Orthocladiinae

Hebert, Torbjørn Ekrem Elisabeth Stur Paul D. N., 2010, Females do count: Documenting Chironomidae (Diptera) species diversity using DNA barcoding, Organisms Diversity & Evolution 10 (5), pp. 397-408 : 401-406

Orthocladiinae

Both Bryophaenocladius and Smittia are taxonomically diverse genera, in which most species probably have terrestrial or semi-terrestrial immature stages ( Cranston et al. 1989). At least some arctic species of Smittia are known to be parthenogenetic, e.g. S. brevipennis (Boheman, 1856) and S. velutina (Lundbeck, 1898) . Only one of three Smittia species, S. nudipennis (Goetghebuer, 1913) , is represented by males in our dataset, but the number of sampled specimens is too low to determine whether the other two species are parthenogenetic or otherwise strongly female-biased.

Three species of an unrecognisable genus of Orthocladiinae (Genus A) were represented by females only. Morphologically, the specimens are similar to described females in the genera Tavastia , Parasmittia and Gymnometriocnemus , but they do not fit any of the present generic diagnoses.

Chaetocladius is a relatively species-rich genus with about 25 recognised species in Europe. It has never been revised, and new findings have often been grouped with the most similar species descriptions. In our collections we have seven different species, of which two are represented by females only. Our Chaetocladius sp. 2 is morphologically similar to C. laminatus Brundin, 1947 in having a broadly triangular gonostylus, but diverges in some details such as a broader anal point, a less crenulate median margin of the gonostylus, and slightly different setation of the anal tergite and gonostylus (see Brundin 1947, fig. 50).

In addition to Gymnometriocnemus brumalis (Edwards, 1929) and G. volitans (Goetghebuer, 1940) , we found two unidentified species of the genus represented only by females. Three of the five known European species have been recorded in Norway ( Saether and Spies 2004), and we have a DNA barcode of the third of these species, G. subnudus (Edwards, 1929) , from a different locality. Since the genetic distances between our unknown females and the identified males range between 6% and 15%, we suspect that the two unidentified female species represent taxa so far unknown from Norway. The genus Gymnometriocnemus is characterised as “probably species-poor” in the Holarctic region ( Cranston et al. 1989). Our data indicate higher species diversity than expected, and we suspect that future investigations of terrestrial and semi-terrestrial habitats will substantially increase the number of species in this genus.

Our single specimen belonging to the genus Krenosmittia keys to K. boreoalpina ( Goetghebuer, 1944) in Tuiskunen and Lindeberg (1986) and is morphologically very similar to two males collected from springs in Luxemburg and the German Alps. However, the former differs from the latter two by COI K2P genetic distances of 8.4% and 7.8%, respectively, while the specimen from Luxemburg differs from the German one by 10%. Thus, these specimens that are currently all assigned to K. boreoalpina likely represent more than a single species. Krenosmittia boreoalpina was originally described from one male collected in the Austrian Alps ( Goetghebuer 1944). We therefore regard the specimen from the German Alps as closest to the true K. boreoalpina and leave the Sølendet specimen undetermined at species level. The larvae of Krenosmittia are cold-stenothermic and typically inhabit springs and cold streams and rivers ( Cranston et al. 1989).

A total of 11 Limnophyes species have been recorded and sequenced in the present study, among which Limnophyes sp. 1 , L. sp. 2 and L. sp. 3 are new to science. The

Fig. 1 (continued)

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genus has a worldwide distribution and larval habitats range from truly aquatic to terrestrial ( Cranston et al. 1989). Most species are small and many occur in the Arctic, high mountains and/or spring habitats. Seventeen valid species have been recorded from the Norwegian mainland and Svalbard ( Saether and Spies 2004). Saether’ s (1990) review of the genus presented 14 new species and provided a key to nearly 40 Holarctic and Afrotropical species. Since then, various new species have been described, especially from Asia and Russia ( Przhiboro and Saether 2007 and references therein).

The genus Metriocnemus is represented by seven species in our dataset. Two of these are based on females which we could not associate to any male or identify to species using the available literature. In his comprehensive study on Metriocnemus, Saether (1989 , 1995) revised numerous Holarctic species. Using the keys and diagnoses in this publication, we could positively identify males of M. albolineatus (Meigen, 1818) , M. beringiensis Cranston & Oliver, 1988 , M. eurynotus (Holmgren, 1883) , M. fuscipes (Meigen, 1818) , and M. tristellus Edwards, 1929 . Previously, M. tristellus had not been recorded from Norway ( Saether and Spies 2004). Comparison with DNA barcodes of Metriocnemus species from other localities revealed some interesting findings for M. fuscipes and M. eurynotus : Both morphospecies include multiple genetically divergent clusters. Specimens fitting the morphological description of M. fuscipes are placed in four different clusters, with the specimens from Sølendet in one cluster, specimens from Rondane ( Norway) in another, and specimens from the German Alps in the remaining two. Saether (1989: 423) stated that “ M. fuscipes is a well defined species and there appears to be no need for designation of a neotype ”. Our results indicate otherwise, i.e. that some of the currently seven junior synonyms ( Saether 1989) might have to be revalidated or that descriptions of new species might even be necessary. A similar situation exists with M. eurynotus . The specimens that fit the most recent morphological description of this species belong to four different COI clusters. The male specimens from Sølendet are almost identical to specimens from Iceland, while specimens from Svalbard group with specimens from Rondane, and M. eurynotus from the German Alps constitute two additional, separate clusters. Saether (1989: 410) introduced eight new synonyms for M. eurynotus but stated that “there is a large variation within the species which might indicate that more than one species is involved.” Our data supports the presence of multiple species within the currently accepted morphospecies M. eurynotus . Even though multiple, well separated clusters are not observed in the species M. albolineatus , one specimen (a hermaphrodite) shows considerable genetic difference (min. 4.7%) from the other sequenced members of this species. Metriocnemus albolineatus is relatively well defined within its genus ( Saether 1989, 1995), and our morphological identification of the hermaphrodite is tentative due to the mixture of male and female characters observed in this specimen.

