Survival of clones of NAN Ophiostoma novo-ulmi around its probable centre of appearance in North America

Mycol. Res. 104 (11) : 1322–1332 (November 2000)

Printed in the United Kingdom.

1322

Survival of clones of NAN Ophiostoma novo-ulmi around its probable centre of appearance in North America

Clive M. BRASIER and Susan A. KIRK Forest Research Station, Alice Holt Lodge, Farnham, Surrey GU10 4LH, UK. E-mail : c.brasier!forestry.gov.uk Received 10 May 1999 ; accepted 1 February 2000.

275 isolates of Ophiostoma novo-ulmi, sampled across the southern Great Lakes region of North America from Wisconsin to Ohio in 1996, were analysed for vegetative compatibility (vc) types. Over 60 % of the sample was a clonal vc component, comprising only two vc types (the AMSG and EUSG) ; the remainder of the sample was highly heterogeneous for vc types. Vc diversity was highest in Indiana and Michigan ; close to where NAN O. novo-ulmi probably first appeared in North America. Each vc clone exhibited a uniform RAPD haplotype and a lower frequency of the A compared with the B mating type. This is consistent with the clones being mainly asexually spread, but with a potential for inbreeding, an unusual feature in a ‘ genetic clone ’. The heterogeneous component, however, exhibited diverse RAPD haplotypes and a near 1 : 1 ratio of A : B mating types, indicating that the mainly novel vc types of this component arise via sexual recombination. In Europe, dominant vc clones have been quickly replaced by novel vc types. In the Great Lakes region, however, vc clones appear to have survived for over 50 years despite a high potential for emergence of new vc types via sexual recombination. No differences were found between the clonal and heterogeneous components with regard to growth rate and pathogenicity, two important fitness parameters. Two other factors, a low level of selection imposed by deleterious d-factor viruses and a density dependent effect associated with vegetative incompatibility, may have favoured prolonged survival of the clones in North America.

INTRODUCTION Two pandemics of Dutch elm disease have occurred in Europe and North America since 1900, caused by the spread of Ophiostoma ulmi and O. novo-ulmi respectively. O. ulmi was introduced from Europe into the eastern seaboard of North America during in the 1920s (Clinton & McCormick 1936), and advanced steadily across the continent (Brasier 1990). O. novo-ulmi has spread as two distinct races, the Eurasian (EAN) and North American (NAN), now considered equivalent to subspecies (Brasier 1991). Their geographical origins remain unknown (Brasier & Mehrotra 1995, Pipe, Buck & Brasier 1997). The NAN race is believed to have first appeared in the Indiana area of the southern Great Lakes in North America during the 1940s, again as a result of an introduction (McNabb 1974, Brasier 1990, Mitchell & Brasier 1994). It has since migrated eastwards and westwards, replacing O. ulmi (Gibbs, Houston & Smalley 1979, Houston 1985, 1991, Hintz et al. 1993, Mitchell & Brasier 1994, Brasier 1996). The EAN race is thought to have appeared first in the Moldova-Ukraine region of Europe in the 1940s. Subsequently, the NAN was introduced from North America into Europe, where it now overlaps in many places with the EAN (Brasier 1990). Analysis of a trans-American sample of NAN O. novo-ulmi showed that this population had very low diversity compared with NAN populations in Europe. Only 16 vegetative

compatibility (vc) types were identified among 112 isolates examined. Three dominant vc types (the AMSG, AM2SG and EUSG vc supergroups ; see Brasier 1996) comprised 58 %, 20 % and 10 % of the sample respectively. The isolates within each of these groups had morphologically uniform colonies. Each of the remaining 13 vc types was represented by a single isolate and these isolates were morphologically variable. The population therefore comprised a large ‘ clonal ’ and a small ‘ heterogeneous ’ component (Brasier 1996). Vc diversity was concentrated in the southern Great Lakes, whereas on the east and west coasts, where O. novo-ulmi had arrived only recently, the samples comprised a single dominant vc type (the AM2SG and AMSG respectively). This pattern was consistent with previous evidence that NAN O. novo-ulmi first appeared in North America in the Great Lakes area. The distribution of the novel vc types across the southern Great Lakes suggested that they arose locally, through independent genetic events. However, the data were insufficient to allow a critical analysis. Crosses carried out subsequently between the dominant AMSG, EUSG and AM2SG groups revealed a much greater potential for vc diversity than that actually observed in nature (Milgroom & Brasier 1997). The AMSG and EUSG differ by at least seven unlinked vic loci, giving a potential to generate a minimum of 128 vc types via recombination. This demonstrated that the low vc diversity observed in nature was not due to limited

C. M. Brasier and S. A. Kirk numbers of polymorphic vic loci. Milgroom & Brasier (1997) proposed restricted sexual recombination as an explanation for the low diversity. It has also been suggested that selection may favour survival of the vc supergroups over recombinant vc types (Brasier 1988 a, 1996). The dynamics of vc diversity in the Great Lakes O. novoulmi population continues to be of special interest for two reasons. First, because the vegetative incompatibility system regulates spread of deleterious fungal viruses, known as dfactors, in O. novo-ulmi (Brasier 1986 a). These d-factors are being studied as natural and artificial biological control agents of the pathogen (Brasier 1988 a, Webber 1993). In Europe, they have spread abundantly in the vc clones at epidemic fronts (Brasier 1988 a). In North America, however, only three overtly d-infected O. novo-ulmi isolates have been identified so far, all in the AM2SG vc supergroup (Brasier 1996). The reasons for such a low d-infection level in North America remain unclear. Second, the genetic structure of the O. novoulmi population in the Great Lakes area may throw further light upon the appearance of the NAN in North America. To address these issues, a detailed sample survey of the southern Great Lakes O. novo-ulmi population was carried out in 1996. The principal objectives were to determine the structure of the population across the region ; and to investigate the role of sexual recombination and natural selection in the maintenance of vc diversity. Other objectives were to search for a major vic gene combination (‘ w reactiongroup ’), the existence of which was predicted from crosses between the AMSG and EUSG vc types by Milgroom & Brasier (1997) ; and to search for additional d-factors for possible use as biological control agents. This paper presents the main results and conclusions of the survey. MATERIALS AND METHODS Isolates studied and survey details The geographical locations of the isolates collected are summarised in Table 1 and Fig. 1. Each isolate was derived from an elm twig with live bark, collected from an individual diseased tree or sapling of Ulmus americana or U. rubra. Each represents a unique elm xylem infection resulting from a vector beetle feeding wound (Brasier 1986 b). Isolates US1US295 were collected by CMB between 23 September and 11 October 1996 across Wisconsin, Illinois, Indiana, Michigan and Ohio adjacent to Lake Michigan and Lake Erie (Fig. 1). Isolates US296-US307 were collected by Prof. E. B. Smalley and forestry officers of Milwaukee, Wisconsin, during October 1997. Owing to time constraints and the distances to be covered, the distribution of the samples largely reflected the density and accessibility of elms along the route. For convenience of the vegetative compatibility type analysis, the isolates were arbitrarily assigned to 13 subsamples, denoted I–XIII (Fig. 1). Each subsample comprised a cluster of sample sites. Isolation method, media and storage of cultures Slivers of xylem from the twig samples showing dark streaks characteristic of infection were removed with a scalpel, plated

