Biodiversity Data Journal :
Taxonomy & Inventories
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Corresponding author: Jostein Kjærandsen (jostein.kjarandsen@uit.no)
Academic editor: Vladimir Blagoderov
Received: 12 Dec 2022 | Accepted: 27 Jan 2023 | Published: 14 Feb 2023
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Kjærandsen J, Kerr PH, Lindemann JP, Kurina O (2023) When details matter: Integrative revision of Holarctic Coelophthinia Edwards (Diptera, Mycetophilidae), including mapping of its mitogenome, leads to the description of four new pseudocryptic species. Biodiversity Data Journal 11: e98741. https://doi.org/10.3897/BDJ.11.e98741
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The small genus Coelophthinia Edwards, 1941 of the subfamily Gnoristinae (Diptera, Mycetophilidae) is so far known to harbour four species from the Palaearctic, Nearctic and Neotropical Regions. Extensive DNA barcoding of fungus gnats of the family Mycetophilidae through the International Barcode of Life project (iBOL) have initiated integrative studies resulting in taxonomic upgrades and a better understanding of many species and their delimitation. The opportunity was also taken to describe the mitogenome of a member of Coelophthinia for the first time.
The integrative studies give evidence for splitting the European species C. thoracica Edwards, 1941 into three different species. Four new species are described from the USA, Japan and the Nordic Region in Europe, Coelophthinia cirra Kerr sp. n., Coelophthinia itoae Kurina sp. n., Coelophthinia lata Kjaerandsen sp. n. and Coelophthinia loraasi Kjaerandsen sp. n., raising the number of Holarctic species from two to six. The mitogenome of Coelophthinia loraasi sp. n. is described and analysed.
Coelophthinia, morphology, DNA barcoding, integrative taxonomy, new species, new mitogenome
The small genus Coelophthinia Edwards, 1941 of the subfamily Gnoristinae (Diptera, Mycetophilidae) is so far known to harbour four species, viz. C. thoracica (Winnertz, 1964) from the Palaearctic Region, C. curta (Johannsen, 1912) from the eastern Nearctic Region and C. accita (Plassmann & Vogel, 1990) and C. flavithorax (Freeman, 1951) from the temperate zone of the Neotropical Region (
Due to their slender appearance (Fig.
Live photo of the type species for the genus, a Coelophthinia thoracica (Winnertz, 1864) male resting on a window in a barn in western Norway. The photographed specimen was subsequently collected, DNA barcoded and assigned to Barcode Index Number (BIN) BOLD:ACJ0721 (Specimen ID TSZD-JKJ-111214). The mid-tibial organ is visible and marked with a red arrow. The 180 deg. torsion of the male terminalia is also visible.
Extensive DNA barcoding of fungus gnats of the family Mycetophilidae through the International Barcode of Life project (iBOL, see
The studied material has accumulated over the last 50 years, the majority during the last decade and is deposited in the insect collections of Tromsø University Museum, Norway (TMU), Estonian University of Life Sciences, Tartu, Estonia (IZBE) and California State Collection of Arthropods, Sacramento, California, USA (CSCA). Additionally, DNA barcoded material was borrowed from the Centre for Biodiversity Genomics, University of Guelph, Canada (BOLD). Being initially stored in 70-95% ethanol, the majority of the fresh specimens were dried through baths of hexamethyldisilazane (HMDS,
The 658 bp fragment of the mitochondrial protein-encoding cytochrome c oxidase subunit I (COI) has been sequenced from a total of 46 Coelophthinia specimens on BOLD, 25 of them submitted by us during this study. One leg from each fresh specimen was sent to the Canadian Centre for DNA barcoding, BIO (Guelph, Ontario, Canada), for DNA extraction and bi-directional Sanger sequencing as a part of the Norwegian Barcode of Life (NorBOL) and Finnish Barcode of Life (FinBOL) initiatives, both branches of the International Barcode of Life project (iBOL). The new sequences are publicly available from The Barcode of Life Data System (BOLD) and referred to below with external links to their Barcode Index Numbers (BINs) on BOLD for each of the barcoded species.
