Genetic and morphological variation among biotypes of Tephritis bardanae

BiochemicalSystematicsand Ecology,Vol. 19, No. 7, pp. 549-557, 1991. Printed in Great Britain.

0305-1978/91 $3.00+0.00 © 1991 PergamonPressplc.

Genetic and Morphological Variation among Biotypes of Tephritis bardanae SABINE EBER, PETRA STURM and ROLAND BRANDL Lehrstuhl fQr Tier6kologie I, Universit~it Bayreuth, Postfach 101251, D-8580 Bayreuth, F.R.G.

Key Word Index--Tephritis bardanae; Tephritidae; Diptera; Arctium; Cardueae; allozymes; behaviour; morphology; biotypes; phytophagous insects. Abstract--Larvae of the tephritid species Tephritis bardanae develop in flower heads of some Arctium species (Cardueae). Fly populations attacking A. tomentosum plants, which have woolly flower heads, are observed to lay their eggs into the plant stem. This contrasts with the usual egg-laying behaviour within the genus Tephritis, where eggs are normally deposited into flower heads. AIIozyme and morphological differences between these biotypes are small, but quite consistent. We compare the genetic distance between these biotypes with other allozyme studies of tephritids, and conclude that the evolution of biotypes is not necessarily matched by genetic differentiation detectable at the allozyme level.

Introduction In plant-herbivore systems host shifts and the formation of biotypes are a common strategy of phytophagous insects to assure host plant availability, to avoid infra- and interspecific competition and to reduce enemy pressure [1]. The frequency of host shifts in some herbivorous insect groups suggests that either the basic genetic mechanisms might be very simple or that host shifts are only cases of non-genetic polyphenism. Consequently, biotypes are not automatically the first step to the evolution of a new species. For example most of the biotypes in tephritids are able to hybridize without any hybrid depression (Brandl and Zw61fer, unpublished results, [2]). Obviously genetic differentiation, if it exists at all, is reversible. Thus, the formation of a new species may require some additional events to generate reproductive isolation. Nevertheless biotypes are preformed break-lines along which populations of phytophagous insects may split into distinct species [1]. Within the genus Tephritis several species are known to form biotypes on different host plants [3]. One of them is Tephritis bardanae, which develops in flower heads of the plant genus Arctium (Cardueae). ZwSIfer [4] mentions A. tomentosum, A. minus and A. nemorosum as hosts of T. bardanae. We will concentrate on the first two host species. Arctium minus and A. tomentosum have an overlapping distribution in Middle and Eastern Europe [5]. Beside the branching pattern, differences in flower heads are important for the phytophagous fly: the flower heads of A. tomentosum are larger than those of A. minus and have a felt-like, woolly outside. During June, females of T. bardanae lay their eggs into the flower heads (but see below) of Arctium plants, where larvae feed on the achenes and pupate during August. Adults emerge in September and hibernate at unknown places (Sturm, unpublished observations). The present paper reports behavioural and genetical differences between biotypes of T. bardanae attacking A. tomentosum and A. minus. We present evidence for a differentiation of egg-laying behaviour in correlation with the head structure of host plants. The genetical differentiation measured by allozymes, however, is rather small, but seems to be host-related. (Received 25 March 1991) 549

