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Population structure and genetic diversity of the threatened quillwort Isoëtes malinverniana and implication for conservation
Aquatic Botany 93 (2010) 147–152
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Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Population structure and genetic diversity of the threatened quillwort Isoëtes malinverniana and implication for conservation Rodolfo Gentili a,∗ , Thomas Abeli b , Graziano Rossi b , Mingai Li c , Claudio Varotto c , Sergio Sgorbati a a b c
Dipartimento di Scienze dell’Ambiente e del Territorio, Università degli Studi di Milano-Bicocca, Piazza della Scienza 1, I-20126 Milano, Italy Dipartimento di Ecologia del Territorio, Università degli Studi di Pavia, Via S. Epifanio, 14, I-26100 Pavia, Italy Environment and Natural Resources Area, IASMA Research and Innovation Centre, Fondazione Edmund Mach, Via Edmondo Mach 1, I-38010 San Michele all’Adige (TN), Italy
a r t i c l e
i n f o
Article history: Received 26 February 2010 Received in revised form 10 May 2010 Accepted 27 May 2010 Available online 8 June 2010 Keywords: Conservation genetics Restoration Isoëtaceae Population Running water Extinction risk
a b s t r a c t The goal of this research was to investigate genetic variation in Isoëtes malinverniana (Isoëtaceae) to select candidate populations for future conservation efforts. To this aim, ISSR and AFLP analyses, carried out using six and four primer combinations, respectively, produced a total of 425 bands, 97.18% of which were polymorphic. Our results suggest that I. malinverniana shows medium to high genetic diversity (mean Nei’s genetic diversity: H = 0.1491 for ISSR data; H = 0.2289 for AFLP data) and a substantial amount of gene flow between the analysed populations (Nm = 1.768, with combined ISSR and AFLP data). The moderate levels of population differentiation support the hypothesis that the fragmentation and isolation of I. malinverniana occurred only recently, probably due to the intensive agriculture practice and water pollution. These results will be used to focus further studies aimed at supporting reintroduction programs within suitable sites of the distribution area. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Habitat destruction and habitat fragmentation represent the major negative driving forces, which can strongly impact ecosystems, populations and species (Lienert, 2004). In particular natural and semi-natural aquatic habitats (canals, streams, rivers, lakes, etc.) are under increasing pressure due to water polluting agriculture practices (e.g. excessive use of pesticides and fertilizers), the incorrect management of waterside areas and the alteration of flow regime (Lenzen et al., 2008). Previous studies provide evidence that numerous species belonging to the genus Isoëtes worldwide have a high risk of extinction (Rhazi et al., 2004; Chen et al., 2005, 2007; Kang et al., 2005; COSEWIC, 2006; Kim et al., 2008). Isoëtes is a heterosporous lycopodsid genus with ancient origins and fossil records dating back to the Paleozoic (Hoot et al., 2004). At present, it is a cosmopolitan genus containing more than 200 species (Schuettpelz and Hoot, 2006); in Italy six species of quillworts are present (Conti et al., 2005): Isoëtes durei Bory, Isoëtes echinospora Durieu, Isoëtes histrix Bory, Isoëtes malinverniana, Ces. & De Not. Isoëtes subinermis (Durieu) Cesca & Peruzzi, Isoëtes velata A. Braun.
∗ Corresponding author. Fax: +39 02 64482996. E-mail addresses: [email protected] (R. Gentili), [email protected] (T. Abeli), [email protected] (G. Rossi), mingai [email protected] (M. Li), [email protected] (C. Varotto), [email protected] (S. Sgorbati). 0304-3770/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2010.05.003
I. malinverniana is a tetraploid (2n = 4× = 44) aquatic quillwort from the plain of the river Po, Piedmont and Lombardy, Italy (Schneller, 1982; Troia, 2001). This strictly endemic species is a perennial growing on sandy substrate within canals for rice fields water supply and in fresh water deriving from rivers and from resurgence spring lines (Soldano and Badino, 1990). Within the genus, I. malinverniana has a high taxonomic significance as it is isolated from the other species and its origin is very uncertain (Hoot et al., 2006). Schneller (1982) suggested that the species could be of Asian origin, accidentally introduced in Italy along with rice seeds, as supported by the fact that it is mainly confined to areas around rice crops. However, the phylogenetic study of Hoot et al. (2006) concluded that it is not closely related to any of the Indian or other Asian species. Other authors hypothesized that the species is an ancient pre-glacial relict biogeographically and phylogenetically isolated from other species of the genus (Mondino, 2007). The range of I. malinverniana is very fragmented and limited to the north-western Po plain. At present only 12 occurrences are known, in very restricted and isolated areas. In the last 30 years the whole population decreased by about 90% due to habitat loss and degradation. For this reason it was listed in the Italian Red Lists under the IUCN category Endangered (EN). Moreover I. malinverniana is included in annexes II and IV of the EU “Habitat” Directive 92/43, that include species of European interest in need of particularly strict protection and in annexe I of the Berne Convention. In this context, the conservation genetics plays an important role for the conservation of I. malinvenriana, because it increases
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Z populations) and Piedmont (Ea, Eb, R, Fa and Fb populations) (Table 1). The distance of the two main growing areas is about 40 km. In the case of population Fa–Fb and Ea–Eb the second letter indicates that a population were upstream “a” and the other one were downstream “b” in the same canal at a distance lower than 1 km. All sampled populations occur in channels around rice fields where the main impact is represented by the agricultural activity (water management, fertilization, etc.). Along canals habitats are characterized by Alnus glutinosa (L.) Gaertn. and by Carex sp. pl.; submerged habitats within channel are characterized by several aquatic species that grow along with Isoëtes: Potamogeton sp. pl., Sagittaria sagittifolia L., Vallisneria spiralis L. 2.2. DNA extraction Genomic DNA from about 0.1 g of frozen young leaves of Isoëtes was isolated using Nucleon PhytoPure plant DNA extraction kit (Amersham Biosciences) as specified by the manufacturer. The pelleted DNA was washed twice with 70% (v/v) ethanol, dried and resuspended in 80–100 L of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). The DNA was stored at −20 ◦ C until use. 2.3. ISSR
Fig. 1. Genetic diversity trend. (a) Divergence from the mean (DFM) of genetic diversity values obtained by means of the different molecular markers used (ISSR, AFLP, merged markers) and (b) comparative trend of genetic diversity.
