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ChromosomalpolymorphisminGalerucellasagittariae(Gyll.)(Chrysomelidae,Coleoptera)inFinland

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Heredilus 111: 189-199 (1989)

Chromosomal polymorphism in Galerucella sagittariae (Gyll.) (Chrysomelidae, Coleoptera) in Finland CHRISTINA NOKKALA and SEPPO NOKKALA

Laboratory of Genetics, Department of Biology, University of Turku, Finland

NOKKALA, C. and NOKKALA, S. 1989. Chromosomal polymorphism in Galerucello sagittariae (Gyll ) (Chrysomelidae, Coleoptera) in Finland. - Hereditas 111: 189-199. Lund, Sweden. ISSN 0018-0661. Received April 4, 1989. Accepted August 8, 1989

B-chromosome polymorphism in 20 populations of Galerucella sagittariae in Finland and one population in Sweden was studied. 13 of the populations were living on Comarum palustre (southern Finland and Sweden), 7 on Rubus chamaemorus (northern Finland), and one on Alchemilla vulgaris. The distributions of the beetles on Comarum and Rubus do not overlap broadly. The standard chromosome number was found to be 2n = 34 (16 + Xy,). In all but one Finnish population, also supernumerary B-chromosomes were found in varying frequencies (1.658.6 YO). In general, the B-chromosome frequency of a population seemed to stay at a constant level from generation to generation. The distribution of the frequencies formed an increasing cline from southwest to northeast, indicating an adaptive significance of B-chromosomes. The size of the B- chromosomes varied from minute-sized to autosome-sized chromosomes. Being largely euchromatic, all B- chromosomes were able to form chiasmatic bivalents in meiosis. They were isopycnotic during meiotic prophase and their segregational behavior was normal. Part of the B-chromosomes were mitotically stable and another part mitotically unstable. The presence of B-chromosomes could not be detected by inspecting the external phenotype of individuals. Obviously some genetic differentiation has taken place between south- ern and northern Galerucella sagittariae. The southern form is dependent on the immediate proximity of water while the northern form is able to live in considerably drier habitats, and thus the variety of suitable food plants is much broader.

Christina Nokkala, Laboratory of Genetics, Department of Biology, University of Turku, SF-20500 Turku. Finland

The taxonomically controversial Chrysomelid Galerucella nymphaeae species complex has been under intensive study over a considerable period of time (see HIPPA and KOPONEN 1986). Recently, an extensive cytological analysis was undertaken in the species complex to clarify the taxonomic situa- tion (see NOKKALA and NOKKALA 1987, 1989). The complex was established to contain at least two independent species in Fennoscandia, viz. Galeru- cella nymphaeae (L.), encountered on aquatic plants, and G. sagittariae (Gyll.), which occurs on several semiaquatic and terrestrial plants (HIPPA and KOPONEN 1986; NOKKALA and NOKKALA 1987). The separation of G. nymphaeae and G. sagittariae was largely based on the finding that, of the two distinct chromosomal polymorphisms encountered, one caused by an unusually large Y chromosome was restricted to G. nyrnphaeae only. B-chromosome polymorphism was found both in G. nymphaeae and G. sagittariae.

The populations of both G . nymphaeae and G.

sagittariae seem to fluctuate in an unregular manner. The latest important mass occurrence of G. sagitta- riae in Fennoscandia took place in the early 1970’s, when severe damage was caused to an economically important natural plant, cloudberry (Rubus chamae- morus L.) and cultivated strawberry (HIPPA and KOPONEN 1975,1976). In Finland, abundant popula- tions were observed on a variety of plants, mostly belonging to Rosaceae (HIPPA and KOPONEN 1975).

