryland, and another haplotype was found in Pennsylvania and Massachusetts. For the 14 USA isolates, only tenpolymorphic AFLP loci were found, whereas 56 loci were polymorphic among the 16 Asian isolates and 29 loci were polymorphic among the 12 isolates from Japan alone (Table 2). In all, 26 unique haplotypes were identified for the 30 isolates in this study (Table 3). Average gene diversity estimates h for the polymorphic loci were 0.223¡0.005 for Asia (including Japan), 0.131¡0.006 for Japan only, and 0.041¡0.004 for the USA. Cluster analysis The neighbour-joining analysis (Fig. 2) and parsimony analysis of AFLP data produced essentially identical results, with only the hypothesized relationships among
the most similar isolates from the USA differing between the two techniques ; all other hierarchical relationships were identical in the two analyses. Both distance and parsimony analyses formed distinct clades that correlated with geographical regions of origin. Among the Japanese isolates, one clade consisted of isolates collected near Tokyo (Chiba and Ibaraki Prefectures) ; three other clades of Japanese isolates correlated to the other three prefectures sampled (Ishikawa, Iwate, and Hiroshima). On the Asian mainland, one clade was formed by the three Chinese isolates. The single isolate available from Russia was used as an out-group to root the tree because it was the most genetically different isolate in an unrooted neighbour-joining tree (not shown). However, we could not calculate bootstrap values from an unrooted tree ; we used it only to choose an out-group. All 14 isolates from the USA were included in one clade (Fig. 2). The US clade is part of the larger Japanese clade, and is in a subclade that includes isolates from Japan outside of the Tokyo area. No correlations between geographical location and clade were found among the USA isolates. Morphology Morphological dimensions of conidia and resting spores of Entomophaga maimaiga and E. aulicae and numbers of nuclei per conidia are listed in Table 4. For E. maimaiga, the length of conidia varied between 33–38 mm, the width between 25–29 mm and the lengthwidth ratio between 1.25 and 1.38. E. maimaiga contained 17–36 nuclei per conidium. The diameter of resting spores was ca 37 mm. The primary conidia of E. aulicae measured 36r25 mm. DISCUSSION The number of polymorphic loci, number of haplotypes found, and gene diversity demonstrate that populations of Entomophaga maimaiga are more genetically diverse in Asia than in the USA. The average gene diversity is approximately five times greater in the Asian sample than the USA sample, and three times greater in the Japanese sample compared to the USA sample. These results are consistent with what would be
C. Nielsen, M. G. Milgroom and A. E. Hajek
947
Table 4. Dimensions of conidia and resting spores (mm¡SE) and number of nuclei per conidium in Entomophaga maimaiga and E. aulicae. All structures were obtained from cadavers of Lymantria dispar injected with an in vitro isolate of fungus. Values given are based on 20 objects per specimen except for number of nuclei per conidium, which is only based on counts from 10 conidia per isolate. Primary conidia, mean length
Primary conidia, mean width
Mean ratio (Primary conidia)
No. of nuclei per conidium
RSa, mean diam
Isolate no. Cadaver no.
1
2
1
2
1
2
1
3
E. maimaiga 89MA1-1-1 (2779)b 03MA3-1-1 (7123) 98MI1-1A (6053) 03PA1-1-5 (7190) 84JP1-1400 (1400) 98JP2-9A (6162) 99RU1-1-1 (7127) 02CN1-1-1 (7139)
34.5¡0.5 35.5¡0.5 38¡0.5 35¡0.5 34.5¡0.5 35.5¡0.5 33.5¡0.5 37.5¡0.5
33¡0.5 37¡0.5 35¡0.5 33.5¡0.5 35.5¡0.5 36¡0.5 34.5¡0.5 37¡0.5
26.5¡0.5 27.5¡0.5 29¡0.5 26¡0.5 25¡0.5 28.5¡0.5 25¡0.5 27.5¡0.5
25¡0.5 27.5¡0.5 26¡0.5 26¡0.5 27¡0.5 28.5¡0.5 26¡0.5 27¡0.5
1.29¡0.01 1.33¡0.01 1.32¡0.01 1.34¡0.01 1.36¡0.01 1.25¡0.01 1.35¡0.02 1.37¡0.01
1.32¡0.03 1.35¡0.01 1.36¡0.01 1.30¡0.01 1.32¡0.01 1.28¡0.01 1.31¡0.01 1.38¡0.01
21.8 [19–27]c – – – 23.3 [19–36] – 19.0 [17–24] 22.6 [18–30]
37¡0.5 – 37¡0.5 – – 37¡0.5 37¡0.5 37¡0.5
1.43¡0.01
–
–
–
E. aulicae 03ME3-1-2 a b c
36¡0.5
–
25¡0.5
–
RS, Resting spores. Number given in parentheses is the ARSEF accession number. Numbers in square brackets give the range.
