morphological separation of aphid species cryptomyzus leonuri bozhko, 1961 and cryptomyzus...

Biologia 67/5: 953—958, 2012Section ZoologyDOI: 10.2478/s11756-012-0084-z

Morphological separation of aphid species Cryptomyzus leonuriBozhko, 1961 and Cryptomyzus alboapicalis (Theobald, 1916)(Hemiptera: Sternorrhyncha: Aphididae)

Jekaterina Bašilova1 & Rimantas Rakauskas1

1Department of Zoology, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21/27, LT-03101, Vilnius,Lithuania; e-mails: [email protected], [email protected]

Abstract: Aphids of the species Cryptomyzus alboapicalis (Theobald, 1916) and Cryptomyzus leonuri Bozhko, 1961 arevery similar morphologically, although exploit different host plants (Lamium album and Leonurus cardiaca, respectively).Morphological characters proposed for the separation of this species couple in the identification key to European Cryptomyzusspecies appeared to be of little discriminatory power when applied to apterous viviparous females from clonal lineages,whilst alate viviparous females of C. leonuri were not included in the key at all. The aim of this study was to findreliable morphological characters and their combinations for the separation of apterous and alate viviparous females ofC. alboapicalis and C. leonuri. Forward stepwise discriminant analysis based on characters without statistically significant(P < 0.05) correlation (|r| ≥ 0.50) with body length resulted in canonical functions enabling correct classification of 95–100% of specimens from clonal lineages involved in the analysis with a priori specified group membership. The post hocclassification gave 95–100% correct identification of individuals from clonal and 85–100% from field-collected samples. Thediscriminative values of single morphological characters and canonical functions are discussed and modified key for themorphological identification of C. alboapicalis and C. leonuri apterous and alate viviparous females is suggested.

Key words: aphids; Cryptomyzus alboapicalis; Cryptomyzus leonuri; morphological separation; morphometric analysis;measurements; discriminant analysis

Introduction

The aphid genus Cryptomyzus Oestlund, 1922 com-prises 18 species of Palaearctic origin (Remaudiére &Remaudiére 1997; Holman 2009). For the present, tenspecies of this genus are registered in Europe (Ni-eto Nafria et al. 2004), three of them, Cryptomyzusalboapicalis (Theobald, 1916), Cryptomyzus leonuriBozhko, 1961 and Cryptomyzus ulmeri Borner, 1952,are reported as monoecious (Guldemond 1991; Bašilova2010). These aphids spend all their life cycle on peren-nial herbs of the family Lamiaceae (Guldemond 1991;Bašilova 2010).Aphids of the species C. alboapicalis and C. leonuri

are very similar morphologically, although their hostplants (Lamium album and Leonurus cardiaca, respec-tively) belong to different genera. Identification key toEuropean Cryptomyzus species (Guldemond 1991) of-fers the characters for the separation of C. alboapi-calis and C. leonuri apterous viviparous females exclu-sively. Morphological differences between other morphsof this species couple were not discussed (Guldemond1991; Blackman & Eastop 2006). Experimental rearingof C. leonuri enabled the description of other morphs,such as alate viviparous females, oviparous females andmales (Bašilova 2010). Morphometric analysis of apter-

ous viviparous females from clonal lineages revealed theoverlapping of the key characters proposed by Gulde-mond (1991) for the separation of C. alboapicalis andC. leonuri (Bašilova 2010).The aim of this study is to find reliable morphologi-

cal characters and their combinations for the separationof apterous and alate viviparous females of C. alboapi-calis and C. leonuri.

Material and methods

Aphid rearing, microscope slide preparation and aphidspecies identificationAphid rearing was carried out in Pakalniškes, Vilnius distr.,Lithuania, in 2007–2008. The field collection data of aphidsused for experimental rearing are given in Table 1. Aphidlineages started from the single aphid individual were iso-lated in the whole plant cages, type II (Adams & van Em-den 1972). Fresh host plants where supplied when neededby transferring 5–10 aphids to new pot cage. The meth-ods of aphid sampling, preservation and slide mounting inCanada balsam were those commonly used (Blackman &Eastop 2000).

