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Neglected native or undesirable alien? Resolution of a

conservation dilemma concerning the pool frog Ranalessonae

TREVOR J.C. BEEBEE1, JOHN BUCKLEY2, IVOR EVANS3,JIM P. FOSTER4,*, ANTONY H. GENT2, CHRIS P. GLEED-OWEN2,GEOFFREY KELLY5, GRAHAM ROWE1, CHARLES SNELL3,JULIA T. WYCHERLEY1 and INGA ZEISSET1,6

1School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK; 2The Her-

petological Conservation Trust, 655a Christchurch Road, Boscombe, Bournemouth BH1 4AP, UK;3School of Chemical and Life Sciences, University of Greenwich, Medway Campus, Central Avenue,

Chatham Maritime, Kent ME4 4TB, UK; 4English Nature, Northminster House, Peterborough PE1

1UA, UK; 520 Buxton Road, Frettenham, Norwich NR12 7NG, UK; 6Present address: Department of

Population Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100

Copenhagen 0, Denmark; *Author for correspondence (e-mail: jim.foster@english-nature. org.uk; fax

+44-1733-568834)

Received 22 July 2003; accepted in revised form 26 January 2004

Key words: Archaeozoology, Bioacoustic analysis, Introduced species, Literature, Microsatellites,

Native species, Randomly Amplified Polymorphic DNA, Ranidae

Abstract. Introduced species often pose serious threats to biodiversity, but occasionally confu-

sion arises as to whether a species really is introduced or is in fact an overlooked native. A

recent UK conservation dilemma has centred on the status of the pool frog Rana lessonae. This

species has been the subject of documented introductions from central and southern Europe

since the early 1800s, the accepted position being that all UK R. lessonae populations are

descended from these introductions. However, a closer examination of early UK literature

sources, and recent discoveries of isolated, native R. lessonae populations in Sweden and Nor-

way, led some herpetologists to question whether the species was in fact present as a native at

some locations prior to the introductions. Research was initiated along four major lines of

enquiry: genetic, bioacoustic, archaeozoological and archival. A high degree of convergence

among the genetic and bioacoustic investigations demonstrated that the potentially native UK

pool frogs were closely related to Scandinavian frogs, thus ruling out introductions from further

south as a potential origin. Subfossil evidence of pool frogs was found from ca. 1000 years

before present, demonstrating that the species occurred in the UK prior to known introductions.

Archival sources produced no historical support for introductions from northern Europe. The

postglacial history inferred for these northern populations is consistent with the known climatic

and geographical conditions. Taken together, the evidence for the native status of the pool frog

is compelling, and furthermore the UK population appears to be part of a distinct northern

clade.

Abbreviations: bp – before present; LGM – Late Glacial Maximum; mtDNA – mitochondrial

DNA; RAPD – Randomly Amplified Polymorphic DNA.

Biodiversity and Conservation 14: 1607–1626, 2005. � Springer 2005

DOI 10.1007/s10531-004-0532-3

Introduction

The movement of animals and plants around the world, and the subsequentestablishment of alien species in environments distant from their origins, isincreasingly recognised as one of the major threats to global biodiversity (e.g.Schoener and Spiller 1996; Manchester and Bullock 2000). However, a corol-lary of this problem is the risk of labelling a species as alien when it might infact be a previously unrecognised native. This judgement has dramatic impli-cations for management, potentially making the difference between pro-grammes of eradication or conservation. Uncertainties of this kind are mostlikely to arise near range edges, and in this paper we describe the outcome ofjust such a dilemma concerning an amphibian in Britain.

Until recently it has been generally accepted that there are only six speciesof amphibians native to Britain, including a single frog, Rana temporaria.However, various other amphibians occur wild in Britain including membersof the ‘green’ or ‘water’ frog group. Several types of water frog inhabitmainland Europe and three are particularly widespread, the pool frog Ranalessonae, the marsh frog R. ridibunda and the edible frog R. esculenta. Waterfrogs are morphologically very variable and are often difficult to distinguishin the field. Hybridisation occurs between some of them; most notably, theedible frog is a fertile hybrid of marsh and pool frogs, and is more accu-rately described as R. kl. esculenta. The hybridogenetic reproduction pro-cesses that occur among these amphibians are highly complex (e.g. Berger1973; Graf and Polls Pelaz 1989; see review in Beebee 1996). Prior to the1970s pool and edible frogs were generally considered to be subspecies ofRana esculenta.

