This article was downloaded by: [University of Wyoming Libraries] On: 25 September 2013, At: 03:11 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Natural History Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnah20 Species status of two colour morphs of Eupelmus vesicularis (Hymenoptera: Eupelmidae) as revealed by allozyme electrophoresis, morphometric and host preference data Lucian Fusu a a Faculty of Biology, “Al. I. Cuza” University, Iaşi, România Published online: 21 Apr 2010. To cite this article: Lucian Fusu (2010) Species status of two colour morphs of Eupelmus vesicularis (Hymenoptera: Eupelmidae) as revealed by allozyme electrophoresis, morphometric and host preference data, Journal of Natural History, 44:17-18, 1113-1129, DOI: 10.1080/00222931003632773 To link to this article: http://dx.doi.org/10.1080/00222931003632773 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms- and-conditions
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This article was downloaded by: [University of Wyoming Libraries]On: 25 September 2013, At: 03:11Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Natural HistoryPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tnah20
Species status of two colour morphs ofEupelmus vesicularis (Hymenoptera:Eupelmidae) as revealed by allozymeelectrophoresis, morphometric andhost preference dataLucian Fusu aa Faculty of Biology, “Al. I. Cuza” University, Iaşi, RomâniaPublished online: 21 Apr 2010.
To cite this article: Lucian Fusu (2010) Species status of two colour morphs of Eupelmus vesicularis(Hymenoptera: Eupelmidae) as revealed by allozyme electrophoresis, morphometric and hostpreference data, Journal of Natural History, 44:17-18, 1113-1129, DOI: 10.1080/00222931003632773
To link to this article: http://dx.doi.org/10.1080/00222931003632773
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
TNAH0022-29331464-5262Journal of Natural History, Vol. 0, No. 0, Feb 2009: pp. 0–0Journal of Natural HistorySpecies status of two colour morphs of Eupelmus vesicularis (Hymenoptera: Eupelmidae) as revealed by allozyme electrophoresis, morphometric and host preference dataJournal of Natural HistoryL. FusuLucian Fusu*
Faculty of Biology, “Al. I. Cuza” University, Iasi, România
(Received 30 September 2009; final version received 18 January 2010)
Eupelmus vesicularis (Retzius) is considered to be a widespread and polyphagousparasitoid recorded from more than 200 hosts. The aim of this study was todetermine whether two colour forms of the putative species represent two differentmorphs of a single species or a pair of cryptic species. Three different methods –morphometric analysis, allozyme electrophoresis and evaluation of host preferences –were used to investigate the existence of two cryptic species. Bivariate and multivariateanalyses clearly indicate two species that are also distinguishable electrophoretically atthe Idh-2, G6pdh, Me, Pgm and Xdh loci. The evaluation of host preferences indicatessome overlap in host range between the two species.
Eupelmus (Macroneura) includes most species of the genus Eupelmus with brach-ypterous females; 13 species of this subgenus are known to occur in the Palaearcticregion (Kalina 1981) and 7 species in America north of Mexico (Gibson 1990). Manymore species likely are present in the tropical regions (especially Afrotropics), butthey are mainly undescribed or wrongly ascribed to genus or subgenus. The hosts areunknown for most species, but where host records are available these indicate apolyphagous behaviour in various concealed situations (Gibson 1990).
Eupelmus (Macroneura) vesicularis (Retzius, 1783) is a well known and wide-spread species that has been recorded as a parasitoid of more than 200 host speciesbelonging to Orthoptera, Hemiptera, Hymenoptera, Coleoptera, Lepidoptera andDiptera (Noyes 2003). Gahan (1933) stated that it is “one of the most polyphagicspecies of all chalcidoids”. It can act as a primary or secondary parasitoid (Martinezet al. 1999) and several of its hosts are of economic importance (Thompson 1955).The species is highly variable in colour, ranging from almost black to brownish oryellowish, which was already noticed by the earlier revisers of the group (Ruschka1921; Ferrière 1954). However, many parasitoid species once considered polyphagousand polymorphic have been more recently shown by molecular, allozyme or cytogeneticstudies to represent complexes of oligophagous or monophagous cryptic species(Dawah 1988; Dawah et al. 2002; Gokhman and Timokhov 2002; Kankare and Shaw2004; Kankare et al. 2005a,b; Smith et al. 2007; Smith et al. 2008; Bernardo et al.2008; Gebiola et al. 2009).