Distance 0.1
96VA9-1A 98MI1-1A 54 97VA10-1A 03PA6-1-4 00NY1-1-2 03PA5-1-5 03PA1-1-5 USA 03MA3-2-1 98MA2-4A 03MA3-1-1 100 89MA1-1-1 79 92NY14-1-1 96MD-B2-6 71 96NY1-13A 1 84JP1-1400 Ishikawa, Japan 84JP1-1392 01JP4-12-1 Iwate, Japan 01JP4-11-1 57
61 92 60 100
86
98
03JP5-1-4 03JP5-1-2 98JP2-9A 98JP3-5A 99 63 98JP3-8A 87 98JP2-14A 98JP2-4A 85 86JP2-2370
02CN1-1-1 100 02CN1-2-2 02CN1-2-1
99RU1-1-1
Hiroshima, Japan
Chiba & Ibaraki,, Japan
China Russia
Fig. 2. Neighbour-joining tree based on AFLP haplotypes of Entomophaga maimaiga. This tree was generated using the Dice coefficient for calculation of the distance matrix. The calculations and the drawing were performed with the software program TreeCon for Windows v. 1.3b. USA isolate numbers are shown in grey, and Asian isolates are shown in black.
expected when comparing an ancestral source population and a founder population (Nei et al. 1975) and support the hypothesis of establishment of only a small number of E. maimaiga individuals in the USA. Consequently, the founder population in the USA lost genetic variability compared to the source population
in Asia. Our data are thus similar to previous reports of plant pathogens recently established on new continents, e.g. Cryphonectria parasitica (Milgroom et al. 1992, Milgroom et al. 1996), and Phytophthora infestans (Fry & Spielman 1991) where a loss of genetic variability was found in the founder population compared to the
AFLP diversity of Entomophaga maimaiga source population. In such studies the sampling scheme must, however, be taken into account especially when interpreting results from a relatively limited number of isolates per population. Because we used roughly comparable sampling schemes in the USA and Japan we are confident that the population of E. maimaiga is more diverse in Japan than the USA population. In other words, the number of isolates included from the USA was greater than for Japan, and the distance between some of the sampling localities in the USA was greater than in Japan (Fig. 1). Among the 30 isolates in this study, both distance and parsimony analyses formed six distinct clades separated from the out-group from Russia (99RU1-1-1). The clade that included all isolates from the USA was in the same clade as all isolates from Japan, which was distinct from isolates from China. High bootstrap support for most clades in Asia may indicate restricted migration, preventing significant genetic exchange between geographically distinct populations. In contrast to the situation in Asia, no correlation between geographical location and clade was found among the US isolates. However, this fungus has been moved between locations within the USA for control purposes (Hajek & Roberts 1991, Smitley et al. 1995, Hajek, Elkington & Witcosky et al. 1996a). For example, E. maimaiga was moved from Massachusetts to Maryland, Michigan, New York, Pennsylvania, Virginia and West Virginia (Smitley et al. 1995, Hajek et al. 1996a) and from central New York to Maryland, Virginia and West Virginia (Hajek & Roberts 1991, Hajek et al. 1996a) in the early and mid-1990s. It is therefore impossible to determine whether the fungus at an individual location is the result of spread from a release or migration from naturally infected areas. Furthermore, because E. maimaiga was introduced into the USA relatively recently, genetically distinct subpopulations may not have evolved yet. Both distance and parsimony analyses of AFLP data support an introduction of E. maimaiga into the USA from a location in Japan other than the Tokyo area (Chiba and Ibaraki), although it has to be kept in mind that the isolate from the Toyo areas released in Massachusetts in 1910 and 1911 was not included in our study. The closest relative to the North American isolates was 84JP1-1400 (ARSEF 1400), the isolate released in New York state and Virginia in 1985 and 1986, respectively (Hajek et al. 1995). However, based on the following four arguments we hypothesize that the population now present in the USA came from an accidental introduction rather than the deliberate release of 84JP1-1400. (1) Isolate 84JP1-1400 is in a clade distinct from the USA clade supported by a boot-trap value of 92%. (2) The amount of variation in AFLP haplotypes seen in one locality over time is relatively small (e.g. isolates 86JP2-2370 and 98JP2-4A/8A/9A/ 14A from Chiba were collected in 1989 and 1998, respectively, but have nearly the same AFLP profile), thus a change of haplotype from isolate 84JP1-1400
