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DivergentcytogeneticevolutioninNearcticandPalearcticpopulationsofsiblingspeciesinChironomus(Camptochironomus)Kieffer

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IyaiIIvanovnaKiknadze

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Divergent cytogenetic evolution in Nearctic and

Palearctic populations of sibling species in

Chironomus (Camptochironomus) Kieffer

Iya I. Kiknadze, Malcolm G. Butler, Karlygash G. Aimanova,Evgenya N. Andreeva, Jon Martin, and Larissa I. Gunderina

Abstract: Chromosomal polymorphism is described for natural populations of Chironomus pallidivittatus in both the

Palearctic and Nearctic regions. The Palearctic populations studied exhibit 24 banding sequences, whereas 10 banding

sequences have been recorded from Nearctic C. pallidivittatus. In total, 29 sequences and 37 genotypic combinations have

been found. Of the 29 sequences known, only 5 are Holarctic (common to both the Nearctic and Palearctic), 19 are

exclusively Palearctic, and 5 are Nearctic. The karyotype of Nearctic C. pallidivittatus is characterized by specific,

homozygous Nearctic sequences in arms B and G and fixed Holarctic inversion sequences in the other arms. Only two

chromosome arms in C. pallidivittatus, but all seven arms in the sibling species Chironomus tentans, differ between Palearctic

and Nearctic forms by the presence of unique, homozygous sequences in the Nearctic karyotype. This indicates a great

difference in the cytogenetic histories of these closely related species; much less karyotypic divergence between continents

has occurred in C. pallidivittatus than in C. tentans. The cytogenetic distance between Palearctic and Nearctic populations of

C. tentans is higher (DN = 1.62) than in C. pallidivittatus (DN = 0.27). Thus, Palearctic and Nearctic C. tentans should be

regarded as sibling species, but Palearctic and Nearctic C. pallidivittatus are best viewed as strongly divergent races of the

same species. A photomap of polytene chromosomes of C. pallidivittatus is presented in which banding sequences are mapped

by using C. tentans as a standard.

Résumé: On trouvera ici la description du polymorphisme des chromosomes chez des populations naturelles paléarctiques et

néarctiques de Chironomus pallidivittatus. Les populations paléarctiques étudiées comptent 24 séquences de bandes, alors que les

populations néarctiques n’en comptent que 10. Au total, 29 séquences et 37 combinaisons génotypiques ont été trouvées. Des 29

séquences connues, seulement cinq sont holarctiques (communes aux populations néarctiques et paléarctiques), 19 sont

exclusivement paléarctiques et 5, exclusivement néarctiques. Le cariotype des C. pallidivittatus néarctiques est caractérisé par des

séquences néarctiques spécifiques, homozygotes, dans les bras B et G et des séquences d’inversion holarctiques fixes dans les autres

bras. Seulement deux bras chromosomiques chez C. pallidivittatus, mais les sept bras de l’espèce apparentée Chironomus tentans

diffèrent, chez les formes paléarctiques et néarctiques, par la présence de séquences homozygotes caractéristiques dans le caryotype

néarctique. Cette condition reflète la différence considérable de l’évolution cytogénétique chez ces deux espèces apparentées : il s’est

produit beaucoup moins de divergence caryotypique d’un continent à l’autre chez C. pallidivittatus que chez C. tentans. La distance

cytogénétique entre les populations paléarctiques et néarctiques de C. tentans (DN = 1,62) est beaucoup plus importante que chez les

populations de C. pallidivittatus (DN = 0,27). Les C. tentans paléarctiques et néarctiques sont donc en réalité deux espèces soeurs,

mais il vaut mieux considérer les C. pallidivittatus des deux continents comme des races très divergentes de la même espèce. Une

carte photographique des chromosomes polytènes de C. pallidivittatus montre les séquences de bandes en utilisant le caryotype de

C. tentans comme référence.

[Traduit par la Rédaction]

Introduction

The banding patterns of polytene chromosomes in Diptera al-low species to be identified accurately, and chromosomal

rearrangements that may have led to divergence of their karyo-types can be observed. One of the most important results ofsuch cytotaxonomic studies has been the discovery of siblingspecies in many polytypic species of Drosophilidae(Dobzhansky 1970; Krimbas and Powell 1992), Chironomi-dae (Beermann 1955, 1960; Keyl 1957; Martin 1979; Wülker1991; Kiknadze 1987), Simuliidae (Rothfels 1979, 1989), andCulicidae (Kitzmiller 1976). Morphologically, sibling speciesare indiscernible or scarcely discernible, but their karyotypesare clearly different, owing to fixed chromosomal rearrange-ments (para- and peri-centric inversions, chromosome arm fu-sions, etc.). Sibling species often live sympatrically withouthybridizing. Comparative karyotypic analysis of siblingspecies is of great value for understanding the cytogenetics ofmicroevolutionary processes (White 1977; Rothfels 1979, 1989).

Several groups of sibling species have been found in the

Can. J. Zool., 76: 361–376 (1998)

Received February 24, 1997. Accepted September 5, 1997.

I.I. Kiknadze, K.G. Aimanova, E.N. Andreeva, and L.I.Gunderina. Laboratory of Cell Biology, Institute of Cytologyand Genetics, Novosibirsk, Russia.M.G. Butler. 1 Department of Zoology, North Dakota StateUniversity, Fargo, ND 58105, U.S.A.J. Martin. Genetics Department, University of Melbourne,Parkville, Victoria 3052, Australia.

1 Author to whom all correspondence should be sent (e-mail:mbutler@plains.nodak.edu).

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Chironomidae. Initially, only two sibling species-pairs ofChironomus were known: Chironomus (Camptochironomus)tentans and Chironomus pallidivittatus (Beermann 1955), plusChironomus piger and Chironomus riparius (thummi) (Keyl1957). Increasingly, larger groups of sibling species are beingrecognized, such as the plumosus group with 11 or 12 species(Kerkis et al. 1989; Shobanov 1989; Kiknadze et al. 1991), theaberratus group with 5 species (Wülker 1991), and the riihi-makiensis group with 9 species (Kiknadze et al. 1992, 1994,1996c). Martin (1979) suggested that the Nearctic speciesChironomus decorus Johannsen might actually comprise asmany as 15 species, and cytogenetic differences among severalsibling species in this decorus group have been noted (Wülkeret al. 1991; Butler et al. 1995). Comparative studies of polytenechromosome banding patterns in these groups have clarifiedthe role of fixed inversions and tandem fusions in the chromo-somal evolution of sibling species, and have aided inter-pretation of phylogenetic relationships.

