Central European Journal of Biology

* E-mail: v.tubelyte@gmf.vdu.lt

Research Article

1Department of Biology, Vytautas Magnus University, LT 44404 Kaunas, Lithuania2Institute of Ecology, Nature Research Centre, LT 08412 Vilnius, Lithuania 3Institute of Biology of Latvian University, LV 2169 Salaspils, Latvia4Department of Biology, Immanuel Kant State University of Russia, 236038 Kaliningrad, Russia5Scientific Practical Centre for Bioresources of Belarusian Academy of Sciences, 220072 Minsk, Belarus

Vaida Tubelyte1,*, Saulius Švažas2, Aniolas Sruoga1,2, Dalius Butkauskas2, Algimantas Paulauskas1, Vykintas Baublys1, Janis Viksne3, Gennady Grishanov4, Alexander Kozulin5

Genetic diversity of tufted ducks (Aythya fuligula, Anatidae) in Eastern Europe

1. IntroductionThe tufted duck (Aythya fuligula, Anatidae) is widespread in the Palearctic across northern Eurasia, generally found between 45º and 70º N [1]. The total European breeding population of the tufted duck is estimated at approximately 730,000 – 880,000 breeding pairs [2]. Russia is home to about 70% of the total European population of tufted ducks [3]. It is one of the most abundant breeding duck species in the Baltic countries and Belarus [3]. Birds breeding in Northern and Eastern Europe are highly migratory, while populations from Western Europe are partially migratory or resident

[4]. In the northern tundra zone of European Russia, the first arrival of tufted ducks occurs in late spring towards the end of May to early June [5], whereas in the Kaliningrad region of western Russia, first arrivals occur at the end of February [6]. In Latvia, spring migration lasts from mid- April to the end of May [3]. Autumn migration of tufted ducks begins in September and lasts until the end of November [4,7]. Tufted ducks breeding in the northern tundra of European Russia both start and finish migrations earlier than birds breeding in the Baltic States, Belarus and Western Russia [3,6,8,9]. Two biogeographic populations of the tufted duck are currently distinguished in Europe

Cent. Eur. J. Biol. DOI: 10.2478/s11535-011-0083-z

Received 18 April 2011; Accepted 13 July 2011

Keywords: Eastern Europe • Genetic diversity • Polymorphism • RAPD • Tufted duck

Abstract: The tufted duck (Aythya fuligula, Anatidae) is widespread in the Palaearctic across Northern Eurasia. Birds breeding in Northern and Eastern Europe are highly migratory, while populations from Western Europe are partially migratory or resident. The aim of this study is to explore genetic variation within and between ducks breeding in Latvia and migrants sampled in North West Russia and Belarus. The technique of random amplified polymorphic DNA was applied using five random primers (ol-1, ol-9-12). Genetic variability was measured for all tufted ducks investigated and for different sub-populations from various regions. Individual genetic structure and genetic variability was higher in ducks collected from Latvia. Gene diversity of amplified DNA bands in birds of Latvian origin was 24% with 80% polymorphism. Means of gene diversity and polymorphism for tufted ducks sampled in other countries varied from 12 to 14% and from 27 to 40%, respectively. A high number of unique bands characterized ducks breeding in Latvia. The oligonucleotide primers used in this study were suitable to analyze differences among tufted ducks of different origin. Possible explanations for the variation observed among the studied ducks are discussed.

© Versita Sp. z o.o.

Genetic diversity of tufted ducks (Aythya fuligula, Anatidae) in Eastern Europe

based on their main wintering areas: the Northwest European population, estimated at 1,200,000 birds and the Black Sea/Mediterranean population, estimated at 700,000 birds [10]. Tufted ducks breeding in Northwest Russia, Belarus and the Baltic States mostly migrate to wintering grounds in Northwestern Europe, particularly along the coasts of the Baltic and North Sea, but there is considerable mixture among individuals from different biogeographic populations in the major staging, moulting and wintering grounds [4,8]. During the non-breeding months, this species is highly gregarious, forming flocks of up to 200,000 individuals in key staging and wintering sites [3]. Ringing recoveries of tufted ducks born in Latvia (402 recoveries of birds ringed as day-old ducklings) indicate a wide dispersal of these birds throughout Europe [3].

