Biol Invasions DOI 10.1007/s10530-013-0486-z
ORIGINAL PAPER
Hybridization and introgression between the exotic Siberian elm, Ulmus pumila, and the native Field elm, U. minor, in Italy Johanne Brunet • Juan E. Zalapa • Francesco Pecori • Alberto Santini
Received: 31 July 2012 / Accepted: 4 May 2013 Ó Springer Science+Business Media Dordrecht (outside the USA) 2013
Abstract In response to the first Dutch elm disease (DED) pandemic, Siberian elm, Ulmus pumila, was planted to replace the native elm, U. minor, in Italy. The potential for hybridization between these two species is high and repeated hybridization could result in the genetic swamping of the native species and facilitate the evolution of invasiveness in the introduced species. We used genetic markers to examine the extent of hybridization between these two species and to determine the pattern of introgression. We quantified and compared the level of genetic diversity between the hybrids and the two parental species. Hybrids between U. pumila and U. minor were common. The pattern of introgression was not as strongly biased towards U. pumila as was previously observed for hybrids between U. rubra and U. pumila in the United States. The levels of heterozygosity were similar between U. minor and the hybrids and both
groups had higher levels of heterozygosity relative to U. pumila. The programs Structure and NewHybrids indicated the presence of first- (F1) and secondgeneration (F2) hybrids and of backcrosses in the hybrid population. The presence of healthy DED resistant U. minor individuals combined with the selfcompatibility of U. minor could help explain the presence of F2 individuals in Italy. The presence of F2 individuals, where most of the variability present in the hybrids will be released, could facilitate rapid evolution and the potential evolution of invasiveness of U. pumila in Italy.
J. Brunet (&) USDA-ARS, VCRU, Department of Entomology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA e-mail:
[email protected];
[email protected]
Introduction
J. E. Zalapa USDA-ARS, VCRU, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706, USA F. Pecori A. Santini Institute of Plant Protection, C.N.R., Via Madonna del Piano, 10, 50019 Sesto Fiorentino, Italy
Keywords Dutch elm disease Field elm Hybridization Introgression Microsatellites Siberian elm
The repeated introduction of exotic species by humans has accelerated the range expansion of many organisms (Crowl et al. 2008). While various species were inadvertently introduced, some introductions were intentional. For example, exotic trees have been planted as ornamental trees in urban areas or to replace native tree species decimated by a disease epidemic (Machon et al. 1997; Cogolludo-Agustin et al. 2000; Zalapa et al. 2010). The introduced species
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J. Brunet et al.
were often close congeners of the native species, which implied a high potential for hybridization between the native and introduced species. While the potential impact of hybridization on the preservation of genetic diversity of the native species was not recognized when most of these exotic species were introduced, it is now clear that hybridization can pose an increased risk of genetic assimilation and eventual loss of the native taxa (Rhymer and Simberloff 1996; Hedge et al. 2006). This is especially true for small populations already at risk from biotic or abiotic stresses (Rieseberg et al. 1989; Ellstrand and Elam 1993; Daehler and Strong 1997; Collin 2002; Burgess et al. 2005; Prentis et al. 2007). In addition, it has been established that hybridization can increase genetic diversity and novel gene combinations which may, in turn, facilitate the process of adaptation and help a species spread into new habitats (Ellstrand and Schierenbeck 2000; Vila et al. 2000; Sakai et al. 2001; Rieseberg et al. 2003; Hedge et al. 2006). Many of the European and American species of elm, Ulmus spp. (Ulmaceae), were decimated by Dutch elm disease (DED) during the 20th century, with a first pandemic caused by the ascomycete fungi, Ophiostoma ulmi (Buisman) Nannf. and a more aggressive species, O. novo-ulmi, responsible for the second pandemic (Brasier 1988, 1991). While European and North American species of elms were very susceptible to DED, with infected trees dying within 1–2 years, several Eurasian species exhibited varying degrees of tolerance to both the first (Smalley and Guries 1993) and the second DED pandemics (Santini et al. 2005; Solla et al. 2005). In response to the two DED pandemics, Siberian elm, U. pumila, was planted to replace the native elms in different countries, including Italy (Goidanich 1936) and the United States (Zalapa et al. 2010). The native Field elm, U. minor Mill., was widely used as living support for grapevine, fodder for cattle, timber for construction, and firewood when the first DED epidemic reached Italy in the 1930s (Sibilia 1930). Field elm was also important as shadow tree in pastures and as an ornamental tree in streets and in parks (Goidanich 1936). Given its wide usage, the progressive disappearance of Field elm was considered disastrous, which stimulated private nurserymen (Ansaloni 1934) and the scientific community (Sibilia 1932; Passavalli 1935) to look for a suitable replacement. Within a few years following the onset of the first DED epidemics, thousands of Siberian elms