Marsh, edible and pool frogs have all been introduced to Britain fromcontinental Europe on numerous occasions over the past 200 years. Many ofthese events have been well documented, the first recorded introductions beingmainly of edible frogs to several sites in Norfolk in 1837 (Smith 1951).Unsurprisingly in light of this history, the accepted position has been that allpopulations of these animals in Britain have resulted from introduction byhumans. Most if not all the water frog populations established in Britain fromintroductions during the 19th century have subsequently disappeared, butdescendants of some recent, known translocations are presently found in anumber of locations (Wycherley et al. 2003).

Although some of the earliest recorded water frog introductions occurred inNorfolk in the 1830s, there is evidence to suggest pool frogs were alreadypresent in the area. The species was ‘pretty generally diffused’ in the area andwas known to have been present by 1820 (Boulenger 1884a). A colony of poolfrogs at Rockland All Saints and Stow Bedon, near Thetford in Norfolk, wasknown in the mid-1800s and is represented in museum specimens collected atthat time (Newton 1859). Illustrations of the Norfolk specimens are given inBoulenger (1897). Gadow (1904) reported the species as abundant at that timein this area and commented that they may have been native, though other

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authors suggested early introduction via various routes, for instance by Italianmonks (Boulenger 1884b).

Another population was discovered in 1843 at Fowlmere (formerly Foul-mire) Fen in Cambridgeshire (Bond 1844a). Museum specimens confirm thatthese animals were pool frogs, and no other species of water frog was repre-sented in the sample. Moreover, it appears that the species occurred in theFenland area before the early 1800s. Thus Bell (1859) wrote

I have to remark that the fact of the esculent frog being native to thiscountry appears to me to rest upon irrefragable testimony. My father,who was a native of Cambridgeshire, has often described to me, as longago as I can recollect, the peculiarly loud and somewhat musical sounduttered by the frogs of Whaddon and Foulmire, which procured for themthe name of ‘Whaddon Organs’. My father was always of the opinion thatthey were of a different species from the common frog and this opinion ofhis, formed nearly a century ago, was confirmed by Mr Thurnall’s dis-covery that the frogs of Foulmire are of the species Rana esculenta’ (nowknown from museum specimens actually to be R. lessonae).

Writing on the Fowlmere frogs, Bond (1844b) noted their unusual vocalsacs, timidity and restriction to water, all consistent with water frog behaviour.Pennant (1776) noted that the croaking of frogs in fen districts earned them thenames of ‘Dutch Nightingales’ and ‘Boston Waites’. Since the only native frog,R. temporaria, has a low amplitude, low frequency call that is rarely detectedby humans distant from the breeding site, these descriptions may well haverelated to water frogs. The frogs at Fowlmere were exterminated by fendrainage by 1847 (Wolley 1847).

However, pool frogs were rediscovered in Norfolk during the 1960s, veryclose to where Newton (1859) documented populations in the mid-1800s(Buckley 1986). A notable feature of this population as well as those identifiednear Thetford and at Fowlmere during the 19th century was that all appear tohave been of pool frogs only (based on coloration and morphology), whereasthe documented introductions in East Anglia (also represented in museumcollections) were mostly of edible frogs. Furthermore, isolated pool frog pop-ulations were discovered elsewhere in northern Europe during the 20th century.A large metapopulation, discovered during the 1940s, occurs in a group ofabout 100 ponds north of Uppsala in Sweden (Forselius 1962) and a muchsmaller set of populations was discovered in southern Norway in the 1980s(Dolmen 1996). In both cases no other water frog species occur anywhere nearthese pool frog populations, which are at the northerly range edge of R. les-sonae. Extensive studies on the Swedish frogs have strongly indicated that theyare long-standing natives of the area (Sjogren 1991). An interesting feature ofthe pool frogs from Norway, Sweden, Thetford and Fowlmere is that they arepredominantly brown in colour whereas pool frogs from elsewhere in Europeare mostly green.

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This accumulation of circumstantial evidence prompted a re-evaluation ofthe pool frog’s status in Britain (Snell 1994; but see Dutt (1906) for a summaryof a debate on the same question occurring almost a century earlier). Were theThetford and Fowlmere pool frogs relict native populations rather than, aspreviously assumed, the descendants of introductions? If true, the pool frogwould be Britain’s most endangered amphibian, rather than an alien whichmight be ignored or even extirpated. Although R. lessonae being an introducedspecies in England would not prompt such major concern as some other non-native amphibians (e.g. R. catesbeiana, Banks et al. 2000), there would still bequestions over potential impacts on native fauna through competition,predation and pathogen transfer.

The question of how to determine native status has been addressed for plantsby Webb (1985) and more generally by Usher (1999). Eight main criteria aresuggested for assisting with a judgement on native status: fossil evidence, his-torical evidence, habitat (natural versus artificial), geographical distribution(continuous versus disjunct), frequency of known naturalisation, geneticdiversity, reproductive pattern (vegetative versus sexual; relevant mainly toplants) and possible means of introduction. In addition, the analysis of similaror shared characters across a species’ range can help to pinpoint the origin ofpopulations; for instance, affinities among allozyme loci have been used as ameans of distinguishing introduced toad populations from native ones(Szymura 1998).