Phylogeographic studies have recently focused on nuclear markers, paying careful attention to the resolution of individual nuclear genotypes [11]. Molecular markers provide an important tool for assessing the genetic variability and structure of natural populations, and for studying biodiversity in general [12,13]. In order to detect genetic changes in recently evolved populations or admixture levels of sub-populations, more variable nuclear markers are necessary [14].

Random Amplified Polymorphic DNA (RAPD) is a highly variable nuclear marker that provides reliable information on genetic diversity, polymorphisms and the relationships among populations of different origins [15]. RAPD generates DNA fingerprints with a single synthetic nucleotide primer [16], which efficiently detects polymorphisms based on comparisons throughout the genome. The main advantages of the RAPD method lie in its speed and applicability to any organism without prior knowledge of a nucleotide sequence [15]. RAPD has been used in the genetic structure analysis of wild and domestic birds [17-25] and has successfully identified hybrid birds [26-28]. Studies using the RAPD method for genetic structure analysis of wild duck populations are scarce, so the number of detected specific markers for wild ducks is small [18,29].

The aim of this study is to assess the use of random primers for tufted ducks and to define possible differences in genetic structure of tufted tucks breeding and migrating in different regions of Eastern Europe.

2. Experimental Procedures2.1 SamplingBlood samples were collected from 43 individual tufted ducks shot by hunters between 2006 and 2007. A total of 23 samples were collected from individuals breeding

in Latvia in August 2006. The remaining samples were collected from migratory birds in eastern Belarus (5 individuals hunted in April 2007), along the Curonian coast of the Baltic Sea in the Kaliningrad Region of Russia (6 individuals hunted in September 2006) and in Lake Ilmen in the Novgorod Region of Russia (9 individuals hunted in September 2006) (Figure 1). Blood samples (400-500 ml) were collected in plastic autoclaved tubes containing the anticoagulant, heparin and refrigerated at -20°C until DNA extraction.

2.2 Extraction of DNA, RAPD-PCR procedures and gel electrophoresis

DNA was extracted from each sample as described in Miller et al. [30] with an additional chloroform extraction step. Extracted DNA was dissolved in water and stored at -20°C. Five random 10-mer primers (Shanghai Sangon Ltd., China), with GC contents of 55-80%, were used for RAPD-PCR analysis (Table 1).

The RAPD-PCR was carried out in a total volume of 25 μl containing 100 ng of genomic DNA, 200 μM (each) dNTPs, 20 pmol (each) primers, 2.5 μM MgCl2, and 1.5 unit of Taq DNA Polymerase (MBI Fermentas, Lithuania). The PCR program was carried out using Mastercycler® Gradient from Eppendorf and the following temperature profile: 95°C for 2 min; 35 cycles of 94ºC for 45 s, 36°C for 45 s, 72°C for 1 min. Each sample was tested three times for all of the selected primers to check the reliability of single RAPD profiles. Only bands present in all replicates were positively scored.

The amplified DNA fragments were separated according to their molecular weights using electrophoresis on 1.7% agarose gel prepared in 1X TBE buffer. A standard molecular marker - Gene Ruler TM 100kb DNA Ladder (MBI Fermentas, Lithuania), was used to determine molecular weights of the amplified bands. Gels were examined visually under UV light using ethidium bromide. The gels were photographed and documented with Gel Doc 1000 (Bio Rad, Germany) system. Analysis was performed using the TotalLab v.1.10 (Nonlinear Dynamics Limited, England) software.

2.3 Statistical analysis RAPD revealed a multi-locus banding pattern. For data analysis, a binary matrix reflecting the presence (1) or absence (0) of the DNA band was generated and analysed as genotypic data. For genotypic data, a band was considered to be a locus with the dominant allele present (allele-based approach).

A band was assumed to be monomorphic if it was detected in all the samples investigated. The percentage of polymorphic bands (P) was calculated by dividing the

V. Tubelyte et al.

Figure 1. Boundaries of different biogeographic populations of tufted duck in the Western Palearctic [4] and sampling areas (indicated by arrows): 1- the Kaliningrad region of Russia, 2 – Latvia, 3 – Belarus, 4 – the Novgorod region of Russia.