Most Paraphaenocladius larvae live in terrestrial or semi-aquatic habitats ( Cranston et al. 1989). The adults are very similar morphologically, and the few features used to differentiate the species show considerable intraspecific variation. It seems that pupae are easier to separate, but only few pupal morphotypes have been associated to any name-bearing males ( Saether and Wang 1995). Due to the lack of adult characters that would be diagnostic at the species level, several subspecies have been erected for taxa that are morphologically variable and geographically widespread. The Sølendet samples contained six species of Paraphaenocladius . A single female was identified to P. impensus s. str. with the key to known females by Saether and Wang (1995) but is slightly different from those authors’ description of the subspecies in being smaller and having fewer setae on the thorax and genitalia. However, the key only contains half of all species and subspecies known in Paraphaenocladius . Hence, with the DNA barcode of our specimen being>10% divergent from males of P. impensus s.str. (Fig. 1), that female probably belongs to a different subspecies. The other taxa recognised in our samples are: P. impensus subsp. contractus , P. exagitans subsp. monticola , P. pseudirritus s. str., and P. irritus s. str. The DNA barcodes indicate considerable genetic distance between the subspecies, so that the latter probably should be raised to species. Comparison of the Sølendet data to the DNA barcodes of Paraphaenocladius species from other projects (not shown) reveal more diversity, with 17 well separated clusters within this genus. Five of these groups contain specimens that fit the current definition of P. impensus sensu lato, and even within the subspecies P. impensus s. str. and P. pseudirritus s. str. there are clusters showing high genetic divergence (>10%).

The genus Paratrichocladius has 11 registered species in Europe, ten of which were partly revised by Rossaro (1990, 1991). Adult males of species in the P. skirwithensis group are extremely similar and unpublished data on presumably intraspecific variation indicate that there could be one or more cases of synonymy (Bruno Rossaro and Peter H. Langton pers. comm.). For one of our species we have a barcode-associated pupa from the Atna watershed ( Norway) which fits the description of P. osellai sensu Langton and Visser (2003) . Specimens of the other Paratricocladius species at Sølendet best fit P. nivalis as described by Rossaro (1991). Both species are new to Norway and associated with cold springs, brooks and streams ( Rossaro 1991).

Pseudorthocladius filiformis (Kieffer, 1908) belongs to the P. filiformis species group, which contains at least six Holarctic species ( Saether and Sublette 1983) some of which are only separable as pupae. There is about 5% difference between the sampled specimens of this species, but the COI sequences of P. filiformis from Sølendet are more than 13% different from those of P. curtistylus (Goetghebuer, 1922) that we have sampled from the Atna watershed. Pseudorthocladius curtistylus and P. filiformis so far are the only two European members of this species group, but they can be difficult to separate as adult males ( Saether and Sublette 1983). Until more material (including immature stages) is examined and barcoded we regard the observed 5% sequence difference as intraspecific variation in P. filiformis . Immatures of Pseudorthocladius are associated with damp habitats such as mosses, seeps and floodplains along stream banks ( Cranston et al. 1989).

The species Rheocricotopus effusus (Walker, 1856) shows the largest intraspecific variation in COI observed by us, with one female specimen slightly less than 6% different from the others. Based on the review by Saether (1985), our specimens fit the description of R. effusus . The barcode from the divergent female from Sølendet is almost identical to the barcode of a male R. effusus from the Atna watershed and the high genetic divergence within this group might indicate cryptic species. Rheocricotopus effusus has been found to inhabit springs, brooks and cold streams ( Lehmann 1969).

Our record of the genus Tavastia is the first from Norway. The genus was first described from northern Fennoscandia by Tuiskunen (1985), but additional species have been recorded from the USA (one species) and northern Europe (two species) ( Brodin et al. 2008). All species are found in wetlands or spring habitats. Our two females fit the generic diagnosis and resemble the only described female in the genus, T. yggdrasilia Brodin et al., 2008 . However, because they do not completely fit the latter species diagnosis and description, we leave them as an undetermined species in the genus Tavastia .