CO1 DNA Barcoding Amphibians: Take the Chance, Meet the Challenge

Molecular Ecology Resources (2008) 8, 235–246 doi: 10.1111/j.1471-8286.2007.01964.x

DNABlackwell Publishing Ltd BARCODING CO1 DNA barcoding amphibians: take the chance, meet the challenge

M. ALEX SMITH,* NIKOLAI A. POYARKOV JR† and PAUL D. N. HEBERT* *Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada N1G 2W1, †Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Moscow 119121, Russia

Abstract Although a mitochondrial DNA barcode has been shown to be of great utility for species identification and discovery in an increasing number of diverse taxa, caution has been urged with its application to one of the most taxonomically diverse vertebrate groups — the amphibians. Here, we test three of the perceived shortcomings of a CO1 DNA barcode’s utility with a group of Holarctic amphibians: primer fit, sequence variability and overlapping intra- and interspecific variability. We found that although the CO1 DNA barcode priming regions were variable, we were able to reliably amplify a CO1 fragment from degenerate primers and primers with G-C residues at the 3′ end. Any overlap between intra- and interspecific variation in our taxonomic sampling was due to introgressive hybridization (Bufo/ Anaxyrus), complex genetics (Ambystoma) or incomplete taxonomy (Triturus). Rates of hybridization and species discovery are not expected to be greater for amphibians than for other vertebrate groups, and thus problems with the utility of using a single mitochondrial gene for species identification will not be specific to amphibians. Therefore, we conclude that there is greater potential for a CO1 barcode’s use with amphibians than has been reported to date. A large-scale effort to barcode the amphibians of the world, using the same primary barcode region of CO1, will yield important findings for science and conservation. Keywords: Anaxyrus fowleri, cox 1, DNA barcode, Triturus Received 1 May 2007; revision accepted 1 August 2007

concerns have been raised (Vences et al. 2005a, b; Lockridge Introduction Mueller 2006) and reported (Cowan et al. 2006; Godfray DNA barcoding has emerged at the forefront of efforts to 2006; Rubinoff et al. 2006; Clare et al. 2007; Little & accelerate the rates of taxonomic discovery and description Stevenson 2007; Waugh 2007), regarding the efficacy of the to meet or exceed rates of biodiversity loss (Janzen et al. CO1 approach to DNA barcoding for the class Amphibia. 2005; Savolainen et al. 2005; Smith et al. 2005). A DNA If accurate, these cautions are indeed unfortunate, as barcode — a short, standardized gene for species identi- amphibian populations around the world are in decline fication (Hebert et al. 2004b; Hebert & Gregory 2005) — could (Alford & Richards 1999; Blaustein et al. 1994; Green 2003). revolutionize how taxonomy and diversity estimates are Our ability to prioritize conservation decisions is further conducted through linking established museum collections compromised by the likelihood that many currently to unknown animals from the field (Janzen et al. 2005), recognized taxon names actually represent cryptic species facilitating the description of new species (Hebert et al. complexes (Kohler et al. 2005). A standardized molecular 2004b), revealing cryptic species (Hebert et al. 2004a; Smith tool to facilitate species identification would then greatly et al. 2006, 2007), linking adult with juvenile (Greenstone alleviate potential problems resulting from taxonomic et al. 2005; Thomas et al. 2005; Raharivololoniainaa et al. synonomy and crypsis. Clearly, amphibians are a taxonomic 2006) or male with female (Willassen 2005). However, group which would benefit from an emerging global initiative to barcode all constituent species. Such a global Correspondence: M. Alex Smith. Fax: 519-824-5703; E-mail: initiative is currently underway for the only more diverse [email protected] vertebrate groups: birds and fishes (Marshall 2005).