∑=

−=n

iii

S ppH1

2log

Primer Primer sequence 5’ → 3’

Molecularweight

range (bp)

Totalnumberof bands

Totalnumber of

polymorphicbands

Percentage ofpolymorphicbands (%)

Number of polymorphic bands per location

Latvia Kaliningrad Belarus Novgorod

ol-1

ol-9

ol-10

ol-11

ol-12

5’-TCCCTTCCTC-3’

5’-TCCGCTCTGG-3’

5’-TGCTCTGCCC-3’

5’-GTGAGGCTGC-3’

5’-GATGACCGCC-3’

310-1100

420-1750

250-1500

60-1450

210-1550

9

11

11

15

7

8

10

9

15

5

89

91

82

100

71

7

10

9

15

5

2

8

1

6

1

2

6

1

6

1

6

7

0

11

0

Sum or range 60-1750 53 47 46 18 16 24

Table 1. Average number of DNA bands in tufted ducks by location and primer.

number of polymorphic bands at the population, region or species level by the total number of bands surveyed.

For phenotypic data, Shannon’s phenotypic diversity index SH was calculated for each primer and sampling site using the following formula:

where pi is the frequency of the ith RAPD band [31]. We chose Shannon’s index over other diversity indices

Genetic diversity of tufted ducks (Aythya fuligula, Anatidae) in Eastern Europe

because it is less sensitive to bias introduced into RAPD data as a result of the inability to detect heterozygotes [32] and because it has been widely used for RAPD analysis [33-38].

Scored bands were also treated as dominant markers of Mendelian loci with two alleles [15], and therefore we were able to obtain estimates of allele frequencies, heterozygosity, genetic diversity and genetic distance. When binary data are used for heterozygosity calculations, the quantity H is the same as gene diversity [39]. Allele frequencies and genetic diversities were derived from the corrected formulae for the dominant marker as in Lynch & Milligan [40]. Gene diversity H was computed as the probability that two randomly drawn genes differ at a given locus i:

where var(qi) = (1-x)/4N, and x is the proportion of the N sampled individuals that do not show the marker.

Genetic distance between populations was calculated from the following formula [41]:

where I is genetic identity among pairs of populations: Iab is the probability that two alleles are identical in populations A and B; Ia and Ib are the probabilities of allele identity in A or in B population.

We used an analysis of the molecular variance (AMOVA) according to Excoffier et al. [42] in GenAlEx v.5[43] to evaluate differentiation within and among the studied groups. The Euclidean distance was used as the basis for the principal coordinate analysis (PCoA) [44], from which graphical relationships between individuals were derived.

3. Results3.1 Number of bands and molecular sizeFive primers (ol-1, ol-9, ol-10, ol-11, ol-12) were used to amplify fragments of the tufted ducks’ DNA and were successful in all individuals. The spectrum of amplification products was unique to each primer. In total, 53 amplified DNA fragments (47 to 53 bands per sample) from different locations, were obtained. The average number of bands amplified by primers varied from 7 (amplified with ol-12 primer) to 15 (amplified with ol-11 primer) (Table 1), with the approximate size of each band ranging from 60 to 1750 bp. A smaller number of DNA bands (3 to 13) was detected in two

genera of domestic duck (Anas and Cairina) [21,45]. However, each of these bands was longer (from 200 to 2800 bp) than tufted duck bands. In a study on dabbling ducks, Kulikova et al. [18] reported that RAPD patterns for Mallard Anas platyrhynchos and spot-billed ducks Anas poecilorhyncha showed high variability and a lack of fixed differences.

The spectrum of amplified DNA products obtained using ol-1, ol-9, ol-11 and ol-12 primers are presented in Figure 2. Individual band numbers produced by the same primer varied among individuals collected in different countries (Table 1). After DNA amplification with all oligonucleotide primers, five bands (ol-1-800,ol-9-900, ol-10-200, ol-10-450, ol-12-580) were detected in the genomes of all individuals.