During a survey of parasitoid complexes of gall inducing insects from Romania,it became evident that in most instances either dark brown or brownish-yellowfemales (here informally named Eupelmus vesicularis dark form and E. vesicularislight form) were obtained from a particular host. Females of Eupelmus vesicularisdark form are almost uniformly dark brown, with a faint green and blue lustre on thehead; mesoscutum often with strong metallic shine: bronze anteriorly, blue mediallyand purple posteriorly. Females of E. vesicularis light form are generally brownish-yellow, somewhat bicoloured, with a darker head (with metallic, mostly green, lustre)and metasoma, and a brownish-yellow or reddish-brown mesosoma; mesoscutumwith faint, mostly bluish-green lustre. This difference in colour led to the assumptionthat E. vesicularis, although considered a single polyphagous and polymorphicspecies, could represent a pair of cryptic species whose phenotypic plasticity hasprevented their recognition as distinct entities. Three different methods were used totest if the two colour forms represent two morphs of a single species or two differentspecies that specialize to a certain degree on different hosts: (1) morphometric com-parison of the two colour forms using characters thought to be useful in the taxonomyof the subgenus; (2) absence of gene flow between the two putative species; and (3)differences in biological traits, i.e. different host preferences.
Beside the theoretical aspects of this taxonomic problem there are also obviouspractical issues. The much-debated issue of the value of plant communities on themargins of agricultural ecosystems, as a sources of economically important parasi-toids on alternative hosts (Menalled et al. 1999; Miliczky and Horton 2005; Stephenset al. 2006; Bianchi et al. 2008; Gebiola et al. 2009), is largely dependent on the theo-retical aspect of how frequent is polyphagy among parasitoid insects. As mentionedearlier, many previously thought polyphagous species have proven to be complexesof more or less specialized cryptic species and this clearly influences the degree towhich the parasitoid communities could make use of alternative hosts. Another prac-tical aspect is the assessment of the influence of introduced parasitoid species, used inbiological control programmes of invasive pest species, on non-target arthropodcommunities. For example, Lozan et al. (2008) showed the influence that the intro-duced wasp Cotesia glomerata (L.) (Ichneumonoidea: Braconidae), a parasitoid ofeconomically important Pieridae (Lepidoptera) species, could have on the endemicbutterfly fauna of the Canary Islands.
Material and methods
Insects
Because males of E. vesicularis are rarely collected, this study is based entirely onfemales. For the electrophoretic and morphometric analysis, sympatric dark andlight females were collected in north-east Romania from two different populationssituated 22 km apart: (1) Botanical Garden of Iasi (N 47°11′10″, E 27°33′03″);and (2) “Poiana cu Schit” Nature Reserve, a glade in the middle of BârnovaForest (N 46°59′06″, E 27°35′04″).
The hosts, galls and other vegetable material were collected by the author, I.E.Popescu, O.A. Popovici, I. Andriescu and M.D. Mitroiu in Romania and the Repub-lic of Moldova, and in Spain by A. Ribes; additional host records were taken fromthe labels of specimens preserved in the collections of the Natural History Museum,
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London, UK (BMNH), Institute for Plant Protection and Ecological Agriculture,Chisinau, Republic of Moldova (IPPM), Zoological Museum, Lund University, Sweden(LUZN) and Naturhistoriska Riksmuseet, Stockholm, Sweden (NHRM).
