Wolbachia Infections in Bees (Anthophila) and Possible Implications for DNA Barcoding

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/233329088

Wolbachia infections in bees (Anthophila) and possible implications for DNA barcoding

Article in Systematics and Biodiversity · December 2011 DOI: 10.1080/14772000.2011.627953

CITATIONS READS 21 254

3 authors, including:

Michael Gerth Christoph Bleidorn University of Liverpool Georg-August-Universität Göttingen

48 PUBLICATIONS 150 CITATIONS 123 PUBLICATIONS 2,303 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Platyzoa View project

All content following this page was uploaded by Michael Gerth on 16 September 2014.

The user has requested enhancement of the downloaded file. This article was downloaded by: [University of Leipzig] On: 16 September 2014, At: 06:02 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Systematics and Biodiversity Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tsab20 Wolbachia infections in bees (Anthophila) and possible implications for DNA barcoding MICHAEL GERTH a , ANNEMARIE GEIßLER a & CHRISTOPH BLEIDORN a a Molecular Evolution and Systematics of Animals, Institute for Biology , University of Leipzig , Talstr. 33, D-04103, Leipzig, Germany Published online: 25 Nov 2011.

To cite this article: MICHAEL GERTH , ANNEMARIE GEIßLER & CHRISTOPH BLEIDORN (2011) Wolbachia infections in bees (Anthophila) and possible implications for DNA barcoding, Systematics and Biodiversity, 9:4, 319-327, DOI: 10.1080/14772000.2011.627953 To link to this article: http://dx.doi.org/10.1080/14772000.2011.627953

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions Systematics and Biodiversity (2011), 9(4): 319–327

Perspective Wolbachia infections in bees (Anthophila) and possible implications for DNA barcoding

MICHAEL GERTH, ANNEMARIE GEIßLER & CHRISTOPH BLEIDORN Molecular Evolution and Systematics of Animals, Institute for Biology, University of Leipzig, Talstr. 33, D-04103 Leipzig, Germany (Received 15 February 2011; revised 24 August 2011; accepted 25 August 2011)

The widespread intracellular bacterium Wolbachia is transmitted exclusively maternally and alters the reproduction of its hosts by different mechanisms. Thereby, inheritance patterns of mitochondrial genomes are modified, possibly confining interpretations of mitochondrial sequence data. Although this phenomenon has been reported before, its conclusions seem to be widely ignored. In the light of recent large-scale barcoding projects relying solely on mitochondrial cox1 sequences, we screened the native German bee fauna (Anthophila) for Wolbachia infections. The screening revealed that 66% of the native German bees and 54% of sphecid wasps are infected by Wolbachia. Many species bore identical or similar infections, suggesting a high rate of horizontal transfer. Supergroup A infections were recovered in most cases; only one species bore a super-group F Wolbachia infection. Because Wolbachia is not only present in 66% of bees but also in the majority of arthropod species, we argue that studies interpreting sequence data of arthropod species cannot rely on mitochondrial data alone – nuclear markers must be incorporated. DNA barcoding using only mitochondrial cox1 will not be sufficient to delimit, identify or discover Wolbachia-infected species, i.e. probably the majority of all animal species. Key words: bees (Anthophila), DNA barcoding, molecular taxonomy, sphecid wasps, Wolbachia

Introduction mitochondrial cytochrome oxidase subunit I gene (cox1)is Bees (Anthophila) are a group of aculeate Hymenoptera used as a barcode (Hebert et al., 2003). The backbone of which include over 16,000 described species (Michener, the initiative is a database including sequence and voucher- 2000). The monophyly of bees is well established based on ing information for curated specimens (Ratnasingham and molecular and morphological characters, however, ingroup Hebert, 2007). Within this initiative, different campaigns relationships are still a matter of ongoing debate (Danforth aim to facilitate massive DNA barcoding for selected an- et al., 2006). Many interesting evolutionary phenomena imal taxa or to barcode the complete fauna of certain ge- have been reported from bees, including evolution of euso- ographic regions. In the case of bees, a ‘Bee Barcode of ciality (Andersson, 1984), cleptoparasitism (Cardinal et al., Life Initiative’ (http://www.bee-bol.org/) has been brought Downloaded by [University of Leipzig] at 06:02 16 September 2014 2010) and coevolution with flowering plants (Johnson to life. As a first step, the bee fauna of Nova Scotia (Canada) & Steiner, 2000). Besides these, bees are known for their has already been extensively barcoded (Sheffield et al., important role as pollinators and as such they contribute 2009). Bees are also flagship species of the Barcoding Fauna to an immense part of the worldwide human food supply Bavarica campaign (http://www.faunabavarica.de/). (Greenleaf & Kremen, 2006). This makes research on bee A central dogma of DNA barcoding is the postulation of a biodiversity, conservation and autecology economically im- ‘barcoding gap’ which means that intraspecific genetic vari- portant topics (Losey & Vaughan, 2006). ability of mitochondrial cox1 is significantly lower than its Unsurprisingly, bees became a major target of the Bar- interspecific variability (Hebert et al., 2003, 2004b). The code of Life initiative (http://www.barcodeoflife.org/). The existence of such a ‘barcoding gap’ has repeatedly been aim of this initiative is to provide highly standardized ge- questioned in studies on different animal groups (Meyer netic marker systems for species identification (Golding & Paulay, 2005; Meier et al., 2006; Wiemers & Fiedler, et al., 2009). In the case of animals, a 648 bp region of the 2007) and for theoretical reasons (Hickerson et al., 2006). For some insects, this gap does not seem to exist at all Correspondence to: Michael Gerth. E-mail: michael.gerth@uni- (Virgilio et al., 2010). Moreover, the reliance on a single mi- leipzig.de tochondrial marker has been regarded as not adequate due to

