REDIA, XCII, 2009: 87-91

ROBERT G. FOOTTIT (*) - H. ERIC L. MAW (*) - KEITH S. PIKE (**)

DNA BARCODES TO EXPLORE DIVERSITY IN APHIDS (HEMIPTERA APHIDIDAE AND ADELGIDAE)

(*) Canadian National Collection of Insects, National Environmental Health Program, Agriculture and Agri-Food Canada, K.W. Neatby Building, 960 Carling Avenue, Ottawa, Ontario K1A 0C6, Canada;[email protected] (**) Washington State University, Irrigated Agriculture Research and Extension Center, 24106 N. Bunn Road, Prosser, WA 99350, U.S.A

Foottit R.G., Maw H.E.L., Pike K.S. – DNA barcodes to explore diversity in aphids (Hemiptera Aphididae and Adelgidae).

A tendency towards loss of taxonomically useful characters, and morphological plasticity due to host and environmental factors, complicates the identification of aphid species and the analysis of relationships. The presence of different morphological forms of a single species on different hosts and at different times of the year makes it difficult to consistently associate routinely collected field samples with particular species definitions. DNA barcoding has been proposed as a standardized approach to the characterization of life forms. We have tested the effectiveness of the standard 658-bp barcode fragment from the 5’ end of the mitochondrial cytochrome c oxidase 1 gene (COI) to differentiate among species of aphids and adelgids. Results are presented for a preliminary study on the application of DNA barcoding in which approximately 3600 specimens representing 568 species and 169 genera of the major subfamilies of aphids and the adelgids have been sequenced. Examples are provided where DNA barcoding has been used as a tool in recognizing the existence of cryptic new taxa, linking life stages on different hosts of adelgids, and as an aid in the delineation of species boundaries. The future use of these DNA barcodes for the routine detection of invasive species, the resolution of pest complexes, and the analysis of diverse faunas is discussed.

KEY WORDS: Aphididae, Adelgidae, species identification, DNA barcodes, COI

INTRODUCTION MATERIALS AND METHODS

Many aphid species are important pests in agriculture and Aphid samples were collected between 1991 and 2008 forestry, both through direct damage and by acting as into liquid nitrogen or into 95% ethanol. Voucher speci- vectors of numerous plant diseases (BLACKMAN and EASTOP, mens from each collection were mounted on microscope 2000; EASTOP, 1977; VAN EMDEN and HARRINGTON, 2007; slides and deposited in the Canadian National Collection MINKS and HARREWIJN, 1987). As a result of ease of of Insects (Agriculture and Agri-Food, Ottawa, Ontario, transport and parthenogenetic reproduction, they are Canada) or at the Irrigated Agriculture Research and important as invasive pests throughout the world (for Extension Center, Washington State University (Prosser, example, FOOTTIT et al., 2006; MESSING et al., 2007; Washington, USA). Samples of aphid DNA (particularly TEULON and STUFKENS, 2002). Reliable identification of Hormaphidinae: Cerataphidini) were provided by D. species is essential for the integrated management of pest Stern (vouchers in natural History Museum, London). aphids and for the early detection and risk analysis of newly DNA extraction, amplification and sequencing for most introduced species (MILLER and FOOTTIT, 2009). However, specimens were done at the Biodiversity Institute of an evolutionary tendency towards the loss of taxonomically Ontario (Guelph, Ontario, Canada) using techniques useful characters, polymorphism, and morphological described by deWAARD et al. (2008). Additional samples plasticity due to host influences and other environmental were processed at Agriculture and Agri-Food Canada factors, complicate the recognition of species and the (Ottawa, Ontario, Canada) or by Nathan Havill at Yale analysis of relationships at all levels (FOOTTIT, 1997). University (New Haven, Connecticut, USA). The primer DNA barcoding, using a short standardized DNA pairs LepF and LepR or M13-tailed alternates, sequence, has been proposed as a approach to the LCO1490_t1 and HCO2198_t2 (primer sequences avail- characterization of life forms in numerous groups of living able in BOLD – the Barcode of Life Data System organisms (HAJIBABAEI et al., 2007; FLOYD et al., 2009). In (RATNASINGHAM and HEBERT, 2007), http://www.barco animals, the selected region is the 5’-terminus of the dinglife.org, “Published projects / View all primers” links) mitochondrial cytochrome c oxidase subunit 1 (COI) gene were used to amplify an approximately 700 bp DNA frag- (HEBERT et al., 2003). ment of mitochondrial COI, which was subsequently This paper updates our efforts to develop a DNA sequenced in both directions using either LepF and LepR barcode library for aphid species (see FOOTTIT et al., 2008 or M13F and M13R (primer sequences given in BOLD). and 2009). We also provide examples of the use of DNA Collection information and sequence data have been barcodes to explore diversity in aphids and use of barcode entered into BOLD – the Barcode of Life Data System. data as a tool to reassess the status of intraspecific forms, Subsets discussed in FOOTTIT et al., (2008, 2009) are detect host associated lineages and cryptic species, and publicly available on BOLD. Additional sets will be made associate morphs on alternate hosts. available as they are validated and published.

