Karyotypes of Chironomus Meigen (Diptera: Chironomidae) species from Africa

Abstract The karyotypes of six African Chironomus species (Chironomus alluaudi Kieffer, 1913, Chironomus transvaalensis Kieffer, 1923, Chironomus sp. Nakuru, Chironomus formosipennis Kieffer, 1908, Chironomus prope pulcher Wiedemann, 1830, Chironomus sp. Kisumu) were investigated; four of these karyotypes were described for the first time (Chironomus sp. Nakuru, Chironomus formosipennis, Chironomus prope pulcher, Chironomus sp. Kisumu). Of the six Chironomus karyotypes, three had “pseudothummi” cytocomplex chromosome arms combinations AE CD BF G (Chironomus alluaudi, Chironomus transvaalensis, Chironomus sp. Nakuru), two had “thummi”cytocomplex arms combinations AB CD EF G (Chironomus formosipennis, Chironomus prope pulcher), and one had “parathummi”armcombinations AC BF DE G (Chironomus sp. Kisumu). Thus, three of the ten main cytocomplexes known were detected in Africa. Detailed photomaps of all chromosome arms, with the exception of arms B and G, were prepared for the karyotypes of Chironomus alluaudi, Chironomus transvaalensis, Chironomus sp. Nakuru, Chironomus prope pulcher; the karyotypes of Chironomus formosipennis, Chironomus sp. Kisumucould only be fragmentarily mapped. Endemic African banding sequences were characteristic for most of the chromosomal arms in all species studied. However, basic sequences, which can be present in different Chironomus species on different continents (Wülker, 1980; Kiknadze et al. 2008), were also detected also in several African species (Chironomus alluaudi, Chironomus sp. Nakuru, and Chironomus formosipennis). The banding sequences of African species studied allow discussion of the derivation of modern banding patterns from hypothetical species, living before separation of cytocomplexes and continents.


introduction
As shown by cytogenetic analysis of chromosomal evolution, the divergence of animal karyotypes during speciation was mainly mediated by para-and pericentric inversions, altering the gene orders in linkage groups (Dobzhansky 1970, White 1977, King 1993, Zdobnov et al. 2002. The other types of chromosomal rearrangements (translocations, fusions, duplications) play an additional role in rearrangements of the linear structure of genome. Alteration of the gene orders in chromosomes during evolution can be visualized in Diptera, which possess polytene chromosomes with distinct banding sequences. The bands of polytene chromosomes, which form species-specific banding sequences, are considered as genetic markers to analyze divergence patterns of the linear genome structure during evolution. The use of the number of chromosomal breakpoints as a divergence measure provided establishment of phylogenetic relationships between species (Kiknadze et al. 2008). Species of the genus Chironomus have four giant chromosomes with seven chromosome arms (A-G). Based on the different combination of the arms, caused by whole-arm translocations, the Chironomus species are grouped into several cytocomplexes (Keyl 1962, Wülker 1980. Cytocomplex is not a taxonomic term. It includes the species with definite chromosome arms combinations, but not similar morphologically. Comparison of banding sequences between species from different cytocomplexes have shown that karyotypes can include species-specific sequences and so called basic sequences, common to more than one cytocomplex and in more than one continent. Such basic sequences were probably present before the separation of species and cytocomplexes (Keyl 1962, Wülker 1980. By global analysis of banding sequences in Eurasia, North and South America, Australia, we have traced banding sequence changes during Chironomus species divergence and continent dispersal (Martin et al. 1974, Wülker 1980, Kiknadze at al. 2003, 2008. It was shown that in Eurasia, North America, and Australia, banding sequence pools of many species were represented mainly by endemic continent-specific sequences. However, basic sequences, common for different continents were also found in karyotypes of some species in addition to the endemic sequences. Such basic sequences were noted also in two African Chironomus species (Ch. alluaudi, and Ch. sp. Nakuru) (Martin 1979, Wülker 1980. It was of interest to study how often such basic sequences can be found among African species. However, the data on Chironomus karyotypes in Africa are very scanty despite there being much information on the morphology of African chironomids. Wülker (1980) has presented photographs of seven chromosome arms of Ch. alluaudi; Martin (1979) has quoted the arm F banding sequence of Ch. transvaalensis;   The presence of further basic banding sequences in the karyotypes of African Chironomus species was discovered, along with endemic continent-specific (Ethiopian region) sequences.