ISSN 1477-2000 print / 1478-0933 online C 2011 The Natural History Museum http://dx.doi.org/10.1080/14772000.2011.627953 320 M. Gerth et al.

reduced effective population size, introgression, maternal Wenseleers et al., 1998; Russell et al., 2009; Lachowska inheritance, inconsistent mutation rate, pseudogenization et al., 2010). and heteroplasmy (Rubinoff et al., 2006; Song et al., 2008; In our study we simply asked: How prevalent are Wol- Galtier et al., 2009; Magnacca & Brown, 2010). Further- bachia infections in the native German bee fauna? We draw more, the presence of cytoplasmic bacteria can dramatically general conclusions for barcoding studies (for which this alter inheritance patterns of mitochondrial genes (Hurst & bee fauna is a major target) and suggest that it is critical to Jiggins, 2005). include nuclear markers in future DNA barcoding studies There are different lineages of symbionts that are capa- of arthropods. ble of inducing such effects, including Cardinium, Rick- ettsia, Spiroplasma (e.g. Duron et al., 2008; Perlman et al., 2008; Weinert et al., 2009) and, probably most widespread, Materials and methods Wolbachia. This genus of intracellular α-proteobacteria is All specimens were collected between August 2009 and found in arthropods and nematodes. Since its first discov- July 2010. The animals were captured with hand nets or ery in ovaries of Culex pipiens in 1924 (Hertig & Wolbach, snap-cap vials in the vicinity of their nests, their host’s 1924), numerous strains were found across many insect or- nest or on their pollen source. To cover a broad range of ders as well as in isopods, amphipods, ostracods, arachnids taxa, sampling took place at different times of the year and and filarial nematodes (Ros et al., 2009). The high inter- at various locations in Germany. Altogether, 126 bees (75 est in and increasing number of studies about Wolbachia species) and 16 sphecid wasps (13 species) were collected is due to its ability to alter the reproduction of its hosts. (Tables 1 and 2). DNA was isolated from flight muscle tissue This is achieved by four mechanisms: (1) feminization of dissected from the thorax. The remains of the animals were genotypic males, (2) induction of parthenogenesis, (3) male stored as vouchers in 96% ethanol. DNA extraction was killing and (4) cytoplasmic incompatibility (CI) between in- performed with NucleoSpinR Tissue Kit (Machery-Nagel) fected males and uninfected females (Werren et al., 2008). following the manufacturer’s protocol. All of those mechanisms result in the accelerated spread of All PCRs were carried out in a total volume of 20 µl, con- Wolbachia throughout the population. Since the infection taining 1 µl of ‘forward’ and 1 µl of ‘reverse’ primer, 0.5 µl is transmitted exclusively maternally, there is positive se- of each dNTP (2mM), 2.5 µlof10× DreamTaqTMGreen lection acting on mitochondrial DNA (mtDNA) of infected Buffer, 0.1 µl of DreamTaqTMGreen DNA Polymerase, 1 µl females, resulting in selective sweeps and consequently ho- of genomic DNA and 12.4 µl of ddH2O. Primers ampli- mogenized mitochondrial haplotypes. Wolbachia-induced fying Wolbachia gene fragments wsp and ftsz were taken selective sweeps can spread through closely related popula- from Jeyaprakash and Hoy (2000), using the PCR conditions or species via hybridization events, leading to misin- tions described therein. For the endosymbiont of Osmia terpretation of data based on mitochondrial DNA (Jiggins, caerulescens, amplification of the Wolbachia MLST genes 2003). Recently, it has been shown that Wolbachia may gatB, coxA, hcpA and fbpA was achieved using the primers also be beneficial to their host by increasing its fertility and PCR conditions from Baldo et al. (2006). Negative (Vavre et al., 1999; Weeks et al., 2007), acting as a nutri- controls containing water instead of template DNA were tional mutualist (Brownlie et al., 2009; Hosokawa et al., used in all PCR reactions. DNA sequencing took place on 2010) or by increasing the host’s resistance against certain an ABI PRISMR 3100 Genetic Analyzer (Applied Biosys- viruses (Hedges et al., 2008; Teixeira et al., 2008). Those tems). All sequences were submitted to NCBI GenBank mechanisms offer another explanation for the rapid spread Downloaded by [University of Leipzig] at 06:02 16 September 2014 under accession numbers JN639215–JN639287. of Wolbachia and its associated mitochondrial haplotype throughout host populations without alterations to their reproduction. Wolbachia infections are estimated to be widespread Results and discussion among arthropods with infection rates ranging from 20% Altogether, we tested 74 bee species and 13 sphecid wasp (Werren et al., 1995) to 76% (Jeyaprakash & Hoy, 2000). A species for the presence of Wolbachia infections by using meta-analysis based on available published data estimated two bacterial genes (wsp and ftsz). We found Wolbachia that 66% of all arthropod species are Wolbachia infected to be present in 49 of the tested bee species (66%) and and that the infection rate within those species is generally in six sphecid wasp species (46%) (Tables 1 and 2). The very high (>90%) or very low (<10%) (Hilgenboecker screening thus covered 16% of the 560 native German bees et al., 2008). However, these theoretical predictions were (Westrich et al., 2008). The coverage of sphecid wasps was based mainly on studies in which few species from se- not as representative: 5% of the 251 native German sphecid lected higher-ranked arthropod taxa (e.g. ‘insect orders’) wasp species were covered (Blosch,¨ 2000). All infections had been investigated. Detailed studies estimating infection were verified by sequencing the bacterial gene fragments. rates for lower-ranked taxa are rare (Bouchon et al., 1998; Of 41 ftsz sequences from bee and sphecid wasp hosts, 31 Wolbachia infections in bees (Anthophila) 321

Table 1. List of bee (Anthophila) species tested for Wolbachia. Infections are indicated by +, double infections by ++.