1323 on a selective medium containing actidione and streptomycin (Brasier 1981) and incubated at 20–22 mC. Elm sapwood agar (ESA) and 2 % Oxoid malt extract agar (MEA) were prepared as described in Brasier (1981). Shortterm stock cultures were maintained on MEA at 20 m and subcultured at 2 wk intervals. Long-term stock cultures were maintained on MEA slopes at k20 m and under liquid nitrogen as a glycerol and spores mixture. Vegetative compatibility tests Vegetative compatibility type tests were carried out on ESA in 9 cm Petri dishes. Isolates were inoculated 1 cm apart and incubated at 20 m in darkness until the colonies had reached the edge of the plate (ca 7 d). They were allowed to develop a further 25 d in diffuse daylight at room temperature (20–23 m) before being scored. The vc system in O. novo-ulmi Vegetative incompatibility reaction types in O. novo-ulmi are determined by the extent of barrage formation and subsequent mycelial penetration in paired cultures (Brasier 1984, 1996). In compatible reactions (c-reactions) isolates either intermingle freely, or produce slight mycelial thickening along the junction line between the two colonies. Two main incompatible reaction types are found : the wide (w) and narrow (n) reactions (see Brasier 1996 : figs 1–4). W-reactions are characterised by a wide, diffuse mycelial barrage that forms along the junction line. This is accompanied by relatively deep (ca 20 mm) mycelial penetration by one isolate into the other’s colony. The extent of the penetration is indicated by the depth of synnematal production (and, in sexually compatible pairings, perithecial production) on either side of the barrage. N-reactions are characterised by a narrow, dense white mycelial barrage accompanied by limited mycelial penetration (3–8 mm), synnematal and perithecial production. Transmission of d-factor viruses (and formation of nuclearcytoplasmic hybrids) is strongly inhibited in pairings giving w-reactions, but less inhibited in those giving n-reactions. Dfactors are transmitted freely across compatible reactions (Brasier 1984, 1986 b, Milgroom & Brasier 1997). Isolates giving compatible reactions are isogenic at all vic loci. W-reactions are epistatic to n-reactions and are determined by genes segregating at two unlinked vic loci. The genetic control of the subordinate n-reactions involves a minimum of five additional vic loci, but no further details are known (Brasier 1984, Milgroom & Brasier 1997). Isolates giving a w-reaction when paired belong to different wgroups ; isolates belonging to the same w-group give either an n-reaction or a c-reaction (Brasier 1996, Milgroom & Brasier 1997). Four w-groups (1–4) were identified in the previous, trans-American survey (Brasier 1996). Of the vc supergroups identified in that survey, the AMSG and AM2SG belonged to w-group 1 and the EUSG to w-group 2. W-group 3 was represented by only a single isolate and w-group 4 by two isolates. A fifth w-group, w-group 5, was generated in crosses between w-groups 1 and 2 and was predicted to occur in North America if the AMSG and EUSG crossed in nature (Milgroom & Brasier 1997).

Clones of Ophiostoma novo-ulmi

1324

Table 1. Distribution of w-groups, vc supergroups and other vc categories among 13 regional subsamples of Ophiostoma. novo-ulmi.

W-reaction group … State WI

WI

WI

IL

IL

IL

IN

MI

MI

MI

MI

OH

OH

VC type or category … Subsample I US69–US88 vc types\isolates

w-group 1 No. of isolates

AMSG

Heterogeneitya (isolates\vc types)

w-group 2 niAMSG

EUSG

niEUSG

– –

–

– –

w-group 4

% AMSG

Overall

HC

– –

85n0

5n0

1n0

– –

78n3

5n8

1n0

20

17(&)b

23

18("!)

1(!)

3(!)

1(")

27

15(#)

4($) 3\4

3(!)

1(!)

4(#) 4\4

– –

55n6

2n7

1n1

Subsample IV US1–US18 ; US89 vc types\isolates

18

10(#)

4(#) 3\4

4(#) 3\4

– –

55n6

2n6

1n3

Subsample V US90–US116 vc types\isolates

26

20(#)

2(") 2\2

4(!)

– –

– –

76n9

6n5

1n0

Subsample VI US117–132 vc types\isolates

15

6(!)

2(#) 1\2

3(#)

3(") 2\3

– –

1(")

40n0

2n5

1n5

Subsample VII US133–155 vc types\isolates

22

7(")

6(%) 5\6

3(#)

3(#) 3\3

2(#) 2\2

31n8

1n7

1n1

Subsample VIII US156–179 vc types\isolates

24

9($)

6(!) 6\6

3(!)

1(!)

5(!) 3\5c

37n5

2n0

1n2

Subsample IX US199–220 vc types\isolates

22

10(!)

2(") 2\2

3(!)

1(")

4($) 4\4

2(!) 2\2

45n5

2n0

1n0

Subsample X US180–198 vc types\isolates

15

6(!)

5(#) 3\5c

1(!)

1(")

1(!)

1(!)

40n0

1n9

1n3

Subsample XI US221–244 vc types\isolates

23

9(!)

4(") 4\4

2(!)

2(#) 2\2

5(!) 2\5d

1(")

39n1

2n1

1n3

Subsample XII US245–262 vc types\isolates

13

5(")

4(%) 3\4

1(!)

1(")

2(!) 2\2

38n5

1n6

1n2

Subsample XIII US263–295 vc types\isolates

33

17($)

3(!) 3\3

8(")

1(!)