3.2 Polymorphism and gene diversityThe genetic variability of each primer used in this study was evaluated. The percentage of polymorphic loci varied from 71 (ol-12) to 100% (ol-11) (Table 1). Ott [46] considered a RAPD genetic marker to be highly polymorphic if it amplified bands with a heterozygosity estimate of more than 70%. All primers used in this study showed low levels of polymorphism; heterozygosity (gene diversity) estimates ranged from 7 (ol-10) to 23% (ol-11) (Table 3). Despite this low variability, there were detectable differences in the levels of polymorphism among individuals collected in different countries. The highest number of polymorphic bands was detected in ducks of Latvian origin, whereas some parts of bands were absent in individuals collected from other countries (Table 1).

We found 47 polymorphic RAPD bands with 89% polymorphism in tufted ducks. By comparison, the estimated percentage of genetic polymorphism using RAPD in wild A. platyrhynchos and domestic ducks varied from 50% to 80% [18,47-49].

The amount of polymorphism differed among tufted ducks sampled at the four locations. The percentage of polymorphic RAPD bands was 80% for ducks of Latvian origin, 31% for migratory ducks sampled in the Kaliningrad Region of Russia, 27% for migratory ducks sampled in Belarus and 40% for migratory ducks sampled in the Novgorod Region of Russia (Table 2). Similarly, higher gene diversity was found within Latvian ducks (0.244), and lower gene diversity was found for migratory ducks collected in Belarus (0.120) and the Kaliningrad Region (0.127) (Table 2).

Shannon’s diversity index [31] ranged from 0.176 in Belarus to 0.380 in Latvia (Table 2). Gene diversity indices within tufted ducks collected at the same sites using different primers are presented in Table 3. Gene diversity values differed significantly among primers

)var(2)1(2)( iii qqqiH +−=

),ln(ba

ab

III

D −= ab

V. Tubelyte et al.

Figure 2. �DNA�fingerprints�from�different�samples�of�the�Tufted�Duck�obtained�by�PCR�with�four�primers.�Sample�marking:�LV�–�individuals�of�Latvian origin, BL – migratory birds collected in Belarus, NG – migratory birds collected in the Novgorod Region of Russia, KG – migratory birds collected in the Kaliningrad Region of Russia, M – Gene Ruler TM 100kb DNA Ladder (MBI ‘Fermentas’).

Parameter of genetic variation (mean values ± S. E.)

Sampling areas

Latvia Kaliningrad Belarus Novgorod

No. of alleles

No. of private alleles*

% of polymorphic loci (P)

Gene diversity

Shannon‘s index

1.851 ± 0.048

0.241

80

0.244 ± 0.027

0.380 ± 0.031

1.333 ± 0.065

0.000

31

0.127 ± 0.025

0.189 ± 0.037

1.296 ± 0.063

0.000

27

0.120 ± 0.026

0.176 ± 0.038

1.444 ± 0.068

0.037

40

0.141 ± 0.024

0.218 ± 0.035

Table 2. Summary polymorphisms generated by primers in tufted ducks.

*Equivalent to the number of alleles unique to a single population in the data set [50]

Primer

Gene diversity within individuals sampled in the same area

Latvia Kaliningrad Belarus Novgorod Average ± S. E.

ol-1

ol-9

ol-10

ol-11

ol-12

0.161

0.212

0.188

0.354

0.230

0.089

0.262

0.044

0.150

0.046

0.086

0.212

0.040

0.167

0.040

0.209

0.162

0.000

0.247

0.000

0.136 ± 0.025

0.212 ± 0.018

0.068 ± 0.036

0.229 ± 0.040

0.079 ± 0.045

Table 3. Genetic diversity indices within tufted ducks sampled in the same area generated by primers.

ranging from 0.068 for bands amplified with ol-10 to 0.229 for bands amplified with ol-11. Gene diversity was not detected for individuals collected in the Novgorod region after amplification with the primers ol-10 and ol-12.

