Biological Control 39 (2006) 9–18 www.elsevier.com/locate/ybcon
Possible geographic origin of beech scale, Cryptococcus fagisuga (Hemiptera: Eriococcidae), an invasive pest in North America
a,b a,b, a a Rodger A. Gwiazdowski , Roy G. Van Driesche ¤, Adrienne Desnoyers , Suzanne Lyon , San-an Wu c, Naotoa Kamata d, Benjamin B. Normark a,b
a Department of Plant, Soil, and Insect Sciences, University of Massachusetts, Amherst, MA, USA b Graduate Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, MA, USA c Beijing Forestry University, Beijing, People’s Republic of China d Kanazawa University, Kanazawa, Ishikawa, Japan
Received 14 November 2005; accepted 14 April 2006 Available online 25 April 2006
Abstract
Beech scale, Cryptococcus fagisuga Lindinger, is invasive in North America. The immediate source of the introduction was Europe, but its native range may be diVerent. Knowledge of the native range is useful when searching for coevolved natural enemies for classical bio- logical control. We report results of a search for the native range of C. fagisuga, using historical records, Weld surveys, and molecular phy- logenetics. Beech scale feeds exclusively on beech. We review historical accounts of movement of species of Fagus between Europe, Asia, and North America and report on extensive surveys for C. fagisuga on Fagus species in China and Japan. We undertook a phylogeo- graphic study of C. fagisuga throughout its known range using sequences of the mitochondrial gene cytochrome oxidase I (COI). We also investigated the phylogenetic relationships of C. fagisuga to other species of Cryptococcus and related species in the Eriococcidae, using ribosomal DNA (18S). For COI sequences within C. fagisuga, we found one widespread, most-common haplotype in North America, Europe, Turkey, and Georgia; a diversity of slightly (0.1–0.5%) divergent haplotypes in Bulgaria; a diversity of moderately (2.2–2.8%) divergent haplotypes in Georgia and Turkey; and a highly (3.6–4.2%) divergent group of haplotypes in Iran. Phylogenetic analysis of 18S places C. fagisuga within a cosmopolitan clade of eriococcids feeding on other temperate trees (ash, maple, and southern beech). Based on the phylogeographic study, we suggest that the subspecies F. sylvatica orientalis is the native host of C. fagisuga and that natural enemies are best sought on oriental beech in northeastern Greece, the Black Sea drainage basin, the Caucasus Mountains, and northern Iran. 2006 Elsevier Inc. All rights reserved.
Keywords: Beech scale; Cryptococcus fagisuga Lindinger; Area of origin; Invasive pest; Fagus grandifolia; Fagus sylvatica; CO1; Haplotype diversity; Phylogeography
1. Introduction over most of the species’ range due to beech bark disease. This disease is caused by an invasive insect, Cryptococcus 1.1. History of beech scale in North America and Europe fagisuga Lindinger, and two associated fungal plant patho- gens, Nectria coccinea var. faginata Lohm. and Watson and From the end of the Nineteenth Century to the present Nectria galligena Bres. An epidemic of tree mortality, day, stands of American beech (Fagus grandifolia Ehrhart), caused by this disease, moved from the Canadian Mari- a common tree species in eastern deciduous forest of North times starting in about 1890 (Hewitt, 1914), west and south America, have been degraded in health and average size (Hawboldt, 1944; Houston, 1994a), reaching Michigan (O’Brien et al., 2001) and North Carolina in the Appala- * Corresponding author. Fax: +1 413 545 2115. chian Mts. (Houston, 1994b) by the end of the 20th century. E-mail address: [email protected] (R.G. Van Driesche). Currently, healthy older trees occur in limited areas, mostly
1049-9644/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.04.009 10 R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18 in the southern and western parts of the tree’s range. Both Steph, and a cecidomyiid in the genus Lestodiplosis (Baylac, the scale and the principal pathogen (N. c. faginata) are 1980). A study of Lestodiplosis sp. indicated it did not invasive species of uncertain origin (Plante et al., 2002). A control C. fagisuga (Baylac, 1986). Beech scale has an comparative molecular study, using nuclear and mitochon- unusually small adult body size (0.5–1.0 mm body length drial markers, found that this pathogen was more closely [Kosztarab, 1996]), but this alone cannot explain the related to European than to American populations of its absence of parasitoids, because Cryptococcus williamsi closest relative, N. coccinea var. coccinea and suggested that Kosztarab and Hale (0.61–0.86 mm) is attacked by parasit- the