The C. tentans – C. pallidivittatus group is a valuablemodel for studying chromosome divergence in sibling speciesand chromosomal polymorphism in natural populations. Thesespecies are distributed across the Holarctic, usually occur sym-patrically, and are widely used experimentally in cytogeneticand ecological studies (Beermann 1955, 1960; Hein 1969;Hein and Schmulbach 1971; Grossbach 1977; Edström et al.1980; Rovira et al. 1993; Ball and Baker 1996). However, onlyfor C. tentans have the karyotype and chromosomal polymor-phism been studied thoroughly (Beermann 1955; Acton 1957,1959, 1962; Kiknadze et al. 1996b; Gunderina et al. 1996).Drawn maps and photomaps of C. tentans polytene chromo-somes have been published, chromosomal polymorphism hasbeen studied in 45 Palearctic and Nearctic populations, andgeographic patterns of karyotype divergence have been estab-lished.

In contrast, C. pallidivittatus received very little detailedcytogenetic study before 1996. Bauer (1936, 1937) reportedup to four heterozygous inversions per C. pallidivittatus larva,but the locality of his population is unknown. Beermann(1955) described the karyotype and chromosomal polymor-phism in a German population of C. pallidivittatus. He estab-lished that the karyotype of this species differed significantlyfrom that of the sibling species C. tentans by fixed inversionsin arms A, B, C, D, E, and G, as well as by the number ofnucleolar organizers. However, further work on populationdifferences in inversion polymorphism and investigation ofchromosomal evolution in Palearctic or Nearctic C. pallidivit-tatus were hindered by the lack of photographic chromosomalmapping for this species. Another obstacle has been the factthat although the two species often occur together, C. pallidi-vittatus larvae are typically scarcer than those of C. tentans.Beermann (1955) reported that C. pallidivittatus constitutedonly 5–10% of total larvae in his collections of the subgenusCamptochironomus in Germany. Development of a photomapfor C. pallidivittatus permitted Kiknadze et al. (1996a) to ana-lyze chromosomal polymorphism in 6 Siberian populations,where this species was also rare relative to C. tentans.

Karyotypes of Palearctic and Nearctic C. tentans arestrongly divergent, most inversion sequences found in the Nearcticdiffering from those found in the Palearctic (Acton 1959,1962). Recent studies (Kiknadze et al. 1996b; Gunderina et al.1996) show that of the 62 banding sequences known in

C. tentans, only 6 occur in populations on both continents(Holarctic banding sequences), 45 are exclusively Palearctic,and 23 have been found only in the Nearctic. This strong di-vergence of the C. tentans karyotype on the two continentssupports the suggestion that Nearctic and Palearctic popula-tions of this species should be regarded as sibling species(Acton and Scudder 1971; Kiknadze et. al. 1996b).

In contrast to the well-studied C. tentans, no data existedon zoogeographic variation in the karyotype of C. pallidivit-tatus, despite the wide distribution of this species in both thePalearctic (Goetghebuer 1937; Shilova 1957; Kalugina 1963)and the Nearctic (Hein 1969; Hein and Schmulbach 1971).Here we report on chromosomal polymorphism in severalnatural Palearctic and Nearctic populations of C. pallidivit-tatus, and describe 13 previously unknown inversion se-quences in this species. We have found the intercontinentalpattern of karyotype variation in C. pallidivittatus to differgreatly from that previously reported for its sibling speciesC. tentans (Kiknadze et al. 1996b; Gunderina et al. 1996).

Materials and methods

Fourth-instar larvae of C. pallidivittatus were collected from naturalpopulations at nine Siberian and four North American locations(Table 1). Beermann’s (1955) data on a German population were alsoused to characterize chromosomal polymorphism in Palearctic C. pal-lidivittatus. In most cases, C. pallidivittatus larvae were found in thesame samples with the more abundant C. tentans. In only four Sibe-rian and three North American collections were the numbers ofC. pallidivittatus larvae sufficient (>10) to permit an assessment ofchromosomal polymorphism. In the other cases, only 1–4 larvae ofC. pallidivittatus occurred among multitudes of C. tentans larvae.These are referred to as small populations in the results. Larvae fromthe SD-V population (Table 1) came from a laboratory stock derivedfrom larvae collected by J. Hein near Vermillion, South Dakota.

For karyological analysis, larvae were fixed in a 3:1 mixture of100% ethanol and glacial acetic acid. Squashes were prepared by theaceto-orcein method (Keyl and Keyl 1959; Kiknadze et al. 1991).

Denotation of chromosome arms follows Keyl (1962), and indi-cates homology of arms between C. pallidivittatus and other Chiro-nomus species. In preparing maps, each chromosome is divided intoregions corresponding to those defined for C. tentans (Beermann1955; Kiknadze et al. 1996b). We mapped the banding pattern ofC. pallidivittatus polytene chromosomes by using the photomap ofC. tentans as a standard. Thus, our mapping of C. pallidivittatus dif-fers from that of Beermann (1955), whose labeling of polytenechromosomes in this species and C. tentans did not always maintainhomology between band groups. Band sequences in chromosome re-gions where our mapping is tentative because of poor band resolutionare given in parentheses in the Appendix.

In Keyl’s (1962) system, chromosome arm sequences known foreach species are numbered consecutively according to the order oftheir discovery, and are prefixed by an abbreviation of the speciesname. For example, C. pallidivittatus would have palA1, palA2, etc.for arm A, palB1, palB2, etc. for arm B, and so forth. Genotypiccombinations of arm sequences are indicated by palA1.1,palB2.2, etc. when homozygous and by palA1.2, palB1.2,palB2.3, etc. when heterozygous.

As in a previous paper comparing Nearctic and Palearctic popula-tions of C. tentans (Kiknadze et al. 1996b), we use a prefix to indicatethe known geographic distribution of each sequence. In addition to theprefixes p′ for sequences known only from Palearctic locations and n′for exclusively Nearctic sequences, here we also use h′ to denotesequences with an established Holarctic distribution. For example,

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p′palD1 is a sequence in arm D of C. pallidivittatus that has beenfound in Germany and Kazakhstan, n′palD3 is an alternative sequenceknown only from North America, and h′palD2 is the most commonsequence found in populations on both continents. The prefix maychange from p′ or n′ to h′ if a sequence is subsequently discovered tooccur on both continents, but the unique, consecutive number is main-tained for every sequence in each arm of a given species.