550

S. EBER ETAL.

Materials and Methods Sampling, electrophoresisand morphometrics Samples of A. minus and A. tomentosumwere collected during June 1989 (localities: A. tomentosurn-Sweden, Nidda and Bayreuth; A. minus--Kiel near 2 or 3 in Fig. 1). To infer egg-laying behaviour on different host plants, flowerheads were dissected and we recorded number and position of clutches. In summer 1989samples of A. minusand A. tomentosumwere taken at different sites in Europe (Fig. 1) to obtain flies for electrophoresis.From the Ahrensee site we have samples from two different years (Ahrenseel, 1987; Ahrensee2, 1989). From these samples mature larvae of T. bardanaewere reared to adults. Flies were collected daily and stored in liquid nitrogen for electrophoresison horizontal starch gels [6]. The homogenate of each individual was analysed for variation at nine scorable enzyme systems coded by 10 presumptive loci: aconitase 1 (ACON-1),aconitase 2 (ACON-2),fumarase (fum), glutamate oxalacetate transaminase (GOT), hexokinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), malate enzyme (ME), peptidase (PEP) and phosphoglucomutase (PGM). Zymograms were scored by giving the most common electromorph at each locus the arbitrary score of 100. For morphometric analyses 24 characters of the wing were measured (Fig. 4), four measurements were made at the ovipositor (oviscapt: length, basal and distal width; acuieus: length) using a binocular microscope. Data were obtained from 20 females per site, except at the site near Bayreuth, where only 10 females were available. From Sweden and Berkenthin no flies were measured.

Statisticalanalyses Genetics. A hierarchical analysis of population differentiation was performed using the approach of Wright [7]. We defined three levels of population Subdivision within the total sample (indexed by "1"): individuals of single populations (indexed by I), sampled sites (S) and biotypes (B). Quantities of variation are estimated as variance components for each level relative to another. The percentage of genetic variation for different levels was calculated by dividing the appropriate variance component by the limiting variance. From allele frequencies a distance matrix between sites was constructed [8]. The resulting matrix was summarized using principal coordinates and cluster analysis [9]. Morphometrics. From a previous study with another tephritid species (Oxyna parietina) we know that different methods of size correction provide almost identical results (Eber et al., submitted). Therefore we simply used the logarithm of the raw data divided by wing length to reduce the impact of size (see [10] and [11] for other possibilities). Similar to allozyme data we performed nested analyses of variance to calculate the relative amount of morphological variance within sites, between sites and between biotypes. Discriminant analysis was used to ordinate sampling sites and for cluster analysis the Mahalanobis distances between sampling sites were calculated. Packages. All calculations were performed using procedures of SPSS/PC+ [12], SAS [13], BIOSYS [14] and NTSYS-pc [15].

Results Egg-laying behaviour The positions of clutches in the t w o Arctium species are s h o w n in Fig. 2. From 108 clutches in A. minus 87 (81%) w e r e located in the b o t t o m o f the f l o w e r heads, as e x p e c t e d for a typical species w i t h i n the g e n u s Tephn'tis (sampling locality Kiel; Fig. 1). For A. tomentosum a totally different picture w a s f o u n d : m o r e than 99% of the 345 dissected clutches w e r e laid into the stem beneath the f l o w e r head (Fig. 2). This result holds for different g e o g r a p h i c a l regions (Sweden, Bayreuth; Fig. 1). O b v i o u s l y T. bardanae uses different strategies of e g g laying in correlation to the attacked host species.

Population genetics From 10 scorable loci ACON-2 and FUMwere m o n o m o r p h i c . ACON-1, GOT, HK, IDH, MDH, ME, PEP and PGM a p p e a r e d w i t h at least t w o and up to five alleles (Table 1). No e n z y m e locus w a s fixed for alternative alleles at different sites o r biotypes. Estimates of the e x p e c t e d h e t e r o z y g o s i t y range f r o m 0.127 to 0.232 (Table 1) w i t h no consistent differences b e t w e e n biotypes. Total limiting variance of the analysed a l l o z y m e s in T. bardanae is 1.56. Local p o p u l a t i o n s contain m o s t of the genetic variation (O~s= 1.45; 93% of the limiting variance). Genetic divergence o f b i o t y p e s (osT = 0.033; 2.1%) as well as of local p o p u l a t i o n s ( = sites; OSB= 0.077; 4.9%) is quite small. Nei-distances b e t w e e n sites range f r o m 0.002 to 0.076 (Table 2). The genetic distance b e t w e e n s a m p l e s of the s a m e site m a d e in different years is 0,001

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FIG. 1. SAMPLING SITES OF 1: BARDANAE.