Morphometric analysis
For the morphometric analysis, females were collected using a sweep net and individ-uals were transported alive to the laboratory in small vials. Females of Eupelminaecontort their body in a characteristic way (Gibson 1986) when killed with variousanaesthetic chemicals, which makes accurate comparative measurements difficult. Toovercome this problem, specimens were killed by gradually cooling the body temper-ature first to about 4°C and then to –20°C in a freezer. After thawing they were trans-ferred to absolute ethanol and chemically dried using hexamethyldisilazane toprevent collapsing or shrivelling (Heraty and Hawks 1998). Fifteen high-quality spec-imens of each colour morph were mounted on card rectangles as described by Noyes(1982) and measurements were taken with a binocular stereomicroscope fitted withan ocular grid. The characters and ratios used were head height (hdH), head width(hdW), head length (hdL), pedicel plus flagellum length (antL), hind tibia length(ti3L), metasoma length (mtL), metasoma width (mtW), ovipositor sheath length(ovL), forewing base length (wbL), forewing apical part length (waL), hdH/hdL,hdW/hdL, ovL/ti3L, ovL/mtL, mtL/mtW, antL/hdW and waL/wbL. Measurementsfollow Gibson (1990) except for forewing and hind tibia; for forewing basal andapical half and hind tibia length see Figure 1.
Because of the great variation in size of females, the normality of data and homo-geneity of variance were first tested using the Shapiro-Wilk’s W test and Levene’s testrespectively. In order to eliminate possible size effects, morphometric data weretreated using two different approaches: log-transformation (base 10) of raw data andratios. Principal component analysis (PCA) was performed using the covariancematrix of log-transformed raw measurements without an a priori constraint ofassigning them to one of the two classes. A PCA based on untransformed data or ona correlation matrix gave very similar results and the data are not presented. Acanonical discriminant analysis (CDA) was performed on log-transformed raw dataor on ratios using the two presumed species as grouping variables. Both a standardand a step-wise CDA were performed in order to find the minimum number ofinformative measurements and ratios that separate the two putative species. All com-putations were made using Statistica 6.0 (StatSoft).
Electrophoresis
An indirect method was used to test for the absence of gene flow between the two col-our forms: allozyme electrophoresis. This is because the species frequently repro-duces by thelytokous parthenogenesis, as is always the case in North Americanpopulations (Gibson 1990), and in Europe, even if males are present, they are veryrare (Fusu, unpublished data). It was not possible to establish a laboratory cultureowing to problems in finding a suitable host, and the electrophoretic study was car-ried out on two wild populations. Using specimens from wild populations alsoremoved the problem of reduced genetic diversity in laboratory lines and offered alarger number of loci. The specimens were anaesthetized using ethyl acetate, and
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ascribed to one of the two colour morphs before protein extraction. Soluble proteinextracts were prepared by grinding single specimens in a microcentrifuge tube withthe aid of a glass rod, in 30 μl ice-cold homogenization buffer (Loxdale et al. 1983;Al-Barrak et al. 2004). If not immediately analyzed, the homogenates were stored at –85°C for at most 1 month.
The following enzymatic activities, known to give good results in other species withsmall individuals, were tested: acid phosphatase (ACP, EC 3.1.3.2), alcohol dehydroge-nase (ADH, EC 1.1.1.1), alkaline phosphatase (ALP, EC 3.1.3.1), aldehyde oxidase(AO, EC 1.2.3.1), carboxylesterase (EST, EC 3.1.1.1) glucose-6-phosphate isomerase
Figure 1. Measurements of forewing rudiment and hind tibia for morphometric analysis ofEupelmus vesicularis (female). Notes: ti3L, hind tibia length; waL, forewing apical part length;wbL, forewing base length.
