SYSTEMATICS Molecular Validation of a Morphological Character for Distinguishing Between the Armored Scale pinifoliae and Chionaspis heterophyllae (Hemiptera: )

DOMINIC E. PHILPOTT, STEWART H. BERLOCHER, ROBERT F. MITCHELL, AND LAWRENCE M. HANKS1

Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

Ann. Entomol. Soc. Am. 102(3): 381Ð385 (2009) ABSTRACT The armored scale insects Chionaspis pinifoliae (Fitch) and Chionaspis heterophyllae Cooley (Hemiptera: Diaspididae) overlap broadly in host range and geographic distribution and are so similar in morphology that they can be distinguished only by a subtle morphological character of the adult females: the form of the median pygidial lobes. However, this character is quite variable for both species. We used allozyme electrophoresis and DNA sequencing of a region that included the mitochondrial genes COI and COII to determine whether two species really exist and, if so, whether the morphology of the pygidial lobes is a reliable character to use to discriminate between them. Material for genetic analysis was collected as third instar females from Þve Illinois populations of each species (as identiÞed by morphology of the pygidial lobes). Chionaspis species were difÞcult to analyze electrophoretically, but Þxed allelic differences were found at three allozyme loci: malic acid dehy- drogenase (Mdh), phosphoglucose isomerase (Pgi), and an esterase (Est). The 572-bp (alignment length) mitochondrial region was invariant within species but differed between the species for both base substitutions (p-distance ϭ 0.091) and insertion-deletion differences. Estimated divergence time was at least 3.8 million yr. The consistent absence of heterozygotes at the three allozyme loci and the large mitochondrial sequence difference conÞrms that the morphology of the pygidial lobes is a reliable and convenient character for identifying C. pinifoliae and C. heterophyllae and supports the hypothesis that they are not only separate, but very old species.

KEY WORDS phytophagous , morphology, systematics, allozyme, mitochondrial DNA

The armored scale insects Chionaspis pinifoliae (Fitch) lae is conÞned to the eastern and midwestern states, and Chionaspis heterophyllae Cooley (Hemiptera: Di- where it co-occurs with C. pinifoliae (Shour and aspididae) are important pests of conifers throughout Schuder 1987, Kosztarab 1996). Both species are bi- North America (Johnson and Lyon 1988). They are so voltine throughout most of their ranges, and their similar in morphology that they can be distinguished crawlers emerge concurrently in spring, but subse- only by a subtle morphological character of the adult quent generations become asynchronous because C. females: the form of the median pygidial lobes (Liu et pinifoliae develops more slowly than C. heterophyllae al. 1989). Mesal margins of the median lobes of C. (Shour 1986). pinifoliae are more or less parallel and are narrowly Morphological and biological similarities between separated, whereas those of C. heterophyllae are C. pinifoliae and C. heterophyllae have led to taxo- widely divergent and more broadly separated. How- nomic confusion in previous publications (Nielsen ever the morphology of the lobes is quite variable in and Johnson 1972, 1973; Walstad et al. 1973; Shour both species (Liu et al. 1989). 1986; Fondren and McCullough 2005; Miller and Da- C. pinifoliae and C. heterophyllae also are very sim- vidson 2005). Such errors may hinder the develop- ilar in their host range and life history, and they over- ment of effective management strategies. In particu- lap broadly in geographical distribution, further com- lar, the timing of crawler emergence is critical for plicating their identiÞcation (Liu et al. 1989). Both managing armored scale insects because the crawler species feed on needles of Abies, Picea, and Pinus stage is the most vulnerable to pesticides (Nielsen and species and can occur on the same individual host Johnson 1972). Pest managers can more accurately plants (Shour and Schuder 1987, Kosztarab 1996). C. predict when crawlers will emerge if they have cor- pinifoliae is distributed throughout much of North rectly identiÞed the scale species. America and Northern Mexico, whereas C. heterophyl- The objective of our study was to use allozymes and mitochondrial DNA sequences to conÞrm that the 1 Corresponding author, e-mail: [email protected]. morphology of the median pygidial lobes is a reliable

