Molecular and Morphometric Data Indicate a New Species of the Aphid Genus Rhopalosiphum (Hemiptera: Aphididae) Author(S): I

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Molecular and Morphometric Data Indicate a New Species of the Aphid Genus Rhopalosiphum (Hemiptera: Aphididae) Author(s): I. Valenzuela, V. F. Eastop, P. M. Ridland, and A. R. Weeks Source: Annals of the Entomological Society of America, 102(6):914-924. 2009. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1603/008.102.0602 URL: http://www.bioone.org/doi/full/10.1603/008.102.0602

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. SYSTEMATICS Molecular and Morphometric Data Indicate a New Species of the Aphid Genus Rhopalosiphum (Hemiptera: Aphididae)

1,2,3 4 2 1 I. VALENZUELA, V. F. EASTOP, P. M. RIDLAND, AND A. R. WEEKS

Ann. Entomol. Soc. Am. 102(6): 914Ð924 (2009) ABSTRACT Here, we provide evidence for a new aphid species from the genus Rhopalosiphum Koch, based upon an Australian survey of variation in the mitochondrial cytochrome oxidase I gene, and subsequently validated by cytochrome b, nuclear microsatellites, nuclear sequence characterized ampliÞed region locus, and karyotypic analyses. Despite the new species being morphologically very similar to Rhopalosiphum padi (L.), there was signiÞcant genetic differentiation, with the new species being closer to the Rhopalosiphum insertum (Walker) group. Karyotypic analyses indicated a putative hybrid origin for the new species, but mitochondrial and nuclear DNA sequence data showed that the available Australian and overseas populations of Rhopalosiphum spp. did not serve as parental lineages. Diagnostic methods were developed that enabled the identiÞcation of the new species based on morphometric data and a polymerase chain reaction-restriction fragment length polymorphism based molecular technique.

KEY WORDS hybridization, cryptic, karyotype, mitochondrial DNA, nuclear DNA

Surveys of molecular variation in nuclear and mito- conditions, molecular techniques, such as karyotype chondrial DNA sequences of invertebrates are in- and DNA studies, have proven more efÞcient in sep- creasingly being used to identify cryptic species even arating morphologically indistinguishable taxa. For ex- in well-studied genera. Examples include the recent ample, two studies on the genetic diversity of Rhopa- discovery of a new species of Drosophila based on losiphum revealed previously unrecorded and microsatellite sequence data (Schiffer et al. 2004), the undescribed cryptic taxa. In Australia, Hales and Co- detection of new taxa of Neotropical skipper butter- wen (1990) characterized allozyme patterns and chro- ßies (Hebert et al. 2004), the molecular characteriza- mosome numbers of Rhopalosiphum maidis (Fitch), tion of two cryptic species of bumblebees (Ellis et al. Rhopalosiphum padi (L.), Rhopalosiphum ruﬁabdomi- 2005), and the discovery of various cryptic parasitoid nale (Sasaki), and Rhopalosiphum near insertum ßies from the genus Belvosia Robineau-Desvoidy (Walker) [see Blackman and Eastop (2006) for R. (Smith et al. 2006). Once new molecular variants are insertum and R. near insertum details] with results established, simple molecular tests also can be devel- conforming to previous overseas records. But, they oped that distinguish between the cryptic taxa. For found a R. padi-like form that had a diploid chromo- example, polymerase chain reaction (PCR)-restric- some number of 2n ϭ 9 (compared with 2n ϭ 8 for the tion fragment length polymorphism (RFLP) analysis other species) and a unique allozyme pattern. They of the mitochondrial genes cytochrome oxidase subunit postulated that this new taxon could be a hybrid be- I (COI) (Carew et al. 2005, 2007) and cytochrome b tween R. padi and R insertum because of morpholog- (Ellis et al. 2006), and the intragenic transcribed ical similarities, but the allozyme patterns did not spacer (ITS) nuclear ribosomal region have been de- support this idea, and the authors suggested the R. veloped recently (Carew et al. 2004). padi-like form was most likely a new undescribed In aphids, groups of closely related taxa can be species. In New Zealand, Bulman et al. (2005) de- particularly difÞcult to identify, mainly because of the tected two undescribed taxa using karyotype, random high plastic nature of aphid morphology and the pres- ampliÞed polymorphic DNA markers, cytochrome b, ence of cryptic species, hybrids, and complex groups and ITS sequence data. Molecular results revealed a R. of species (Blackman et al. 1987a, Blackman and padi-like form that had signiÞcant molecular differ- Spence 1994, Blackman and Eastop 2007). Under these ences from local and overseas populations of R. padi. Analysis of ribosomal RNA sequence data, however, 1 Centre for Environmental Stress and Adaptation Research, The related this taxon (called R. padi T) to the R. insertum University of Melbourne, Parkville, Victoria 3010, Australia. group that included a European taxon and a New 2 Department of Primary Industries, KnoxÞeld Centre, Private Bag Zealand taxon called R. near insertum. The authors 15, Ferntree Gully Delivery Centre, Victoria 3156, Australia. concluded that R. padi T, was an undescribed species 3 Corresponding author, e-mail: [email protected]. 4 Natural History Museum, Cromwell Rd., London SW7 5BD, of Rhopalosiphum. Moreover, the same DNA se- United Kingdom. quence data also found that New Zealand R. near

