Mirov NT, 1967. The genus Pinus. New York: Roland Press. Mitchell A, 1996. Trees of Britain.

London: Harper Collins. Mu ller-Starck G, Baradat P, and Bergmann F, 1992. Genetic variation within European tree species. New For 6:2347. Politov DV, Krutovskii KV, and Altukhov YuP, 1989. Genetic variability in Siberian stone pine, Pinus sibirica Du Tour. III. Linkage relationships among isozyme loci [in Russian with English summary]. Genetika (Moscow) 25:16061618. Poulik MD, 1957. Starch gel electrophoresis in a discontinuous system of buffers. Nature 180:14471448. Shaw CR and Prasad R, 1970. Starch gel electrophoresis of enzymesa compilation of recipes. Biochem Genet 4:297320. Shurkhal A, Podogas A, and Zhivotovsky L, 1992. Allozyme differentiation in the genus Pinus. Silvae Genet 41: 105109. Sokal R and Rohlf FJ, 1995. Biometry, 3rd ed. San Francisco: W.H. Freeman. Vidakovic M, 1991. Conifers. Morphology and variation. Zagreb: Gracki Zavod Hrvatske. Received September 17, 2001 Accepted November 26, 2001 Corresponding Editor: James L. Hamrick

investigation across a broader range of colubroid snakes. Both allozyme ( King and Lawson 1997) and DNA studies ( Bushar et al. 1998; Gibbs et al. 1997, 1998; Prior et al. 1997) indicate that snake populations are microgeographically structured, but allozyme studies of the grass snake (Natrix natrix) reveal insufcient genetic variation for ne-scale spatial patterns ( Hille 1997). Long-term ecological monitoring of the endangered grass snake population in the city of Amsterdam, The Netherlands ( Zuiderwijk et al., 1998) has shown the need for molecular markers that can reveal the impact of species-inherent inuences like population size, individual vagility, and philopatric behavior on the population pattern of genetic variation. Highly variable loci, such as microsatellites, have the potential to resolve genetic relationships at all levels of population structure (among individuals, demes, and metapopulations) (Jarne and Lagoda 1996). However, the use of microsatellites is compromised by the time, expense, and difculty of isolating these short tandem repeats and their anking regions from the genome of the target organisms. An alternative approach to de novo development is to exploit the available information by cross-species amplications among a range of phylogenetically related species ( Blanquer-Maumont and Crouau-Roy 1995; Coltman et al. 1996; Pepin et al. 1995; Scribner et al. 1996). Here we cross-amplied 21 published microsatellite primers derived from advanced colubroid snakes [loci Hb2, Hb20, Hb48, Hb65, and Hb70: Hoplocephalus bungaroides, Elapidae (Burns and Houlden 1999); locus Ts1-4: Thamnophis sirtalis, Thamnophiinae (McCracken et al. 1999); loci Ns1-8 and Ns9b: Nerodia sipedon, Thamnophiinae (Prosser et al. 1999); loci Ch5A, Cg7-150, Ch5-183: Crotalus horridus, Crotalidae (Villarreal et al. 1996)] in European natricines and additional snake species rather than isolating and characterizing new microsatellites by screening speciesspecic genomic libraries. We report on the amplication and genotyping protocols using a temperaturegradient block cycling [polymerase chain reaction (PCR)] machine and automated DNA analysis system based on near-infrared ( IR) uorescence technology. Our long-term objectives are twofold. First, we wanted to cross-validate lines of evidences stemming from long-term eld surveillance, such as mark-recapture programs in an endangered metapopulation of N. natrix

in Amsterdam ( Zuiderwijk et al. 1998), with independent molecular data. Second, we sought to explain the patterns of gene ow between the demographic subunits by considering the inuences of the fragmented landscape structure of city habitats on the population genetic structure.