Like many Chironomus species, C. pallidivittatus exhibits avery high level of chromosomal polymorphism, with many complex(nested or overlapping) inversions. Thus, cytogenetic analysis ofchironomid populations involves study of whole-arm sequences ratherthan of individual inversions (Beermann 1955; Acton 1957, 1959,1962; Acton and Scudder 1971; Keyl 1962; Wülker 1980, 1991;Kiknadze et al. 1991). The karyotype of each larva studied was re-corded as the genotypic combination of sequences in all chromosomearms. The number and frequency of different banding sequences ineach population were then determined from the genotypic data ob-tained from each sample (Dobzhansky 1970; Ayala and Kiger 1984).

Banding patterns for each arm sequence found in our samples orby Beermann (1955) are listed in the Appendix. Underlining of bandgroups indicates regions with simple or complex inversions relativeto the standard band sequence described by Beermann (1955). Otherconventions used in describing Chironomus banding patterns are de-tailed in Kiknadze et al. (1996b).

Heterozygote frequencies within each population were tested forconformity to expectation under Hardy–Weinberg equilibrium withthe χ2 test. The significance of differences among populations in thefrequencies of alternative sequences was tested with Fisher’s test(Snedecor and Cochran 1967). Cytogenetic distances between thepopulations examined were calculated with Nei’s method (Nei 1972)and a dendrogram was constructed by means of UPGMA clusteranalysis (Sneath and Sokal 1973).

Results

Karyotype and chromosomal polymorphism inPalearctic C. pallidivittatus

Chironomus pallidivittatus belongs to the Camptochironomus

cytocomplex (Keyl 1962; Martin 1979) because it shares withC. tentans a unique combination of chromosome arms. Chromo-some I consists of arms C(1L)2 and F(1R), chromosome II ofarms B(2L) and A(2R), and chromosome III of D(3L) andE(3R), and arm G(4) constitutes the telocentric chromosomeIV (Fig. 1). As in C. tentans, the centromeric bands in C. pal-lidivittatus are not heterochromatinized (Beermann 1955;Rovira et al. 1993). The single nucleolus typically present inthe karyotype of C. pallidivittatus is located in chromo-some BA (Figs. 1, 2). In contrast, C. tentans carries two nu-cleoli borne in chromosomes BA and DE in Palearcticpopulations and chromosomes BA and CF in Nearctic popula-tions (Fig. 2).

The karyotypes of C. tentans and C. pallidivittatus differby fixed inversions in arms C, B, A, E, D, and G, and only inarm F is the banding pattern identical in these two species(Figs. 2a, 2c). When comparing a C. pallidivittatus sequencewith a sequence in C. tentans, we underline the inverted regionin C. pallidivittatus; it is important to note that this does notimply any direction of evolutionary change, as we do not knowthe ancestral sequence. In describing various sequences found

Locality

Latitude and

longitude Population

Collection

date

No. of larvae

sampled

Siberia

Yakutsk

Suburb pond 62°00′N, 129°45′E Y-O June 1994 34

Pond near leather-dressing factory 62°00′N, 129°45′E Y-K June 1993 13

Nurba region

Lake at Antonovka village 66°00′N, 118°00′E Y-N August 1994 11

Kazakhstan

Ust-Kamenogorsk suburban pond 50°00′N, 83°00′E K-U October 1994 60

Delta of Ili River 45°30′N, 74°00′E K-I December 1991 3

Nura River 50°30′N, 70°10′E K-N December 1991 1

Novosibirsk region

Berdsk Pond 54°46′N, 83° 02′E N-Br June 1982 2

Altai region

Gorkoe Lake 51°26′N, 81°30′E A-Go October 1993 2

Irkutsk region

Telminka Pond 52°4l′N, 103°42′E I-T May 1993 4

Germany

Plön 1 (Beermann 1955) 54°10′N, 10°20′E G-Pl 1 1934 19

Plön 2 (Beermann 1955) 54°10′N, 10°20′E G-Pl 2 1949–1950 20

U.S.A.

Ditch near Sioux Falls, S.D. 43°52′N, 96°65′W SD-SF March 1994 16

Pond near Vermillion., S.D. 42°78′N, 96°93′W SD-V December 1968 31

Pond near Cleveland, N.D. 46°89′N, 99°12′W ND-C May 1995 39

Canada

Pond near Theodore, Saskatchewan 50°25′N, 102°53′W C-S June 1968 2

Table 1.Collection sites, dates of collection, and numbers of specimens in Chironomus pallidivittatus populations.

2 Beermann’s (1955) terminology for chromosome arms is givenin parentheses.

Kiknadze et al. 363

© 1998 NRC Canada

Fig. 1. Karyotype of Siberian Chironomus (Camptochironomus) pallidivittatus (Palearctic C. pallidivittatus). Arrows indicate centromeric bands. Specific banding sequences

within homologous pairs of chromosome arms are labeled p′palC1.1, h′palF1.1, etc. N, nucleolus; BR, Balbiani ring.

Can.

J.

Zool.

Vo

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6,

199

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998

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CC

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a

within C. pallidivittatus (Appendix), we underline regions inalternative sequences that are inverted relative to the sequencethat was first described for the species.

Chromosome I(CF)Arm C(1L) shows four inversion sequences in the Palearctic

populations studied (Table 2, Figs. 3a–3b, 3j–3k, Appendix).

Sequence p′palC1 (Figs. 1, 3a) can be related to p′tenC2 in

C. tentans by a fixed simple inversion: p′tenC2: 1ABC 2ABC

3ABC 4ABC 8A 7CBA 6CBA 5CBA 8BC 9ABC 10ABC;

p′palC1: 1A 6ABC 7ABC 8A(B) 4CBA 3CBA 2CBA1CBA

5CBA 8BC 9ABC 0ABC.