With a view to determining the status of the pool frog in Britain, a researchprogramme was initiated as part of a Species Action Plan (English Nature1998). Four primary lines of evidence were pursued to this end: geneticanalyses, archaeozoology, mating call analyses and historical written records.The outcomes of these investigations, together with an assessment of nativestatus criteria, are summarised here.

Genetic analyses

Genetic methods can be employed as powerful tools for elucidating the originsof individuals and populations (Jehle and Arntzen 2002). We investigated thephylogeographic relationships among pool frog populations sampled acrossthe species’ range (Figure 1) using both nuclear and mitochondrial DNAmarkers. Our expectation was that molecular genetic distances should suggestphylogeographic links between British frogs and mainland European popula-tions, and that these distances would indicate how much time has elapsed sincetheir separation. A population of pool frogs known to have been introducedinto southern England (Surrey) about 100 years ago (Gillett 1988) wasincluded as a control for the genetic distance comparisons.

Six nuclear microsatellite markers developed for R. lessonae (Garner et al.2000; Zeisset et al. 2000) were employed with samples of between five and 41individuals from 10 widely separated populations (Zeisset and Beebee 2001).

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Only small numbers (five from Norway and 12 from Sweden) were availablefrom Scandinavia because R. lessonae is rare in both countries. DNA wasobtained (non-destructively) from the single surviving individual of potentiallynative R. lessonae in Britain, but also from four museum specimens taken inthe same area during the 19th century to give a sample size of five. The frogssampled from Norway, Sweden and the potentially native British specimenswere homozygous at all six loci and fixed for the same alleles at five of them. Bycontrast, frogs sampled from elsewhere in Europe (Figure 1) were polymorphicand had mean expected heterozygosities ranging between 0.311 and 0.611, andmeans of between 3 and 7 alleles per locus. Furthermore, the introduced Surreypopulation had mean expected heterozygosities and alleles per locus of 0.736and 5.2, respectively. Pool frog populations along the northern range edge(Norway, Sweden and ‘native’ British) were therefore both genetically depau-perate and closely related. We also successfully amplified DNA from a singlemuseum specimen taken during the 19th century from an introduced popula-tion, long since extinct, near the area of Norfolk occupied until recently by thepotentially native frogs. Alleles from this individual were quite different from

Figure 1. European distribution of Rana lessonae (shaded area shows core range). Dark circles

indicate sampling sites for microsatellite analysis: F, France; NL, The Netherlands; CH, Switzer-

land; I, Italy; P, Poland; H, Hungary; S, Sweden; N, Norway; i, UK introduced; n, UK potential

native (Thetford, Norfolk). Figure from Zeisset and Beebee (2001).

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those found in the potentially native British frogs but similar to those in westEuropean animals. Tree construction using the neighbour-joining method withCavalli-Sforza chord distances (Figure 2) confirmed this phylogeographicpattern with a distinct northern clade more closely related to east than to westEuropean animals. We also used the STRUCTURE program (Pritchard et al.2000) as an independent method to assign the potentially native British poolfrogs to the most closely related European populations. On this basis theBritish frogs were unequivocally (p=0.998) allocated to the Scandinavian area,indicating on the basis of this test that the northern clade had a commonorigin. Introduced frogs in Surrey, by contrast, clustered quite separately.

In a separate study, Randomly Amplified Polymorphic DNA (RAPD)techniques were used to compare loci throughout the genome (Snell and Evansin preparation). The populations examined (with sample numbers parenthes-ised) were from Norway (7), Norfolk (1), Sweden (21), Poland (24), Hungary(43), The Netherlands (52), France (72; three populations: Cherbourg, Dijon,and Paris), Switzerland (44) and Italy (39). In initial work, 107 10-mer primerswere screened, and six deemed the most useful on the basis of polymorphicband resolution and reproducibility were selected and used in later analyses.Band sizes and identities were determined electronically from digitally capturedgel images. Results showed levels of polymorphism which were lowest in theNorwegian and Swedish populations and highest in the Italian population.Analysis of the extensive data set (covering 109 polymorphisms) using clusterand dendrogram techniques has generated estimates of the genetic distancesbetween the populations (Figure 3).

The results concur with those of the microsatellite work in showing a closeaffinity between Norfolk, Swedish and Norwegian individuals with, for the

Figure 2. Consensus phylogeographic tree of R. lessonae based on microsatellite allele frequen-

cies. Numbers at branch points are percentages based on 1000 bootstrap replicates. Figure from

Zeisset and Beebee (2001).

