The genus Xenochironomus Kieffer has one Holarctic species, Xenochironomus xenolabis (Kieffer, 1916), and several purportedly undescribed species in the eastern Palaearctic (Pinder and Reiss, 1983). For those species with known immature stages, the larvae typically occur in, or are otherwise associated with, freshwater sponges. Adult males conforming well to X. xenolabis (as in Townes, 1945) have been collected sporadically in light traps near localities in Kansas where sponge colonies are known to occur, and larvae conforming well to X. xenolabis have been retrieved from sponges (Ferrington, 1987); however, one badly damaged male was collected with a sweep net near the outlet at the dam breastworks of Kanopolis Lake in Kansas that differs from X. xenolabis in terms of superior volsella structure, shape and proportional size of anal point (Ferrington, personal observation). This adult likely represents a new species of the genus; however, a satisfactory description cannot be developed with this damaged specimen, and no other specimens similar to it have been collected elsewhere in Kansas. Although no larvae have been collected at this locality, larvae collected at other two localities in Kansas, which differ in both karyotype and larval morphology from X. xenolabis, are described here.

It is well known that species of the family Chironomidae possess polytene chromosomes which can be used to provide characteristics for studying both taxonomy and phylogeny. Butler et al. (1999) and Kiknadze et al. (2000) analyzed banding patterns of the polytene chromosomes of some chironomid species from Palaearctic and Nearctic locations, which showed strong karyotype divergence between populations on the two continents. In addition, Kiknadze et al. (1996) found that three North American populations previously considered to be Camptochironomus tentans Fabricius differ strongly from European and Siberian populations in both banding patterns and localization of nucleoli. These cytogenetic differences, together with some subtle but consistent morphological characteristics of the Nearctic populations, justified them being described as the new Nearctic species Camptochironomus dilutes Shobanov, Kiknadze and Butler (Shobanov et al., 1999).

The first karyotype described for the genus was of X. xenolabis by Belyanina and Durnova (2002) from material collected in Russia. They constructed a detailed and comprehensive chromosome map, and documented that this species has a chromosome set of 2n = 8, with seven chromosome arms jointed in chromocentre. Durnova (2009) subsequently described another species from Russia, Xenochironomus sp., which differs from X. xenolabis by banding patterns and larval morphology. This second species also has 2n = 8 and different degrees of stability relative to what was previously observed in the chromosomes of X. xenolabis.

Our analysis of salivary gland chromosomes and morphology of larvae collected from two localities in Kansas shows substantive differences from Xenochironomus sp. and X. xenolabis from Russia. Consequently, the goal of this contribution is: (1) to describe the larval morphology; (2) to present a detailed description of salivary gland chromosomes and make a chromosome map of the species of the larvae; and (3) to present a comparative karyological analysis with Xenochironomus sp. from Russia and X. xenolabis.

Material and Methods

The larval material was collected from two different localities in Kansas, USA: Cedar Creek, south of K-10 Highway, Johnson County (27.IX.1991) and Soldier Creek, Jackson County (15.VIII.1984). Combined, these collections consist of 35 larvae belonging to the genus Xenochironomus (Pinder and Reiss, 1983). Larvae used for this study were fixed immediately upon collection in a 3:1 solution of ethyl alcohol (96%): glacial acetic acid. For cytogenetic analysis we only used IV instar larvae (17 larvae). From each larva, separate preparations were produced of the larval head capsule, and of salivary gland chromosomes (thorax) and chromosomes from tissues of developing gonads (abdomen). The chromosomes of salivary glands and gonads have been examined by applying routine acet-orcein staining (Michailova, 1989). The slides of larval parts used to describe diagnostic morphology, and all slides of chromosome preparations, were deposited in the collections of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences. Identification to genus, followed by determination of the species, has been done using the external larval morphology (Pankratova, 1983, Pinder and Reiss, 1983). The names of chromosome arms are given on the basis of the comparative karyological analysis with respect to the chromosome map of X. xenolabis (Belyanina and Durnova, 2002), and confirmed using some common band patterns with chromosome arms of the Russian species, which are indicated in figures by an asterisk. In the chromosome map for this North American species, every chromosome was conditionally divided into sections, and then the morphology of the chromosomes was established by the morphology of the metaphase chromosomes. Chromosome aberrations found in the species have been calculated and are reported as percentages.

Results

Larval Morphology

Larva: The IV instar is of moderate length, about 7–8 mm and red color.