During quiescence, populations of any signifi- cance are found almost exclusively on Comarum palustre L. (Rosaceae) and Lysimachia thyrsiflora L. (Primulaceae) in southern parts of Finland, and on Rubus chamaemorus (Rosaceae) in northern Finland, though the distribution of all three plants covers the whole country. During the years of mass occurrence, G. sagittariae has been found in the whole country, up to the northernmost Lappland (see HIPPA and KOPONEN 1975, 1976), but during more quiescent years, the border of the distribution range seems to be drawn some 200-300 km south-

wards. Distributions of the beetles on different food plants seem t o overlap only on a comparative- ly narrow zone. where beetles may occur sympa- trically on different food plants. Consequently, possible differentiation o f southern and northern forms of G. sugitruriue cannot be totally excluded. although taxonomists arc content to find a uniform G. sugitturiae on different food plants all over the country (HIPPA and K o ~ o u r u 1986).

In the present paper. a comprehensive survey and characterization of the occurrence of B-chro- mosomes in Galerucella .sugirtariue (Gyll.) is repor- ted. The distribution of the beetles on different food plants has been examined carefully. and an attempt has been made to establish the evolutio- nary and taxonomic relations between beetles living on different food plants.

Material and methods The life cycle of Gulerucella sugirturiae is similar to that o f G. riymphueae (see NOKKALA and NOKKAL.A 1989). A slight difference is found in egglaying, the eggs of G. nvmphueae being laid on the upper sur- face of leaves. whereas those of G. sagirrariae are preferably laid on the lower side of leaves or on the stalks of the food plant.

Population samples of beetles living on Comarim palustre were collected from twelve Finnish and one Swedish locations (Fig. 1 and Table 1). The only sample collected from Alchemilla vulgaris coll. (Rosaceae) is dealt with together with the Comarum samples. Beetles living on Rubus chamaemorus were sampled at seven locations (Fig. 1 and Table 3). two of which are the same as the Comarum sample sites, i .e . . beetles occur sympatrically on both food plants. In addition, the only sample site for Akhemilfc is situated less than 1 km apart from one of these sites. In the present study. the term sympatric has been used in the sense of Wiiirr (1978), who regards 'two populations as sympatric ,.. if intermating could occur with a genetically significant frequency. unless prevented by genetic isolating mechanisms'.

Six o f the Comarum populations and three of the Rubus populations were sampled more than once. and part of the samples represent the old, over- wintered generation (collected at early summer). whereas another part represents the new genera- tion (collected at late summer). The location of Finnish sample sites is given by the uniform grid system (see H E I K l s H E i M o and R A A n K , u N E s 1971). and for each sample,a code is given. which is used later in the paper.

l€ip+l,

0 Comarum '15 \

I I 5

Fig. I. Locations of Finnish Gulerucella sagitturiue popula- tions. 1 Parainen, Mustfinn; 2 Virolahti, Virojoki; 3 Sakyla, Kolvaa: 4 Forssa, Matku, Halinanjarvi; 5 Vammala, Houhajirvi; 6 Sulkava. Halmejarvi; 7 Maalahti, Ders- trask: 8 Juuka, Panjavaara; 9 Sievi, Syyry; 10 Sievi, Vaha-Juurikka: 11 Vuolijoki, Salinsuo; 12 Paltarno, Is0 Teerisuo: 13 Vaala. Pelso; 14 Oulu, Kuivasjarvi; 15 Rovaniemi, Apukka; 16 Kolari, Teuravuoma; 17 Kolari, Venej irvi.

Since the majority of population samples analyzed were collected by the authors, comparative obser- vation of the sampling sites was also possible. In addition, several suitable habitats which were not sampled were checked for the presence of the beetles on different food plants. especially on the border zone of Comarum-feeding and Rubus-feeding populations.

Adult males were used for chromosomal prepa-

Heredilas I 1 I (1989) CHROMOSOMAL POLYMORPHISM IN G SAGITTARIAE 19 1

Table I . Locations of population samples of Galerucella sagittariae collected from Comarum and Alchemilla