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Fig. 1. Representative pygidia of adult female scale insects identiÞed by morphology of the median lobes (enclosed in black boxes). (A) C. pinifoliae (mesal margins of median lobes Ϸ parallel, space between lobes narrow). (B) C. heterophyllae (mesal margins of median lobes divergent, space between lobes broader). MagniÞcation, 40ϫ. character to use to distinguish between the two spe- 120, Thermo Fisher ScientiÞc, Waltham, MA) and cies. Allozyme electrophoresis has for decades been stored them in a Ϫ80ЊC freezer. We later removed the used to discriminate between difÞcult species because pygidium of each scale and slide-mounted it by placing it allows rapid search for Þxed allele differences, the it in a drop of mineral oil, covering it with a glass criterion for species status under the biological species coverslip, and applying clear Þngernail polish around concept (Murphy et al. 1996, Avise 2004). More re- the edges to seal the coverslip. The remainder of each cently, sequencing of mitochondrial DNA has become then was transferred to a well of an eight- a standard taxonomic method because of the relative well sample plate (all electrophoresis equipment from ease of obtaining DNA sequences (Avise 2004). Helena Laboratories, Beaumont, TX). We assigned a species name (C. pinifoliae or C. heterophyllae) to each scale insect individual by using the pygidial lobe char- Materials and Methods acter (Liu et al. 1989; Fig. 1). We collected scale insects from pine trees that were Enzyme electrophoresis of individual scale insects scattered in urban habitats of the cities of Champaign used the cellulose acetate method of Herbert and and Urbana, IL (Champaign County), and used the Beaton (1989) because it allows analysis of very small pygidial lobe character to assign populations to either insects. For each run, we assigned four individuals of C. pinifoliae or C. heterophyllae. We selected Þve of one presumed species from one population to the Þrst these populations per scale species for this study (Phil- four wells of the sample well plate, and four individ- pott 2007), and each population apparently comprises uals of the other species from one population to the a single species based on pygidial morphology. Pop- remaining wells. This process was repeated until 48 ulations that were identiÞed as C. pinifoliae were on individuals had been assayed per population. Insects Pinus mugo Turra (N ϭ 2 trees), Pinus sylvestris L. in the wells were homogenized with the bulb of an (N ϭ 2), and Pinus nigra Arnold (N ϭ 1), whereas all insect pin. The homogenate was stamped onto cellu- populations that were identiÞed as C. heterophyllae lose acetate plates (Titan III, Helena Laboratories) at were on P. mugo. Nearest neighbor distances among two positions with an applicator (model Super Z, study trees were 2.1 Ϯ 1.1 km (mean Ϯ SD). eight-sample applicator, Helena Laboratories) several We collected Ͼ50 third-instar female scale insects times to transfer sufÞcient homogenate. Runs were per- from each population during summer 2007. The in- formed for 20 min with an ice pack on top of the sects were collected from the most heavily infested apparatus. We screened nine loci: phosphoglucose parts of conifers by collecting entire needles and re- isomerase (Pgi, EC 5.3.1.9), malic acid dehydrogenase moving the bodies from beneath their tests under a (Mdh, EC 1.1.1.37), alcohol dehydrogenase (EC dissecting microscope. We placed the insects individ- 1.1.1.1), isocitrate dehydrogenase (EC 1.1.1.42), ually into microcentrifuge tubes (catalog no. 5-408- phosphoglycerate mutase (EC 5.4.2.1), glycerol-3- May 2009 PHILPOTT ET AL.: MOLECULAR VALIDATION OF A MORPHOLOGICAL CHARACTER 383

Table 1. Allele frequencies for two species of Chionaspis that were identified using morphology of the median pygidial lobe

Site Chionaspis species N Mdh90 Mdh100 N Pgi80 Pgi100 N Est100 Est110 1 Pinifoliae 44 0 1 0 4 1 1 2 Pinifoliae 48 0 1 0 0 3 Pinifoliae 43 0 1 20 0 1 0 4 Pinifoliae 36 0 1 0 0 5 Pinifoliae 27 0 1 8 0 1 18 1 0 6 Heterophyllae 47 1 0 0 0 7 Heterophyllae 44 1 0 0 4 0 1 8 Heterophyllae 35 1 0 0 0 9 Heterophyllae 44 1 0 20 1 0 0 10 Heterophyllae 27 1 0 8 1 0 20 0 1