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S. EBER ETAL

552

bracts i head st/h

stem

!00

80

60

40

clutches

20

0

0

20

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100

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FIG. 2. POSITION OF 7?BARDANAECLUTCHES ON BOTH INVESTIGATED HOST PLANT SPECIES.

(Ahrenseel, Ahrensee2; Table 2). The mean genetic distance between biotypes is 0.03 (range: 0.012-0.076), whereas the mean genetic distance between populations of the same biotype is 0.005 (0.002-0.008) within A. tomentosum and 0.027 (0.001-0.053) within A. minus. With one exception (population of Berkenthin) the biotypes of T. bardanae are separated by a UPGMA cluster and a principal coordinates analysis (Fig. 3). Morphometrics Morphometric data sets from wing (Fig. 4) and ovipositor were analysed separately. The relative amount of morphological variation between biotypes is around 10% for wing and 8% for ovipositor measurements; values for individual characters differ considerably. Because most morphological measurements are intercorrelated, the above values are only rough guides to the differentiation between biotypes. The results of canonical discriminant and cluster analyses are illustrated in Fig. 5. For wing measurements the four canonical discriminant functions are at least marginally significant (canonical variate I to III, P<0.004; variate IV, P=0.068). Functions 1 and 2 summarize 53% and 26%, respectively of total variation. 83% of individuals were classified correctly by the discriminant analysis. For ovipositor rneasurements only the first two canonical discriminant functions are significant (both P<0.003). These two axes include 99% of total variance, 40% of individuals were assigned to the correct site. Individuals from different host plants are well separated along the first canonical variate for both morphological data sets. Cluster analyses provide a separation of 7?.bardanae populations from A. tomentosum and A. minus, except for Bayreuth within the analysis of ovipositor measurements. Both morphometric distance matrices are correlated significantly, but only the wing distance matrix shows a significant correlation with genetic distances between sites (Table 3). Discussion Most tephritids, which attack flower heads, lay their eggs into the heads of the host plants [16]. In this way the A. minus biotype of T. bardanae is typical for this genus (see also [17]). The populations associated with A. tomentosum, however, are an exception within the species as well as within the whole genus Tephritis. This behavioural difference could be a consequence of the flower head morphology of A. tomentosum.

VARIATION AMONG TEPHRITIS BARDANAE

553

TABLE 1. ALLELE FREQUENCIESOF POLYMORPHICLOCI AND VALUES OF EXPECTED HETEROGENEITYFOR EIGHT SITES OF T. BARDANAE

ACON

GOT

HK

IDH

MDH

ME

PEP

PGM

Pop

1

2

3/1

3/2

4

5

6

7

n 80 100 129 n 75 100 114 124 132 n 100 107 n 77 85 90 100 110 n 60 70 100 147 n 100 110 n 100 107 116 n 82 92 100 119

