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Journal of the Lepidopterists' Society 36(3), 1982, 185-191

EVOLUTIONARY STUDIES ON CTENUCHID MOTHS OF THE

GENUS AMATA: 2. TEMPORAL ISOLATION AND NATURAL

HYBRIDIZATION IN SYMPATRIC POPULATIONS OF

AMATA PHEGEA AND A. RAGAZZII

V. Sbordoni,1 L. Bullini,2 P. Bianco,3 R. Cianchi,2 E. De Matthaeis1 and S. Forestiero1

ABSTRACT. Amata phegea and A. ragazzii are two sibling species which occur sympatrically in several areas of Central and Southern Italy. The occurrence of certain enzyme loci, electrophoretically diagnostic (PGM, HK, EST-5), allowed the correct identification of all individuals, including hybrids. Population studies in the field carried out in sympatric areas of Central Italy revealed the occurrence of seasonal displacement in the flight period acting as a premating isolating mechanism between the two species, in spite of a partial overlapping. Premating behavior of homospecific and heterospecific pairs investigated in the laboratory showed the existence of isolating mechanisms also at the etiological level, which lowers the mating success of the heterospecific pairs. The frequency of hybrids in different localities varies from 0 to 0.053; the highest rates of hybridization were found in biotopes recently altered by man. Hybrid frequencies, similar in larval and adult stages, suggest a normal viability of the hybrids.

The ctenuchid moths Amata phegea (Linnaeus, 1758) and A. ragazzii (Turati, 1917) are two sibling species, so far scarcely investigated from the genetic, ecological, ethological and zoogeographical points of view. A. phegea is widely distributed in Central Western Europe (with the exception of the Iberian peninsula) and is particularly abundant and widespread in Italy; while A. ragazzii is endemic in Central Southern Italy. The latter species has to date been observed in Calabria, Campania, Lucania, Molise, Lazio and Umbria.

The two species presumably diverged through geographical isolation in warm refugia during the Pleistocene. They successively expanded their range in the post-glacial era following the spread of the deciduous broad-leaved forest. Areas of sympatric occurrence have been identified in the Alban Hills, in the Fioio Valley (Simbruini Mountains), near Leonessa (Rieti), at S. Polo dei Cavalieri (Sabine Mountains), at Mount Faito (Campania), at Viggiano (Lucanian Ap-pennines) and in other areas of Central and Southern Italy.

At the morphological level identification of the adult of the two species may be difficult, particularly in females. On the other hand, allozyme separation by electrophoresis enables us to identify all individuals, both larvae and adults (Bullini et al., 1981). Using this

1  Institute of Zoology, University of Rome.

2  Institute of Genetics, University of Rome.

3 Institute of Zoology, University of L'Aquila.

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method we were able to discover some hybrids in the overlapping areas although no evidence of introgression has been detected. This seems to indicate the existence of highly effective isolating mechanisms.

We are investigating these mechanisms both in the laboratory and in the field. In this paper we will present data showing patterns of temporal displacement in the flight period of the two species, probably representing an effective premating isolating mechanism; a further premating mechanism is also reported operating at the etiological level. In addition, we will present data on the occurrence of natural hybridization in some areas.

Study Sites, Materials and Methods

As previously reported, a number of localities have now been identified in Central Italy where A. phegea and A. ragazzii occur sym-patrically. In most of them the phenology of the two species was investigated by frequent observations over several years. More detailed information came from five localities near Rome, where periodic observations and counts were carried out.

The first locality is situated at a height of 850 m near Camerata Nuova, a village 50 km north east of Rome at the foot of the Simbruini Mountains. This site is characterized by a Quercetum association and represents the middle-lower part of the biotope described in detail by Sbordoni et al. (1979). The remaining four sites, namely Zagarolo, San Cesareo, Rocca Priora and Monte Cavo, were selected along an altitudinal transect 14 km long from 300 to 900 m above sea level within an area including the Alban Hills about 30 km south east of Rome. Most of this area is characterized by chestnut woods.

Observations were carried out from 1967 to 1980; reported data refer to 1974, which can be considered representative of the overall period. Direct counts of adult moths were employed to describe the phenology of A. phegea and A. ragazzii. Mark-release-recapture methods were also utilized to estimate absolute populations sizes of the two species (see Sbordoni et al., 1979).