Specimens were collected from individual trees at Þve different study sites per scale insect species. phosphate dehydrogenase (EC 1.1.99.5), lactate at least an approximate molecular clock does exist dehydrogenase (EC 1.1.2.3), glucose-6-phosphate (Arbogast et al. 2002). We used the widely cited clock dehydrogenase (EC 1.1.1.49), and a presumed a calibration for insect mitochondria of Brower (1994) carboxylic esterase (Est, EC 3.1.1.1; e.g., Mouche` set because it is based on several parts of the mitochon- al. 1986). Plates were imaged with a digital camera. drial genome (no direct calibration of the COI-COII We calculated the relative mobility of each band per intergenic spacer for insects is available). We used insect species by dividing the average mobility for C. both uncorrected p-distance and an HKY-gamma cor- heterophyllae by that for C. pinifoliae and then aver- rection for multiple substitutions suggested by a re- aged the relative mobilities of the bands for each allele cent study (Meraner et al. 2008) that included another across all populations. Alleles were named by their noncoding mitochondrial segment, the control region. average relative mobilities. We extracted genomic DNA from a single scale of Results and Discussion each population (for a total of 10 scales, Þve per species) by using DNeasy blood & tissue kits (QIAGEN, Valen- Chionaspis proved to be difÞcult material for en- cia, CA). Primers c1-J-2753ywr (Provencher et al. zyme electrophoresis; only three of the nine tested 2005) and c2-N-3662 (Simon et al. 1994) were used to loci produced strong, clear bands in at least some amplify and sequence initial fragments of the mi- individuals, and many insects had to be eliminated tochondrial genes COI and COII. Fragments pro- from the data. These resolution problems may have duced by these primers ampliÞed inconsistently, but been because of enzyme inhibition by precursors in from them we designed the more speciÞc primers the wax glands. However, for the loci that did produce C3 (5ЈCATTAGGATCAATAATAACAAT3Ј) and satisfactory bands, the results were unambiguous: the C4 (5ЈCATGAATGAATTACATCTATAG3Ј) that two presumed species were completely Þxed for dif- very consistently produced strong polymerase chain ferent alleles (Table 1). Staining by the cathodally reaction (PCR) ampliÞcations. migrating Mdh was the most consistent, so that it could PCR was performed in 100 ␮l with Þnal concentra- serve as a molecular marker for identifying these spe- ϫ ␮ tions of 1 PCR buffer, 1.5 mM MgCl2, 100 M dNTPs, cies in future studies, for example, in crawlers. The 400 nM of each primer,1UofTaq Polymerase (In- results for the DNA analysis are also unambiguous: the vitrogen, Carlsbad, CA), and 1 ␮l of template. The two species show no intraspeciÞc variation but show reaction was heated at 94ЊC for 2 min, followed by 40 multiple Þxed differences between the species. In the cycles of 1 min at 94ЊC (denaturation), 1 min at 39ЊC 572 bp of aligned sequence there were 51 base differ- (annealing), and 1 min at 76ЊC (extension). For se- ences and 2 indel differences (Fig. 2). This corre- quencing, we concentrated the product to 40 ng/␮l sponds to an uncorrected divergence of 0.091 base and used 1 ␮l in a second reaction with a BigDye differences per base (p-distance) or 0.113 bases per terminator kit (Applied Biosystems, Foster City, CA) base when corrected for multiple substitutions (MKY- performed at 96ЊC for 1 min and 40 cycles of 30 s at gamma distance). Even accepting the irregularities in 96ЊC,30sat41ЊC, and 4 min at 60ЊC. Product from the the molecular clock, this implies that these are very old BigDye reaction was sequenced with an automatic species, with a divergence date of 3.8 million yr ob- DNA sequencer (3730xl DNA analyzer, Applied tained from applying the Brower (1994) calibration to Biosystems). Sequences have been submitted to uncorrected p-distance. GenBank/European Molecular Biology Laboratory Our experiment unambiguously supports the hy- (accession number for P. pinifoliae, FJ498792; for P. potheses that 1) C. pinifoliae and C. heterophyllae are heterophyllae, FJ498791). good species and 2) that the morphological character We aligned sequences using ClustalW (Thompson that has been used to distinguish between (the form et al. 1994) and conÞned our data to 574 bp (aligned of the median pygidial lobe) is reliable. Agreement length) that sequenced clearly across all 10 samples. between the allozyme and DNA sequence results and Estimation of divergence times from DNA sequence the morphology was complete, with no exceptions. data has been controversial, but the consensus is that These Þndings conÞrm that entomologists can make 384 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 3

