Three new Scandinavian species of Culicoides (Culicoides): Culicoides boyi sp. nov., Culicoides selandicus sp. nov. and Culicoides kalix sp. nov. (Diptera: Ceratopogonidae)

Abstract Background In the context of a major monitoring program of Culicoides in Denmark and Sweden due to the appearance of bluetongue disease in 2007–2008, a large number of specimens were collected by light traps and sorted morphologically, with COI barcodes generated for selected specimens. New information Three species are described as new to science based on both morphological and molecular data: Culicoides (Culicoides) boyi sp. nov. (Denmark: Jutland), C. (C.) selandicus sp. nov. (Denmark: Zealand) and C. (C.) kalix sp. nov. (Sweden: Norrbotten). All are diagnosed morphologically as well as by molecular barcoding. A key to slide-mounted females of all Scandinavian species of Culicoides (Culicoides) is presented.


Introduction
Following the outbreak of bluetongue disease in 2007-2008, a major entomological monitoring program of Culicoides was implemented in Denmark and Sweden to collect a large material of Culicoides (Nielsen et al. 2010, Nielsen et al. 2014. Most specimens were sorted morphologically and some were selected for subsequent molecular barcoding in order to develop an efficient method for correct species identification (Pagès et al. 2009, Lassen et al. 2012a, Lassen et al. 2012b. One group of specimens was morphologically similar to Culicoides pulicaris (Linnaeus, 1758) but showed divergent cytochrome c oxidase subunit 1 (COI) barcode sequences; and two groups of specimens were morphologically similar to C. newsteadi (Austen, 1921) but showed divergent COI barcode sequences (Lassen et al. 2011). The three groups of specimens were denoted with informal names as Culicoides 'dk1', with a COI barcode diverging by 14-17% from other species of subgenus Culicoides, and Culicoides 'dk3' and Culicoides 'Kalix', which diverged in their COI barcodes by 5.9% from each other and by 13-18% from other species of subgenus Culicoides (Lassen et al. 2012b). A phylogenetic analysis clearly separated each of these three groups of specimens from all named species of Culicoides for which COIbarcodes were known (Lassen et al. 2012b), and based on this as well as on a detailed morphological study, the three fully diagnosable clusters of specimens were considered by Nielsen and Kristensen 2015 to represent separate species and provided with formal, scientific names. Evidence was given in Nielsen and Kristensen 2015 that a registration of the work had been done in the Official Register of Zoological Nomenclature (ZooBank), which after the latest amendment (International Commission on Zoological Nomenclature 2012) is required for nomenclatural acts in a digital work to be potentially available. However, the naming was not compliant with the current edition of the International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature 1999) by lacking explicit fixation of name-bearing types, i.e., holotype or syntypes, for each nominal taxon (Article 16.4.1). We are here providing formal scientific names for all three species, accompanied by evidence for ZooBank registration, details of type material, and diagnostic descriptions in agreement with the current Code. Descriptions are kept to a minimum as more elaborate descriptions are found in Nielsen and Kristensen 2015. Furthermore, we provide a key to females of all Scandinavian species of Culicoides ( Culicoides) (based on slide-mounted material).

Materials and methods
Biting midges were collected in [2007][2008] in Sweden (Nielsen et al. 2010) and 2008in Denmark using blacklight suction traps (Lassen et al. 2012b, Nielsen et al. 2014. The entire material of Culicoides was identified to species level by the first author.
For documentation of the identification, the head, wings and the posterior abdominal segments were removed from the female individuals and slide mounted, and the remaining parts of the animal were processed for DNA analysis as described by Nielsen and Kristensen 2015. All morphological measurements and molecular comparisons used in the present circumscriptions of species were carried out by Nielsen and Kristensen 2015.
The holotypes and paratypes of the three new species are deposited in the collection of the Natural History Museum of Denmark, University of Copenhagen (ZMUC).
Wing length 1,641 ± 10 µm (Fig. 1d, e). The dark hourglass-shaped mark in the centre of cell r is broadest above the longitudinal fold above M . Wing markings light brownish, distinct from the remaining, hyaline wing membrane. Cubital cell usually with a distinct spot (Fig. 1d, e; observed in about two thirds of the specimens), but this spot may be very small or entirely absent.
Two normal-sized spermathecae, ovoid, of almost equal size, with a short neck and a third rudimentary one (Fig. 1f).

Etymology
Named as a tribute to Boy Overgaard Nielsen, an outstanding Danish entomologist and current emeritus at Aarhus University.

Taxon discussion
The length divided by the width of the first flagellomere is significantly higher in C. boyi compared to all other Scandinavian species of this subgenus.
Wing length 1,339 ± 33 µm (Fig. 2d, e). The dark hourglass-shaped mark in the centre of cell r is broadest above the longitudinal fold above M . The dark areas on the wing are extensive and encompassing vein M and sometimes M . Small pale spots may be found at the tip of veins M and M . Wing with a large dark spot in cell cu separated from the dark areas bordering Cu and Cu (Fig. 2d, e).

Etymology
The species epithet refers to the name of the major Danish island Sjaelland (Latin = Selandia; English = "Zealand" or more rarely "Sealand"), where the type series was collected.