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(Ulmus pumila L.) were distributed by authorities and nurseries throughout the whole Italian territory (Goidanich 1936). In addition, seedlings of Siberian elm were used as rootstocks (Goidanich and Azzaroli 1941), or scions were grafted onto Field elms (Ansaloni 1934). Moreover, two clones of Field elm, ‘Christina Buisman’ and ‘Villagrappa 3’, were planted as trials because they showed enough resistance to O. ulmi. Following the first DED pandemic, Goidanich (1941), an Italian plant pathologist, affirmed that the impact of DED was successfully controlled, partly as a result of the increased use of U. pumila and the selection of tolerant U. minor clones. While the widespread distribution of Siberian elms stopped shortly after World War II, many thousands of Siberian elms remained in the landscape, mainly along roads, rivers and streams or in abandoned fields. Elm species are known to cross-hybridize (Mittempergher and La Porta 1991; Goodall-Copestake et al. 2005) and the ability of U. pumila to produce fertile offspring when crossed with various DED susceptible elm species has been exploited to develop DED tolerant varieties (Smalley and Guries 1993; Santamour and Bentz 1995; Ware 1995; Santini et al. 2002, 2007; Mittempergher and Santini 2004). The ability for elm species to cross-hybridize suggests, however, a strong potential for the formation of hybrid seeds between Siberian elms and the native elm species. To date, widespread hybridization has been documented between the exotic Siberian elm, U. pumila, and Field elm, U. minor, in Spain (CogolludoAgustin et al. 2000) and between U. pumila and the native red elm, U. rubra Muhl in the Midwestern United States (Zalapa et al. 2009, 2010). A pattern of introgression biased towards U. pumila, with few backcrosses between hybrid individuals and the native species, was detected in both Spain and the United States. This pattern of biased introgression has been attributed to the lower abundance of the DED susceptible native elm species over the landscape (Cogolludo-Agustin et al. 2000; Zalapa et al. 2009, 2010). In addition, the biased introgression has been considered a threat for the conservation of the genetic diversity of the native elm, U. rubra in the United States (Zalapa et al. 2009) and U. minor in Spain (Cogolludo-Agustin et al. 2000). Furthermore, hybridization between U. pumila and U. minor or between U. pumila and U. rubra has been shown to increase the genetic diversity (Cogolludo-Agustin
Hybridization and introgression
et al. 2000; Zalapa et al. 2009, 2010) and, at least in the Midwestern United States, to affect the genetic structure of U. pumila populations (Zalapa et al. 2010). Hybridization could, therefore, have contributed to the increased range of habitats where U. pumila can establish in the United States compared to its native range (Zalapa et al. 2010). Moreover, hybridization may help explain the fact that Siberian elm has become an invasive species in 41 out of 50 states in the United States (USDA, NRCS 2002; Ding et al. 2006). In the current study, we use genetic markers to examine the extent of hybridization between the native Field elm, U. minor, and the exotic Siberian elm, U. pumila, in Italy. We examine the pattern of introgression between these two species to determine whether introgression is biased towards U. pumila, as was previously found between U. pumila and U. minor in Spain (Cogolludo-Agustin et al. 2000) and between U. pumila and U. rubra in the United States (Zalapa et al. 2009). We examine the level of genetic diversity of the hybrids relative to the two parental species and quantify the degree of genetic differentiation between these three groups. We discuss the similarities and differences between the patterns of hybridization between U. pumila and U. minor in Italy relative to U. pumila and U. minor in Spain and U. pumila and U. rubra in the Midwestern United States. Finally, we address some of the potential consequences of hybridization for the conservation of the native elm species, U. minor, in Italy and for the potential spread of naturalized populations of U. pumila throughout Italy.
Materials and methods Sampling of plant material Leaf material was collected from 96 elm trees at 61 different locations throughout Italy and some parts of France (Table 1). Twelve specimens were collected at the ex situ elm clonal collection of the Institute of Plant Protection (C.N.R.) located in Antella (43°430 N 11°220 E; altitude: 170 m), in the province of Florence, Italy. All other samples were collected in fields throughout Italy, with one to three accessions sampled per site (Table 1). The following criteria were used to assign each tree to a putative species at the time of collection: Siberian elm possesses symmetrical, onceserrate, small leaves (3–7 cm long); slender, smooth,