Fig. 2. Sequence alignments for 572 bases of a mitochondrial region including COI and COII for the scale species C. heterophyllae (ChH) and C. pinifoliae (ChP). Asterisks represent identical nucleotides and dashes represent gaps. simple slide mounts of the pygidia of scale insect inferred from patterns of mitochondrial DNA evolution. specimens and assign them to C. pinifoliae or C. het- Proc. Natl. Acad. Sci. U.S.A. 91: 6491Ð6495. erophyllae with a high probability of accuracy. Fondren, K. M., and D. G. McCullough. 2005. Horticultural oil for control of Chionaspis heterophyllae (Homoptera: Diaspididae) in Christmas tree plantations. J. Econ. En- Acknowledgments tomol. 98: 1603Ð1613. Herbert, P.D.N., and M. J. Beaton. 1989. Methodologies for We thank L. E. Graham, E. C. Kluger, E. S. Lacey, A. M. allozyme analysis using cellulose acetate electrophoresis. Ray, P. F. Reagel, M. L. Richardson, H. M. Robertson, M. Helena Laboratories, Beaumont, TX. Allen, and K.K.O. Walden for assistance with various aspects Johnson, W. T., and H. H. Lyon. 1988. Insects that feed on of this study; and J. B. WhitÞeld for comments on an early trees and shrubs. Cornell University Press, Ithaca, NY. draft of the manuscript. We gratefully acknowledge funding Kosztarab, M. 1996. Scale insects of northeastern North support from the Alphawood Foundation. America: identiÞcation, biology, and distribution. Vir- ginia Museum of Natural History, Special Publication 3. References Cited Virginia Museum of Natural History, Martinsville, VA. Liu, T., M. Kosztarab, and M. Rhoades. 1989. Biosystematics Arbogast, B. S., S. V. Edwards, J. Wakeley, P. Beerli, and J. B. of the adult female of the genus Chionaspis (Homoptera: Slowinski. 2002. Estimating divergence times from mo- Coccoidea: Diaspididae) of North America, with empha- lecular data on phylogenetic and population genetic sis on polymorphism. Virginia Polytechnic Institute and timescales. Annu. Rev. Ecol. Syst. 33: 707Ð740. State University, Virginia Agricultural Experiment Sta- Avise, J. C. 2004. Molecular markers, natural history, and tion Bulletin 88-2: 1Ð126. evolution, 2nd ed. Sinauer, Sunderland, MA. Meraner, A., A. Brandsta¨tter, R. Thaler, B. Aray, M. Unter- Brower, A.V.Z. 1994. Rapid morphological radiation and lechner, H. Niedersta¨tter, W. Parson, R. Zelger, J. Dalla convergence among race of the butterßy Heliconius erato Via, and R. Dallinger. 2008. Molecular phylogeny and May 2009 PHILPOTT ET AL.: MOLECULAR VALIDATION OF A MORPHOLOGICAL CHARACTER 385

population structure of the codling moth (Cydia Provencher, L. M., G. E. Morse, A. R. Weeks, and B. B. pomonella) in central Europe: I. Ancient clade splitting Normark. 2005. Parthenogenesis in the Aspidiotus nerii revealed by mitochondrial haplotype markers. Mol. Phy- complex (Hemiptera: Diaspididae): a single origin of a logenet. Evol. 48: 825Ð837. worldwide, polyphagous lineage associated with Car- Miller, D. R., and J. A. Davidson. 2005. Armored scale insect dinium bacteria. Ann. Entomol. Soc. Am. 98: 629Ð635. pests of trees and shrubs (Hemiptera: Diaspididae). Com- Shour, M. H. 1986. Life history studies of the pine scale, stock Publishing Associates, Cornell University Press, Chionaspis heterophyllae Cooley, and the pine needle Ithaca, NY. scale, Chionaspis pinifoliae (Fitch). Ph.D. dissertation, Mouche`s, C., N. Pasteur, J. B. Berge´, O. Hyrien, M. Raymond, Purdue University, West Lafayette, IN. B. R. de Saint Vincent, M. de Silvestri, and G. P. Shour, M. H., and D. L. Schuder. 1987. Host range and Georghiou. 1986. AmpliÞcation of an esterase gene is geographic distribution of Chionaspis heterophyllae responsible for insecticide resistance in a California Culex Cooley and C. pinifoliae (Fitch) (Homoptera: Diaspidi- mosquito. Science (Wash., D.C.) 233: 778Ð780. dae). Indiana Acad. Sci. 96: 297Ð304. Murphy, R. W., J. W. Sites, Jr., D. G. Buth, and C. H. Haufler. Simon, C., F. Frati, A. Beckenbach, B. Crespi, H. Liu, and P. 1996. Proteins: isozyme electrophoresis, pp. 51Ð120. In Flook. 1994. Evolution, weighting, and phylogenetic D. M. Hillis, C. Mortiz, and B. K. Mable [eds.], Molecular utility of mitochondrial gene sequences and a compila- systematics. Sinauer, Sunderland, MA. tion of conserved polymerase chain reaction primers. Nielsen, D. G., and N. E. Johnson. 1972. Control of the pine Ann. Entomol. Soc. Am. 87: 651Ð701. needle scale in central New York. J. Econ. Entomol. 65: Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. 1161Ð1164. CLUSTAL W: improving the sensitivity of progressive Nielsen, D. G., and N. E. Johnson. 1973. Contribution to the multiple sequence alignment through sequence weight- life history and dynamics of the pine needle scale, Phe- ing, positions-speciÞc gap penalties and weight matrix nacaspis pinifoliae, in central New York. Ann. Entomol. choice. Nucleic Acids Res. 22: 4673Ð4680. Soc. Am. 66: 34Ð43. Walstad, J. D., D. G. Nielsen, and N. E. Johnson. 1973. Effect Philpott, D. E. 2007. Validation of a morphological charac- of the pine needle scale on photosynthesis of Scots pine. ter for distinguishing between the armored scale insects For. Sci. 19: 109Ð111. Chionaspis pinifoliae (Fitch) and C. heterophyllae Cooley (Hemiptera: Diaspididae). M.S. thesis, University of Illi- nois at Urbana-Champaign, Urbana. Received 28 November 2008; accepted 10 February 2009.