understanding of the spatial organization of genetic diversity and the patterns of population differentiation. The knowledge and ability to predict the extent of the genetic variation of a species might be applied in future and more successful conservation efforts (Hedrick, 2001; Heywood and Iriondo, 2003). Molecular markers are commonly used to define the genetic structure of natural plant populations and for the estimation of biodiversity levels (Bouzat, 2001; Manel et al., 2007). In particular ISSR (Inter Simple Sequence Repeat) and AFLP (Amplified Fragment Length Polymorphism) markers have been successfully used for plant population characterization and for genetic diversity estimation in several studies on Isoëtes and other species (e.g. Sgorbati et al., 2004; Chen et al., 2005; Bonin et al., 2007). The aim of this study was to assess the genetic structure and the level of genetic variation within and among the extant populations of I. malinverniana using ISSR and AFLP markers: such information could provide a better understanding of the genetic relationships among the populations and will also be crucial to identify plant sources for local reintroduction plans within suitable habitats to facilitate the conservation management of this species. 2. Materials and methods 2.1. Sampling DNA analyses were performed on 72 accessions from seven natural populations in the Po Plain (Fig. 1, Table 1). Within each population from 7 to 14 individuals were sampled and used for analyses. For conservation reasons, in some cases the number of samples is low due to the small population size. Sampled populations can be assigned into two distinct biogeographic areas: lower and upper Po plain (Andreis et al., 2005), corresponding to the administrative districts of Lombardy (B and
PCR was carried out in a 25 L total reaction volume, using 30 ng of genomic DNA as template. The primers were purchased from Operon Technologies (Alameda, CA, USA) Series Band C. Primer sequences are reported in Table 2. Taq DNA polymerase (Pharmacia, Karlsruhe, Germany) was used at 1U per reaction. Amplification was performed in a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) under the following temperature profile: 94 ◦ C for 2 min, 35 cycles at 94 ◦ C for 30 s, 55 ◦ C for 1 min, 72 ◦ C for 1.5 min; final extension was carried out at 72 ◦ C for 7 min. ISSR products were separated by electrophoresis in TBE buffer with 2% agarose gels. Pictures of the ethidium bromide-stained gels were taken with a Gel Doc 2000 imaging system (Biorad, USA). 2.4. AFLP Genomic DNA (80 ng) was digested (2 h at 37 ◦ C) with EcoRI (0.5 U) and MseI (0.5 U), and ligated with EcoRI- (5 pMol) and MseIadapters (50 pMol). Primer pairs used in the pre-amplification reaction were M01 and E01. The analysis of DNA fingerprints was based on the detection of EcoRI/MseI genomic restriction/ligation fragments by PCR amplification with four different primer combinations (choose among a screening panel of 15 different combinations of MseI/EcoRI primer combinations) having three selective nucleotides (Table 2). Primers were purchased from Sigma–Aldrich (St. Louis, USA). The EcoRI primers were fluorescently labelled 5 -end with 6carboxyfluorescein (6-FAM). Amplifications were performed using a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) with the following cycle profile: 30 s at 94 ◦ C, 30 s at 65 ◦ C and 1 min at 72 ◦ C. The annealing temperature of the first cycle (65 ◦ C) was subsequently reduced by 0.7 ◦ C at each cycle for the next 11 cycles, and kept at 56 ◦ C for the last 24 cycles. For detection of fluorescently labelled DNA fragments, PCR products (1 L) were mixed with GeneScan® LIZ size standard (0.2 L; Applied Biosystems, Carlsbad, USA) and formamide (8.8 L; Sigma–Aldrich, St. Louis, USA). The fragments were separated on a 3730xl DNA Analyzer sequencer (Applied Biosystems, Carlsbad, USA).
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Table 1 Provenance of Isoëtes malinverniana samples collected for this study and site characteristics. Populations E and F were divided in two sub-populations, one upstream (a) and the other downstream (b). Location
Biogeographic district
Population code
Sample size
Population size
Population area (m2 )
Latitude (N)
Longitude (E)
Arborio, Roggia Molinara Arborio, Dondoglietto Roggia dell’Avvocato Lenta Lenta 2 Piccolini (PV) Vigevano (PV)
Upper Po plain Upper Po plain Upper Po plain Upper Po plain Upper Po plain Lower Po plain Lower Po plain
Ea Eb R Fa Fb B Z
7 7 9 14 10 17 8
≈100 ≈9 ≈500 ≈1000 ≈500 ≈200 ≈50
78.3 23.4 61.6 183.4 107.2 50.0 17.7
45◦ 30 33 45◦ 29 41 45◦ 33 32 45◦ 33 21 45◦ 33 33 45◦ 19 34 45◦ 19 43
8◦ 22 46 8◦ 23 11 8◦ 21 28 8◦ 22 02 8◦ 22 02 8◦ 48 55 8◦ 47 58
Table 2 ISSR and AFLP primers, number of total and polymorphic (NPB) bands with percentage of polymorphic bands (PPB%) for each primer used in this study. Primers
Sequences (5 –3 )
ISSR UBC808 UBC810 UBC825 UBC826 UBC827 UBC862
AGA GAG AGA GAG AGA GC GAG AGA GAG AGA GAG AT ACA CAC ACA CAC ACA CT ACA CAC ACA CAC ACA CC ACA CAC ACA CAC ACA CG AGC AGC AGC AGC AGC AGC Tot
AFLP (pairs) E40/M44 E42/M44 E42/M37 E40/M35
E-AGC/M-ATC E-AGT/M-ATC E-AGT/M-ACG E-AGC/M-ACA Tot
ISSR + AFLP
No. of Bands
NPB
PPB (%)
15 20 18 18 19 17
15 20 16 15 19 17
100.00 100.00 88.89 83.33 100.00 100.00
107
102
95.33
49 88 95 86
47 86 94 84
95.92 97.73 98.95 97.67
318 425
311 413
97.80 97.18
2.5. Data analyses
3.1. ISSR
In the case of ISSR, each DNA band was amplified and visually scored on agarose gels for presence (1) or absence (0). AFLP electropherograms were collected, analyzed and scored with the internal size standard using the ABI Prism GeneScan® ver. 3.7 analysis software (Applied Biosystems, 1989–2001). Analyses by means of the software were performed with default manufacturer parameters with the exception of bin width (set at 2 bp). Only peaks in the 100–800 bp size range were scored. All scoring data were validated by visual peak inspection. The percentage of polymorphic bands (PPBs), the Shannon index of diversity (Sh), the gene diversity parameters (H, Nei’s gene diversity; HT , genetic diversity over all the groups; HS within population; GST = 1 − (HS /HT ), proportion of genetic diversity among populations) according to Nei (1973) and gene flow (Nm ) were calculated to estimate genetic variation. The binary matrix was analysed using the software POPGENE version 1.32 (Yeh et al., 1997) assuming Hardy–Weinberg disequilibrium. Analysis of molecular variance (AMOVA) was performed using the Genalex version 6.1 software (Peakall and Smouse, 2006) to estimate genetic structure and degree of genetic differentiation within populations, among populations and among biogeographic districts. The significance of the estimates was obtained through 999 data replications. To visualize genetic relationships among populations a dendrogram was constructed on the basis of the unweighted pair-group method (UPGMA) and the Nei’s genetic distance using the software POPGENE.