hairless twigs; small, blunt, hairless buds; shallowly furrowed, gray or brown bark; and comparatively small, smooth samaras (Wyman 1951); the crown is wide from roundish to vase-shaped, secondary shoots are generally pendulous. In contrast, Field elm leaves are oblanceolate to nearly circular, coarse and pubescent when young, smooth and glossy above at maturity, glabrous beneath with axillary pubescence, base uneven, corky wings (Santini et al. 2008); the crown shape varies from cylindrical to conical. Based on these morphological descriptors, eleven trees were classified in the field as U. pumila, 41 as U. minor, and 42 as putative hybrids between U. pumila and U. minor while two trees could not be identified (Table 1). In addition to these 96 samples, to facilitate the genetic identification of hybrid individuals, we used as a reference population for U. pumila, 49 accessions from China whose genetic composition had been previously described (Zalapa et al. 2008a). Genotyping using microsatellites We extracted DNA from leaf tissue using a E.Z.N.A.Ò kit (Omega Biotek, Norcross, GA, USA) and measured DNA concentrations with a BioPhotometer (Eppendorf AG, Hamburg, D). All 96 samples were genotyped using the following ten microsatellite primer-pairs (UR138, UR141, UR153, UR158, UR159, UR175, UR188a, ULM-2, ULM3, and Ulmi1-98). These 10 primer pairs amplified alleles in both U. pumila and U. minor and were originally developed in either U. rubra (UR- primers: Zalapa et al. 2008b), U. laevis (ULM- primers: Whiteley et al. 2003) or U. minor (Ulmil-98 primer: Collada et al. 2004). A total volume of 15 ll was used for the PCR reactions which included 1.5 ll 10 9 PCR buffer, 1.8 ll 25 mM MgCl2, 2.4 ll dNTPs (1.25 mM of each dATP, dGTP, dTTP, and dCTP), 1.0 ll 5 lM primer, 2 ll 10 ng ll genomic DNA, 1 U Taq DNA polymerase (Lucigen, Middleton, Wisconsin, USA), and 6.2 ll H2O. The thermocycling conditions consisted of an initial melting step at 94 °C for 3 min, followed by 30 cycles each of 94 °C for 15 s, 55/60 °C for 90 s and 72 °C for 2 min, and an elongation step of 72 °C for 20 min. We used fluorescent labeled primers 50 end 6-FAM [6-carboxyfluorescein] fluorophore; IDT Coralville, Iowa, USA. Microsatellites were run on an ABI 3730 fluorescent sequencer (POP-6 and a 50 cm array; Applied Biosystems, Foster City, California,
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J. Brunet et al. Table 1 Collected samples of putative Ulmus pumila (P), U. minor (M) and their Hybrids (H) with the classification based on morphology and the classification based on the genetic profile (genotype) determined from the program Structure
123
Sample ID
Province
Morphology
Genotype
Latitude
M100
Torino
M
M
45.13
7.47
M101
Cosenza
M
M
39.49
16.30
M102
Massa
M
M
44.10
10.11
M103
Palermo
M
M
37.54
13.26
M104
Oristano
M
H
40.80
8.47
M105
Salerno
M
M
40.24
15.35
M106
Frosinone
M
M
41.38
13.17
M107 M108
Crotone Avellino
M M
M M
39.10 40.58
17.70 15.12
M109
Agrigento
M
M
37.36
12.56
M110
Lodi
M
M
45.45
9.17
M111
Teramo
M
M
42.40
13.42
M112
Benevento
M
M
41.54
12.30
M113
Piacenza
M
M
45.20
10.00
M114
Bolzano
M
M
46.33
11.10
M115
Vercelli
M
M
45.22
8.27
M116
Bergamo
M
M
45.43
9.52
M117
Modena
M
M
44.32
10.54
M118
Pordenone
M
M
45.52
12.56
M120
Matera
M
M
40.10
16.37
M121
Imperia
M
–
44.60
7.53
M122
Padova
M
M
45.23
11.50
M123 M124
Sassari Foggia
M M
M M
41.30 41.29
9.11 15.10
M126
Piacenza
M
–
44.48
9.50
M128
Venezia
M
H
45.46
12.50
M130
Seine et Marne (FR)
M
H
48.57
3.74
M131
Calvados (FR)
M
M
49.43
2.92
M132
Charente (FR)
M
–
45.30
1.57
M133
Finistere (FR)
M
–
48.26
3.40
M134
Loiret (FR)
M
M
47.50
2.44
M135
Finistere (FR)
M
–
47.59
4.25
M136
Calvados (FR)
M
–
49.43
2.92
M137
Orne (FR)
M
H
48.55
1.23
M138
Somme (FR)
M
–
49.48
2.50
M139
Firenze
M
M
43.41
11.25
M225
–
–
H
M227 M80
– Catania
– M
M M
– 37.54
– 14.54
M81
Enna
M
M
37.23
14.18
M87
Treviso
M
M
45.47
12.35
M97
Vibo Valentia
M
M
43.47
11.15
M98
Genova
M
H
44.21
9.14
MP01
Aquila
H
M
42.02
13.56
MP02
Aquila
H
H
41.46
14.06
–
Longitude
–
Hybridization and introgression Table 1 continued
Sample ID
Province
Morphology
Genotype
Latitude
Longitude
MP03
Aquila
H
H
41.46
14.06
MP04
Aquila
H
P
41.46
14.06
MP05
Aquila
H
H
41.46
14.06
MP06
Aquila
H
H
41.46
14.06
MP07
Aquila
H
H
41.46
14.06
MP08
Aquila
H
H
42.02
13.56
MP09
Aquila
H
P
42.02
13.56
MP10 MP11
Aquila Aquila
H H
M H
42.02 42.02
13.56 13.56
MP12
Aquila
H
H
42.02
13.56
MP13
Aquila
H
H
42.02
13.56
MP14
Aquila
H
H
42.02
13.56
MP15
Aquila
H
H
42.02
13.56
MP16
Aquila
H
H
42.02
13.56
MP17
Aquila
H
P
42.02
13.56
MP18
Aquila
H
P
42.02
13.56
MP19
Aquila
H
H
42.02
13.56
MP20
Siena
H
H
43.25
11.10
MP21
Firenze
H
M
43.29
11.09
MP22
Firenze
H
M
43.29
11.08
MP23
Firenze
H
M
43.36
11.11
MP24
Firenze
H
M
43.36
11.11
MP25 MP26
Firenze Firenze
H H
M M
43.36 43.36
11.11 11.11
MP27
Firenze
H
M
43.36
11.11
MP28
Firenze
H
H
43.36
11.10
MP29
Firenze
H
H
43.36
11.10
MP30
Firenze
H
H
43.36
11.10
MP31
Firenze
H
H
43.36
11.10
MP32
Firenze
H
H
43.36
11.10
MP33
Firenze
H
–
43.42
11.24
MP34
Firenze
H
H
43.42
11.24
MP35
Firenze
H
M
43.47
11.27
MP36
Firenze
H
H
43.49
11.29
MP37
Firenze
H
–
43.49
11.29
MP38
Firenze
H
M
43.51
11.20
MP39
Firenze
H
H
43.46
11.13
MP40 MP41
Firenze Firenze
H H
H H
43.46 43.46
11.13 11.13
MP42
Firenze
H
H
43.46
11.13
P25
Firenze
P
H
43.49
11.29
P26
Firenze
P
H
43.49
11.29
P27
Firenze
P
H
43.49
11.29
P28
Firenze
P
H
43.49
11.29
P29
Firenze
P
P
45.45
13.18
123
J. Brunet et al. Table 1 continued
The provinces are in Italy unless otherwise noted where FR indicates France
Sample ID
Province
Morphology
Genotype
P30
Gorizia
P
P
45.50
13.30
P31
Trento
P
H
45.55
11 .00
P32
Lecco
P