Aphid species identification was based on the life cy-cle and host specificity. These characters appear to be themost reliable for the present. Holocyclic monoecious aphidlineages on Leonurus cardiaca were taken as C. leonuri,whilst those holocyclic monoecious on Lamium album as

c©2012 Institute of Zoology, Slovak Academy of Sciences

954 J. Bašilova & R. Rakauskas

Table 1. Clonal samples of Cryptomyzus alboapicalis and Cryptomyzus leonuri used for rearing and morphometric analysis.

Lineagecode

Collection sites of aphids used for rearing andtheir coordinates

Number of individuals (n) and collection dates

Cryptomyzus alboapicalis from Lamium album

H1 Vingis park, Vilnius, Lithuania54◦41′1′′ N, 25◦14′28′′ E

n = 40, 07.06.–19.09.2007 – apteraen = 28, 07.06.–15.09.2007 – alatae

H2 Truskava, Kedainiai distr., Lithuania55◦25′52 N, 24◦13′30′′ E


H3 Pakalniškes, Vilnius distr., Lithuania54◦38′50′′ N, 25◦22′20′′ E

n = 22, 15.07–12.09.2007 – apteraen = 20, 19.07.–15.08.2008 – apteraen = 20, 21.07.–22.08.2007 – alatae

H4 Narva, Ida-Virumaa, Estonia59◦23′13′′ N, 28◦12′9′′ E


Cryptomyzus leonuri from Leonurus cardiaca

L1 Truskava, Kedainiai distr., Lithuania55◦25′59′′ N, 24◦13′30′′ E


L2 Skaidiškes, Vilnius distr., Lithuania54◦36′36′′ N, 25◦23′17′′ E


L1, L2 Skaidiškes, Vilnius distr., Lithuania54◦36′31′′ N, 25◦23′19′′ E

L1, n = 37, 01.07.–17.09.2008 – apteraeL2, n = 20, 20.07.–28.09.2008. – apteraeL1, n = 11, 01.07–17.09.2008 – alataeL2, n = 10, 06.07.–28.09.2008 – alatae

Table 2. Characters used for the morphometric analysis and their acronyms.

Full character name Acronym

Body length, excluding cauda, mm BODYHind tibia length, mm HTIBIALength of antennal segment 3, mm ANT3Length of antennal segment 4, mm ANT4Length of basal part of antennal segment 5, mm ANT5ALength of processus terminalis, mm PTLength of distal part of antennal segment 5 from the upper edge of primary rhinarium, mm ANT5BLength of the base of antennal segment 6, mm BANT6Length of cauda, mm CAUDALength of siphunculus, mm SIPHONMinimal width of the proximal part of siphunculus, mm MIWSIMaximal width of the distal part of siphunculus, mm MAWSILength of the last rostral segment, mm LRSLength of segment 2 of hind tarsus, mm TARS2Diameter of the base of antennal segment 3, mm DBANT3Length of the longest hair on antennal segment 1, mm HANT1Length of the longest hair on antennal segment 3, mm HANT3Length of the longest hair on abdominal tergites 2–4, mm ABHAIRNumber of dorsal hairs on abdominal segments 1–4 DHAS1–4Number of secondary rhinaria on antennal segment 3–5 SRHIN3–5Number of additional hairs on the last rostral segment HLRS

C. alboapicalis. The common morphological key characterswere also checked for the possible morphological and eco-logical congruence (Guldemond 1991). Both mounted andethanol-preserved specimens are stored at the Departmentof Zoology, Vilnius University (Vilnius, Lithuania).

Morphometric analysisCharacters for morphometric analysis (Table 2) were se-lected from the identification keys and morphological de-scriptions of both species (Hille Ris Lambers 1953; Bozhko1961; Guldemond 1991; Heie 1994; Bašilova 2010). Measure-ments were performed with the Olympus BX40 microscopeand the interactive measuring system MicroImage (Olympu-

sOptical Co. GmbH). Both clonal and field-collected sam-ples of C. alboapicalis and C. leonuri were used in this study(Tables 1, 3).