The cellulose acetate electrophoresis system with all the equipment supplied byHelena Laboratories, Beaumont, Texas, USA (Hebert and Beaton 1993) was used.All the reagents were high purity grade, provided by Sigma-Aldrich. The stainingmixtures for the 16 enzymatic activities tested were taken mainly from Hebert andBeaton (1993), but for EST from Al-Barrak et al. (2004). For ACP, HBDH and SDHthe recipes were from Pasteur et al. (1987) except as adapted as follows. For ACP thecellulose acetate plate was incubated for 10 min at 5°C in acetate buffer, pH = 5.0previous to staining; the staining mixture consisted of 2 ml 1-Naphthyl phosphatesolution (50 ml acetate buffer pH = 5.0, 200 mg NaCl, 10 mg 1-Naphthyl phosphatedisodium salt) and 5 drops of Fast blue BB saturated solution. For HBDH the stain-ing mixture consisted of 0.6 ml 0.1 M Tris-HCl buffer, pH = 8.0, 10 drops of D,L-Sodium 3-hydroxybutyrate solution (50 mg/ml), 5 drops MTT solution (10 mg/ml)and 5 drops PMS solution (2 mg/ml). For SDH used were 1 ml 0.1 M Tris-HCl bufferpH = 8.0, 10 drops L-iditol solution (100 mg/ml), 1.5 ml NAD+ solution (2 mg/ml), 5drops of MgCl2 (20 mg/ml), 5 drops MTT solution (10 mg/ml) and 5 drops PMSsolution (2 mg/ml). Melted agarose (2 ml agarose 2% at 60°C) was added to all stain-ing mixtures before pouring on the plates. All plates were run for 15–20 minutesusing 50 mM Tris-citrate buffer, pH 7.8.
Results
Morphometric analysis
All the univariate measures used failed to separate the two putative species, theonly significant difference being the length of the forewing apical part (waL), butthis feature largely overlapped in the two forms (Table 1). By combining variablesas ratios (bivariate analysis), two putative species were distinctly separated,although only the ratio wbL/waL was non-overlapping and fully discriminatory.All other ratios (except antL/hdW), although statistically significant (t-test, p <0.01), were overlapping and cannot be used directly to differentiate the two puta-tive species (Table 1). Although showing some overlap, the dark form was sepa-rated from the light form of E. vesicularis in all bivariate plots, except for hdWversus antL (Figure 2).
The PCA extracted 10 factors based on the 10 analyzed variables. In Figure 3 allspecimens are projected on the first two principal components (which explain 96.24 %of the total variance: 91.75 and 4.48 % respectively), resulting in two distinct clustersthat correspond to the two colour forms. Table 2 presents the proportion of variance,eigenvalues and each of the 10 variables’ contribution to the first 2 principal compo-nents (PC I and PC II). Along PC I, all of the variables have a similar contribution,with waL, hdH and ti3L having slightly higher weights. Along PC II, waL and mtWhave the highest weight and ti3L almost none.
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A standard CDA performed on log-transformed raw data produced a single func-tion capable of discriminating 100% of all specimens (p < 0.000001; df = 10). Thestep-wise CDA also produced a single function (p < 0.001, df = 7) also capable of cor-rectly discriminating all the specimens on the base of seven traits: ovL, waL, wbL,hdL, hdH, antL, mtL.
The CDA performed on ratios, which are much more important in the taxon-omy of chalcid wasps than individual measurements, gave a less exact separation.CDA was applied to a dataset that excluded wbL/waL which, per se, is 100%informative. A standard analysis produced a single function capable of discriminat-ing 96.66 % of all specimens (p < 0.001, df = 7). The step-wise analysis also pro-duced a single function (p < 0.001, df = 3) capable of correctly discriminating 96.66% of the specimens on the basis of only two traits: hdH/hdL, ovL/mtL. The func-tion coefficients (raw and standardized) for log-transformed data and ratios arepresented in Table 3.
Enzyme electrophoresis
No staining occurred or the bands were poorly resolved for 7 enzymatic activities(ACP, ADH, ALP, EST, HBDH, HEX, LDH) from the 16 tested. The other nine(AO, G-6-PDH, GPI, IDH, MDH, ME, PGM, SDH, XDH,) produced clear bands(Figure 4) and these were used for a detailed study.
Table 1. Mean, minimum and maximum for the 10 characters and 7 ratios in the dataset fordark and light colour forms of Eupelmus vesicularis (measurements in mm).
Notes: ti3L, hind tibia length; ovL, ovipositor sheath length; waL, forewing apical part length;wbL, forewing base length; hdW, head width; hdL, head length; hdH, head height; antL, pedi-cel plus flagellum length; mtW, metasoma width; mtL, metasoma length.