51n5

3n3

1n0

281

149(#*) 52n0

39(")) 14n8

34(&) 11n3

19("") 7n6

30("!) 11n1

10(%) 3n3

19n5

46n2

14n7

57n9

33n3

40n0

Subsample II US19–US33 ; US56–US68 vc types\isolates Subsample III US34–US52 ; US296–US307 vc types\isolates

Total no. of isolates % vc type or category overall % A mating type overalle

– –

–

– –

3(") 3\3

w-group 5

– –

– –

1(!)

– –

– – 4(#) 4\4

a vc categories niAMSG, niEUSG, w-group 4 and w-group 5 ; b No. of A mating types shown in parentheses ; c Three out of five isolates are the same vc type ; d Four out of five isolates are the same vc type ; e A types in clonal component (AMSGjEUSG) l 18n6 % ; A types in heterogeneous component (HC) l 43n9 %.

Determination of vc types The individual vc type of every isolate within each of the regional subsamples I-XIII was determined as follows. Initially, all 281 isolates were paired against a tester isolate of the AMSG, representing w-group 1. Isolates giving a compatible reaction were assigned to the AMSG. Within each subsample, isolates giving an n-reaction against the AMSG tester were paired in all possible combinations to determine the individual

vc types. All those that gave a w-reaction with the AMSG were paired with a tester isolate of the EUSG, representing wgroup 2. Those giving a compatible reaction were assigned to the EUSG. Those giving an n-reaction were again paired against each other on a subsample basis to determine the individual vc types. Thirty-five isolates gave w-reactions against both the AMSG and the EUSG testers. These were divided into two geographical sets of 18 and 17 isolates respectively. The

mn

1325

L. Huron

C. M. Brasier and S. A. Kirk

WI X

pe

Milwaukeee

n = 2.0 h = 5.0 md

wa

II

III

n =23 h = 5.8

MI

V

ma sy

yi

XI

en

n= 23 h=2.1

ss

Chicago

IV

wr

bk

n=2.4 h=2.0

an

Toledo

n=18 h=2.6

bg

VII n= 22 h=1.7

ln

fe

XII n= 13 h=1.6

hn

VI

Peoria bn

pn

Detroit

se

rd ga

iy

py

lg

VIII

n = 26 h = 6.5

IX n= 22 h=2.0

n=27 h=2.7

bt

n= 15 h=1.9

L. Erie

I

L. Michigan

ws

XIII wd sh

n= 33 h= 3.3

mf sg

n=15 h=2.5

IN IL

OH Columbus

cn

Indianapolis

Fig. 1. Distribution of subsamples I–XIII of Ophiostoma novo-ulmi across the southern Great Lakes area. Dashed lines denote the subsample clusters. n, total number of isolates in subsample ; h, overall heterogeneity (isolates\vc types) of subsample. Within the subsamples, the circle sizes (smallest to largest) represent 1, 2–4, 5–9, 10–14 and 17 isolates sampled respectively. Towns and cities : an, Adrian ; bg, Bowling Green ; bk, Battle Creek ; bn, Bloomington ; bt, Beloit ; cn, Champaign ; en Elgin ; fe, Fort Wayne ; ga, Genoa ; hn, Huntingdon ; iy, Imlay City ; lg, Lansing ; ln, Lacon ; ma, Mendota ; md, Madison ; mf, Mansfield ; mn, Mauston ; pe, Portage ; pn, Port Huron ; py, Perry ; rd, Rockford ; se, Stockbridge ; sg, Strasburg ; sh, Shenandoah ; ss, Sturgis ; sy, Spring Valley ; wa, Waukesha ; wd, Willard ; wr, Windsor ; ws, Wisconsin Dells ; yi, Ypsilanti. Scale bar l 50 km.

isolates within each set were paired in all possible combinations. This led to the identification of two additional wgroups. The occurrence of the same two w-groups in each set was confirmed by inter-set pairings. Representatives of the additional w-groups were then paired against representative testers of w-groups 3 and 4. Sexual compatibility types The single-locus, two-allele sexual compatibility system of O. novo-ulmi operates independently of the vegetative incompatibility system (Brasier 1984, 1998 b). The two sexual compatibility types, termed A- and B-types, were determined from perithecial production in the vc pairings. Assessment of colony morphologies, growth rates and d-infection levels Mean radial growth rates and colony characteristics of the isolates were assessed on MEA following the methods of Gibbs & Brasier (1973) and Brasier (1981). A test for the presence of a deleterious, cytoplasmically transmissible virus (d-factor) was carried out on any overtly unstable, and therefore potentially d-infected, isolate. Each unstable isolate was paired, as a donor, with a healthy MBC

(benomyl) tolerant recipient isolate of the same vc type on ESA at 20 m, as described by Brasier (1983, 1986 a). Each pairing was then monitored for the development of a ‘ dreaction ’ (Brasier 1986 a), indicating transfer of a d-factor to the recipient. Subcultures were taken from any visibly dinfected portions of the recipient’s colony. These were tested for MBC tolerance ; and for overt d-infection as indicated by growth instability on MEA. Pathogenicity tests Pathogenicity tests were carried out on 4-year old clonal UlmusiCommelin at the Forestry Commission Research Nursery, Headley, Hampshire by inoculating 0n2 ml of a 1i10& ml−" suspension of yeast phase cells of an isolate into the xylem as described by Brasier (1981, 1986 c). O. ulmi isolate GOLB4, and medium and high pathogenicity EAN O. novo-ulmi tester isolates H413a and H327 (Brasier 1986 c, Sutherland et al. 1997) were used as controls. There were 4 replicate trees per isolate, except for the controls for which there were 8. Percent defoliation of inoculated trees was scored at 12 wk by a team of three independent assessors (Brasier 1986 c). Inoculated trees were destroyed after assessment.

Clones of Ophiostoma novo-ulmi AMSG

EUSG

1326 RESULTS

Heterogeneous component

Vegetative compatibility types W-groups 1000 bp

AMSG

EUSG

Heterogeneous component

W-groups 1, 2 and 4 identified in the trans-American survey (Brasier, 1996) were confirmed in the present analysis. Also identified was a new group, w-group 5, the existence of which was predicted by Milgroom & Brasier (1997). No other wgroups were found. Elimination of w-group 3

300 bp

Fig. 2–3. Agarose gel electrophoresis of RAPD products of DNA from Ophiostoma novo-ulmi isolates with two different primers. Fig. 2. Primer OPA9. Fig. 3. Primer OPC18. Lanes : 1, 1 kb ladder ; 2–7, AMSG isolates US140, US271, US296, US91, US123, US181 ; 8–12, EUSG isolates US117, US36, US59, US208, US274 ; 13–14, niAMSG isolates US122, US136 ; 15–16, niEUSG isolates US120, US300 ; 17–18, w-group 4 isolates US135, US221a ; 19–20, w-group 5 isolates US290, US275 ; 21, 1 kb ladder. Arrows indicate fragments characteristic of the EUSG (OPA9) and AMSG (OPC18).