pathogen was introduced from Europe (Mahoney et al., oids (Coccophagoides sp., Eulophidae) (Kosztarab and 1999). In this research, we have examined the hypothesis Hale, 1968). that beech scale was invasive Wrst in Europe and secondar- From a review of the early literature, Ehrlich (1934) ily in North America. An alternative hypothesis not dealt noted that there was a severe, tree-killing epidemic of beech with in our research is the possibility that Europe might bark disease in England between 1850 and 1900. In the have been invaded not by the scale but by the pathogen, 1840s, beech scale was described as a “rapidly spreading” N. c. var. faginata, perhaps brought in on horticultural pest (M’Intosh, 1849). The Rev. Wilks wrote that “We fear material. the Beech is doomed all over the country” (Ehrlich, 1934). The scale is believed to have entered Nova Scotia about A Mr. Walker from near London stated that “1847 was I 1890 on seedling beech trees brought to the Halifax Public think the Wrst year in which a cottony substance appeared Garden (Hewitt, 1914), most likely from Europe, where the on the trunks of beech trees” (Hardy, 1850). These reports scale has been known since 1832 (Fries, 1832), feeding on the imply that beech scale was a new problem not previously European beech species Fagus sylvatica L. It is unclear, how- familiar to people in England in the last half of the 19th ever, whether Europe is the native range of C. fagisuga, or century (Ehrlich, 1934). In France, beech scale was not perhaps just a previously invaded region. Because knowledge recorded until 1908 (Viennot-Bourgin, 1935), which is of the native range of an invasive pest is useful in classical rather late for a native species, given that it had been killing biological control programs, we conducted surveys on Asian trees in England for over 50 years. In 1921, it was noted beech species and molecular investigations of the scale and that beech scale was increasing its range in Holland (Anon, its relatives to assess the possibility of a non-western Euro- 1921) and at approximately the same time (Anon, 1926) pean origin of beech scale. We explored three alternative pos- was known from only one location in Ireland. These sibilities, all based on the geography of the host genus, Fagus, reports seem inconsistent with C. fagisuga being a wide- and the historically earliest known host, Fagus sylvatica. C. spread native European species. Indeed, an 1862 letter read fagisuga feeds on more than one species of beech, but is not before the Botanical Society of Edinburgh on deaths of known to feed regularly on any other plant genera. We con- beech trees asked “Can it [the beech scale] have been intro- sidered three other possible origins: (1) China, where the duced from abroad on foreign beech plants?” (Anon, 1862). genus Fagus apparently originated and where its species Movement of beech trees is known to be a mechanism diversity is greatest (Peters, 1997); (2) Japan, where the sister able to move beech scale. Seven instances are recorded in species (Fagus crenata Blume) of Fagus sylvatica is found which national or regional beech scale infestations were (Denk, 2003); and (3) western Asia, where a subspecies Wrst noted in a botanical garden or park: Canada (Hewitt, (Fagus sylvatica orientalis Lipsky) of F. sylvatica is found. 1914), the United States, both in Massachusetts (Ehrlich, We also investigated the phylogenetic aYnities of beech 1932) and Ohio (Houston, 1994a), Ireland (Carpenter, scale to other scales currently placed in the genus Crypto- 1903), Scotland (Ehrlich, 1934), Germany (Baerensprung, coccus to see if beech scale might be particularly close to 1849), and Spain (Soria et al., 1993). American beech was any other species in the genus, since the members of the taken to Britain as early as 1766 and F. sylvatica orientalis genus do not cluster geographically. We placed our results had likely been imported from western Asia by 1880 (Bean, within the framework of a previous phylogenetic analysis 1976). East Asian beeches are not recorded as present in of the Eriococcidae by Cook and Gullan (Cook et al., 2002; Britain before 1892 (David Gardner, Kew Gardens, UK, Cook and Gullan, 2004). pers. comm.), but earlier introductions are possible because extensive planting of several exotic beech species was 1.2. Is western Europe the native range of C. fagisuga? underway by 1750 (Ehrlich, 1934). If not native to Europe, where exactly beech scale came from is open to speculation. In western Europe, natural enemies of beech scale are all The scale is unknown from East Asia, but in Western Asia generalist