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IDH
For the Idh-2 locus (mitochondrial form of IDH), all females showed an electro-phoretic mobility pattern characteristic of heterozygotes for homodimer formingenzymes. The dark phenotype females have the alleles 85/100, whereas the lightphenotype females have the alleles 82/93, with no 82/85, 85/93, 82/100 or 93/100hybrids observed. Dark phenotype females, instead of presenting a three-bandpattern with a staining intensity of 1:2:1, present only two bands with the inten-sity 2:1. The most likely explanation is that homodimers 100/100 are unstable orlack enzymatic activity (null allele) and only the 85/100 and 85/85 dimers arestained.
Figure 2. Scaterplots of pairs of variables for Eupelmus vesicularis light colour and dark colourforms. Note: All measurements in mm.
`E. vesicularis’ dark form
`E. vesicularis’ light form0,4
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G-6-P DH
For the G6pdh locus, the dark phenotype females are homozygous for allele 100 (100/100) and the light phenotype females for allele 95 (95/95), with no 95/100 hetero-zygous specimens found.
ME
For the Me locus, the dark phenotype females are polymorphic, with all individualshomozygous and having 93/93, 96/96 or 100/100 alleles; the light phenotype femalesare all homozygous for allele 90.
PGM
For the Pgm locus, all dark phenotype females are homozygous for allele 77and light phenotype females are either homozygous for allele 100 or hetero-zygous (82/100).
XDH
For the Xdh locus, all dark phenotype females are homozygous for allele 100,whereas light phenotype females are homozygous for allele 90, with no 90/100 hetero-zygous specimens found.
MDH
For both the Mdh-1 and Mdh-2 loci all females presented a single monomorphicband. When samples are loaded towards the cathodal end of the plate or near the
Table 2. Proportion of variance, eigenvalues and variablecontribution for the first two principal components (PCs).
Notes: ti3L, hind tibia length; ovL, ovipositor sheathlength; waL, forewing apical part length; wbL, forewingbase length; hdW, head width; hdL, head length; hdH, headheight; antL, pedicel plus flagellum length; mtW, metasomawidth; mtL, metasoma length.
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centre, both isozymes migrate anodally; when loaded beyond centre, slightly towardsthe anodal end of the plate, Mdh-1 (cytosolic malate dehydrogenase) migrates anod-ally and Mdh-2 (mitochondrial malate dehydrogenase) migrates cathodally.
AO, GPI and SDH
All females produced a single monomorphic band.The previous data indicate that G6pdh, Idh-2, Me, Pgm and Xdh loci are polymorphic
and the rest (Ao, Gpi, Mdh-1, Mdh-2 and Sdh) are monomorphic. All polymorphic lociare drastically heterogeneous and with statistically significant deviation from Hardy–Weinberg equilibrium (p = 0.00) (Table 4). If the specimens of this heterogeneous datasetare grouped (according to phenotype and observed allozyme pattern) in two distinct datasets, only the Me locus of the dark phenotype and the Idh-2 locus are not in Hardy–Wein-berg equilibrium (computation not shown). This indicates that females of the dark phe-notype and those of the light phenotype represent two distinct gene pools, characterizedby two different allele sets in their multilocus genotypes: (1) Idh-282, Idh-293, G6pdh95,Me90, Pgm82, Pgm100 and Xdh90, characteristic for the light form; (2) Idh-285, Idh-2100,G6pdh100, Me93, Me96, Me100, Pgm77 and Xdh100, characteristic for the dark form. Not asingle specimen of the 61 females screened had a hybrid enzyme pattern, indicating thecomplete absence of gene flow between the 2 sympatric colour “phenotypes”.
Figure 3. Plot of the first two factors from the principal component analysis of the log-trans-formed raw measurements.
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The discrimination of two putative species by morphological characters (colourand forewing rudiment length) is in accordance with their genetic separation,although separation is not absolute using colour alone. One small female was attrib-uted to the dark form using colour, but to the light form by allozyme pattern.