RAPD analysis of genomic DNA Mycelium was harvested from 10-day-old colonies grown on MEA overlain with cellophane (Rogers, Buck & Brasier 1986). Genomic DNA was extracted using freeze dried ground mycelium. A primary extraction buffer containing 10 m TrisHCl [pH 8n0], 10 m EDTA and 0n5 % SDS was followed by a minimum of four phenol\chloroform\isoamyl alcohol [25 : 24 : 1] and one chloroform\isoamyl alcohol [24 : 1] extractions. After precipitation the DNA was subjected to an additional extraction with ribonuclease (RNase One, Promega) to remove any extraneous RNA, and then dried down under vacuum and stored at k80 m. A Perkin–Elmer GeneAmp 9700 thermal cycler was used to amplify the DNA in 25 µl reaction volumes comprising 10 m Tris-HCl [pH 9n0 at 25 m], 50 m KCl, 0n1 % Triton X100, 2 m MgCl , 0n2 m each of dATP, dCTP, dGTP, dTTP, # 5 pmoles Operon primer and 50 ng DNA. The template DNA was denatured at 95 m for 4 min, then held at 72 m while 2n5 units of Taq DNA polymerase [Promega] were added. The amplification conditions were : denaturing at 94 m for 15 s, annealing at 36 m for 1 min, followed by extension at 72m for 1 min. This sequence was repeated for 40 cycles after which samples were held at 72 m for a further 9 min. Amplified products were run on horizontal 1n2 % agarose gels (Molecular biology grade agarose, Sigma) prepared in 1iTAE electrophoresis buffer (prepared from ultrapure grade Promega 40iTAE buffer). Gels were run at 90 V for 3 h and stained with 0n5 µg ml−" ethidium bromide and visualized under shortwave UV light. A one kilobase ladder (Promega) was used as the size marker.

In the previous trans-American survey (Brasier 1996), wgroup 3 was represented by only a single isolate, H967. This isolate gave normal w-reactions when paired with isolates of w-groups 1 and 2, but an anomalous w-reaction (described as having an ‘ n-like barrage ’) with w-group 4 (Brasier 1996). In the present survey, two isolates, (US200 and US261) were found to be fully vegetatively compatible with H967. However, both these isolates also gave a normal n-reaction against a range of w-group 4 testers. Evidently the reaction between H967 and w-group 4 isolates is actually an anomalous, ‘ w-like ’ n-reaction. H967 should therefore be reassigned to w-group 4, and w-group 3 eliminated. Frequency and distribution of vc supergroups and other vc types The distribution of w-groups 1–5, their breakdown into component vc types and the level of vc heterogeneity (isolates\vc types) in each of the 13 regional subsamples, is shown in Table 1. The number of A mating types is indicated in parentheses. The AMSG was predominant across the entire survey area, comprising 52n0 % of the overall sample (Table 1). Also present at high frequency was the EUSG, at 11n3 %. The AM2SG was absent. No other ubiquitous vc groups were detected. The remainder of the sample comprised mainly unique vc types belonging to w-group 1 (i.e. niAMSG), wgroup 2 (niEUSG), or w-groups 4 and 5 (Table 1). Approximately 63 % of the sample could therefore be considered a clonal vc component (i.e. the AMSGjEUSG) and 37 % a highly heterogeneous component. Overall, wgroup 1 represented 66n8 %, w-group 2 18n9 %, w-group 4 11n1 % and w-group 5 3n3 % of the sample. The frequency of A mating types was significantly lower in the clonal than in the heterogeneous component. Thus A types represented only 19n5 % of the AMSG and 14n7 % of the EUSG ; compared with 46n2 % of the niAMSG, 57n9 % of the niEUSG, and 33n3 % and 40 % of the w-group 3 and w-group 4 categories respectively (Table 1). The diversity of the heterogeneous component was consistently high across the entire sample. Thus, on the basis of the simple index h l number of isolates\number of vc types, the diversity of this component ranged from only 1n0 to 1n5 (average 1n15 ; Table 1). No significant difference was detected between the more westerly group of subsamples (I–VI) from Wisconsin and Illinois, or the more easterly group (VII–XIII) from Indiana, Michigan and Ohio (Fig. 1). The frequency of the AMSG, however, was significantly higher in

C. M. Brasier and S. A. Kirk the westerly group of subsamples, at 66n7 %, than in the easterly group at 41n4 % (Table 1 and Fig. 1 ; Mann–Whitney test : U l 3, P l 0n004). It was highest, at 85 % and 78 %, in the two most northwestern subsamples, I and II. Correspondingly, overall levels of vc heterogeneity were significantly higher in eastern subsamples (VII–XIII ; h l 1n6–3n3) than in the western subsamples (I–VI ; h l 2n5–6n5) (Table 1 ; Mann–Whitney test : U l 3, P l 0n004). This overall difference in vc diversity between the western and eastern subsamples was also reflected at a local level. At some sample sites, where small elm saplings were plentiful, beetle vector activity and disease pressure was very high. At such sites, multiple twig samples were collected when it could be deduced, from the vertical distribution of streaking in the xylem, that each represented a unique, beetle-initiated infection. To the west, isolates collected at such sites were sometimes exclusively of the AMSG (e.g. subsample II, isolates US 20–30, South Beloit to Magnolia, Wisconsin and subsample V, US 91–100, Scarboro to Troy Grove, Illinois). In contrast, to the east, high levels of local vc diversity sometimes occurred. For example in Kingswood, Indiana, six twig samples along a 400 m transect at the same location (US 135–140, subsample VII) yielded six different vc types ; 1 AMSG, 1 niAMSG, 1 EUSG, 1 niEUSG, 1 w-group 4 and 1 w-group 5. At Mansfield, Ohio 13 twig samples along a 50 m roadside transect (subsample XIII, US 266–278) also yielded six vc types : 5 AMSG (B-type), 1 AMSG (A-type), 1 niAMSG, 3 EUSG and 3 different w-group 5 vc types. Growth-rate and d-infection tests All 281 isolates were initially screened to assess whether they had a normal, wild-type colony morphology or the unstable growth indicative of a d-infection. In repeat subcultures, only two isolates (US193 l AMSG, subsample X ; and US246 l EUSG, subsample XII) showed severely debilitated growth. When these isolates were tested as donors for their ability to transfer the degenerate phenotype to a healthy (benomyl tolerant) recipient, only US246 was confirmed to have a cytoplasmically transmissible phenotype, and was therefore considered overtly d-infected. Two other isolates (US254 and US290) exhibited non wild-type colonies consistent with their having mutated since isolation (Brasier 1982). No other unusual (i.e. non wild-type) colony types were observed. In the trans-American survey (Brasier 1996) no differences in growth rate were detected between the AMSG, the EUSG, the AM2SG and the heterogeneous component. In the present study, growth rates of the different heterogeneous component vc categories, namely the niAMSG, niEUSG, w-group 4 and w-group 5 categories (represented by 27, 16, 22 and 7 wild-type isolates respectively) were compared on MEA. The A and B mating types were roughly equally distributed in each category. These isolates were also chosen to represent the geographic range of each vc category across the Great Lakes survey area. Again, no significant differences in growth rate were found between the vc categories. Their mean growth rates (mm day−") and s were : niAMSG isolates, 4n18p0n32 ; niEUSG isolates, 4n10p0n36 ; w-group 4 isolates,