Mentum (Fig. 1a): dark brown with central median tooth, but lacking smaller, recessed lateral pair of teeth adjacent to central tooth. Seven pairs of lateral teeth: first pair large, light, higher than the median tooth, second pair very small, sometimes not seen, remaining pairs consistently decreasing in size. The outermost pair of lateral teeth light brown. In a few larvae the mentum is slightly worn or damaged and the characteristics of the outermost teeth are difficult to interpret. Ventromental plates are broad anteriorly, with edges and striae radiating from common base posteriorly. S1, S2, S3 are very short and simple. Premandible without inner teeth and reduced.

Fig. 1. 

Larval morphology of Xenochironomus sp. a. Mentum – the arrow indicates the location of the small tooth; b. Mandible; c. Antenna. Bar 100 µm

Mandible (Fig. 1b): Dark, strong apical tooth, and three small, light brown inner teeth. Seta subdentalis simple, long, and reaching to the basal part of the apical mandibular tooth. It is elongated and looks like a “point of sword” toward the apex.

Antenna (Fig. 1c): Composed of 5 segments. Ring organ located in basal half of first segment. Lauterborn organs on segment 2. Flagellum is long and ends at the beginning of the fourth segment.

Larval body: Lateral and ventral tubules absent.

Differential characteristics: The larva has features similar to the other two species, Xenochironomus sp. and X. xenolabis from Russia, but in different combinations. In the studied species, the small lateral teeth adjacent to the central tooth are lacking, as recorded for X. xenolabis (Fig. 2a, Durnova, 2009), but are present in Xenochironomus sp. from Russia (Fig. 2b, Durnova, 2009). The length of the flagellum of the antenna reaches the beginning of fourth segment of the antenna. In Xenochironomus sp. (Russia), it reaches the end of third segment (Fig. 2f, Durnova 2009), but in X. xenolabis it reaches the proximal half of the fourth segment (Fig. 2e, Durnova, 2009).

Fig. 2. 

Salivary gland chromosomes of Xenochironomus sp. a. Chromosome arm A + chromosome arm G with a heterozygous inversion in section 7; a’ Metaphase chromosomes; p – a puff; * - the band patterns similar to that of X. xenolabis: sections 11–13 of arm A are similar to those of 20–23 in X. xenolabis. b. Chromosome arm B; * - the band patterns similar to that of X. xenolabis: sections 10 of arm B are similar to those of 16–18 in X. xenolabis. The arrows indicate the localization of the centromeres which are combined in chromocentre. Bar: 100 µm

Ecology and distribution: Larvae of Xenochironomus xenolabis are obligate miners in freshwater sponges in both standing and flowing water (Pinder and Reiss, 1983) and are encountered in several species of sponges (Roback, 1968; Ferrington, 1987). The larvae of this new North American species also were only collected within sponges, and are likely to be obligate miners like X. xenolabis. Habitat at the collection locality in Cedar Creek (Johnson County) consisted of riffle with large broken layers of bedrock and smaller cobble. Substrate in riffle areas of Soldier Creek was mostly coarse gravels to small sand. Larvae at both collection locations were only found within the tissues of freshwater sponges. The sponge colonies at Cedar Creek were dense and large, some exceeding 30 cm in longest dimension, whereas the sponge colonies in Soldier Creek were smaller and less abundant. At both sites the sponge colonies only occurred on or under rocks in riffles.

Karyotype

The chromosome set is 2n = 8. There are seven chromosome arms: A, B, C, D, E, F, G. Three chromosomes are metacentric and one is telocentric (Fig. 2a′). Very rarely the chromosomes can be shown individually because the chromosome arms are combined either in chromocentre (Fig. 4) or they are in different arm combinations: AB or CA (Fig. 6c).

Fig. 4. 

Arms A B C D E F G jointed in chromocentre (NOR- Nucleolar Organizer; BR- Balbiani ring). Bar 100 µm

Fig. 5. 

Complex heterozygous inversions. a. Heterozygous inversions in arm A, sections 8-10 and arm B, sections 6-10; b. Heterozygous inversion in arm C, sections 6-8; c. Heterozygous inversion in arm D, sections 8-11. bar 100 µm

Fig. 6. 