DNA extraction, library preparation and DNA sequencing
One specimen of C. loraasi sp. n. (TSZD-JKJ-105417) was selected for further DNA sequencing. DNA was extracted from the whole individual, except its terminalia, using the E.Z.N.A. Insect DNA Kit (Omega bio-tek), following the manufacturer’s protocol. The terminalia was dissected and preserved in glycerine as voucher. DNA library preparation and sequencing were carried out at The Norwegian Sequencing Centre (NSC). The DNA sample was fragmented to a target of 350 bp average size. Library preparation was performed using the Kapa Hyper library prep kit (Roche), with cycles of PCR with the Kapa Library amplification mix (Roche) and two rounds of bead clean-up (both on PE Sciclone). The average fragment length after library preparation was measured to 538 bp. The samples were sequenced on the HiSeq 4000 system (Illumina inc.) with 150 bp paired-end sequencing following the manufacturer’s recommendations.
Assembly and gene annotation
Filtering of raw reads was carried out in Trimmomatic 0.39 (
A Gnoristinae genus with slender and medium-sized (2.8–4.3, 3.8 mm) species (Figs
Habitus of Coelophthinia lata sp. n. Abbreviations: Cext = Extension of costa beyond R5; clyp = clypeus; CuA = anterior branch of cubital vein; CuP = posterior branch of cubital vein; fr fur = frontal furrow; fr tub = frontal tubercle; h = humeral vein; iCu = intercubital fold (not a vein); lbl = labrium; l oc = lateral ocellus; M = medial veins (numbered); om = ommatid; plp (numbered) = palpal segment; R = radial veins (numbered); r–m = radial–medial crossvein; Rs = radial sector; Sc = subcostal vein; scp = scapus; sc-r = crossvein between subcosta and radius; vrt = vertex;
Male terminalia (Fig.
Structure and terminology of male terminalia of Coelophthinia: a, b C. thoracica (Winnertz, 1864); c–f C. loraasi sp. n. Abbreviations: aed = aedeagus; aed gd = aedeagal guide; cerc = cercus; gc = gonocoxite; gc ap = gonocoxal apodeme; gc lobe = posterolateral lobe of gonocoxite; gc spat = spathulate gonocoxal lobe; gs = gonostylus; gst dbr = dorsal branch of gonostylus; gst vbr = ventral branch of gonostylus; hyp pr = hypoproct; lb 1 = lobe 1 of ventral gonostylar branch; lb 2 = lobe 2 of ventral gonostylar branch; med elev T10 = medial elevated & protrusive projection from tergite 10; T9 = tergite 9; T10 = tergite 10.
Female terminalia (Fig.
Males of the genus Coelophthinia are easily distinguished from all other Gnoristinae genera by the characteristic shape of their torsioned terminalia (Figs
Phthinia thoracica Winnertz, 1964
Coelosia thoracica sensu Edwards (1925)
Coelophthinia thoracica sensu Edwards (1941)
Male (Figs
Coloration and most body characteristics as in the genus description. Body length 3.6–4.2 mm. Wing length 2.8–3.1 mm; ratio of length to width 3.1–3.3. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 6.3–8.1 times longer than wide, length 0.26–0.29 that of tibial length.
Terminalia. Tergite 9 rectangular in dorsal view, about 1.3 times as long as wide, medially somewhat constricted, curved like a hood in lateral view. Medial protrusion of tergite 10 short and rounded, about as high as wide in posterior view, densely setose. Gonocoxites in lateral view with a somewhat elongated outline, with straight or slightly convex ventral margin. Posterolateral lobe of gonocoxite long and narrow, constricted section about 3x longer than wide, rounded apically, laterally bare for about 3x apical width. Spathulate gonocoxal lobe 7–7.5 times as long as wide, curved slightly medially, with constricted base, basad of base with a group of 3–5 longer setae. Dorsal branch of gonostylus small, simple, semicircular, dorsally setose with one extra long seta deviating from others. The broad lobe 1 (cf. Fig.