The six ISSR primers generated a total of 107 bands ranging from 300 bp to 4000 bp. Of these, 102 (95.33%) were polymorphic across 72 individuals. The most informative ISSR primer was UBC810 with the production of 20 bands all of which polymorphic (100%, Table 2). The percentage of polymorphic loci ranged from 25.33% in population Z to 57.94% in population R, with a mean value of 42.07%. Populations R and Fa (Fig. 1, Table 3) exhibited the highest genetic diversity (H = 0.1842 and 0.1677, respectively). The mean value of H across the seven populations was 0.1491.
3. Results The seven populations surveyed for population size and area varied in size from about 9 plants to more than 1000 plants and the population areas ranged from 18 m2 to 183 m2 (Table 1).
3.2. AFLP The four AFLP primer pairs produced a total of 318 bands ranging from 100 bp to 800 bp, 311 of which were polymorphic. The most informative AFLP primer pair was E42/M37 (E-AGT/M-ACG) with the production of 94 polymorphic bands (98.95%, Table 2). At the population level the percentages of polymorphic bands ranged from 56.92 in population Eb to 87.74% in population Fa. Populations Ea and Fa exhibited the highest genetic diversity (H = 0.2620 and 0.2510, respectively) (Fig. 1, Table 3). The mean value of H across the seven populations was 0.2289. Combined analysis (AFLP + ISSR) produced a total of 425 bands, 413 of which (97.18%) were polymorphic (Table 2). The percentage of polymorphic loci ranged from 51.33% in population Eb to 77% in population Fa. The highest values of Nei’s genetic diversity (H) were found for the populations “Ea” (VC) and “Fa” (PV). The highest values of Shannon Index (Sh) were 0.3519 and 0.3505 for Ea and Fb populations, respectively (Table 3). No correlations were found between genetic variability and population sizes according to ISSR, AFLP or the combined dataset. However the smallest populations (Eb ≈ 9 individuals) showed the lowest values of genetic variability, while the largest populations
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Table 3 Genetic diversity (H) within populations according to Nei‘s statistics (1973) on the basis of analyses carried out on ISSR, AFLP and combined datasets. Abbreviations: GST = proportion of genetic diversity among populations; Nm = gene flow; NPB = number of polymorphic bands; %PPB = percentage of polymorphic bands; Sh = Shannon index. Population
Pop Ea Pop Eb Pop R Pop Fa Pop Fb Pop B Pop Z Mean Species level
ISSR
AFLP
Total (ISSR + AFLP)
NPB
%PPB
H
NPB
%PPB
H
NPB
%PPB
H
Sh
GST
Nm
40 37 62 49 46 54 27
37.38 34.58 57.94 45.79 42.99 50.47 25.33 42.07 95.33
0.1483 0.1314 0.1842 0.1678 0.1558 0.1677 0.0887 0.1491 0.2676
255 181 210 279 218 257 248 235.43 311
80.19 56.92 66.04 87.74 68.55 80.82 77.99 74.04 97.8
0.2620 0.1920 0.2030 0.2510 0.2160 0.2370 0.2410 0.2289 0.2667
288 212 268 318 258 304 273 274.43 413
69.73 51.33 64.89 77.00 62.47 73.61 66.10 66.45 97.18
0.2322 0.1763 0.1996 0.2273 0.2005 0.2182 0.2066 0.2087 0.2630
0.3519 0.2649 0.3060 0.3509 0.3034 0.3347 0.3169 0.3184 0.4113
0.2204
1.7685
102
(Fa ≈ 1000 individuals) showed the highest values of genetic variability. The divergences from the mean (DFM) of the H values obtained with ISSR, AFLP and the combined dataset are shown in Fig. 1a. Population Fa and population B showed positive DFM in relation to ISSR, AFLP and combined dataset; population Eb showed negative DFM values for all three methods. The different markers used produced contrasting trends concerning genetic diversity (Fig. 1b). 3.3. Population genetic structure Population genetic structure was assessed for the combined dataset. The coefficient of genetic differentiation (GST ), was 0.2204. The level of gene flow (Nm ) was estimated to be 1.7685. AMOVA analyses showed that most of the total genetic variation (82%) is attributed to individuals within populations, while 16% and 2% are due to differences among populations (P < 0.001) and between regions (lower and higher Po plain; P < 0.018), respectively (Table 4). Population pairwise relationships showed the lowest genetic distance, measured according to the combined ISSR and AFLP dataset, between Fa and Fb populations and the highest between Z and Fb populations (Fig. 2). Mantel test revealed a nonsignificant correlation between the genetic and geographical distance matrixes (Table 5; r2 = 0.4025; P > 0.05). 4. Discussion The amount of genetic diversity of a species is the result of the interplay among relatively recent evolutionary processes, historical events and ecological factors. Our genetic analyses on I. malinverniana populations revealed medium to high levels of
Fig. 2. Genetic distance. UPGMA dendrogram among the 7 populations of I. malinverniana based on Nei’s distance using ISSR and AFLP markers.