Taxon Wolbachia infection No. of individuals tested/infected with Wolbachia

Colletidae Colletinae Colletes cunicularius (LINNAEUS 1761) + 1/1 Colletes daviesanus SMITH 1846 + 1/1 Hylaeinae Hylaeus signatus (SCHENCK 1853) + 2/2 Andrenidae Andreninae Andrena barbilabris (KIRBY 1802) + 1/1 Andrena florea FABRICIUS 1793 + 2/2 Andrena haemorrhoa (FABRICIUS 1781) − 2/0 Andrena hattorfiana (FABRICIUS 1775) + 2/2 Andrena labiata FABRICIUS 1781 − 2/0 Andrena nigroaenea (KIRBY 1802) + 1/1 Andrena pilipes/nigrospina∗ + 2/2 Andrena nitida (MULLER¨ 1776) + 1/1 Andrena ovatula (KIRBY 1802) + 1/1 Andrena pandellei PEREZ´ 1895 + 2/2 Andrena proxima (KIRBY 1802) − 1/0 Andrena ventralis IMHOFF 1832 + 1/1 Andrena viridescens VIERECK 1916 ++ 2/2 Andrena wilkella (KIRBY 1802) + 1/1 Panurginae Panurgus calcaratus (SCOPOLI 1763) + 2/2 Halictidae Rophitinae Rophites quinquespinosus SPINOLA 1808 ++ 2/2 Rhophitoides canus (EVERSMANN 1852) + 2/2 Halictinae Halictus maculatus SMITH 1848 − 1/0 Halictus quadricinctus (FABRICIUS 1776) + 1/1 Halictus scabiosae (ROSSI 1790) + 1/1 Halictus subauratus (ROSSI 1792) + 1/1 Lasioglossum nitidulum (FABRICIUS 1804) + 1/1 Lasioglossum pauxillum (SCHENCK 1853) + 2/2 Lasioglossum sexnotatum (KIRBY 1802) + 1/1 Sphecodes albilabris (FABRICIUS 1793) + 2/2 Sphecodes crassus THOMSON 1870 + 1/1 Sphecodes ephippius (LINNAEUS 1767) + 1/1 Specodes gibbus (LINNAEUS 1758) + 1/1 Sphecodes pellucidus SMITH 1845 + 7/7 Sphecodes puncticeps THOMSON 1870 + 1/1 Downloaded by [University of Leipzig] at 06:02 16 September 2014 Melittidae Melittinae Macropis europaea WARNCKE 1973 + 1/1 Melitta haemorrhoidalis (FABRICIUS 1775) ++ 1/1 Melitta leporina (PANZER 1799) + 1/1 Megachilidae Megachilinae Osmia aurulenta (PANZER 1799) − 2/0 Osmia bicornis (LINNAEUS 1758) ++ 6/2 Osmia caerulescens (LINNAEUS 1758) + 2/2 Osmia campanularum (KIRBY 1802) − 1/0 Osmia claviventris THOMSON 1872 − 2/0 Osmia florisomne (LINNAEUS 1758) ++ 2/2 Osmia rapunculi (LEPELETIER 1841) − 1/0 Osmia truncorum (LINNAEUS 1758) − 2/0 Anthidium manicatum (LINNAEUS 1758) − 1/0 Anthidium punctatum LATREILLE 1809 − 1/0 Anthidium strigatum (PANZER 1805) − 1/0 (Continued on next page) 322 M. Gerth et al.

Table 1. (Continued)

Taxon Wolbachia infection No. of individuals tested/infected with Wolbachia

Stelis punctulatissima (KIRBY 1802) − 1/1 Coelioxys aurolimbata FORSTER¨ 1853 ++ 1/1 Coelioxys conica (LINNAEUS 1758) − 2/0 Coelioxys conoidea (ILLIGER 1806) − 1/0 Megachile circumcincta (KIRBY 1802) + 1/1 Megachile ericetorum LEPELETIER 1841 − 1/0 Megachile maritima (KIRBY 1802) − 1/0 Megachile pilidens ALFKEN 1924 − 1/0 Megachile rotundata (FABRICIUS 1787) − 1/0 Megachile versicolor SMITH 1844 ++ 1/1 Apidae Nomadinae Nomada alboguttata HERRICH-SCHAFER¨ 1839 + 9/9 Nomada ﬂavopicta (KIRBY 1802) ++ 1/1 Nomada fucata PANZER 1798 − 2/0 Nomada goodeniana (KIRBY 1802) ++ 5/5 Nomada ruﬁcornis (LINNAEUS 1758) + 1/1 Nomada succincta PANZER 1798 ++ 3/3 Nomada zonata PANZER 1798 + 1/1 Epeolus variegatus (LINNAEUS 1758) ++ 2/2 Apinae Eucera nigrescens PEREZ´ 1879 + 2/2 Anthophora furcata (PANZER 1798) − 1/0 Anthophora plumipes (PALLAS 1772) + 2/2 Anthophora quadrimaculata (PANZER 1798) − 1/0 Melecta luctuosa (SCOPOLI 1770) ++ 2/2 Melecta punctata (FABRICIUS 1775) − 1/0 Bombus hypnorum (LINNAEUS 1758) − 1/0 Bombus pascorum (Scopoli 1763) ++ 1/1 Bombus rupestris (FABRICIUS 1793) − 1/0 74 species 49 (66%) 123 / 88 (72%)

Table 2. List of sphecid wasp species tested for Wolbachia. Infections are indicated by +, double infections by ++.