predators and this lack of more specialized para- it is recorded from Iran, Turkey, and the Caucasus Mts. sitoids argues against this being the native range. Natural (Adeli and Soleimanì, 1976), where it infests F. sylvaticus enemies found in Germany were the coccinellids Chilocorus ssp. orientalis. stigma Say, Chilocorus renipustulatus Scriba, and Exocho- mus quadripustulatus (L.) (Schwenke, 1972). Natural ene- 1.3. Hypotheses about the location of a species’ native range mies in France were these last two coccinellids, plus the anthocorid Temnosthetus gracilis Horváth, the neuropter- Knowing the native range of an invasive pest is an ans Chrysopa Xavifrons Br. and Sympherobius elegans important clue as to where coevolved natural enemies asso- R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18 11 ciated with the pest might exist. However, determining the in Britain it would have to have been introduced a few native range of an invader is not straightforward. If the pest decades earlier, but such an earlier introduction that has is known from multiple locations, molecular tools, allow not been recorded is at least plausible. comparison between samples from the invaded range and other locations (e.g., Williams et al., 1994; Biron et al., 2000; 1.3.2. The native range is where the closest relatives of the Goolsby, 2004). Using this approach, hemlock woolly adel- pest species occur gid (Adelges tsugae Annand), which is invasive in the east- A second approach to understanding the geographic ori- ern United States and known from the western United gin of a species is to identify either the area with the most States, Japan, and China was determined to have invaded closely related taxa or the location where sister species from Japan (Havill et al., 2006). Other suggestions as to occur. However, the genus Cryptococcus contains only Wve where an insect’s native range might occur include (1) other species, which do not cluster in any single region (one where the host plant evolved, (2) where the insect species’ being found in Europe, one in China, one in the Russian closest relatives occur, and (3) where the species shows the Far East, one in New Zealand, and one in eastern North greatest genetic diversity. America). Also, the genus has few diagnostic characters (Miller and Miller, 1993) and may be an artiWcial grouping 1.3.1. The native range is the location where the host plant of unrelated species (Miller et al., 1998). One of our objec- group evolved tives was a molecular phylogenetic analysis of Cryptococcus Of the 13 species of Fagus worldwide, 11 occur in East species and of related eriococcid genera to see which species Asia compared to only one in western Asia and Europe (F. might be closest to beech scale. sylvatica) and one in North America (F. grandifolia) (Peters, 1997). There are three species in Japan (F. crenata 1.3.3. The native range is where the pest species shows the Blume, F. japonica, Maximowicz, F. okamotoi Shen) and greatest genetic diversity eight in China (F. hayatae Palibin, F. engleriana Seemen, F. A third approach to Wnding the native range of species is longipetiolata Seeman, F. brevipetiolata Hu, F. tientaiensis to determine where the species shows greatest genetic varia- T. N. Liou, F. bijiensis C. F. Wei and Y. T. Chang, F. lucida tion (e.g., ScheVer and Grissell, 2003). To assess geographic Rehder and Wilson, and F. chienii Cheng). China is patterns of genetic diversity in beech scale, beech scale were believed to be the area of origin of the genus (Denk, 2003). collected from across the invaded range in North America In Europe, Fagus sylvatica, is the host of beech scale and and the variation of the COI gene in those populations was this species has two subspecies, F. sylvatica sylvatica and F. compared to that from beech scales from Europe and west- sylvatica orientalis (Denk, 2003). F. s. sylvatica occurs ern Asia. Our assumption was that variation would be low- throughout most of Europe. F. s. orientalis is found from est in the invaded range(s) and highest in the native range. northern Greece and Bulgaria to northern Iran, including most of the Black Sea drainage basin and the Caucasus 1.4. Objectives of our study Mountains (Davis, 1982). Assuming that beech scale is not native to western This study’s goal was to determine the most likely native Europe, an East Asian origin should be considered, given range of beech scale. The null hypothesis is that the native the diversity of East Asian beeches and the early contact range is within Europe, as generally assumed. The hypothe- between East Asia countries and European plant collectors, ses