0013-8746/09/0914Ð0924$04.00/0 ᭧ 2009 Entomological Society of America November 2009 VALENZUELA ET AL.: MOLECULAR CHARACTERIZATION OF AN APHID SPECIES 915 insertum was signiÞcantly different from the European ical studies have been conducted on these taxa that taxon. These studies show how karyotypic and mo- addressed speciÞc questions on their taxonomy and lecular techniques are capable of characterizing new phylogenetic relationships. Thus, our study intended taxonomic entities that were previously unknown be- Þrstly to detect Rhopalosiphum cryptic species that cause of their cryptic morphology and habitat with had previously been recorded by allozymes and karyo- more common and widespread forms. type studies in Australia (Hales and Cowen 1990) and Rhopalosiphum Koch has Ϸ17 species, which are maintain cultures of these in the laboratory so further mainly associated with rosaceous trees as primary molecular and morphological studies could be under- hosts and monocotyledonous plants as secondary taken. We used genetic markers, including karyotype hosts (Remaudie`re and Remaudie`re 1997, Blackman counts, mitochondrial COI, and cytochrome b se- and Eastop 2006). Native species from the northern quencing as well as nuclear microsatellites and a temperate zones of both the New World and the Old nuclear sequence characterized ampliÞed region World have been found; however, the geographic or- (SCAR) marker (Simon et al. 1999) to reveal phylo- igins of some species remain uncertain because of genetic relationships between Australian and overseas the worldwide propagation with agricultural practices populations of R. padi and investigate putative hybrid (Halbert and Voetglin 1998). Taxonomic surveys have origins for cryptic taxa. We searched for new mor- been conducted in Canada and North America (Rich- phological characters that could discriminate between ards, 1960, 1962), Britain (Stroyan 1984), Fennoscandia cryptic species and their close relatives, and con- and Denmark (Heie 1986), the Iberian peninsula (Nieto ducted a canonical discriminant analysis from a range Nafrõ´a et al. 2005), northeastern India (Raychaudhuri of morphometrics in apterous and alate aphids. Addi- 1980), eastern Siberia (Pashchenko 1988), Japan (Taka- tionally, mitochondrial COI sequence data were used hashi 1965, Torikura 1991), and Australia (Eastop 1966), to develop a simple and accurate PCR-RFLPÐbased and more recent molecular studies have addressed ques- diagnostic method for species identiÞcation in the tions regarding levels of genetic diversity found between genus. Rhopalosiphum species (Hales and Cowen 1990, Bulman et al. 2005, Yeh et al. 2005, Valenzuela et al. 2007). Materials and Methods Molecular markers have been decisive in detecting cryptic variation in the genus. For example, Delmotte Aphid Collections. Species from the genus Rhopa- et al. (2003) characterized two mitochondrial cyto- losiphum were collected in 2004Ð2007 in Victoria, chrome b haplotypes of R. padi as two genetically southeastern Australia (Fig. 1), from a range of dif- distinct lineages that diverged 0.4 MYA, an obligate ferent locations and host plants (Table 1). They were asexual lineage of hybrid origin with a mitochondrial individually placed into Solo cups (Highland Park, IL) (mt)DNA haplotype I, and a second group containing containing a barley, Hordeum vulgare L., seedling and sexual and asexual populations with an mtDNA hap- were reared at 20 Ϯ 1ЊC under a photoperiod of 16:8 lotype II. Nuclear markers showed that R. padi hap- (L:D) h (Ridland et al. 1988). Solo cupsÕ lids were lotype I originated from a hybridization event be- air-tight, avoiding problems of cross-contamination tween European R. padi and an unknown closely between clonal lineages, whereas a mesh allowed wa- related species. Likewise, genetic studies in New Zea- ter evaporation. Alate (arising from crowding) and land showed that R. insertum lineages are signiÞcantly apterous aphids were stored in 70% ethanol for the different from the European lineages, hence the name morphological identiÞcation, whereas the rest of the R. near insertum being used in Australasia (Ridland colony was stored in absolute ethanol for the molec- and Carver 1987, Bulman et al. 2005). These differ- ular analysis. Cultures of aphids were kept for 8 mo ences also have been validated with morphology under controlled laboratory conditions before pres- (V.F.E., unpublished data). However, unlike R. padi, ervation. Embryos from adults were used for karyo- there are no genetic studies that address the origins of typic studies (Blackman 1980). The morphological R. near insertum. identiÞcations were conducted using dichotomous The presence of cryptic species in a genus that keys (Cottier 1953; Eastop 1966; Stroyan 1984; comprises signiÞcant agricultural pests represents a Blackman and Eastop 1994, 2000). Aphid species, serious gap in the management of aphid pest species. localities, dates of collections, and host plants are For example, R. padi is a known vector of a group of listed in Table 1. luteoviruses (barley yellow dwarf) that affect mem- DNA Extraction and PCR Ampliﬁcations. Single bers of the Poaceae family, including cereals and aphid DNA was extracted using a Chelex protocol grasses (Kennedy et al. 1962, Plumb 1983). To monitor following Carew et al. (2003). Brießy aphids were aphid movements and virus epidemiology on cropping dried on tissue paper letting excess ethanol evaporate areas, trap surveys are conducted that generate a large and then transferred into a microcentrifuge tube con- number of alate aphids that are identiÞed using mor- taining 100 ␮l of 5% Chelex resin (Bio-Rad laborato- phological keys. However, morphological identiÞca- ries, Hercules, CA) and 2 ␮l of proteinase K from tion cannot reveal the presence of cryptic populations Roche (Penzberg, Germany). The aphids were such as those found in Australia and New Zealand crushed with a sterile pestle, and this was followed by (described above). incubation for 45 min at 65ЊC and boiling at 95ЊC From the time when cryptic taxa were discovered immediately afterward for 8 min. The extractions were in Australasia, no additional molecular or morpholog- stored in the freezer. 916 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 6

Fig. 1. Rhopalosiphum spp. collection sites in Victoria, southeastern Australia. Three collection sites (Parkville, Brunswick, and Northcote) are indicated by a single (larger) point.

Mitochondrial DNA from the cytochrome oxidase ATCCGATCTTTCCATGC-3Ј, RpCytb-R 5Ј-CTGGT- subunit I and cytochrome b genes were ampliÞed using TGTATCCCAATTCAGG-3Ј(designed by the authors the universal primers LCO1490 and HCO2198 from from partial cytochrome b sequence in GenBank). The Folmer et al. (1994) and primers RpCytb-F 5Ј-TA- PCR conditions for COI and cytochrome bina25-␮l

Table 1. Rhopalosiphum species (clonal lineages), localities, GPS, dates of collections, and host plants

Rhopalosiphum Locality GPS latitude/longitude Collection date Host plant species R. near insertum (1) Ararat Ϫ37 17/142 55 23 March 2004 Poa annua L. R. near insertum (2) KnoxÞeld Ϫ37 53/145 14 19 Oct. 2004 P. annua R. near insertum (3) Fyansford Ϫ38 08/144 17 19 Oct. 2004 Poaceae R. near insertum (4)b Northcote Ϫ37 46/144 60 5 Oct. 2006 Dactylis glomerata L. R. maidis (1) Anakie Ϫ23 33/147 44 29 July 2004 Hordeum vulgare L. R. maidis (2) Mildura Ϫ24 11/142 09 31 Aug. 2004 Poaceae R. maidis (3) Orbost Ϫ37 42/148 27 6 April 2005 Zea mays var. rugosa L. R. maidis (4) Boisdale Ϫ37 53/146 59 4 April 2006 Poaceae R. musae (1) Silvan Ϫ37 49/145 25 1 Jan. 2005 Cyperus papyrus L. R. musae (2) Parkville Ϫ37 47/144 57 8 Oct. 2006 Strelitzia sp. Ait R. nymphaeae (1) Fyansford Ϫ38 08/144 17 12 Oct. 2004 Prunus domestica L. R. padi (1)a,b Lindenow Ϫ37 50/147 51 4 April 2006 Z. mays var. rugosa R. padi (2)a,b Lindenow Ϫ37 50/147 51 Dec. 2006 Lolium sp. L. R. padi (3) Ararat Ϫ37 17/142 55 May 2006 P. annua R. padi (4) Ararat Ϫ37 17/142 55 6 Aug. 2006 P. annua R. padi (5)a Ararat Ϫ37 17/142 55 6 Aug. 2006 P. annua R. padi (6) Hamilton Ϫ37 44/142 01 4 Oct. 2004 Triticum aestivum L. R. padi (7) Hamilton Ϫ37 44/142 01 4 Oct. 2004 T. aestivum R. ruﬁabdominale (1) Balliang Ϫ37 50/144 20 9 Oct. 2004 T. aestivum R. ruﬁabdominale (2) KnoxÞeld Ϫ37 53/145 14 21 Oct. 2004 Rye ϩ wild oats crop R. sp. x (1) Orbost Ϫ37 42/148 27 6 April 2005 Z. mays var. rugosa R. sp. x (2)a,b Lindenow Ϫ37 50/147 51 4 April 2006 Z. mays var. rugosa R. sp. x (3) Brunswick Ϫ37 46/144 57 14 Feb. 2007 Z. mays var. rugosa