It has been proposed that the main problems with using DNA polymerase using a thermocycling profile of one CO1 as a DNA barcode in amphibians will be due to ‘(i) the cycle of 2 min at 94 °C, five cycles of 40 s at 94 °C, 40 s at high variability of priming sites that hinder the application 45 °C, and 1 min at 72 °C, followed by 35 cycles of 40 s at of universal primers to all species and (ii) the observed 94 °C, 40 s at 51 °C, and 1 min at 72 °C, with a final step of distinct overlap of intraspecific and interspecific diver- 5 min at 72 °C. Products were visualized on a 2% agarose gence values, which implies difficulties in the definition E-Gel 96-well system (Invitrogen) and samples containing of threshold values to identify candidate species.’ (p. 1859, clean single bands were bidirectionally sequenced using Vences et al. 2005a). Additionally, as amphibians are often BigDye version 3.1 on both an ABI 3730 and an ABI 3730xl dispersal-limited (although see Vences et al. 2003; Smith DNA Analyser (Applied Biosystems). Contigs were assembled & Green 2005), there is expected to be sufficient phylo- using either sequencher version 4.0.5 (Gene Codes) or the geographic signal within a mitochondrial barcode as to ContigExpress module of vector nti advance (version obfuscate species identification (Vences et al. 2005a). Based on 10.1, Invitrogen) and were subsequently aligned by eye in preliminary testing with other taxa, they found that the CO1 bioedit (Hall 1999). barcode would not perform as efficiently for amphibians as The proposed size for a full-length CO1 barcode is 648 bp for all other groups of animal life and that other genes (Hebert et al. 2003a, b). A CO1 sequence greater than 500 bp should be gathered to complement CO1. This seemed, to in the 5′ end of the CO1 gene, with appropriate collateral us, to place undue emphasis on the challenges which we information (primer sequences, trace files, collection latitude felt were not restricted to amphibians, but rather exist in and longitude), can be categorized in GenBank (Benson all animal taxa. Therefore, we gathered a small group of et al. 2005) as a DNA barcode. We have found that a 500 bp, Holarctic amphibians from six families, 11 genera and 34 or shorter, barcode gives identical species identification results species to test the efficacy of a CO1 DNA barcode for species to analyses of 648 bp sequences (Hajibabaei et al. 2006; identification. Our intent was not to compile an authorita- Smith et al. 2006). Sequences, electropherograms (for both tive database of amphibian CO1 sequences (a task already successful and unsuccessful amplifications) and other underway in Canada; the Canadian Barcode of Life specimen information for the individuals analysed here are Network, www.bolnet.ca), but rather to test the ease available in the ‘CO1 Barcoding Amphibians’ file in the with which such a database would be compiled, and Completed Projects section of the Barcode of Life website how successful a CO1 barcode would be for species (www.barcodinglife.org), and all sequences have been identification. deposited in GenBank (Accession nos EF525707–EF526063) and are detailed in Table S1, Supplementary material. Methods Primer fit Total genomic DNA was extracted from small pieces (~1 mm long) of toe or tail clip using the NucleoSpin 96 Tissue kit The variation in priming sites for several standard and (Macherey-Nagel Duren), following the manufacturer’s degenerate CO1 barcode oligonucleotides was calculated protocols. Extracts were resuspended in 40 μL of distilled for 83 amphibian species in GenBank using dnasp (version water, and a 658-base pair (bp) region near the 5′ terminus 4.1) (Rozas et al. 2003). The internal forward primer for a of the CO1 gene was amplified following standard protocol smaller CO1 fragment (or minibarcode — MLepF1) was (Hebert et al. 2003a). Briefly, full length CO1 sequences were tested using individuals from GenBank, and a further 301 amplified using primers LepF1 (5′-ATTCAACCAATCA- individuals examined in this study. A comparison of primer TAAAGATATTGG-3′) and LepRI (5′-TAAACTTCTGGA- fit within published Lepidoptera was completed by com- TGTCCAAAAAATCA-3′, Hebert et al. 2004a), and/or VF1-d piling sequences using the Taxonomy Browser function (5′-TTCTCAACCAACCACAARGAYATYGG-3′) and VR1-d of bold (www.barcodinglife.org; Ratnasingham & Hebert (5′-TAGACTTCTGGGTGGCCRAARAAYCA-3′, Ivanova 2007). et al. 2006). Internal primer pairs (mLepF1 5′-GCTTTCC- CACGAATAAATAATA-3′, Hajibabaei et al. 2005; LepRI Saturation and LepF1 ANTMR1-d, Smith et al. 2006) were employed to generate shorter, overlapping sequences in some cases. Increasing genetic distance not reflected by an increase in A very small amplicon (~160 bp) could also be generated the number of mutational differences, or saturation, of the using LepF1 and C113Rdeg (5′-GGYATWACTATRAAR- CO1 barcode region, was tested by plotting the estimated AARATTAT-3′, Hajibabaei et al. 2006). Polymerase chain number of transitions and transversions against genetic reactions (PCRs) were carried out in 96 well plates in 12.5 μL divergence [Kimura 2-parameter (K2P); Kimura (1980)] reaction volumes containing: 2.5 mm MgCl2, 5 pmol of each using dambe (version 4.2.13; Xia & Xie 2001). Third codon primer, 20 μm dNTPs, 10 mm Tris HCl (pH 8.3), 50 mm positions and the first two codon positions were tested KCl, 10–20 ng (1–2 μL) of genomic DNA, and 1 U of Taq separately and together.