H
45.45
9.18
P33
Trento
P
M
45.53
11.10
P34
Trento
P
H
45.42
10.55
P35
Parma
P
H
44.48
10.20
USA) using a Gensize Rox 650 ladder (GENPAK Ltd., Brighton, UK), at the UW Biotechnology Center DNA Sequence Facility. Alleles were scored using GeneMarker Software version 1.5 (SoftGenetics, State College, Pennsylvania, USA). Identification of parental species and hybrids We used three different methods to classify individuals as hybrids or as belonging to one of the two parental species (U. pumila or U. minor), based on their multilocus genotypic information. These methods included the Bayesian clustering method available in Structure (v. 3.1) (Pritchard et al. 2000), the Bayesian algorithms provided in NewHybrids v. 1.0 (Anderson and Thompson 2002) and Principal Coordinate Analyses (PCoA). We used the 49 accessions from China as reference U. pumila population in these analyses (Zalapa et al. 2008a). We could not do a manual identification of hybrids based on the genotypic profiles of U. minor and U. pumila, as was previously done to separate U. rubra from U. pumila in the United States, because we did not find sufficient species-specific alleles between U. minor and U. pumila (Zalapa et al. 2009). This could reflect a more distant relationship between U. pumila and U. rubra relative to U. pumila and U. minor as suggested by phylogenetic analyses (Wiegrefe 1992). In the Bayesian clustering method available in the program Structure (v. 3.1) (Pritchard et al., 2000), we used a priori value of K = 2 (2 genetic clusters) to account for the two parental species. We ran Structure using 50,000 Markov chain Monte Carlo iterations with 50,000 burn-in iterations and 10 replicates per run. Analyses were performed with no a priori taxonomic information and used the genetic admixture option and the correlated allele frequencies model. The program Structure calculates an admixture coefficient (q) for each individual, where q represents the
123
Latitude
Longitude
proportion of an individual’s genotype that originated from each reference population. Therefore, with K = 2, q values for the two parental species are expected to be close to 1 and first-generation hybrids (F1) are expected to have q values of 0.5. Similarly, individuals with q values of 0.75 from species 1 and 0.25 from species 2 likely represent first-generation backcrosses (BC1) to species 1, while individuals with q values of 0.25 species 1 and 0.75 species 2 most likely are BC1 to species 2. A posterior probability of 0.92 or greater was used to assign a genotype to one of the two parental species. We first associated a genotypic profile to the morphological phenotype when running Structure. To optimize data presentation, we ran Structure a second time and associated a genotypic profile with the categorical species classification provided by the first Structure run (the genetic classification of an individual as U. pumila, U. minor or as hybrid). Finally, we ran Structure for K = 1–8 to confirm the use of K = 2 as the optimal value. We used PCoA to examine the clustering of individuals. These analyses were based on the genetic distances calculated by GeneAlEx 6.0 (Peakall and Smouse 2006) for PCoA analyses (Appendix 1 of GeneAlEx). We first ran PCoA where genotypic profiles were associated to the morphological phenotypic classification of an individual as U. pumila, U. minor or hybrid; we then ran a second PCoA where genotypic profiles were associated to the genetic classification obtained from the Structure results. Finally, the program NewHybrids v. 1.1beta (Anderson and Thompson 2002) permitted an independent classification of an individual as U. minor, U. pumila or hybrid, based on its genotypic profile, and it helped further classify the hybrids into specific categories. We considered the following hybrid classes: first- (F1) and second- (F2) generation hybrids and first- (BC1) and second-generation (BC2) backcrosses. The NewHybrids algorithm was run without prior information for
Hybridization and introgression Fig. 1 Structure results for the classification of a reference population of Ulmus pumila from China, U. pumila, U. minor and their hybrids from Italy. A Structure results for K = 2, with genotypes grouped based on their morphology; B structure results for K = 2 with genotypes grouped based on genetic profiles. Each individual is represented by a thin vertical line; the black lines separate the groups. The colored segments of each line illustrate the individual’s estimated membership fractions to each of the two genetic clusters (K = 2)
A
B
China
Europe
Reference U. pumila (n=49)
Putative U. pumila (n=11), U. minor (n=36), hybrids (n=40)
China
Europe
Reference U. pumila (n=49)
BayesianU. pumila (n=6), U. minor (n=42), hybrids (n=39)