1327 Table 2. Pathogenicity of Ophiostoma novo-ulmi vc categories in UlmusiCommelin. % defoliation AMSG Vc category

A-type

B-type

niAMSG w-groups 4 and 5 B-type B-type

No. of isolates 15 15 14 14 % defoliation Isolate range 14n6–39n1 18n6–31n1 17n1–32n4 19n4–34n8 Mean and  27n1p6n5 25n0p3n0 25n2p4n1 26n8p4n7

4n22p0n32 ; w-group 5 isolates, 4n11p0n38. Likewise, no growth rate differences were obtained between the two mating types either within each category or overall. The overall mean growth rate of the 35 A-types was 4n17p0n32 and of the 37 B-types, 4n17p0n34 mm day−". European A-type isolates of O. novo-ulmi are intrinsically more fertile than B-types. They typically produce numerous protoperithecia (L) on ESA medium, whereas B-types produce a small number of protoperithecia or ascogonial initials (Brasier 1986 b). Sixty A- and sixty B-type isolates representing the vc categories across the Great Lakes area were compared for fertility on ESA medium. Female reproductive structures developing after 21 d were scored on the basis of k, nil to jjj, abundant. A similar pattern of fertility was observed to that in Europe. The A types produced numerous dark protoperithecia (common frequency jj to jjj) ; the B types only a few pale protoperithecia or ascogonial initials (common frequency j to jj). Pathogenicity tests To compare the pathogenicity of a vc supergroup with that of the heterogeneous component, 15 AMSG isolates, 14 niAMSG isolates and 14 w-group 4 and w-group 5 isolates, all B mating types, were inoculated into clonal U.iCommelin in June 1997. To compare A and B mating types, 15 A-type AMSG isolates were also tested. The EUSG was omitted because of the limited numbers of trees available and because, in the trans-American survey, it was shown to be no different in pathogenicity from the AMSG (Brasier 1996). The results are summarised in Table 2. No significant differences were detected between the mean pathogenicity of the AMSG, niAMSG or w-group 4 plus w-group 5 categories. Within the AMSG, the B-types were slightly less pathogenic and slightly less variable than the A-types (25n0p3n0 versus 27n1p6n5 % defoliation respectively), but again not significantly so. RAPD banding patterns Wild isolates To examine the underlying genetic diversity of the supergroup and heterogeneous components, PCR amplification of DNA from 70 isolates representing the AMSG, EUSG, niAMSG, niEUSG, w-group 4 and w-group 5 vc categories was carried out. Fifty-six of the isolates were chosen to represent the broad geographic range of the Great Lakes sample and the

Clones of Ophiostoma novo-ulmi approximate frequency of A and B mating types in the vc categories. An additional 14 isolates from other areas of North America (Brasier 1996) and from Europe were included to further widen the geographic range. Initially, genomic DNA of a sample of 16 isolates was amplified with 32 random primers : OPA 1–20 ; OPC7, 10, 16, 18 and 19 ; OPJ 14, 16, 18 and 20 ; OPK 2, 4 and 10. The products were then separated by gel electrophoresis. Five primers (OPA 8, 9, 14, OPC 18 and OPK 10) each resulted in a single, clear, intense band that, on a presence or absence basis, distinguished AMSG from EUSG isolates and was reproducible in multiple independent assays (Figs 2–3). These five primers were then used to generate PCR products for the remaining 54 isolates, to establish whether they would consistently discriminate between the AMSG and EUSG ; and to discover whether heterogeneous component isolates exhibited different RAPD patterns. The distribution of these markers among all 70 isolates is shown in Table 3. All 18 AMSG isolates exhibited a uniform and distinctive banding pattern. This applied whether the isolates were from the current survey, or from geographic locations well outside the Great Lakes area, such as Oregon, California, Kansas, Quebec and Maine. It also applied to both A and B mating types. Likewise, a uniform and distinct marker pattern was exhibited by all 16 EUSG isolates whether from North America or Europe, or of A or B mating type. In contrast, 36 heterogeneous component isolates showed a wide assortment of marker patterns. No predominant associations of markers occurred. Nor was there a tendency for these isolates to exhibit either AMSG or EUSG – like marker combinations. For example, only three of the 36 isolates exhibited both of the OPA 8 and OPA 9 generated markers that were representative of the EUSG. Likewise, only five isolates exhibited all three markers (generated by OPA 14, OPC 18 and OPK 10) that characterised the AMSG. On a presence\absence basis, none of the markers occurred in a near 1 : 1 ratio. Segregation of markers in an AMSGiEUSG cross To investigate whether the pattern of RAPD markers among the heterogeneous component would be consistent with recombination between the AMSG and EUSG, 14 F progeny " of a cross between AMSG isolate H1064 (A-type) and EUSG isolate H975 (B-type) (see Milgroom & Brasier 1997 for further details) were analysed. The progeny examined reflected those still available in long term storage. They included representatives of the two novel w-groups, 4 and 5, generated by the cross ; and the only two progeny of a parental (AMSG) vc type obtained. RAPD markers were generated for these isolates using the same five primers as for the wild isolates. The results are shown in Table 4. The parent isolates exhibited the marker patterns that characterise the AMSG and EUSG. The progeny isolates showed apparently random combinations of these markers, similar to the patterns observed in wild heterogeneous component isolates. The two progeny, F P and F P , that " $& " %( were of AMSG (i.e. a parental) vc type each exhibited a unique marker profile distinct from that of the parental AMSG