Population number is in parentheses. GPS latitude and longitude values are in degrees and minutes. a Karyotypic analysis. b Morphological analysis. November 2009 VALENZUELA ET AL.: MOLECULAR CHARACTERIZATION OF AN APHID SPECIES 917

reaction were as follows: 1ϫ bovine serum albumin Phylogenetic Analyses. Mitochondrial and nuclear (BSA) from Bioline (Tauton, MA), 1ϫ Immobuffer SCAR products were puriÞed and sequenced in ␮ (Bioline), 3.5 mM MgCl2, 1 mM of each dNTP, 10 M both directions on an ABI3730 XL automatic DNA of each primer, 0.2 U of Immolase (heat-activated sequencer by Macrogen Inc. (Applied Biosystems, thermostable DNA polymerase [Bioline]), and 2 ␮lof Foster City, CA). Aligned sequences were edited template DNA. The cycling program for COI was 95ЊC manually using Sequencher 3.2.1 for Macintosh for 6.30 min (long denaturation required to activate (Genecodes, Ann Arbor, MI) and analyzed using polymerase), 40 cycles of 94ЊC for 30 s, 50ЊC for 30 s, the Clustal W algorithm (Thompson et al. 1994). 72ЊC for 40 s, followed by 3 min at 72ЊC. The cycling MODELTEST 3.7 was used to select the model of program for cytochrome b was 95ЊC for 6.30 min, 35 DNA substitution that best Þtted the data by using cycles of 94ЊC for 30 s, 52ЊC for 30 s, 72ЊC for 45 s, the hierarchical likelihood ratio test (Posada and followed by 3 min at 72ЊC. All products were separated Crandall 1998), and subsequent maximum likeli- in 1.2% agarose gel in 1ϫ Tris-EDTA stained with hood and parsimony phylogenetic analyses were ethidium bromide, and visualized and photographed performed in PAUP 4.0*b10 (Swofford 2003). Phy- over UV light. Mitochondrial COI was used for de- lograms were constructed and supports for nodes veloping a PCR-rRFLP identiÞcation method and for were determined through bootstrap analyses (1,000 ϩ comparative phylogenetic analyses between Austra- replicates). For COI, the TrN G model, with sub- ϭ ϭ lian taxa, whereas cytochrome b was used for compar- stitution rate matrices [A-C] 1.000, [A-G] ϭ ϭ ϭ ative phylogenetic analyses between Australian and 2.4646, [A-T] 1.0000, [C-G] 1.0000, [C-T] ϭ overseas taxa. 18.3080, [G-T] 1.0000, nucleotide frequencies A ϭ 0.3410, C ϭ 0.1337, G ϭ 0.0995, T ϭ 0.4259 and The nuclear SCAR was ampliÞed using primers ϭ from Simon et al. (1999). The cycling program was gamma distribution shape parameter 0.0083, was 95ЊC for 6:30 min, 35 cycles of 94ЊC for 30 s, 58ЊC for used for heuristic searches. For cytochrome b, the Њ Њ GTRϩG model, with substitution rate matrices 30 s, 72 C for 45 s, followed by 3 min at 72 C. Then, 2.5 ϭ ϭ ϭ ␮l of the PCR product was ligated into a pGEM-T easy [A-C] 6.9970, [A-G] 33.1427, [A-T] 7.3323, [C-G] ϭ 3.3975, [C-T] ϭ 92.4830, [G-T] ϭ 1.0000, vector (Promega, Madison, WI) and transformed into ϭ ϭ Escherichia coli XL1-Blue MRFÕ competent cells. nucleotide frequencies A 0.35590, C 0.11160, G ϭ 0.08410, T ϭ 0.44840 and gamma distribution Transformed cells were plated onto agar/ampicillin shape parameter ϭ 0.0858 was used. For SCAR lo- plates and left to incubate for 18 h at 37ЊC. Six white cus, the K81uf model, with substitution rate matri- colonies were harvested and dissolved in 50 ␮lof ces [A-C] ϭ 1.0000, [A-G] ϭ 3.4637, [A-T] ϭ sterile Milli-Q water (ultrapure water from Millipore, 0.5155, [C-G] ϭ 0.5155, [C-T] ϭ 3.4637, [G-T] ϭ Billerica, MA). Two microliters of the solution was 1.0000, nucleotide frequencies A ϭ 0.3891, C ϭ 0.1121, then used for PCR with SCAR primers. For all ampli- G ϭ 0.0971, T ϭ 0.4017 and an estimated gamma distri- Þcations, 5 ␮l of the PCR product was separated in a ϫ bution shape parameter was used for heuristic searches. 1.2% agarose gel in 1 Tris-EDTA stained with For the maximum parsimony analyses 100 heuristic ethidium bromide, and visualized and photographed searches with random additions and tree bisection-re- over UV light. SCAR products were used for compar- connection were performed. Pairwise distances based on ative phylogenetic analyses. the Kimura two-parameter model were calculated in Aphids also were genotyped at six microsatellite MEGA version 3.0 (Kumar et al. 2004). Mitochondrial loci: R5.10, R2.73, R5.29b, R3.171, R5.138, and R5.50 haplotypes and nuclear alleles have been deposited (Simon et al. 2001). Polymerase chain reaction am- in GenBank, COI accession numbers DQ499047- pliÞcations for microsatellite loci were carried out in DQ499050, DQ499056, DQ499057, EU179242ÐEU179244; ␮ ϫ ϫ a 10- l volume containing 5 BSA (Bioline), 10 cytochrome b accession numbers EU179245ÐEU179252; thermopol buffer (Bioline), 2.5 mM MgCl2,1mMof and nuclear SCAR accession numbers EU179253Ð ␮ each dNTP, 10 M of each primer (radiolabeled F EU179258, EU190480, and EU190481. 33 primer with [␥ P]ATP radioisotope), 0.3 U of Taq Karyology and Morphological Measurements. Chro- polymerase (Bioline), and 2 ␮l of template DNA. The mosome squash slides were prepared from fresh em- cycling program for loci R5.29b, R3.171, and R5.50 was bryos following Blackman (1980). The slide prepara- 94ЊC for 3 min, 40 cycles of 94ЊC for 30 s, 60ЊC for 30 s, tions were examined under phase contrast on an 72ЊC for 30 s, followed by 3 min at 72ЊC; and for loci Olympus BH-2 microscope. Four clonal lineages were R5.10, R2.73, and R5.138 was 94ЊC for 2 min, 40 cycles assessed for karyotypes and morphological analysis of 94ЊC for 20 s, 60ЊC for 20 s, 72ЊC for 20 s, followed (Table 1). Approximately 10 slides per morph and by 2 min at 72ЊC. After denaturation, 3 ␮l of the Þnal species were prepared following Blackman and Eastop product was loaded on a 5% polyacrylamide urea gel (2000) and mounted with permanent Euparal (Aus- and subjected to electrophoresis at a constant 65 W in tralian Entomological Supplies Pty. Ltd., Bangalow, 1ϫ Tris-bromide EDTA buffer. After electrophoresis NSW, Australia). These also were reared on barley the gel was dried on blotting paper and exposed to seedlings at 20 Ϯ 1ЊC under a photoperiod of 16:8 autoradiography Þlm (Biomax Kodak, Sigma-Aldrich, (L:D) h. Measurements were taken following Ilharco St. Louis, MO). The size of alleles for each locus was and van Harten (1987) except that the body was mea- estimated using a sequencing size ladder ␭gt11 vector sured from the front of the head to the end of the (fmol DNA Cycle Sequencing System, Promega). eighth abdominal segment. Morphological characters 918 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 6 were measured on both sides of the specimen and containing both sexual and asexual taxa (Delmotte et were as follows: Body length (bl), length of antennal al. 2003). The genetic proÞle was based on two distinct segment III (ant III), length of antennal segment IV cytochrome b haplotypes, named hI and hII for each (ant IV), length of antennal segment V (ant V), length group, respectively. These haplotypes also were de- of antennal segment VI (ant VI), length of processus tected in Australian populations and the same desig- terminalis (pt), antennal ßagellum (af), length hair nation as Delmotte et al. (2003) is followed here, i.e., antennal segment III (hair ant III), diameter of base of R. padi hI and R. padi hII. Furthermore, we found a antennal segment III (base ant III), length ultimate third mitochondrial haplotype, named R. padi hIII rostral segment (urs), length hind femur (hf), length (Fig. 2). longest hair on hind femur (hair hf), length II segment Variation at Mitochondrial and Nuclear Markers. hind tarsus (ht2), length siphunculus (siph), length Individuals representing the six species and R. sp. x cauda (cau), and length setae eighth abdominal seg- were ampliÞed and sequenced for COI and a subset of ment (hair eighth seg). Alatae measurements also in- these also were sequenced for cytochrome b with the cluded the following: no secondary rhinaria on anten- ampliÞcation products being 709 bp for COI and 795 nal segments III, IV, and V (rhi ant III, rhi ant IV, and bp for cytochrome b. Maximum likelihood and parsi- rhi ant V). Slides have been deposited in the Agricul- mony analyses resulted in similar trees for COI, with tural Insect Collection (Department of Primary In- clear separation of the different species (Fig. 2a). dustries) KnoxÞeld, VIC, Australia, and the Natural Rhopalosiphum sp. x grouped with R. near insertum, History Museum in London, United Kingdom. Mor- even though morphologically it is most similar to R. phological measurements were analyzed using a dis- padi. A similar result was also found for cytochrome b, criminant analysis on SPSS 15.0 for Windows (SPSS with the maximum likelihood tree similar to the COI Inc., Chicago, IL). tree (Fig. 2b), despite the inclusion of several other PCR-RFLP on COI. The virtual NEB cutter pro- taxa from GenBank. Again, R. sp. x grouped with the gram (New England Biolabs, Ipswich, MA) (http:// R. insertum group and was clearly differentiated from tools.neb.com/NEBcutter2/index.php) was used to R. padi. COI intraspeciÞc sequence difference varied determine restriction cut sites for each species for from 1.1% (between R. maidis haplotypes) to 1.4% the mitochondrial COI region. Then, eight individ- (between R. padi haplotypes), whereas the levels of uals per population were used for PCR-RFLP in interspeciÞc variation ranged from 2.5% (between R. which 10 ␮l of PCR product was cut in separate sp. x and R. near insertum) to 9.4% (between R. nym- reactions with the endonucleases TaqI, FokI, HinfI, phaeae and R. maidis). For cytochrome b, the levels of and ApoI in a total volume of 20 ␮l for each reaction intraspeciÞc sequence divergence varied from 1.3% and run on a 2.5% agarose gel along with a 50-bp (between R. insertum haplotypes) to 2.4% (between DNA ladder (Promega). The gels were stained in R. maidis haplotypes), whereas interspeciÞc variation ethidium bromide, and visualized and photographed was from 2.7% (between R. sp. x and R. insertum- over UV light. France) to 7.3% (between R. padi-Washington and R. The diagnostic method should combine the results maidis). from all four restriction enzymes to avoid possible The SCAR marker was used to investigate a putative misidentiÞcations from ambiguous fragments that are hybrid origin for the undescribed Rhopalosiphum spe- too small (fragments that are Ͻ80 bp have a poor cies (R. sp. x). We considered a range of putative resolution in agarose gels) or differ only by a few parents after a number of molecular and morpholog- base pairs (see HinfI proÞles for R. nymphaeae and ical criteria that could explain the observed patterns. R. maidis). Based on morphological keys, R. sp. x was identiÞed as R. padi. Thus, because of their morphological resem- blance, we chose R. padi as one of the parental lin- Results eages. However, mitochondrial results seemed to in- Samples of Rhopalosiphum were collected from a dicate that R. sp. x was maternally related to R. near variety of locations and host plants (Table 1). Using insertum. Thus, we explored the idea that this taxon taxonomic keys (Cottier 1953; Eastop 1966; Stroyan could represent the maternal lineage. Furthermore, 1984; Blackman and Eastop 1994, 2000), we identiÞed both taxa have the appropriate chromosome numbers six different species in our samples [R. padi, R. near 2n ϭ 8 for R. padi and 2n ϭ 10 for R. near insertum that insertum, R. ruﬁabdominale, Rhopalosiphum musae could explain the karyotype observed in R. sp.xof (Shouteden), Rhopalosiphum nymphaeae L., and R. 2n ϭ 9. A single copy nuclear SCAR marker was maidis]. Karyotypic analysis of several R. padi samples sequenced in the three R. padi cytochrome b haplo- revealed a unique strain of R. padi collected from corn types (hI, hII, and hIII), R. near insertum, and R. sp. x (Zea mays L.) in several locations. These samples had to investigate the origins of R. sp. x. R. padi haplotypes a karyotype of 2n ϭ 9, whereas other R. padi samples hI and hII were directly sequenced without cloning had the expected karyotype for this species of 2n ϭ 8 (there was no evidence of heterozygous sites when (Hales and Cowen 1990). We refer to this new strain the initial direct sequencing was conducted) How- as R. sp. x (Table 1). ever, R. sp. x, R. near insertum and R. padi haplotype Previous molecular studies in France detected the hIII were Þrst cloned, and the cloned products were presence of two divergent R. padi groups, one obligate sequenced (initial direct sequencing produced mul- asexual group with hybrid origin and another group tiple peaks in several locations indicating heterozy- November 2009 VALENZUELA ET AL.: MOLECULAR CHARACTERIZATION OF AN APHID SPECIES 919