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most part, an increasing, incremental divergence away from these populationsthe further south in Europe the sampled populations are situated.

Finally, we also investigated sequence variation in pool frog mitochondrial(mt) DNA. We hoped that a greater number of museum specimens would yielddata with this marker, because of its high copy number relative to nuclearsequences, than was possible with microsatellites. A total of 17 museumspecimens from potentially native British pool frogs were available for analysis,but only four yielded microsatellite genotypes. Attempts to sequence smallregions of D-loop, usually the most polymorphic region of mtDNA, failed forunknown reasons possibly related to the repetitive nature of this region in Rana(Sumida et al. 2000). We therefore amplified and sequenced a 451 base pairsection of the cytochrome b gene (using data from Tanaka et al. (1996) todesign primers) from a total of 49 frogs sampled across the biogeographicalrange (Figure 1). Having identified a single polymorphic site among thesefrogs, a subfragment of this section (177 base pairs) was successfully amplifiedand sequenced from five museum specimens, including three that wereunsuccessful with microsatellites (unpublished data). However, only twocytochrome b haplotypes were discovered across the whole range (Genbankaccession numbers: AY057099 and AY057100) and these differed by just asingle base change (A-G transition at the second codon position of amino acid75 in the gene) corresponding to a conservative amino acid substitution.Haplotype A was the only sequence found throughout most of Europeincluding the northern populations, whereas haplotype B occurred in all thesamples from France and was at 50% abundance in those from the Nether-lands. The mtDNA analysis therefore made it unlikely that the potentiallynative British frogs originated in France (from whence came at least some ofthe known introductions in the 19th century) but could not discountintroductions from elsewhere.

Figure 3. Inferred relationships between European populations of R. lessonae. Consensus phy-

logram created using band frequency data from 109 RAPD polymorphisms. Distance estimation by

Cavalli-Sforza chord measure (Cavalli-Sforza and Edwards 1967). Tree inferred by the ‘Neighbour

Joining’ method (Saitou and Nei 1987). Figures at nodes represent percentage of 1000 bootstrap

cycles. Scale bar represents a 10% variation in frequency data.

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Archaeozoology

Evidence for native status can be gained from retrieving bones in archaeo-logical deposits that pre-date known or suspected introductions. Amphibianbones are commonly encountered in deposits where they have entered bypitfall, predation or death during hibernation. As a result, frog bones areroutinely recovered by archaeologists from programmes of soil-sieving, butthese remains are generally not studied any further. A survey of such materialfrom 55 archaeological sites in eastern England was carried out between 1996–2000 (Gleed-Owen 1999, 2001). All amphibian remains were diagnosed to thehighest taxonomic level possible, by morphological comparison with modernskeletons. Although frontoparietals (cranial bones) have been recommended insubfossil frog identification (Bohme 1977; Bohme and Gunther 1979), thesewere infrequently recovered, so ilia (pelvic bones) were mostly relied uponhere. Two modern British specimens including the potentially native individ-ual were used, but most expertise was gained from studying the GottfriedBohme collection in Berlin (see Bohme and Gunther 1979) containing 318water frog skeletons. Diagnostic criteria based on ilial shape were developedand tested blind on a random selection of 48 water frog pelves. The mor-phology of 19 specimens was ambiguous, but specific identifications weremade for the other 29. Of these, 27 were correctly identified, corresponding toa success rate (after rejecting ambiguous specimens) of 93% and suggestingthat specific diagnosis is possible using water frog ilia. The ilium of water frogsis distinctly thicker along the junctura ilioischiadica than in brown frogs. Inaddition, the tuber superior (dorsal prominence of some authors) has a rela-tively flat lateral facet in water frogs, which is distinct from the uneven orridged surface in brown frogs. In R. lessonae, the tuber is narrower antero-posteriorly, sometimes more sinuous and always more laterally produced thanin R. ridibunda. The posterior margin of the tuber is also steeper in R. lessonae,and the dorsal edge of the vexillum tends to be straight as opposed to convexin R. ridibunda. R. esculenta is intermediate between R. lessonae and R. rid-ibunda in ilial morphology, but can generally be separated from either species.The shape of the corpus and the angles of its margins, in combination withtuber and vexillum shape, allow the distinction of R. lessonae from R. escul-enta in most cases.