we test are (1) a Chinese origin, (2) a Japanese origin, which started as early as 1542 when the Portugese gained and (3) a western Asian origin. For any non-European ori- access to Nagasaki, Japan. In 1830, Philipp von Siebold, an gin, we see transportation on beech specimens shipped by employee of the Dutch East India Company, returned to plant collectors for use in Europe as the most likely means Holland in 1830 with a cargo of live Japanese plants (von by which the scale reached Europe. We tested each of these Siebold, 1856). To ascertain if beech scale might be a low origin hypotheses by direct surveys seeking C. fagisuga density, non-pest species in East Asia that had been over- populations on beech, followed (where C. fagisuga was looked, one of us (Dr. Sanan Wu) visited stands of various found) by sequencing of mitochondrial DNA to assess species of beech in China to survey for beech scale. Another genetic diversity. The mitochondrial sequence diversity of of us (Dr. Naoto Kamata) did the same in Japan. That such Asian populations of beech scale was then compared to scales might exist and have been overlooked seemed possi- that found in Europe and North America, using an intra- ble, given that another species of Cryptococcus, C. ulmi speciWc phylogenetic analysis. Tang and Hao, was discovered and described in China in 1995 (Tang and Hao, 1995). 2. Materials and methods A second possible non-western European origin that can be hypothesized based on Fagus biogeography is that the 2.1. Surveys and collection of beech scale scale originated in eastern Europe or western Asia on F. sylvatica orientalis. Beeches of this subspecies were likely 2.1.1. China present in Britain by 1880 (Bean, 1976). For F sylvatica ori- From 4 April to 6 October, 1999, we surveyed all com- entalis to have been the source of the C. fagisuga infestation mon Fagus species (F. hayatae, F. engleriana, F. longipeltio- 12 R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18 lata, and F. lucida) in 19 Weld sites in 11 provinces in China. locations and details of stand composition and elevation The four rare Fagus species (F. brevipetiolata, F. tientaien- are available from the second author (RVD). sis, F. bijiensis, and F. chienii) were not included. Fagus hayatae was examined at four locations: Tianmushan (Zhe- 2.1.3. Western Asia, Europe, and North America jiang Province), Jiulongshan (Zhejiang), Wuyishan (Fuj- Specimens of C. fagisuga were collected during 2001– ian), and Tangjiahe Natural Reserve Area (Sichuan). Fagus 2003, targeting localities throughout the known range as engleriana was examined at seven locations: Huangshan reXected in the literature (Hoy, 1963; Parker, 1974; Koszta- (Anhui), Cenwanglaoshan of Langping (Guangxi), Tie- rab and Kozár, 1988; Miller and Miller, 1993; Stimmel, changba Tree Farm of Lueyang County (Shaanxi), Tangj- 1993; Kosztarab, 1996; Kozár et al., 1996; Miller and Gim- iahe Natural Reserve Area (Sichuan), Emeishan (Sichuan), ple, 2005). Localities are listed in Table 1. Adults, or whole Maotoushan (Yunnan), and Sanjiangkou Natural Reserve infestations on bark fragments, were Weld collected directly Area, Shoatong (Yunnan). Fagus longipeltiolata was exam- into 100% ethanol and stored at 80 or 20 °C. These spec- ined at eight locations; Wuyishan (Fujian), Lushan imens were collected by our colleagues¡ ¡ (see acknowledge- (Jiangxi), Hengshan (Hunan), Maoershan Natural Reserve ments). Voucher specimens have been deposited in the Area (Guangxi), Daloushan (Guizhou), Fanjingshan Natu- University of Massachusetts Insect Collection. ral Reserve Area (Guizhou), Subaoding (Hunan), and the Zhangijiajie National Forest Park (Hunan). Fagus lucida 2.2. Selection and collection of other taxa was examined at three locations: Hengshan (Hunan), Maoershan Natural Reserve Area (Guangxi) in Daloushan In addition to beech scale (C. fagisuga), there are Wve (Guizhou), and the Fanjingshan Natural Reserve Area other species in the genus Cryptococcus worldwide: (1) C. (Guizhou). Coordinates of sample locations and details of aceris Borchsenius, which was Wrst recorded in Germany on stand composition and elevation are available from the sec- Acer pseudoplantanus L. (Schwenke, 1972) and in eastern ond author (RVD). Europe (Hungary, Russia, Azerbaijan, and the Oblast Holdings of scale insects were also examined by visiting region of the former USSR) on species of Acer, Pyrus, and seven institutional insect collections, representing locations Tilia (Kosztarab and Kozár, 1988); (2) C. integricornis in six provinces: (1) Institute of Entomology, Chinese Danzig, reported only from the