(a) R. near insertum (1, 2, 3, 4) (b) R. near insertum (1, 2, 3, 4) 76 55 79 R. insertum-AJ315894-France R. sp. x (1, 2, 3) R. sp. x (1, 2)

R. musae (1) R. rufiabdominale (1, 2) 88 70 52 R. rufiabdominale (2) 55 R. padi (1, 3, 6) R. cerasifoliae-AJ315896

94 R. padi (3) hI 99 96 79 R. padi (2, 4, 7) 100 70 98 R. padi-AJ315891-France 99 R. padi (5) 98 R. padi (4) hII 100 61 100 R. padi-AJ315883-USA R. musae (1, 2) R. padi-AJ315889-Australia 93 97 R. nymphaeae (1) R. padi-AJ315884-France

R. padi (5) hIII R. maidis (1, 2) R. maidis (4)

R. maidis (France) R. maidis (3, 4) 0.01 substitutions/site 0.01 substitutions/site

R. near insertum (3) allele 2

R. padi (3) 98 98 R. padi-clone B69-AJ010774-France

66 R. padi-clone B71-AJ010774-France

53 R. padi (4)

R. padi (5) allele 1

100 100 R. padi (5) allele 2

R. padi-clone L112-AJ010771-France

65 R. padi-clone B79-AJ010772-France 58 69 R. padi-clone B69 and B71-AJ010773-France

R. sp. x (2) allele 2 100 100 R. sp. x (2) allele 1 0.005 substitutions/site Fig. 2. Phylogenetic relationships among Rhopalosiphum species using a maximum likelihood model for the mitochondrial cytochrome oxidase I gene (a), cytochrome b (b), and nuclear SCAR region (c). Bootstrap values for maximum likelihood (ML) (above) and parsimony (below) are based on a 1,000 replicates with bootstrap Ͻ50% not shown. Numbers in parentheses refer to populations shown in Table 1. Sequences taken from GenBank and used in the analyses are shown with their accession numbers. 920 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 6

