A review of Norwegian Gymnometriocnemus (Diptera, Chironomidae) including the description of two new species and a new name for Gymnometriocnemus volitans (Goetghebuer) sensu Brundin

Abstract Examination of the syntypes of Metriocnemus volitans Goetghebuer, 1940 revealed that these specimens belong to the genus Chaetocladius and are not con-specific with Gymnometriocnemus volitans (Goetghebuer, 1940) sensu Brundin (1956) and Sæther (1983). A literature search showed that Gymnometriocnemus kamimegavirgus Sasa & Hirabayashi, 1993 fits well with the species figured and diagnosed by Brundin (1956) as well as with specimens of this species from Norway. We present arguments for Chaetocladius volitans (Goetghebuer) comb. n. and for the use of Gymnometriocnemus kamimegavirgus for Gymnometriocnemus volitans sensu Brundin. In addition, we provide DNA barcode data that indicate the presence of at least seven Gymnometriocnemus species in Norway of which six are collected as male adults. Two of these, Gymnometriocnemus (Gymnometriocnemus) pallidus sp. n. and Gymnometriocnemus (Raphidocladius) autumnalis sp. n. are regarded as new to science and diagnosed based on adult male morphology and DNA barcodes. The species Gymnometriocnemus (Gymnometriocnemus) marionensis Sæther, 1969 is re-established and a key to all Holarctic species is provided.


Introduction
The orthoclad genus Gymnometriocnemus was suggested by Goetghebuer (1932), but without designation of a type species. Edwards (1932) designated Gymnometriocnemus subnudus (Edwards, 1929) as type species and made the name available according to the International Code of Zoological Nomenclature. Edwards is thus credited authorship of the genus (Spies and Saether 2004). The genus was revised by Saether (1983) who distinguished two subgenera based on adult male and pupal morphology. There currently are 15 Gymnometriocnemus species recognized (Ashe and O'Connor 2012). Larvae of most species have previously been regarded as terrestrial (Andersen et al. 2013), but there is evidence for at least semi-aquatic lifestyle in larvae of subgenus Raphidocladius from Norway (own data).
Metriocnemus volitans was described by Goetghebuer (1940) based on material collected in Abisko, Sweden by Thienemann in 1939. The species was placed in genus Gymnometriocnemus by Brundin (1956), probably without consulting the type material. Since then, chironomid workers have used Brundin's interpretation of this species and even Saether (1983) in his revision of Holarctic Gymnometriocnemus relied on the characters presented by Brundin (1956). He stated explicitly, however, that he had not examined Goetghebuer's types. Saether (1983) erected the subgenus Raphidocladius for Gymnometriocnemus species possessing an extremely long virga with needle-like sclerotization in the adult males. The species G. brumalis (Edwards, 1929) and G. acigus Saether, 1983 were listed as members of the group, while G. volitans was considered a possible member since immatures were unknown and virga had not been examined (Saether 1983). Two species in this subgenus were later described from Japan, but both Gymnometriocnemus kamimegavirgus Okazawa, 1994 andG. tairaprimus Sasa &Okazawa, 1994 were diagnosed without considering G. volitans sensu Brundin (1956) and Saether (1983) (Sasa and Hirabayashi 1993;Sasa and Okasawa 1994). Cranston and Oliver (1988) argued for a synonymy of the type species for Raphidocladius, G. (R.) acigus, with G. (R.) brumalis claiming that the characters used by Saether (1983) to separate these species were not reliable and interspecifically variable even within the same population. This leaves four valid species in subgenus Raphidocladius in the World Catalogue of Chironomidae: G. Subgenus Gymnometriocnemus is characterised by a short virga and a weakly developed crista dorsalis in the adult male hypopygium (Saether 1983). Cranston and Oliver (1988) synonymized Gymnometriocnemus marionensis Saether, 1983 with G. subnudus, leaving 11 valid species in subgenus Gymnometriocnemus the World Catalogue of Chironomidae: G. (G.) ancudensis (Edwards, 1931), G. (G.) benoiti (Freeman, 1956), G. (G.) brevitarsis (Edwards, 1929), G. (G.) johanasecundus Sasa & Okazawa, 1994, G. (G.) lobifer (Freeman, 1956), G. (G.) longicostalis (Edwards, 1931), G. (G.) subnudus, G. (G.) terrestris Krüger, Thienemann & Goetghebuer, 1941, G. (G.)  DNA barcoding using partial cytochrome c oxidase subunit 1 sequences (COI) (Hebert et al. 2003) has been shown to perform well for species identification of many Chironomidae groups (Brodin et al. 2012;Ekrem et al. 2010;Ekrem et al. 2007;Stur and Ekrem 2011). We have therefore had a strong focus on developing a barcode library of chironomids for use in taxonomy, life stage association and future environmental monitoring in Norway. In connection with biosurveillance projects in central, eastern and northern Norway, we have barcoded adult specimens of Gymnometriocnemus species from various habitats.
The motivation for this study was to clarify the identity of Gymnometriocnemus volitans (Goetghebuer), describe hitherto unknown species of Gymnometriocnemus and to present the DNA barcodes of Norwegian Gymnometriocnemus as a resource for future studies of this genus.

