Institute for Biodiversity and Ecosystem Dynamics / Zoological Museum Amsterdam



Speciation in Dichrorampha

Dr Sandrine A. Ulenberg



This study is part of a research program in which biological data are incorporated into phylogenetic studies in an attempt to formulate effect analyses for processes of speciation.

The genus Dichrorampha belongs to the lepidopteran family of Tortricidae, subfamily of Olethreutinae, tribe of Grapholitini. The genus Dichrorampha consists of approximately 115 species, of which 14 occur in the Netherlands. The distribution of the genus is Holarctic

D. acuminatana

D. obscuratana

D. aeratana

D. petiverella

D. agilana

D. plumbagana

D. alpinana

D. plumbana

D. consortana

D. sedatana

D. flavidorsana

D. simpliciana

D. gueneeana

D. sylvicolana

Tabel 1: Pictures of species of Dichrorampha occurring in the Netherlands


Most of the 14 Dichrorampha species occurring in the Netherlands are sympatric and have overlapping flight periods. The larvae feed on a restricted number of host plant species, all of which belong to the family Asteraceae. For many closely related sympatric tortricids host plant association shows to be the key to reproductive isolation. However, the host plant associations amongst the Dichrorampha species are not mutually exclusive, so that they cannot function as isolating mechanisms (Ulenberg et al., 1993). The daily sexual activity patterns of the different species are also overlapping (data unpubl.). Thus, the answer to the question how reproductive isolation is maintained points to the pheromone system. We, therefore, propound female sex pheromone variation between the 14 different species of Dichrorampha most probably is the only isolating mechanism suggesting that female sex pheromone variation played a major role in the speciation events within Dichrorampha.

The aim of this study is to characterise the female sex pheromone variation among these 14 species of Dichrorampha and to interpret the pheromone variation in view of the genus' phylogeny.

Various projects in this study:





  • Description of projects:

Sexual communication in the genus Dichrorampha (Lepidoptera, Tortricidae)

by Jeroen J.N. van den Boezem1, Sandrine A. Ulenberg1 and Teris A. van Beek2

Co-operation of:

1 University of Amsterdam, Institute for Systematics and Population Biology, Department of Entomology, Plantage Middenlaan 64, 1018 DH, Amsterdam.

2 Wageningen Agricultural University, Laboratory of Organic Chemistry, Phytochemical Section, Dreijenplein 8, 6703 HB, Wageningen.



Many moth species have reproductively isolating host plant associations, but the genus Dichrorampha (Olethreutinae, Grapholitini) does not. Also flight period, circadian rhythm, lifecycle and morphology of the sympatric Dichrorampha species show striking similarities and do overlap.A Therefore, we propound sex pheromone variation is the only isolating mechanism suggesting that sex pheromone changes played a major role in the events from which new species originated (speciation) within the genus Dichrorampha.


Fig. 1: Dichrorampha and Epiblema : a.D. alpinana; b. D. simpliciana ; c. D. flavidorsana.; d.E. foenella



The aim of this project is the determination of the female sex pheromones of 14 Dichrorampha species, occurring in the Netherlands, and the deduction of the most probable sequence of changes in the composition of the sex pheromone on basis of the reconstruction of their evolution (phylogeny). We have chosen Epiblema foenella (Eucosmini) as outgroup species.



Pheromone is isolated by solvent extraction from the pheromone gland, Solid Phase Micro Extraction (SPME) or Thermal Desorption (TD) from females from both the field and rearing. Methods for optimisation SPME and TD are still under investigation.

By 2-dimensional Gas Chromatography (2d-GC) in combination with Electro-Antennographical Detection (EAD) the pheromone gland extracts are analysed (figure 2).B

Figure 2. The 2d-GC/EAD output of an extract of the pheromone glands of female D. plumbana/D. sedatana. Horz.: 1.0min/div. Channel 1: EAG response; 0.5mV/div. (Filt. 8), Channel 2: 2d-GC output; 0.5mV/div. (Filt. 8).


Major EAD-response is found at retention time (37.51 min.) of E9,11-dodecadien-1-yl acetate (E9,11-12:Ac) for the species complex D. plumbana/D. sedatana. Other EAD-responses correspond to retention times of other possible components (figure 2). Similar experiments on D. simpliciana showed the same results. Off-line Electro-Antennography (EAG) experiments are conducted with reference chemicals (figure 3).