Sampling Code Population Grid 27"E time n

KOS3 PRFR PRS9 PRSO PRSl PRS2 VIFl SAF9 SAFO FHS9 FHSO VASO VAFl SUF3 MAFO MAF3 JCS3 SSF3 s v s 3 v c s 2 v c s 3 OUFl

Kolingared, Vastergotland, Sweden Parainen, Mustfinn

Virolahti, Virojoki Sakyla, Kolvaa

Forssa, Matku, Halinanjarvi

Vammala, Houhajarvi

Sulkava, Halmejarvi Maalahti, Derstrask

Juuka, Panjavaara Sievi, Syyry Sievi, Vaha-Juurikka Vuolijoki, Salinsuo

Oulu, Kuivasjarvi

669:23

67153 676~24

676:31

680:28

68656 698:22

70259 707:39 707:39 71151

722142

VI 83 1x78 VI 79 V, VI 80 VI 81 V 82 VIII 81 IX 79 VIII 81 VI 19 VI 80 VI 80 VIII 81 VI 83 VIII 80 VI 83 VI 83 VIII 83 VII 83 VI 82 VI 83 VIIIRl

34 37 51 43 46 14 57 24 41 53 29 18 54 57 25 57 63 50 16 29 58 60

JAS3 Juuka, Panjavaara Alchemilla

70259 VI 83 61

Table 2. Locations of population samples of Calerucellu sagittariae collected from Rubus

Code Population Sampling

Grid 27"E time n

JRS3 VRS2 VRS3 PLS0 VLS9 VLSO VLSl ROF8 ROS9 KTFO KVFl

Juuka, Panjavaara 70259 VI 83 Vuolijoki, Salinsuo 71151 VI 82

VI 83 Paltamo, IsoTeerisuo 71456 VI 80 Vaala, Pelso 715:47 VI 79

VI 80 VI 81

VI 79 Rovaniemi, Apukka 738:45 VIII 78

Kolari, Teuravuoma 746:36 VII 80* Kolari, Venejarvi 146:37 VIIl8l

61 50 65 43 47 46 36 44 49 36 67

*Collected as larvae and reared in laboratory

rations, made according to the method of PURO and Results NOKKALA (1977) or that of NOKKALA and NOKKALA (1983). Both spermatogonial and meiotic metaphases Cytological observations

were scored to determine the chromosome consti- tution of each individual. Special thoroughness was undertaken to reveal all possible intraindividual variation in the number of B-chromosomes.

The karyotype of Galerucella sagittariae (Fig. 2 a-c) appears to be identical with that of G. nymphaeae possessing a small Y chromosome (cf. NOKKALA and NOKKALA 1989). Standard chromosome number is

Fig. 2 a-h. Mitotic and meiotic chromosomes of Gderircrllti sqirruriue. a Spermatogonial metaphase, 2n = 34. The nega- tively heteropycnotic region o n an autosome is visible (arrow). (Individual collected from Comarum). b Spermatogonial mctaphasc. 2n = 31 + 2B. Thc negativelv heteropycnotic regions on the largest autosome pair are visible (arrows). (From Kithus) . c Spermatogonial metaphase. Zn = 34 + 3B. (From Riibus). d Pachytene, 2n = 34 + 3B. A univalent B-chromosome and a B-chromosome bivalent are shown. (From Rubits). e Diffuse stage, 2n = 34 + 2-3B. Only the positivcl! heteropycnotic XY pseudobivalcnt and heterochromatic regions on autosomes and H-chromosomes are visible. (From Rubus). f Diakinesis. 211 = 34. Three ring bivalents with two chiasmata are visible (arrows). (From Huhlrr). g Metaphase I. 2n = 34 + 2B Large B-chromosomes cannot be identified. (From Cornarum). h Metaphase 11, n = 16 + y,, + B and n = 16 + X + B . Large B-chromosome3 cannot be identified. (From Rithus). Bar = 10 pm.

Hereditus I I 1 (19x91 CHROMOSOMAL POLYMORPHISM IN G. SAGITTARIAE I93

2n=34, and the only identifiable chromosomes in spermatogonial metaphases are the small Y chromo- some and, in fortunate cases, also the largest auto- some pair, carrying a negatively heteropycnotic region (Fig. 2a and 2b).

Meiotic prophase (Fig. 2d-f) proceeds as in G. nyrnphaeae (cf. NOKKALA and NOKKALA 1989). A chiasma is most often formed in only one chromo- somal arm but also ring bivalents - usually t\vo per cell - are encountered, indicating chiasma forma- tion in both arms.