genetic diversity. The total genetic diversity from all populations (H = 0.2087, mean among all sampling sites and 0.2630 at the species level) was in general higher than that from comparable analyses carried out with ISSR, AFLP and RAPD markers for other species of the genus Isoëtes. RAPD markers detected a low degree of genetic diversity in the hexaploid Isoëtes coreana Chung and Choi (mean within population HE = 0.061) (Kim et al., 2008). Relatively to ISSR markers, on average, a higher degree of within population Nei’s genetic diversity was found in I. malinverniana (H = 0.1491) with respect to the diploid (2n = 22) Isoëtes hypsophila Hand.-Mazz. (mean within population, Nei’s genetic diversity = 0.0507; value calculated from Chen et al., 2005). Relatively to AFLP markers, I. malinverniana had a higher genetic diversity (mean within population H = 0.2289) with respect to the tetraploid (2n = 4× = 44) Isoëtes sinensis Palmer (mean within population H = 0.118) (Kang et al., 2005). Under the hypothesis of Schneller (1982) that I. malinverniana could be an Asian species accidentally introduced in Italy along with rice seeds one would expect low genetic diversity due to a recent bottleneck caused by founder effects (Nei et al., 1975). On the contrary, given its relatively high genetic variability compared to other Isoëtes, our results are more in agreement with the hypothesis put forward by Mondino (2007) that I. malinverniana could be an ancient pre-glacial relict species. Several factors, namely its ploidy level (2n = 4× = 44), its mainly outcrossing behaviour, long-lived individuals and overlapping generations (Aegisdóttir et al., 2009; Gulsen et al., 2009) all possibly concurred to some extent to maintain until today the observed medium to high within population genetic diversity in this rare quillwort. Analysis of molecular variance (AMOVA) revealed that most of the total genetic differentiation occurred among populations or between regions for I. hypsophila, I. coreana and I. sinensis (Chen et al., 2005; Kang et al., 2005; Kim et al., 2008) unlike I. malinverniana, where most of the total genetic differentiation occurred within populations. This homogeneity may indicates that the relatively low distance among populations (ranging from 0.356 km to 44.144 km) does not exceed the dispersal ability of this species, thus allowing ongoing gene flow among populations. Even if the presence of dense and isolated stands of the species may suggest that spores are typically dispersed mainly to short distances (Kang et al., 2005), in fact, water current and periodic events as flooding and predation by waterfowls may favour spore dispersal at longer distance (COSEWIC, 2006; Hoot et al., 2006; Pollux et al., 2009). One has however to notice that another possibility is that habitat fragmentation and isolation of I. malinverniana occurred only so recently that the moderate population differentiation (particularly evidenced by means of AFLP analysis) indicated by GST and Nm values and AMOVA does not reflect the contemporary, but the historical gene flow among populations. In support of this hypothesis there is the fact that, according to previous descriptions on historical distribution data, this species was relatively abundant in
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Table 4 Analyses of molecular variance (AMOVA). Differences among region are referred to upper and lower Po plain (biogeographic district) populations. Abbreviations: df = degrees of freedom; SSD = sum of square deviations; MSD = mean square deviations. Source of variation
df
SSD
MSD
Estimated variance
Total variance (%)
P-value
Among regions Among pops Within pops Total
1 5 65 71
225.723 764.768 3407.713 4398.203
225.723 152.954 52.426
1.207 10.521 52.426 64.155
2 16 82 100
<0.018 <0.001 <0.001
Table 5 Genetic (above the diagonal) and geographical (km; under the diagonal) distance matrices. Pop Ea Pop Ea Pop Eb Pop R Pop Fa Pop Fb Pop B Pop Z
Pop Eb 0.0546
1.680 5.799 5.280 5.630 38.485 39.682
7.479 6.948 7.296 37.224 38.431
Pop R 0.0698 0.0866 0.816 0.738 42.988 44.144
the W-Po Plain around 1950 and until 1990s (Soldano and Badino, 1990). Since 1990, however, five populations disappeared, possibly due to habitat loss/degradation, agriculture practice or water pollution or other unidentified causes. It is currently not possible to distinguish among these two hypotheses without specific studies aimed at this purpose. It is therefore important to plan conservation strategies that may be crucial mainly if the actual gene flow among populations should result lower than expected. As already evidenced in previous studies (Bahulikar et al., 2004; Ci et al., 2008), AFLP and ISSR analyses may result in differences in the absolute estimates of genetic variation and divergent results in some populations. Also in the case of I. malinverniana, the total level of genetic diversity among populations estimated by ISSRs was higher than that by AFLPs. The two methods, in fact, amplify different types of genomic regions: while AFLPs are designed to randomly sample regions from the whole genome, ISSR markers specifically detect pre-identified repeat regions (Karp et al., 1998). Moreover, genomic regions sampled by ISSR and AFLP markers may be subjected to different selective pressures as they result from basically different mutational processes. AFLP markers tend to overestimate the number of loci and absolute population diversity but to underestimate the number of alleles (DeHaan et al., 2003). On the contrary, ISSR markers tend to emphasize among population diversity (Ci et al., 2008). With the exclusion of populations R and Ea, the overall trends for genetic diversity and population structure from ISSR and AFLP markers were comparable. The most diverse populations using ISSR were R and Fa. The most diverse populations using AFLP were Ea and Fa. This last trend was confirmed with the dataset obtained by the combination of both marker types. The level of genetic diversity (from ISSR and AFLP markers) in the Eb population is the lowest among the extant populations of I. malinverniana. This is likely related to the small size of Eb population, with fewer than 15 individuals. Small populations are more subject to genetic drift and inbreeding, which are processes that can cause a reduction of genetic diversity. In this study, the upstream populations (Ea and Fa) from the same water canal showed a higher genetic diversity than downstream populations (Fb and Eb). This trend is in contrast with data of Pollux et al. (2009), which described an increasing genetic diversity in downstream populations for the aquatic species Sparganium emersum Rehmann. This discrepancy could be due to the following reasons: (a) founder effect; (b) recent colonization of downcanal populations. Further analyses are necessary to distinguish these two possibilities, as the hypothesis of recent colonization may con-
Pop Fa 0.0653 0.0686 0.0416 0.356 42.187 43.341
Pop Fb 0.0868 0.0698 0.0579 0.0263 42.398 43.549
Pop B
Pop Z
0.1005 0.0858 0.0574 0.0321 0.0477
0.1135 0.134 0.0956 0.0812 0.1251 0.0858
1.262
tribute to a better definition of the ecological requirements of I. malinverniana. Around the world several naturally occurring species belonging to the genus Isoëtes are rare and on the brink of extinction due to water quality deterioration (Chen et al., 2005; Kim et al., 2008). As for I. malinverniana, the causes of population reduction require further investigations that are still ongoing. Detecting genetic diversity by means of different markers could facilitate the choice of conservation-priority populations (Fallon, 2007). In particular, those populations with the highest values of genetic diversity (H) and a positive trend of DFM for the different markers (ISSR, AFLP and merged markers) could be mostly considered as a source of individuals to employ in reintroduction or reinforcement plans: in particular Fa population. Such population showed the highest value of population size (≈1000 individuals). On the other hand, those populations with negative or discordant values of DFM could be mostly considered as populations to be reinforced: Eb and Z. Indeed, these populations showed the lowest values of population size (9 and 50 individuals, respectively). Moreover, the low genetic diversity among populations facilitates the translocation of individuals between populations without concerns on outbreeding and it could have a positive effect on population genetic structure. Some agricultural practices of rice crops around populations (e.g. draining or over-pumping of ground water in canals, herbicide use, discharge of liquids from rice fields in canals), along with many other threats (invasion of exotic species, urbanization), jeopardize the existing populations. Indeed, most of I. malinverniana populations typically grow close to the resurgence spring line of the Po plain, where water pollution is lower. The use of herbicides in the crop areas close to I. malinverniana occurrences should, therefore, be strongly limited awaiting specific studies about their effect on the species. Acknowledgments This study was funded by University of Milano-Bicocca, University of Pavia, by Natural Resources Area of the Fondazione Edmund Mach (San Michele all’Adige, Trento) and by the project “CORINAT”, D.G. Agricoltura, Regione Lombardia. References Aegisdóttir, H.H., Kuss, P., Stöcklin, J., 2009. Isolated populations of rare alpine plant show high genetic diversity and considerable population differentiation. Ann. Bot. 104, 1313–1322.