Identification Sequence divergences were calculated using the K2P distance model, and a neighbour-joining (NJ) tree of distances (Saitou & Nei 1987) was created to provide a graphic representation of the among-species divergences using mega3.1 (Kumar et al. 2004), and bold (Ratnasingham & Hebert 2007). Unless otherwise stated, all genetic distances are corrected.

Intraspecific and interspecific divergence values Divergence values for intra- and interspecific pairwise comparisons were calculated with bold.

Results COI sequences were recovered from 80% (301/377) specimens analysed. Full-length PCR products (> 500 bp) were amplified from 88.7% of these (267). Shorter amplicons (< 500 bp) were recovered from 11.3% (34) of the specimens. Many of the specimens which produced shorter fragments or which failed to amplify had been fixed in formalin or were collected more than 15 years ago.

Primer fit As reported by Vences et al. (2005a), we found that CO1 primer sites were variable among the taxa tested here (Fig. 1): LepF1/VF1-d π = 0.13389, MLepF1 π = 0.17800, LepRI/ VR1-d π = 0.14589. However, we note that specifically at G- C positions, there is less, or no, variability (Fig. 1). As a comparison, values of π for all Lepidoptera CO1 sequences covering the LepF1 and LepRI regions were 0.069 and 0.07, respectively (LepF1–710 sequences, LepRI–1311 sequences; data not shown). Although we tested six primer pairs, it is important to note that by using only one primer pair (LepF1/LepRI) we would have successfully amplified 25 species from six families; while the combination of two pairs (LepF1/LepRI and VF1/VR1d) would have yielded all 34 species from 11 genera and six families. Trace files for all amplifications are available in the ‘CO1 Barcoding Amphibians’ file in the Completed Projects section of the Barcode of Life website (www.barcodinglife.org).

Fig. 1 Primer site variation with commonly used barcode primers Panels A–D were made with 83 CO1 profiles from GenBank. The and amphibians. Variation in primer sites for four full length primer pair MLepF1 (E) and LepRI is a minibarcode designed for (~650 bp) barcode primer pair combinations. LepF1 (panel A) and insects (Hajibabaei et al. 2006). Panel E is made from 430 sequences LepRI (panel C) were designed for insects and Lepidoptera generated in this work and from GenBank. For both VR1-d and specifically. VF1-d (panel B) and VR1-d (panel D) are a degenerate LepRI, the reverse compliment of the primer is shown. Note the primer pair designed for use with vertebrates (Ivanova et al. 2006). reduced y-axis scale in panel A and B.

Fig. 2 Transition and transversions plotted against the pairwise sequence divergence [Kimura 2-parameter (K2P) distance] for amphibians using 650 bp of the 5′ end of the cytochrome c oxidase (CO1) barcode. All three positions used. Qualitatively, saturation appears evident at 10–13% sequence divergence with departure from an x–y relationship.