Materials and Methods

The following list represents the animals examined in the present study with emphasis on natricine snakes: N. natrix (Amsterdam, The Netherlands; n 60; blood), N. tesselata ( Lake Balaton, Hungary; b 23; blood), and N. maura (Pyrenees, Spain; Atlas mountains, Morocco; n 9; liver, blood). Single individuals of the New World natricines, Thamnophis sirtalis and Nerodia fasciata (commercially obtained by pet trade, shed skins), were used as positive amplication controls for the primers routinely used in the focus study. Furthermore, single specimens of a variety of snake species, including Vipera berus ( Viperidae; Greece, collection of the University of Bielefeld, Germany; muscle), Coronella austriaca (Colubridae; location unknown, collection of the University of Bielefeld, Germany; muscle), Elaphe longissima (Colubridae; Hirschberg, Taunus, Germany; muscle), Coluber nummifer (Colubridae; Troodos, Cyprus; muscle), Eunectes murinus and E. notaeus ( Boinae; Brazil, Bolivia; blood), Boa constrictor ( Boinae; commercially obtained by pet trade; blood), and Typhlops vermicularis ( Typhlopidae; Israel, collection of the University of Bielefeld, Germany; muscle) completed the tests on cross-species amplications. Individual blood samples were collected from the caudal vessels and stored in lysis buffer (White and Densmore 1992) or 96% ethanol. Tissue samples ( liver, muscle) from museum specimens were transferred to 96% ethanol and kept at 4C until processing. DNA was extracted with the Nucleospin CT kit (Macherey & Nagel, Du ren, Germany) according to protocols supplied by the manufacturer. A total of 18 primer pairs were screened for cross-species amplication ( Table 1). Primers were synthesized by MWG-Biotech (Germany) and the forward primers were end-labeled (5) with an IR800 infrared uorescence label (Roy et al. 1996). We modied a touchdown program ( LiCOR Genotyping) to run with gradient function on the T-Gradient thermocycler (Whatman-Biometra) for quick screening of optimal amplication cycle proles of

Heterologous Amplication of Microsatellite Markers From Colubroid Snakes in European Natricines (Serpentes: Natricinae)
A. Hille, I. A. W. Janssen, S. B. J. Menken, M. Schlegel, and R. S. Thorpe
Eighteen microsatellite loci developed for a range of snake species (New World natricines, elapids, crotalids) were tested against European natricines (Natrix natrix, N. maura, and N. tessellata) in cross-species amplication experiments. Five loci were polymorphic (average expected heterozygosity 0.749 for a population of N. natrix in Amsterdam, mean sample size 47.8) and three loci were monomorphic. The remainder could not be consistently scored or failed to amplify. Further tests on single individuals of a diverse set of eight species of colubroid snakes showed that 15 of the 18 loci could be cross-amplied in at least one of these species. We conclude that our results show promise for the utilization of these markers for experimental assessments of genetic variation in the phylogenetically closely related group of European natricine snakes with emphasis on N. natrix. The full suite of microsatellite markers now available for snakes may show additional potential for subsequent

Brief Communications 63

Table 1. Results from 18 microsatellite markers designed for thamnophiine, elapid, and crotalid snakes that were tested in cross-species amplications in a variety of primitive and advanced colubroid snakes with emphasis on European natricines Locus Reference PCR conditions Ta (C) [MgCl2] [primer] Cycles Natricines N. natrix N. tesselata N. maura N. fasciata T. sirtalis Colubroids V. berus C. austriaca E. longissima C. nummifer E. murinus E. notaeus B. constrictor T. vermicularis Ns2 a 55 2.0 mM 0.6 M 30 Ns3 a 51 2.5 mM 0.6 M 30 Ns4 a 52.5 2.5 mM 0.6 M 40 x x x x x x x x Ns6 a 53 3.0 mM 0.6 M 40 Ns7 a 50 3.5 mM 0.5 M 30 Ns9b a 54 3.5 mM 0.6 M 45 Ts1 b 56 3.0 mM 0.6 M 40 Ts2 b 55 2.0 mM 0.6 M 30 x x x x x x x x

a, b, c, d: Prosser et al., 1999; McCracken et al., 1999; Burns and Houlden, 1999; Villarreal et al. 1996, respectively. , a smear, a multiple banding pattern, or no fragment was detected. , successful amplication products of one or two bands of expected size close to those of the original species for whom the primer was designed. x, not tested.