Table 2. Microsatellite alleles for Australian R. near insertum, R. padi, and R. sp. x

Rhopalosiphum Locus R 5.29b Locus R 5.10 Locus R 2.73 Locus R 5.50 Locus R 5.138 Locus R 3.171 species R. near insertum (1) 181/189 278/296 272/284 319/349 199/203 232/236 R. padi (3) 161/171 256/260 264/286 307/317 238/249 227/227 R. padi (4) 165/165 260/260 266/272 329/331 211/247 236/282 R. padi (5) 175/179 268/272 246/264 327/341 244/246 Ña R. sp. x (2) 183/185 272/278 268/292 315/351 248/250 214/216

The population numbers shown in Table 1 are in parentheses. Microsatellite lengths are given in base pairs (e.g., 181 bp/189 bp), and all loci contained two alleles. a Alleles did not amplify. gosity). The SCAR product sizes varied from 724 bp to Fig. 3, whereas means, standard deviations, and num- 740 bp. ber of replicates/clonal lineage are shown in the Sup- Heterozygous SCAR alleles were identiÞed for R. plemental Table A.1. near insertum, R. padi haplotype hIII, and R. sp. x, The main discriminant characters that separate apter- whereas R. padi haplotype hI and hII were homozy- ous and alate individuals of R. sp. x and R. padi are: the gous. Fragment (genotype) sizes were, R. near inser- length of the longest measurable hair on the hind femur tum (724/729), R. padi hI (735/735), R. padi hII (736/ (apterous and alate), and the number of secondary rhi- 736), R. padi hIII (737/739), and R. sp. x (739/740). Therefore, no alleles were shared between R. near insertum and R. sp. x, although one allele was shared between R. padi hIII and R. sp. x. The results from the Table 3. Eigenvalues and standardized coefﬁcients for the ﬁrst maximum likelihood and parsimony analyses grouped two canonical discriminant functions based on all morphological alleles within species, with three clear clades repre- characters for apterous and alate clonal lineages of R. near inser- senting the different species (Fig. 2c). Despite sharing tum, R. padi, and R. sp. x an allele of the same size, allele 739 from R. padi hIII and R. sp. x, these taxa were unrelated. The estimated Function 1 2 sequence difference between R. sp. x and the two most Apterae (n ϭ 76) divergent R. padi alleles varied from 4.7 to 6.6% and R. Eigenvalue 98.013 24.569 % of variance 72.296 18.123 near insertum alleles from 2.7 to 3%. Thus, SCAR nu- bl 0.840 Ϫ0.450 clear data indicated that R. sp. x is more closely related ant III ϩ IV 0.505 0.313 to R. insertum than R. padi. ant V Ϫ0.922 1.315 We also determined the genotypes of R. near ant VI 5.038 4.294 pt Ϫ3.205 Ϫ4.742 insertum, the three R. padi clones, and R. sp. x at six pt/ant VI 3.527 2.128 microsatellite loci to further explore the origins of hair ant III Ϫ0.941 Ϫ0.362 R. sp. x. All microsatellite loci ampliÞed in each base ant III 0.103 Ϫ0.750 Ϫ species, with only one locus (R 3.171) not amplify- urs 1.012 0.987 hf 0.452 Ϫ0.294 ing in R. padi hIII. Genotypes across loci clearly hair hf 0.109 Ϫ0.509 differentiated R. near insertum from the R. padi ht2 0.044 Ϫ0.030 clones, with different allelic distributions at most siph Ϫ0.041 0.150 Ϫ loci (Table 2). Rhopalosiphum sp. x was heterozy- cau 0.281 0.118 hair eighth seg Ϫ0.478 Ϫ0.291 gous at all loci and alleles were generally distinct Alatae (n ϭ 62) from R. padi and R. near insertum. Alleles at locus R Eigenvalue 106.84 12.79 5.10 were shared with both R. near insertum (allele % of variance 86.5 10.3 Ϫ 278) and R. padi hIII (allele 272). bl 0.499 0.077 rhi ant III Ϫ1.909 1.501 Morphometrics. Canonical discriminant analysis rhi ant IV Ϫ0.323 Ϫ0.154 showed that apterous and alate R. sp. x are morpho- rhi ant V Ϫ1.380 Ϫ1.024 logically different from R. padi and R. near insertum ant III 2.720 Ϫ1.106 Ϫ clones and that the same morphological characters ant IV 2.338 0.454 ant V 0.182 0.376 differentiated between two haplotypes of R. padi. We ant VI 6.947 3.737 also found that R. near insertum apterae had Þve an- pt Ϫ5.449 Ϫ2.965 tennal segments instead of the usual six found in the rhi ant III/ant III 1.510 Ϫ1.786 genus, with segments III and IV being fused as shown pt/ant VI 4.815 3.056 urs Ϫ0.641 0.758 in Blackman et al. (1987b). Thus, here, we used the hf Ϫ3.340 Ϫ0.150 sum of both antennal segments for all species and hair hf Ϫ1.086 Ϫ2.768 clones (ant III ϩ IV). Eingenvalues and coefÞcients of ht2 0.500 0.533 the Þrst two factors are shown in Table 3. These siph 1.114 0.407 cau 1.010 0.802 accounted for 90.4 and 97% of the total variance for hair eighth seg Ϫ1.438 0.284 apterae and alatae, respectively. A plot of the mean scores of the Þrst two canonical functions is shown in n is the number of measurements per character. November 2009 VALENZUELA ET AL.: MOLECULAR CHARACTERIZATION OF AN APHID SPECIES 921 a R. padi haplotypes hI and hII also can be distin- guished, although differentiation is greater in apterous R. padi hII 5 (Fig. 3a) than alate (Fig. 3b) aphids. Discriminant characters in apterous aphids are the ratio between R. sp. x the processus terminalis and the base of the last antennal segment and, in alate aphids, the density of 0 secondary rhinaria on the third antennal segment. R. near insertum Diagnostic PCR-RFLP of the COI Mitochondrial Gene. Aphid COI sequences (709 bp) were imported

-5 into the virtual restriction digest program from New

Function 2 England Biolabs (NEB cutter) and four enzymes TaqI, FokI, HinfI, and ApoI were selected according to the R. padi hI size and number of DNA fragments scorable on a gel. -10 The predicted restriction proÞles were conÞrmed by running the digest reactions on eight aphids per population. The enzymes differentiated eight haplotypes -20 -15 -10 -5 0 5 10 that corresponded to seven species. The combined Function 1 diagnostic proÞles are shown in Table 4. R. padi and Group Centroid R. maidis were the only species that showed signiÞcant sequence divergence between haplotypes (1.4 and b 1.1%, respectively; Fig. 2a). However, the selected 7.5 restriction enzymes separated R. maidis haplotypes only and not R. padi (hI and hII). R. maidis showed a R. sp. x 5.0 difference in the number and size of the fragments when digested with FokI and ApoI. The diagnosis of species requires the combination of 2.5 all four restriction enzymes so as to avoid misidenti- Þcations of small fragments that have a poor resolution R. near insertum Ͻ Function 2 Function 0.0 in an agarose gel ( 80 bp) and fragments that differ R. padi hI on only a few base pairs. The combination of all DNA fragments leads to a speciÞc identity. -2.5