600,000 iterations following a 100,000-iteration burnin. We combined the information obtained from Structure and NewHybrids to determine the specific hybrid class an individual tree was most likely to belong. Parental species and hybrid genetic diversity After classifying individuals as U. minor, U. pumila or hybrids, based on their genotypic profiles, we used GeneAlEx 6 (Peakall and Smouse 2006) to separately describe the genetic diversity of U. pumila from China, U. pumila from Italy, U. minor and the hybrids between the two species. The genetic data from the 10 microsatellite loci were used to estimate the average observed (A) and effective (Ae) number of alleles per locus, the number of loci with allele frequencies greater than 0.05, the Shannon’s information index (I), the number of private alleles, where a private allele is defined as the number of alleles unique to a group, and the levels of observed (Ho) and expected (He) heterozygosity for each of the 4 groups. After describing the genetic diversity within each group, we compared groups by calculating the degree of genetic differentiation among groups. We performed an analysis of molecular
variance (AMOVA) (Excoffier et al. 1992) to estimate the degree of genetic variation within and among groups. We first considered 4 groups including both U. pumila from China and U. pumila from Italy and then examined the variation among 3 groups considering only U. pumila from Italy. Lastly, we calculated pairwise FST values between the four groups, U. pumila from China, U. pumila from Italy, U. minor and the hybrids using the AMOVA method of GenAlEx with 9999 bootstrap iterations. This approach brings the FST estimates in line with the Weir and Cockerham estimates (Peakall and Smouse 2006).
Results Identification of parental species and hybrids We obtained the genotypic profiles for 87 out of the 96 elm trees sampled. Based on their morphology, 11 of these trees were classified at the time of collection as U. pumila, 36 as U. minor and 40 as hybrids (Table 1). We confirmed using Structure that 2 genetic clusters best explained the genetic diversity (Appendix). Using K = 2, the Structure results identified 6 trees as U.
123
J. Brunet et al. Fig. 2 Principal coordinates analyses (PCoA 1 and 2) based on 10 microsatellite markers of a reference population of Ulmus pumila from China, and of U. pumila, U. minor, and their hybrids from Italy. A Grouping designations based on morphology; B Grouping designations based on genetic profiles
A
B
U. pumila(P1) = 56
U.minor (P2) = 43 F1= 0 F2= 35 BCP1 = 2 BCP2= 0
Fig. 3 NewHybrids analyses based on 10 microsatellite markers for a reference population of Ulmus pumila from China, and for U. pumila, U. minor, and their hybrids from Italy. Each individual is represented by a thin horizontal line divided into
123
colored segments that represent the individual’s estimated membership fractions to each of the six categories, U. pumila, U. minor, first- (F1) or second- (F2) generation hybrids, backcross to U. pumila (BCP1) or to U. minor (BCP2)
Hybridization and introgression
pumila, 42 trees as U. minor and 39 trees as hybrids (Table 1; Fig. 1). In many instances, however, the classification based on the genotype using Structure (Pritchard et al. 2000) did not correspond to the classification based on the morphology (Table 1; Fig. 1). For example, only two of the individuals originally classified as U. pumila based on their morphology were identified as U. pumila based on their genotypic profile, with the majority being identified as hybrids and one as U. minor (Table 1; Fig. 1A). In addition, six of the individuals originally classified as U. minor based on their morphology were identified as hybrids based on their genotypic profile (Table 1; Fig. 1A). Finally, a number of individuals Table 2 Genetic diversity characteristics based on 10 microsatellite loci for Ulmus pumila, U. minor, and their hybrids collected in Italy and for a U. pumila reference population from China Population
U. pumila China
U. pumila Italy
U.minor
Hybrids
N
49
6
42
39
A
4.90
2.70
5.70
7.20
Afreq. C 5 %
2.90
2.70
3.60
4.20 3.10
Ae
2.46
2.09
2.66
I
0.79
0.60
1.17
1.28
No. private alleles
0.70
0.00
0.60
0.60
Ho He
0.34 0.36
0.35 0.32
0.54 0.59
0.56 0.59
N number of individuals, A average number of alleles per locus, Afreq. C 5 % mean number of alleles with frequency greater than 0.05 per locus, Ae average effective number of alleles per locus, I Shannon index of diversity, Ho average observed heterozygosity per locus, He average expected heterozygosity per locus Table 3 Analysis of molecular variance (AMOVA) based on 10 microsatellite loci for A. four groups: Ulmus pumila, U. minor, and their hybrids from Italy and U. pumila reference Source of variance
originally classified as hybrids based on their morphology actually represented U. pumila or U. minor based on their genotypic profile (Table 1; Fig. 1A). The morphological traits typically used to identify elm species in the field did not reliably distinguish the genetic parental species and hybrids. The PCoA results supported the greater reliability of the genotypic profiles relative to morphology to group individual trees into categories representing the two parental species and the hybrids (Fig. 2). Moreover, the PCoA results illustrated a bilateral pattern of introgression, with backcrosses to both parental species (Fig. 2B), as was further supported by the Structure results. When using the admixture coefficients (q) generated in Structure to further classify the 39 hybrid individuals, we detected 21 first-generation hybrids (F1), and 18 backcrosses (BC). The 18 backcrosses were further categorized as 6 backcrosses to U. minor and 12 backcrosses to U. pumila (Fig. 1B). The NewHybrids results identified 7 individuals as U. pumila, 43 as U. minor and 37 as hybrids (Fig. 3).