Saturation Discussion The 5′-CO1 fragment is highly variable across the taxa we In our preliminary sampling of Holarctic amphibians, we tested. For all 483 sequences tested, the average nucleotide found that a single mitochondrial gene DNA barcode cor- diversity (π) was 0.20343. Saturation was qualitatively evident rectly identified 94% of species. Amplicons for the 5′ CO1 at 12–13% when all codon positions were compared (Fig. 2). region were straightforward to generate using standard primers designed for insects and vertebrates — we experienced no more difficulty with amphibian samples Identification than with insects (Hebert et al. 2004a; Smith et al. 2005), fish The CO1 barcode correctly identified 94% (37 of 39) of the (Ward et al. 2005) birds (Hebert et al. 2004b), or bats (Clare species in this work (Fig. 3). Identification failures occurred et al. 2007). Specific problems with amplification were where the CO1 barcode identified Bufo fowleri specimens frequently solved through the design of degenerate from Canadian edge of range populations as B. americanus. oligonucleotides, or the use of M13-tailed primers. In the Previous analysis suggests that these populations are fixed Holarctic species represented in our sampling, any overlap for introgressive mitochondrial DNA (mtDNA) hybrids between intra- and interspecific variation was due to (see Smith & Green 2004). hybridization (Bufo fowleri, B. americanus — Smith & Green Deep divisions were evident within the Eurasian salaman- 2004), complex genetics (gynogenesis of the Ambystoma ders included here. Triturus (Mesotriton) alpestris alpestris laterale–jeffersonium complex; Bogart et al. 2007) or in- collected in Serbia Montenegro and Western Ukraine, complete taxonomy (Triturus salamanders, e.g. García-París respectively, were divergent and Triturus (Ommatotriton) et al. 2004a), not reflecting deep intraspecific divergences. vittatus vitattus was found to contain two distinct geno- Estimating rates of hybridization specific to a taxonomic types between Israel and southern Turkey. groups is a challenge (Mallet 2005); however, there is no Individuals of the Ambystoma laterale–jeffersonium com- reason to expect that hybridization will affect barcode plex exhibited extremely large (9–14%) pairwise sequence identification of species more frequently with amphibians divergences. (Kraus & Miyamato 1990; Hedges et al. 1992; than for other vertebrate groups [6–9% rates of hybrid- Spolsky et al. 1992; Bogart & Klemens 1997; Bogart et al. 2007). ization for fish and birds even with variation in life history, sex determination, etc. (Mallet 2005), and just over 21% of surveyed amphibian species were found to be polyphyletic Intraspecific and interspecific divergence values by Funk & Omland (2003), while 16 and 17% of bird and Intraspecific pairwise divergences were beneath 2% with three mammal species, respectively, were polypyletic]. However, notable exceptions: comparisons within B. fowleri (Fig. 4-Aa), rates of species discovery within amphibians are expected T. (Mesotriton) alpestris, and T. (Ommatotriton) vittatus (Fig. 4- to be ‘explosive’ (Kohler et al. 2005; Fouquet et al. 2006; Ab) and for the A. laterale–jeffersonium complex (Fig. 4-Ac). Frost et al. 2006). If a sequence threshold value alone was Inter-specific divergences varied greatly between genera. used for species discovery, then large intraspecific diver- Within Rana, for instance, values ranged from 4 to 8%, while gences would confound decisions. However, such issues within Triturus (sensu lato) values ranged from 4 to 20%. are also likely not specific to amphibians (Clare et al. 2007),