The inversion sequence h′palC2 differs from p′palC1 by asimple inversion of a major part of the arm (Figs. 3a–3b, Ap-pendix), and is identical with inversion IL–1 reported byBeermann (1955). Sequence p′palC3 (Fig. 3j, Appendix),found only in the Yakutian population Y–O (Table 2), carriesa small inversion near the centromeric region that is apparentlynot pericentric. The morphology of heterozygous chromo-somes bearing the sequence p′palCk (Fig. 3k) suggests itsidentity with Beermann’s (1955) 1L–k1, but to date we havebeen unable to map this complex pericentric inversion.

Arm F(1R) is typically monomorphic in the populationsstudied (Table 2) and has a banding pattern, h′palF1, that is

identical with p′tenF1 in C. tentans (Fig. 1). A complexpericentric inversion (Fig. 3k) occurred at low frequency as theheterozygote p′palF1.k in two Yakutian populations, and wasalso found in populations in Germany by Beermann (1955),who referred to it as inversion 1R–K1. This arm F inversionappears in association with p′palC1.k in arm C. Beermann(1955) also found two rare sequences in arm F (p′palF2 andp′palF3min) in the German populations he analyzed.

Chromosome II (BA)Arm B(2L) shows a banding pattern that differs strongly from that

of arm B in C. tentans in having complex, non-overlapping inver-

sions and the transposition of the nucleolus from region 9A to

region 12BC in arm A (Fig. 1): p′tenB1: 1ABC 2ABC 3ABC

4ABC 5ABC 6ABC 7AB C 8ABC 9ABC 10ABC 11ABC;

p′palB1: 1A 3BC 4ABC 5ABC 6ABC 7ABC 8ABC 9ABC 10A

1BAC 2ABC 3AB 10ABC 11ABC.

Arm B is extremely polymorphic in C. pallidivittatus.Eight inversion sequences and 13 genotypic combinationshave been found in this arm in the Palearctic populations stud-ied (Table 2, Figs. 1, 3c–3d, 3l–3q, Appendix).

Sequences p′palB2 and p′palB5 are derived from p′palB1by different simple inversions occupying a major part of thearm (Figs. 3c, 3d, 3m, 3n, 3p, Appendix). A very small inversion

Fig. 2. Comparison of C. tentans and C. pallidivittatus karyotypes in the Palearctic and Nearctic, showing standard sequences of Palearctic

C. tentans (a) and sequences for Nearctic C. tentans (b), Palearctic C. pallidivittatus (c), and Nearctic C. pallidivittatus (d). Notation is as in

Fig. 1. Brackets indicate chromosome regions involved in simple and complex inversions.

Kiknadze et al. 365

© 1998 NRC Canada

near the distal end of the arm leads to sequence p′palB3 orp′palB4, depending on whether it occurs with p′palB1 orp′palB2 (Fig. 3d, Appendix). These sequences can be difficultto detect when in the heterozygous state, as they strongly re-semble a mere nonpairing of homologs. Sequence p′palB6 (Fig. 3l)also differs from p′palB1 by a small inversion, in this case nearthe centromeric region (Appendix).

Sequences p′palB7 and p′palBk2 were formed by complex

inversions. These sequences have only been found as hetero-zygotes with complex morphology (Figs. 3o, 3q) and we have failedto map them. Only four of the above sequences, p′palB1(=2L),p′palB2(=2L–1),p′palB3(=2L–K1), andp′palB4(=2L–1+K1),hadbeen described previously by Beermann (1955) (see Appendix).

Arm A(2R) in C. pallidivittatus deviates from sequence p′tenA2

in C. tentans by two fixed, overlapping inversions that occupy a

major portion of the arm (Fig. 1): p′tenA2: 20CBA 19CBA

Banding

sequence

Siberia Germany U.S.A.

Y-O Y-K Y-N K-U G-Pl 1* G-Pl 2* SD-SF SD-V ND-C

h′palA1 1.00 1.00 1.00 0.925 0.844 0.850 1.00 1.00 1.00

p′palA2 0 0 0 0.075 0.156 0.150 0 0 0

Hob 0.150 0.316 0.300

Hex 0.139 0.263 0.255

p′palB1 0.294 0.269 0.364 0.384 0.633 0.475 0 0 0

p′palB2 0.603 0.614 0.636 0.450 0.367 0.525 0 0 0

p′palB3 0 0.039 0 0.008 0.105† 0.075† 0 0 0

p′palB4 0.103 0.078 0 0.033 † † 0 0 0

p′palB5 0 0 0 0.092 0 0 0 0 0

p′palB6 0 0 0 0.008 0 0 0 0 0

p′palB7 0 0 0 0.008 0 0 0 0 0

p′palBk2 0 0 0 0.017 0 0 0 0 0

n′palB8 0 0 0 0 0 0 0.656 0.823 0.641

n′palB9 0 0 0 0 0 0 0.344 0.177 0.359

Hob 0.677 0.692 0.363 0.517 † † 0.687 0.354 0.718

Hex 0.539 0.546 0.463 0.648 † † 0.452 0.291 0.461

p′palC1 0.162 0.192 0 0.725 0.500 0.575 0 0 0

h′palC2 0.764 0.769 1.00 0.275 0.474 0.425 1.00 1.00 1.00

p′palC3 0.015 0 0 0 0 0 0 0 0

p′palCk 0.059 0.039 0 0 0.026 0 0 0 0

Hob 0.441 0.308 0.317 0.421 0.550

Hex 0.386 0.333 0.398 0.499 0.488

p′palD1 0 0 0 0.225 0.368 0.225 0 0 0

h′palD2 1.00 1.00 1.00 0.775 0.632 0.775 0.969 1.00 0.897

n′palD3 0 0 0 0 0 0 0.031 0 0.103

Hob 0.317 0.421 0.350 0.062 0.208

Hex 0.348 0.466 0.348 0.060 0.184

h′palE1 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

h′palF1 0.941 0.923 1.00 1.00 0.920 0.950 1.00 1.00 1.00

p′palFk 0.059 0.077 0 0 0.026 0 0 0 0

p′palF2 0 0 0 0 0.026 0 0 0 0

p′palF3min 0 0 0 0 0.026 0.050 0 0 0

Hob 0.119 0.154 0.104 0.100

Hex 0.112 0.142 0.153 0.094

p′palG1 0.382 0.808 0.864 0.900 0.422 0.350 0 0

p′palG2 0.603 0.192 0.136 0.100 0.578 0.650 0 0

p′palG3 0.015 0 0 0 0 0 0 0

n′palG4 0 0 0 0 0 0 0.938 1.00 0.792

n′palG5 0 0 0 0 0 0 0.062 0.208

Hob 0.471 0.385 0.273 0.200 0.632 0.500 0.125 0.416

Hex 0.490 0.310 0.236 0.180 0.488 0.545 0.116 0.330

*Beermann’s (1955) collections from Plön, Germany: G-Pl 1 in 1934 and G-Pl 2 in 1949–1950.