-5.0 R. padi hII Discussion The results indicate that an undescribed species -20 -15 -10 -5 0 5 10 exists in Rhopalosiphum that is morphologically very Function 1 similar to R. padi but has signiÞcant genetic differen- Group Centroid tiation. Mitochondrial and nuclear regions indicated Fig. 3. Plot of mean scores on the Þrst two canonical that the species, here called R. sp. x, is closely related discriminant functions, based on all morphological charac- to the R. insertum group, which includes taxa from ters for apterous (a) and alate (b) clonal lineages of R. near Europe and Australasia (not included in this study), insertum, R. padi, and R. sp. x. Two cytochrome b mitochon- and karyotypic analysis found the species to be diploid drial haplotypes are shown for R. padi. with 2n ϭ 9. The common Rhopalosiphum diploid chromosome naria on the third antennal segment (alate). It is impor- numbers are 2n ϭ 8 and 2n ϭ 10 (Blackman and Eastop tant to mention that apterous R. padi (hII) and R. sp. x 2006); thus, it seems possible that 2n ϭ 9 could be a are morphologically very similar (Fig. 3a). hybrid. However, after examining mitochondrial COI

Table 4. PCR-RFLP–based method for the discrimination of Rhopalosiphum species

TaqI FokI HinfI ApoI Rhopalosiphum species n 89, 620 56, 111, 197, 345 270, 439 79, 630 Rhopalosiphum near insertum (1Ð4) 28 89, 620 56, 111, 197, 345 79, 270, 360 192, 517 Rhopalosiphum padi (1Ð7) 56 89, 620 111, 253, 345 270, 439 79, 192, 438 Rhopalosiphum musae (1,2) 16 89, 620 253, 456 270, 439 79, 192, 438 Rhopalosiphum sp. x (1,2,3) 24 88, 95, 526 111, 253, 345 117, 270, 322 78, 92, 539 Rhopalosiphum nymphaeae (1) 8 89, 95, 525 111, 253, 345 280, 429 78, 79, 92, 100, 360 Rhopalosiphum maidis (1,2) 16 89, 95, 525 345, 364 280, 429 78, 79, 192, 360 Rhopalosiphum maidis (3,4) 16 89, 303, 317 111, 253, 345 280, 429 78, 79, 192, 360 Rhopalosiphum ruﬁabdominale (1,2) 16

The diagnostic method requires the combination of all DNA fragments from enzymes TaqI, FokI, HinfI, and ApoI, which will lead to a single taxon. n is the total number of aphids screened for RFLP. The population numbers shown in Table 1 are in parentheses. All DNA fragments are given in base pairs. 922 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 6 and cytochrome b sequence data from Australian and chrome oxidase subunit I gene that separated all seven overseas populations, we found no evidence that sup- species of Rhopalosiphum in Australia. IntraspeciÞc ported a recent hybridization event. First, the level of sequence divergence was greatest in R. padi with 1.4% sequence divergence between R. sp. x and putative differentiation between two haplotypes (hI and hII). maternal lineages in the R. insertum group was higher However, the restriction enzymes chosen in this study than levels of intraspeciÞc difference in the group did not separate them and species identiÞcation could itself, i.e., 2.7% between species versus 1.3% within the be reliably resolved with the results from four restric- R. insertum lineages for cytochrome b. Second, the tion enzymes TaqI, FokI, HinfI, and ApoI (Table 3). nuclear SCAR and microsatellite data did not clearly Sequence divergence within R. maidis was 1.1%, and indicate the origins of R. sp. x. For instance, the excess the restriction enzyme FokI separated the two hap- of heterozygous alleles displayed by microsatellite lotypes. It is possible that these haplotypes represent markers can be explained by hybridization or by pro- the two karyotype races present in south east Australia longed parthenogenetic reproduction, as over time, (Hales and Cowen 1990, de Barro 1992). The remain- microsatellites can accumulate mutations that lead to ing Rhopalosiphum species did not show intraspeciÞc genetic differentiation from ancestral alleles (Wilson genetic variation despite different host plants and geo- et al. 2003). Unfortunately, neither microsatellite nor graphical origins. Thus, restriction enzymes TaqI, SCAR markers showed any particular pattern of evo- FokI, HinfI, and ApoI were able to identify Rhopalo- lution from which we could assign R. sp. x origins siphum species, many of which are commonly found in based on the available data. It is important to note that agricultural surveys. only a selected number of taxa were available in this Discriminant function analysis showed that R. padi, study. Consequently, other species and populations R. sp. x and R. near insertum are morphologically not sampled here could still act as putative parental distinct. A range of characters were identiÞed that lineages. allowed their separation for both apterous and alate Several examples have been found in aphids where forms. However, the differences are subtle and only new chromosomal races arise from a single lineage, visible under high magniÞcation on a slide (e.g., the e.g., Sitobion aphids in Australia and New Zealand length of the longest hair on the hind femur). Also, we (Hales et al. 1990, Sunnucks et al. 1996, Wilson et al. only measured specimens derived from a single female 1999), R. maidis (Brown and Blackman 1988), and collected in the Þeld (one clonal lineage per species) Myzus antirrhinii (Macchiati) (Hales et al. 2000). and reared at a constant temperature of 20ЊC and Based on its cryptic morphology and its karyotype, R. photoperiod of 16:8 (L:D) h. Therefore, the analysis sp. x could have been treated as a chromosomal race does not reßect the potential degree of variation of R. padi. But, as shown by the high levels of mito- present in natural populations, but is a valuable Þrst chondrial and nuclear sequence divergence between step toward understanding the role of various mor- the two species (5.2Ð6% for cytochrome b and 4.7Ð6.6% for SCAR), we concluded that R. sp. x is not a new phological characters in species separation. Given that chromosomal race of R. padi but a distinct unde- the data available were only based on one clonal lin- scribed species more genetically similar to the R. in- eage per species, variation in traits from additional sertum group than to R. padi (based on mitochondrial clones would Þrst need to be considered when de- and nuclear data). scribing the species or developing a dichotomous key Reexamination of microscopic slides from earlier for the identiÞcation of R. sp. x. Australian studies on Rhopalosiphum spp. have re- The Þnding of a new cryptic species of Rhopalosi- vealed that R. sp. x might have been in Australia for phum inhabiting corn also has potential implications some time. The current collections are morphologi- for agriculture. Abundant colonies were found in cally indistinguishable from specimens recognized to three consecutive years (2004Ð2007) on sweet corn in transmit the banana mosaic virus recorded by Magee south eastern Australia (I.V., unpublished data). Pre- (1940) as Aphis sp. but corrected to Rhopalosiphum sp. vious records date from Queensland (Magee 1940) by Hughes and Eastop (1991). It is also indistinguish- and New South Wales (1973) on sweet corn (V.F.E., able from the taxon that Hales and Cowen (1990) unpublished data) and from wheat, Triticum aestivum characterized as having a diploid chromosome num- L., and barley grown in a glasshouse (Hales and Co- ber of 2n ϭ 9 and allozyme pattern similar to R. near wen 1990). At present, there is very little biological insertum. The data presented here show a similar pro- information on this cryptic species, including its geo- Þle to that presented in Bulman et al. (2005). New graphic distribution, genetic origins, its capacity to Zealand “R. padi T” and Australian “R. sp. x” share a vector important plant viruses, and its host range. We cryptic morphology and habitat with R. padi and both have conducted a range of preliminary experiments on mitochondrial DNA sequences reveal an association the effects of host plant and temperature on Þtness with the R. insertum group, although their karyotype traits such as fecundity, longevity, time from birth to is different (R. padi Tis2nϭ 8). Despite their simi- onset of reproduction, and intrinsic rate of increase larities, we found that these two taxa showed signiÞ- under controlled laboratory conditions (Valenzuela cant mitochondrial sequence divergence at cyto- 2008). These preliminary experiments seemed to cor- chrome b(Ϸ5.4%). roborate the Þndings from the Þeld where R. sp. x We were able to develop a PCR-RFLP diagnostic clones performed better on sweet corn than on cereals method, based on the genetic variation in the cyto- compared with R. padi clones. November 2009 VALENZUELA ET AL.: MOLECULAR CHARACTERIZATION OF AN APHID SPECIES 923