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Contents lists available at ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Population structure and genetic diversity of the threatened quillwort Isoëtes malinverniana and implication for conservation Rodolfo Gentili a,∗ , Thomas Abeli b , Graziano Rossi b , Mingai Li c , Claudio Varotto c , Sergio Sgorbati a a b c
Dipartimento di Scienze dell’Ambiente e del Territorio, Università degli Studi di Milano-Bicocca, Piazza della Scienza 1, I-20126 Milano, Italy Dipartimento di Ecologia del Territorio, Università degli Studi di Pavia, Via S. Epifanio, 14, I-26100 Pavia, Italy Environment and Natural Resources Area, IASMA Research and Innovation Centre, Fondazione Edmund Mach, Via Edmondo Mach 1, I-38010 San Michele all’Adige (TN), Italy
a r t i c l e
i n f o
Article history: Received 26 February 2010 Received in revised form 10 May 2010 Accepted 27 May 2010 Available online 8 June 2010 Keywords: Conservation genetics Restoration Isoëtaceae Population Running water Extinction risk
a b s t r a c t The goal of this research was to investigate genetic variation in Isoëtes malinverniana (Isoëtaceae) to select candidate populations for future conservation efforts. To this aim, ISSR and AFLP analyses, carried out using six and four primer combinations, respectively, produced a total of 425 bands, 97.18% of which were polymorphic. Our results suggest that I. malinverniana shows medium to high genetic diversity (mean Nei’s genetic diversity: H = 0.1491 for ISSR data; H = 0.2289 for AFLP data) and a substantial amount of gene flow between the analysed populations (Nm = 1.768, with combined ISSR and AFLP data). The moderate levels of population differentiation support the hypothesis that the fragmentation and isolation of I. malinverniana occurred only recently, probably due to the intensive agriculture practice and water pollution. These results will be used to focus further studies aimed at supporting reintroduction programs within suitable sites of the distribution area. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Habitat destruction and habitat fragmentation represent the major negative driving forces, which can strongly impact ecosystems, populations and species (Lienert, 2004). In particular natural and semi-natural aquatic habitats (canals, streams, rivers, lakes, etc.) are under increasing pressure due to water polluting agriculture practices (e.g. excessive use of pesticides and fertilizers), the incorrect management of waterside areas and the alteration of flow regime (Lenzen et al., 2008). Previous studies provide evidence that numerous species belonging to the genus Isoëtes worldwide have a high risk of extinction (Rhazi et al., 2004; Chen et al., 2005, 2007; Kang et al., 2005; COSEWIC, 2006; Kim et al., 2008). Isoëtes is a heterosporous lycopodsid genus with ancient origins and fossil records dating back to the Paleozoic (Hoot et al., 2004). At present, it is a cosmopolitan genus containing more than 200 species (Schuettpelz and Hoot, 2006); in Italy six species of quillworts are present (Conti et al., 2005): Isoëtes durei Bory, Isoëtes echinospora Durieu, Isoëtes histrix Bory, Isoëtes malinverniana, Ces. & De Not. Isoëtes subinermis (Durieu) Cesca & Peruzzi, Isoëtes velata A. Braun.
∗ Corresponding author. Fax: +39 02 64482996. E-mail addresses: [email protected] (R. Gentili), [email protected] (T. Abeli), [email protected] (G. Rossi), mingai [email protected] (M. Li), [email protected] (C. Varotto), [email protected] (S. Sgorbati). 0304-3770/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2010.05.003
I. malinverniana is a tetraploid (2n = 4× = 44) aquatic quillwort from the plain of the river Po, Piedmont and Lombardy, Italy (Schneller, 1982; Troia, 2001). This strictly endemic species is a perennial growing on sandy substrate within canals for rice fields water supply and in fresh water deriving from rivers and from resurgence spring lines (Soldano and Badino, 1990). Within the genus, I. malinverniana has a high taxonomic significance as it is isolated from the other species and its origin is very uncertain (Hoot et al., 2006). Schneller (1982) suggested that the species could be of Asian origin, accidentally introduced in Italy along with rice seeds, as supported by the fact that it is mainly confined to areas around rice crops. However, the phylogenetic study of Hoot et al. (2006) concluded that it is not closely related to any of the Indian or other Asian species. Other authors hypothesized that the species is an ancient pre-glacial relict biogeographically and phylogenetically isolated from other species of the genus (Mondino, 2007). The range of I. malinverniana is very fragmented and limited to the north-western Po plain. At present only 12 occurrences are known, in very restricted and isolated areas. In the last 30 years the whole population decreased by about 90% due to habitat loss and degradation. For this reason it was listed in the Italian Red Lists under the IUCN category Endangered (EN). Moreover I. malinverniana is included in annexes II and IV of the EU “Habitat” Directive 92/43, that include species of European interest in need of particularly strict protection and in annexe I of the Berne Convention. In this context, the conservation genetics plays an important role for the conservation of I. malinvenriana, because it increases
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Z populations) and Piedmont (Ea, Eb, R, Fa and Fb populations) (Table 1). The distance of the two main growing areas is about 40 km. In the case of population Fa–Fb and Ea–Eb the second letter indicates that a population were upstream “a” and the other one were downstream “b” in the same canal at a distance lower than 1 km. All sampled populations occur in channels around rice fields where the main impact is represented by the agricultural activity (water management, fertilization, etc.). Along canals habitats are characterized by Alnus glutinosa (L.) Gaertn. and by Carex sp. pl.; submerged habitats within channel are characterized by several aquatic species that grow along with Isoëtes: Potamogeton sp. pl., Sagittaria sagittifolia L., Vallisneria spiralis L. 2.2. DNA extraction Genomic DNA from about 0.1 g of frozen young leaves of Isoëtes was isolated using Nucleon PhytoPure plant DNA extraction kit (Amersham Biosciences) as specified by the manufacturer. The pelleted DNA was washed twice with 70% (v/v) ethanol, dried and resuspended in 80–100 L of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). The DNA was stored at −20 ◦ C until use. 2.3. ISSR
Fig. 1. Genetic diversity trend. (a) Divergence from the mean (DFM) of genetic diversity values obtained by means of the different molecular markers used (ISSR, AFLP, merged markers) and (b) comparative trend of genetic diversity.