Material and methods
We examined eight syntypes of Metriocnemus volitans Goetghebuer from the Royal Belgian Institute of Natural Sciences (RBINS), five male and three female adults mounted between cellophane strips on two separate pins. Both pins bear the label "Env. d. Abisko, Aout 1939, Dr. Thienemann" and "Metriocnemus volitans n sp" (Figs 1A-B). Thienemann (1941) uses Goetghebuer's name and state that only adults were collected in a groundwater spring area at the beginning of the Njulja road 29.viii.1939.
Additional material of Gymnometriocnemus was collected using a variety of methods in different biosurveillance projects: Malaise traps, sweep netting and fogging of oak canopies (Supplementary file 1). This material is deposited in the NTNU University Museum insect collection (NTNU-VM). One to three legs were dissected off the specimens and submitted to the Canadian Centre for DNA Barcoding. Metadata, photos, sequences and trace-files are available in the Barcode of Life Data Systems (BOLD, www.boldsystems.org) through the dataset DS-GYMNO with doi: 10.5883/ DS-GYMNO. GenBank accessions are given in Supplementary file 1.
DNA extracts and partial COI gene sequences were generated using standard primers and bi-directional Sanger sequencing with BigDye 3.1 termination at the Canadian Centre for DNA Barcoding in Guelph. Protocols and original trace-files are available through the dataset DS-GYMNO in BOLD. Alignments were done on amino acid sequences and was trivial as indels were absent; only sequences > 300bp were used in the final alignment. The taxon ID-tree was generated using neighbour joining analysis and 1000 bootstrap replicates on Kimura 2-parameter (K2P) genetic distances in MEGA 6 (Tamura et al. 2013).
Morphological terminology and abbreviations follow Saether (1980). Antennal and fore leg ratios of Norwegian Gymnometriocnemus are given in Table 1. Measurements are given as ranges followed by the mean. Anal point lengths were measured from posterior margin of anal tergite to tip of anal point.

Remarks.
Several of the type specimens are quite damaged and many characters are difficult or impossible to observe (Figs 1-2). However, it is obvious that they do not belong to Gymnometriocnemus, and the better preserved male specimens show characters that fits the diagnosis of Chaetocladius: wings with coarse punctuation and without macrotrichia on the membrane (Fig. 1D), divergent lateral spinules on the tibial spurs (Fig. 1C) and a fore leg ratio of 0.75. We are confident that this species should be placed in Chaetocladius, but due to the state of the specimens and the unrevised nature of the genus, we have not attempted to compare C. volitans with other species in Chaetocladius. Edwards, 1932 Remarks. We have only seen two females from eastern and central Norway. The two specimens fit Edwards' description for G. brevitarsis and represent the first records of this species in Norway. The wing and antenna are photographed (Fig. 3A, B) and leg ratios of fore-mid-and hind legs are measured to be LR 1 0. partial COI gene sequences do not reveal close relationships to any other species in Gymnometriocnemus. Subgeneric placement of G. brevitarsis is therefore not possible.

Gymnometriocnemus (Gymnometriocnemus) marionensis Saether, 1969
Remarks. Only one specimen of this species from Norway has been available to us. It    Diagnosis. Gymnometriocnemus (G.) pallidus can be separated from other Gymnometriocnemus species by the following combination of characters in the adults: body pale yellow-green; male with short inconspicuous virga, gonostylus with convex outer margin and weakly developed crista dorsalis. Female with setae on most of wing surface, including numerous in cell m; antenna with apical flagellomere pointed and longer than flagellomere 4; genitalia with long rami, about the same length as notum. Description. Male adult (n = 5 unless otherwise stated). Wing length 1.21-1.30, 1.28 mm. Colouration pale yellow-green body, legs and antennae; slightly darker bands on scutum; postnotum, dorsal side of head, ventral part of preepisternum pale brown; eyes dark brown.
Immature stages unknown Remarks. The species is morphologically similar to G. (G.) subnudus and G. (G.) johanasecundus, but paler (see whole specimen figures in BOLD dataset DS-GYMNO). Males and females are almost completely yellow-green with pale brown posterior side of head and postnotum; pale brown scutal bands and ventral side of preepisternum. Gymnometriocnemus (G.) pallidus is also similar to these species in having a short, triangular anal point and a small virga, but the hypopygium of G. (G.) pallidus has a more prominent inferior volsella than G. (G.) johanasecundus and considerably stronger anal tergite setae than G. (G.) subnudus (Figs 4B, C). Comparison with DNA barcode data in BOLD indicates that the species also has records from Germany and France.

