C 1999 The Japan Mendel Society Cytologia 64: 401-409, 1999

Karyotype Diversity in the Family (Symphyta, ): New Data and Review

M. Westendorff 1, V. G. Kuznetsova 2, A. Taeger 1 and S. M. Blank 1 1Deutsches Entomologisches Institut Eberswalde , 2Zoological Institute , RussianAcademy of Sciences, St. Petersburg,Russia AcceptedJuly 12, 1999

Summary Karyotypes of 34 species referred to 9 genera of 6 tribes and 4 subfamilies of Tenthre- dinidae are reported. Variation in karyotypes at different taxonomic levels is discussed. The haploid chromosome numbers are shown to range from 6 to 20 in the species studied whereas they range from 5 to 22 in the family as a whole. An ancestral chromosome number for Tenthredinidae is sug- gested to lie between n=6 and n=10. Some genera are discussed in terms of karyotype diversity. Possible pathways, in which karyotype evolution has proceeded in these genera and in Tenthre- dinidae as a whole, are discussed.

The family Tenthredinidae includes some 1000 species in Europe. So far chromosome num- bers were known for about 40 of them (Sanderson 1933,1970, Comrie 1938, Peacock and Sander- son 1939, Smith 1941, Benson 1950, Waterhouse and Sanderson 1958). The Japanese fauna has been studied in more detail (Nogusa 1965, Naito 1978-1996, Abe 1988). Naito (1982) reported chromosome numbers of some 200 species and considered the karyotype data in terms of systemat- ics and evolution. Saini and Grewal (1986) published chromosome numbers for 5 Indian species. The current paper presents karyotypes of tenthredinid species from Germany. Karyotype vari- ability at different taxonomic levels within Tenthredinidae is discussed.

Materials and methods A total of 34 species belonging to the subfamilies Selandriinae, , Allantinae and Tenthredininae were studied. Species karyotyped are presented in the table. The systematic arrange- ment and nomenclature are those proposed by Taeger and Blank (1998: 337-338 and unpublished. data). Females have been collected at different localities in Germany between 1995 and 1997. Un- fertilized eggs were incubated in vitro and 2-5 days old parthenogenetic male embryos used to ob- tain haploid metaphase plates (Naito 1971). Chromosome preparations were made by an air-drying technique (Imai et al. 1977). Slides were stained with 4% Giemsa, rinsed with water and air dried. Karyotypes were analyzed and photos were made using microscope BX 50, camera OM-4, Olym- pus, Japan. The fundamental number (FN) was estimated on the basis that metacentrics (M), sub- metacentrics (SM) and subtelocentrics (ST) display a value of two, while acrocentrics (A) a value of one. In some cases comparison was made with the data published previously by other authors. If a description of the karyotype characters was absent in the original paper we judged the chromo- some morphology on the base of the figures presented. Chromosome preparations and the remains of adult specimens have been deposited in the collection of the Deutsches Entomologisches Institut,

1 E-mail: [email protected] 402 M. Westendorff et al. Cytologia 64

Table 1. Karyotypes of sawfly species

(n: haploid chromosome number, FN: fundamental number, Fe: numbers of studied females, E: embryos, M: metaphas- es. 1 Sanderson (1933), 2Naito (1982), 3 Sanderson (1970).

Eberswalde.