understanding of the spatial organization of genetic diversity and the patterns of population differentiation. The knowledge and ability to predict the extent of the genetic variation of a species might be applied in future and more successful conservation efforts (Hedrick, 2001; Heywood and Iriondo, 2003). Molecular markers are commonly used to define the genetic structure of natural plant populations and for the estimation of biodiversity levels (Bouzat, 2001; Manel et al., 2007). In particular ISSR (Inter Simple Sequence Repeat) and AFLP (Amplified Fragment Length Polymorphism) markers have been successfully used for plant population characterization and for genetic diversity estimation in several studies on Isoëtes and other species (e.g. Sgorbati et al., 2004; Chen et al., 2005; Bonin et al., 2007). The aim of this study was to assess the genetic structure and the level of genetic variation within and among the extant populations of I. malinverniana using ISSR and AFLP markers: such information could provide a better understanding of the genetic relationships among the populations and will also be crucial to identify plant sources for local reintroduction plans within suitable habitats to facilitate the conservation management of this species. 2. Materials and methods 2.1. Sampling DNA analyses were performed on 72 accessions from seven natural populations in the Po Plain (Fig. 1, Table 1). Within each population from 7 to 14 individuals were sampled and used for analyses. For conservation reasons, in some cases the number of samples is low due to the small population size. Sampled populations can be assigned into two distinct biogeographic areas: lower and upper Po plain (Andreis et al., 2005), corresponding to the administrative districts of Lombardy (B and
PCR was carried out in a 25 L total reaction volume, using 30 ng of genomic DNA as template. The primers were purchased from Operon Technologies (Alameda, CA, USA) Series Band C. Primer sequences are reported in Table 2. Taq DNA polymerase (Pharmacia, Karlsruhe, Germany) was used at 1U per reaction. Amplification was performed in a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) under the following temperature profile: 94 ◦ C for 2 min, 35 cycles at 94 ◦ C for 30 s, 55 ◦ C for 1 min, 72 ◦ C for 1.5 min; final extension was carried out at 72 ◦ C for 7 min. ISSR products were separated by electrophoresis in TBE buffer with 2% agarose gels. Pictures of the ethidium bromide-stained gels were taken with a Gel Doc 2000 imaging system (Biorad, USA). 2.4. AFLP Genomic DNA (80 ng) was digested (2 h at 37 ◦ C) with EcoRI (0.5 U) and MseI (0.5 U), and ligated with EcoRI- (5 pMol) and MseIadapters (50 pMol). Primer pairs used in the pre-amplification reaction were M01 and E01. The analysis of DNA fingerprints was based on the detection of EcoRI/MseI genomic restriction/ligation fragments by PCR amplification with four different primer combinations (choose among a screening panel of 15 different combinations of MseI/EcoRI primer combinations) having three selective nucleotides (Table 2). Primers were purchased from Sigma–Aldrich (St. Louis, USA). The EcoRI primers were fluorescently labelled 5 -end with 6carboxyfluorescein (6-FAM). Amplifications were performed using a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) with the following cycle profile: 30 s at 94 ◦ C, 30 s at 65 ◦ C and 1 min at 72 ◦ C. The annealing temperature of the first cycle (65 ◦ C) was subsequently reduced by 0.7 ◦ C at each cycle for the next 11 cycles, and kept at 56 ◦ C for the last 24 cycles. For detection of fluorescently labelled DNA fragments, PCR products (1 L) were mixed with GeneScan® LIZ size standard (0.2 L; Applied Biosystems, Carlsbad, USA) and formamide (8.8 L; Sigma–Aldrich, St. Louis, USA). The fragments were separated on a 3730xl DNA Analyzer sequencer (Applied Biosystems, Carlsbad, USA).
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Table 1 Provenance of Isoëtes malinverniana samples collected for this study and site characteristics. Populations E and F were divided in two sub-populations, one upstream (a) and the other downstream (b). Location
Biogeographic district
Population code
Sample size
Population size
Population area (m2 )
Latitude (N)
Longitude (E)
Arborio, Roggia Molinara Arborio, Dondoglietto Roggia dell’Avvocato Lenta Lenta 2 Piccolini (PV) Vigevano (PV)
Upper Po plain Upper Po plain Upper Po plain Upper Po plain Upper Po plain Lower Po plain Lower Po plain
Ea Eb R Fa Fb B Z
7 7 9 14 10 17 8
≈100 ≈9 ≈500 ≈1000 ≈500 ≈200 ≈50
78.3 23.4 61.6 183.4 107.2 50.0 17.7
45◦ 30 33 45◦ 29 41 45◦ 33 32 45◦ 33 21 45◦ 33 33 45◦ 19 34 45◦ 19 43
8◦ 22 46 8◦ 23 11 8◦ 21 28 8◦ 22 02 8◦ 22 02 8◦ 48 55 8◦ 47 58
Table 2 ISSR and AFLP primers, number of total and polymorphic (NPB) bands with percentage of polymorphic bands (PPB%) for each primer used in this study. Primers
Sequences (5 –3 )
ISSR UBC808 UBC810 UBC825 UBC826 UBC827 UBC862
AGA GAG AGA GAG AGA GC GAG AGA GAG AGA GAG AT ACA CAC ACA CAC ACA CT ACA CAC ACA CAC ACA CC ACA CAC ACA CAC ACA CG AGC AGC AGC AGC AGC AGC Tot
AFLP (pairs) E40/M44 E42/M44 E42/M37 E40/M35
E-AGC/M-ATC E-AGT/M-ATC E-AGT/M-ACG E-AGC/M-ACA Tot
ISSR + AFLP
No. of Bands
NPB
PPB (%)
15 20 18 18 19 17
15 20 16 15 19 17
100.00 100.00 88.89 83.33 100.00 100.00
107
102
95.33
49 88 95 86
47 86 94 84
95.92 97.73 98.95 97.67
318 425
311 413
97.80 97.18
2.5. Data analyses
3.1. ISSR
In the case of ISSR, each DNA band was amplified and visually scored on agarose gels for presence (1) or absence (0). AFLP electropherograms were collected, analyzed and scored with the internal size standard using the ABI Prism GeneScan® ver. 3.7 analysis software (Applied Biosystems, 1989–2001). Analyses by means of the software were performed with default manufacturer parameters with the exception of bin width (set at 2 bp). Only peaks in the 100–800 bp size range were scored. All scoring data were validated by visual peak inspection. The percentage of polymorphic bands (PPBs), the Shannon index of diversity (Sh), the gene diversity parameters (H, Nei’s gene diversity; HT , genetic diversity over all the groups; HS within population; GST = 1 − (HS /HT ), proportion of genetic diversity among populations) according to Nei (1973) and gene flow (Nm ) were calculated to estimate genetic variation. The binary matrix was analysed using the software POPGENE version 1.32 (Yeh et al., 1997) assuming Hardy–Weinberg disequilibrium. Analysis of molecular variance (AMOVA) was performed using the Genalex version 6.1 software (Peakall and Smouse, 2006) to estimate genetic structure and degree of genetic differentiation within populations, among populations and among biogeographic districts. The significance of the estimates was obtained through 999 data replications. To visualize genetic relationships among populations a dendrogram was constructed on the basis of the unweighted pair-group method (UPGMA) and the Nei’s genetic distance using the software POPGENE.