– Received 30 July 2009 Accepted 2 September 2009 Published 1 December 2009 88 R.G. FOOTTIT ET AL. REDIA, Vol. XCII, 2009

Pairwise distances were calculated using the Kimura 2- californicum (Clarke), Myzus cerasi (Fabricius), Rhopalosiphum parameter model of base substitution (KIMURA, 1980) and padi (Linnaeus), Cinara curvipes (Patch), Cinara hottesi (Gillette patterns of variation among samples were examined using & Palmer), and Cinara obscura Bradley. neighbor-joining (SAITOU & NEI, 1987) as implemented Minimum distances between representatives of congeneric on BOLD. species varied from 0 to 16.6% with a mean of 7.4%. However Classification and nomenclature of aphid taxa follows most species (92%) diverge from congeners by more REMAUDIÈRE and REMAUDIÈRE (1997) and NIETO NAFRÍA than 2%. Nominal species not distinguished by barcode et al. (1998). include members of the Aphis helianthi group (helianthi Monell, asclepiadis Fitch and nigratibialis Robinson), Chaetosiphon fragaefolii (T.D.A. Cockerell) versus Ch. RESULTS thomasi Hille Ris Lambers, Hyperomyzus sandilandicus (Robinson) versus H. nabali (Oestlund) and Illinoia richardsi GENERAL RESULTS Robinson versus I. goldamaryae (Knowlton) . In addition, A total of 3630 specimens of 568 species in 169 genera Aulacorthum dorsatum Richards and Ericaphis wakibae have been sampled, representing the families Adelgidae, (Hottes) have identical sequence (see FOOTTIT et al., 2008). Phylloxeridae and 18 of the 24 subfamilies of Aphididae Species differing by less than 1% include Aphis (Bursaphis) (Table 1). About one third of the species are represented varians Patch versus A. manitobensis Robinson and by at least 3 samples and one fifth by 10 or more samples. Rojanavongse, members of the Acyrthosiphon kondoi group The majority of the specimens are from North America, (kondoi Shinji, caraganae (Cholodkovsky) and churchillense but samples of Aphididae from Micronesia and New Robinson), Ericaphis fimbriata (Richards) versus E. scam- Zealand, and Adelgidae from Japan, China and Europe melli (Mason), and certain groups of Uroleucon species, are also included in the current data set. Illinoia species, Macrosiphum species and Kakimia (genus Maximum within-species pairwise distances varied from Nasonovia) species. Although some of these species may 0 to 5.5% in Aphididae and 0 to 8.75% in Adelgidae, with prove to be synonyms, most are distinguishable by host a mean maximum of 0.58%. However, maximum within- association and by morphology (albeit with some difficulty), species distance was less than 2% in 94% of the species rep- and thus likely represent recently separated species. Additional resented by more than 3 samples, and mean maximum pairwise sampling of low divergence species pairs is required to deter- distance in these species is 0.35%. Within the more vari- mine whether the small observed differences are fixed able species, distribution of pairwise distances was distinctly sequence differences or artefacts of the sample set. bimodal or trimodal, suggesting possible cryptic species, although this may also be explained as a sampling artefact or STATUS OF FORMS OF PENTALONIA NIGRONERVOSA evidence of introgression. In particular the maximum intraspe- (APHIDIDAE: APHIDINAE) cific divergence of 8.87 % observed in Adelges tsugae Annand Pentalonia nigronervosa Coquerel, the banana aphid, is is due to the occurrence of three very distinct tight clusters known as a pest of banana and various crop plants in within this nominal