Female (Fig.
Coloration as for male. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 7.4 times longer than wide, length 0.28 that of tibial length.
Terminalia as described for genus. Tergite 8 short, posteriorly emarginated in dorsal view, bare, except 1–2 small setae posterolaterally. Tergite 9 short, posteriorly emarginated in dorsal view, bare, except apical margin with row of 7–8 long setae, which are longer than those on cercus. Cercus about two times as long as wide medially, evenly covered with setae. Sternite 8 longer than tergites 8 and 9 together, posterolaterally rounded, posterior half setose, in ventral view hypogynal valves separated by V-shaped deep incision, about one-third as deep as segment length. Gonapophysis 8 elongate, about 6 times as long as wide, extending beyond mid-cercus, ventrally setose, with one deviating strong subapical seta.
Coelophthinia thoracica can be distinguished from other species of the genus by the combination of having a somewhat elongated shape of the gonocoxite as seen in lateral view, with a straight or slightly convex ventral margin, a long and slender posterolateral lobe and a short and rounded medial protrusion of tergite 10. The aedeagus is uniquely shaped, long, with ventrally curved hook not longer than width of medial section of aedeagus. The length of the mid-tibial organ is about 0.3 of tibial length.
Male (Fig.
Coloration and general body characteristics as in the genus description. Body length 3.5–4.1 mm. Wing length 2.9–3.1 mm; ratio of length to width 2.6. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 7.5 times longer than wide, length 0.25 that of tibial length.
Terminalia. Tergite 9 rectangular in dorsal view, about 1.5 times as long as wide, curved like a hood in lateral view. Medial protrusion of tergite 10 short and rounded, about as high as wide in posterior view, densely setose. Gonocoxites in lateral view with a rounded outline, with distinctly convex ventral margin. Posterolateral lobe of gonocoxite short and wide, constricted section about 1.5x longer than wide, evenly rounded apically, laterally bare for about 2x apical width. Spathulate gonocoxal lobe about seven times as long as wide, without constricted base, basad of base with a row of 3 long setae. Dorsal branch of gonostylus small, simple, oblong and semicircular, dorsally setose with some extra long setae. The broad lobe 1 (cf. Fig.
Female unknown.
Coelophthinia lata can be distinguished from other species of the genus by the combination of having a rounded shape of gonocoxite in lateral view, with distinctly convex ventral margin, broad and short posteriolateral lobe and short and broad, semicircular medial protrusion of tergite 10. The aedeagus is uniquely shaped with apical hook not longer than the height of the base of aedeagus. The length of the mid-tibial organ is about 1/4 of tibial length.
The species epithet is from the Latin word lata, which means broad, denoting the broad caudal extension from the gonocoxite. This feature is diagnostic for this species.
Nordic, so far known only from Norway, Sweden and Finland.
Male (Fig.
Coloration and general body characteristics as in the genus description. Body length 3.5–3.6 mm. Wing length 2.8–2.8 mm; ratio of length to width 2.6. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 6.1 times longer than wide, length 0.20 that of tibial length.
Terminalia. Tergite 9 rectangular in dorsal view, about 1.5 times as long as wide, curved like a hood in lateral view. Medial protrusion of tergite 10 thin, height approximately 3x width in posterior view, curved posteriad, densely setose on apex and ventral side. Gonocoxites in lateral view with a narrow subsquare outline, with nearly straight ventral margin and posterolateral lobe situated along the ventral edge. Posterolateral lobe of gonocoxite long, slender and evenly tapering, about 4x longer than width at apex, rounded apically, laterally bare for about 2x apical width. Spathulate gonocoxal lobe about five times as long as wide, rounded, with distinctly constricted base, basad of base with a row of 5 long setae. Dorsal branch of gonostylus small, simple, semicircular, laterally setose with some extra long setae. The broad lobe 1 (cf. Fig.
Female (Fig.
Coloration as for male. Body length 3.9–3.9 mm. Wing length 2.9–3.0 mm; ratio of length to width 2.8. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 5.5 times longer than wide, 0.32 times of tibial length.