The six ISSR primers generated a total of 107 bands ranging from 300 bp to 4000 bp. Of these, 102 (95.33%) were polymorphic across 72 individuals. The most informative ISSR primer was UBC810 with the production of 20 bands all of which polymorphic (100%, Table 2). The percentage of polymorphic loci ranged from 25.33% in population Z to 57.94% in population R, with a mean value of 42.07%. Populations R and Fa (Fig. 1, Table 3) exhibited the highest genetic diversity (H = 0.1842 and 0.1677, respectively). The mean value of H across the seven populations was 0.1491.
3. Results The seven populations surveyed for population size and area varied in size from about 9 plants to more than 1000 plants and the population areas ranged from 18 m2 to 183 m2 (Table 1).
3.2. AFLP The four AFLP primer pairs produced a total of 318 bands ranging from 100 bp to 800 bp, 311 of which were polymorphic. The most informative AFLP primer pair was E42/M37 (E-AGT/M-ACG) with the production of 94 polymorphic bands (98.95%, Table 2). At the population level the percentages of polymorphic bands ranged from 56.92 in population Eb to 87.74% in population Fa. Populations Ea and Fa exhibited the highest genetic diversity (H = 0.2620 and 0.2510, respectively) (Fig. 1, Table 3). The mean value of H across the seven populations was 0.2289. Combined analysis (AFLP + ISSR) produced a total of 425 bands, 413 of which (97.18%) were polymorphic (Table 2). The percentage of polymorphic loci ranged from 51.33% in population Eb to 77% in population Fa. The highest values of Nei’s genetic diversity (H) were found for the populations “Ea” (VC) and “Fa” (PV). The highest values of Shannon Index (Sh) were 0.3519 and 0.3505 for Ea and Fb populations, respectively (Table 3). No correlations were found between genetic variability and population sizes according to ISSR, AFLP or the combined dataset. However the smallest populations (Eb ≈ 9 individuals) showed the lowest values of genetic variability, while the largest populations
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Table 3 Genetic diversity (H) within populations according to Nei‘s statistics (1973) on the basis of analyses carried out on ISSR, AFLP and combined datasets. Abbreviations: GST = proportion of genetic diversity among populations; Nm = gene flow; NPB = number of polymorphic bands; %PPB = percentage of polymorphic bands; Sh = Shannon index. Population
Pop Ea Pop Eb Pop R Pop Fa Pop Fb Pop B Pop Z Mean Species level
ISSR
AFLP
Total (ISSR + AFLP)
NPB
%PPB
H
NPB
%PPB
H
NPB
%PPB
H
Sh
GST
Nm
40 37 62 49 46 54 27
37.38 34.58 57.94 45.79 42.99 50.47 25.33 42.07 95.33
0.1483 0.1314 0.1842 0.1678 0.1558 0.1677 0.0887 0.1491 0.2676
255 181 210 279 218 257 248 235.43 311
80.19 56.92 66.04 87.74 68.55 80.82 77.99 74.04 97.8
0.2620 0.1920 0.2030 0.2510 0.2160 0.2370 0.2410 0.2289 0.2667
288 212 268 318 258 304 273 274.43 413
69.73 51.33 64.89 77.00 62.47 73.61 66.10 66.45 97.18
0.2322 0.1763 0.1996 0.2273 0.2005 0.2182 0.2066 0.2087 0.2630
0.3519 0.2649 0.3060 0.3509 0.3034 0.3347 0.3169 0.3184 0.4113
0.2204
1.7685
102
(Fa ≈ 1000 individuals) showed the highest values of genetic variability. The divergences from the mean (DFM) of the H values obtained with ISSR, AFLP and the combined dataset are shown in Fig. 1a. Population Fa and population B showed positive DFM in relation to ISSR, AFLP and combined dataset; population Eb showed negative DFM values for all three methods. The different markers used produced contrasting trends concerning genetic diversity (Fig. 1b). 3.3. Population genetic structure Population genetic structure was assessed for the combined dataset. The coefficient of genetic differentiation (GST ), was 0.2204. The level of gene flow (Nm ) was estimated to be 1.7685. AMOVA analyses showed that most of the total genetic variation (82%) is attributed to individuals within populations, while 16% and 2% are due to differences among populations (P < 0.001) and between regions (lower and higher Po plain; P < 0.018), respectively (Table 4). Population pairwise relationships showed the lowest genetic distance, measured according to the combined ISSR and AFLP dataset, between Fa and Fb populations and the highest between Z and Fb populations (Fig. 2). Mantel test revealed a nonsignificant correlation between the genetic and geographical distance matrixes (Table 5; r2 = 0.4025; P > 0.05). 4. Discussion The amount of genetic diversity of a species is the result of the interplay among relatively recent evolutionary processes, historical events and ecological factors. Our genetic analyses on I. malinverniana populations revealed medium to high levels of
Fig. 2. Genetic distance. UPGMA dendrogram among the 7 populations of I. malinverniana based on Nei’s distance using ISSR and AFLP markers.