species, supporting the suggestion by families Araceae and Zingiberaceae. VAN DER GOOT Havill et al. (2006) that this taxon consists of three species. (1917) described P. caladii, distinguished by differences in Other multimodal species include Pineus coloradensis (Gillette), length of the ultimate rostral article and siphunculus. Morwilkoja vagabunda (Walsh), Pemphigus betae Doane, However, the status of this entity has not been adequately Pachypappa sacculi (Gillette), Brachycaudus helichrysi (Kaltenbach) addressed. Later authors treated it a synomym or ‘forma’ (also noted by COEUR D’ACIER et al. 2007), Jacksonia papil- of P. nigronervosa. REMAUDIÈRE and REMAUDIÈRE (1997) lata (Theobald), Neomyzus circumflexus (Buckton), Macrosiphum continue to list it as a form, and biological studies often make no explicit distinction (for example, ROBSON et al., 2007). Barcode data for sixty P. nigronervosa samples from Table 1 – Summary of taxonomic distribution of material Polynesia, Australia and Florida (USA) falls into two sampled. Classification follows REMAUDIÈRE and REMAUDIÈRE uniform groups differing by about 3% sequence (1997) and NIETO NAFRÍA et al. (1998). divergence. All samples from one group (1 exception) Genera Species occur on banana, and all samples from the other group on Araceaea and Zingiberaceae (again 1 exception only). Adelgidae 2 17 Subsequent investigation showed that the groups Phylloxeridae 3 3 correspond exactly to fixed differences in sequences for Aphididae nuclear elongation factor 1 alpha introns, and to non- Anoeciinae 1 2 Aphidinae 77 323 overlapping morphometric differences. Given the Calaphidinae 23 55 economic importance of this aphid, more formal Chaitophorinae 3 14 recognition and definition of the form caladii is desirable Drepanosiphinae 2 6 (manuscript in preparation). Eriosomatinae 20 44 Greenideinae 2 3 GEOGRAPHIC AND HOST-ASSOCATIATED VARIATION IN Hormaphidinae 9 13 Lachninae 12 59 CHAITOPHORUS POPULICOLA (APHIDIDAE: CHAITOPHORINAE) Lizeriinae 1 1 Chaitophorus populicola Thomas is widespread in North Mindarinae 1 2 America on various species of Populus. There is Phloeomyzinae 1 1 considerable variation in coloration, body size and setal Phyllaphidinae 2 3 shape, but the extent to which the observed variation is of Pterocommatinae 2 6 genetic or environmental origin is unclear. HILLE RIS Saltusaphidinae 5 9 Tamaliinae 1 4 LAMBERS (1960) described subspecies patchae for Taiwanaphidinae 1 1 specimens with relatively longer, acuminate setae. Thelaxinae 1 1 Chaitophorus brunneri Williams is currently considered a total 169 568 synonym. DNA BARCODES TO EXPLORE DIVERSITY IN APHIDS (HEMIPTERA APHIDIDAE AND ADELGIDAE) 89

Maximum pairwise distance among barcodes for 237 collections from across Canada and north-central United States is 3.16%, or if two samples from Populus deltoides in north-central USA are excluded, 2.01%. The minimum pairwise distance to the most similar Chaitophorus species (Ch. nudus Richards) is 7.46%. Forty-two haplotypes were observed, of which 16 were replicated. Each replicated haplotype (along with similar unique haplotypes) is restricted to a particular geographic area (eastern, central or western part of the continent) and is specific to a particular Populus section (sections Populus, Tacamahaca and Aigeros) (4 exceptions). Thus, in each region, sympatric host-specific haplotypes occur. We are currently examining nuclear genes and morphometric characters to confirm and assess the taxonomic significance of this variation.