Acknowledgments Carew, M. E., V. Pettigrove, and A. A. Hoffmann. 2005. The utility of DNA markers in classical taxonomy: using cy- We are grateful to Dinah Hales who provided information tochrome oxidase I markers to differentiate Australia on the hosts and localities from an earlier karyotypic and Cladopelma (Diptera: Chironomidae) midges. Genetics enzyme electrophoresis study (Hales and Cowen 1990). We 98: 587Ð594. thank Michael Nash for the supply of aphids from sweet corn Carew, M. E., V. Pettigrove, R. L. Cox, and A. A. Hoffmann. from Brunswick, VIC, Australia. Funding for the study was 2007. DNA identiÞcation of urban Tanytarsini chirono- provided by the Victorian government Our Rural Land- “ mids (Diptera: Chironomidae). J. N. Am. Benthol. Soc. 26: scape initiative through the award of a postgraduate schol- ” 586Ð599. arship to I.V. Cottier, W. 1953. Aphids of New Zealand. New Zealand Department of ScientiÞc and Industrial Research Bulletin 106: 1Ð382. References Cited Delmotte, F., B. Sabater-Mun˜ oz, N. Prunier-Leterme, A. Latorre, P. Sunnucks, C. Rispe, and J. C. Simon. 2003. de Barro, P. J. 1992. Karyotypes of cereal aphids in South Phylogenetic evidence for hybrid origins of asexual lin- Australia with special reference to Rhopalosiphum maidis eages in an aphid species. Evolution 57: 1291Ð1303. (Fitch) (Hemiptera: Aphididae). J. Aust. Entomol. Soc. Eastop, V. F. 1966. A taxonomic study of Australian 31: 331Ð334. Aphidoidea (Homoptera). Aust. J. Zool. 14: 399Ð592. Blackman, R. L. 1980. Chromosome numbers in the Aphi- Ellis, J. S., M. E. Knight, and D. Goulson. 2005. Delineating didae and their taxonomic signiÞcance. Syst. Entomol. 5: species for conservation using mitochondrial sequence 7Ð25. data: the taxonomic status of two problematic Bombus Blackman, R. L., and V. F. Eastop. 1994. Aphids on the species (Hymenoptera: Apidae). J. Insect Conserv. 9: worldÕs trees: an identiÞcation and information guide. 75Ð83. CAB International in association with the Natural History Ellis, J. S., M. E. Knight, C. Carvell, and D. Goulson. 2006. Museum, Wallingford, United Kingdom. Cryptic species identiÞcation: a simple diagnostic tool for Blackman, R. L., and V. F. Eastop. 2000. Aphids on the discriminating between two problematic bumblebee spe- worldÕs crops: an identiÞcation and information guide, cies. Mol. Ecol. Notes 6: 540Ð542. 2nd ed. Wiley Ltd., Chichester, United Kingdom. Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrijenhoek. Blackman, R. L., and V. F. Eastop. 2006. Aphids on the 1994. DNA primers for ampliÞcation of mitochondrial worldÕs herbaceous plants and shrubs, 1st ed. Wiley Ltd., cytochrome c oxidase subunit I from diverse metazoan Chichester, United Kingdom. invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294Ð299. Blackman, R. L., and V. F. Eastop. 2007. Taxonomic issues, Halbert, S. E. and D. J. Voetglin. 1998. Evidence of North pp. 1Ð22. In H. F. van Emden and R. Harrington [eds.], American origin of Rhopalosiphum and barley yellow Aphids as crop pests. CAB International, Wallingford, dwarf virus, pp. 351Ð356. In J. M. Nieto Nafrõ´a and A.F.G. United Kingdom. Dixon [eds.], Aphids in natural and managed ecosystems. Blackman, R. L., P. A. Brown, and V. F. Eastop. 1987a. Prob- Secretariado de publicaciones, Universidad de Leo´n, lems in pest aphid taxonomy: can chromosomes plus mor- Leon, Spain. phometrics provide some answers?, pp. 233Ð238. In J. ´ Hales, D. F., and R. Cowen. 1990. Genetic studies of Rho- Holman, J. Pelikan, A.G.F. Dixon, and L. Weismann palosiphum in Australia. Acta Phytopathol. Entomol. [eds.], Proceedings, Symposium: Population Structure, Genetics and Taxonomy of Aphids and Thysanoptera, Hung. 25: 283Ð288. 9Ð14 September 1985, Smolenice Czechoslovakia. SPB Hales, D. F., R. L. Chapman, R. M. Lardner, R. Cowen, and Academic Publishing, The Hague, The Netherlands. E. Turak. 1990. Aphids of the genus Sitobion occurring Blackman, R. L., V. F. Eastop, and P. A. Brown. 1987b. The on grasses in southern Australia. J. Aust. Entomol. Soc. 29: biology and taxonomy of the aphids transmitting barley 19Ð25. yellow dwarf virus, pp. 197Ð214. In P. A. Burnett [ed.], Hales, D. F., A.C.C. Wilson, J. M. Spence, and R. L. Blackman. Proceedings, Workshop: World perspectives on Barley 2000. ConÞrmation that Myzus antirrhinii (Machiati) Yellow Dwarf, 6Ð11 July 1987, Udine, Italy. International (Hemiptera: Aphididae) occurs in Australia, using mor- Maize and Wheat Improvement Center, Mexico. phometrics, microsatellite typing and analysis of novel Blackman, R. L., and J. M. Spence. 1994. The effects of karyotypes by ßuorescence in situ hybridisation. Aust. J. temperature on aphid morphology, using a multivariate Entomol. 39: 123Ð129. approach. Eur. J. Entomol. 91: 7Ð22. Hebert, P.D.N., E. H. Penton, J. M. Burns, D. H. Janzen, and Brown, P. A., and R. L. Blackman. 1988. Karyotype variation W. Hallwachs. 2004. Ten species in one: DNA barcoding in the corn leaf aphid, Rhopalosiphum maidis (Fitch), reveals cryptic species in the Neotropical skipper but- species complex (Hemiptera: Aphididae) in relation to terßy Astraptes fulgerator. Proc. Nat. Acad. Sci. U.S.A. 101: host-plant and morphology. Bull. Entomol. Res. 78: 351Ð 14812Ð14817. 363. Heie, O. E. 1986. The Aphidoidea (Hemiptera) of Fen- Bulman, S. R., M.A.W. Stufkens, V. F. Eastop, and D.A.J. noscandia and Denmark. III. Family Aphididae: subfamily Teulon. 2005. Rhopalosiphum aphids in New Zealand. II. Pterocommatinae and tribe Aphidini of subfamily DNA sequences reveal two incompletely described spe- Aphidinae. Fauna Entomol. Scand. 17: 324. cies. N.Z. J. Zool. 32: 37Ð45. Hughes, M. A., and V. F. Eastop. 1991. Aphids associated Carew, M. E., V. Pettigrove, and A. A. Hoffmann. 2003. with damage to banana plants and a corrected identity for Identifying chironomids (Diptera: Chironomidae) for bi- a banana mosaic virus vector (Hemiptera: Aphididae). J. ological monitoring with PCR-RFLP. Bull. Entomol. Res. Aust. Entomol. Soc. 30: 278. 93: 483Ð490. Ilharco, F. A., and A. van Harten. 1987. Systematics, pp. Carew, M. E., M.A.D. Goodisman, and A. A. Hoffmann. 2004. 51Ð76. In A. K. Minks and P. Harrewijn [eds.], Aphids. Species status and population genetic structure of grape- Their biology, natural enemies and control, vol. A. vine eriophyoid mites. Entomol. Exp. Appl. 111: 87Ð96. Elsevier, Amsterdam, The Netherlands. 924 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 6