different primers. In these exible preruns the gradient along the thermoblock was adopted to the spectrum of annealing temperatures given by the selection of up to 12 different primers to be tested. The amplication trials started with a temperature of 5C above the highest annealing temperature suitable for the primer selection. PCR reactions (10 l) were conducted in Mlti Ultrastrips (Roth, Stuttgart) containing 100 ng genomic DNA, 0.26 mM dNTPs, 0.2 units of SigmaTaq (Sigma), and nal MgCl2 and primer concentrations as outlined in Table 1. The KCl concentration of the 10 PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, and 0.01% gelatin) supplied with the enzyme was not altered. Optimized PCR programs (the number of cycles and annealing temperature for each primer are listed in Table 1) consisted of an initial denaturation step at 94C (5 min), 2540 cycles of denaturation (1 min at 94C), annealing (30 s), and extension (45 s at 72C), and 20 min at 72C. Gel electrophoresis and visualization of PCR products was accomplished using a LI-COR Model 4000 automated DNA sequencer ( LI-COR, Inc.). Gels, 25 cm long and 0.25 mm thick and prepared with 6.5% Long Ranger ( Biozym) polyacrylamide matrices, were run in 1 TBE electrophoresis buffer. Electrophoresis was controlled by the Quick SequencIR software

that automates the process of focusing and autogaining the uorescence signals from the gel and setting the electrophoresis parameters (1500 V, 40 mA, 40 W, 50C gel back-plate heating, reading seven image frames). After initially identifying the optimal PCR conditions for each primer pair, individuals were again screened for all primer pairs at the optimized annealing temperature to produce clear and robust DNA band amplication patterns as estimated from the screen image (one or two bands indicating homozygotes and heterozygotes, with sizes close to those of the original species in which the primer was designed; three and more secondary bands of different sizes around the expected allele size were considered stutter bands). In most cases amplied products had to be serially diluted (dilution factor varied between 8 and 20) with loading buffer for optimal automated detection. Loading of the samples was repeated, including a minimum of two or three STR size standard (50350 bp STR Size Standard, LI-COR supplied by MWG-Biotech) lanes along with 12 sample lanes. Allelic patterns displayed as autoradiogram-like images were scored visually and quantitated by Gene ImagIR software (Schwengel et al. 1994). On completion of the run after a separation time of 45120 min, the image area was captured and the individual DNA pro-

les were analyzed by computerized fragment analysis (RFLPScan, ScanAnalytics, Billerica, MA). Accurate fragment sizes were attained by a matching bands option that generates allele bin classes with less than 0.5% standard deviation of sizing precision across all gels analyzed. Observed and expected heterozygosities were analyzed using BIOSYS-1 [Swofford and Selander (1981); modied and updated to BIOSYS-2 by Black IV (1997), Colorado State University, Ft. Collins, CO, ftp://ftp.lamar.colostate.edu/pub/wcb4]. Genotypic frequencies at each polymorphic locus were checked for conformance to the HardyWeinberg principle using the exact probability test provided by BIOSYS-2.

Results
All but three of the loci tested ( Ns8, Ns9, and Ns10) produced amplicon size ranges in the European natricines similar to those allele sizes from potentially homologous loci in those species (especially thamnophiine snakes as controls) for which the primers were originally developed ( Table 1). Five of the ampliable STR loci ( Ns2, Ns3, Ts2, Ts3, and Hb30) were polymorphic for N. natrix, three loci were monomorphic ( Ts1 with a fragment size of 120 bp, Ts4 with a fragment size of 172 bp, and Ns6 with a product size of 198 bp), while most others could not be

64 The Journal of Heredity 2002:93(1)

Table 1. Extended.