1328 pattern. On a presence\absence basis, the distribution of EUSG versus AMSG markers among the progeny was close to a 1 : 1 ratio in four of the five primers, suggesting the involvement in each case of two alleles at a single locus. That for OPK 10, at 11 : 3, deviated further from a 1 : 1. It was nonetheless within the expected binomial distribution for 5 samples of 14 randompevents. Overall, the results for the progeny were consistent with genetic recombination of unlinked parental markers. DISCUSSION A previous study showed that in North America the vc diversity of O. novo-ulmi was highest in the Indiana-Great Lakes region, where it purportedly first appeared, whereas the pathogen was nearly clonal on the east and west coasts, where it is a relatively recent arrival (Brasier 1996). This study has investigated the structure of the Southern Great Lakes population in detail. Approximately 63 % of this population could be assigned to the AMSG and EUSG clonal lineages ; the remaining 37 % being mainly unique vc types. The frequency of the AMSG was significantly lower in the more easterly Great Lakes states of Indiana, Michigan and Ohio (mean 41n4 %) than in Wisconsin and Illinois to the west of the region (mean 66n7 %). Vc diversity overall was also significantly greater in the Indiana–Michigan–Ohio area. This pattern supports the earlier proposal that clonality increases with the distance from Indiana, reaching its maximum on the west and east coasts (Brasier 1996). This pattern of variability is also consistent with the view that the O. novo-ulmi founder population in the Indiana area was largely clonal, but has steadily increased in diversity ; and that the AMSG and EUSG were a significant part of the founder population (Brasier 1996). The behaviour of O. novoulmi populations in Europe, where dominant vc clones are found at epidemic fronts, but where the population subsequently becomes heterogeneous, further supports this interpretation. Indeed, the dominant frontal vc clone found in many regions of Europe is the EUSG (Brasier 1988 a). However, European populations change from clonal to
90 % heterogeneous in only ca 6–10 years (Brasier 1988 a, Brasier & Kirk 1992), whereas the Indiana–southern Great Lakes population has reached only 37 % heterogeneity in ca 50 years. In Europe, also, the resulting diversity in the heterogeneous component is greater. For example, over five years an increase from 2 to 14 w-groups was recorded at a single sample site in central Spain (Brasier & Kirk 1992) ; yet only 4 w-groups have been identified so far across the whole of North America. An obvious question is why the vc clones have survived longer in North America. Using an AMSGiEUSG cross, Milgroom & Brasier (1997) showed that the potential vc diversity in North America is much higher than that observed. They suggested limited sexual recombination as a cause. Another possibility is that the dominant vc clones have a fitness advantage over recombinants (Brasier, 1988 a, 1996). In the present survey, three pieces of evidence indicate that the novel vc types do arise via sexual recombination. First, the ratio of A : B mating types is close to the 1 : 1 expected of

C. M. Brasier and S. A. Kirk ascospore progeny, in both the niAMSG and niEUSG vc categories. It also approaches a 1 : 1 in the heterogeneous component as a whole (44 % A-types). In contrast, the A : B ratio in the clonal component deviates strongly from a 1 : 1, the AMSG and EUSG combined exhibiting only 18 % Atypes. Second, the heterogeneous component exhibits highly diverse RAPD haplotypes, resembling the progeny of AMSGiEUSG crosses ; whereas the haplotypes of the AMSG and EUSG are characteristically uniform. Third, the vc types of heterogeneous component isolates tend to be unique even at a local level. This is consistent with their arising locally, via recombination. The RAPD analysis has also confirmed that the vc supergroups are ‘ true ’ genetic clones, disseminated mainly via asexual reproduction. Thus EUSG isolates, whether from Indiana, Michigan, the UK or Portugal, exhibit an identical RAPD profile. Indeed, this supports an earlier conclusion that NAN O. novo-ulmi was introduced into Europe from the Great Lakes area (Brasier & Gibbs 1973, Bates 1990). Even the scarce A-mating types of the EUSG or AMSG exhibit the same RAPD profile as their respective B-types, indicating that the two mating types are near isogenic. In contrast, two A-type AMSG progeny generated in an AMSGiEUSG cross (Table 4) exhibited unique RAPD profiles. This suggests that a degree of sexual inbreeding is occurring within the supergroups in nature. A propensity for inbreeding is an unusual phenomenon in a genetic ‘ clone ’, and could reflect the influence of virus pressure (see below). To examine the possibility that the vc clones have a fitness advantage over heterogeneous components vc types, they were compared for two major fitness variables (Brasier 1986 c, 1999), growth rate and pathogenicity. No differences were detected between them, confirming a similar result obtained in a previous study (Brasier 1996). Likewise, no differences in growth rate or pathogenicity were found between A and B mating types. The A-types, however, were shown to have a higher intrinsic fertility, comparable to that in European Atypes. In Europe, this higher fertility is associated with reduced growth and pathogenic fitness and with a consequent scarcity of A-types in the field (Brasier 1984, 1986 c, 1999, Brasier & Webber 1987). Over many generations, it may also have led to a lower frequency of A-types in the North American vc clones. In America, however, selection against Atypes is likely to be less intense than in Europe because American elms are more susceptible to the pathogen (Brasier 1996). Two other factors could account for the prolonged survival of the vc clones in North America. One is a density-dependent phenomenon associated with vegetative incompatibility. In O. novo-ulmi, antagonistic, inter-mycelial invasion occurs between adjacent, vegetatively incompatible colonies ; this is known as the ‘ penetration effect ’ (Brasier 1984, 1986 d, 1999, Mitchell 1988). Studies in Europe show that O. novo-ulmi populations in elm bark are highly dynamic. During the initial colonization of the bark (along with the vector bark beetles), a mosaic of competing vc types develops. Later, a further turnover of vc types occurs : (1) when perithecia are produced and the resulting ascospore germlings colonise the bark in competition with the resident vc types ; and (2) when the genotypes in the