Data analysisCharacters and their acronyms used in the text are listedin Table 2. Pearson’s correlation coefficients (r) were cal-culated for the whole dataset of each studied morphand the correlation of characters with the length of thebody was evaluated. Characters with statistically signifi-cant (P < 0.05) correlation (|r| ≥ 0.50) with body lengthwere removed from the further analysis. These characters

Morphological separation of Cryptomyzus leonuri and C. alboapicalis 955

Table 3. Field-collected samples of Cryptomyzus alboapicalis (from the collection of the Department of Zoology, Vilnius University)and Cryptomyzus leonuri (from the collection of Institute of Entomology, Biological Centre of Czech Academy of Sciences) used formorphometric analysis.

Sample code, host plant, locality, number of individuals (n) Collection date

Apterous viviparous females

Cryptomyzus alboapicalis

B08-7, Lamium album, Narva, Estonia, n = 2 23.06.2008B03-64, Lamium album, Skaidiškes, Vilnius distr., Lithuania, n = 1 29.07.2003B03-88, Lamium album, Pakalniškes, Vilnius distr., Lithuania, n = 4 21.08.2003B04-35, Lamium album, Pervalka, Kuršiu Nerija National Park, Lithuania, n = 2 10.07.2004B04-49, Lamium album, Salininkai, Vilnius, Lithuania, n = 1 31.07.2004B05-19, Lamium album, Pakalniškes, Vilnius distr., Lithuania, n = 3 25.07.2005

Cryptomyzus leonuri

581, Leonurus quinquelobatus, Dedinovo, Moskva, Russia, n = 6 20.06.19551480, Leonurus cardiaca, Jičín, Czech Republic, n = 2 20.10.19578380, Leonurus cardiaca, Pavlovské vrchy, Czech Rebuplic, n = 2 27.05.19644912, Leonurus cardiaca, Somotor, Slovakia, n = 1 07.07.19613261, Leonurus cardiaca, Čelakovice, Czech Republic, n = 1 26.05.19609/57, Leonurus cardiaca, Praha – Lhotka, Czech Republic, n = 1 09.06.1957

Alate viviparous females

Cryptomyzus alboapicalisB05-19, Lamium album, Pakalniškes, Vilnius distr., Lithuania, n = 3 25.07.2005

Cryptomyzus leonuri

581, Leonurus quinquelobatus, Dedinovo, Moskva, Russia, n = 1 20.06.19551480, Leonurus cardiaca, Jičín, Czech Republic, n = 1 20.10.19574912, Leonurus cardiaca, Somotor, Slovakia, n = 1 07.06.1961

Table 4. Morphological characters removed from the data set due to their statistically significant (P < 0.05) correlation |r| ≥ 0.50with the body length and their Pearson’s correlation coefficient (r) values. Character acronyms are the same as in Table 2.

Character name Apterous viviparous females Alate viviparous females

BODY r = 1 r = 1HTIBIA r = 0.87 r = 0.82ANT3 r = 0.75 r = 0.77ANT4 – r = 0.66ANT5A – r = 0.61PT r = 0.65 r = 0.58

BANT6 r = 0.69 r = 0.55CAUDA r = 0.68 r = 0.64TARS2 r = 0.60 –DBANT3 r = 0.67 r = 0.72

are listed in Table 4. The data set containing measure-ments, counts and ratios of aphids from reared lineageswas divided into two subsets. One subset was used forthe construction of canonical function through the for-ward stepwise discriminant analysis, while the other onefor the post hoc evaluation of its effectiveness. Field sam-ples from the collections of Vilnius University (Vilnius,Lithuania) and Institute of Entomology of Biological Cen-tre of the Czech Academy of Sciences (České Budějovice,Czech Republic) (Table 3) were also included in the posthoc evaluation. Canonical scores were visualised as scat-ter plots. Statistica 8.0 software was used for calcula-tions.