Material and methods
Forth instar larvae of African Chironomus species were used for karyotype study. 35 years ago, one of us (W.W.) had the opportunity to visit Kenya (22.12.1975Kenya (22.12. -16.01.1976. From a base at the house of relatives in Nairobi, he went with family (wife and 3 sons) to collect chironomids to the west to Lake Nakuru and Lake Victoria, to the north to Abrader Mount Ca. 3000 m above N.N., and to the southeast to Tsavo National park, Mombasa and vicinity. Other material was contributed by colleagues: Mount Elgon and Lake Naivasha (Peter N. Cox), Mount Kenya, near 4350 m (scientific excursion of University Erlangen, Germany, under Prof. Dr. Rüppell), Zigi River, Tanzania (Dr. J. Grunewald). The list of collection sites of Chironomus larvae is presented in Table 1. We have not identified species Ch. sp. Nakuru and Ch. sp Kisumu, but the study of the banding sequences of their karyotypes was very important for purpose of our paper.
Larvae were fixed in ethanol-glacial acetic acid (3:1). The technique of chromosome preparation was as usual (Keyl, 1962). The identification of chromosome banding sequences follows by Keyl (1962) for arms A, E, and F, and by Dévai et. al., (1989) for arms C and D.
To trace the relationship of African Chironomus banding sequences with sequences from other continents, we compared them with known basic sequences; if basic sequences for some of species were unknown, we compared them with Chironomus piger standard (ST).
We have pointed to previous literature on morphological characteristics of species studied at the beginning of each species description. Most part of the material (larvae, pupa, adults and karyotype slides) is now deposited in Zoologische Staatssammlungen in Münich (Germany).
Equipment of the Center of Microscopy Analysis of Biological Objects of SB RAS in the Institute of Cytology and Genetics (Novosibirsk) was used in accomplishment of this work: microscope "Axiokop" 2 Plus, CCD camera AxioCam HRc, software package AxioVision 4 (Zeiss, Germany). Kieffer, 1913 http://species-id.net/wiki/Chironomus_alluaudi Previous reports: Kieffer 1913, imago. Freeman 1957, imago. Wülker 1980, photo of arms A-G Wülker et al. 1989, phylogenetic position Kiknadze et al. 2004, list of banding sequences of arms A, C, D, E, and F Karyotype (Fig. 1a). Haploid number n=4, arm combination AE CD BF G ("pseudothummi" cytocomplex), centromere bands not heterochromatinized, nucleolus in arm G (terminal), at least 3 Balbiani Rings (BRs) on arm G, inversion polymorphism in arms C and G.

Chironomus alluaudi
Banding sequences ( Fig. 1b-g) Arm A (Fig. 1b) has the sequence all A1 identical with the main sequence of arm A found in many Chironomus species (Ch. holomelas Keyl, 1961, Ch. melanescens Keyl, 1961 and it is considered a cosmopolitan basic sequence (holA1). Arm E (Fig. 1, c, 7, b) has the sequence allE1 identical with Chironomus piger ST (cosmopolitan basic sequence). allE1 1a -13g Arm C (Fig. 1, d) has two sequences, allC1 and allC2, differing by a simple inversion. The sequence allC1 differs greatly from the basic sequence in arm C; therefore we have compared it with Chironomus piger ST: differing by seven inversion steps from pigST: Arm D (Fig. 1, e) has single sequence allD1 differing by one inversion step from pigST: Arm B (Fig. 1, a) not mapped, monomorphic. The common BR is not developed.  Arm F (Fig. 1, f ) has the sequence allF1, identical with pigST (cosmopolitan basic sequence).
allF1 1a-23f Arm G (Figs 1, a, g) not mapped, has two sequences allG1 and allG2 differing by one simple inversion in the central part of arm G, including two of the Balbiani rings.
In total, the banding sequence pool of Ch. alluaudi contains 9 sequences. Six of them endemic for Africa (Ethiopian sequences), three of them (allA1, allE1, allF1) belong to the category of cosmopolitan basic sequences. Ch. alluaudi can be considered as a Chironomus species with a primitive karyotype (Wülker, 1980(Wülker, , 2010. Larva: "thummi-type" (no tubuli laterales) on abdominal segment VII). Mentum with high lateral tooth, median tooth as in other Chironomus species, pectin epipharyngis about 11 teeth, antenna black with 4 segments, paralabial plates about 40 striae.