†Beermann combined sequence B3 with B4 in this arm, so expected frequencies were not calculated.

Table 2.Frequencies of banding sequences in C. pallidivittatus populations.

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18CBA 12C 13ABC 14ABC 15ABC 16ABC 17ABC (18A)

12(C)BA; h′palA1: (20C) 18C 19A 16A 15CBA 14CBA13CBA

12C 18AB 20CBA 19CBA 16ABC 17ABC (18A) 12(C)BA.

Arm A is monomorphic in most of the populations studied,with heterozygotes p′palA1.2 found only in one population inKazakhstan and in the German populations (Table 2). The al-ternative sequence p′palA2 differs from the predominant se-quence by a simple inversion (Fig. 3r, Appendix). Both arm Asequences were previously described from German popula-tions by Beermann (Table 2).

Chromosome III(DE)Arm D(3L) has two sequences in Palearctic C. pallidivittatus.

The less common sequence, p′palD1, differs from p′tenD1 in

C. tentans by a fixed simple inversion (Fig. 1): p′tenD1:

1ABC 2ABC 3ABC 4ABC 5ABC 6ABC 7ABC 8ABC 9ABC

10ABC; p′palD1: 1ABC 2ABC 3ABC 4ABC 5A 9A 8CBA

7CBA 6CBA 5CBA 9(A)BC 10ABC.

The predominant arm D sequence in all populations studiedis h ′palD2, which differs from p ′palD1 by a complexrearrangement (Table 2, Figs. 3e, 3f, 3s, Appendix). Rareheterozygotes p′palD1.2 were found in small populations.Both these sequences were described by Beermann (1955).

Arm E(3R) in C. pallidivittatus bears the single sequenceh′palE1 (Fig. 1, Appendix), which differs from p′tenE1 by threeoverlapping inversions: p′tenE1: 16CBA 15CBA 14CBA 13CBA12CBA 11CBA; h′palE1: 16CB 13A 11C 12ABC 16A 15CBA14CBA 13CBA 11CBA.

Although Arm E was monomorphic in all populations stud-ied (Table 2), it is noteworthy that nonpairing of homologs wasobserved in some larvae.

Chromosome IV(G)Arm G(4) of C. pallidivittatus differs from arm G of C. tentans

by a complex inversion (Fig. 1): p′tenG1: 6CBA 5CBA 4CBA

3CBA 2CBA 1CBA; p′palG1: 6CBA 5CBA 2CBA 4CBA 3CBX

1CBA.

We propose that the region denoted by Beermann (1955)as X includes the sequence 3A 4C 5A 2A (Figs. 3g, 5d). Asecond sequence, p′palG2, differs from p′palG1 by a simpleinversion (Table 2, Figs. 3g, 3h, Appendix). Both these se-quences were described earlier by Beermann (1955). In addi-tion to these two common sequences, we found the heterozygotep′palG1.3 with an unknown sequence p′palG3 in populationY–O (Fig. 3i). Sequence p′palG1 was the only sequence foundin small populations.

On the whole, Palearctic populations of C. pallidivittatusdisplay a high level of chromosomal polymorphism (Table 2).In the populations studied, 64–82% of larvae were hetero-zygous for inversions, although the average number ofheterozygous inversions per individual was low (0.6–1.5;Table 3). The number of sequences in each population variedbetween 9 and 15 (Table 3), with a total of 30 inversion se-quences and 37 genotypic combinations found in the Palearcticpopulations.

The overall level of chromosomal polymorphism inPalearctic C. pallidivittatus results both from diversity of se-quences within chromosomal arms and from differencesamong populations in their expression of this polymorphism.The most polymorphic arm is B, which carries 10 sequences

and is polymorphic in all populations (Table 2). The leastpolymorphic is arm E, which is represented in all populationsby the single sequence p′palE1. Other arms are intermediate,with 2–4 sequences and polymorphism in at least half thepopulations studied (Table 2). Thus, arm C carries 4 sequences,and was polymorphic in 4 out of the 5 populations studied.Arms F and G each have 3 sequences, but arm G was poly-morphic in all Palearctic populations, whereas arm F was po-lymorphic in only 3 populations. Arms A and D have 2sequences each and are polymorphic in 2 of the 5 Palearcticpopulations (Table 2). Thus, the seven arms can be ranked bytheir degree of chromosomal polymorphism according to twocriteria. Based on the number of alternative sequences in eacharm, the ranking is E < A = D < F = G < C < B. When basedon the number of populations showing polymorphism for eacharm, the ranking becomes E < A = D < F < C < G = B. Thedifference between these rankings reflects the independenceof the two criteria used to express the level of chromosomalpolymorphism. Although arms F and G have the same numberof sequences, G is polymorphic in all 6 populations, whereasarm F is polymorphic in only 4. Taking both criteria into ac-count, we can rank the arms for overall polymorphism asfollows: E < A = D < F < C < G < B.

Karyotype and chromosomal polymorphism in NearcticC. pallidivittatus

Nearctic C. pallidivittatus has a karyotype similar to itsPalearctic counterpart (Figs. 1, 4), but differs in having spe-cific Nearctic sequences in arms B and G and fixed Holarcticinversion sequences in arms A, C, F, D, and E.

Chromosome I(CF)Arm C is monomorphic (Table 2, Fig. 4), the one sequencefound being identical with h′palC2. As this sequence occurs inpopulations on both continents, we refer to it as Holarctic(h′palC2).

Arm F is monomorphic (Table 2, Fig. 4), with a bandingpattern identical with h′palF1. Thus, this sequence is also re-ferred to as Holarctic.

Chromosome II(BA)Arm B is polymorphic in the Nearctic populations. It has two

sequences, n′palB8 and n′palB9, both endemic to the Nearctic

region (Table 2, Figs. 4, 5a, Appendix). The sequence n′palB8

differs from p′palB1 by two non-overlapping inversions:

p′palB1: 1A 3BC 4ABC 5ABC 6ABC 7ABC 8ABC 9ABC

10A 1BAC 2ABC 3AB 10ABC 11ABC; n′palB8: 1A 9CBA

8CBA 7CBA 6CBA 5CBA 4CBA 3CBA 2CBA 1CAB

10ABC 11ABC.