Starch gel electrophoresis applied to three diagnostic enzyme loci: phosphoglucomutase (Pgm), hexokinase (Hk) and esterase (Est-5) was utilized to identify morphologically doubtful specimens and to discover hybrids (see Fig. 3). Electrophoretic techniques were, with minor modifications, those described by Ayala et al. (1972) for phosphoglucomutase and hexokinase, and by Selander et al. (1971) for esterase.

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Fig. 1. Phenology of adult populations of Amata phegea (black circles) and A. ragazzii (white circles) at Camerata Nuova, Simbruini Mountains, 850 m, showing temporal displacement in the flight period.

Results and Discussion

Temporal Displacement in Flight

By scattared observations and preliminary collection data we noted that the two species emerge at different times. This trend is clearly apparent from the data reported here.

At Camerata Nuova Amata phegea emerges about one month before A. ragazzii (Fig. 1). In the Alban Hills the emergence dates of the two species are separated by about 20 days, regardless of altitude (Fig. 2). Similar phenological data were observed in other localities in Central Italy. The degree of overlapping depends on the relative population sizes of the two species, which vary according to the localities. At Camerata Nuova A. phegea greatly outnumbers A. ragazzii and the ratio between the population sizes is approximately 30:1 (Sbordoni et al., 1979). More balanced situations were found in the localities of the Alban Hills. Here, consistent overlapping is limited to 10-15 days.

In both species males are first to emerge. Females are generally inseminated a few hours after their emergence (Stauder, 1927; Ob-raztsov, 1941). Female sex attractants are probably related to the anal papillae, which are rhythmically displayed by the virgin females (Ob-raztsov, 1966). Several males generally attempt to mate a single virgin female (Rasetti and Rasetti, 1921). Among adults the sex ratio is def-

188                                           Journal of the Lepidopterists' Society

ZAGAROIO 300 m                                                                               S. CESAREO 450 m

ROCCA PRIORA 650 m                                                                           MONTE CAVO 900 m

JUNE                              JULY                                               JUNE                               JULY

Fig. 2. Phenologies of adult populations of Amata phegea (black circles) and A. ragazzii (white circles) in 4 localities along an altitudinal transect south east Rome.

initely in favor of the males (80-85% in both species). According to these behavioral traits and the sex ratio, it seems improbable that a female of A. phegea could be inseminated by a male of A. ragazzii. On the other hand, old males of both species show an erratic behavior, and they tend to wander from their usual habitat in forest edges, open woods, etc. to open fields; also, this behavior limits the opportunities of mating between males of A. phegea and emerging females of A. ragazzii.

These observations led us to conclude that seasonal displacement may act as a premating isolating mechanism between A. phegea and A. ragazzii, although partial temporal overlapping occurs. This barrier would be particularly effective if monogamy were present in both species. However, this latter point needs to be tested.

Similar temporal displacement occurs also in other sympatric combinations of related Amata species. We observed this phenomenon in Istria and Dalmatia between A. marjana (Stauder, 1913) and A. phegea. In such instances, A. marjana is the first to fly, and the emergences of the two species are separated by about one month.

Premating Behavior

Besides the temporal displacement, an ethological isolating mechanism was also detected by preliminary laboratory experiments. A.

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PGM

1 2 3 4 5 6 7 8 9 10 11 12 13 14

FlG. 3. Phosphoglucomutase (PGM) electrophoretic patterns of Amata phegea, A. ragazzii and their hybrid. A. ragazzii (specimens 1-4 and 10-14, from left to right) shows an anodic band migrating faster than A. phegea (specimens 5 and 7-9), whereas the hybrid (specimen 6) shows the two bands characteristic of the parental species.

phegea males isolated with conspecific virgin females are strongly attracted and approach the partner with a characteristic zig-zag flight. When the male touches the female she stops flying and the male lands on her. He repeatedly touches the costal area of her anterior wings with his antennae; the female, keeping her wings spread, raises her abdomen. Then copulation takes place. When A. ragazzii males are isolated with virgin A. phegea females, precopulatory behavior always begins later. Furthermore, the female often doesn't stop when touched and moves away. This generally interrupts a male's precopulatory behavior. Even when copulation takes place, the female frequently lays unfertilized eggs.