A number of bands unique to tufted ducks collected in Latvia were detected. Primer ol-10 detected fragments

in the range of 400 and 1000 bp which were specific to Latvian samples. Also, primers ol-1 detected 2 fragments in the range of 550-600 bp and 850-1100 bp and primer ol-12 detected 1 fragment located in the area of350-420 bp. These fragments were not amplified in birds collected from other regions. The mean number of

Genetic diversity of tufted ducks (Aythya fuligula, Anatidae) in Eastern Europe

Source df SS* MS est. var. % P

Among individuals sampled in different countries 3,000 11.570 3.857 0.241 13 < 0.001

Among individuals sampled in the same area 39,000 64.494 1.654 1.654 87 < 0.001

Total 42 76.064 1.895 100

Table 4. Summary�of�AMOVA�results.

*SS – sum of squares; MS – mean squares; est. var. – estimated variability; % – proportion of genetic variability; p – significance level.

Pairs of samples*Genetic distance

ol-1 ol-9 ol-10 ol-11 ol-12 Overall

LV ↔ KG

LV ↔ BL

LV ↔ NG

KG ↔ BL

KG ↔ NG

BL ↔ NG

0.014

0.022

0.027

0.001

0.026

0.033

0.042

0.055

0.065

0.015

0.051

0.048

0.033

0.057

0.072

0.026

0.015

0.010

0.134

0.123

0.135

0.077

0.304

0.145

0.037

0.216

0.440

0.428

0.487

0.350

0.060

0.089

0.125

0.082

0.158

0.102

Table 5. Nei’s�genetic�distance�[41]�estimates�calculated�from�DNA�fingerprints�from�different�samples�of�tufted�duck.

*LV – individuals of Latvian origin; BL, KG, NG – migratory birds sampled in Belarus, the Kaliningrad and Novgorod regions, respectively

unique alleles was high in the Latvian ducks (0.241) and low in migratory individuals collected from Novgorod (0.037). In migratory birds sampled in the Kaliningrad and Belarus, unique alleles were not detected at all (Table 2). The average value of unique alleles is equivalent to the number of alleles unique to a single population in the data set [50].

AMOVA results revealed significant variation in randomly amplified bands within and among samples collected in different locations (P<0.001). Most of the total variance (87%) was found within birds sampled in the same population and the remaining part (13%) was attributed to variation among individuals sampled in different countries (Table 4).

3.3 Genetic distance and principal coordinate analysis

Analysis of pair-wise genetic distance, using Nei’s [41] calculations, revealed that the minimum genetic distance was between flocks of birds from Latvia and those sampled in Kaliningrad (0.060). Meanwhile, the maximum genetic distance was established between flocks of migratory birds sampled from Kaliningrad and Novgorod (0.158) (Table 5).

PCoA analysis showed that individuals sampled from Belarus and Novgorod fell into two distinct groups,

while Latvian ducks shared some genetic similarities with individuals from all sampling locations. The power of discrimination with the first two coordinates is not so high (total value was 40%), however the tendency was informative as well (Figure 3). The PCoA analysis was corroborated by the AMOVA results and showed the genetic differentiation of East European Tufted Ducks, correlating with geographical localities of investigated populations.

4. DiscussionThe use of molecular methods in studies of migratory birds has increased over the last decade. In many cases, the aim of conducting genetic analysis of migratory birds is to reconstruct their recent evolutionary history [51], search for the genetic evidence of past population expansions [52] and/or assess the degree of divergence in population genetic structure across a breeding range [53]. Genetic analysis is useful for inferring patterns of connectivity between breeding and overwintering sites as well as population mixing during the winter season [54]. As migratory water birds are sources of various pathogens [55], the detection of species range expansions and connectivity patterns among wintering

V. Tubelyte et al.

Figure 3. �Plot�of�the�first�two�principal�coordinates�for�each�individual�tufted�duck�sampled�in�different�locations.�Between�14,73%�and�26,88%�of�the�variation�is�explained�by�the�first�two�axes.�

and breeding grounds is of great importance. Due to potential mixing of birds from different regions of Asia and Europe in key wintering and staging grounds, the Organization of International Epizootics (http://www.oie.int) put the tufted duck on the list of candidate bird species that are long distance vectors of avian influenza.