Results and discussion Data on the material, including haploid chromosome numbers (n) and FN of the species as well as the numbers of studied females, embryos and metaphase plates are summarized in the table. In Selandriinae 4 Dolerus species were karyotyped (Figs. 1-4). All of them show 8 biarmed chromosomes in the haploid sets. The karyotypes of D. nigratus and D. varispinus are similar dis- playing only M and SM. The karyotypes of D. haematodes and D. niger include several ST, one in 1999 Karyotype Diversity in the Sawfly 403 the first and three in the second species. In D. haematodes, D. nigratus and D. varispinus the chro- mosomes decrease in size more or less gradually, while in D. niger there is one very small chromo- some. Naito (1982) reported haploid chromosome numbers of 8 other Dolerus species: n=8 in D. ephippiatus, D. gessneri and D. yokohamensis, n=9 in D. japonicus and D. subfasciatus, n=11 in D. genucinctus insulicola, and n=14 in D. lewisii and D. eversmanni obscurus. Naito considered the latter two as polyploid species, since their chromosomes can be arranged in pairs on the basis of their relative length and morphology. If so, the haploid chromosome number of these species should be determined as 7 rather than 14. Based on present knowledge, n=8 is the modal number in Dolerus. The distribution of chromosome numbers does not correspond with the subgeneric arrangement of Dolerus as proposed by Goulet (1986) and Zhelochovtsev (1988). At present the karyotypes are known for some 50 Selandriinae species and n=7 has been found in 16 of them. Following the system of Ross (1937), Naito (1982, 1988, 1996) proposed n=7 to be the ancestral state for the subfamily. Both lower and higher numbers probably have been de- rived from it secondarily. In Nematinae, only Cladius pectinicornis was karyotyped (Fig. 5). This species shows n=6 with M and SM gradually decreasing in size. Sanderson (1933) found the same chromosome num- ber in the second spermatocytes of this species, however, chromosome morphology can not be seen in the figure presented. C. pectinicornis shares n=6 with two other Cladiini, Priophorus spp., previ- ously studied by Comrie (1938) and Naito (1982). Karyotype morphology of Priophorus seems to be similar in detail to that of C. pectinicornis (Naito 1982). In Nematinae the data available concern 16 species belonging to 8 genera and two tribes, Cladiini and Nematini. Chromosome numbers range from n=6 to n=9 without a clear mode. Based on the morphological evidence, Naito (1982) is inclined to accept n = 8 as the ancestral number for the subfamily. In Allantinae karyotypes of 4 species were studied (Figs. 6-9). 3 of them belong to Athalia (Athaliini) and one to Taxonus (Allantini). In Athalia 2 species show n=6 and one n=8. A. bicolor with n=6 has only biarmed chromosomes, which are M and SM. Although the karyotype of A. cor- data includes also 6 chromosomes, it is different showing one large acrocentric chromosome. The third species, A. rosae rosae, displays n=8 with one small acrocentric chromosome. This karyotype pattern has already been described by Naito (1982) in A. rosae japonensis, which is a synonym of A. rosae ruficornis. 3 other Athalia species were found to have the same range of chromosome numbers, n= 8 in A. japonica and A. lugens infumata, and n=6 in A. kashmirensis (Naito 1982, Abe 1988). Accordingly, by the chromosome number Athalia can be divided into two groups, which cor- respond with those accepted on the basis of morphological features and food habits (Abe 1988). In Taxonus alboscutellatus 9 biarmed chromosomes gradually decreasing in size were found (Fig. 9). Two other Taxonusspecies, T minomensis and T japonicus, display n=9 and n=11 respec- tively, the latter species having one small acrocentric chromosome (Naito 1982). The genus Tax- onus is fairly heterogeneous in morphology and coloration pattern and perhaps the species currently included belong to various groups. In Allantinae, the data available concern 20 species referred to 9 genera in 4 tribes. Chromo- some numbers range from n = 6 to n =16. Naito (1982) suggested n=8 to be the ancestral state for this subfamily, in which more specialized forms have higher or lower chromosome numbers. The subfamily Tenthredininae is the most species-rich in the Tenthredinidae. 25 species be- longing to the tribes Tenthredopsini (1), Macrophyini (5), and Tenthredinini (19) were karyotyped. aucupariae (Tenthredopsini) shows n=12 with eleven biarmed and one acrocentric chromosomes, which all gradually decrease in size (Fig. 10). This chromosome number has not been known previously in this genus, for which a wide range of chromosome numbers (n=9, 10, 15, 16, 17, 22) has been reported by Naito (1982). Aglaostigma is a morphologically heterogeneous 404 M. Westendorff et al. Cytologia 64