Natural Hybridization Between A. phegea and A. ragazzii

In this section we will present data demonstrating the occurrence of natural hybrids between the two Amata species occurring sym-patrically in areas of Central Italy.

As reported in the materials and methods section, detection of hybrids by electrophoresis was made at some enzyme loci, which are diagnostic for A. phegea and A. ragazzii (Pgm, Hk, Est-5). At these loci no common allele is shared between the two Amata species studied. Hybrids are characterized by heterozygous patterns at each of these loci.

In Fig. 3 an example of a zymogram is shown, illustrating the pattern of A. phegea, A. ragazzii and their hybrid, at the Pgm locus.

Table 1 shows the numbers of A. phegea, A. ragazzii and hybrids in samples from some sympatric areas in Central Italy. Samples tabulated were collected during periods of temporal overlapping of the two species. However, if samples are collected over the whole period of flight of the two species, rates of hybridization may appear lower,

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Table 1. Rates of natural hybridization between A. phegea and A. ragazzii, as revealed by electrophoresis on samples of adult moths from some sympatric areas of Central Italy.

because all the hybrids detected were in flight relatively later, together with A. ragazzii.

Table 2 shows the data obtained from samples of larvae collected in three sites of the Alban Hills.

A comparison between the two tables do not reveal substantial differences in the frequency of hybrids between adult and larval stages from the same locality, suggesting normal viability of hybrids.

The frequency of hybrids varies from 0 to 0.053, but several values are around 0.004. The high rate of hybridization detected at San Cesareo, both from larval and adult samples observed in two distinct generations, may be tentatively related to the man-made alteration of their biotope. At the collection sites a wide zone of chestnut wood was replaced by Rubus sp.

Cases of sympatric hybridization are often associated with habitat alteration (Woodroof, 1973). In the case of A. phegea and A. ragazzii habitat alteration could affect the rate of hybridization and even the occurrence of sympatry between the two species. This working hypothesis requires further investigation.

Table 2. Rates of natural hybridization between A. phegea and A. ragazzii, as revealed by electrophoresis on samples of larvae from some sympatric areas of Central Italy.

Number of specimens scored                Relative

frequency

Study site                               Date of sampling              A. phegea A. ragazzii Hybrids          of hybrids

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Literature Cited

Ayala, F. J., J. R. Powell, M. L. Tracey, C. A. Mourao & S. Perez-Salas. 1972. Enzyme variability in the Drosophila willistoni group. IV. Genie variation in natural populations of Drosophila willistoni. Genetics 70:113-139.

Bullini, L., R. Cianchi, C. Stefani & V. Sbordoni. 1981. Biochemical taxonomy of the Italian species of the Amata phegea complex (Ctenuchidae, Syntominae). Nota Lepid. 4: in press.

OBRAZTSOV, N. S. 1941. Die Artdifferenzierung der phegea-Gruppe des Subgenus Syntomis O. der europaischen Fauna. Univ. Kijev. Acta Mus. Zool. 1:103-164.

--------- 1966. Die palaearktischen Amata-Arten (Lepidoptera, Ctenuchidae). Veroff.

Zool. Staatssamml. 10:1-383.

Rasetti, E. & F. Rasetti. 1921. Note entomologiche. Boll. Soc. Entomol. Ital. 53:19-27.

Sbordoni, V., L. Bullini, G. Scarpelli, S. Forestiero & M. Rampini. 1979. Mimicry in the burnet moth Zygaena ephialtes: population studies and evidence of a Mullerian-Batesian situation. Ecol. Entomol. 4:83-93.

Selander, R. K., M. H. Smith, S. Y. Yang, W. E. Johnson & J. B. Gentry. 1971. Biochemical polymorphism in the genus Peromyscus. I. Variation in the old field mouse (Peromyscus polionotus). Studies in Genetics, 6, Univ. Texas Publ. 7103:49-90.

Stauder, H. 1927. Uber Zucht siiditalienischer Syntomiden. Lepid. Rundsch. 1:57-59.

WOODROOF, D. S. 1973. Natural hybridization and hybrid zone. Syst. Zool. 22:213-217.