Sequence n′palB9 (Fig. 5a) is very unusual. It differs fromn′palB8 by a complex inversion, but its most unique feature isthat it carries several nucleoli, not one nucleolus as is found inchromosome BA of all Camptochironomus species known todate. We have found this sequence in the material sampledfrom North American sites recently as well as back in 1968.Sequence n′palB9 occurs with high frequency in all the Nearcticpopulations studied, although only in the heterozygousstate. It is important to note that arm B is a Y chromosomein some Palearctic individuals (Beermann 1955) and so a pos-sible explanation for the lack of B9.9 homozygotes could bethat the sequence is associated with a Y chromosome in these

Kiknadze et al. 367

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Fig. 3. (a–i) Homozygous and heterozygous inversion sequences in Palearctic C. pallidivittatus.

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Fig. 3. (concluded) (j–s) Heterozygous inversion sequences in Palearctic C. pallidivittatus. Notation is as in Fig. 1.

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Fig. 4. Karyotype of Nearctic C. pallidivittatus. Notation is as in Fig. 1.

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Nearctic populations. Only a small proportion of our sampleswere sexed, but B8.9 was observed only in male larvae and nofemales, adding support to this hypothesis.

Arm A is monomorphic (Table 2, Fig. 4), with a sequenceidentical with h′palA1, so this sequence should also be consid-ered Holarctic.

Chromosome III(DE)Arm D is nearly monomorphic (Table 2, Fig. 4), with a pre-dominant sequence that is identical with h′palD2, and shouldtherefore be considered Holarctic. In addition to h′palD2, an-other sequence, n′palD3, differing by a simple inversion hasbeen found as a heterozygote (Table 2, Fig. 5b, Appendix).Sequence h′palD2 was the only sequence found in small popu-lations.

Arm E is monomorphic, with the Holarctic sequenceh′palE1 (Table 2, Fig. 4).

Chromosome IV(G)Arm G(4) is dominated by the sequence n′palG4 (Table 2,

Fig. 4), which differs from p′palG1 by a simple inversion:

p′palG1: 6CBA 5CBA 2CBA 4CBA 3CBX 1CBA; n′palG4:

6CBA 5C 4ABC 2ABC 5AB 3CBX 1CBA.

An additional sequence, n′palG5, was found as a hetero-zygote (Table 2, Fig. 5c). This sequence differs from n′palG4by a simple inversion (Appendix). Sequence n′palG4 was theonly sequence found in small populations.

Ten banding sequences and 10 genotypic combinations ofthese sequences have been found in the 3 better sampledNearctic populations of C. pallidivittatus (Table 3). Larvaefrom the Vermillion laboratory stock collected in 1968(Table 1) produced 8 of these 10 sequences (h′palA1,n′palB8, n′palB9, h′palC2, h′palD2, h′palE1, h′palF1, andn′palG4), as did the 2 larvae collected in Saskatchewan in1968. Arms A, C, D, E, and F are monomorphic in these 1968samples, whereas the polymorphic arm B has 2 sequences. Thechromosomal arms in Nearctic populations fall into twogroups with respect to overall chromosomal polymorphism:A = C = E = F < B = D = G.

Cytogenetic differentiation of Palearctic and Nearcticpopulations in C. pallidivittatus

On the basis of the data available (Tables 2, 3), Palearcticpopulations of C. pallidivittatus appear to be much more poly-morphic than Nearctic populations. The number of bandingsequences in each of the 4 Siberian populations ranged from 9to 19, whereas the 2 wild Nearctic populations both had 10sequences (Table 3), and no additional sequences were foundin other Nearctic material examined (Table 1). The cyto-genetic structure of the Palearctic populations is also moreheterogeneous, in that the Nearctic populations did not differin the number of polymorphic arms (3) or the number of band-ing sequences per arm (1 or 2), and sequence frequencies werenearly identical (Table 2). Consequently, the cytogenetic dis-tance between these two Nearctic populations is very small(DN = 0.04; Table 4).

In contrast, Palearctic populations differ in all the criteriamentioned above (Tables 2, 3). Chromosomal polymorphismwas encountered in six of the seven arms. The number of al-ternative banding sequences per arm ranged from 8 (arm B) to3 (arms C, F, and G) to 2 (arms A and D), with only arm Emonomorphic. Whereas both of the Nearctic populations ex-

hibited the same 10 sequences, the 24 sequences found in thePalearctic region were distributed differentially among theSiberian and European populations. Nine of these sequenceswere common to all Palearctic populations, but 8 were uniqueto single populations.

The frequencies of several sequences differed significantly(p < 0.001) among the Palearctic populations. In the Kazakhstanpopulation K–U, p′palC1 was dominant in arm C, but this se-quence was observed at low frequency in populations Y–O andY–K and was absent in population Y–N. Population Y–O hada frequency of p′palG1 significantly lower than in otherPalearctic populations. Such differences in cytogenetic struc-ture are reflected in the calculated cytogenetic distances be-tween the 4 Siberian populations (Table 4). These cytogeneticdistances range from a low of 0.009 between the Yakutianpopulations Y–K and Y–N to a high of 0.123 between popu-lation K–U in Kazakhstan and population Y–O inYakutia. Thecollecting sites in Kazakhstan and Yakutia are separated by over3500 km, so it is not surprising that the average cytogeneticdistance between Kazakhstan and Yakutian populations(0.099 ± 0.032) is higher than the distances among theYaku-tian populations themselves (0.029 ± 0.011). The averagecytogenetic distance between all 4 Siberian populations ofC. pallidivittatus is 0.064 ± 0.018.

Nearctic populations of C. pallidivittatus differ most nota-bly from Palearctic C. pallidivittatus in the set of inversionsequences they carry. Of the 30 banding sequences known forthis species, only 5 (1 each in arms A, C, D, E, and F) areHolarctic, having been found in both Palearctic and Nearcticpopulations. Another 20 sequences are exclusively Palearcticand 5 are Nearctic. The cytogenetic distance betweenPalearctic and Nearctic populations of C. pallidivittatus was0.272 ± 0.023, greatly exceeding distances among populationswithin either continent (Table 4). The cytogenetic relationshipsbetween the various Siberian and North American populationsare summarized as a dendrogram in Fig. 6, which shows thatthe cytogenetic distances between populations increase in ap-proximately the same order as geographic distance.