Complex heterozygous inversions and arm combinations. a. Heterozygous inversion in arm E, sections 9-12; b. Heterozygous inversion in arm F, sections 8-16. c. Association of arm C with arm A. Bar 100 µm. NOR – Nucleolar Organizer; BR- Balbiani ring

Arm A (Fig. 2a): This arm is divided into 14 sections. As in X. xenolabis the telomere of this arm is active and the band sequences in sections 11–13 are similar to those in sections 20–21 and 22–23 of X. xenolabis respectively. Markers of this arm are the puff near the telomere and the band sequences in sections 4, 5, 6, 7. Especially conspicuous diagnostic markers are the groups of double bands after an active region in section 4 and before the puff in section 7. A complex heterozygous inversion occurred at the telomere, sections 8–10 in 47% of karyotypes examined (Fig. 5a).

Arm B (Fig. 2b): The arm is divided into 13 sections. At the telomere there is a darkband, which serves as a marker of the arm. In all studied individuals, near this band there is a puff. The bands in section 10 are similar to those of section 16–18 of X. xenolabis. The telomere of the arm B is similar to the telomere of arm B of X. xenolabis. The dark bands in sections 5, surrounded by dark bands on both sides, are characteristic of this arm. A complex heterozygous inversion was found in sections 6–10 in 41% of karyotypes analyzed (Fig. 5a).

Arm C (Fig. 3a): This arm has 17 sections. A Nucleolar Organizer (NOR) near the chromocentre and Balbiani Ring (BR), and located in section 12, are specific for this arm. Bands in sections 1–2 and 15 correspond to those of 3 and 24–25 of X. xenolabis. This arm has a constriction (section 8) similar to one occurring in section 18 of X. xenolabis and in Xenochironomus sp. (Durnova, 2009). At the telomere there is a complex heterozygous inversion in sections 6–8 in 82% of karyotypes analyzed (Fig. 5b).

Fig. 3. 

Salivary gland chromosomes of Xenochironomus sp. a. Chromosome arm C; b. Chromosome arm D; c. Chromosome arms EF; d. Chromosome arm F. NOR – Nucleolar Organizer; BR- Balbiani ring, p – a puff; * - the band patterns similar to that of X. xenolabis: sections 1-2, 15 of arm C are similar to those of 3, 24-25 in X. xenolabis. Section 13 in arm D is similar to sections 24-25 of X. xenolabis. Section 15 of arm E is similar to those of 1-4 in X. xenolabis. The constriction and active region in section 12 of arm F can be seen in X. xenolabis, and in sections 6-11 in the same arm of Xenochironomus sp. from Russia. The arrows indicate the localization of the centromeres which are combined in chromocentre. The small arrow indicates the localization of somatic heterozygous inversion, section 7 in arm F. Scale bar 100 µm

Arm D (Fig. 3b): This arm is divided into 14 sections. The constriction in section 13 is similar to that of section 24-25 of X. xenolabis. The arm can be recognized by the band patterns in sections 1, 2, and 4. A useful marker is also the active region in section 5. A complex heterozygous inversion can be seen at the telomere, sections 8–11 in 65% of karyotypes analyzed (Fig. 5c).

Arm E (Fig. 3c): This arm has 15 sections. Band patterns in section 15 are similar to those of sections 1-4 of X. xenolabis. The bands in sections 6, 9, 10, 13 are good markers for this arm. A complex heterozygous inversion in this arm occurred in sections 9–12 in 35% of karyotypes (Fig. 6a).

Arm F (Fig. 3d): This arm is divided into 16 sections. The active region in section 12 and the constriction near it are well seen in X. xenolabis (6-11) and in the same chromosome arm of Xenochironomus sp. (Durnova, 2009). Markers of the arm are the dark bands at the telomere (16) and two bands in section 8. A complex heterozygous inversion appears in sections 8-16 in 41% of karyotypes examined (Fig. 6b).

Arm G (Fig.2a): Nine sections can be seen in this arm. One BR occurs near the chromocentre. The band patterns in section 3 are similar, but in inverted position, in comparison with sections 3-4 of X. xenolabis (Belyanina and Durnova, 2002). The two dark bands in section 7 are very similar to those in section 19 on arm G of X. xenolabis. There is a complex heterozygous inversion in section 7 which occurred in a high frequency (94%) of karyotypes examined (Fig. 2a).

This is a very polymorphic species, and no individuals with homokaryotype were found among the larvae examined. There are complex heterozygous inversions observed in all arms of the chromosomes and two types of somatic inversions (as reported in Sella et al., 2004) affecting a few cells of individual larvae occurred in arms D and F at section 7 (Fig. 3c).