Terminalia as described for genus. Cercus slightly narrower than in other species, with fewer setae on ventral side. Gonapophysis 8 forming an equilateral triangle in ventral view. Sternite 9 forming a distinct circle in ventral view.
Coelophthinia loraasi can be distinguished from other species of the genus by the combination of having a narrow, rectangular shape of the gonocoxite as seen in lateral view, with a near straight ventral margin, a narrow and long posteriolateral lobe and a long, narrow and curved medial protrusion of tergite 10. The aedeagus is uniquely shaped with apical hook much longer than the height of the base of aedeagus. The length of the mid-tibial organ is about 1/6 of tibial length.
Named in honour of Professor Jostein Lorås, the local biologist who, for many years, has worked hard for and succeeded to protect areas of old growth coniferous forests in Grane Municipality of Nordland County, including those from where the type materials originate.
So far known only from northern Norway.
Male. (Figs
Coloration and most body characteristics as in the genus description. Body length 3.2 mm. Wing length 2.70–2.77 mm; ratio of length to width 1.9–2.5. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 6.7 times longer than wide, length 0.34 that of tibial length.
Terminalia. Tergite 9 rectangular in dorsal view, about 1.25 times as long as wide, curved like a hood in lateral view. Medial protrusion of tergite 10 remarkably long, height approximately 4-5x width in posterior view, curved posteriad, densely setose. Gonocoxites in lateral view with a rounded outline, with slightly convex ventral margin. Posterolateral lobe of gonocoxite short and wide, only somewhat longer than wide, truncated apically, laterally bare for about 1.5x apical width. Spathulate gonocoxal lobe about 3 times as long as wide, curved slightly medially, without constricted base, basad of base with a group of 3–4 longer setae. Dorsal branch small, simple, wider than long, apically rounded, dorsally setose, with one extra long seta deviating from others. The broad lobe 1 (cf. Fig.
Female. (Fig.
Coloration as for male. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 9.2 times longer than wide, length 0.32 that of tibial length.
Terminalia as described for genus. Tergite 8 short, posteriorly emarginated in dorsal view, bare. Tergite 9 short, posteriorly emarginated in dorsal view, bare, except posterior margin with row of 7–8 long setae, which are longer than those on cercus. Cercus about two times as long as wide medially, evenly covered with setae. Sternite 8 longer than tergites 8 and 9 together, posterolaterally rounded, posterior half setose, in ventral view hypogynal valves separated by V-shaped deep incision, about one-third as deep as segment length. Gonapophysis 8 elongate, about 8 times as long as wide, extending to mid-cercus, setose, with one deviating strong subapical seta.
Coelophthinia itoae can be distinguished from other species of the genus by the combination of having a rounded shape of the gonocoxite as seen in lateral view, with a slightly convex ventral margin, a short and wide posterolateral lobe and a long and posteriad curved medial protrusion of tergite 10. The ventral branch of the gonostylus with broad lobe transversally extended. The aedeagus is uniquely shaped, short, with delimited swelling medially and with apical hook about three times longer than the width of the base of aedeagus. The length of the mid-tibial organ is about 0.3 of tibial length.
Named after Tomiko Ito, who kindly guided JK and OK on a collecting trip around Hokkaidō, Japan, in 2006, when this species was collected and discovered.
Eastern Palaearctic: Japan (Hokkaidō, Honshū).
Phthinia curta Johannsen, 1912
Male (Fig.
Coloration and general body characteristics as in the genus description. Body length 2.8–3.7 mm. Wing length 2.5–3.0 mm; ratio of length to width 2.8. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 10 times longer than wide, length 0.3 times that of tibial length.