Gymnometriocnemus (Gymnometriocnemus) subnudus (Edwards, 1929)
Remarks. We have seen specimens from eastern and central Norway that fit well with the original and later descriptions of the species, except for having a lower AR (1.0-1.1) compared to what Edwards (1929) reported. The specimens group in a well-defined barcode cluster (Fig. 6)   Etymology. The species is named "autumnalis", Latin adjective meaning belonging to autumn, referring to the time of the year when the type material was collected.
Diagnosis. Gymnometriocnemus (R.) autumnalis can be separated from other Gymnometriocnemus species by the following combination of characters in the adult male: body brown, dark brown; virga long and conspicuous with strong lateral sclerotization, anal tergite without dorsal anal point or ridge, gonostylus with convex outer margin and well-defined median crista dorsalis.
Female and immature stages unknown. Remarks. The species is morphologically similar to G. (R.) brumalis and G. (R.) kamimegavirgus, but different in lacking an anal point or ridge on the anal tergite. This character is similar to characters reported for G. terrestris and G. tairaprimus, but these two species can according to original descriptions be separated by having a higher AR (1.4 in G. terrestris) and a different shape of the superior volsella (Krüger and Thienemann 1941;Sasa and Okasawa 1994). The virga of G. terrestris has not been described, but the species is listed as a member of subgenus Gymnometriocnemus in the World Catalogue of Chironomidae (Ashe and O'Connor 2012). We have examined two males and one female between cellophane strips on two separate pins from the Goetghebuer collection; labels reading "Allemagne, Dr Thienemann, Bonn 1939". Only one pin with a female bears a type label, but all specimens are likely part of the type series and regarded as syntypes. A small virga is visible in a rather well mounted and cleared hypopygium on one of the specimens. We can confirm placement in subgenus Gymnometriocnemus and that none of the specimens we have collected in Norway belong to this species. G. (R.) autumnalis has so far only been recorded from the very north of mainland Norway and the Rondane mountains (1280 m a.s.l.) in Central Norway. (Edwards, 1929) Remarks. We have barcoded specimens from eastern, central and northern Norway that fall within the same genetic cluster although with quite large intraspecific divergence (0-6.8%, mean 3.42% K2P-distance) (Fig. 6). We are at present not able to find morphological differences that fully correspond to the internal groupings inside the G. (R.) brumalis cluster and all our specimens fit the description by Saether (1983) with    Cranston and Oliver (1988). There is some variation observed in the length of the virga in relationship with the length of the gonocoxite, one of the characters used to separate G. (R.) acigus from G. (R.) brumalis (Cranston and Oliver 1988;Saether 1983), but we have not examined or barcoded a sufficient number of specimens to confidently conclude if the divergent genetic clusters in our G. (R.) brumalis can be regarded as separate species. We have examined the male holotype and a male paratype of G. (R.) acigus and can confirm that the species fits our and Cranston and Oliver's (1988) interpretation of G. (R.) brumalis. The species has a Holarctic distribution.
Diagnosis. Gymnometriocnemus (R.) kamimegavirgus can be separated from other species of the genus Gymnometriocnemus by having well-developed, long virga (about the length of the gonocoxite); AR 0.9-1.1 (n=5); LR 1 about 0.53-0.56 (n=3); wing membrane with setae at the apex only, occasionally with 1-2 setae proximally in cell an; R 2+3 situated in the middle between R 1 and R 4+5 ; dark brown almost blackish thorax and head, slightly paler abdomen and legs. Remarks. Our examined material is from eastern, central and northern Norway, frequently collected near streams, rivers and moors. Male adults fit well with Brundin's description of G. volitans, and Sasa & Hirabayashi's description of G. kamimegavirgus except for slightly fewer setae on the abdominal tergites (Brundin 1956;Sasa and Hirabayashi 1993;Sasa and Okasawa 1994). The species is Holarctic in distribution.

Key to Holarctic male adults of the genus Gymnometriocnemus
The species Gymnometriocnemus brevitarsis is only known as female and therefore not included in the key.

Final remarks
As a result of this study, there are now 17 species of Gymnometriocnemus registered worldwide and the genus is present in all major biogeographical regions except Antarctica. Our findings through moderate sampling in Norway indicate that the number of species could be considerably higher also on a global scale and show that molecular data can be a great advantage in diversity assessments of targeted groups. Moreover, our study also highlights the importance of consulting type material for correct identification of Chironomidae if we are to avoid long term misconceptions of species.
Bergen for loan of specimens of G. (G.) marionensis and types of G. (R.) acigus. We are grateful for the help provided by Martin Spies in obtaining copies of relevant literature from the library at the Zoologische Staatssammlung München. Thanks to Rick Jacobsen and two anonymous reviewers for comments on the submitted manuscript.