Since the sex chromosomes are seen as the typical coleopteran parachute bivalent (see SMITH and VIRKKI 1978) at metaphase I (Fig. 2g), the meiotic chromosome number can be expressed as 2n = 16 + Xy,. At metaphase 11, strongly condensed and small chromsomes are to be found (Fig. 2h).

In all but one of the 21 populations sampled, supernumerary B-chromosomes were found in addition to the standard members of the karyotype (Fig. 2b-e, 2g, and 2h; Tables 3 and 4). Usually, one to five B-chromosomes per cell were found, most common number being two B’s per cell. The highest number encountered was ten B’s in a few cells of one individual. The number of B’s showed intra- individual variation, both between and inside cysts, indicating an unstable nature of B-chromosomes.

However, in most individuals carrying B-chromo- somes, the number of B’s was the same in all cells inspected.

As in Gaterucellcl nymphaeae (see NOKKALA and NOKKALA 1989), unambiguous classification of B- chromosomes is impossible, despite obvious varia- tion in their size. The smallest B-chromosomes are even smaller than the small Y-chromosome (Fig. 2b), the majority of B’s are about the size of the Y (Fig. 2c), and the largest B’s are indistinguishable from autosomes or the X-chromosome (Fig. 2g and 2h). When B’s can not be told apart from auto- somes, they have been classified as large, all remai- ning B’s being classified as small (Tables 3 and 4).

Though the size of B-chromosomes varies, all seem to contain a considerable amount of euchro- matin and being able to pair and form chiasmata if several B’s are present in a cell (Fig. 2d and 2g). Multivalent B’s were not observed. All B-chromo- somes are isopycnotic during meiotic prophase (Fig. 2d and 2e) and large B-chromosomes also at metaphase I (Fig. 2g), while smaller B-chromo- somes tend to be slightly negatively heteropycnotic at this stage. The behavior of B’s in meiosis is normal.

Inspection of the external phenotype of an individual did not in any way indicate whether the individual in question was carrying B-chromosomes or not.

Table 3. B-chromosome frequencies in Galerucella sagittariue samples collected from Comarum and Alchemilla

Sample B-chromosome type and frequency

n Small (stable) Large (stable) Unstable B-chr

1 2 3 4 1 2 Small Large 2 (”/.I

KOS3 PRFR PRSY PRSO PRSl PRS2 VIFl SAFY SAFO FHS9 FHSO VASO VAFl SUF3 MAFO MAF3 JCS3 SSF3 svs3 v c s 2 vcs3 OUFl JAS3

34 37 51 43 46 14 57 24 41 53 29 I8 54 57 25 57 63 50 16 29 58 60 61

- 1

- -

5 -

- -

2 -

1 - 2 -

12 -

8 - 21 3

7 1 3 - 3 -

23 I 13 2 17 I

- -

- -

11 - -

4 1 I 3

18 3

11 29 16 5 7

34 20 26

14.7 2.1 2.0 2.3 0.0 0.0

19.3 0.0 0.0 7.5 3.4 5.6 5.6

31 6 12.0 19.3 46.0 32.0 31.3 24.1 58.6 33.3 42 h

Iferediras / I I (1989)

Tahir 4 B-chromi)some irequencie\ in Galeruwllo sogrtrurirrt. samples collected from Rubits

R-chromosome type and frequency Sample n Small (stable 1 Large (stable) Unstable B-chr

- I 2 3 4 5 1 2 3 Small Large X (Yo)

JRF3 VRS2 VRS? PLFO \ LSS VLFO V I F I ROFX ROS9 KTFO KVFl

18 2 8 1

15 1 5 - 4 -

10 -

I? - 8 1 4 -

I 1 4 5 1

28 45.9 17 34.0 25 38.5 I I 25.6 19 40.4 23 50.0 I9 52 8 19 43.2 22 44.9 19 28.3 10 27 8

Comparison of populations

The frequency of B-chromosome- carrying individuals showed considerable variation between samples and populations (Tables 3 and 4). Differences were tested by using either Fisher exact test (preferably) or xz test.