Fig. 3 NJ tree for 200 amphibian specimens.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 240 DNA BARCODING and we feel that a multidimensional approach, involving stricto, Lissotriton, Mesotriton and Ommatotriton). The latter morphology and a multigene approach, will always be species was previously grouped in one genus together required to formally describe new species following the with large-bodied newts of Triturus cristatus–T. marmoratus discovery of provisional species using a DNA barcode. species groups (Titus et al. 1995; Larson et al. 2003; García- In the end, any problems with the utility of using a single París et al. 2004b; Carranza & Amat 2005), but our data mitochondrial gene for species identification will not be support its position within a monotypic clade of Ommatotriton. specific to amphibians, and we therefore conclude that These results are in line with data on morphology, allozymes there is more hope for a CO1 barcode’s use for amphibians and cytochrome b sequences of mtDNA (Litvinchuk et al. than has been reported to date. Indeed, the conclusions of 2005; Steinfartz et al. 2006) and contradict some other Vences et al. (2005a, b), while stressing the utility of 16S (an previously published mtDNA data (Zajc & Arntzen 2000). mtDNA gene for which there are currently about an order Recently published extensive mtDNA phylogenies for of magnitude more GenBank sequences than CO1), were Salamandridae suggest paraphyly (Weisrock et al. 2005, not entirely negative regarding the use of CO1 as a DNA 2006) or polyphyly (Steinfartz et al. 2006) of Triturus newts. barcode. Unfortunately, and importantly, the subsequent The deep divergence shown between different Triturus reporting and referencing of their results has focused on lineages and their affinities with other representatives of communicating that amphibians are a problem group for Eurasian newts (Neurergus, Euproctus and Calotriton) support CO1 DNA barcoding (Cowan et al. 2006; Godfray 2006; the generic status of Triturus lineages. CO1 data gives a Rubinoff et al. 2006; Clare et al. 2007; Little & Stevenson good resolution for identifying these genera, all studied 2007; Waugh 2007). Based on our Holarctic data set, we Triturus species were grouped in four monophyletic clades. suggest that amphibians are more frequently discussed However, CO1 fragment saturation prevents the use of this as a problem group for CO1 DNA barcoding than is marker for resolving phylogenetic interrelationships between warranted by the data. genera. Many other investigations have demonstrated that there are certain Triturus species groups that consist of deeply Primer and barcode variation differentiated lineages (e.g. T. vittatus (Poyarkov 2005; Universal, or near universal, primers may be an important Steinfartz et al. 2006), T. alpestris (Alcobendas et al. 2003), T. component of an envisioned ‘hand-held barcoder’ (Janzen vulgaris (Babik et al. 2005), T. marmoratus (García-París et al. 2004). If variation in amphibian mtDNA was sufficiently 2004a) and T. cristatus (Wallis & Arntzen 1989; Steinfartz high to preclude the use of universal primers for CO1, this et al. 2006)). The taxonomic status of these lineages is chal- would reduce the likelihood of amphibians being included lenging to any operational species identification technique. in a global push towards a DNA barcode for every animal In cases of parapatric distributions gene flow in the contact species (Marshall 2005). However, we found that a primer zone could be tested which would allow the measurement pair designed for insects and a degenerate pair designed of gene flow and a more in depth evaluation of the taxo- for vertebrates amplified the vast majority of the speci- nomic status (García-París et al. 2004a; Wallis & Arntzen mens tested here — 34 species from six families. This may 1989). However, if distributions are allopatric the situation have to do with the important G-C positions at the 3′ end becomes more difficult (Poyarkov 2005). Moreover, genetic of the primer (where for instance in the forward primers differentiation in newts may not coincide with morpho- tested here, there is no variability (Fig. 1)). G-C pairs are very logy (Babik et al. 2005). The deep divergence evident in the stable as they form three hydrogen bonds, and therefore barcodes of these Triturus species are worthy of further annealing efficiency is increased by including a double examination and are likely evidence of cryptic species — and G-C position at the 3′ end of an oligonucleotide. therefore, any barcode ‘misidentifications’ are indicative of incomplete taxonomy and not a failure of the approach, or the gene region. Identification and intraspecific divergences: case studies Within the group of crested newts (T. cristatus, T. dobro- with three difficult groups gicus, T. carnifex and T. karelinii), all four species form Triturus. The CO1 barcode provides further support for the well resolved monophyletic clades. Some specimen with paraphyly of European newts, the genus Triturus (Titus & intermediate T. cristatus–T. dobrogicus morphologies from the Larson 1995 Larson et al. 2003). Recently, splitting of this contact zone area in Western Ukraine had only T. dobrogicus genus into three genera, Mesotriton, Lissotriton and Triturus mtDNA indicating the maternal parent of this likely hybrid (Titus et al. 1995; Larson et al. 2003; García-París et al. 2004b; (see also Wallis & Arntzen 1989). Carranza & Amat 2005), was proposed. CO1 data indicate In this study, two species were shown to be deeply diver- deep divergence between four Triturus evolutionary lineages gent. Ommatotriton vittatus was considered to represent (large-bodied Triturus, small-bodied Triturus, T. alpestris two allopatric species — O. vittatus in the southern and O. and T. vittatus) of probably generic status (Triturus sensu ophryticus in the northern part of the range (Litvinchuk