1329 xylem migrate into the bark (Webber & Brasier 1984, Brasier 1986 b, Webber, Brasier & Mitchell 1988). In North America, however, much (60–100 %) of the bark is likely to be colonized by one or other of the dominant vc clones. Often, colonies of the resident clone may fuse to form a sizeable thallus (Brasier 1996). To become established, a germinating ascospore of a novel (recombinant) vc-type would have to overcome the territorial dominance of the clone. It would also have to compete with sibling ascospores of other vc types (Brasier 1986 d, 1999). Experiments show that O. novo-ulmi individuals with larger territories tend to be stronger penetrators of adjacent colonies (J. F. Webber, unpubl.). Therefore, embryonic ascospore-derived colonies of a novel vc-type are likely to be unilaterally penetrated and so displaced by larger, resident AMSG or EUSG colonies. Unless they have some other advantage over the clones, the establishment of novel vctypes in the bark could be a relatively infrequent event. Another factor likely to influence survival of a particular vc type is virus pressure. In Europe, vc clones become highly dinfected (Brasier 1988 a). In North America, however, dinfection levels are remarkably low. In the present survey, only one overtly d-infected isolate was found (in the EUSG). Likewise in the previous survey, only three overtly d-infected isolates were found, all in the AM2SG vc supergroup and all from Millinocket, Maine (Brasier 1996, unpubl.). D-infected isolates are poor elm colonisers (Webber 1987, 1993, Sutherland & Brasier 1997). Also, like other weakly growing colonies, they tend to be unilaterally penetrated by other vctypes (Brasier 1984). In Europe, selection pressure exerted on the vc clones by high levels of d-infection is believed to have favoured the survival of the largely ‘ d-factor free ’ novel vc types of the heterogeneous component (Brasier 1988 a). In North America, however, pressure exerted by d-factors could be too low to confer a comparable survival advantage to novel vc types. The above conclusion has considerable implications for proposals to release ‘ wild-type ’ d-factors as biological control agents (Brasier 1996). Were this to be attempted in areas such as the Great Lakes, where both the AMSG and EUSG are present, and therefore multiple vic loci are available, it is likely that the heterogeneous component would simply increase in frequency at the expense of the clonal component. Such releases would have a better chance of success if carried out in areas having only a single vc clone. For example, in Oregon and Washington DC, which currently have only the AMSG and AM2SG clones respectively ; and New Zealand, where only the EUSG is present (Brasier 1996, unpubl., Brasier & Gadgil, 1992). In areas where vc diversity is already high or potentially high, a virus deployment strategy that bypasses the vc system would be needed. One such strategy involves the insertion of viruses into the pathogen’s nuclei or mitochondria (Choi & Nuss 1992, Anagnostakis et al. 1998, Brasier 1998). The four d-factors identified so far in North American O. novo-ulmi isolates are being studied for their molecular properties and for their effectiveness as potential biocontrol agents. The reasons why the levels of d-infection in America are so low compared with Europe also require investigation (Brasier 1996). Ascospores of O. novo-ulmi are typically free

Clones of Ophiostoma novo-ulmi

1330

Table 3. Distribution of RAPDs markers among vc categories of wild-type Ophiostoma. novo-ulmi isolates. Primer and fragment size (bp) VC type and isolate

State or Provincea

Mating type

OPA8 1300

OPA9 1000

OPA14 1900

OPC18 300

OPK10 300

AMSG US140 US271 US296 US58 US91 US123 US158 US181 US204 US222 US267

IN OH WI WI IL IL IN MI MI MI OH

A A A B B B B B B B B

# # # # # # # # # # #

# # # # # # # # # # #

$ $ $ $ $ $ $ $ $ $ $

$ $ $ $ $ $ $ $ $ $ $

$ $ $ $ $ $ $ $ ND $ ND

QU QU NS KA ME OR CA

A A B B B B B

# # # # # # #

# # # # # # #

$ $ $ $ $ $ $

$ $ $ $ $ $ $

$ $ $ $ $ ND ND

IL IN WI WI IL IN MI MI MI OH OH OH

A A B B B B B B B B B B

$ $ $ $ $ $ $ $ $ $ $ $

$ $ $ $ $ $ $ $ $ $ $ $

# # # # # # # # # # # #

# # # # # # # # # # # #

# # # # # # # # # ND ND #

QU

B

$

$

#

#

#

B PG UK

A B B

$ $ $

$ $ $

# # #

# # #

# # #

niAMSG US122 US211 US254 US302 US306 US136 US218 US225 US269 US304

IL MI OH WI WI IN MI MI OH WI

A A A A A B B B B B

# $ # # # # # # # #

# # # # # $ $ # # #

$ # $ $ $ # $ $ # $

$ $ $ # # # # $ # $

# ND $ $ $ $ $ $ ND $

niEUSG US11 US120 US139 US300

IL IL IN WI

A B B B

# # # #

# $ $ #

$ $ # #

# $ # #

$ # $ #

w-group 4 US135 US203 US216 US298 US303 US305

IN MI MI WI WI WI

A A A A A A

# $ # # $ #

$ $ # # # #

$ # $ $ # $

# # $ # $ #

$ # $ $ # $

Trans American survey H1064 H961 H161 H667 H937 H2001 H2002 EUSG US117 US137 US36 US59 US103 US157 US193 US208 US238 US260a US270 US274 Trans American survey H975 European isolates H351 PG402 W2

C. M. Brasier and S. A. Kirk

1331

Table 3. (cont.) Primer and fragment size (bp) VC type and isolate

State or Provincea

Mating type

OPA8 1300

OPA9 1000

OPA14 1900

OPC18 300

OPK10 300

IL IL MI MI MI WI

B B B B B B

# $ # # # #

$ $ # # # #

$ $ # $ $ $

# # # $ # $

# # # $ $ $

Trans American survey H967b H2094 H2092

QU OH OH

A A B

$ # #

$ # $

$ $ $

# # $

ND ND ND

w-group 5 US266 US290 US138 US213 US217 US273 US275

OH OH IN MI MI OH OH

A A B B B B B

$ # # # ND # #

# # $ $ # # $

# $ $ # # # $

# $ # $ $ $ #

# ND $ $ $ $ #

29 : 6

23 : 13

24 : 12

15 : 21

20 : 10

US13 US14 US219 US221a US221b US229

Ratio AMSG : EUSG marker

Open circles, no fragment ; closed circles, fragment present. a B l Belgium, CA l California, IL l Illinois, IN l Indiana, KA l Kansas, ME l Maine, MI l Michigan, NS l Nova Scotia, OH l Ohio, OR l Oregon, PG l Portugal, QU l Quebec, WI l Wisconsin. b Previously assigned to w-group 3. ND l no data. bp l base pairs. Table 4. Distribution of RAPD markers among parents and progeny of an AMSGiEUSG cross. Primer and fragment size (bp) VC type and isolate