948 since its release in 1985 and 1986 to the haplotypes obtained for the USA isolates does not seems likely, although comprehensive sampling within population was not done. (3) Despite intensive sampling, E. maimaiga was never found from the release sites in 1987, 1989 or 1990 (Hajek et al. 1995). And (4) during an epizootic, the maximum geographic spread of E. maimaiga is about 100 km per season (Dwyer, Elkinton & Hajek 1998), and even shorter distances are expected in endemic situations. In 1989, E. maimaiga was documented o300 km from the release sites of isolate 84JP1-1400 in 1985 and 1986, which is probably farther than it could have migrated in 3–4 yr. This study further demonstrates that AFLPs are a reliable genotyping technique for estimating intraspecific variation of entomophthoralean fungi. Previously, RAPDs have been the predominant method for fingerprinting in insect pathology (e.g. Hodge, Sawyer & Humber 1995, Rohel et al. 1997, Nielsen et al. 2001), but this method has often been criticized for lack of reproducibility, because the fragment patterns are highly sensitive to the polymerase, the primers and the cycling parameters used (Takamatsu 1998). In contrast, AFLP assays are more robust and reliable for genotyping due to the stringent reaction conditions for primer annealing (Vos & Kupier 1998). We found that the best results were obtained using a combination of MseI primers with three selective bases and EcoRI primers with two or three selective bases. The length of the primers chosen in this study is thus more similar to protocols recommended for plants and invertebrates rather than protocols used for most fungi (Vos & Kupier 1998). This is, however, not surprising because the genome size of E. aulicae previously was estimated as y8.0r106 kb (Murrin et al. 1986) which is the same order of magnitude of most plants and about 100 times larger than most fungi (Murrin et al. 1986, Vos & Kupier 1998). For each primer combination, 65–91 loci were scored and generated 14–19 polymorphic loci, enabling us to differentiate 26 out of the 30 E. maimaiga isolates included in this study. As expected, more loci were scored for AFLP assays employing EcoRI with two compared to three base extensions. However, the shorter primers did not reveal a greater number of scorable polymorphisms possibly because several loci had to be excluded due to the closeness of the peaks in the chromatogram, which made it impossible to determine whether some of the loci consisted of one or two amplification products. Morphological and cytological characters of the primary conidia are of taxonomic importance in the E. aulicae species complex, which includes E. maimaiga (Soper et al. 1988). Our measurements of conidial length and width made it possible to distinguish E. maimaiga isolates from the only E. aulicae isolate included in this study, whereas morphological measurements could not distinguish between E. maimaiga isolates from different geographical locations. Morphometric dimensions of both conidia and resting
C. Nielsen, M. G. Milgroom and A. E. Hajek spores were slightly larger in our study than those given in the original description of E. maimaiga in Soper et al. (1988), even for isolate 84JP1-1400 (ARSEF 1400) which was included in both studies; our results were similar to dimensions given by Andreadis & Weseloh (1990) for in vivo isolates collected in Connecticut. However, it has previously been shown, for the closely related entomophthoralean fungus Entomophthora schizophorae, that in vitro growth history can significantly affect the dimensions of conidia and number of nuclei per conidium, at least until these fungi have been cycled in the host twice (Eilenberg, Bresciani & Latge´ 1990). It is therefore likely that previous in vitro growth history could have affected both our morphometric measurements as well as those given in the literature. Unfortunately, neither our study nor that published by Soper et al. (1988) have recorded the growth history of the isolates included in their studies. In summary the most significant finding in this study, that the overall genetic diversity of E. maimaiga is considerably greater in Japan than the USA, is consistent with what would be expected when comparing an ancestral source population and a founder population. Furthermore, E. maimaiga appears to have been introduced to the USA from Japan, probably from a location outside the Tokyo area.
ACKNOWLEDGEMENTS We are grateful to Stephen Kresovich, Institute of Genomic Diversity at Cornell University, who placed his laboratory at our disposal. We also thank Sharon E. Mitchell, Rebecca S. Bennet and Italo Delalibera jr for their kind help, technical advice and constructive discussions throughout this project. We thank Joshua E. Izzo, Hong Sun, Michael M. Wheeler and Joshua J. Hannan for technical laboratory support. For collection of soil we are indebted to the following people: George Boettner, Joseph S. Elkinton, Shota Jikumaru, Beth Knoblauch, Gary Laudermilch, Zengzhi Li, Timothy Marasco, Galina Markova, Max McFadden, Mitsuaki Shimazu and Nathan Siegert. We thank Alison E. Burke, Michael M. Wheeler, and Richard S. Soper for isolation of several of the isolates included in this study. Finally, we thank The Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF) for providing and maintaining all the fungal isolates included in this study. This project was principally funded by USDA NRICGP grant no. 2002-01968 with additional funding from The Danish Research Agency grant no. 23-03-0009.
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Corresponding Editor: R. A. Humber