Host preferences
The hosts recorded for E. vesicularis in this study are presented in Table 5. Althoughthere appears to be some overlap in hosts, a clear difference in the biology of the twocolour forms is apparent. Only the light form was reared, with a single exception,from stems of Poaceae inhabited by larvae of various Tetramesa species (Chalcidoi-dea: Eurytomidae). The single record of the dark form from a Tetramesa gall(T. brevicornis) is based on one specimen reared from a large number of galls andlikely represents an accidental parisitization because the light form was always rearedin fairly large numbers from this host. An accidental event is indicated because thedark form was frequently collected using a sweep net in almost all habitats whereTetramesa galls were gathered but otherwise never reared from the galls. Apart from
Table 3. Function coefficients of canonical discriminant analysis (CDA) on log size-adjustedraw data and ratios, performed on dark colour and light colour form of Eupelmus vesicularis.
Notes: ti3L, hind tibia length; ovL, ovipositor sheath length; waL, forewing apical part length;wbL, forewing base length; hdW, head width; hdL, head length; hdH, head height; antL, pedi-cel plus flagellum length; mtW, metasoma width; mtL, metasoma length.
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this, it is difficult to find any other correlation and the two colour forms apparentlyshare at least some of their hosts (e.g. Yponomeuta malinellus, Neodiprion sertifer andAndricus quercuscalicis).
Figure 4. Enzyme electromorphs of Eupelmus vesicularis light colour form (left) and dark col-our form (right). Notes: The direction of current flow is indicated by +; the specimen loadingpoint is shown by an arrow.
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Table 4. Observed (O) and expected (E) frequency of genotypes in heterogeneous samples ofsympatric individuals.
Locus Genotip Botanical Garden; n = 29: 15 light form,
Notes: n, total number of females; genotypes characteristic for the dark colour form are in boldtext; those characteristic for the light colour form are in regular font.
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Journal of Natural History 1125T
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tw
o sp
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ens.
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1126 L. Fusu
Discussion
The combined use of morphometric and genetic analyses of the two colour forms ofEupelmus vesicularis of authors, considered merely varieties of the same species,clearly show that the two morphs represent two different species that also have, tosome extent, different host preferences.
The morphometric analysis clearly separates two species. Although the univariateanalysis was not useful, the bivariate analysis showed that the waL/wbL ratio is fullydiscriminatory for the two species. The initial separation of the two species by colouris also supported by the clustering of the two groups using PCA (Figure 3) and CDA.Considering that the PCA was done without an a priori assumption of group mem-bership, the clustering of points is a strong indication of the morphological distinc-tiveness of the two species. The significance of this separation is further supported bysimilar morphometric studies (Heraty and Polaszek 2000; Polaszek et al. 2004;Bernardo et al. 2008) where sibling species or species complexes were successfullyresolved using multivariate analysis.
The analysis of the staining pattern for the nine enzymatic activities (Figure 4)also clearly indicates that individuals of the dark phenotype and those of the lightphenotype represent two distinct gene pools (different species). The two species areclearly distinguishable electrophoretically at the Idh-2, G6pdh, Me, Pgm and Xdh loci.If both the dark and light females of E. vesicularis are considered part of the sameinterbreeding population, all these loci significantly deviate from Hardy–Weinbergequilibrium. When this heterogeneous dataset is split into two distinct datasets, onlythe Me locus of the dark form and Idh-2 locus deviate. A likely explanation for all the61 investigated females being heterozygous for the Idh-2 locus (not a singlehomozygous individual was found for either of the alleles) is a duplication of the Idh-2 gene rather than a true heterozygosity for this locus. This also explains the presencein the dark form (as fixed heterozygosity) of the null (lethal) allele Idh-2100 that showsno enzymatic activity. Gene duplication by polyploidization is ruled out because allother enzymatic activities show a staining pattern characteristic for single locus genesand the karyotype of E. vesicularis has five pairs of chromosomes as found in manyother Eupelmus species (Fusu 2008). Furthermore, as recently shown by Gokhman(2009), polyploidization is extremely rare in Hymenoptera. A similar situation wasreported for another chalcid wasp, Trichogramma marylandense Thorpe (Tricho-grammatidae) by Hung (1985), who detected a duplication of the Me locus.