Russian Far East (southern Academy of Science (Shanghai) (813 homopteran speci- Primorye, Ussuri Sanctuary and Tigrovoy) by Danzig mens from Fagaceae), (2) Zhejiang University, Zhejiang (76 (1986) on Tilia amurensis Rupr; (3) C. nudatus Brittin, specimens from Fagaceae), (3) Research Center for Scale reported from New Zealand on Hoheria sp. (Brittin, 1914); Insects, Shanxi University (780 specimens), (4) Insect (4) C. ulmi Tang and Hao, reported from Shanxi Province Museum of Northwestern University of Agriculture and Beijing, China from Ulmus pumilla L. (Tang and Hao, (Shanxi) (365 specimens), (5) Institute of Plant Protection, 1995; Wu, 2000); and (5) C. williamsi Kosztarab and Hale, Agricultural Academy of Sichuan (289 specimens), (6) reported from several parts of the eastern United States, Institute of Zoology, The Chinese Academy of Science, Ontario, and Quebec from species of Acer (Kosztarab and Yunnan (collection contained Margarodidae only), and (7) Hale, 1968; Miller and Miller, 1993). Of these, three could Shandong Agricultural University, Tai-an, Shandong (1438 be obtained (C. nudatus, C. ulmi, and C. williamsi), which specimens). A total of 3761 scales collected from beech were together with C. fagisuga gave us four of the six species in individually examined to determine their family and, if erio- the genus for our analyses. Some researchers recognize a coccids, their genus. Further details on these collections are separate family, Cryptococcidae (Kosztarab, 1968; Hodg- available from the second author (RVD). son, 1997; Koteja, 2000), comprising the genus Cryptococ- cus and the monotypic European genus Pseudochermes 2.1.2. Japan Nitsche (Kosztarab and Kozár, 1988; Kosztarab, 1996). We From 29 June to 27 October, 2000, we surveyed stands obtained specimens of the only species of Pseudochermes, of Japanese Fagus (either F. crenata or F. japonica) in 15 P. fraxini (Kaltenbach), and included these in the phyloge- prefectures, checking at least 100 trees per site. Stands of F. netic study. In 2001, live specimens of P. fraxini were col- crenata were examined at 12 sites: Oudaigahara (Nara Pre- lected in Hungary by F. Kozár and live specimens of fecture), Hakusan (Ishikawa), Esan (Hokkaido), Hakkouda Cryptococcus species by R. Henderson, J. Janis, and SW. (Aomori), Shibisan (Kagoshima), Shiragadake (Kuma- Specimens were collected into 100% ethanol and stored at moto), Seburiyama (Fukuoka), Takanawayama (Ehime), 20°C, as above. Other eriococcid 18S sequences (the Ounogahara (Ehime), Ibukiyama-Ishizuchi (Ehime), majority¡ in our analysis) were obtained from a recent Wakasugi-Ushiroyama (Okayama), and Men’noki (Aichi). molecular phylogenetic study of Eriococcidae (Cook and Stands of F. japonica were examined at six sites: Chichibu Gullan, 2004). (Saitama Prefecture), Takaharayama (Tochigi), Yaita (Tochigi), Tegurayama (Miyagi and Fukushima), Rokkou- 2.3. DNA preparation, PCR, and sequencing san (Hyogo), and Men’noki (Aichi). Scale insects found on trees were collected and sent to Dr. Sadao Takagi of Hok- DNA was extracted from individual scales, using any of kaido University for identiWcation. Coordinates of sample three diVerent extraction methods: a salting-out protocol R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18 13
Table 1 Localities and haplotype designations for sampled populations of C. fagisuga Population locality Host # Ind. Number of individuals in COI haplotype groups Country Region, locality Haplotype 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1BelgiumBrabant, Alsemberg ? 6 6 2 Belgium Brabant, Braine l’Alleud ? 4 3 1 3 Bulgaria Kondolovo, Strandza Mts. O 2 1 1 4 Bulgaria Malko Tarnovo, Strandza Mts. O 2 1 1 5 Bulgaria SoWa, Vitosha Mountain D576 S 6 6 6 Canada Nova Scotia, Halifax, Waverly ? 14 14 7 Georgia Batumi, Botanical Gardens O 4 2 1 1 8 Georgia Batumi, Mtirala mountain O 5 3 1 1 9GeorgiaSakhalwasho, O 5 5 10 Iran Gilan, Sari-Dehsang O 17 17 11 Iran Gilan, Rasht-Shaft O 8 8 12 Iran Gilan, Rasht-Siakal O 19 7 12 13 Italy Veneto, Bosco del Cansiglio S 7 7 14 Switzer. Delémont, S 2 2 15Switzer. Undervelier S 9 9 16 Turkey Kalinçam O 3 2 1 17 Turkey Uzungol O 3 3 18 UK Buckinghamshire, Burnham Beeches S 15 15 19 USA Massachusetts, Huntington G 6 6 20 USA Massachusetts, Dayville G 12 12 21 USA New Hampshire, Thornton G 3 3 22 USA Virginia, Rockingham G 17 17 23 USA West Virginia, Tucker G 5 5 Totals 174 85 25 3 2 2 1 1 1 1 6 1 1 1 32 12 Host abbreviations are Fagus grandifolia (G), F. sylvatica sylvatica (S), F. sylvatica orientalis (O), and unknown (?). Haplotype designations correspond to those presented in Figs. 1 and 2.