Fig. 4 Intra- (A) and interspecific (B) pairwise divergences [Kimura 2-parameter (K2P)] calculated with bold (3058 pairwise intraspecific and 10061 intrageneric comparisons). Pairwise comparisons within Bufo fowleri are in white, within Triturus (Mesotriton) alpestris and Triturus (Ommatotriton) vittatus are in grey. Interspecific comparisons in panel B in white contain both Bufo and Triturus comparisons.

et al. 2005) — with four subspecies of variable morphology tion to T. vittatus cilicensis from Cilicia of Turkey. This is not (see also Arntzen & Olgun 2000). The question of their tax- surprising as the variability of southern T. vittatus populations onomic status remains unresolved. Previous studies found is poorly studied and our data might indicate presence of deep intraspecific divergences (6–11%) between different an unrecognized subspecies in the area. T. vittatus populations, suggesting ancient split 16–18 million In the monotypic genus of Mesotriton, we have also found years ago (Poyarkov 2005; Steinfartz et al. 2006) most likely deep divergence in barcode sequences. T. (Mesotriton) alpestris caused by Tethys dynamics and Taurus mountains forma- is morphologically uniform species with trans-European tion in Asia Minor. Intraspecific differentiation within these distribution and consist of several subspecies, some of two lineages differs in the south and the north. The northern them are known as ‘one-lake subspecies’ found only in form contains two subspecies (T. (Ommatotriton) vittatus local lakes. The nominative form T. alpestris alpestris has the nesterovi and T. (Ommatotriton) vittatus ophryticus); how- largest range from Western to Central Europe and south- ever, they form one clade with poor differentiation within, wards to the Balkans. According to the CO1 barcode data, which might indicate relatively recent dispersal in the there are two main clades within T. alpestris. One of them northern part of the range. However, deep divergences joins populations from the western part of the distribution, were found in southern populations of T. vittatus vittatus. and the other from the southeast (which supports the Populations here formed two well-defined clades for Israel preliminary results of Alcobendas et al. 2003). These clades and southern Turkey which seem to be paraphyletic in rela- probably correspond to Iberian and Balkan landmasses