Discussion

Previous karyological comparisons of Palearctic and NearcticC. tentans uncovered great cytogenetic differentiation betweenpopulations east and west of the Atlantic Ocean (Acton 1959,1962; Kiknadze et al. 1996b; Gunderina et al. 1996). Before asimilar analysis of the sibling species C. pallidivittatus couldbe made, it was necessary to prepare a detailed photomap ofthe polytene chromosomes of this species. A karyotypic stand-ard is normally used for comparative cytogenetic analysis ofdifferent species. For example, the standard for the subgenusChironomus (Chironomus) is C. piger (Keyl 1962). In the sub-genus Chironomus (Camptochironomus) we have used thekaryotype of C. tentans as a standard for mapping the chromo-somes of its sibling species C. pallidivittatus. These maps per-mit one to trace all inversion steps involved in the cytogeneticdivergence of the respective karyotypes of the two species. Sixof the seven chromosome arms differ between these species byfixed inversions (Fig. 2). These are simple inversions inarms C, D, and G and complex inversions in arms A, B, and E(Fig. 1). Our results are in agreement with those reported by

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Beermann (1955), who was the first to describe differencesbetween the karyotypes of C. tentans and C. pallidivittatus.However, Beermann’s drawn maps do not maintain homology

between chromosomal regions that are inverted between thetwo species, thus obscuring evolutionary patterns.

In addition to differences in banding patterns, the karyotype

Fig. 5. (a–d) Heterozygous inversion sequences in Nearctic C. pallidivittatus. Notation is as in Fig. 1.

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of C. pallidivittatus differs from that of C. tentans by areduction in nucleoli from two to one, and transposition of theremaining nucleolus from region 9 to region 12 of chromo-some BA (Fig. 2).

Cytogenetic diversity among Palearctic populations ofC. pallidivittatus results from differences in levels of heterozy-gosity, as well as in the numbers, proportions, and genotypiccombinations of inversion sequences (Tables 2, 3). Beermann(1955) first studied a European population of this species fromPlön, Germany, where he identified 18 banding sequences. Noadditional populations from Europe have been investigated

since. We have found 22 banding sequences in Siberia, includ-ing all but 2 of those found by Beermann in Germany (p′palF2and p′palF3). We have also found 6 new sequences in theSiberian populations, including 4 in arm B and 1 each inarms C and G. Most of these new sequences are rare and occuronly as heterozygotes, whereas the predominant sequences arecommon to both the European and Siberian populations. As arule, between-population differences in sequence frequenciesare minor in Palearctic C. pallidivittatus. In arms C and D, how-ever, Beermann’s German population resembled the Kazakhstanpopulation more than it resembled the Yakutian populations.

Population

Frequency of

heterozygous

larvae (%)

Avg. no. of

heterozygous

inversions/ larva

No. of inversion

sequences in

population

No. of genotypic

combinations of

inversion sequences

Siberia

Y-O 79 1.5 15 18

Y-K 69 1.5 15 16

Y-N 64 0.6 9 10

K-U 82 1.5 19 24

U.S.A.

SD-SF 81 0.9 10 10

SD-V 30 0.3 8 8

ND-C 87 1.3 10 10

Table 3.General characteristics of chromosomal polymorphism in C. pallidivittatus populations.

Yakutia Kazakhstan Germany North America

Population Y-K Y-N K-U G-Pl1 G-Pl2 SD-SF SD-V ND-C

Y-O 0.324 0.462 0.094 0.056 0.052 0.234 0.253 0.221

Y-K 0.009 0.187 0.089 0.083 0.251 0.270 0.239

Y-N 0.244 0.121 0.116 0.243 0.263 0.230

K-U 0.038 0.036 0.385 0.405 0.372

G-Pl1 0.002 0.330 0.350 0.315

G-Pl2 0.328 0.349 0.314

SD-SF 0.005 0.004

SD-V 0.012

Table 4.Cytogenetic distances between C. pallidivittatus populations.

Fig. 6. Dendrogram showing Nei’s genetic distances between populations of C. pallidivittatus, based on chromosomal polymorphism. For

details of populations see Table 1.

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Nonetheless, inspection of the sequence frequencies inTable 2 shows the general similarity between the Germanand Siberian populations.

Comparing the karyotypes of Palearctic and Nearcticpopulations tells another story. Cytogenetic differentiationon opposite sides of the Atlantic Ocean exceeds that withineither continent, primarily because of different sets ofbanding sequences in Palearctic and Nearctic populations.Of the 29 sequences known from both continents, only 5 areHolarctic, 19 are Palearctic, and 5 are Nearctic, with spe-cific Nearctic sequences found in three arms (B, D, and G).The levels of divergence of these arm sequences differ.Arms B and G show complete cytogenetic differentiation,in that no Holarctic sequences are found in either arm. Thedivergence of arm D is not complete, because a Holarcticsequence (h′palD2) is universally present in addition to en-demic Palearctic and Nearctic alternatives (p′palD1 andn′palD3). It is important to note that there were no signifi-cant differences between Palearctic and Nearctic popula-tions of C. pallidivittatus when judged by level of heterozygosityor other criteria of chromosomal polymorphism listed inTable 3.

The cytogenetic relationship between Palearctic andNearctic populations of C. pallidivittatus is similar to thatof its sibling species C. tentans, but differs in importantdetails. As in C. tentans, European and Siberian C. palli-divittatus differ from North American populations primarilyby having different sets of fixed banding sequences. Of the62 inversion sequences, known for C. tentans, only 6 areHolarctic, whereas 39 are Palearctic and 17 are Nearctic.However, isolation on different continents has produced greaterdivergence between Palearctic and Nearctic forms of C. ten-tans than has occurred within C. pallidivittatus. All sevenchromosome arms of North American C. tentans have spe-cific Nearctic sequences, but this is true for only threearms of C. pallidivittatus. Divergence is complete for threechromosomal arms in C. tentans and nearly complete in twoothers, whereas only two arms in C. pallidivittatus show nosequences that are shared between both Nearctic andPalearctic populations. The cytogenetic distance betweenPalearctic and Nearctic C. tentans (DN = 1.618) (Gunderinaet al. 1996) was six times greater than that in C. pallidivit-tatus (DN = 0.272). These cytogenetic differences suggestthat Palearctic and Nearctic C. tentans should be regardedas well-diverged sibling species (Kiknadze et al. 1996b;Gunderina et al. 1996), whereas Palearctic and NearcticC. pallidivittatus represent highly differentiated populationsof a single species.