Not all molecular markers are suitable for the detection of genetic differences in migratory and resident birds [56]. However the choice of suitable genetic markers provides significant insights into the migratory behavior and gene flow among bird populations [17,57]. What is more, molecular markers are able to identify different geographical populations of migratory species and could potentially be used to track migratory birds throughout their annual cycle [54].

In this study we evaluated genetic variability of tufted duck individuals sampled in Eastern Europe by using highly variable nuclear markers (Random Amplified Polymorphic DNA). Successful use of randomly amplified bands for distinguishing between bird populations has been reported in native breeds of Fujian ducks [58], woodpigeons Columba palumbus [59] and red-cockaded woodpeckers Picoides borealis [60]. Although the primers used in our study had low levels of polymorphism, the analysis of polymorphism and gene diversity in tufted tucks revealed significant differences among the studied groups. This result demonstrates the efficiency of RAPD primers in the assessment of genetic specificity and reflects genetic diversity in the studied duck populations.

The level of genetic variability was assessed in birds breeding in Latvia and in migratory individuals collected in Belarus and two regions of Russia: Kaliningrad and Novgorod. Ducks in Kaliningrad were hunted in mid-September, when birds of Northern European origin clearly dominate the large staging flocks of tufted ducks

in the Curonian Lagoon [3]. We assumed that most of the birds hunted in Kaliningrad were transit migrants from North Russia, as birds of the eastern Baltic region (including Latvia) only occur at these sites in October - November. Most birds sampled in Belarus and Novgorod are likely to have been breeders of North Russian origin. In Belarus, tufted ducks were hunted in April at the stop-over site located close to the Russian border, which, in spring, was almost exclusively used by migrants to Russia [3].

Genetic analysis of tufted ducks revealed that birds of Latvian origin were more genetically variable than birds breeding in Northern Russia. The different genetic structures of tufted ducks inhabiting different areas of the breeding range can possibly be explained by the phylogenetic peculiarities of the species. The tufted duck is a relatively new breeding species in the Baltic region and in Western Europe having colonized this area after a very marked westward expansion of its breeding range, recorded since the late 19th century [1]. This range expansion was likely caused in part by impacts due to global climate change [61,62]. The size of the Latvian breeding population increased throughout the early 20th century, from several breeding pairs observed by the end of the 19th century to up to 1,500 pairs recorded in the 1970s [3]. In certain regions of northwestern Europe (Great Britain, southern Sweden, Denmark, the Netherlands, etc.) abundant breeding populations of the tufted duck have settled earlier than in Latvia [1,63]. Therefore, the Latvian breeding population of this species is likely to have been formed both by individuals from the core breeding area located in Northern and Eastern Europe, and by individuals from the newly established breeding populations in Northwestern Europe. A similar colonization pattern was recorded in the neighboring Lithuania. The first

Genetic diversity of tufted ducks (Aythya fuligula, Anatidae) in Eastern Europe

groups of breeding tufted ducks were recorded in the early 20th century at the western and eastern borders of Lithuania simultaneously, with further expansion of breeding birds into the central part of the country [64,65]. The quick expansion of a species range due to climate change or natural barriers often leads to a rise in genetic variability in recently established sub-populations [51,53]. The history of the establishment of the tufted duck as a breeding species in the eastern Baltic region is consistent with the results of the genetic analysis obtained in our study. It is likely that higher levels of genetic variability in ducks breeding in Latvia was caused by a mixture of individuals of different origins during its initial phases of establishment in this country. Meanwhile, a more monotypic genetic structure is characteristic of ducks breeding in Northern Russia, the core breeding range of this species.

The results of our study indicate that RAPD can be effectively used for the identification of tufted ducks of different origins and should be used in future investigations of tufted ducks throughout their breeding range.

AcknowledgementsThe authors are very grateful to Inga Ribelyte for assistance with the PCR analysis. This work was part of the “Migratory Duck Populations and Avian Influenza” program funded by the International Association “Migratory Birds of the Western Palearctic”. Investigations were also supported by the Lithuanian Science Council PhD Student Research Fellowship Award.

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