Arm A (Fig. 2, b) has the sequence trvA1, differing by only one inversion step from the basic sequence holA1.
Arm F (Fig. 2, f ) has the banding sequence trvF1 differing from cosmopolitan basic pigST by three inversion steps.
In total, the banding sequence pool of Ch. transvaalensis contains 10 sequences, all of them are Ethiopian endemic sequences.

Chironomus sp. Nakuru
Previous report: Wülker, 1980, banding pattern of arms A, E, and F. This species was not identified as well as Ch. sp. Kisumu because there was no additional possibility to collect larvae for rearing. However, the study of Ch. sp. Nakuru karyotype was very important for comparative analysis of Ethiopian Chironomus banding sequences with Chironomus sequences of the other continents.
Arm F (Fig. 3, d) has the banding sequence nakF1 formed by four inversion steps from pigST.
The arm F of Chironomus sp. Nakuru has a nucleolus in region 17-19.
Arm G (Fig. 3, a) has the banding sequence nakG1. It differs from the most of Chironomus species arm G by numerous Balbiani rings. It is possible to suggest that some of them can be nucleoli. But it is often impossible to differentiate nucleoli and Balbiani rings without electron microscopy or in situ hybridization.
In total, seven banding sequences are found in sequence pool of Ch. sp. Nakuru, six chromosomal arms have Ethiopian endemic sequences, and one arm (A) a cosmopolitan basic sequence. Larva: long tubuli laterales at abdominal segment VII, extremely long antenna, gula light, no dark stripe on clypeus.
The association to this species is based on one male adult from the collecting sites of the larvae. Karyotype (Fig. 5, a). Haploid number n=3, arm combination AB CD FEG (modified "thummi" cytocomplex), centromeric bands not heterochromatinized, nucleolus in arm F (at the very telomeric end) and nucleolus-like bodies at the ends of arms A, B, E; Balbiani rings are in arms G and B. Chromosomal polymorphism in arm C (Fig. 5, a).
Arm C (Fig. 5, a) not mapped. It has two banding sequences pulC1 and pulC2 differing by a simple inversion, which involved practically the whole central part of arm C.
The characteristic of arm F in Ch. prope pulcher is the presence of the nucleolus at the telomeric end, which is a rare event among Chironomus species.
Arm G (Fig. 5, e) is joined with arm E. There is large Balbiani ring near the site of fusion, and a small Balbiani ring or puff in the center of arm G. A small nucleolus is possibly developed at the telomeric end of arm G.
In total, eight banding sequences were recorded in the Ch. prope pulcher banding sequence pool. All of them are endemic for Ethiopia. There are no basic sequences. Larva: long tubuli laterales on abdominal segment VII. Other characters - Dejoux, 1968.
Distribution: two pools within a short distance, River Athi south of Nairobi, Kenya.
Arm B (Fig. 6, a) not mapped. It has one sequence -kisB1. The common BR is well developed.
Arm F (Fig. 6, f ) has the sequence kisF1. It was mapped only fragmentarily because of complex inversions in comparison with Chironomus piger ST. The presence of a large Balbiani ring situated just near the centromeric band is a characteristic of arm F in the Ch. sp. Kisumu karyotype. There is pericentric inversion in the chromosome BF (Fig. 6, a, f ).
Arm G (Fig. 6, a) is longer than usual in Chironomus species. There is a nucleolus and four Balbiani Rings on arm G. One of Balbiani Rings, noted by the black dot in Fig. 6, a, was developed only in some cells of the salivary gland cells.
In total, seven Ethiopian endemic banding sequences are found in the sequence pool of Ch. sp. Kisumu. All these sequences differ from Ch. parathummi Keyl, 1961 sequences. Larva: long tubuli laterales on abdominal segment VII. Distribution: near Victoria lake, Kenya.