genetic diversity. The total genetic diversity from all populations (H = 0.2087, mean among all sampling sites and 0.2630 at the species level) was in general higher than that from comparable analyses carried out with ISSR, AFLP and RAPD markers for other species of the genus Isoëtes. RAPD markers detected a low degree of genetic diversity in the hexaploid Isoëtes coreana Chung and Choi (mean within population HE = 0.061) (Kim et al., 2008). Relatively to ISSR markers, on average, a higher degree of within population Nei’s genetic diversity was found in I. malinverniana (H = 0.1491) with respect to the diploid (2n = 22) Isoëtes hypsophila Hand.-Mazz. (mean within population, Nei’s genetic diversity = 0.0507; value calculated from Chen et al., 2005). Relatively to AFLP markers, I. malinverniana had a higher genetic diversity (mean within population H = 0.2289) with respect to the tetraploid (2n = 4× = 44) Isoëtes sinensis Palmer (mean within population H = 0.118) (Kang et al., 2005). Under the hypothesis of Schneller (1982) that I. malinverniana could be an Asian species accidentally introduced in Italy along with rice seeds one would expect low genetic diversity due to a recent bottleneck caused by founder effects (Nei et al., 1975). On the contrary, given its relatively high genetic variability compared to other Isoëtes, our results are more in agreement with the hypothesis put forward by Mondino (2007) that I. malinverniana could be an ancient pre-glacial relict species. Several factors, namely its ploidy level (2n = 4× = 44), its mainly outcrossing behaviour, long-lived individuals and overlapping generations (Aegisdóttir et al., 2009; Gulsen et al., 2009) all possibly concurred to some extent to maintain until today the observed medium to high within population genetic diversity in this rare quillwort. Analysis of molecular variance (AMOVA) revealed that most of the total genetic differentiation occurred among populations or between regions for I. hypsophila, I. coreana and I. sinensis (Chen et al., 2005; Kang et al., 2005; Kim et al., 2008) unlike I. malinverniana, where most of the total genetic differentiation occurred within populations. This homogeneity may indicates that the relatively low distance among populations (ranging from 0.356 km to 44.144 km) does not exceed the dispersal ability of this species, thus allowing ongoing gene flow among populations. Even if the presence of dense and isolated stands of the species may suggest that spores are typically dispersed mainly to short distances (Kang et al., 2005), in fact, water current and periodic events as flooding and predation by waterfowls may favour spore dispersal at longer distance (COSEWIC, 2006; Hoot et al., 2006; Pollux et al., 2009). One has however to notice that another possibility is that habitat fragmentation and isolation of I. malinverniana occurred only so recently that the moderate population differentiation (particularly evidenced by means of AFLP analysis) indicated by GST and Nm values and AMOVA does not reflect the contemporary, but the historical gene flow among populations. In support of this hypothesis there is the fact that, according to previous descriptions on historical distribution data, this species was relatively abundant in
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Table 4 Analyses of molecular variance (AMOVA). Differences among region are referred to upper and lower Po plain (biogeographic district) populations. Abbreviations: df = degrees of freedom; SSD = sum of square deviations; MSD = mean square deviations. Source of variation
df
SSD
MSD
Estimated variance
Total variance (%)
P-value
Among regions Among pops Within pops Total
1 5 65 71
225.723 764.768 3407.713 4398.203
225.723 152.954 52.426
1.207 10.521 52.426 64.155
2 16 82 100
<0.018 <0.001 <0.001
Table 5 Genetic (above the diagonal) and geographical (km; under the diagonal) distance matrices. Pop Ea Pop Ea Pop Eb Pop R Pop Fa Pop Fb Pop B Pop Z
Pop Eb 0.0546
1.680 5.799 5.280 5.630 38.485 39.682
7.479 6.948 7.296 37.224 38.431
Pop R 0.0698 0.0866 0.816 0.738 42.988 44.144
the W-Po Plain around 1950 and until 1990s (Soldano and Badino, 1990). Since 1990, however, five populations disappeared, possibly due to habitat loss/degradation, agriculture practice or water pollution or other unidentified causes. It is currently not possible to distinguish among these two hypotheses without specific studies aimed at this purpose. It is therefore important to plan conservation strategies that may be crucial mainly if the actual gene flow among populations should result lower than expected. As already evidenced in previous studies (Bahulikar et al., 2004; Ci et al., 2008), AFLP and ISSR analyses may result in differences in the absolute estimates of genetic variation and divergent results in some populations. Also in the case of I. malinverniana, the total level of genetic diversity among populations estimated by ISSRs was higher than that by AFLPs. The two methods, in fact, amplify different types of genomic regions: while AFLPs are designed to randomly sample regions from the whole genome, ISSR markers specifically detect pre-identified repeat regions (Karp et al., 1998). Moreover, genomic regions sampled by ISSR and AFLP markers may be subjected to different selective pressures as they result from basically different mutational processes. AFLP markers tend to overestimate the number of loci and absolute population diversity but to underestimate the number of alleles (DeHaan et al., 2003). On the contrary, ISSR markers tend to emphasize among population diversity (Ci et al., 2008). With the exclusion of populations R and Ea, the overall trends for genetic diversity and population structure from ISSR and AFLP markers were comparable. The most diverse populations using ISSR were R and Fa. The most diverse populations using AFLP were Ea and Fa. This last trend was confirmed with the dataset obtained by the combination of both marker types. The level of genetic diversity (from ISSR and AFLP markers) in the Eb population is the lowest among the extant populations of I. malinverniana. This is likely related to the small size of Eb population, with fewer than 15 individuals. Small populations are more subject to genetic drift and inbreeding, which are processes that can cause a reduction of genetic diversity. In this study, the upstream populations (Ea and Fa) from the same water canal showed a higher genetic diversity than downstream populations (Fb and Eb). This trend is in contrast with data of Pollux et al. (2009), which described an increasing genetic diversity in downstream populations for the aquatic species Sparganium emersum Rehmann. This discrepancy could be due to the following reasons: (a) founder effect; (b) recent colonization of downcanal populations. Further analyses are necessary to distinguish these two possibilities, as the hypothesis of recent colonization may con-
Pop Fa 0.0653 0.0686 0.0416 0.356 42.187 43.341
Pop Fb 0.0868 0.0698 0.0579 0.0263 42.398 43.549
Pop B
Pop Z
0.1005 0.0858 0.0574 0.0321 0.0477
0.1135 0.134 0.0956 0.0812 0.1251 0.0858
1.262
tribute to a better definition of the ecological requirements of I. malinverniana. Around the world several naturally occurring species belonging to the genus Isoëtes are rare and on the brink of extinction due to water quality deterioration (Chen et al., 2005; Kim et al., 2008). As for I. malinverniana, the causes of population reduction require further investigations that are still ongoing. Detecting genetic diversity by means of different markers could facilitate the choice of conservation-priority populations (Fallon, 2007). In particular, those populations with the highest values of genetic diversity (H) and a positive trend of DFM for the different markers (ISSR, AFLP and merged markers) could be mostly considered as a source of individuals to employ in reintroduction or reinforcement plans: in particular Fa population. Such population showed the highest value of population size (≈1000 individuals). On the other hand, those populations with negative or discordant values of DFM could be mostly considered as populations to be reinforced: Eb and Z. Indeed, these populations showed the lowest values of population size (9 and 50 individuals, respectively). Moreover, the low genetic diversity among populations facilitates the translocation of individuals between populations without concerns on outbreeding and it could have a positive effect on population genetic structure. Some agricultural practices of rice crops around populations (e.g. draining or over-pumping of ground water in canals, herbicide use, discharge of liquids from rice fields in canals), along with many other threats (invasion of exotic species, urbanization), jeopardize the existing populations. Indeed, most of I. malinverniana populations typically grow close to the resurgence spring line of the Po plain, where water pollution is lower. The use of herbicides in the crop areas close to I. malinverniana occurrences should, therefore, be strongly limited awaiting specific studies about their effect on the species. Acknowledgments This study was funded by University of Milano-Bicocca, University of Pavia, by Natural Resources Area of the Fondazione Edmund Mach (San Michele all’Adige, Trento) and by the project “CORINAT”, D.G. Agricoltura, Regione Lombardia. References Aegisdóttir, H.H., Kuss, P., Stöcklin, J., 2009. Isolated populations of rare alpine plant show high genetic diversity and considerable population differentiation. Ann. Bot. 104, 1313–1322.
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