POSSIBLE CRYPTIC SPECIES WITHIN PEMPHIGUS BETAE (APHIDIDAE: ERIOSOMATINAE) Pemphigus betae Doane, the sugar-beet root aphid is widespread in North America. Barcode sequences reveal three distinct sympatric groups among 190 samples identified as this species. Pairwise genetic distances among the groups are of the same magnitude as distances among other Pemphigus species. The barcode-defined groups correspond exactly to groups previously identified using several nuclear markers. Morphometric analysis of this group is now being pursued.

EXPLORATION OF PATTERNS OF VARIATION AMONG CINARA SPECIES (APHIDIDAE: LACHNINAE) Conifer aphids in the genus Cinara are a diverse group in North America and appear to have undergone extensive postglacial radiation on a number of hosts. While there is a wide range in morphological characters, many of these characters form a continuum across putative species. It is thus unclear which components of the observed variation are genetically based, and thus of taxonomic value, and which are the result of environmental effects. We have begun to use DNA barcoding as a tool for the exploration of species diversity in this group. A neighbour-joining tree summarizing preliminary results for Cinara species is shown in Figure I. Species are well differentiated: within-species pairwise distances range from 0 to 2.03% (mean 0.52%) (assumes C. obscura groups discussed below are separate species) in contrast to minimum between-species differences of 2.7 to 13.2% (mean 7.91%). Clusters based on barcode sequences correspond to major host groups and to morphologically defined species groups. Three potential morphologically cryptic species are suggested within Cinara hottesi (Gillette and Palmer) on Picea, C. obscura Bradley on Picea and C. curvipes (Patch) on Abies. The separation of the C. obscura groups shows an especially deep sequence divergence (labelled C. obscura and C. sp. nr obscura in Figure I). The taxonomic status of these potential new species is currently being investigated using molecular and morphometric approaches.