Discussion
Karyotypes of six African Chironomus species were studied. Four of these karyotypes were described for the first time (Ch. sp. Nakuru, Ch. formosipennis, Ch. prope pulcher, Ch. sp. Kisumu). Detailed photomaps of arms A, C, D, E, and F were presented, also for the first time, for Ch. alluaudi, Ch. transvaalensis, and Ch. sp. Nakuru. Among the species studied, three species (Ch. transvaalensis, Ch. prope pulcher, Ch. sp. Kisumu) have only endemic Ethiopian banding sequences in their karyotypes, while cosmopolitan basic banding sequences were discovered in the karyotypes of the other species, along with endemic sequences (Ch. alluaudi, Ch. sp. Nakuru, Ch. formosipennis). The presence of these basic sequences indicates a relationship of African Chironomus species to Chironomus species from other continents before their separation (Kiknadze et al. 2008).
The results on African species are relevant the problem whether or not the chromosome arm combination of the "thummi" cytocomplex is rare in Southern continents. At the moment, one species in South America (Chironomus sp. Las Brisas, Wülker, Morath, 1989), one species in India (Ch. javanus Kieffer, 1924), and two species in Australia (Ch. javanus, Ch. queenslandicus Martin, 2005) are known to have this "thummi" cytocomplex chromosome arm combination (Martin 2010, Martin, pers. comm. and this paper).
Earlier it was demonstrated (Wülker 1980), that the presence of basic sequences in arms A, E, F of some Chironomus species of the "thummi" and "pseudothummi" cytocomplexes supports an idea that the basic sequences existed in hypothetical stem species before the separation of the complexes. The results of this paper contribute to the understanding of chromosome arms C and D in phylogeny in both cytocomplexes, in addition to data on arms A, E and F published earlier (Wülker 1980, Kiknadze et al. 2008. Keyl (1962) established the hypothesis, that "the hypothetical species, which crossed the border between "thummi" and "pseudothummi" cytocomplexes" had most probably three banding patterns in arm E (in Keyl's terms): standard as Ch. piger Strenzke, 1959, pattern as Ch. aprilinus Meigen, 1838 and others, pattern as Ch. aberratus Keyl,1961 and others. We can ask, whether these three patterns are known today in both cytocomplexes. This is indeed so (Fig. 7, a) with the exception of the fact that Relations of recent species and hypothetical "basic" species before separation of the cytocomplexes in arm E (a), arm C (b), and arm D (c). The data of Keyl (1962) and Kiknadze (unpublished) were also used. Dots -inversion steps between banding sequences; st -piger standard after Keyl (1962) and Dévai et al. (1989). all -alluaudi, atr -atrella, apr -aprilinus, abe -aberratus, cra -crassicaudatus, fro -frommeri, hol -holomelas, hpi -heteropilicornis, kii -kiiensis, pil -pilicornis, plu -plumosus, pst -pseudothummi, sax -saxatilis, trv -transvaalensis. the pattern of Ch. aberratus itself is not known in the "pseudothummi" cytocomplex, but there are the sequences trvE1 and nakE1 which differ only by 1-2 inversions from abeE (Fig. 7, a).
In arms C and D, an accumulation of species with the identical sequences was previously observed only in the "thummi" cytocomplex (Wülker, 2010). With the data of this paper we can propose that chromosome arms C and D had also two patterns before separation of the cytocomplexes (Chironomus piger ST sequence sensu Keyl, 1962 and basic pattern sensu Wülker, 1980Wülker, , 2010. Fig. 7, b shows that pattern ST and basic themselves are not found in "pseudothummi" cytocomplex (question marks in Fig. 7, b), but there are several species, which have banding patterns differing only by a few inversions from ST and basic. The African species (Ch. alluaudi, Ch. transvaalensis, Ch. sp. Nakuru, Ch. formosipennis) play an important role in the development of the arm C and D phylogeny. Fig. 7, c demonstrates that there are ST and basic patterns in the 'thummi' cytocomplex, but only patterns close to ST were found in the "pseudothummi" cytocomplex: allD1 only by one, yosD1 by two, nakD1 by three, and trvD1 by four inversions from ST.
A great peculiarity of some African Chironomus karyotypes is the presence of large numbers of functionally active chromosome sites, especially Balbiani rings. For example 5 BRs were found in Ch. transvaalensis (Figs 2, a, 2, g-i), 6 BRs in Ch. sp. Nakuru (Fig. 3, a). Most Chironomus species have two or three visible BRs since e.g. many species have the gene for BR4 but do not express it, and the number seen may also vary with developmental stage.