After examining tens of thousands of amphibian bones from Britisharchaeological sites, two pool frog ilia were identified (Figure 4). One incom-plete bone from a Middle Saxon house foundation at Gosberton, Lincolnshire,was associated with a radiocarbon date of AD 880–1040 (Gleed-Owen 1999,2000). It was referred to R. cf. lessonae before study of the Berlin collection. Avirtually complete second bone came from a stratum dated archaeologically to850–1050 AD (R. Mortimer, personal communication) at a Saxon settlement inEly, Cambridgeshire (Gleed-Owen 2001). Neither of the bones showed anyambiguous characters. G. Bohme (personal communication) agreed with theidentifications, especially noting the well-developed characters of the more

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Figure 4. (a) Subfossil pool frog bone (left ilium) dating from ca. 850–1050 AD, excavated at

West Fen Road, Ely, Cambridgeshire. Scale bar,10 mm. (b) Subfossil pool frog bone (incomplete

right ilium) dating from ca. 880–1060 AD, excavated at Chopdike Drove, Gosberton, Lincolnshire.

Scale bar, 3 mm.

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complete Ely specimen. Both ilia have particularly prominent tubers, and donot overlap the morphological range observed in R. esculenta.

Bioacoustics

Male vocalisation is a recognised part of mating behaviour in many species ofmammals, birds and amphibians. Interspecific call differences have been usedto determine species identity (Schneider and Haxhiu 1994; Kaya and Simmons1999) and intraspecific call variation is clearly recognised for many speciesincluding mammals (Terhune et al. 2001), birds (Haftorn and Hailman 1997),and frogs (Hasegawa et al. 1999). However, previous studies on pool frog callcharacteristics did not find regional differentiation (Sinsch and Schneider1996). We considered the possibility that differences may indeed exist as aconsequence of biological range expansion and subsequent isolation, and thatfrog dialects may evolve through founder and drift effects.

Initial studies evaluated analytical techniques for separating three of theEuropean water frog species using characteristics of their male advertisement(mating) calls. We found that the calls of R. ridibunda, R. esculenta andR. lessonae could be distinguished by the number, position and characteristicsof the peaks in the call frequency spectrum (Wycherley 2000). It was apparentusing this approach that the Norfolk frogs were R. lessonae. However this byitself did not give any indication of the origin of the Norfolk population.Techniques were required that would enable this population to be distin-guished from other pool frog populations.

We therefore developed new analytical techniques, based on Fourier trans-formation of individual subunits of the call, and used these to compare matingcalls of separate populations of pool frogs from selected sites across Europe,including Estonia (Wycherley et al. 2001; Wycherley et al. 2002a). These in turnwere compared to historical recordings of the Norfolk frogs (source: BritishLibrary Sound Archives and original recordings; Wycherley et al. 2001). Fre-quency analysis of the call subunits showed two major peaks and our analysesquantified features of these peaks, notably their frequencies, relative height ofmajor peak to minor peak, and frequency band widths at half maximumheight. We also included the number of subunits per call as a 6th variable.

As the pool frogs tend to call in chorus, sample size was limited to thenumbers of ‘clean’ calls where individual frogs were identified. All variableswere quantified and the data were checked for normal distribution of residualsprior to statistical analysis. Temperature may affect some variables so we testedfor temperature effects using recordings made over a range from 16 to 21.5 �C.A weak, but significant, temperature relationship was observed for the fre-quency of the upper peak but not for any of the other variables. We thereforecorrected measurements of this variable to 20 �C, using our temperaturecalibration, for all sites except Norfolk and Estonia (where contemporarytemperature data were not recorded).

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Discriminant analyses of pairwise comparisons of populations showedhighly significant differentiation of mating call characteristics (Wycherley et al.2002b). The data sets (cases) of call variables from all countries were alsoclassified using discriminant analysis. Classification success was 63.8%, and foreach country nearly all the remaining cases were assigned to an adjacentcountry. The populations were then grouped into regions in order to minimisevariable background noise effects from different sites and to increase com-parative sample size. Discriminant analysis again showed highly significantdifferentiation. The ‘northern group’ comprised data from Estonia, Norwayand Sweden, the ‘central group’ had data from Poland and Hungary, and the‘western group’ from France and the Netherlands. These were then comparedto the data from Norfolk so that an independent assessment of phylogeneticrelationships could be made. Classification success after this grouping rose to71–100% in the original groups with a mean of 77.3%. We therefore consid-ered that regional accents were clearly discernible in the pool frog. The pooleddata sets were also used for hierarchical cluster analysis and the resultingdendrogram is shown in Figure 5. This shows clear differentiation between thegroups, and places the Norfolk population close to other north Europeanpopulations (Wycherley et al. 2002b).

Call characters in the pool frog therefore varied systematically between pop-ulations sampled across the biogeographical range. This intraspecific call vari-ation can be used to infer phylogeny. As the populations of the northern groupand Norfolk are particularly distinct from the central and western groups, itseems unlikely that the Norfolk frogs were introduced from these latter groups.