Table 4 Pairwise genetic differentiation (FST) based on 10 microsatellite loci for Ulmus pumila, U. minor, and their hybrids from Italy and a U. pumila reference population from China U. pumila China
U. pumila Italy
U. minor
Hybrids
U. pumila China U. pumila Italy
0.05
U. minor
0.37
0.32
Hybrids
0.11
0.08
0.14
Values in bold are statistically significant (P \ 0.01) using the AMOVA method of GenAlEx with 9,999 permutations
population from China and for B. three groups: U. pumila, U. minor and their hybrids from Italy
d.f.
MS
Variance components
Total variance (%)
F-Stat
Value
3 132
100.01 5.99
3.03 5.99
34 66
FST
0.22
Among populations
2
63.84
2.33
25
FST
0.16
Within populations
84
6.89
6.89
75
A. Four groups Among populations Within populations B. Three groups
Significance level of P \ 0.01 for FST using the AMOVA method of GenAlEx with 9,999 permutations is in bold
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J. Brunet et al.
The programs Structure and New Hybrids classified individuals quite similarly as belonging to one of the two parental species or as representing a hybrid but the two programs differed into how the hybrids were further classified. Structure more commonly classified individuals as F1 and backcrosses (Figs. 1B, 2B). The majority of hybrid individuals were identified as F2 by the program NewHybrids; 9 individuals had a posterior probability of 0.94 or greater of belonging to the F2 category and 13 had a posterior probability of 0.90 or greater; probabilities were lower for the other hybrid individuals (Fig. 3). Genetic diversity and genetic differentiation for parental species and hybrids The hybrid individuals had the largest number of alleles, followed by U. minor and then U. pumila (Table 2). The lower number of alleles in the U. pumila individuals sampled from Italy relative to China reflected the low sample size for this group. The level of heterozygosity was greater for the hybrids and U. minor relative to U. pumila from China. The number of private alleles was similar for these three groups. Overall, genetic diversity was greatest for the hybrids, followed by U. minor and lastly U. pumila. When all four groups were considered, the AMOVA indicated that 66 % of the genetic variation was found within each group with 34 % of the variation detected among groups (Table 3A). When only U. pumila from Italy was considered, 75 % of the variation was identified within groups and 25 % among groups (Table 3B). Such a pattern would be expected if the U. pumila individuals in Italy were involved in hybrid formation. As expected, we observed a greater level of genetic differentiation between the two parental species than between each of the parental species and the hybrids (Table 4). Similar levels of genetic differentiation were observed between the hybrids and each of the two parental species, supporting bilateral introgression (Table 4). A low level of genetic differentiation was observed between U. pumila from China and U. pumila from Italy (Table 4).