Ts3 b 59 3.5 mM 0.5 M 30

Ts4 b 54 2.0 mM 0.6 M 35

Hb2 c 54 2.0 mM 0.6 M 35 x x

Hb30 c 59 2.5 mM 0.45 M 25 x x

Hb48 c 59 2.0 mM 0.6 M 35 x x

Hb65 c 53 2.0 mM 0.6 M 35 x x

Hb70 c 54 2.0 mM 0.6 M 35 x x x x x x x x x x

Ch5A d 54 3.5 mM 0.6 M 45 x x

Ch7-150 d 54 3.5 mM 0.6 M 45 x x

Ch5-183 d 55 3.5 mM 0.6 M 45 x x

consistently scored and interpreted ( Ns4, Ns7, Ns9b, Ts3, Hb2, Hb48, Hb65, Hb70, Ch5A, Ch7-150, and Ch5-183) either in natricines or other taxa screened ( Table 1). N. maura was polymorphic at four loci ( Ns2, Ns3, Ts2, and Ts3; locus Hb30 was not scorable), while N. tessellata was polymorphic at three ( Hb30, Ns3, and Ts2; Ns2 and Ts3 amplied only in one specimen). Expected heterozygosity for the sample of 60 individuals of N. natrix varied from 0.545 for locus Ns3 to 0.907 for locus Ts3, with an unbiased average

expected heterozygosity of 0.749 (SE 0.083). Genotypic frequencies did not signicantly deviate from HardyWeinberg equilibrium (P values; see Table 2), and no evidence of null alleles was found. The mean number of alleles per locus was 9.2 (SE 2.7). Average repeat counts of microsatellite arrays could not be given because alleles were not sequenced. Allele ranges of N. natrix, however, were high for the simple dinucleotide-type locus Ns2 (allele range 363418), the trinucleotide locus

Table 2. Characterization of the subset of ve polymorphic microsatellite markers successfully amplied in Natrix natrix and the other European natricines Locus Natrix natrix Sample size Allele sizes No. of alleles H( DC) H( HW) P Natrix tessellata Sample size Allele sizes No. of alleles Natrix maura Sample size Allele sizes No. of alleles Ns2 Ns3 Ts2 Ts3 Hb30

Ts3 (allele range 282341), and the compound tetranucleotide locus Ns3 (allele range 625638). Moderate numbers of allele bins were found for the perfect dinucleotide locus Hb30 (allele range 252256) and low numbers were found for the compound microsatellite Ts2 (allele range 164306). When the same panel of STR markers was tested on eight additional snake species covering a wide spectrum of primitive and modern taxa ( Table 1), amplicons were produced for 15 loci for at least one species. There was no greater success in cross-amplication in advanced colubroid snakes compared to primitive species (worm snake and boid), even though the primers were developed from colubroids.

Discussion
57 363418 11 0.930 0.843 .087 1 152 1 9 146154 4 37 625638 4 0.622 0.545 .320 23 192242 11 9 186222 6 60 164306 18 0.964 0.901 1.000 23 191220 9 9 165221 6 28 282341 12 0.750 0.907 .861 1 150 1 9 140165 6 57 252256 3 0.737 0.553 .067 23 239253 2 9

H( DC) direct count estimate of mean heterozygosity per locus. H( HW) sample-size corrected estimate of mean heterozygosity per locus. P exact probability of heterozygotes deciencies. , a smear, a multiple banding pattern, or no fragment was detected.