ASSOCIATION OF MORPHS ON ALTERNATE HOSTS IN ADELGES TSUGAE (ADELGIDAE) Figure I – Neighbour-joining tree summarizing mitochondrial cytochrome oxidase subunit 1 sequence (DNA barcode) dis- Adelges tsugae Annand, the hemlock woolly adelgid, is tances for species of Cinara. Host-plant groups are indicated on found in east Asia and in western North America, and has the right (Cup = Cupressaceae). Identical haplotypes are col- been introduced to eastern North America where it is a lapsed to a single node with number (n) of identical samples serious pest. As a result of morphological differences indicated for n>1. 90 R.G. FOOTTIT ET AL. REDIA, Vol. XCII, 2009 between stages of the complex life cycles of adelgids, V., BARBAGALLO S., 2008 – Molecular phylogeny and combined with the reduced number of characters for systematics in the genus Brachycaudus (Homoptera: delineating species, it is often difficult to link life cycle Aphididae): insights from a combined analysis of nuclear forms of a species on different hosts. Host alternation in and mitochondrial genes. - Zoologica Scripta, 37: 175- A. tsugae has been reported previously in Japan based on 193. circumstantial evidence (Inouye 1953, McClure 1996), but DEWAARD J.R., IVANOVA N.V., HAJIBABAEI M., HEBERT this species has otherwise been collected only on Tsuga. P.D.N., 2008 – Assembling DNA barcodes: analytical Barcodes sequences for samples of Adelges collected from protocols. In: Environmental Genomics, Methods in galls on spruce (Picea) in Japan and China match barcodes Molecular Biology, Vol. 410, Martin C.C. (Ed.), for A. tsugae populations on Tsuga in the respective region, Humana Press, Totowa, New Jersey, pp. 275-293. supporting the presence of host alternation in this species. EASTOP, V.F., 1977 – Worldwide importance of aphids as virus vectors. In: Aphids as virus vectors, Harris K.F., Maramorosh K. (eds.). In Harris KF, Maramorosh K. DISCUSSION (eds.), Academic Press, New York pp. 4-62. FLOYD R.M., WILSON J.J., HEBERT P.D.N., 2009 – DNA The current expanded data set confirms the trends reported barcodes and insect biodiversity. In: Insect Biodiversity: earlier by FOOTTIT et al. (2008, 2009), namely that in general Science and Society, Foottit R.G., Adler P.H. (Eds), species of aphids and adelgids correspond to clusters of sim- Wiley-Blackwell, Oxford, pp 417-432. ilar barcode sequences distinct from neighbouring clusters. FOOTTIT R.G., 1997 – Recognition of parthenogenetic Thus barcode sequences are useful as an identification tool insect species. In: Species. The units of biodiversity, in these groups. Congeneric species of aphids and adelgids Claridge M.F., Dawah H.A., Wilson M.R. (Eds), usually form cohesive barcode assemblages, indicating that Chapman & Hall, London, pp. 291-307. DNA barcodes can also be used for genus level identification FOOTTIT R.G., HALBERT S.E., MILLER G.L., MAW E., of species not included in the barcode library. There are, how- RUSSELL L.M., 2006 – Adventive aphids (Hemiptera: ever, some limitations for species identification in some spe- Aphididae) of America North of Mexico. - Proceedings of ciose recently radiated groups such as Illinoia, Uroleucon and the Entomological Society of Washington, 108: 583-610. Macrosiphum. FOOTTIT R.G., MAW H.E.L., VON DOHLEN C.D., HEBERT Beyond simple identification, DNA barcodes can also pro- P.D.N., 2008 – Species identification of aphids (Insecta: vide a means for detecting and resolving taxonomic problems. Hemiptera: Aphididae) through DNA barcodes. - In the case of banana aphid (Pentalonia nigronervosa), the use Molecular Ecology Resources, 8: 1189–1201. of barcodes has helped to clarify the status of host specific FOOTTIT R.G., MAW H.E.L., HAVILL N.P., AHERN R.G., forms. Even in well-studied aphid groups, cryptic species are MONTGOMERY M.E., 2009 – DNA Barcodes to Explore present. For example, cryptic entities within the economically Diversity and Life Cycles in the Adelgidae (Insecta: important sugar-beet root aphid (Pemphigus betae) are clearly Hemiptera: Aphidoidea). Molecular Ecology Resources 9 evident through DNA barcode analysis. Barcodes provide a (Suppl. 1), 188-195. simple method of identifying members of this group and thus HAJIBABAEI M., DEWAARD J.R., IVANOVA N.V., will be useful in the monitoring and management of this pest. 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HAVILL N.P., MONTGOMERY M.E., YU G., SHIYAKE S., ACKNOWLEDGEMENTS CACCONE A., 2006 – Mitochondrial DNA from hemlock woolly adelgid (Hemiptera: Adelgidae) suggests cryptic This work was funded by Agriculture and Agri-Food speciation and pinpoints the source of the introduction in Canada, the USDA Forest Service and USDA CSREES, eastern North America. - Annals of the Entomological and through grants to the Canadian Barcode of Life Society of America, 99:195-203. Network from Genome Canada (through the Ontario HAVILL N.P., FOOTTIT R.G., 2007 – Biology and evolution Genomics Institute), NSERC, the Moore Foundation and of the Adelgidae. - Annual Review of Entomology, 52: other sponsors listed at www.BOLNET.ca. 325-349. We thank the staff of the Biodiversity Institute of HEBERT P.D.N., CYWINSKA A., BALL S.L., DEWAARD J.R., Ontario for processing samples and data management. 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