Kennedy, J. S., M. F. Day, and V. F. Eastop. 1962. A con- satellite loci in the aphid species, Rhopalosiphum padi. spectus of aphids as vectors of plant viruses. Common- Mol. Ecol. Notes 1: 4Ð5. wealth Institute of Entomology, London, United King- Smith, M. A., N. E. Woodley, D. H. Janzen, W. Hallwachs, and dom. P. D. Hebert. 2006. DNA barcodes reveal cryptic host- Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated speciÞcity within the presumed polyphagous members of software for molecular evolutionary genetics analysis and a genus of parasitoid ßies (Diptera: Tachinidae). Proc. sequence alignment. Brief. Bioinform. 5: 150Ð163. Nat. Acad. Sci. U.S.A. 103: 3657Ð3662. Magee, C.J.P. 1940. Transmission of infectious chlorosis or Stroyan, H.L.G. 1984. Aphids-Pterocommatinae and Aphidi- heart-rot of the banana and its relationship to cucumber nae (Aphidini). Handbooks for the identiÞcation of British mosaic. J. Aust. Inst. Agric. Sci. 6: 44Ð47. insects, vol. 2, part 6. Royal Entomological Society of Lon- Nieto Nafrı´a, J. M., M. P. Mier Durante, F. Garcı´a Prieto, and don. London, United Kingdom. N. Pe´rez Hidalgo. 2005. Hemiptera, Aphididae III. Fauna Sunnucks, P., P. R. England, A. C. Taylor, and D. F. Hales. Ibe´rica 28: 1Ð326. 1996. Microsatellite and chromosome evolution of par- Pashchenko, N. F. 1988. Aphidinea-aphids, pp. 546Ð686. In thenogenetic Sitobion aphids in Australia. Genetics 144: P. A. Ler [ed.], Keys for the identiÞcation of insects of the 747Ð756. Soviet Far East, vol. 2, Hemiptera and Heteroptera. Swofford, D. L. 2003. PAUP*: phylogenetic analysis using Nauka, Leningrad, USSR. parsimony (* and other methods). Version 4. Sinauer, Plumb, R. T. 1983. Barley yellow dwarf virusÑa global Sunderland, MA. problem, pp. 185Ð198. In R. T. Plumb and J. M. Thresh Takahashi, R. 1965. Some new and little-known Aphididae [eds.], Plant virus epidemiology: the spread and control of Japan. Insecta Matsumurana 28: 19Ð61. of insect born viruses. Blackwell ScientiÞc Publications, Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Oxford, United Kingdom. CLUSTAL W: improving the sensitivity of progressive Posada, D., and K. A. Crandall. 1998. Modeltest: testing the multiple sequence alignment through sequence weight- model of DNA substitution. Bioinformatics 14: 817Ð818. ing, position-speciÞc gap penalties and weight matrix Raychaudhuri, D. N. 1980. Aphids of north-east India and choice. Nucleic Acids Res. 22: 4673Ð4680. Bhutan. Zoological Society, Calcutta, India. Torikura, H. 1991. Revisional notes on Japanese Rhopalo- Remaudie`re, G., and M. Remaudie`re. 1997. Catalogue des siphum, with keys to species based on the morphs of the Aphididae du Monde Homoptera: Aphidoidea. Institut primary host. Jpn. J. Entomol. 59: 257Ð273. National de la Recherche Agronomique, Paris, France. Valenzuela, I., A. A. Hoffmann, M. B. Malipatil, P. M. Rid- Richards, W. R. 1960. A synopsis of the genus Rhopalosi- land, and A. R. Weeks. 2007. IdentiÞcation of aphid spe- phum Koch in Canada. Can. Entomol. 92: Suppl. 13. cies (Hemiptera: Aphididae: Aphidinae) using a rapid Richards, W. R. 1962. A new species of Rhopalosiphum PCR-RFLP method based on the cytochrome oxidase sub- Koch. Can. Entomol. 94: 969Ð972. unit I gene. Aust. J. Entomol. 46: 305Ð312. Ridland, P. M., and M. Carver. 1987. Rhopalosiphum inser- Valenzuela, I. 2008. A molecular analysis of aphids tum (Walker) (Homoptera: Aphididae) newly recorded (Hemiptera: Aphididae) in south eastern Australia. Ph.D. from Australia. J. Aust. Entomol. Soc. 26: 128. dissertation, The University of Melbourne, Melbourne, Ridland, P. M., R. J. Sward, and R. B. Tomkins. 1988. A Australia. simple rearing system for cereal aphids especially suited Wilson, A.C.C., P. Sunnucks, and D. F. Hales. 1999. Micro- to transmission studies with barley yellow dwarf viruses. evolution, low clonal diversity and genetic afÞnities of Australas. Plant Pathol. 17: 18Ð19. parthenogenetic Sitobion aphids in New Zealand. Mol. Schiffer, M., M. E. Carew, and A. A. Hoffmann. 2004. Mo- Ecol. 8: 1655Ð1666. lecular, morphological and behavioural data reveal the Wilson, A.C.C., P. Sunnucks, and D. F. Hales. 2003. Heri- presence of a cryptic species in the widely studied Dro- table genetic variation and potential for adaptive evolu- sophila serrata species complex. J. Evol. Biol. 17: 430Ð442. tion in asexual aphids (Aphidoidea). Biol. J. Linn. Soc. 79: Simon, J. C., N. Leterme, and A. Latorre. 1999. Molecular 115Ð135. markers linked to breeding system differences in segre- Yeh, H. T., C. C. Ko, T. C. Hsu, and C. J. Shih. 2005. PCR- gating and natural populations of the cereal aphid Rho- RFLP technique for identiÞcation of Rhopalosiphum palosiphum padi L. Mol. Ecol. 8: 965Ð973. (Aphididae). Form. Entomol. 25: 33Ð46. Simon, J. C., N. Leterme, F. Delmotte, O. Martin, and A. Estoup. 2001. Isolation and characterization of micro- Received 27 October 2008; accepted 9 April 2009.