Results and discussion

Apterous viviparous femalesTo discriminate between apterous viviparous females ofC. alboapicalis and C. leonuri the following canonicalfunction (abbreviated onwards as AL-apt, for characteracronyms see Table 2) was obtained: –0.630× (DHAS1–4) + 0.381 × (SRHIN3) + 4.360 × (SIPHON/CAUDA)– 52.150 × (LRS) – 0.369 × (PT/BANT6) – 58.093 ×(MAWSI) + 11.814. The values of canonical scores wereAL-apt < 0 for C. alboapicalis and AL-apt > 0 for C.leonuri. This combination of canonical variables sepa-


Fig. 1. Canonical scores of AL-apt for apterous viviparous females used in analysis with a priori specified group membership. ♦ –holocyclic monoecious on Lamium album, C. alboapicalis; AL-apt < 0; – holocyclic monoecious on Leonurus cardiaca, C. leonuri;AL-apt > 0. Solid line indicates 0 value of AL-apt.

Fig. 2. Canonical scores of AL-apt for apterous viviparous females used for post hoc classification. ♦ – holocyclic monoecious onLamium album, C. alboapicalis; – holocyclic monoecious on Leonurus cardiaca, C. leonuri; + – field samples from Lamium album,C. alboapicalis; × – field samples from Leonurus spp., C. leonuri. Solid line indicates 0 value of AL-apt.

rated 100% of C. leonuri (n = 38) and 95% of C. al-boapicalis (n = 60) specimens involved in the analysiswith a priori specified group membership (Fig. 1). Thepost hoc classification of clonal samples gave 95% cor-rect identification of C. alboapicalis (n = 60) and 97%of C. leonuri (n = 39) specimens (Fig. 2). The posthoc classification of field-collected apterous viviparousfemales (Table 3) revealed 100% classification of indi-viduals from L. album (n = 13) as C. alboapicalis and

85% classification of individuals from Leonurus spp. (n= 13) as C. leonuri (Fig. 2).Morphological characters proposed for the separa-

tion of C. alboapicalis and C. leonuri in the identifi-cation key to European Cryptomyzus species (Gulde-mond 1991) appeared to be of little discriminatorypower. The character SRHIN3 resulted in 98% classifi-cation of C. leonuri aphids from clonal lineages as C. al-boapicalis. The ratio CAUDA/SIPHON enabled correct

Morphological separation of Cryptomyzus leonuri and C. alboapicalis 957

Fig. 3. Canonical scores of AL-al for alate viviparous females used in analysis with a priori specified group membership. ♦ – holocyclicmonoecious on Lamium album, C. alboapicalis; AL-al > –0.5; – holocyclic monoecious on Leonurus cardiaca, C. leonuri; AL-al < –0.5.Solid line indicates –0.5 value of AL-al.

Fig. 4. Canonical scores of AL-al for alate viviparous females used for post hoc classification. ♦ – holocyclic monoecious on Lamiumalbum, C. alboapicalis; – holocyclic monoecious on Leonurus cardiaca, C. leonuri; + – field samples from Lamium album, C. alboapi-calis; × – field samples from Leonurus spp., C. leonuri. Solid line indicates –0.5 value of AL-al.

identification of 90.9% of C. alboapicalis and 57.1% ofC. leonuri specimens from reared lineages, while the ra-tio PT/BANT6 – 48.8% of C. alboapicalis and 76.6%of C. leonuri.

Alate viviparous femalesAlate viviparous females of C. leonuri are not includedin the identification keys to European species of genusCryptomyzus (Guldemond 1991). When these keys werecompiled, the only published morphological description

of C. leonuri was that of apterous viviparous femalegiven by Bozhko (1961). To discriminate between alateviviparous females of C. alboapicalis and C. leonuri thefollowing canonical function (abbreviated onwards asAL-al, for character acronyms see Table 2) was ob-tained: –65.464 × (ANT5B) + 57.515 × (TARS2) +208.394 × (MAWSI) – 0.185 × (SRHIN3) – 86.360 ×(HANT1) – 2.450. The values of canonical scores wereAL-al > –0.5 for C. alboapicalis and AL-al < –0.5 forC. leonuri. This combination of canonical variables sep-


arated 96% of C. leonuri (n = 26) and 100% of C. al-boapicalis (n = 35) specimens involved in the analysiswith a priori specified group membership (Fig. 3). Thepost hoc classification of clonal samples gave the 100%correct identification of C. alboapicalis (n = 34) andC. leonuri (n = 26) (Fig. 4). The post hoc classifica-tion of field-collected alate viviparous females (Table 4)showed that 100% of individuals from L. album (n = 3)and Leonurus spp. (n = 26) were classified as C. al-boapicalis and C. leonuri, respectively (Fig. 4).