Discussion

All species of the genus Xenochironomus studied cytogenetically show common cytogenetic characteristics of the 2n = 8 and some shared common band sequences in the polytene chromosomes. As reported for the other two species of Xenochironomus (X. xenolabis and Xenochironomus sp. from Russia), this North American species has the chromosomes very often combined in the chromocentre, and sometimes different arm combinations can be observed. The appearance of a chromocentre is a rare event in the family Chironomidae and is considered by some authors (e.g., Stegney, 1993) as a primitive state. The formation of a chromocentre has only been documented in few species of the subfamilies Chironominae, Prodiamesinae and Orthocladiinae (Michailova, 1989; Jablonska-Barna et al., 2013). It could be suggested that the chromocentre in the polytene chromosomes has been formed as a result of a number of ectopic contacts arising between separate replicates of mobile genetic elements in the precentromere regions of the chromosomes. Also, it is known that associated regions have similar and highly repeated AT–rich DNA sequences (Michailova, 1989).

In the studied species we found several levels of cytogenetic changes. The first level of cytogenetic changes are species-specific banding patterns and fixed homozygous inversions which occur in all studied individuals. The common banding sequences among X. xenolabis and Xenochironomus sp. from Russia and this species have different positions in the polytene chromosomes which might have resulted through many steps of homozygous inversions. They indicate differences in the karyotype which are well known as markers of species divergence (Keyl, 1962). On the basis of karyotypic differences and specific morphological characteristics of larvae, this species can be considered as a new Xenochironomus species. It is important to note that some of the same morphological differences in the larval stage have been described for Xenochironomus sp. from Russia (Durnova, 2009); however, this North American species and the Xenochironomus sp. species from Russia are readily distinguished from each other by band patterns of the salivary gland chromosomes, and all three of the species have species–specific band sequences. Additional analysis of adults and/or pupa stages could provide supporting morphological characters to define and help clarify the taxonomic positions of the two species known only from karyotypes.

The second category of cytogenetic changes are the heterozygous inversions which occurred in high frequency in different chromosome arms (between 35–94%), which may reflect the local adaptations to specific environmental conditions.

The third level of karyotype changes are the somatic inversions which affect small regions of the polytene chromosomes, occurred only in few cells, and with lower frequency. These rearrangements very often are associated with some anthropogenic factors (Caceres et al., 1997). As somatic chromosome rearrangements can be caused by different environmental stresses (e.g., trace metals, organic pollutants) (Sella et al., 2004; Michailova et al., 2012) we suggest that these types of somatic alterations observed in the studied species likely indicate the existence of pollution induced stress and as such can be used as biomarkers at the cytogenetic level for assessment of environmental conditions. Although they occurred only in a few cells in the larvae from Kansas, they may indicate genotoxic agents in the water basins which have affected the genome of this new species.

The comparative cytogenetic analysis of the new species from Kansas with respect to Xenochironomus sp. (Durnova, 2009) and X. xenolabis (Belyanina and Durnova, 2002) shows banding sequences in common in arms C (section 8) and F (section 12). In the genus Chironomus, Wűlker (1980) also established similar common band patterns in more than one of the cytocomplexes, which he considered plesiomorphic. It is quite possible that the above observed common banding patterns found in the three species studied cytologically also should be interpreted as plesiomorphic and, consequently, present in these species before their separations into distinct cytospecies. Further cytogenetical studies of other still undescribed species of Xenochironomus will be necessary to confirm this interpretation.

Another interesting contrast between the Russian and North American Xenochironomus species relates to the extent of chromosome polymorphism. Xenochironomus xenolabis is monomorphic (Belyanina and Durnova, 2002), but in the Russian species of Xenochironomus sp. a few larvae with small heterozygous inversions were discovered (Durnova, 2009). By contrast, this new North American species is polymorphic, with complex heterozygous inversions observed in all arms.

The studied North American species is here interpreted as a new species because it has small, but specific, larval external morphological differences, as well as species-specific banding sequences. It is also possible that the larvae correspond to the adult collected at the outlet of Kanopolis Lake; however, further field work to collect and rear larvae to the adult would be necessary to confirm this presumption. Studies on all life history stages of the species would be preferred in order to provide a standard, comprehensive taxonomic description and formal name for this species from Kansas.