Archive and literature review

Evidence for native status can be gleaned from references to species occurrencepre-dating introductions; qualitative information contained in old referencescan also assist in elucidating status. A search for archive and literature material

Figure 5. Hierarchical cluster analysis of pool frog call characters. Dendrogram from the pooled

data sets for four sample groups: Norfolk, Northern Europe, Central Europe and Western Europe

(see text for details). Figure redrawn from Wycherley, Doran and Beebee (2002b).

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relating to water frogs in Britain was undertaken to determine whether anymaterial relating to pool frogs in the East Anglian region had been overlookedpreviously. This process involved searching for references to water frogs andtheir historical sites. Museums, libraries, record offices, conservation organi-sations and local naturalists were contacted, resulting in the identification of129 published and archival sources, of which 97 contained relevant information(Kelly 2004). However, there was a dearth of relevant material in the manu-script sources. This was even true of papers by naturalists associated with theThetford and Fenland areas. Few naturalists recorded water frogs prior to1880, and little relevant work was undertaken from 1914 through to the late1960s. Though the paucity of records in this latter period may initially elicitdoubts about the presence of the species, it is notable that two Norfolk pop-ulations of the natterjack toad Bufo calamita were not formally recorded untilthe 1960s, and one of the largest British B. calamita populations was onlyconfirmed in north-west England in 1993 (Beebee and Buckley 2001). It alsoseems likely that the native R. lessonae populations had declined by this stageand so would have been less likely to be detected. On this basis, a scarceliterature record does not argue for the absence of the species.

Writers considering that the pool frog was introduced outnumbered thosewho either believed that it was native or that the matter could not be proved.An early proponent of the view that the edible frog (as the R. lessonae was thenknown) was a native was Charles Kingsley (Kingsley 1867). He considered thatthe early Holocene land bridge with the continent enabled such species tocolonise. No evidence was found to suggest that water frogs were introduced asa food source by Romans, Huguenots, or particular religious communities.Indeed there was no evidence of any kind of introductions pre-dating thosestarting in the early 1800s.

Discussion

Evidence for native status

Confident identification of two pool frog bones, from 9th to 11th century ADarchaeological contexts in adjacent counties (Lincolnshire and Cambridge-shire) provides conclusive evidence that the species was present in Britainaround a 1000 years ago. The number of finds might seem small against thevolume of work carried out, but water frogs are not normally associated withhuman habitation, and any finds are remarkable. A similar situation arises inthe Netherlands where water frogs are abundant today. Water frog remains arerarely encountered in archaeozoological studies despite the more or lessubiquitous occurrence of the more commensal species R. temporaria andB. bufo (Gleed-Owen 1999). Importantly, the evidence presented here pre-datesthe documented 19th and 20th century introductions by about 1000 years.Webb (1985) regarded plant fossil evidence as conclusive if it can be attributed

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to a date between the last glaciation and the arrival of Neolithic agriculture (i.e.between ca. 20,000 and 6000 years before present (bp); note that all dates givenhere are the current best estimates of real calendar ages, from Bjork et al. 1998,rather than those based on uncalibrated radiocarbon dates). Webb (1985)points out, however, that the fossil record is far from complete, and inferringfrom absences is unwise.

With respect to the other native status criteria proposed by Webb (1985),there is no mention in the historical record of pool frogs being introduced fromlocalities supporting genotypes consistent with the genetic results presentedhere. The Romans and Vikings were potential agents for an early introduction,but there is no historical or archaeological evidence that they did. AlthoughDutt (1906) referred to Baron von Hugel’s claim that water frog bones wereknown from Roman middens, this may refer to continental sites where frogshave been eaten since pre-history (Bailon 1993), or to a misinterpretation of thenatural occurrence of native frog bones in archaeological deposits. Compre-hensive oral and literature searches (by C.G.O.) have failed to reveal anyevidence that frogs were a food resource for Romans or Vikings in Britain.Furthermore, none of the 24 Roman sites studied during this project (Gleed-Owen 1999, 2001), nor Roman and Viking sites in York (C.G.O. and P. Piperunpublished notes) have produced any water frog bones. Clearly this lack ofevidence does not exclude the possibility of pre-19th century importation forfood, but it makes it a highly unlikely origin for British water frog populations.In any case, results from both the genetic and bioacoustic studies are notconsistent with a Roman introduction as they demonstrate that western, cen-tral and southern European frogs could not have been the founders. Webb(1985) notes that the record of an introduction can prove introduced status,and that the absence of a species from historical records is suggestive ofintroduction, but that demonstrating native status through such evidence isproblematic. We therefore conclude that the historical evidence supports nativestatus as there is no indication of an introduction, but there are suggestions ofearly (18th and 19th century) sightings of R. lessonae and archaeologicalconfirmation of much earlier occurrence.