Key for the morphological identification of C. alboapi-calis and C. leonuri

Apterous viviparous females(1) Number of dorsal hairs on abdominal tergites 1–4is 14–18; the length of siphunculus is 0.10–0.12 timesthe length of the body; the length of cauda is 0.70–0.80times the length of siphunculus; canonical scores of AL-apt (–0.630× (DHAS1–4) + 0.381× (SRHIN3) + 4.360× (SIPHON/CAUDA) – 52.150 × (LRS) – 0.369 ×(PT/BANT6) – 58.093 × (MAWSI) + 11.814) are lessthan 0; on Lamium album – Cryptomyzus alboapicalis;(–) Number of dorsal hairs on abdominal tergites

1–4 is 12–14; the length of siphunculus is 0.12–0.14times the length of the body; the length of cauda is0.60–0.70 times the length of siphunculus; canonicalscores of AL-apt (–0.630 × (DHAS1–4) + 0.381 ×(SRHIN3) + 4.360 × (SIPHON/CAUDA) – 52.150 ×(LRS) – 0.369 × (PT/BANT6) – 58.093 × (MAWSI) +11.814) are more than 0; on Leonurus cardiaca – Cryp-tomyzus leonuri.

Alate viviparous females(1) Number of dorsal hairs on abdominal tergites 1–4 is 14–18; the length of distal part of antennal seg-ment 5 from the upper edge of primary rhinarium is0.040–0.060 mm; canonical scores of AL-al (–65.464 ×(ANT5B) + 57.515 × (TARS2) + 208.394 × (MAWSI)– 0.185 × (SRHIN3) – 86.360 × (HANT1) – 2.450) aremore than –0.5; on Lamium album – Cryptomyzus al-boapicalis;(–) Number of dorsal hairs on abdominal tergites

1–4 is 12–14; the length of distal part of antennal seg-ment 5 from the upper edge of primary rhinarium is0.060–0.080 mm; canonical scores of AL-al (–65.464 ×(ANT5B) + 57.515 × (TARS2) + 208.394 × (MAWSI)– 0.185 × (SRHIN3) – 86.360 × (HANT1) – 2.450)are less than –0.5; on Leonurus cardiaca – Cryptomyzusleonuri.

Concluding remarks

The present study confirmed that precise morphologi-cal discrimination between C. leonuri and C. alboapi-calis remains a difficult task (Guldemond 1991; Bašilova2010) and canonical functions are still needed for theproper morphological separation of these two species.In addition to the common key characters (SRHIN3,SIPHON/CAUDA, PT/BANT6), DHAS1–4, LRS and

MAWSI were included for more precise morphologicalidentification of C. alboapicalis and C. leonuri apterousviviparous females. Nonetheless, this procedure did notensure a 100% correct classification of this species cou-ple and host plant information still remains the basiccharacter. The same concerns alate viviparous femalesof C. alboapicalis and C. leonuri, although DHAS1–4and ANT5B seem to give rather satisfactory results ontheir own (see the key above).

Acknowledgements

This study was partly supported by the Lithuanian StateScience and Study Foundation, Grant No. C – 08023. Thekind permission of Dr. J. Holman and Dr. J. Havelka (Bi-ological Centre of the Czech Academy of Sciences, ČeskéBudějovice, Czech Republic) to access aphid collection en-abled the analysis of C. leonuri field samples. The authorsare grateful to E. Kananovičiute (Vilnius University, Lithua-nia) for providing aphid material from Kedainiai district(Lithuania).

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Received 16 March 2012Accepted 6 July 2012

morphological separation of aphid species cryptomyzus leonuri bozhko, 1961 and cryptomyzus...

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