Intrapopulation comparisons of samples collected at different times are presented in Table 5 for the Sakyla. Forssa, Vammala. Vuolijoki-Comarcrm. Vuolijoki-Rubus, Vaala, and Rovaniemi popula- tions. Comparisons for the Parainen population, which was sampled during five successive years. are presented in Table 6. Different samples for each population were pooled in all cases where statisti- cally significant differences were not found (Table 7). and the pooled sarnplcs were used in all further comparisons.

The two samples of the Vuolijoki-Comaritm population differ significantly from one another. In further comparisons, the sample from 1983 is used. since the 1982 sample was collected so early in summer that it consisted of the very first individuals found after overwintering - actually all individuals encountered were collected -and it may thus be an unrepresentative sample.

When comparing differcnt populations collected from Comarum. the Vuolijoki population stands out as the one with the clearly highest B-chromo- some frequency (Tables 3 and 8). In Oulu. both Sievi, Juuka-Comarum. Juuka-Alchemilla and Sulkava populations, the frequencies are also high (31.146.0 YO). These populations d o not differ from each other, but in most comparisons the fre- quencies are statistically significantly higher than in

Tarrhlr 3. Comparison of B-chromosome trsquencles of Galeriicello sagiffarrue samples collected at different times from one popula- tion. Fisher exact teSt (I-tail. Yo)

B-chromos Samples compared p=lY")

SAFY vsSAFO FHSY vs FHSO VASO vs V A F l MAFO vs MAF3 VCS2 v5 VCS3 VRS2 vs VRS3 VLS9 vs VLSO VLS9 vs VLSl VLS0 VS\'LSl ROF8 vs ROSY

' Tested with x 2 test

100.00 79.57 68.08 32.09 0.22

38.41 35.36' 18.d9 48.93 51.72

Tnhk 6 Comparison of €3-chromosome frcquencies of Galrrucella sagiftarfoe samples collected at different times from Parainen popu- lation. Fisher exact test (I-tail, Yo)

PRFR PRSY PRSO PRSl

PRS? 72.55 78.46 75.44 1iK1.00

PRSl 34.58 52.58 48 31

PRSO 71.42 70 83

PRS9 66 69

Herediras I I ! (1989) CHROMOSOMAL POLYMORPHISM IN G SAGIVARIAE 195

Tabie 7. B-chromosome frequencies in pooled samples from eight Galerucella sagittariae populations

Code Population 9-chromosome type and frequency

1 2 3 4 5 1 2 3 Small Large Z (Yo) n Small (stable) Large (stable) Unstable B-chr.

PRP SAP FHP VAP MAP VRP VLP ROP

Parainen, Mustfinn Sakyla, Kolvaa Forssa, Matku, Halinanj. Vammala, Houhajarvi Maalahti, Derstrask Vuolijoki. Salinsuo Vaala, Pelso Rovaniemi, Apukka

3 1.6 - 0.0

2 - 5 6.1 3 - 4 5.6 8 - 14 17.1

1 9 1 1 2 - - - _ - - - - - - - - - - _ - - 65 -

8 2 - 2 1 - - _ - - 72 1 - - - - 8 2 1 4 - I - _ _ _

- - _

115 2 6 4 2 1 - 1 1 23 2 42 36.5 129 9 1 3 6 5 2 - - - 26 - 61 47.3 9 3 6 9 4 7 1 - 1 - 12 - 41 44.1

Table 8. Comparison of B-chromosome frequencies of Galerucella sagittariae populations collected from Comarum and Alchemilla. Fisher exact test (1-tail, %). +: the frequency of the population on the left column is significantly higher. -: the frequency of the population on the left column is significantly lower

PRP VIFl SAP FHP VAP SUF3 MAP JCS3 JAS3 SSF3 SVS3 VCS3

OUFl +O.OO v c s 3 +O.OO s v s 3 +0.01 SSF3 +o.oo JAS3 +O.OO JCS3 f0.00 MAP +0.00 SUF3 +O.OO VAP 9.17 FHP 5.55 SAP 41.37 VIFl +O.W