Webb (1985) proposed, with some caveats, that plant species restricted toartificial habitats are likely to be introduced while those found extensively innatural habitats are probably native. This principle is more problematic foranimals, given that few are now restricted to natural habitats. Due to a longhistory of intensive human impacts, Britain now has very few naturalamphibian breeding ponds, with the majority of sites effectively being by-products of human activity, notably agriculture (Williams et al. 2000). How-ever, it is intriguing that some of the potentially native pool frog populationsoccurred in pingos, one of the few surviving natural pond types in Britain andnow highly restricted in extent. Pingos are periglacial features and in Britainwere formed as ground ice melted at the end of the last glaciation. Theytypically occur at extremely high densities in the landscape, one of severalfeatures which distinguish them from artificial ponds. The notion that pool

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frogs may be long-standing natives of this ancient landscape is given credenceby parallel discoveries in entomology (see below).

The geographical distribution of possibly native pool frogs is ostensiblydisjunct, thereby indicating alien status according to Webb’s (1985) criteria.However, given the high degree of habitat modification in lowland Britain inrecent centuries, combined with the narrow habitat preferences exhibited bynorthern clade pool frogs in Scandinavia, this criterion does not assist greatlywith the determination of native status. Webb’s (1985) ‘frequency of knownnaturalisation’ criterion provides limited support for the native status of thepool frog since there are no records of northern clade pool frog populationsbeing introduced and becoming established in similar habitats in Britain. Hisgenetic diversity criterion appears to apply expressly to plants of open ground,but Usher (1999) extends the reasoning to propose that a species exhibitinggreater genetic diversity is more likely to be native. However, two caveatsmentioned in Usher (1999) apply to pool frogs in Britain: species present assmall relict populations and populations at range edges. Pool frogs in Norway,Sweden and England exhibited very low genetic diversity at microsatellite locias well as sharing most of the alleles present. This situation is most readilyexplained by common descent from a small group of range-edge founders.

Webb (1985) stressed that the decision on native status is unlikely to besettled by any single line of enquiry, and that a balanced view may be arrived atwhen several lines point in the same direction. In the case of the pool frog, anassessment of the evidence is best made after setting it in context, allowing thepossibility of native status to be examined from a biogeographical andphylogeographical perspective.

Biogeography and phylogeography

Following the peak of the Late Glacial Maximum (LGM), around 22,000 yearsbefore present, the climate of northern Europe oscillated between warmer andcolder states over the next 10,000 years (Bjork et al. 1998). Thermophilic faunaand flora, previously restricted to southern refugia, dispersed northwards dur-ing warmer phases (interstadials). Amphibians and reptiles were among the firstBritish colonists (Gleed-Owen and Keen in preparation). Intervening coldphases (stadials) would have extirpated thermophiles in northern areas andcaused range contractions once more. Of all the stadials which have occurredsince the LGM, the Younger Dryas (ca. 12,600–11,500 bp) was the most severe(Bjork et al. 1998; Lowe et al. 1999). After ca. 11,500 bp, temperatures increasedrapidly and have remained relatively stable and high ever since. This back-ground of climatic events has strongly influenced the biogeography of allEuropean amphibians and reptiles.

During the LGM, sea levels were ca. 130 m lower than today, andthroughout the late glacial period Britain formed a continuous landmass withEurope (Figure 6). Increasing global temperatures melted the ice sheets and led

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to sea level rise which eventually drowned the North Sea bed. For about3,000 years after the end of the Younger Dryas, thermophilic terrestrial faunacould reach Britain before it was severed from the continent ca. 8500 bp(Lambeck 1995; Pelletier 1998) and further terrestrial immigration was pre-vented. During the Atlantic period (ca. 9000–5000 bp), average summer tem-peratures in Britain were about 2–3 �C warmer than today. Thermophilicspecies such as the European pond terrapin Emys orbicularis reached EastAnglia and Sweden, but died out later apparently due to climatic deteriorationat the end of the Atlantic period (Isberg 1929; Stuart 1979).

All the evidence from call characters, microsatellites and RAPDs is con-cordant with a distinct northern clade of R. lessonae currently extant in Nor-way and Sweden but recently extinct in Britain. Phylogeographic interpretationof both the microsatellite and RAPD analyses (Zeisset and Beebee 2001; Snell

Figure 6. Northwestern Europe showing the current landmass (dark shading) and that which

existed during the early Holocene period up to ca. 8500 bp (extensions shown as lighter shading).

Based on Lambeck (1995).