19

33

26

14

14

35

32

1.00

0.98 0.02 23 0.02 0.96

1.00

42 0,01 0.99

1.00

1.00

1.00

61

8

21

0.98

1.00

1.00

35 0.01 0.94 0.01 0.01 0.01 35 1.00

0.98 0.02 32

19 1.00

17 0.03 0.97

0.02

0.02 0.01 45 1.00

19 0.92 0.08 19

23 0.83 0.17 23

26

1.00

0.04 0.96

0.06 0.94

19

23 0.04

26

1.00

0.89 0.07 23 1.00

1.00

0.97

26 1.00

45 1.00

0.76 0.24 27

23 0.37 0.30 0.33 23

26 0.35 0.29 0.37 26

45 0.37 0.30 0.33 37

0.10 0.83 0.07 15 0.97 0.03 15 0.13 0.13 0.73 15

0.59 0,41

0.63 0.37

0.65 0.35

0.69 0.31

0.27 0.73

0,127

0.232

0.163

0.172

0.195

Hexp

19 1.00 19

26 1.00

15 1.00

45 0.01 0.03

15 0.17

0.92 0.03 43 0.03

21 0.90 0.10 21

0.03 0.80

0.02 0.98

15

20 0.08

35 0.01

0.94 0.06 32 0.95 0.05 32

0.03 0.94 0.01 35 0.07

0.05 0.95

0.89 0.04 35 1.00

0.91 0.03 32 1.00

35 0.17 0.63 0.20 35

32 0.17 0.55 0.28 32

0.67 0.31

0.56 0.44

0.02 0.52 0.47

0.177

0.185

0.204

0.93 21 0.98 0.02 21 0.12 0.64 0.24 21 0.02

32 0.06

1 = Sweden, 2 - Stodthagen, 3/1 = Ahrenseel, 3/2 = Ahrensee2, 4 = Berkenthin, 5 - Nidda, 6 = Bayreuth, 7 - Neumarkt. 1,5,6,7 with Arctium tomentosum as host plant, 2,3/1,3/2,4 with Arctium minus, n Number of individuals, H,xp= unbiased expected heterozygosity.

TABLE 2. NEI'S GENETIC DISTANCE BETWEEN SITES OF 7; BARDANAE

Sweden Stodthagen Ahrenseel Ahrensee2 Berkenthin Nidda Bayreuth Neumarkt

Sweden

Stodthagen

Ahrenseel

0.022 0.022 0.023 0.058 0.003 0.005 0.006

-0.004 0.005 0.039 0.012 0.046 0.010

-0.001 0.035 0.013 0.012 0.009

Ahrensee2

Berkenthin

Nidda

Bayreuth

0.057 0.046 0.036

-0.004 0.005

0.001

m

0,040 0.013 0,013 0.010

Neumarkt

554

S. EBER ETAL Niddo Sweden

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i

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i

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i

-0.08

0.0

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t

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•

Bayreuth

o

Stodthagen

o

Ahrensee

o

Ahrensee 2

/~

Berkenfhin

1

Principal coordinate I FIG. 3. PRINCIPAL COORDINATE AND UPGMA CLUSTER ANALYSIS OF THE GENETIC DISTANCE MATRIX GIVEN IN TABLE 2. Closed symbols: host plant A. tomentosum, open symbols: host plant A. minus.

A

f' B o

FIG. 4. (A) WING MEASUREMENTS MADE ON /3 BARDANAE: L=WlNG LENGTH, B=WING BREADTH; NUMBERS INDICATE DISTANCES ALONG WING VEINS; F1 TO F4 ARE MEASUREMENTS OF COLOUR PATTERNS; A TO C ARE COUNTS OF WHITE SPOTS IN DARK WING AREAS. (B) TYPICAL WING OF T. BARDANAETO GIVE A VISUAL IMPRESSION.

The woolly flower heads may be an obstacle against the usual egg-laying behaviour: adults switch to the stem near the flower head. In consequence the first instar larvae have to find their way to the feeding places, the achenes. The larvae of the A. minus biotype, which hatch within the bottom of the flower head, also have to move upwards to find the achenes. In that way larvae of both biotypes have to move in the same direction. Thus, we suggest that this difference does not need a genetical-based adaptation, as larvae of both biotypes use the same general movement pattern. It is unlikely, however, that egg-laying behaviour itself is a mere phenotypic adaptation. Firstly, the egg-laying behaviour differs between biotypes irrespective of the sampled locality. Secondly, flies from the A. tomentosum site in Bayreuth, which

555

VARIATION AMONG TEPHRITISBARDANAE

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FIG. 5, PLOT OF INDIVIDUAL DISCRIMINANT SCORES ALONG THE FIRSTTWO CANONICAL VARIATES AS WELL AS UPGMA CLUSTER ANALYSIS OF THE MAHALANOBIS-DISTANCES BETWEEN GROUP CENTROIDS. (A) Wing and (B) ovipositor measurements. Closed symbols: host plant A. tomentosum; open symbols: host plant A. minus.