Discussion We detected many hybrid individuals between U. minor and U. pumila in Italy, a pattern similar to what
123
was previously found between U. minor and U. pumila in Spain (Cogolludo-Agustin et al. 2000) and between U. rubra and U. pumila in the Midwestern United States (Zalapa et al. 2009, 2010). Although hybrids were common in Italy, the pattern of introgression was not as strongly biased towards U. pumila as was previously observed in Spain for hybrids between U. pumila and U. minor (Cogolludo-Agustin et al. 2000) and in the Midwestern United States for hybrids between U. pumila and U. rubra (Zalapa et al. 2009). In fact, approximately 33.3 % of the observed backcrosses in Italy were towards U. minor and 66.6 % towards U. pumila, in contrast to 12.5 % and 87.5 % respectively for backcrosses to U. rubra or U. pumila in the United States (Zalapa et al. 2009). No distinctions were made between different classes of hybrids in the Spanish study (Cogolludo-Agustin et al. 2000). The biased pattern of introgression towards U. pumila suggests that U. minor will likely become assimilated over time, but the process will be slower than between U. pumila and U. rubra in the Midwestern United States where introgression is more strongly biased towards U. pumila (Zalapa et al. 2009). While the threat of genetic assimilation of U. minor may be less imminent in Italy, planting U. minor trees over the landscape would increase the probability of backcross with U. minor and help prevent its genetic assimilation. The stronger pattern of biased introgression towards U. pumila observed in previous studies was attributed to the low abundance of healthy U. minor over the landscape in Spain (Cogolludo-Agustin et al. 2000) and of healthy U. rubra in the United States (Zalapa et al. 2009). The high susceptibility of U. rubra to DED, with many populations consisting of only young or diseased trees with little pollen production (Lester and Smalley 1972a, b), could explain the low abundance of U. rubra over the landscape in the United States. The species U. minor is, however, less susceptible to DED relative to U. rubra (Heybroek 1968). In fact, some DED resistant U. minor clones were planted in Italy as part of the efforts to reduce the impact of DED on elms, following the first DED pandemic (Goidanich 1941). In addition, some resistance to the more aggressive O. novo-ulmi species, responsible for the second DED epidemics, has also been observed in Field elm (Santini et al. 2005; Solla et al. 2005). Therefore, resistance to DED could help explain the maintenance of healthy Field elms over the landscape in Italy. The differences in the patterns of introgression
Hybridization and introgression
observed between Italy and Spain remain, however, difficult to explain. The pattern of biased introgression in Spain was, however, based on patterns of allele distributions and genetic distances (Cogolludo-Agustin et al. 2000) and not on direct observations of genetic backcrosses as was done in Zalapa et al. (2009, 2010) and in the current study. The grouping of trees into U. pumila, hybrids and U. minor in CogolludoAgustin et al. (2000) was based on morphological characteristics and was not adjusted once the genetic results were obtained. In fact, the authors claim that 20.5 % of the hybrids had only alleles from U. pumila which suggests that these individuals were genetically U. pumila although they remained classified as hybrids in future analyses, based on their morphology. The First Canonical Discriminant Function analyses suggested introgression towards U. minor (Fig. 3 in Cogolludo-Agustin et al. 2000). The fact that the UPGMA results and allele frequency showed the hybrids closer to U. pumila than U. minor could simply reflect the fact that some morphological hybrids were misclassified and really represented U. pumila based on their genetic profile. Under such circumstances, there would be no evidence for a strong pattern of biased introgression towards U. pumila in Spain and the pattern would be more similar to what we observed in Italy. If U. pumila was only introduced once in Spain in the sixteenth century as an ornamental tree during the rule of King Philip the Second (Kamen, 1997), constant biased backcrosses towards U. pumila over close to 400 years would make such individuals difficult to identify without a large number of loci distributed over the genome. We must therefore conclude, based on the data currently available, that there is no evidence for a strong pattern of biased introgression towards U. pumila in Spain. The programs Structure (Pritchard et al. 2000) and NewHybrids (Anderson and Thompson 2002) both identified a similar number of hybrid individuals, although the classification of these individuals into specific hybrid classes differed significantly between the two programs. Structure identified hybrid individuals as first-generation hybrids (F1) and backcrosses (BC) and did not distinguish between first- (F1) and second-generation (F2) hybrids. With K = 2, the expected proportion of the genome originating from each of the two parental species (the q values from Structure) is expected to be 0.5 and similar in the F1 and F2 hybrid individuals. While NewHybrids can
theoretically distinguish between F1 and F2 hybrid individuals, the lack of species-specific alleles and the relatively low number of loci can render the inference of the specific genotype frequency classes to which an individual belongs and the classification of individuals into specific categories difficult (Anderson and Thompson 2002). However, NewHybrids identified 9 individuals with a greater than 0.94 posterior probability of belonging to the F2 category. Combining the information obtained from NewHybrids and Structure, we concluded that the hybrid population consisted of a mixture of F1, F2 and BC individuals. The availability of more loci in the future would improve the classification of hybrids into distinct categories for these populations. Second-generation (F2) hybrids between U. pumila and U. minor were likely present in Italy although they were not detected between U. pumila and U. rubra in the Midwestern United States (Zalapa et al. 2010). The low number of allozyme loci used in an earlier hybridization study between U. minor and U. pumila in Spain did not permit the detection of F2 hybrids in that study (Cogolludo-Agustin et al. 2000). Differences in the mating system of these elm species may help explain differences in the likelihood of producing F2 hybrids between U. pumila and U. rubra relative to between U. pumila and U. minor. While U. minor can set some seeds when selfed (3.4 % seed set from Mittempergher and La Porta 1991) and has been classified as self-compatible, U. pumila and likely U. rubra have been reported as self-incompatible (Zalapa et al. 2009). We expect a low probability of F2 hybrid production from crosses between U. pumila and U. rubra because both parental species may carry selfincompatibility (SI) alleles that are transmitted to the F1 hybrids and can prevent mating between F1 individuals carrying similar SI alleles (Zalapa et al. 2009). The lack of self-incompatibility alleles in U. minor should increase the probability that F1 individuals mate with one another and form F2, relative to a situation where both parental species carry selfincompatibility alleles. An increase in the frequency of matings between F1 individuals would, in turn, tend to reduce the overall frequency of matings between F1 and the parental species, including U. pumila. This reduced frequency of backcrossing with U. pumila will further reduce the probability of genetic assimilation of U. minor through hybridization in Italy (Rhymer and Simberloff 1996; Hedge et al. 2006).