Taxon Wolbachia infection No. of individuals tested/infected with Wolbachia

Sphecidae Sphecinae Ammophila campestris Latreille 1809 − 1/0 Crabronidae Downloaded by [University of Leipzig] at 06:02 16 September 2014 Astatinae Dinetus pictus (FABRICIUS 1793) − 2/0 Crabroninae Crabro cribrarius (LINNAEUS 1758) − 2/0 Crossocerus quadrimaculatus (FABRICIUS 1793) + 1/1 Ectemnius continuus (FABRICIUS 1894) + 1/1 Lestica alata (PANZER 1797) + 1/1 Trypoxylon sp. LATREILLE 1796 − 1/0 Bembicinae Cerceris rybyensis (LINNAEUS 1771) − 1/1 Gorytes laticinctus (LEPELETIER 1832) + 1/1 Mellinus arvensis (LINNAEUS 1758) ++ 2/2 Nysson spinosus (FORSTER 1771) − 1/0 Nysson trimaculatus (ROSSI 1790) − 1/0 Philanthus triangulum (FABRICIUS 1775) + 1/1 13 species 6 (46%) 16 / 8 (50%) Wolbachia infections in bees (Anthophila) 323

were similar to at least one other sequence. However, only This result is in concordance with the ﬁnding that super- 10 of those Wolbachia strains also showed identical wsp group A infections are common in hymenoptera (e.g. Ros sequences. The evolutionary history of Wolbachia in bees et al., 2009; Russell et al., 2009; Stahlhut et al., 2010). There and sphecid wasps cannot be resolved with these data alone. was, however, one exception: the Wolbachia endosymbiont Nevertheless, there is evidence for a high level of horizon- of Osmia caerulescens belonged to supergroup F, a result tal transfer between the hosts, which seems to be the rule which was veriﬁed by multilocus sequence typing (MLST) in some insect communities (Stahlhut et al., 2010). In 14 (Baldo et al., 2006). Supergroup F is widespread across cases, the presence of multiple infections could be detected many different host taxa, as in various insect orders, in by double peaks in chromatograms (Tables 1 and 2). The scorpions and even in nematodes (Ros et al., 2009). Al- recovered infection frequencies are in concordance with es- though supergroup F infections have been found in many timations from a theoretical meta-analysis (Hilgenboecker different taxa, it is still relatively rare within groups (e.g. et al., 2008). Because wsp is prone to recombination and Russell et al., 2009). Because it is the only Wolbachia thus not ideal for reconstructing phylogenetic relationships strain to be found in arthropods and nematodes, its po- (Jiggins et al., 2001; Baldo et al., 2005), we used ftsz to tential role in horizontal transmission between these needs assign Wolbachia strains or supergroups. In most cases, in further exploration. In bees and sphecid wasps, the effects of supergroup A, Wolbachia infections could be recovered. Wolbachia infections are unknown. However, male killing, Downloaded by [University of Leipzig] at 06:02 16 September 2014

Fig. 1. Wolbachia induced selective sweeps and their consequence for population genetics. Scenario (A) a Wolbachia infection (indicated by arrow) leads to a selective sweep and, consequently, to homogenized mitochondrial haplotypes (depicted as barcodes) across the population. Possible misinterpretations of mitochondrial data from such a population include bottleneck or founder effects. Scenario (B) two Wolbachia infections and subsequent selective sweeps affect one population, leading to two distinct mitochondrial haplotypes. Mitochondrial data suggest limited gene ﬂow or even reproductive barriers. This is one possible scenario for one species bearing two different cox1 barcodes. 324 M. Gerth et al.

parthenogenesis and feminization do not seem likely be- independently, subsequent selective sweeps might generate cause we found infected males as well as infected females a pattern of distinct cox1 barcodes within a single species in many cases. CI is often regarded as the prevailing induced (Fig. 1B). This has been shown for nymphalid butterfly phenotype of Wolbachia, but mutualistic relationships can- species (Jiggins, 2003; Charlat et al., 2009). A phenomenon not be ruled out. which might be an even bigger problem is selective sweeps followed by hybrid introgression into other species (Fig. 2), causing different species to bear identical cox1 barcodes. Implications for DNA barcoding Hybrid introgression seems to be common in arthropods (Ballard, 2000; Funk & Omland, 2003; Gompert et al., The presence of Wolbachia and other intracellular sym- 2008; Galtier et al., 2009; Raychoudhury et al., 2009) and bionts can be regarded as a major challenge for DNA several examples for the confounding effect on DNA bar- barcoding. Wolbachia-induced selective sweeps within in- coding efforts have been shown (Whitworth et al., 2007; fected populations can produce different patterns of haplo- Gompert et al., 2008; Langhoff et al., 2009). All this has type distributions (Fig. 2). Whereas the homogenization of been discussed in the literature before. However, when look- haplotypes (Fig. 1) may even be an advantage for barcoding in actual publications applying DNA barcoding to cer- ing efforts, as it reduces intraspecific variability and may tain groups of insects, these kinds of problems are often enhance existing ‘barcoding gaps’ (Galtier et al., 2009), ignored or seem to be largely misunderstood. For exam- two other possible scenarios are problematic for reliable ple, some recent studies (Linares et al., 2009; Ekrem et al., species identification based on a single cox1 sequence bar- 2010) discuss the problem of conserved primer pairs am- code. Firstly, when populations are infected by two strains plifying bacterial Wolbachia loci instead of mitochondrial genes. This type of error, though, is easily identifiable by simple BLAST searches and is not the main problem with Wolbachia. Frequently, potential Wolbachia-induced problems are simply not addressed. Most insect barcoding studies published so far focused on Lepidoptera disagree in their performance on species identification (Hebert et al., 2004a, 2010; Brower, 2006; Elias et al., 2007). However, when nuclear genes were incorporated in insect barcoding studies, contradictory results between mitochondrial and nuclear data were recovered – potentially as a consequence of Wol- bachia infections (Smith et al., 2007; Whitworth et al., 2007). Given that of the nearly 1 million barcoded specimens in BOLD (Cesari et al., 2009), 75% are arthropods, and given that 66% of the described arthropod species har- bour Wolbachia infections (Hilgenboecker et al., 2008), the potentially confounding effects should be taken seriously. The main goal of DNA barcoding is species (re-) identification, not the discovery of cryptic species. How- ever, mismatches between current taxonomy and DNA Downloaded by [University of Leipzig] at 06:02 16 September 2014 barcoding are usually explained by the presence of as yet undiscovered cryptic species – in many cases without any reference to additional nuclear markers (Hebert et al., 2004a; Burns et al., 2008). The line of argument is usually as follows: (i) take a group of organisms with a well-established taxonomy, (ii) perform a barcoding survey, (iii) estimate success of species identification by comparing with current taxonomy, and (iv) declare all other cases as so far unrecognized cryptic species or ‘bad taxonomy’ (Hebert et al., 2004a; Lukhtanov et al., 2009; Sheffield et al., 2009). Fig. 2. A Wolbachia infection leads to homogenized mitochon- Using this approach, barcoding has proven to be a reliable drial haplotypes in species A as described in Fig. 1. Hybridiza- approach of species identification and discovery (Hebert tion is accompanied by mitochondrial introgression, leading to the spread of the Wolbachia infection and to homogenized mito- et al., 2010). However, a more representative test would chondrial haplotypes in species B. This scenario may explain the be to use groups with difficult taxonomic backgrounds phenomenon of two species sharing one cox1 barcode. and to include additional nuclear markers for comparison. Wolbachia infections in bees (Anthophila) 325