TABLE 3. CORRELATIONCOEFFICIENTS(ABOVE) AND LEVELS OF SIGNIFICANCE (BELOW; CALCULATED BY A PERMUTATIONAL STRATEGY [15]; ONE-TAILED) FOR MATRIX CORRELATIONS OF MORPHOMETRIC DATA FROM WINGS AND OVIPOSITORAS WELL AS NEI'S GENETIC DISTANCES Wing Wing Ovipositor Nei

-0.07 0.04

Ovipositor 0.57 ->0.1

Nei 0.65 --0.01 --

attacked nearby A. minus plants only occasionally, showed the typical A. tomentosum egg-laying behaviour at those A. minus plants (Sturm, personal observation). To clarify the genetic basis of the behavioural differences between biotypes, breeding experiments would be the first choice. As flies of the genus Tephritis hibernate as adults, it is very difficult to keep flies in the laboratory over several generations. Therefore we looked for further possibilities to demonstrate differences between biotypes. The allozyme differences found proved to be quite small and fit in the range of geographic differentiation known from other tephritid populations (Fig. 6). We extracted some morphological differences between fly populations matching the host type, but this could be due to our geographic sampling scheme. All samples of flies from A. tomentosum are from the southern part of Germany, whereas all flies from A. minus were collected in the north. Our allozyme samples have a similar geographic bias, except the Swedish sample of A. tomentosum. This sample did not cluster

556

S. EBER ETAL. Orellia Anasfrepha G Rhagolefis Rhagolefls G Eurosfa G Neaspilofa

--I --7 m

Anastrepha

]

Oxyna G

]

Tephrifis G Orellia G Cerofifis G Urophoro Tephriiis G Tephrifis

o.~5

0'1

o.'ls

Nei's genetic disfance FIG. 6. EXAMPLES OF MINIMAL GENETIC DISTANCES BETWEEN SPECIES IN SOME TEPHRITIDGENERA (CLOSED BARS) AS WELL AS MAXIMUM GEOGRAPHIC GENETIC DIFFERENCES WITHIN TEPHRITIDSPECIES (OPEN BARS AND G AFTER THE NAME; SOURCESOF DATA[19-23] NEI DISTANCESGIVEN FOR TEPHRITIS, UROPHORA AND OXYNA ARE OWN UNPUBLISHED RESULTS. For geographical samples of Tephritis we present data for two species /7 bardanae (low value) and 77conura.

according to its geographic position but according to the host (Fig. 3). In a previous study on the tephritid fly Oxyna parietina, which attacks Artemisia vulgaris, we found a shallow cline in allozyme data from Austria to the northern part of Germany, but wing measurements did not match this cline. There was no correlation between the morphological and allozyme distance matrices. In T. bardanae, however, morphometric and allozyme distance matrices are correlated (Table 3), which suggests some consistency between genetic and morphometric data sets. Therefore one may assume that biotypes of T. bardanae are in a process of developing consistent morphological variation between biotypes. Maximum genetic distances between geographic samples of tephritids and minimum genetic distances between species in some tephritid genera are summarized in Fig. 6. As mentioned above our results for T. bardanae are quite typical for geographic samples in tephritids. Furthermore minimum differences between species are well within the range of maximum genetic differences of geographic samples within species. This illustrates that the formation of species from biotypes may be a continuous process on the level of allozymes. Additionally, Fig. 6 suggests that allozymes are an unreliable guide to the classification of biotypes or sibling species in tephritids. This statement has an important implication for biological control projects. Many tephritids were used for biological control of weeds [18]. Despite the low genetic differentiation, biotypes can differ considerably in host specifity. Screening tests are necessary for the selection of an appropriate biological control agent. It would be unwise to exclude populations from screening only because they have a small genetic distance to already tested populations. Our observations illustrate the review of Zw61fer and Romst6ck-V61kl [1], that biotypes are ecological templates for the speciation process and a flexible attribute of herbivorous insect species. The small genetic and morphometric differences suggest that differentiations of biotypes are reversible. Under certain conditions reproductive isolation may evolve during phases of geographic isolation or other processes which deepen the genetic gap between biotypes. This step from a biotype to a full species is not necessarily matched by genetic variation detectable on the allozyme level. We agree with Morgante et al. [19] that tephritids may speciate and evolve more rapidly