We can suggest several possible reasons for suchdifferent patterns of cytogenetic evolution in C. tentansand C. pallidivittatus. Chironomus pallidivittatus mayhave arrived in North America more recently than C. ten-tans, or it may occupy a narrower ecological niche in theNearctic than in the Palearctic region. Both of these fac-tors appear to have played a role in speciation withinDrosophila, especially in species that have recently in-vaded the Nearctic region (Krimbas and Powell 1992).Considerable evidence supports a role for transposableelements in generating chromosomal inversions in Droso-

phila (e.g., Engels and Preston 1984; Krimbas and Powell1992), and in explaining different levels of polymorphism

between sibling species (Lemeunier and Aulard 1992). Thismechanism may therefore play a role in the differences notedbetween these Chironomus species.

Acknowledgements

We thank Dr. I. Borovkova, Dr. A. Borovkov, andV. Filonenko for help with manuscript preparation. This re-search was supported by grants from the Russian Fund forFundamental Research, the North Dakota Water ResourcesResearch Institute, Region VIII of the U.S. EnvironmentalProtection Agency, and the Research and Consulting Commit-tee at North Dakota State University.

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Arm Ap′palA2 [2R] (20C) 18C 19A 16A 15CBA 14CBA 13CBA 12C 18AB 20CBA 19CBA 16ABC 17ABC (18A) 12CBA

p′palA2 [2R–1] (20C) 18A 19A 16A 15C 16CBA 19ABC 20ABC 18BA 12C 13ABC 14ABC 15AB 17ABC 12CBA

Arm B

p′palB1 [2L] 1A 3BC 4 ABC 5ABC 6ABC 7ABC 8ABC 9ABC 10A 1BAC 2ABC 3AB 10ABC 11ABC

p′palB2 [2L–1] 1A 3BC 4ABC 5ABC 6AB 2CBA 1CAB 10A 9CBA 8CBA 7CBA 6CB 3AB 10ABC 11ABC

p′palB3 [2L–k1] 1A 3B 1A 3C 4ABC 5ABC 6ABC 7ABC 8ABC 9ABC 10A 1BAC 2ABC 3AB 10ABC 11ABC

p′palB4[2L–1+k1] 1A 3B 1A 3C 4ABC 5ABC 6AB 2CBA 1CAB 10A 9CBA 8CBA 7CBA 6CB 3AB 10ABC 11ABC

p′palB5 1A 1CAB 10A 9CBA 8CBA 7CBA 6CBA 5CBA 4CBA 3CB 2ABC 3AB 10ABC 11ABC

p′palB6 1A 3BC 4ABC 5ABC 6ABC 7ABC 8ABC 9ABC 10A 1BAC 2CBA 3AB 10ABC 11ABCp′palB7 Not mapped

p′palBk2 Not mapped

n′palB8 1A 9CBA 8CBA 7CBA 6CBA 5CBA 4CBA 3CBA 2CBA 1CAB 10ABC 11ABC

n′palB9 1A 4ABC 5ABC 6AB 9CBA 8CBA 7CBA 6C 3CBA 2CBA 1CB 10AB 10C 11ABC

Table A1. List of banding sequences in Palearctic and Nearctic Chironomus (Camptochironomus) pallidivittatus. Sequence names used by

Beermann (1955) are given in brackets. Inverted regions are underlined. Parentheses indicate uncertainty in band identity or location.

Appendix

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© 1998 NRC Canada

Arm C

p′palC1 [1L] 1A 6ABC 7ABC 8A(B) 4CBA 3CBA 2CBA 1CBA 5CBA 8BC 9ABC 10AB

h′palC2 [1L–1] 1A 6CBA 7CBA 8A(B) 9BA 8CB 5ABC 1ABC 2ABC 3ABC 4ABC 9BC 10ABp′palC3 1A 6ABC 7ABC 8A(B) 4CBA 3CBA 2CBA 1CBA 5CBA 8BC 9A 10A 9CBA 10Bp′palCk [1L–k1?] Not mapped

Arm Dp′palD1 [3L] 1ABC 2ABC 3ABC 4ABC 5A 9A 8CBA 7CBA 6CBA 5CBA 9(A)BC 10ABC

h′palD2 [3L–k1] 1AB 6ABC 7ABC 8ABC 9ABC 10A 5CBA 9A 5A 4CBA 3CBA 2CBA 1C 10ABCn′palD3 1AB 6ABC 7ABC 8AB 1C 2ABC 3ABC 4ABC 5A 9A 5ABC 10A 9CBA 8C 10ABC

Arm Eh′palE1 [3R] 16B 13A 11C 12ABC 16A 15CBA 14CBA 13CBA 11CBA

Arm Fh′palF1 [1R] 20CBA 19CBA 18CBA 17CBA 16CBA 15CBA 14CBA 13CBA 12CBA 11CBA 10CB

p′palF2 [1R–1] 20CBA 19CBA 18CBA 17CBA 16CBA 15CBA 13BC 14ABC 15A 13A 12CBA 11CBA 10CBp′palFk Not mapped

p′palF3min [Ins. 1R] Microinsertion in region 14A

Arm G

p′palG1 [4] 6CBA 5CBA 2CBA 4CBA 3CB {2A 5A 4C 3A}3 1CBA

p′palG2 [4–1] 6CBA 5CBA {5A 2A}3BC 4ABC 2ABC {4C3A}1CBA

p′palG3 6CBA 5C 3CB{2A 5A}5ABC 3C 4ABC 2ABC {4C 3A}1CBA

n′palG4 6CBA 5C 4ABC 2ABC 5AB 3CB {2A 5A 4C 3A}1CBA

n′palG5 6CBA 3BC 5AB 2CBA 4CBA 5C {2A 5A 4C 3A}1CBA

Table A1 (concluded).

3 Braces bound the chromosomal region of arm G designated “X” by Beermann (1955).

Can. J. Zool. Vol. 76, 1998376

© 1998 NRC Canada