6.50 +O.OOI 24.27 9.92

+0.54 +0.16 45.23

9.82 -1.60 - 1.72 -0.01

+o.oo +o.oo +0.02 +o.oo +o.oo +o.oo +0.02 +o.oo

7.33 5.11

' Tested with x2 test

+O.OO +0.00 +0.96 +0.01 +0.00 +o.oo +2.43 +0.01 58.16

+O.W 49.82 +2.09 15.05' 29.26' 52.30 56.41 -0.58'

+0.88 61.73 16.54 21.80 29.82 60.64 +0.00 +0.36' +O.OO 16.61' 8.10' +0.57' +4.82

+0.01 56.34 +3.94 9.31 17.08 +O.W 21.51' +0.07 70.25' +O.OO 10.53' +2.26 -3.11 +0.01

the remaining populations. There are still two populations, Maalahti (17.1 YO) and Virolahti (19.3 YO), where B-chromosomes are compara- tively common. In the last group of populations (Vammala, Forssa, Sakyla and Parainen), B-chro- mosome frequencies are very low ( O . M . 1 YO), and these populations differ significantly from all popu- lations with higher frequencies.

In all populations collected from Rubus, the fre- quency of B-chromosome- carrying individuals is quite high (25.W7.3 YO), and there is much less variation than in populations collected from Coma- rum or in populations of Galerucella nymphaeae (see NOKKALA and NOKKALA 1989). Statistical differences are few, and there is one population (Vuolijoki) which does not differ from any other population (Table 9).

In contrast to Galerucella nymphaeae (see NOKKALA and NOKKALA 1989), the B-chromosome frequency of G. sagittariae shows an increasing cline from

south to north or from southwest to northeast (Table 10). This tendency is especially clear in populations collected from Comarum. When populations from Rubus are analyzed separately, no cline is found. This may, however, reflect the fact that populations differ comparatively little from one another and that all populations sampled are situated on a rela- tively narrow zone on the southwest-northeast axis. In addition, isophenes do not necessarily run straight from southeast to northwest.

Finally, a comparison between all populations collected from Comarum and all collected from Rubus is presented (Table 11). The B-chromosome frequencies of almost all Rubus populations are significantly higher than those of Comarum popula- tions located southwestwards from Sievi-Sulkava line. The frequencies of only two Comarum popu- lations (Vuolijoki and Juuka) are in some compari- sons significantly higher than in populations collected from Rubus. Thus in general, B-chromosome fre-

196 C NOKKALA AND S NOKKALA Hereditas I I I (1989)

Table 9. Comparison of B-chromosome frequencies of Galerucalla sagitrurrae populations collected from Ruhrrs. Fisher exact test (1- tail. %). f ' the frequency of the population on the left column is significantly higher -: the frequency of the population on the left column is significantly lower

-

JRS3 VRP PLSO VLP ROP KTFO

KVFl -3.97' 26.OS' 46.33 -1.05' -4.26' 57 02 KTFO s.94 22.44 51.27 -2.73 6.56 ROP 82 46' 26.80' +2 90 ti3.6R' VLP 85.R2' 8.92' +0.2Y PISO -2.77 13.36 VRP 22 63'

' Tested with $test -

quency is higher in populations originated from Rubus as compared to populations coming from Comarum. An exception to this is the pair of popu- lations of Vuolijoki, where the B-chromosome fre- quency of the Comarum population is higher than that of the Rubus population. In Juuka, the samples collected from three different food plants were all alike in their B-chromosome frequencies.

Discussion The characteristics of B-chromosomes in Gallerucella sagitturiae are identical with those of G. nyrnphaeae (see NOKKALA and NOKKALA 1989). They are largely cuchromatic. their size varies. and in each size group, both mitotically stable and unstable B-chro- mosomes are encountered. In fact, it may be con- cluded that in these two species, B-chromosomes are of common origin and have evolved before the separation of the species, especially so, since in all other species of Galerucella possessing B-chromo- somes, B-chromosomes are different (NOKKALA and NOKKA1.A 1987).