(Sunnucks and Hales, 1996); a CTAB Phenol/Chloroform ampliWed PCR product, a touch-down procedure was protocol (Doyle and Dickson, 1987); or using the DNEasy used for ampliWcation in an MJ Research, Inc., program- kit, following the manufacturer’s protocol (Qiagen, Valen- mable thermocycler. An initial annealing temperature of cia, CA, USA). 54 °C was decreased by 3 °C every 5 cycles until reaching A fragment of cytochrome oxidase I (COI) was ampli- 49 °C and decreased by 2 °C every 5 cycles until reaching Wed from 174 individuals of C. fagisuga, from the locali- 45 °C which was held for 30 cycles. After an initial dena- ties listed in Table 1. A fragment of the internal turation for 60 s at 95 °C, each cycle denatured at 95 °C transcribed spacer of the ribosomal DNA array (ITS-2) for 30 s, annealed at the appropriate temperature accord- was ampliWed from a subset of nine of these individuals, ing to the touchdown procedure for 30 s, and extended at representing localities in Iran, Canada, and the United 72 °C for 60 s. The program Wnished with a Wnal extension States. A fragment of the large ribosomal subunit (18S) at 72 °C for 10 min. was ampliWed from C. fagisuga (24 individuals) as well as ITS-2 and 18S PCRs contained 0.2 !M each primer, from P. fraxini (2), C. nudatus (2), C. williamsi (3), and C. 0.2 mM each dNTP, 2.5 mM MgCl2, 1.25 U of Taq polymer- ulmi (2). PCR primers for all reactions appear in Table 2. ase (Qiagen Inc., Valencia, CA), 1 buVer supplied by the £ For COI, PCRs contained 0.2 !M each primer, 0.2 mM manufacturer (Qiagen) and 1 !l of genomic DNA, in a total each dNTP, 2.5 mM MgCl2, 2 U of Taq polymerase volume of 50 !l. After an initial denaturation at 95 °C for (Qiagen Inc., Valencia, CA), 1 buVer supplied by the 3min, each cycle included denaturation at 95°C for 30s, £ manufacturer (Qiagen) and 1 !l of genomic DNA, in a annealing at 52 °C for 60 s and an extension at 72 °C for total volume of 50 !l. To obtain greater speciWcity of the 2min, repeated for 29 cycles with a Wnal extension of 72 °C
Table 2 PCR primers used for nuclear and mitochondrial DNA ampliWcations Gene/Region Primer Direction Sequence Reference COI C1-J-2195 (LP–C1) Forward 5Ј-TTTATTTTTTGGTCATCCAGAAGT-3Ј Roehrdanz, 1993; Simon et al., 1994 COI a2761asp Reverse 5Ј-GGTATNCCATTTAATCC-3Ј this paper ITS its-2AM-58T Forward 5Ј-CTAAGCGGTGGATCACTCGC -3Ј Weekers et al., 2001 ITS its-2AM-28T Reverse 5Ј-GCACTATCAAGCAACACGACTC-3Ј Weekers et al., 2001 18s 2880 Forward 5Ј-CTGGTTGATCCTGCCAGTAG-3Ј Tautz et al., 1988 18s B- Reverse 5Ј-CCGCGGCTGCTGGCACCAGA-3Ј von Dohlen and Moran, 1995 14 R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18 for 10 min. All PCR products were puriWed using either a 4.0b10 (SwoVord, 2003). Sequence divergences were calcu- Qiagen PuriWcation Kit (Valencia, CA.) or using Exosap-IT lated using PAUP*’s pairwise distance matrix function. (U.S. Biochemicals, Cleveland, OH). Products were sequenced directly and analyzed using either an ABI-3700 2.5. Analysis of phylogenetic relationships of Cryptococcus automated high-throughput DNA analyzer at Kansas State spp. to other eriococcids (18S) University or a 373DX1 DNA Analyser at Brigham Young University. The 18S sequences of C. fagisuga, C. nudatus, C. wil- liamsi, C. ulmi, and P. fraxini were edited using Sequencher 2.4. Analysis of genetic diversity in C. fagisuga (COI and 4.2 (Gene Codes Corporation) and manually aligned to a ITS-2) pre-existing alignment of scale insect sequences used in a recent analysis by Cook and Gullan (2004). The E10 and Sequences were edited using Sequencher 4.2 (Gene E10-1 helices in the V2 region of C. fagisuga, C. nudatus, C. Codes Corporation). ITS-2 regions were invariant between williamsi and P. fraxini were found to be highly expanded all samples and were not analyzed further; the sequence has as observed for other scale insects (Cook et al., 2002); these been deposited in GenBank under Accession No. and other regions that were not able to be unambiguously DQ125268. There were no insertions or deletions in COI; aligned were excluded from the analysis. Parsimony analy- therefore, alignment was trivial. COI sequences were ana- ses were conducted using PAUP* 4.0b10 (SwoVord, 2003) lyzed phylogenetically using a branch-and-bound maxi- with 1000 bootstrap replicates, using a heuristic search with mum parsimony algorithm as implemented in PAUP* 10 random-addition-sequence starting trees per step.