123
J. Brunet et al. Table 5 Results of the structure analysis and the calculations to infer the number of genetic clusters (K) for a reference population of Ulmus pumila from China, and of U. pumila¸ U. minor and their hybrids from Italy, based on 10 microsatellite loci K
Reps
Mean LnP(K)
Stdev LnP(K)
Ln’(K)
1
10
-3,588.440000
0.490351
2
10
-3,024.940000
0.745654
563.500000
526.010000
3
10
-2,987.450000
2.229723
37.490000
10.210000
4.579043
4
10
-2,960.170000
1.411894
27.280000
37.070000
26.255514
–
|Ln’’(K)| –
Delta K – 705.434353
5
10
-2,969.960000
7.316526
-9.790000
9.880000
1.350368
6
10
-2,969.870000
16.828286
0.090000
10.690000
0.635240
7
10
-2,959.090000
18.650436
10.780000
5.480000
0.293827
8
10
-2,953.790000
16.701726
5.300000
47.443333
2.840624
Besides the potential genetic swamping of the native species, a second concern with continued hybridization between an introduced and a native species is the increase in genetic diversity and creation of new genotypes which can stimulate the evolution of invasiveness (Ellstrand and Schierenbeck 2000; Sakai et al. 2001; Hedge et al. 2006). In this study, the hybrids had more alleles than either of the parental species, were quite heterozygous (H0 = 0.56) and equally differentiated from each of the two parental species (FST = 0.11 with U. pumila and 0.14 or 0.08 with U. minor). The level of heterozygosity in the hybrids (0.56) was higher than in U. pumila (0.34) but similar to U. minor (0.59). In this study, Structure identified 46 % of the hybrids as backcrosses (BC) while NewHybrids identified some hybrids as second generation hybrids (F2). While the level of heterozygosity is important and reflects the stage of introgression, most of the variability present in the hybrids will be expressed in the F2 individuals. The maintenance of genetic variability in the hybrid population combined with the variability expressed in an F2 population would increase the expression of novel gene combinations which may in turn set the stage for rapid evolution (Keim et al. 1989), facilitate the process of adaptation and help U. pumila spread into new habitats and become invasive in Italy. The level of heterozygosity observed in this study was lower than the heterozygosity detected for the hybrids between U. pumila and U. rubra in the Midwestern United States (H0 = 0.90) (Zalapa et al. 2009). The level of heterozygosity in the hybrid population is expected to be greatest with F1 hybrids. While Structure identified 69 % of the hybrid individuals as first-generation hybrids (F1) in the Zalapa
123
et al. (2009) study; fewer hybrids were identified as F1 in the current study (54 %). The different stages of introgression identified between these two studies, with a lower frequency of F1 individuals and the presence of F2 individuals in the current study could explain some of the difference in the level of heterozygosity of the hybrid populations observed between these two studies. In addition, the level of heterozygosity observed in U. minor in Italy (H0 = 0.54) was greater than the levels reported in Spain (H0 = 0.22) (Cogolludo-Agustin et al. 2000). This may reflect the fact that allozymes were used in the 2000 study while microsatellites were used in this study and microsatellites are known to be more variable than allozymes (Collevatti et al. 2001). When similar sets of microsatellite loci were used, the level of observed heterozygosity in U. minor was within the range previously detected for the native U. rubra in the Midwestern United States (Zalapa et al. 2009). While the introduction of U. pumila in Italy was strongly encouraged by the local authorities during the 1930s as a barrier against the DED epidemic (Passavalli 1935) this species may have become an even more dangerous threat to the native Field elm than DED. Although the disastrous effects of the DED epidemic on Field elm populations are evident, the impact of hybridization with U. pumila is more difficult to evaluate, partly due to the fact that the two species are difficult to discriminate based on morphology alone. The current study indicates that hybridization and introgression between the native Field elm and the exotic Siberian elm are causing irreversible changes in the genetic structure of the indigenous species. A potential advantage of introgression toward U. minor would be the transmission of DED resistance genes
Hybridization and introgression
from U. pumila. This would increase the survival of U. minor over the landscape in Italy. Introgression toward U. pumila could, however, facilitate the acquisition of useful genes from the native U. minor that would enhance the ability of U. pumila to invade U. minor habitats and could increase its ability to spread. In order to limit these risks, the use of Siberian elm should be restricted, while the use and spread of U. minor genotypes that prove more tolerant to DED should be promoted. Acknowledgments The authors wish to thank Ignazio Graziosi for providing some samples. Eric Collin, Luisa Ghelardini and Francesca Bagnoli commented on the manuscript. We gratefully acknowledge the National Science Foundation Minority Post-doctoral Fellowship to J.E. Zalapa (NSF award #0409651) and support from the USDA-ARS to J. Brunet.
Appendix See Table 5
.
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