Figs_ 1-20. Haploid metaphases. 1) Dolerus haematodes. 2) D. niger 3) D. nigratus. 4) D. varispinus. 5) Cladius pectinicornis. 6) Athalia bicolor 7) A. cordata. 8) Athalia rosae rosae. 9) Taxonus alboscutel- latus. 10) . 11) albipuncta. 12) M. montana. 13) M. punctumalbum. 14) M. rufipes. 15) M. ribis. 16) Elinora koehleri. 17) viridis. 18) atra. 19) T bipunctula. 20) T colon. Bar equals 5 pm in Figs. 1-20. 1999 Karyotype Diversity in the Sawfly 405 genus. The high number of (sub-)generic names proposed for several groups of Aglaostigma reflect its morphological diversity (Taeger and Blank 1996). In Macrophyini (Figs. 11-15) the karyotypes of Macrophya albipuncta and M montana are quite similar, including eight biarmed chromosomes, 7 M/SM and 1ST. The karyotypes however differ in size and morphology of the largest chromosome. In M montana this chromosome is meta- centric and of outstanding size, whereas in M. albipuncta it is submetacentric and differs only slightly in size from the second chromosome of the set. It seems evident that a rise of the chromo- some arm occurred during evolution of the genus probably through an increase in the amount of heterochromatin. The morphologically rather different M. punctumalbum and M. rufipes share n=10. However they are distinct in chromosome morphology showing 10 M/SM in the former, with 9 M/SM and one ST in the latter species. The karyotypes seem to differ also in the length and morphology of the first chromosome, which is comparatively larger in M rufipes. The karyotype of M. ribis diverges markedly from the above described. It displays 12 chromosomes including 5M/SM, 6ST and 1A. In Macrophyini, this chromosome number has been known previously only in M fascipennis, but this species is quite different in chromosome morphology (Naito 1978b). Besides our data, 13 species have been karyotyped in Macrophya (Naito 1982). All the species have n=8, 9, 10, and 12, n=10 being predominant. Like Aglaostigma, this large genus is fairly variable both morphologically and cytologically. Based on the comparatively few chromosome data, some species groups within Macrophya can be distinguished according to their karyotype pattern. For example, karyotypes of M. rufipes, M coxalis, M. falsifica, and M. rohweri are quite similar showing n=10 with a very large metacentric chromosome. Another group includes M. imitator, M. exilis and M. albipuncta. These species display n=8 with gradual size seriation of chromosomes. The karyotype similarity of M. imitator and M. exilis has already been noted by Naito (1978b). In Macrophyini chromosome data are available for 52 species belonging to Macrophya (18) and Pachyprotasis (34) (Naito 1982, Saini and Grewal 1986, present paper). Chromosome numbers vary from n= 8 to n=12 without a gap, the mode being at n=10. In Tenthredinini we karyotyped 19 species, which all belong to the genus Tenthredo, except for Elinora koehleri and (Figs. 16-34). Data on the genus Elinora were obtained for the first time. E. koehleri shows n=10 with only M and SM (Fig. 16). Sanderson (1970) report- ed n=14 for Rh. viridis, but she did not provide further information on the karyotype. Our data con- firm this number and show that the karyotype includes as many as 5A (Fig. 17). In Tenthredo, 13 of 17 studied species show a karyotype of n=10 consisting of biarmed chro- mosomes. There are from 1 to 4 ST in these karyotypes, 4 ST occurring only in T ferruginea. In most species chromosomes gradually decrease in size. Naito (1982) reported n=12 for Japanese T. ferruginea, while we found n=10 in a German specimen. It is noteworthy that the Japanese speci- mens of "T ferruginea" are fairly homogeneous in coloration in contrast to the highly variable Eu- ropean specimens. European and Japanese specimens are most likely not conspecific. In the Ten- thredo species with n=10 studied by Naito (1978a, 1982), all chromosomes seem to be also biarmed, though the centromere position is uncertain in some cases. The karyotypes of T colon and T crassa are characterized by a very large SM (Figs. 20, 21), which can also be observed in T con- tusa, T viridatrix, and T goliath (Naito 1982). Judging from the Fig. 78 of Naito (1982), the kary- otype of T colon nigriventris is quite different from that of T colon. According to Japanese speci- mens from the DEI collection, identified by A. Shinohara as T colon nigriventris, they most likely represent T decens. It suggests that the above distinct karyotype is characteristic of T decens rather than T colon nigriventris. The karyotype of T solitaria shows two very large chromosomes (Fig. 28), one of them is M while the other is SM. This karyotype pattern has not been described so far in Tenthredo. T marginella displays 11 chromosomes including 7M/SM, 3ST and 1A (Fig. 31). Sanderson (1970) reported the same chromosome number for this species from England, however, she did not 406 M. Westendorff et al. Cytologia 64

Figs. 21-34. Haploid metaphases. 21) Tenthredo crassa. 22) T ferruginea. 23) T livida. 24) T

mandibularis. 25) T mesomela. 26) T obsoleta. 27) T omissa. 28) T solitaria. 29) T temula. 30) T velox. 31) T marginella. 32) T amoena. 33) T.brevicornis. 34) T notha. Bar equals 5 ƒÊm in Figs. 21-31, 33, and 4ƒÊm in Fig. 32,34. present further characters of the karyotype. T fuscoterminata has also 11 chromosomes, but none of them is acrocentric (Naito 1982). In T amoena and T brevicornis n=18 was found, in T notha n=20 (Figs. 32-34). Morpholog- ically, the latter two species are closely related and members of the T arcuata-group, whereas T. amoena belongs to the T zonula-group (Taeger 1985, 1991). All 3 karyotypes share a common pe- culiarity showing a large number of small chromosomes. Unfortunately, identification of the mor- phology of each chromosome in the complements based on the conventionally stained preparations fared poorly. It is however evident that the acrocentric chromosomes clearly predominate, whereas the biarmed chromosomes are not more than 5-8 in number in the karyotypes. In T amoena and T brevicornis FN could preliminarily be determined as 46-52, in T notha as 50-56. Contrarily, Sanderson (1970) reported n=18-21 for T brevicornis (cited as T acerrima) and n=18 for T notha (cited as T perkinsi). These differences possibly result from the examination of different species. 1999 Karyotype Diversity in the Sawfly 407

Fig. 35. Distribution of chromosome numbers in Tenthredinidae.