Mating type

OPA8 1300

OPA9 1000

OPA14 1900

OPC18 300

OPK10 300

Parents AMSG-H1064 EUSG-H975

A B

# $

# $

$ #

$ #

$ #

F progeny " AMSG FP " $& FP

A A

# #

$ #

$ $

# #

$ $

A A A B B B

# $ $ # $ #

# $ $ $ # #

# # $ $ $ #

# $ # $ $ $

$ $ $ # $ $

A A A B B B

$ # # $ $ #

# $ # $ # #

# # # $ $ #

# # # $ # $

$ $ $ # $ #

8:6

8:6

7:7

6:8

11 : 3

" %(

w-group 4 FP " & FP " '% FP " "!' FP " '& FP " ""& FP " "%(

w-group 5 FP " $# FP " %& FP " ""$ FP " # FP " #" FP " "'&

Ratio AMSG : EUSG marker bp l base pairs. Open circles, no fragment ; closed circles, fragment present.

from d-factors (Brasier 1986 a, Rogers et al. 1986), so the frequency of sexual reproduction could be one important factor. In North America, A-types occur at ca 13 % in the vc clones, whereas in Europe they are usually absent (Brasier

1988 a). This suggests a much greater potential for sexual reproduction within the American vc clones. There may therefore be a correspondingly greater restriction in d-factor spread.

Clones of Ophiostoma novo-ulmi

1332

A C K N O W L E D G E M E N TS

Brasier, C. M. & Mehrotra, M. D. (1995) Ophiostoma himal-ulmi sp. nov., a new species of Dutch elm disease fungus endemic to the Himalayas. Mycological Research 99 : 205–215. Brasier, C. M. & Webber, J. F. (1987) Positive correlation between in vitro growth rate and pathogenesis in Ceratocystis ulmi. Plant Pathology 36 : 462–466. Choi, G. H. & Nuss, D. L. (1992) Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA. Science 257 : 800–803. Clinton, G. P. & McCormick, F. A. (1936) Dutch elm disease – Graphium ulmi. Connecticut Agricultural Experiment Station Bulletin 389 : 701–752. Gibbs, J. N. & Brasier, C. M. (1973) Correlation between cultural characters and pathogenicity in Ceratocystis ulmi from Europe and North America. Nature 241 : 381–383. Gibbs, J. N., Houston, D. R. & Smalley, E. B. (1979) Aggressive and nonaggressive strains of Ceratocystis ulmi in North America. Phytopathology 69 : 1215–1219. Hintz, W. F., Jeng, R. S., Yong, D. Q. & Hubbes, M. M. (1993) A genetic survey of the pathogenic fungus Ophiostoma ulmi across a Dutch elm disease front in Western Canada. Genome 36 : 418–426. Houston, D. R. (1985) Spread and increase of Ceratocystis ulmi with cultural characteristics of the aggressive strain in north eastern North America. Plant Disease 69 : 677–680. Houston, D. R. (1991) Changes in non-aggressive and aggressive subgroups of Ophiostoma ulmi within two populations of American elm in New England. Plant Disease 75 : 720–722. McNabb, H. S. (1974) Further speculation on the origin of the aggressive strain of Ceratocystis ulmi. 6th Session of the Iowa Academy of Sciences, April 19–20 1974. Abstract. Milgroom, M. G. & Brasier, C. M. (1997) Potential diversity of vegetative compatibility types of Ophiostoma novo-ulmi in North America. Mycologia 89 : 722–726. Mitchell, A. G. (1988) Population structure and interaction between the aggressive and non-aggressive subgroups of Ophiostoma ulmi. PhD thesis, University of Bath. Mitchell, A. G. & Brasier, C. M. (1994) Contrasting structure of European and North American populations of Ophiostoma ulmi. Mycological Research 98 : 576–582. Pipe, N., Buck, K. W. & Brasier, C. M. (1997) Comparison of the cerato-ulmin (cu) gene sequences of the recently identified Himalayan Dutch elm disease fungus Ophiostoma himal-ulmi with those of O. novo-ulmi and O. ulmi suggests that the cu gene of O. novo-ulmi is unlikely to have been acquired recently from O. himal-ulmi. Mycological Research 101 : 415–421. Rogers, H. J., Buck, K. W. & Brasier, C. M. (1986) Transmission of doublestranded RNA and a disease factor in Ophiostoma ulmi. Plant Pathology 35 : 277–287. Sutherland, M. L. & Brasier, C. M. (1997) A comparison of thirteen d-factors as potential biological control agents of Ophiostoma novo-ulmi. Plant Pathology 46 : 680–693. Sutherland, M. L., Pearson, S. & Brasier, C. M. (1997) The influence of temperature and light on defoliation levels of elm by Dutch elm disease. Phytopathology 87 : 576–581. Webber, J. F. (1987) Influence of the d# factor on survival and infection by the Dutch elm disease pathogen Ophiostoma ulmi. Plant Pathology 36 : 531–538. Webber, J. F. (1993) D-factors and their potential for controlling Dutch elm disease. In Dutch elm disease research, cellular and molecular approaches. (M. B. Sticklen & J. L. Sherald, eds) : 322–332. Springer-Verlag, New York. Webber, J. F. & Brasier, C. M. (1984) The transmission of Dutch elm disease : a study of the processes involved. In Invertebrate-microbial Interactions (J. M. Anderson, A. D. M. Rayner & D. Walton, eds) : 271–306. Cambridge University Press, Cambridge, UK. Webber, J. F., Brasier, C. M. & Mitchell, A. G. (1988) The role of the saprophytic phase in Dutch elm disease. In Plant Infecting Fungi (G. F. Pegg & P. G. Ayers, eds) : 298–313. Cambridge University Press, Cambridge, UK.

We wish to thank Professor E. B. Smalley for logistical assistance with the collection of samples in Wisconsin and Dr M. Milgroom for valuable comments on the manuscript.

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Corresponding Editor : M. Levy