Taxon discussion
Culicoides selandicus may be confused with C. kalix but differs habitually by the extensive dark areas on the wings. The head/proboscis ratio of C. selandicus (1.16 ± 0.06) is smaller than in C. kalix (1.29 ± 0.07), although a small overlap should be expected when more specimens are measured. The average number of antennal sensilla coeloconica (12.29±0.95) is higher than in C. kalix (10.70 ± 0.82), and the first flagellomere has a higher average of sensilla (4.1) compared to C. kalix (3.0). The P3/ P2 ratio (0.96 ± 0.06) differs from that of C. kalix (0.87 ± 0.08), but with a large overlap. Second maxillary palp segment is about as long as the third, while in C. kalix the second palp segment is longer than the third. Third maxillary palp segment is more slender (PR = 3.2 ± 0.3) than that of C. kalix (PR = 2.9 ± 0.2). Maxillary palp segments four and five of equal length provides a difference from C. kalix, where the fifth palp segment is longer than the fourth. The ratio of mandibular vs. maxillary teeth is significantly higher in C. selandicus (1.31 ± 0.12) than in C. kalix (1.17 ± 0.12).
Wing length 1,423 ± 39 µm (Fig. 3d, e). The shape of the dark hour-glass formed mark in the middle of r is broadest above the longitudinal fold above M . The dark areas on the wings are extensive and surrounding vein M and M . Wings have a large dark spot in cell cu, which is separated from the dark areas bordering Cu and Cu (Fig. 3d, e).
Two functional spermathecae, ovoid, of almost equal size, with a short neck; a third rudimentary one present (Fig. 3f).

Etymology
The species epithet refers to the municipality Kalix in northern Sweden, in which the type series was collected.

a b
c d e f

Taxon discussion
Culicoides kalix could be confused with C. selandicus but may be separated from this as already discussed above under the description of the latter species and as outlined in the key.

Key to females of Scandinavian species of Culicoides (Culicoides)
The problems in constructing reliable keys to adults of the European species of Culicoides ( Culicoides) due to overlapping morphometric measures are well known (e.g., Campbell and Pelham-Clinton 1960). Lane 1981 showed how a combination of wing pattern elements and (other) quantitative characters would increase the taxonomic resolution, but we acknowledge that the present key may not be able to allocate every adult individual unambiguously to its 'true' taxonomic species. Note that we are following Borkent 2014 in treating C. deltus and C. lupicaris as synonyms. Lassen et al. 2012a treated C. halophilus as a species distinct from C. newsteadi based on molecular data, but as we have been unable to find morphological features separating these taxa, they will key out together. Wing with distinct dark markings; the dark hour-glass mark in the middle of cell r broadest above the longitudinal fold above the longitudinal fold above cell M

Analysis
Relevant comparisons for separating the three new species from their morphologically most similar Scandinavian congeners are given in Table 1 based on data provided by Nielsen and Kristensen 2015.  Table 1.
Pairwise morphometric comparisons between three new species of Culicoides (Culicoides) and the morphologically most similar Scandinavian species, including divergence in COI sequences. The significance of differences between measurements was determined by multiple comparison test after Kruskal-Wallis (P < 0.05) followed by a Conover-Inman test for all pairwise comparisons (Nielsen and Kristensen 2015). Comparisons that are significantly different are shown in yellow highlight.

Discussion
The recent arrival of bluetongue virus in northern and western Europe ) brought an increased interest in re-evaluating the capacity and importance of European/Palaearctic Culicoides species in transmission, which again put increased focus on delimiting and identifying the species found in Europe ). Several studies have pointed to the existence of cryptic species, which were indicated primarily by molecular 'barcoding' techniques (e.g., Pagès et al. 2009, Ander et al. 2012, Lassen et al. 2012b, Augot et al. 2013, Sarvašová et al. 2014, and a growing amount of morphological data has brought support to their validity (e.g., Augot et al. 2013, Nielsen andKristensen 2015). Molecular and morphological data have been shown to be widely congruent for northern European species (Stur and Borkent 2014), and where highly divergent barcode clusters are found within morphological species, this has usually been interpreted as potentially cryptic species (Ander et al. 2012, Lassen et al. 2012b, Wenk et al. 2012, Stur and Borkent 2014. The European fauna of Culicoides (Culicoides) is in need of a thorough taxonomic revision based on both morphological and molecular data, but this will be a massive undertaking that reaches far beyond the scope of the present paper. The Scandinavian fauna of Culicoides (Culicoides) has been sampled extensively as a result of the recent bluetongue epidemic, and the current study is based on thousands of specimens examined [by SAN] from many localities widely distributed through Denmark and Sweden. The three species here described as new do not match any of the European species as keyed by Mathieu et al. 2012 nor any of the species in the key provided by Glukhova 2005 for the Russian fauna.
Revisionary taxonomy of European Ceratopogonidae suffers from the well-known constraints of old names and insufficient or missing type material. A number of nominal species currently listed in synonymy under C. newsteadi and C. pulicaris (see Borkent 2014) could in principle provide the valid name for one or more of the taxa described in the present paper. However, no types appear to exist for the nominal species of Culicoides described by Jean-Jacques Kieffer (B. Mathieu, personal communication June 2015), which represent the majority of these synonyms, and neotypifications of these old nominal species would seem to have little justification without a much more complete European sampling than what has been available to us.
The molecular data from the three species named in the present paper were analysed by Lassen et al. 2012b in a study incorporating barcode data from specimens across Europe. Their resulting cladogram is here redrawn and shown with clear indication of the country of origin for each specimen (Fig. 4). All three species described in the present paper show an evolutionary distance to their nearest neighbour much above the 3% suggested by Hebert et al. 2003 to indicate specific separation.
It is noteworthy that the three new species were collected at single locations or from a few locations in close proximity in spite of a very large sampling. These species are most likely more widely distributed, as are the majority of the well-known biting midge species, and what may look like a restricted geographical occurrence may be due to either a patchy distribution or a very short adult flying period.