Figure 3. Off-line EAG results on D. flavidorsana (green), D. simpliciana (red) and D. alpinana (blue).



Remarkable in the genus Dichrorampha is the high and general response to E9,11-12:Ac. E. foenella's major sex pheromone components and other components of many other members of the Olethreutinae are unsaturated at positions 8 and 10 (D8D10, D8 and D10).C Other mono- (D9 and D8) and double-unsaturated (D8D10) -12:Ac's and -12:OH's seem to play a crucial role in the sex pheromone variation among Dichrorampha species. These differences are subtle, but probably the key to reproductive isolation within the genus Dichrorampha (figure 2 and 3). In the future sex pheromones of all 14 Dichrorampha species will be investigated with these techniques and others.



  • Ulenberg, Minks & van der Pers, Proc. of the Section Exp. and Applied Entomology of the NEV, Vol. 4, p. 211-216, 1993
  • Van den Boezem & Ulenberg, Proc. of the Section Exp. and Applied Entomology of the NEV, Vol. 8, p. 109-113, 1997
  • Witzgall, Chemoecology, 7:13-23, 2555, 1996



Adoxophyes orana as a model insect for Dichrorampha species

Jeroen J.N. van den Boezem, Sandrine A. Ulenberg and Teris A. van Beek



Adoxophyes orana (Lepidoptera, Tortricidae) has been chosen as a model insect to optimise the analytical techniques. Adoxophyes orana's sex pheromone has been fully elucidated from extracts, volatile collections and sex glands1 and its components are commercially available. Adoxophyes orana can be reared. These are all characteristics Dichrorampha does not have.


Electro-Antennographical Detection

2 HP 5890 Series II GC's, equipped with FID's and respectively a DB-1 and DB-wax column, are connected to a Syntech Electro-Antennographical Detector (EAD)2. The connection between the second GC and the EAD is made through a retention reservoir. Female sex glands of Adoxophyes orana are thermally desorbed at 150°C in the Cooled Injection System (CIS) of the first GC.

From the electro-antennographical signals (Ch.1) of peak 1 and 2 in the chromatogram (Ch.2) we can conclude that Z9-14:Ac and Z11-14:Ac respectively induce a physiological response in male Adoxophyes orana.


figure 1: DB-wax column, 2 sex glands. Ch.1 (black); 1mV/div, Ch.2 (blue); 50mV/div, Horz.; 50s/div, filtered (8)



Thermal desorption

A HP 5890 GC, equipped with FID, is connected to a Gerstel Thermal Desorption System (TDS 2). Female sex glands and male abdomen are thermally desorbed in TDS 2 at 50-100°C and volatiles are injected into the CIS. Experiments are also conducted with whole insects, alive just before flash heating3.

Male abdomen are compared to female sex glands. From these chromatograms we can conclude that peaks 1 and 2 are the major components of the sex pheromone of Adoxophyes orana, respectively Z9-14:Ac and Z11-14:Ac in a ratio of 11 to 1.

figure 2 and 3: DB-1 and DB-wax column, male abdomen (black) and 2 sex glands (blue).

figure 2: TDS 2; 25ºC -> 25ºC/min, 100ºC, 5min, splitless: CIS; -150ºC -> 12ºC/s

figure 3: 350ºC, 5min, splitless: Oven; 150ºC, 4min -> 10ºC/min, 238ºC, 10 min.


Mass selective detection

A HP 6890 Series GC is connected to a HP 5973 Mass Selective Detector (MSD). Female sex glands are thermally desorbed in the liner of the GC at 30 to 150°C, while at the beginning of the column some focusing takes place at an oven temperature of 40°C.

From the mass spectra we can conclude that peaks 1-5 are: 1. Z9-14:OH, 2. Z9-14:Ac, 3. E11-14:Ac, 4. Z11-14:Ac and 5. Z11-16:Ac. These compounds are known to be among the largest of the components of the sex pheromone of Adoxophyes orana.



figure 4: HP-5 column, 2 sex glands. Liner; 30ºC -> ca. 25ºC /min, 150ºC, 2min, splitless: Oven; 40ºC, ca. 5min ->10ºC/min, 240ºC, 23min.