Doing this, the DNA barcoding approach seems to be at Acknowledgements least problematic (Whitworth et al., 2007). We thank public authorities in Brandenburg, North Rhine- Only few barcoding studies on bees or sphecid wasps Westphalia and Saxony for permitting the collection of pro- have been published (Hastings et al., 2008; Gibbs, 2009a, tected insect species. We acknowledge assistance in the 2009b; Sheffield et al., 2009; Droege et al., 2010; laboratory work by Franziska A. Franke, Nicole Liebing Magnacca & Brown, 2010). None of them included addi- and Stefan Schaffer. Christian Venne kindly supported field tional nuclear genes or addressed potential problems with work. Wethank two anonymous reviewers whose comments Wolbachia. In a study on the differentiation of a colletid bee improved the quality of this manuscript. species complex, elongation factor 1-α and ITS2 were revealed as promising nuclear markers, whereas cox1 failed to give any resolution (Kuhlmann et al., 2007). For future stud- References ies we strongly suggest to include those or other additional ANDERSSON, M. 1984. The evolution of eusociality. Annual Review of Ecology and Systematics 15, 165–189. nuclear markers to test the performance of the cox1 marker. BALDO,L.,HOTOPP, J.C.D., JOLLEY, K.A., BORDENSTEIN, S.R., DNA barcoding is regarded as a method to democratize BIBER, S.A., CHOUDHURY, R.R., HAYASHI,C.,MAIDEN, M.C.J., taxonomy in a way that anybody will be able to identify TETTELIN,H.&WERREN, J.H. 2006. Multilocus sequence typ- species from a single gene fragment (Golding et al., 2009; ing system for the endosymbiont Wolbachia pipientis. Applied Packer et al., 2009). This attitude of oversimplification and Environmental Microbiology 72, 7098–7110. BALDO,L.,LO,N.&WERREN, J.H. 2005. Mosaic nature of of scientific problems has been already harshly criticized the Wolbachia surface protein. Journal of Bacteriology 187, by others (Brower, 2010; Ebach & De Carvalho, 2010). 5406–5418. Moreover, given the range of problems when using DNA BALLARD, J.W.O. 2000. When one is not enough: Introgression of barcoding as indicated above (introgression, heteroplasmy, mitochondrial DNA in Drosophila. Molecular Biology and pseudogenization, confounding effects due to Wolbachia Evolution 17, 1126–1130. BLOSCH¨ , M. 2000. Die Grabwespen Deutschlands – Tierw. infections, etc.) it is obvious that still much specialist Deutschlds., 71. Goecke & Evers, Keltern. background knowledge is needed to interpret and apply BOUCHON,D.,RIGAUD,T.&JUCHAULT, P. 1998. Evidence the results of this approach. for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization. Proceedings of the Royal Society of London Series B – Biological Sciences 265, 1081–1090. Conclusions BROWER, A.V.Z. 2006. Problems with DNA barcodes for species delimitation: ‘ten species’ of Astraptes fulgerator reassessed In this study, we do not present any evidence for actual (Lepidoptera: Hesperiidae). Systematics and Biodiversity 4, discordance of mitochondrial and nuclear markers in bees 127–132. and sphecid wasps. However, we believe that given the high BROWER, A.V.Z. 2010. Alleviating the taxonomic impediment of prevalence of Wolbachia in these and many other groups, DNA barcoding and setting a bad precedent: names for ten species of Astraptes fulgerator (Lepidoptera: Hesperiidae: Eu- there is reasonable doubt in the performance of a sin- daminae) with DNA-based diagnoses. Systematics and Bio- gle mitochondrial marker. To prove the reliability of cox1 diversity 8, 485–491. barcodes, these doubts must be resolved, which can only BROWNLIE, J.C., CASS, B.N., RIEGLER,M.,WITSENBURG, J.J., be achieved by thoroughly testing the marker of choice. ITURBE-ORMAETXE,I.,MCGRAW,E.A.&O’NEILL, S.L. 2009. The large datasets of DNA barcoding studies often comprise Evidence for metabolic provisioning by a common invertebrate endosymbiont, Wolbachia pipientis, during periods of many, if not all morphospecies of one taxon or geographic