Fig. 1. Phylogram of one of three most parsimonious trees of C. fagisuga haplotypes. Branch lengths correspond to the number of changes between sequences as stated in the numbers along each branch. Haplotype designations (e.g., H1) correspond to localities and number of samples in each haplotype shown in Table 2. R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18 15
3. Results 2175–2778 in the Drosophila melanogaster mitochondrial genome (GenBank Accession No. AF200829). Of these, 39 3.1. Surveys for beech scale in East Asia bases (6.9%) were variable. There were thirteen distinguish- able haplotypes, which have been deposited in GenBank 3.1.1. China under Accession Nos. DQ125269–DQ125283. Phylogenetic Nineteen sites with beech, distributed over 11 provinces, analysis of the haplotypes yielded three most-parsimonious were visited. Specimens of F. hayatae, F. engleriana, F. lon- trees, one of which is presented in Fig. 1. All three trees gipetiolata, and F. lucida were examined at multiple loca- have a similar topology, diVering slightly in the placement tions. Fagus bevipetiolata, F. tientaienis, and F. chienii were of Haplotype H6. not encountered in the survey. At these 19 sites, 2112 trees Relationships among the 13 observed haplotype are (in 145 local patches) were examined. During this survey illustrated using a minimum-spanning network (Fig. 2). work, eriococcids were observed only once, on F. engleriana In Fig. 2, three main groupings of haplotypes may be dis- in Huagnshan (Anhui Province, May 17–21). No insects in tinguished. The Iranian Group comprises two haplotypes the genus Cryptococcus were found at any of the 19 sites. (H14, H15) unique to Iran; the East Black Sea group Also, seven institutional insect collections in six provinces comprises Wve haplotypes (H3, H5, H11, H12, and H13) were visited and 3761 specimens of scales associated with found in Georgia and Turkey; and the Western Group species of Fagaceae were checked. Eriococcids were found comprises eight haplotypes (H1, H2, H4, H6, H7, H8, H9, only in two collections (Institute of the Chinese Academy of and H10). Every individual sampled in Europe and Science in Shanghai and Research Center for Scale Insects, North America had a haplotype belonging to the West- Shanxi Agricultural University). Of 813 scale specimens ern Group. Of the haplotypes in the Western Group, half examined from the Shanghai collection, 16 were eriococcids, (H7, H8, H9, and H10) were found only in Bulgaria and and from 780 specimens in the Shanxi collection, one was an two others were found only in Western Europe (H4 in eriococcid. None were in the genus Cryptococcus. Switzerland, H6 in Belgium). The Western Group also includes the most widespread and abundant haplotype, 3.1.2. Japan H1. H1 is found in every sampled country but Iran and is On F. japonica, one margarodid (Drosicha sp.) was found in nearly half (49%) of individuals sampled. The observed. On F. crenata, unidentiWed mealybugs (Pseudo- remaining Western Group haplotype, H2, was found coccidae) were noted at three sites. No eriococcids were only in the United States. The Iranian haplotypes diVer found. Insect collections in Japan were not examined. from all other European and American localities by 3.5–4.2%; the East Black Sea Group haplotypes diVer 3.2. Genetic diversity in C. fagisuga from the Iranian Group haplotypes by 3.5–4.1% and from the Western Group haplotypes by 2.0–2.5%. The The resulting COI sequences from C. fagisuga comprised Western Group haplotypes diVer from each other by less 564 base pairs, corresponding approximately to bases than 0.6%.
