Identification of Chironomus ( Chironomus ) melanescens Keyl , 1962 in North America

Chironomus melanescens was originally described by Keyl (1962) on the basis of the morphology of polytene chromosome arms A, E, and F in populations from Germany. Keyl ascribed the name to Strenzke, but although Strenzke collected and reared the specimens, he died before he was able to describe them. Keyl also had used the name in a 1961 paper but there was no information that could be considered a species description (i.e. it was a nomen nudum). The type localities in Keyl (1962) are given as Duemer Lake and a pool south of Clauthal-Zellerfeld. Wülker et al. (1981) quoted a personal communication from Keyl in which he nominated the type specimen as slide S1149 B2 (misread as S1149 82), and giving the locality as Harz, Acker, which is a loose reference to Clauthal-Zellerfield. The latter paper gave a more full description of the chromosomes as well as some information about the adult male and the larva of material from Germany and Switzerland, although specifically aimed at distinguishing C. melanescens from the closely related C. holomelas Keyl, 1962 and C. saxatilis Wülker et al., 1981. Kiknadze et al. (1991) illustrated the larva and redescribed the cytology from Russian populations.


Jon Martin
Genetics, Genomics & Development, School of Biosciences, University of Melbourne VIC 3010, Australia.E-mail: j.martin@unimelb.edu.auChironomus melanescens was originally described by Keyl (1962) on the basis of the morphology of polytene chromosome arms A, E, and F in populations from Germany.Keyl ascribed the name to Strenzke, but although Strenzke collected and reared the specimens, he died before he was able to describe them.Keyl also had used the name in a 1961 paper but there was no information that could be considered a species description (i.e. it was a nomen nudum).The type localities in Keyl (1962) are given as Duemer Lake and a pool south of Clauthal-Zellerfeld. Wülker et al. (1981) quoted a personal communication from Keyl in which he nominated the type specimen as slide S1149 B2 (misread as S1149 82), and giving the locality as Harz, Acker, which is a loose reference to Clauthal-Zellerfield. The latter paper gave a more full description of the chromosomes as well as some information about the adult male and the larva of material from Germany and Switzerland, although specifically aimed at distinguishing C. melanescens from the closely related C. holomelas Keyl, 1962and C. saxatilis Wülker et al., 1981. Kiknadze et al. (1991) illustrated the larva and redescribed the cytology from Russian populations.
Larvae from Ontario and Wisconsin, labelled as 'Species e' in Martin (2015) were found to belong to the pseudothummi-cytocomplex, which is uncommon in North America.Analysis of the mitochondrial cytochrome c oxidase subunit 1 (COI) barcode sequence from two larvae indicated that they differed by only 2.6-3.4% from the European C. melanescens sequence of Guryev et al. (2001) (GenBank accession number AF 192204).The conspecificity of the specimens from the two regions was confirmed by a comparison of the available morphological data and the banding patterns of the polytene chromosomes.
The purpose of this note is to provide information on North American specimens, compared to Palearctic descriptions, which are in German or Russian, so they will be more accessible for North American workers.Terminology generally follows Saether (1980), larval characters essentially as Proulx et al. (2013).VMR is the ratio of the anterior marginal band of the ventromentum to the distance to the base of the striae (X/Y in Fig. 3d).

Description
Adult male: Some adults were also collected and some are in the collection of J.E. Sublette in the museum of the University of Minnesota, for which limited data was obtained (e.g.hypopygium, Fig. 1).However, one reared male from Wisconsin was available for study and details are listed here with comparison to characters of Palearctic specimens (in brackets) where these are available from Wülker et al. (1981).
About 51 recurved hooks on posterior margin of tergite II, central hooks with a small spine dorsally; hook row approximately half of width of the segment.Light shagreen pattern particularly near the centerline towards the rear of the segments, small adhesion marks on segments I-III; obvious pedes spurii B on segment III and large pedes spurii A on segment IV; posterolateral spur of segment VIII with 1 -2 spines (Fig. 1c).
Hair fringe on each side of the anal lobe with about 75 filamentous setae.
Gular region pale to slightly dark on posterior third, frontoclypeus pale to slightly darkened.Mentum (Fig. 3c) with pointed teeth; 4th laterals hardly reduced (type I); c1 tooth long and narrow with c2 teeth well separated (type III).Ventromental plate (Fig. 3d) with about 37 -43 not very obvious striae; VMR about 0.35-0.41 of distance to base of striae.Pecten epipharyngis (Fig. 3a) with about 13 -16 moderately broad sharp teeth, although larvae from the Clarence Creek population had the pectin epipharyngis and its teeth somewhat deformed.Premandible (Fig. 3b) with teeth about equally long, unless outer more worn, inner tooth about 1.6-2.3times the width of outer tooth.Antenna (Fig. 3e) with relatively long, narrow basal segment, about 4 times as long as wide, with ring organ between a third and half way up from base; AR about 1.88 -2.3; ratio of segments (in µm) about 183:43:13:15:9.Mandible (Fig. 3f) with 3rd inner tooth only slightly darkened and partly to nearly completely separated (type II-IIIB), and with about 11 -14 furrows on the outer surface at the base.Ventral tubules of Wisconsin specimens were much longer than those from Ontario.

Conclusions
The morphology and polytene chromosome patterns confirm that the North American taxon provisionally called "species e" is conspecific with C. melanescens.Emphasis in this and previous studies of Palearctic material has been on the larvae.It seems likely that they can be identified by the combination of a medium sized bathophilus type larva with anal tubules over 6x longer than wide; pale or slightly darkened gula and frontoclypeus; a mentum of type I with the central trifid tooth of type III; and a mandible with the third inner tooth partially to completely separated, but only slightly darkened.Other characters may also be useful, but they can be variable.It also cannot be guaranteed that there are no other currently insufficiently studied species whose larvae share this combination of characters.

Figure 2 .
Figure 2. Anal tubule of North American larva.
These larval characters are similar to those shown in the excellent figures inKiknadze et al. (1991) and compatible with the few characters given byWülker et al. (1981) Cytology: Four polytene chromosomes with pseudothummi-cytocomplex arm combination AE, BF, CD, G (Fig.4).Sequences are as in Palearctic populations except in arm B, which is inverted compared with the Palearctic sequence.Polymorphism for simple paracentric inversions is recorded for arms A, C and G, the inversions also being present in the Palearctic.Arm G is generally paired unless heterozygous, with a sub terminal nucleolus and 2 Balbiani rings which vary in position depending on the sequence.No nucleoli occur in the other arms.DNA Barcodes: A COI barcode sequence of Palearctic C. melanescens was published byGuryev et al. (2001) (GenBank accession number AF192204), and at least partial sequences have been obtained from two North American populations (Clarence Creek, Carleton Co., Ontario, Canada (45.50° N, 75.22°W); Arboretum, Madison, Dane Co., Wisconsin (43.08°N, 89.42° W) using the same primers asGuryev et al. (2001).A further 19 sequences from Ontario and one from Nova Scotia, from GenBank and the BOLD database, have been included in the comparison of uncorrected pairwise genetic distance.The 21 Nearctic sequences represented 7 haplotypes which varied by 0.2-1.3%.The distance between the Palearctic sequence and those from the Nearctic range from about 2.6-3.7%.Since the morphology and cytology confirm that all refer to a single species, the COI divergence is regarded as intraspecific variation.Speculation about the significance of the Palearctic/Nearctic difference is best left until data on variation across the Palearctic are available -at present even the locality of the existing specimen is uncertain, although it likely to be from Russia.