Downloaded by [University of Leipzig] at 06:02 16 September 2014 nutritional stress. PLoS Pathogens 5, e1000368. region. This kind of extensive sampling is hardly being BURNS, J.M., JANZEN, D.H., HAJIBABAEI,M.,HALLWACHS,W.& achieved in other studies. Therefore, barcoding studies are HEBERT, P.D.N. 2008. DNA barcodes and cryptic species of ideal for testing the problems we address in this study. skipper butterflies in the genus Perichares in Area de Conser- Moreover, this is the only way to comprehensibly proof vacion Guanacaste, Costa Rica. Proceedings of the National Academy of Sciences USA 105, 6350–6355. the usefulness of cox1 as a barcode. We do not question CARDINAL,S.,STRAKA,J.&DANFORTH, B.N. 2010. Comprehen- the usefulness of sequence data for taxonomic purposes in sive phylogeny of apid bees reveals the evolutionary origins general, as this kind of data is well suited to address specific and antiquity of cleptoparasitism. Proceedings of the National problems which simply cannot be tackled with morphology Academy of Sciences USA 107, 16207–16211. (Meier, 2008). We also do not claim that cox1 sequences CESARI,M.,BERTOLANI,R.,REBECCHI,L.&GUIDETTI, R. 2009. DNA barcoding in Tardigrada: the first case study on Macro- are inappropriate to resolve species boundaries in all cases. biotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, However, in our opinion the cox1 marker should not be used Macrobiotidae). Molecular Ecology Resources 9, 699–706. as a ‘one for all solution’ and additional genetic (nuclear) CHARLAT,S.,DUPLOUY,A.,HORNETT, E.A., DYSON, E.A., DAV I E S , markers should be applied to at least severely test the util- N., RODERICK, G.K., WEDELL,N.&HURST, G.D.D. 2009. The ity of DNA barcodes. We hope that this suggestion will be joint evolutionary histories of Wolbachia and mitochondria in Hypolimnas bolina. BMC Evolutionary Biology 9. addressed by future barcoding campaigns such as the ‘Bee DANFORTH, B.N., SIPES,S.,FANG,J.&BRADY, S.G. 2006. The Barcode of Life Initiative’. history of early bee diversification based on five genes plus 326 M. Gerth et al.

morphology. Proceedings of the National Academy of Sci- HERTIG,M.&WOLBACH, S.B. 1924. Studies on Rickettsia-like ences USA 103, 15118–15123. micro-organisms in insects. Journal of Medical Research 44, DROEGE,S.,RIGHTMYER, M.G., SHEFFIELD,C.S.&BRADY,S.G. 329–374. 2010. New synonymies in the bee genus Nomada from North HICKERSON, M.J., MEYER,C.P.&MORITZ, C. 2006. DNA barcod- America (Hymenoptera: Apidae). Zootaxa, 1–32. ing will often fail to discover new animal species over broad DURON,O.,BOUCHON,D.,BOUTIN,S.,BELLAMY,L.,ZHOU,L., parameter space. Systematic Biology 55, 729–739. ENGELSTADTER,J.&HURST, G. 2008. The diversity of repro- HILGENBOECKER,K.,HAMMERSTEIN,P.,SCHLATTMANN,P., ductive parasites among arthropods: Wolbachia do not walk TELSCHOW,A.&WERREN, J.H. 2008. How many species are alone. BMC Biology 6, 27. infected with Wolbachia? – a statistical analysis of current EBACH,M.C.&DE CARVALHO, M.R. 2010. Anti-intellectualism in data. FEMS Microbiology Letters 281, 215–220. the DNA barcoding enterprise. Zoologia 27, 165–178. HOSOKAWA,T.,KOGA,R.,KIKUCHI,Y.,MENG,X.-Y.&FUKATSU, EKREM,T.,STUR,E.&HEBERT, P.D.N. 2010. Females do count: T. 2010. Wolbachia as a bacteriocyte-associated nutritional Documenting Chironomidae (Diptera) species diversity us- mutualist. Proceedings of the National Academy of Sciences ing DNA barcoding. Organisms, Diversity and Evolution 10, USA 107, 769–774. 397–408. HURST, G.D.D. & JIGGINS, F.M. 2005. Problems with mitochon- ELIAS,M.,HILL, R.I., WILLMOTT, K.R., DASMAHAPATRA, K.K., drial DNA as a marker in population, phylogeographic and BROWER, A.V.Z., MALLLET,J.&JIGGINS, C.D. 2007. Limited phylogenetic studies: the effects of inherited symbionts. Pro- performance of DNA barcoding in a diverse community of ceedings of the Royal Society of London Series B – Biological tropical butterflies. Proceedings of the Royal Society of Lon- Sciences 272, 1525–1534. don Series B – Biological Sciences 274, 2881–2889. JEYAPRAKASH,A.&HOY, M.A. 2000. Long PCR improves Wol- FUNK,D.J.&OMLAND, K.E. 2003. Species-level paraphyly and bachia DNA amplification: wsp sequences found in 76% of polyphyly: Frequency, causes, and consequences, with in- sixty-three arthropod species. Insect Molecular Biology 9, sights from animal mitochondrial DNA. Annual Review of 393–405. Ecology, Evolution and Systematics 34, 397–423. JIGGINS, F.M. 2003. Male-killing Wolbachia and mitochondrial GALTIER,N.,NABHOLZ,B.,GLEMIN,S.&HURST, G.D.D. 2009. DNA: Selective sweeps, hybrid introgression and parasite Mitochondrial DNA as a marker of molecular diversity: a population dynamics. Genetics 164, 5–12. reappraisal. Molecular Ecology 18, 4541–4550. JIGGINS, F.M., VON DER SCHULENBURG, J.H.G., HURST, G.D.D. & GIBBS, J. 2009a. Integrative taxonomy identifies new (and old) MAJERUS, M.E.N. 2001. Recombination confounds interpreta- species in the Lasioglossum (Dialictus) tegulare (Robertson) tions of Wolbachia evolution. Proceedings of the Royal Society species group (Hymenoptera, Halictidae). Zootaxa, 1–38. of London Series B – Biological Sciences 268, 1423–1427. GIBBS, J.J. 2009b. New species in the Lasioglossum petrellum JOHNSON,S.D.&STEINER, K.E. 2000. Generalization versus spe- species group identified through an integrative taxonomic ap- cialization in plant pollination systems. Trends in Ecology & proach. Canadian Entomologist 141, 371–396. Evolution 15, 140–143. GOLDING, G.B., HANNER,R.&HEBERT, P.D.N. 2009. Preface. KUHLMANN,M.,ELSE,G.,DAWSON,A.&QUICKE, D. 2007. Molec- Molecular Ecology Resources 9, IV–VI. ular, biogeographical and phenological evidence for the exis- GOMPERT,Z.,FORISTER, M.L., FORDYCE,J.A.&NICE, C.C. 2008. tence of three western European sibling species in the Colletes Widespread mito-nuclear discordance with evidence for in- succinctus group (Hymenoptera: Apidae). Organisms, Diver- trogressive hybridization and selective sweeps in Lycaeides. sity & Evolution 7, 155–165. Molecular Ecology 17, 5231–5244. LACHOWSKA,D.,KAJTOCH,Ł.&KNUTELSKI, S. 2010. Occurrence GREENLEAF,S.S.&KREMEN, C. 2006. Wild bees enhance honey of Wolbachia in central European weevils: correlations with bees’ pollination of hybrid sunflower. Proceedings of the host systematics, ecology, and biology. Entomologia Experi- National Academy of Sciences USA 103, 13890–13895. mentalis et Applicata 135, 105–118. HASTINGS, J.M., SCHULTHEIS, P.J., WHITSON,M.,HOLLIDAY, C.W., LANGHOFF,P.,AUTHIER,A.,BUCKLEY, T.R., DUGDALE, J.S., RO- COELHO,J.R.&MENDELL, A.M. 2008. DNA barcoding of new DRIGO,A.&NEWCOMB, R.D. 2009. DNA barcoding of the world cicada killers (Hymenoptera: Crabronidae). Zootaxa, endemic New Zealand leafroller moth genera, Ctenopseustis 27–38. and Planotortrix. Molecular Ecology Resources 9, 691–698.