Fig. 2. A minimum spanning network for C. fagisuga COI haplotypes. Lengths of lines connecting haplotype circles are proportional to number of charac- ter state changes (mutations) between haplotypes (number of changes give on each branch). Circle area is proportional to the number of specimens of each haplotype in the study’s sample pool. The connection pathways shown presents the simplest set of connections among haplotypes. Solid dots represent presumed hypothetical ancestral haplotype forms. Haplotype localities are listed in Table 2. 16 R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18
3.3. Phylogenetic relationships of Cryptococcus species to only distantly related to the others, belonging instead other eriococcids (again with >90% bootstrap support) to Cook and Gullan’s (2004) “BSE” clade, along with Beesoniidae, Stictococci- The 18S Sequences generated for this study from C. dae, and a few eriococcids. fagisuga, C. nudatus, C. ulmi, C. williamsi, and P. fraxini are deposited in GenBank under Accession Nos. DQ125262– 4. Discussion DQ125267. The resulting phylogenetic analysis is shown in Fig. 3. The analysis strongly (>90% bootstrap) supports a Several lines of evidence weigh against the idea of clade consisting of C. fagisuga, C. nudatus, C. williamsi, P. recent East Asian origin of C. fagisuga: (1) the phyloge- fraxini, and the New Zealand species Madarococcus totarae netic distance of C. fagisuga from C. ulmi, (2) the Euro- (Maskell). This clade forms part of Cook and Gullan’s pean populations’ genetic distance from those in Iran, and (2004) “Gondwanan” clade. The analysis further indicates (3) the apparently complete absence of C. fagisuga from that the other Cryptococcus species sampled—C. ulmi—is both China and Japan. Instead, the very high haplotype
Fig. 3. A phylogenetic tree for Ericoccidae and related scale insects, based on 18S sequences. This tree is based on the data matrix of Cook and Gullan (2004), with the Cryptococcus and Pseudochermes sequences added. Bootstrap support values are shown above the nodes. Overall tree topology and sup- port values are comparable to those of Cook and Gullan (2004). A clade containing representative members of the genus Cryptococcus, including the type species C. fagisuga, is relatively well-supported (boostrap 92). C. ulmi is only distantly related to the other species of Cryptococcus. R.A. Gwiazdowski et al. / Biological Control 39 (2006) 9–18 17 diversity of C. fagisuga in Western Asia and southeastern References Europe (Bulgaria) suggest an ancient presence of the spe- cies in this region, in association with the only beech in Adeli, E., Soleimanì, P., 1976. Insects on oriental beech (Fagus orientalis the region, F. sylvatica orientalis. We suggest that the best ssp. macrophylla) in Iran and their importance for forestry practices and wood utilization. Z. Angew. Entomol. 80, 132–138. place to look for co-evolved predators and parasitoids Anon., 1862. “Botanical Society of Edinburgh” letters read before the that might serve as biological control agents is in this meeting by Prof. Balfour, item entitled “Notice of a Diseased Condi- region. tion of Beeches at Tynninghame, the seat of the Earl of Haddington” The Iranian haplotypes are too divergent from the Gardeners’ Chronicle and Agricultural Gazette January 25, 1862, p. 70. widespread H1 haplotype for Iran to be a plausible imme- Anon., 1921. Beukenwolluis (Cryptococcus fagi Dougl.) [The beech scale]. Tijdschr. Plantenziekten, Wageningen 27 (5), 61–62. diate source of the beech scale invasion in Europe. Indeed, Anon., 1926. The felted beech coccus (Cryptococcus fagi Bärensp.). LeaX. so are most of the Georgian and Turkish haplotypes. The For. Comm. No. 15, London. area with a high density of haplotypes closely related to Baerensprung, F.V., 1849. C. fagi. In, Beobachtungen über einige einheimi- H1 is Bulgaria. Thus, southeasternmost Europe, in the sche Arten aus der Familie der Coccinen. (Schluss). III. Coccus. Stg. area where hybrids between F. sylvatica sylvatica and F. Zool. Zootom., Palaeozool. 1, 174. Baylac, M., 1980. Fauna associated with Cryptococcus fagi (Baer.) sylvatica orientalis are found, is the most likely source of (Homoptera: Coccoidea) in some beech forests in northern France. the beech scale invasion of Europe. In the broader phylo- Acta Oecol. Oecol. Appl. 1, 199–208. genetic analysis, C. fagisuga is part of a clade that Baylac, M., 1986. Observations sur la biologie et l’écologie de Lestodiplosis includes one other Palearctic species (P. fraxini), one sp. 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The occurrence in the United States of Cryptococcus fagi (Baer.) Dougl., the insect factor in a menacing disease of beech. J. We are most grateful to those who provided crucial Arnold Arboretum 13, 75–80. samples from foreign and far Xung localities without Ehrlich, J., 1934. The beech bark disease, a Nectria disease of Fagus, fol- lowing Cryptococcus fagi (Baer.). Can. J. Res. 10, 593–692. which this study could not have been done: Valiollah Ban- Fries, E., 1832. Psilonia Fries. 1. P. nivea. In, Systema mycologicum, sistens iameri, Lerzan Erkiliç, Eric Georgeson, Rosa C. Hender- fungorum ordines generum, et species. Gryphiswaldae: subtibus Ern- son, J. Janis, Marc Kenis, Ferenc Kozár, Jon Martin, esti Mauritii 3, 450–452. Debbie Miller, Douglass L. Miller, Tim Tigner, D. W. Goolsby, J.A., 2004. Potential distribution of the invasive old world climb- Unrue and Douglas J. Williams. We thank Lyn G. Cook ing fern, Lygodium microphyllum, in North and South America. Nat. 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