There are only few follow-up studies concerning B-chromosome frequencies in natural populations (see NOKKALA and NOKKALA 1989). In the present study, one population was checked during five suc- cessive years, one population during three succes- sive years, and several populations were checked twice. There were no significant changes in the fre- quencies over the years. Though the present data are not comprehensive for a thorough follow-up study, one may at least conclude that the B-chro- mosome frequency in a population of G. sagittariae seems to stay at a constant level from generation t o generation. In this respect, B-chromosome frequen- cies in Galerucella sagittariae behave similarly as in G. nymphaeae (NOKKALA and NOKKALA 1989). In other species, there are also examples of B-chro- mosomes the frequencies of which change steadily over the years (see JONES and RELS 1982).

The highest and lowest B-chromosome frequen- cies are similar in Galerucella nyrnphaeae (see NOKKALA and NOKKALA 1989) and G. sagittariae. However, while it was shown that the geographical distribution of frequencies is totally random in G. nymphaeae (NOKKALA and N O K K 4 L A 1989). the B- chromosome frequencies in G. sagittariae form an increasing cline from southwest to northeast. Con- sequently, there is every reason to believe that, in G. sagittariae, B-chromosomes d o have an adaptive significance and that selection determines the level of the frequency in each population.

An interesting question is the taxonomic and evolutionary relation between beetles living on dif- ferent food plants. There are no clearcut morpholo- gical differences between southern (Comarum- feeding) and northern (Rubus-feeding) representa- tives of Galerucella sagittariae, though the size of the beetles increases towards north (HIPPA and KOPONEN 1986). Also larvae and eggs are morphologi- cally identical in the two groups (HIPPA and KOPONEN 1986). However, an overall impression from scan-

Tahle 10. Spearman rank correlation coefficients of 8-chromosome frequencies of Finnish Gale- rucella sugirrarioe populations on south-north. west-east and southwest-northeast axes

Food plant B-chromosome frequency versus

South-North West-East SW-NE

Hiihri.! 1, = 7

Cvmurrim I1 = 12

Ruhu.s+ C'omurrrmi A lchenrrlla

n = 20

-0.1622 N.S 4 . 1 7 8 6 N.S . - n . m N.S .

0.8035' * 0.45 I i N. S. 0.8731'"'

0.5709' 0.2374 N. S 0.5545'

Heredifas I I I (19x9) CHROMOSOMAL POLYMORPHISM IN G SACI'TTARIAE 197

o c b m r J m

0303000 + + + + + + + S S S " N S 8

g 8 b o N N - ? ? N O 8 0000000 + f + f + + +

8 S 8 8 " 0 8 O o o d d d O + + + + + + +

8 8 g e 8 8 8 0 0 0 0 0 0 0 + + + + + + +

ning population samples is that the southern popu- lations seem to be quite uniform in coloration, all individuals being yellowish brown, whereas the northern populations show a considerable degree of variation from relatively light to almost black individuals.

In the three Juuka samples collected from diffe- rent food plants, the frequencies of B-chromosome- carrying individuals are similar, which may indicate these samples to be representatives of one panmictic population. On the other hand, in the two strictly sympatric Vuolijoki samples, frequencies differ significantly. Thus, in Vuolijoki, samples collected from different food plants represent two independent populations.

The seemingly contradictory results obtained from sympatric populations in Vuolijoki and Juuka led us to study the habitats of the beetles more closely. In southern Finland, beetles are encountered on food plants growing out from water but they leave the same plant species unoccupied on land, even if it was growing only a few meters away. This is especially conspicuous in early summer, when plants growing on dry land have already fully deve- loped leaves while in plants growing out from water, leaves are just budding. Similarly, in late summer, if populations are very dense, plants growing out from water are heavily damaged, yet adjacent plants on dry land have been left untouched. The beetles are encountered only in these kinds of "wet" habitats up to the border where the southern- most Rubus-feeding populations are found. North of this border, beetles are met both on Comarum growing out from water and on Comarum growing on dry land as well as on Rubus, and occasionally also on some other terrestrial plants, e.g., on Alchemilla.