Discussion and References

All these experiments confirm the composition of the female sex pheromone of Adoxophyes orana. The same techniques will be applied for the elucidation of the sex pheromones of Dichrorampha species. After optimisation it should be possible to identify the minor, as well as the major, components with 2d-GC/EAD. Surely the male antenna is sensitive enough. GC/MSD and TDS/GC can be used for conformation. GC/MSD can lead to identification of both minor and major pheromone components from its mass spectra. TDS/GC shows the components that only the female insect produces. These are strong hints that compounds are in fact pheromone components.

  • P. M. Guerin, H. Arn, H. R. Buser and P. J. Charmillot. Sex pheromone of Adoxophyes orana: additional components and variability in ratio of Z-9- and Z-11-tetradecenyl acetate. Journal of Chemical Ecology, Vol. 12, No. 3, pp. 763-772, 1986.
  • 2J. J. N. van den Boezem and S. A. Ulenberg. A methodological approach to determination of female sex pheromone of the genus Dichrorampha Guenée. Proceedings of the section experimental and applied entomology of the Netherlands Entomological Society, Vol. 8, pp. 109-113, 1997.
  • 3T. A. van Beek, F. P. Drijfhout and J. J. N. van den Boezem. On-line collection and GC analysis of pheromones from intact insects. Proceedings of the 2nd International Symposium on Insect Pheromones 30 March-3 April 1998.



Sex pheromones of Dichrorampha species

Jeroen van den Boezem, Sandrine Ulenberg and Teris van Beek



Since hostplant association, flight periods and circadian rhythm overlap, sex pheromone variation is the only plausible isolating mechanism suggesting that sex pheromone changes played a major role in speciation within the sympatric genus Dichrorampha (Lepidoptera, Tortricidae, Olethreutinae, Grapholitini).

The possible pheromone composition of D. flavidorsana and D. simpliciana are discussed here. For more information, click here.

The aim of this project is the determination of the female sex pheromones of 14 Western-European Dichrorampha species and the deduction of the most probable sequence of changes in the composition of the sex pheromones on basis of their phylogeny.


Electro-Antennographical Analysis


Figure 1. On-line GC/EAD experiment with extract of single calling 2-5 day old, virgin female D..flavidorsana on DB-wax, 60m, id 0.25mm in Varian 6000 GC connected to Syntech EAG equipment. Ch.1 (EAD) 1mV/div, Ch.2 (FID) 2mV/div, Horz. 15s/div

Figure 2. Off-line EAG experiments with single antennae of 2-5 day old male D. simpliciana with Syntech EAG equipment. Experiments are conducted with 20(l of solution on filterpaper in a Pasteur pipet.


The relative amounts of the pheromone components in the extract of D. flavidorsana are: E10-12:Ac 10%, Z10-12:Ac 85% and E- or Z9,11-12:Ac 5%. The structure of the components is verified with GC/MSD. Sensitivity is highest for E/Z9,11-12:Ac. D. flavidorsana does show low sensitivity to compounds mono unsaturated at the 9th position. No distinction can be made between the 2 isomers of ~9,11-12:Ac, since they have not yet been separated.

D. simpliciana antennae are mainly sensitive to E-, 26%Z, and Z9,11-12:Ac, 40%E (white). Also to compounds mono unsaturated at the 9th and 11th position (blue). Typically compounds mono-unsaturated at the 7th and 8th position (yellow) do not reach their equilibrium in this concentration range, while all others do (black). E- and/or Z9,11-12:Ac has been shown with GC/EAD in earlier experiments in gland extracts of D. simpliciana (click here for more information).


Conclusions & Discussion

The phylogeny on the tribal level of the Olethreutinae is not very clear. The sensitivity to E/Z9,11-12:Ac, however, is restricted to the genus Dichrorampha and thus can be regarded as apomorphic character that separates Dichrorampha from other Olethreutinae. Almost all other Olethreutinae use unsaturated compounds at the 8th and/or 10th position.

The fact that the D. flavidorsana female extract holds E- and Z10-12:Ac as its pheromone components and D. simpliciana does not show any significant sensitivity to these compounds in off-line EAG experiments should be reagarded as a plesiomorphic character in D. flavidorsana and could account for the separation at that specific moment of speciation of the two species in the phylogeny of the genus Dichrorampha.

The phylogeny of Dichrorampha will thouroughly be investigated. The sex pheromones of more Dichrorampha species need to be elucidated and tested in the field to make further comments about speciation events within the genus.