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 242 DNA BARCODING which were separated several times during Oligocene and which could result in the quick fixation of the maternal Miocene and later acted as glacial refugia during Pleistocene mtDNA of the hybridizing species pair has not, as at Long glaciations. After the glaciations, T. alpestris most likely Point, occurred at this location. If true, this suggests that spread from the southern refugia to Central and Western populations within the Niagara phylogroup are not as well Europe following two main routes of recolonization pro- connected as we had previously thought. Alternatively, the posed for European biota (Hewitt 1999) — from Iberia and occurrence of the B. fowleri mtDNA signature at Nickel from the Balkans. The nominative subspecies T. alpestris Beach might be due to human-mediated translocation. B. alpestris is accordingly polyphyletic in relation to southern fowleri is a COSEWIC-listed species in Canada, and therefore forms (T. alpestris cyreni in the west and T. alpestris reiseri in its breeding sites are federally protected. In nearby Fort the east). CO1 data do not support validity of one-lake sub- Erie, recent shoreline developments have been delayed species T. alpestris serdarus and T. alpestris piperianus as they and altered due to the presence of breeding populations are in one clade with surrounding populations of T. alpestris (Robbins 2002). Additionally, provincial officials have had alpestris. T. alpestris is an interesting example of discordance reason to suspect individuals of importing Fowler’s toads between mtDNA and morphological data, we found sig- from other locations in order to impede shoreline develop- nificant CO1 sequences divergence of morphologically ments (OMNR official, personal communication). similar populations. Ambystoma. Unisexual Ambystoma salamanders of the Bufo/Anaxyrus. In 2004, Smith and Green tested phylo- laterale–jeffersonium complex have a complex reproductive geographic relations amongst all known Canadian popu- system where bisexual parents can combine to produce lations of Bufo (Anaxyrus) fowleri using the hypervariable offspring which have an mtDNA signature unrelated to control region of the mitochondria. They found shallow either parent (Hedges et al. 1992). These independent clonal phylogeographic structuring approximately coincident lineages can be as much as 5% divergent from either of their with lake-basin structure and a deep split between the closest possible ancestors (Spolsky et al. 1992). Nuclear entire lake and one population at Long Point Ontario. combinations between the four bisexual species in the They concluded that this deep split was a result of ancient complex (A. jeffersonium, A. laterale, A. texanum, and A. introgression of B. (Anaxyrus) americanus mitochondrion tigrinum) can range across 20 different combinations from into all Canadian populations outside of Long Point. We diploid to pentaploid (Bi & Bogart 2006; Bogart et al. 2007). were therefore not surprised to find here that, except for It is not, therefore surprising that a single mitochondrial individuals from the population at Long Point, a CO1 gene barcode was unable to unambiguously identify the mtDNA barcode misidentifies Canadian B. fowleri as B. Ambystoma salamanders in our collections. However, it americanus. From strictly an identification perspective, would allow the differentiation between A. maculatum and we feel that this sort of misidentification is of small A. laterale–jeffersonium tadpoles or embryos (data not shown), consequence — an mtDNA barcode of two hybridizing species which co-occur in this area. The Ambystoma genetic species takes identification from certainly over a million system is highly intricate (Bogart et al. 2007), and as yet, species to a choice between two. For instance, if one were no single method of species identification can reliably interested in identifying a digested mass in a snake gut differentiate members of this complex. along the Lake Erie shoreline — the barcode approach would In conclusion, we found that a CO1 DNA barcode cor- here winnow your choices down to two species instead of rectly identified 94% of amphibian species tested and cases one, while secondary genetic marker would allow you to of intra- and interspecific pairwise divergences overlap select between these two choices. Utilizing this emerging were a result of unresolved taxonomy. An important caveat molecular database reduces your search from ‘digested we wish to add here is that making conclusions regarding vertebrate’ to either B. fowleri or B. americanus. intra- or interspecific variation based on a single mitochon- Our barcode analysis of B. fowleri included one popula- drial locus assumes sampling from the largest effective tion not included in Smith & Green (2004). Geographical population size could be interpreted as an upper bound location and the genetic identity of all neighbouring popu- to intraspecific divergence. However, because of the wide lations predicted that toads from Nickel Beach would be variance around coalescence times in mtDNA (Hudson & introgressed B. americanus mtDNA (the site is located in the Turelli 2003), this may not be true. The CO1 barcode failed middle of the Niagara phylogroup discovered using control the test of species identification, as would any mitochondrial region mtDNA — Fig. 5). However, Nickel Beach toad CO1 gene, where the specimen in question was suspected to be barcodes are of B. fowleri (i.e. identical to Long Point toads the result of introgressive hybridization (Bufonidae, Smith and unlike neighbouring populations, Fig. 5B). This sur- & Green 2004). Primer sites were variable, but amplifica- prising finding may be explained by one of two hypotheses: tion using standard and degenerate oligonucleotides was the rare coincidence of hybridization and population resilient across all six families tested. Indeed, one of the bottlenecking speculated upon by Smith & Green (2004), most successful primer pairs used here (LepF1/LepRI) was