Downloaded by [University of Leipzig] at 06:02 16 September 2014 HEBERT, P.D.N., DEWAARD,J.R.&LANDRY, J.F. 2010. DNA bar- LINARES, M.C., SOTO-CALDERON, I.D., LEES,D.C.&ANTHONY, codes for 1/1000 of the animal kingdom. Biology Letters 6, N.M. 2009. High mitochondrial diversity in geographically 359–362. widespread butterflies of Madagascar: A test of the DNA HEBERT, P.D.N., PENTON, E.H., BURNS, J.M., JANZEN,D.H.& barcoding approach. Molecular Phylogenetics and Evolution HALLWACHS, W. 2004a. Ten species in one: DNA barcoding 50, 485–495. reveals cryptic species in the neotropical skipper butterfly As- LOSEY,J.E.&VAUGHAN, M. 2006. The economic value of ecolog- traptes fulgerator. Proceedings of the National Academy of ical services provided by insects. Bioscience 56, 311–323. Sciences USA 101, 14812–14817. LUKHTANOV, V.A., SOURAKOV,A.,ZAKHAROV,E.V.&HEBERT, HEBERT, P.D.N., RATNASINGHAM,S.&DEWAARD, J.R. 2003. Bar- P.D.N. 2009. DNA barcoding Central Asian butterflies: in- coding animal life: cytochrome c oxidase subunit 1 diver- creasing geographical dimension does not significantly re- gences among closely related species. Proceedings of the duce the success of species identification. Molecular Ecology Royal Society of London Series B – Biological Sciences 270, Resources 9, 1302–1310. S96–S99. MAGNACCA,K.N.&BROWN, M.J.F. 2010. Mitochondrial hetero- HEBERT, P.D.N., STOECKLE, M.Y., ZEMLAK,T.S.&FRANCIS,C.M. plasmy and DNA barcoding in Hawaiian Hylaeus (Neso- 2004b. Identification of birds through DNA barcodes. PLoS prosopis) bees (Hymenoptera: Colletidae). BMC Evolution- Biology 2, 1657–1663. ary Biology 10. HEDGES, L.M., BROWNLIE, J.C., O’NEILL,S.L.&JOHNSON,K.N. MEIER, R. 2008. DNA sequences in taxonomy: opportunities and 2008. Wolbachia and virus protection in insects. Science 322, challenges. In: WHEELER, Q.D., Ed., The new taxonomy. CRC 702. Press, Boca Raton, pp. 95–127. Wolbachia infections in bees (Anthophila) 327