Species identification in the T arcuata-group is uncertain in several aspects (Taeger 1985). One other explanation could be polymorphism which is however a rare phenomenon in (Crozier and Taschenberg 1972, Naito 1982). Tenthredo s. 1. is the largest sawfly genus with great morphological differentiation. Although chromosome numbers vary within a wide range (n= 8-21), the genus is characterized by a clear mode at n=10 (Naito 1982), which is confirmed by our data. Among approximately 75 karyotyped species only few show some deviating chromosome numbers, n=8, 9, 11, 13, 15, 18, 20, 21, 22. The higher the chromosome number in Tenthredo species the more acrocentrics are present in their karyotypes. This indicates that Robertsonian rearrangements have occurred in the karyotype evolu- tion of the group. In Tenthredininae the data available concern approximately 150 species. Chromosome num- bers range from n=8 to n=22, a karyotype of n=10 being clearly predominant. Naito (1982) sug- gested that the phylogenetically lower groups Sioblini, Perineurini and Tenthredopsini are character- ized by n=9, whereas the higher groups Macrophyini and Tenthredinini have n=10 as the modal number, the karyotype of n=9 being the ancestral state in the subfamily as a whole. Although our data on the higher groups support the above conclusion, more data are required to suggest the an- cestral karyotype in Tenthredininae. Although present knowledge on the karyotype diversity and evolution in Tenthredinidae as a whole is still obviously inadequate, some preliminary generalizations can be drawn. Within the family chromosome numbers range from n= 5 (in Pseudohemitaxonus dryopteridis, Selandriinae) to n=22 (in Aglaostigma sp., Tenthredininae) (Naito 1982). Statistically dominant is n=10, which occurs in about half of the studied species. The next in frequency are n=9 and n=8, found each in about 13% of the species (Fig. 35). It is well known that the modal numbers can derive from some large groups which contain a large proportion of the karyotyped species. In Tenthredinidae such a group is the largest subfamily Tenthredininae, in which two-thirds of the species and two-thirds of the genera show n=10. A range from n=6 to n=10 was found to be characteristic of the family as a 408 M. Westendorff et al. Cytologia 64 whole with n=9 and n=10, which have been found each in one third of studied genera, clearly dominant. Based on phylogenetic considerations, Naito (1982) suggested that evolution of the karyotype of Tenthredinidae started from n=7. Although the ancestral number most likely is between n=6 and n=10, 9 or 10 linkage groups are the most appropriate candidates to be considered as the an- cient genomic organization for the family. However additional evidence is still needed to prove this hypothesis. The genera show clear differences in terms of karyotype variability. Although a great variation in chromosome number occurs in some of them, a remarkable constancy is found in others. In Aglaostigma, each of seven karyotyped species shows its peculiar chromosome number, n = 9, 10, 12, 15, 16, 17 and 22. On the other hand, all five karyotyped species of Siobla display n=9. Like- wise, all 5 studied species of Perineura have n=17, while an overwhelming majority of Tenthredo and Macrophya species have n=10. Although at present there is no clear idea of the pathways in which the karyotype evolution has proceeded in most taxa, suggestions could be made for some of them. As indicated earlier, in Ten- thredo the karyotypes consisting of many chromosomes (n=18 and 20 in T amoena, T brevicornis and T notha) tend to show a higher number of acrocentrics compared to karyotypes with a lower chromosome number, in which acrocentrics are rare, if any, in occurrence. Robertsonian rearrange- ments were clearly responsible for these kinds of karyotype changes. Naito (1982. Fig. 103) sug- gested that fissions dominated over fusions in the evolution of Tenthredinidae, however this hypoth- esis needs further evidence. In many genera, for example in Dolerus, Pachyprotasis and Tenthredo, the closely related species were found to share the same chromosome number but to differ in chro- mosome morphology. It indicates that a great deal of intrachromosomal rearrangements rather than Robertsonian changes have been at work in the evolution of these genera. In the near future, new techniques, presently being developed for chromosome studies, will en- able us to obtain more detailed information on the pathways of karyotype evolution in Tenthre- dinidae.

Acknowledgements We gratefully acknowledge our colleagues of the Deutsches Entomologisches Institut, Ebers- walde for their assistance in this work. We thank A. Shinohara (Tokyo) for the gift of collection ma- terial, and A. D. Liston (Frontenhausen), who kindly corrected the English. This research was partly supported by the Deutsche Forschungsgemeinschaft, Bonn (V G. Kuznetsova and S. M. Blank).

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