Fig. 5 Neighbour-joining tree and map illustrating the phylogeographic structuring of Bufo fowleri CO1 DNA barcodes. (A) Red text identifies B. fowleri populations discussed in the text. Individual specimens are identified by their collection locality and bold process numbers. (B) Map of Lake Erie where coloured shapes correspond to mitochondrial phylogroups identified in Smith & Green (2004). Black circles are Niagara phylogroup 1a, yellow circle is Presque Isle phylogroup 1b, blue circles are phylogroup 1c and red circles are Long Point phylogroup 2b. Our working hypothesis is that phylogroup 1 corresponds to Bufo americanus mtDNA and is a result of an ancient introgression. Phylogroup 2 corresponds to B. fowleri mtDNA. Potential hypotheses to explain this new phylogroup 2 population within the Niagara phylogroup 1 populations are expanded upon in the text.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 244 DNA BARCODING designed for lepidopterans (Hebert et al. 2004a). In the end, Bogart JP, Klemens MW (1997) Hybrids and genetic interactions we conclude that, although there will undoubtedly be dif- of mole salamanders (Ambystoma jeffersonianum, and A. laterale) ficulties (as in the family Mantellidae; Vences et al. 2005a), (Amphibia: Caudata) in New York and New England. American Museum Novitates, 3218, 1–78. our data set suggests that on the class level, we should not Carranza S, Amat F (2005) Taxonomy, biogeography and evolution approach this group with an a priori expectation that they of Euproctus (Amphibia: Salamandridae), with the resurrection will be larger or more serious, than for any other taxonomic of the genus Calotriton and the description of a new endemic group of animals. Furthermore, these difficulties will species from the Iberian Peninsula. Zoological Journal of the Linnean expose questions for further scientific inquiry. It is our con- Society, 145, 555–582. clusion that it is far more advantageous that amphibians Clare EL, Kim BK, Engstrom MD, Eger JL, Hebert PDN (2007) are included in the global effort to utilize a standardized DNA barcoding of Neotropical bats: species identification and discovery within Guyana. Molecular Ecology Notes, 7, 184– gene region for initial species recognition than to exclude 190. them because we wish for more evidence. Cowan RS, Chase MW, Kress WJ, Savolainen V (2006) 300 000 species to identify: problems, progress, and prospects in DNA Acknowledgements barcoding of land plants. Taxon, 55, 611–616. Fouquet A, Vences M, Salducci M-D et al. (2007) Revealing cryptic This research was supported by funding from the Canada Foun- diversity using molecular phylogenetics and phylogeography dation for Innovation, Genome Canada, Ontario Innovation Trust, in frogs of the Scinax ruber and Rhinella margaritifer species groups. NSERC and the Gordon and Betty Moore Foundation to P.D.N.H. Molecular Phylogenetics and Evolution, 43, 567–582. and a Fonds québécois de la recherche sur la nature et les techno- Frost DR, Grant T, Faivovich J et al. (2006) The amphibian tree of life. logies B3 postdoctoral fellowship to M.A.S. We thank the students Bulletin of the American Museum of Natural History, 297, 1–376. in the Ontario Universities Program in Field Biology course ‘Ecology Funk DJ, Omland KE (2003) Species-level paraphyly and of the Kawarthas’ 2004–07 for their enthusiasm for amphibian polyphyly: frequency, causes, and consequences, with insights collection and identification, and also thank the James McLean from animal mitochondrial. DNA, 34, 397–423. Oliver Ecological Centre for permission to conduct research on García-París M, Arano B, Herrero P (2004a) Molecular charac- their property. We are grateful to many colleagues at Guelph, espe- terization of the contact zone between Triturus pygmaeus and cially Sujeevan Ratnasingham, Pia Marquardt, Greg Downs and T. marmoratus (Caudata: Salamandridae) in Central Spain and Rob Dooh who provided valuable IT support and Alex Borisenko, their taxonomic assessment. Revista Española de Herpetologia, 15, Jeremy deWaard, Angela Holliss, Nataly Ivanova and Taika von 115–126. Königslöw for their advice and role in analysis, David Green for García-París M, Montori A, Herrero P (2004b) Amphibia, Lissamphibia. assistance in the preparation of Fig. 4 and Anne Yaggi for speci- Museo de Ciencias Naturales, Madrid. men tissue. This manuscript benefited from the comments of Dirk Godfray HCJ (2006) To boldly sequence. 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