Terminalia. Tergite 9 nearly quadrate in dorsal view, slightly longer than wide, curved like a hood in lateral view. Medial protrusion of tergite 10 short, height approximately 3-4x width in posterior view, densely setose. Gonocoxites in lateral view with a rounded outline, with approximately straight ventral margin. Posterolateral lobe of gonocoxite short and wide, constricted section only approximately 2x apical width, apex more smoothly rounded ventrally, laterally bare for about 1.5x apical width. Spathulate gonocoxal lobe about five times as long as wide, with slightly constricted base, basad of base with 3 long setae not arranged in single line. Dorsal branch of gonostylus small, simple, oblong and semicircular, dorsally setose with some extra long setae. The broad lobe 1 fan-shaped, carrying 7–8 blunt-tipped setae along rim and 4 normal setae subapically on inner side. The narrow, acute tipped lobe 2 with a tiny, practically imperceptible seta subapically; with strong, sharp-tipped seta at approximately its length from the apex of the lobe, followed by one fine seta of similar length and one longer seta in mid-section. Aedeagus 1.2x longer than spathulate gonocoxal lobe in lateral view, with rounded cutout at base of long apical down-curved hook, hook approx. 0.5x length of rest of aedeagus; with a short, inconspicuous sessile projection at the outer side below the curving point of hook.
Coelophthinia curta can be distinguished from other species of the genus by the combination of the following features: Posterolateral lobe of gonocoxite situated along straight ventral margin, broad, nearly as broad as constricted length; spathulate lobe on ventral medial margin of gonocoxite short oblong, mid-tibial organ clearly much longer than width of tibia.
Male. (Fig.
Coloration and most body characteristics as in the genus description. Body length 2.8 mm. Wing length 2.5 mm; ratio of length to width 2.8. Sensory organ dorsally on the basal half of mid-tibia elongate oval, 3.5 times longer than wide, 0.1 times of tibial length.
Terminalia. Tergite 9 setose, except basal fifth medially, nearly quadrate, slightly longer than wide, medially slightly constricted, basal margin concave, curved like a hood in lateral view. Medial protrusion of tergite 10 a modest bulbous swelling, densely setose. Cerci medially fused except for apical third, setose. Gonocoxites in lateral view with a rounded outline, with straight ventral margin. Posterolateral lobe of gonocoxite long, slender and evenly tapering, about 3-4x longer than wide at apex, rounded apically, laterally bare for about 2x apical width. Gonocoxal lobe spathulate, long and slender, with slightly constricted base, basad of base lacking row of distinctive setae, evenly tapered along dorsoventral margins, external surface bare on apical length approximately 3x apical width, apically rounded. Dorsal branch of gonostylus small, simple, oblong and semicircular, dorsally setose. The broad v br lobe 1 fan-shaped, carrying 6–7 blunt-tipped setae along rim and 4 normal setae on inner side. The narrow, acute tipped v br lobe 2 with a tiny, practically imperceptible seta subapically; with thickened, sharp-tipped seta at approximately its length from the apex of the lobe, followed by one fine seta of similar length and one longer seta near the base of the lobe approximately 1.5x length of v br lobe 2. Aedeagus of same length or slightly longer than spathulate gonocoxal lobe in lateral view, slender throughout, ventral margin entire along length, without rounded cutout at base of hook; with long apical down-curved hook, hook approx. 0.4x length of rest of aedeagus; with a short, inconspicuous sessile projection at the outer side below the curving point of hook.
Coelophthinia cirra can be distinguished from other species of the genus by the combination of the following features: mid-tibial organ only slightly longer than width of tibia, gonocoxal lobe situated along straight ventral margin, slender and evenly rounded apically, thinner than constricted length; aedeagus slender throughout, without cutout at base of apical hook.
Adjective taken from the Latin word cirrus, meaning "tendrils" and descriptive of clouds. This species is thin and leggy and found at high elevations, reminiscent of the clouds.