VARIATIONAMONG TEPHRITISBARDANAE

557

than other Diptera (e.g. saprophagic Drosophila), yet undergo less structural gene change. Most importantly these changes predominantly affect loci which influence behavioural patterns. References 1. Zw61fer, H. and Romst6ck-VSIkl, M. (1991) in Plant-Animal Interactions: Evolutionary Ecology in Tropicaland Temperate Regions, p, 487. John Wiley, New York. 2. MSIler-Joop, H. (1988) Biosystematische Untersuchungen an Urophora solstitialis (Tephritidae): Wirtskreis, Biotypen und Eignung zur biologischen Bekdmpfung von Cardus acanthoides L. (Compositae) in Kanada. Doctoral Thesis, University of Bayreuth. 3. Romst6ck, M and Arnold, H. (1987) Zool. Anz. 219, 83. 4. ZwSIfer, H. (1965) Commonw. Inst. Biol. Control, Tech. Bull. 6, 81. 5. Hult~n, E. and Fries, M. (1986) Atlas of North European Vascular Plants. Koeltz Scientific Books, K6nigstein. 6. Hillis, D. M. and Moritz, C. (1990) Molecular Systemadcs. Sinauer, Sunderland. 7. Wright, S. (1978) Evolution and Genetics ofPopulaUons. Vol. 4. University of Chicago Press, Chicago. 8. Nei, M. (1972) Am. Nat. 106, 283. 9. Digby, P. G. N. and Kempton, R. A. (1987) Multivariate Analysis of EcologicalCommunities. Chapman & Hall, London. 10. Reist, J. D. (1985) Can. J. Zool. 63, 1429. 11. Reist, J. D. (1986) Can. J. Zoo/. 64, 1363. 12. Noru,~is, M. J. (1986) SPSS/PC,+Advanced Statistics", SPSS Inc,, Chicago. 13. SAS Institute Inc. (1988) SAS/STAT" User's Guide, Release 6.03 Edition. SAS Institute Inc., Cary NC. 14. Swofford, D. L. and Selander, R. B. (1989) BIOSYS-1, Release 1.7, Illinois Natural History Survey. 15. Rohlf, F. J. (1990) NTSYS-pc. Vers 1.60. Exeter Software, New York. 16. Romst6ck, M. (1987) Tephritis conura Loew (Diptera:Tephritidae) und Cirsium heterophyllum (L.) Hill (Cardueae): Struktur und Funktionsanalyse eines &kologischen Kleinsystems. HoschschulSammlung Nautrw., Biologie Band 18, Hochschuleverlag, Freiburg. 17. Straw, N. A. (1985) Proc. Vlln~ Symp. Biol. Contr. Weeds 1985, 479. 18. Harris, P. (1989) BiocontrolNews Info. 10, 7. 19. Morgante, J. S., Malavasi, A. and Bush, G. L. (1980) Ann. Entomol. Soc. Am. 73, 622. 20. Berlocher, S. H. and Busch, G. L. (1982) Syst. Zool. 31,136. 21. Steck, G. J. (1981) North American Terelliinae (Diptera: Tephritidae): Biochemical Systematics and Evolution of Larval Feeding Niches and Adult Life Histories. PhD Thesis, Univ. Texas, Austin. 22. Milani, R., Gasperi, G, and Malacrida, A. (1989) in Fruit Flies their Biology, Natural Enemies and Control, Vol. 3B, p. 33. Elsevier, Amsterdam. 23. Waring, G. L,, Abrahamson, W. G. and Howard, D. H. J. (1980) Evolution44, 1648.