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and Evans, in preparation) as well as the call data suggests how this distri-bution may have arisen. A first wave of colonists (ancestors of the northernclade) probably moved out of LGM refugia during the initial warming phasesome 18,000–19,000 bp. In central Europe, R. lessonae appears to be a pioneerspecies, frequently colonising newly formed habitats before other water frogs(Grossenbacher personal communication). Tree pollen analyses have shownthat northward postglacial colonisations were generally slower in westernEurope than in the east Hewitt 1999). Northern clade pool frogs are moreclosely related to eastern than to western European frogs (Zeisset and Beebee2001), hinting at the branching off of western colonists from the main eastEuropean route. However, the more northerly populations would subsequentlyhave suffered isolation and bottlenecking during the Younger Dryas, perhapsexacerbated by topographical or geographical barriers.

After the final postglacial warming some 11,500 bp, the survivors from thesenorthernly populations could have reached Scandinavia and Britain beforerising sea levels drowned North Sea and Baltic Sea land-bridges. Later post-Younger Dryas migrations of pool frogs from southerly refugia spread throughmuch of mainland Europe but did not reach Britain or Scandinavia beforemarine isolation. Finally, on the basis of microsatellite andRAPDdata there aresmall but significant differences between northern clade pool frogs of all threecountries where they recently survived. This makes it highly unlikely that Britishfrogs were recently descended from Scandinavian frogs, for example by Vikingimports. Since the Vikings that settled in East Anglia were of Danish origin, it isin any case improbable that they could have introduced R. lessonae because thespecies is absent in that country. As the archaeological evidence refutes a 19thcentury origin and also argues against a Roman introduction, taken together theevidence suggests that R. lessonae was indeed a native British species.

Further support for this phylogeographic interpretation comes from otherspecies. In western Eurasia, the water beetle Hydroporus glabriusculus occurs inisolated fenland habitats (Bilton 1992). Using allozyme electrophoresis, Bilton(1992, 1994) showed that Norfolk populations of this beetle (including one at aknown R. lessonae site) were all genetically depauperate and were also moreclosely related to a Swedish population than to Scottish and Irish populations.The Norfolk and Swedish beetles were estimated to have last shared a commonancestor ca. 13,000–11,000 bp (Bilton 1994), a date consistent with the end ofthe Younger Dryas. Evidently it is not just frogs that show historicalconnectivity between Britain and Scandinavia.

Conclusions

The genetic and bioacoustic studies together demonstrate that the potentiallynative British populations are most closely related to Scandinavian popula-tions. A high level of convergence is apparent between the inferred phylogenyresulting from the microsatellite work and that produced by analysis of call

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characters (Wycherley et al. 2002b); this is further supported by the RAPD andto a lesser extent the mitochondrial DNA results. The hypothesis that poolfrogs at the possibly native sites were introduced from western, central orsouthern Europe can therefore be rejected. An introduction from Scandinaviais highly unlikely given the genetic differences between northern clade rangestates and the lack of a plausible introduction pathway. Archive sources pro-vide no evidence of introductions pre-1800, and archaeozoological finds con-firm the species was present well before known 19th century imports in areassupporting ancient landscape types, consistent with recognised pool froghabitat requirements.

Applying the criteria of Webb (1985) and Usher (1999), there are severallines of evidence that strongly point towards native status. No evidence hasbeen discovered to support an introduction that would be consistent with thegenetic and bioacoustic studies. Taken together, the analyses summarised hereprovide compelling evidence for the pool frog as a native of Britain. Fur-thermore, it appears to be part of a distinct northern clade which is of con-siderable conservation importance, given the small number of isolatedpopulations. Unfortunately, the last surviving native R. lessonae population inBritain became extinct in the 1990s. Plans are currently underway to reintro-duce northern clade pool frogs from Scandinavia into Britain, at sites wherehabitat management has been initiated to redress the presumed reasons forextinction. Since northern clade R. lessonae now breed in only about 80 pondsin Sweden and just two in Norway, this strategy should prove of internationalimportance for the conservation of this highly threatened amphibian.

Acknowledgements

Financial support was received from English Nature and Anglian Water as acontribution to the Species Recovery Programme. We thank Richard Griffithsand an anonymous reviewer for helpful comments on the manuscript. C.G.O.received financial and logistical support from Coventry University. The adviceand help of Gottfried Bohme and the Humboldt University Museum fur Na-turkunde (Berlin), Richard Mortimer and the Cambridge Archaeological Unit,James Rackham and other archaeologists is much appreciated. Kate Wellam atBournemouth University prepared the bone photographs. C.S. and I.E. thankLabImage Software, Labsoft Diagnostics, and Rob Edwards for assistancewith the development of specialised software to sort and display gel data.

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