MEIER,R.,SHIYANG,K.,VAIDYA,G.&NG, P.K.L. 2006. DNA are coamplified. Proceedings of the National Academy of barcoding and taxonomy in diptera: A tale of high intraspecific Sciences USA 105, 13486–13491. variability and low identification success. Systematic Biology STAHLHUT, J.K., DESJARDINS, C.A., CLARK, M.E., BALDO,L.,RUS- 55, 715–728. SELL, J.A., WERREN,J.H.&JAENIKE, J. 2010. The mushroom MEYER,C.P.&PAULAY, G. 2005. DNA barcoding: Error habitat as an ecological arena for global exchange of Wol- rates based on comprehensive sampling. PLoS Biology 3, bachia. Molecular Ecology 19, 1940–1952. 2229–2238. TEIXEIRA,L.,FERREIRA, A.&A´ SHBURNER, M. 2008. The bacterial MICHENER, C.D. 2000. The bees of the world. Johns Hopkins symbiont Wolbachia induces resistance to RNA viral infec- University Press, Baltimore. tions in Drosophila melanogaster. PLoS Biology 6, e1000002. PACKER,L.,GIBBS,J.,SHEFFIELD,C.&HANNER, R. 2009. DNA bar- VAV R E ,F.,GIRIN,C.&BOULETREAU´ , M. 1999. Phylogenetic status coding and the mediocrity of morphology. Molecular Ecology of a fecundity-enhancing Wolbachia that does not induce the- Resources 9, 42–50. lytoky in Trichogramma. Insect Molecular Biology 8, 67–72. PERLMAN, S.J., KELLY,S.E.&HUNTER, M.S. 2008. Population VIRGILIO,M.,BACKELJAU,T.,NEVADO,B.&DE MEYER, M. 2010. biology of cytoplasmic incompatibility: Maintenance and Comparative performances of DNA barcoding across insect spread of Cardinium symbionts in a parasitic wasp. Genet- orders. BMC Bioinformatics 11. ics 178, 1003–1011. WEEKS, A.R., TURELLI,M.,HARCOMBE, W.R., REYNOLDS,K.T. RATNASINGHAM,S.&HEBERT, P.D.N. 2007. BOLD: The Bar- &HOFFMANN, A.A. 2007. From parasite to mutualist: Rapid code of Life Data System (www.barcodinglife.org). Molec- evolution of Wolbachia in natural populations of Drosophila. ular Ecology Notes 7, 355–364. PLoS Biology 5, e114. RAYCHOUDHURY,R.,BALDO,L.,OLIVEIRA, D.C.S.G. & WERREN, WEINERT, L.A., WERREN, J.H., AEBI,A.,STONE,G.N.&JIGGINS, J.H. 2009. Modes of acquisition of Wolbachia: Horizontal F.M. 2009. Evolution and diversity of Rickettsia bacteria. transfer, hybrid introgression, and codivergence in the Naso- BMC Biology 7,6. nia species complex. Evolution 63, 165–183. WENSELEERS,T.,ITO,F.,VAN BORM,S.,HUYBRECHTS,R.,VOL- ROS, V.I.D., FLEMING, V.M., FEIL,E.J.&BREEUWER, J.A.J. 2009. CKAERT,F.&BILLEN, J. 1998. Widespread occurrence of How diverse is the genus Wolbachia? Multiple-gene sequenc- the micro-organism Wolbachia in ants. Proceedings of the ing reveals a putatively new Wolbachia Supergroup recov- Royal Society of London Series B – Biological Sciences 265, ered from spider mites (Acari: Tetranychidae). Applied and 1447–1452. Environmental Microbiology 75, 1036–1043. WERREN, J.H., BALDO,L.&CLARK, M.E. 2008. Wolbachia: master RUBINOFF,D.,CAMERON,S.&WILL, K. 2006. A genomic per- manipulators of invertebrate biology. Nature Reviews Micro- spective on the shortcomings of mitochondrial DNA for ‘bar- biology 6, 741–751. coding’ identification. Journal of Heredity 97, 581–594. WERREN, J.H., WINDSOR,D.&GUO, L.R. 1995. Distribution of RUSSELL, J.A., GOLDMAN-HUERTAS,B.,MOREAU, C.S., BALDO,L., Wolbachia among neotropical Arthropods. Proceedings of the STAHLHUT, J.K., WERREN,J.H.&PIERCE, N.E. 2009. Special- Royal Society of London Series B – Biological Sciences 262, ization and geographic isolation among Wolbachia symbionts 197–204. from ants and lycaenid butterflies. Evolution 63, 624–640. WESTRICH,P.,FROMMER,U.,MANDERY,K.,RIEMANN,H.,RUHNKE, SHEFFIELD, C.S., HEBERT, P.D.N., KEVAN,P.G.&PACKER, L. 2009. H., SAURE,C.&VOITH, J. 2008. Rote Liste der Bienen DNA barcoding a regional bee (Hymenoptera: Apoidea) fauna Deutschlands (Hymenoptera, Apidae). Eucera 1, 33–87. and its potential for ecological studies. Molecular Ecology WHITWORTH, T.L., DAWSON, R.D., MAGALON,H.&BAUDRY,E. Resources 9, 196–207. 2007. DNA barcoding cannot reliably identify species of the SMITH, M.A., WOOD, D.M., JANZEN, D.H., HALLWACHS,W.& blowfly genus Protocalliphora (Diptera: Calliphoridae). Pro- HEBERT, P.D.N. 2007. DNA barcodes affirm that 16 species ceedings of the Royal Society B – Biological Sciences 274, of apparently generalist tropical parasitoid flies (Diptera, Ta- 1731–1739. chinidae) are not all generalists. Proceedings of the National WIEMERS,M.&FIEDLER, K. 2007. Does the DNA barcoding Academy of Sciences USA 104, 4967–4972. gap exist? – A case study in blue butterflies (Lepidoptera: SONG,H.,BUHAY, J.E., WHITING,M.F.&CRANDALL, K.A. 2008. Lycaenidae). Frontiers in Zoology 4,8. Many species in one: DNA barcoding overestimates the

Downloaded by [University of Leipzig] at 06:02 16 September 2014 number of species when nuclear mitochondrial pseudogenes Associate Editor: Andrew Brower

View publication stats