In order to explore the potential utility of heterologous microsatellite primers designed for a diverse taxonomic array of snake species, we performed cross-species amplication experiments with published primers to detect variable loci between the three species of the natricine subfamily of European water snakes. Success in amplication transferability of microsatellite loci between closely related species in general is thought to be a consequence of the homology of anking regions of the simple sequence repeats. The transfer rate in our heterologous amplication trials increased from 0% (no crossamplied locus derived from a crotalid

Brief Communications 65

species), to 20% (one of ve loci with primers designed for an elapid species), to more than 40% (three of seven loci tested where primers were designed for the thamnophiine snake N. sipedon), nally to 100% transferability for the four loci amplied in the target species N. natrix and their closest European sister species N. tesselata and N. maura with primers that were developed for T. sirtalis. The monomorphism of the three loci ( Ns6, Ts1, and Ts4) must be considered carefully. Since the homologous loci have evolved independently in related species, there could be cases in which microsatellite alleles occasionally fail to yield a visible amplication product. Such nonamplifying alleles due to mutations in anking sequences of the simple repeats can produce many homozygous electromorphs which might actually be null heterozygotes. The level of observed multiallelism at the polymorphic loci for the European natricines was well within the range of polymorphism that was reported for the species for which the primers were originally developed. Thus the variable microsatellite loci are expected to hold great potential as markers for population studies with a focus on the target species N. natrix, which shows little genetic variation using other techniques such as allozymes (e.g., Hille 1997). These markers should provide adequate variation to identify and assign individuals to local subpopulations. They can be used to quantify gene ow patterns and to determine the impacts of habitat loss on the genetic structure of the Amsterdam metapopulation. The high level of genome homology found, especially between the vicariant sister groups of the Nearctic Thamnophiine and the Palearctic Natricine taxa corroborated the view of a relatively ancient split between these lineages of water snakes. The detection of much higher fragment sizes for the dinucleotide Ns2, the trinucleotide Ts3, and the highly complex locus Ns3 in N. natrix compared to those found in its relatives, for example, poses a problem to explain STR array expansions as a consequence of the neutrality theory and population dynamics. The number of alleles must have been generated by a greater average number of expansion mutations since the most recent common ancestor, and the lengths of microsatellites are correlated to the persistence of a high long-term effective population size (Amos 1999; FitzSimmons et al. 1995; Karhu et al. 2000). To conclude, though the colubroid

snakes perhaps diverged some 65 million years ago (Greene 1997), one locus identied in an elapid snake still shows polymorphism in European natricines. Moreover, snakes may have evolved 140 million years ago (Greene 1997), yet the majority of loci developed from advanced colubroid snakes still cross-amplify in at least one of the primitive snakes tested. The previous study predominantly showed that the success of heterologous primer amplication and the detection of polymorphic loci was found to be directly related to the phylogenetic closeness among related species. Consequently the microsatellite loci cross-amplied and characterized in this study provide useful molecular markers to address the research goal mentioned. Cross-amplication, rather than de novo development, appears to be a useful strategy in this case.
From the Zoologische Staatssammlung Mu nchen, Muenchhausenstrasse 21, D-81247 Munich, Germany ( Hille), Faculty of Biosciences, Zoology Department, University of Leipzig, Leipzig, Germany ( Hille, Janssen, and Schlegel), Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands (Janssen and Menken), and School of Biological Sciences, University of Wales, Bangor, Gwynedd, United Kingdom ( Thorpe). The authors wish to thank those who helped in the grass snake eld studies. We also thank the anonymous referees for improvements to the manuscript. Blood samples from the Amsterdam population were collected in full compliance with specic federal permits (permit no. 1441 of the Dutch foundation RAVON to I.A.W.J.). This work was funded in part by Stiching ter Bevordering van Herpetologie ( Dutch SBH grant to I.A.W.J.) and the German Science Foundation ( DFG Hi 595/1-2 grant to A.H.). Address correspondence to Axel Hille at the address above or e-mail: axel.hille@zsm.mwn.de. 2002 The American Genetic Association

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