The observations of the present study allow the conclusion that a southern and a northern form of Galerucella sagittariae exist. Field observations suggest that the difference lies in the beetles' dependence on water. The southern form of G. sagittariae is dependent on the immediate vicinity of water, which secondarily restricts the choice of food plant. However, the northern G. sagittariae lives on plants growing on dry land and seems to get all the water it needs from the food plant. It is thus able to live in considerably drier habitats than the southern form and, consequently, also to utilize a broader variety of food plants. Thus, the choice of food plant cannot be considered by any means as the primary dzfference between southern and northern forms. Obviously, on the border zone of distri- butions, among beetles consuming Comarum, both

198 c ~ O K K A L A A U D S WKKALA Heredita., I I ! (1989)

southern and northern G. sagiitariae can be found. This gives an explanation to the contradictory conclusions obtained when comparing B-chromo- some frequencies in sympatric populations in Juuka and Vuolijoki. In Juuka. the beetles were collected from Comarum growing on dry land. In this case, B-chromosome frequencies among sympatric samples of northern G. sagittariae collected from different food plants have been compared. However, in Vuolijoki, beetles were collected from Comarum growing out from water, so in this case B-chromo- some frequencies of sympatrically occurring popula- tions of southern and northern forms of G. sagznariae have been compared. B-chromosome frequency data are compatible with the idea that within the northern form, beetles collected from different food plants are not isolated from each other, whereas the southern and northern forms seem to be repro- ductively isolated in nature.

Further evidence of the isolation of southern and northern forms of Galerucella sagittariae is provi- ded by the inspection of fluctuation cycles. The size of populations show considerable fluctuation over the years. However, fluctuations of southern and northern populations d o not occur parallelly; e.g., in 1980, abundant populations on Rubus were found in northern Finland, whereas it was very hard to find enough beetles for sampling on Comarum in southern Finland. In 1988 the situation was the opposite - there was no difficulty in collecting beetles from Comarum in southern Finland, while populations on Rubus and on Comarum growing on dry land in northern Finland seemed to be almost nonexistent.

According to our observations, the distribution of Galerucella sagiitariae on Cornarum reaches farther north and the distribution on Rubus farther south than earlier recorded (cf. H i w A and KOPOSEN 1975, 1976). Beetles on Comariim growing out from water have been observed almost 100 km north of Oulu. but the border of the distribution is not known very accurately. However, the border of the distribution on Rubus seems to be quite sharp. there being no observations of beetles on Rubus south of Sievi-Juuka line.

The distribution of Galericcelln sagitiariae on Comarum in northern Finland is obviously restricted b!; shorter growth period. which influences the availahility of the food plant. By gaining the ability to live in drier habitats. the utilization of Rubus (availiihlc some 2-3 weeks earlier than Coninrum) has hecome possible. which has further advanced quick expansion towards north and also the infesta- tion ot cultivated strawberry. It is possible. even

probable, that some hybridization occurs in regions where both southern and northern G. sagittariae consume Comarum, but the direction of gene flow will be mainly from north to south, since it is un- likely that southern G. sagittariae would wander on plants growing on drier habitats. This kind of unidi- rectional gene flow does not interfere with the pro- gress of genetic differentiation of the two forms.

It seems obvious that some degree of genetic dif- ferentiation has passed between southern and northern forms of Galerucella sagittariae, which is demonstrated by their adaptation to different habi- tats. Although morphologically and cytologically very similar, the southern and northern forms of Galerucella sagittariae d o differ from one another clearly enough to be considered as subspecies. As a matter of fact, they might even fulfil the criteria of the species as determined by the biological species concept (e.g., MAYR 1988), but to actually confirm the possible existence of reproductive isolation needs further evidence, and this is the most impor- tant topic for future research.

Acknowledgements. -Our thanks are due to Heikki Hippa, Seppo Koponen. Taina Lakkala-Paranko, Seppo Neuvonen, Olli Osmonen, Jaakko Puro and Rauno Roine for helping us to collect the material. Ms. Paula Jokela and Ms. Taina Lakkala-Paranko made part of the preparations. Financial support was given by the University of Turku Foundation. Emil Aaltonen Foundation, Turku Finnish University Association, Jenny and Antti Wihuri Foundation, Niilo Helander Foundation, Finnish Academy of Sciences, Finnish Federation of University Women, and grants from the University of Turku.

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