The host records also suggest the presence of two distinct species because theyindicate a certain specialization. Whereas the light form is frequently reared in afairly large number from stems of Poaceae inhabited by larvae of different Tetramesaspecies, the dark form clearly does not prefer these hosts. The host specializationmight also be reflected in the length of the ovipositor (third valvulae as compared tohind tibia). Ovipositor sheaths are longer in the dark form and shorter in the lightform and this could indicate a certain adaptation of the dark form to more concealedand difficult to reach hosts.
An interesting issue concerns the importance that should be given to body colourin the taxonomy of parasitic wasps. As suggested earlier by Kerrich (1962), colour asa taxonomic character at the species level is much more important than one wants tobelieve, although there are many exceptions. In some instances, such as in the genusPnigalio (Chalcidoidea: Eulophidae), colour pattern was concluded not to be reliable
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Journal of Natural History 1127
for species identification (Bernardo et al. 2007), whereas in a species group ofAleiodes (Ichneumonoidea: Braconidae) the only discriminating character betweenobvious, genetically distinct species is colour pattern (Quicke et al. 2006). In the caseof Eupelmus vesicularis species group, body colour is evidently a very useful, butoverlooked, taxonomic character. Because the difference in colour between the twocryptic species included so far under the name E. vesicularis consists mainly in differ-ent intensities of the brown tint and differences in the metallic hue rather than a dif-ferent colour pattern, this character should be used cautiously. The colour intensityand even the metallic hue of a specimen may be influenced by various factors such assize differences, temperature during the ontogenetic development or whether aspecimen is teneral or several days old; in collection specimens colour can be furthermodified by the collection technique or if specimens are faded by light. In the case ofEupelmus vesicularis species group, colour is mostly influenced by the size of thespecimen. Small females of E. vesicularis dark form (about 1 mm in length) arebrownish yellow with a lighter thorax and could be confused with E. vesicularis lightform, however, the faint metallic lustre on the mesoscutum is still bluish or purpleand not greenish; conversely, some large females of E. vesicularis light form aredarker, but at least parts of the thorax are yellowish brown and the metallic lustre onmesoscutum is mostly green, not blue or purple.
The decision about the names that should be applied to the two colour forms ofE. vesicularis depends on the complex taxonomic problem of ascribing all thesynonyms of E. vesicularis to one form or another. There are six currently recognizedsynonyms for the species (Gibson 1990), but there are eight possible synonyms ifthose listed by Ruschka (1921) are also counted. The taxonomy of E. vesicularis willbe clarified in a revision of European Eupelmus (Macroneura) (Fusu, in preparation).
Acknowledgements
The author is particularly grateful to I.E. Popescu, O.A. Popovici, I. Andriescu, M.D. Mitroiu(“Al. I. Cuza” University, Iasi) and A. Ribes (Lleida, Spain) for the donation of specimens forthe morphological and host preference studies. The allozyme electrophoresis laboratory wasset up and the reagents obtained with the kind help of D. Cojocaru, C. Maniu, C. Cîmpeanu(“Al. I. Cuza” University) and P. Rotinberg (Biological Research Institute, Iasi). I am verythankful to G.A.P. Gibson (CNC, Ottawa), whose very useful comments instigated severalimprovements to this paper. I acknowledge with gratitude J. Noyes and A. Polaszek for theirkind support during my stay at the Natural History Museum (London, UK), G.F. Dyurichand S. Eliseev (Institute for Plant Protection and Ecological Agriculture, Chisinau, Republic ofMoldova), R. Danielsson (Zoological Museum, Lund University, Sweden), H. Vårdal,B. Viklund and G. Lindberg (Naturhistoriska Riksmuseet, Stockholm, Sweden) for their helpduring my work in the collections of the respective institutions. This research received partialsupport from the SYNTHESYS Project (http://www.synthesys.info/) which is financed byEuropean Community Research Infrastructure Action under the FP6 “Structuring the Euro-pean Research Area” Programme (application GB-TAF-4479).
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