Key to males of Holarctic Coelophthinia |
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1 | Protrusive middle section of tergite 10 long, narrow and curved, elevated well above top of tergite 9 (Figs |
2 |
– | Protrusive middle section of tergite 10 short, semicircular, not elevated above top of tergite 9 (eg. Fig. |
3 |
2 | Posterolateral lobe of gonocoxite broad, as broad as constricted length; spathulate lobe on ventral medial margin of gonocoxite short oblong; aedeagus short and deeply downcurved (Fig. |
Coelophthinia itoae sp. n. |
– | Posterolateral lobe of gonocoxite narrow, much thinner than constricted length; spathulate lobe on ventral medial margin of gonocoxite long with constricted base; aedeagus long and less deeply downcurved (Fig. |
Coelophthinia loraasi sp. n. |
3 | Posterolateral lobe of gonocoxite situated midway between dorsal and ventral edges, with distinctly convex ventral margin (Figs |
4 |
– | Posterolateral lobe of gonocoxite situated along straight ventral margin (Figs |
5 |
4 | Posterolateral lobe of gonocoxite broad, nearly as broad as constricted length; spathulate lobe on ventral medial margin of gonocoxite short oblong (Fig. |
Coelophthinia lata sp. n. |
– | Posterolateral lobe of gonocoxite narrow, much thinner than constricted length; spathulate lobe on ventral medial margin of gonocoxite long with constricted base (Fig. |
Coelophthinia thoracica |
5 | Posterolateral lobe of gonocoxite broad, nearly as broad as constricted length; spathulate lobe on ventral medial margin of gonocoxite short oblong (Fig. |
Coelophthinia curta |
– | Posterolateral lobe of gonocoxite narrow, much thinner than constricted length; spathulate lobe on ventral medial margin of gonocoxite long with constricted base (Figs |
Coelophthinia cirra sp. n. |
The mitochondrial genome of C. loraasi Kjaerandsen (Fig.
Map of the mitogenome of Coelophthinia loraasi. The outer circle displays the genes on the + (outer) and - (inner) strands with coding sequences coloured in yellow, rRNA in red and tRNA in blue. Arrows indicate direction of transcription. The inner circle shows the GC content (brown) as a deviation from the avarage of the total mitogenome.
There are ten cases of overlapping genes and 21 cases where intergeneric nucleotides are present (Suppl. material
The protein coding genes constitute 11211 bp of the mitochondrial genome. The most frequently encoded amino acids (Suppl. material
The enormous success of DNA barcoding has accumulated a substantial amount of sequenced insects on BOLD, which is very useful for the kind of new and integrative taxonomic studies that we present here. More than 73,000 specimens belonging to the family Mycetophilidae have been successfully sequenced (
In the Nordic Region, however, strong ties between The Norwegian and Swedish Biodiversity Information Centres, including their Taxonomy Initiatives and NorBOL and FinBOL, are ensuring that the best taxonomic expertise is building up the reference library of the local fauna in the BOLD archive. Hence, the majority of some 15,000 DNA barcoded fungus gnats (Sciaroidea) from the Nordic Region have been identified to species level upon submission and the reference library is profoundly and repeatedly quality-checked and curated after barcodes and BINs are assigned. This has resulted in a high-quality reference library, now covering about 84% of the known Mycetophilidae fauna including more than 150 additional species considered to be new to science (
The male terminalia in species of Coelophthinia are quite similar in general appearance, yet uniquely shaped for the genus. This has probably contributed to the diversity of species previously being overlooked only to be revealed when DNA barcoding started to split them into distinct clades. The morphological differences are, nevertheless, clear and unambiguous when the taxa were re-examined in detail. Morphological differences were just simply overlooked previously due to a convenient lumping of specimens that formed a unique taxon widely different from other species. For this reason, we consider these species pseudocryptic. They were morphologically recognised as distinct only after other methods unveiled their existence. Our concept here is in agreement with
A 180-degree torsion is always seen where the ventral side of the terminalia is turned to the dorsal side in live specimens (see Fig.
JK was supported and funded by UiT—The Arctic University of Norway, the Norwegian Biodiversity Information Centre (NBIC, projects nos. 45–14 and 27–17) and the Norwegian Barcode of Life initiative (NorBOL). OK was supported by institutional research funding from the Ministry of Education and Research of Estonia and funding from the Estonian Research Council (TT14). The publication charges for this article have been funded by a grant from the publication fund of UiT—The Arctic University of Norway. We are very thankful to Prof. Toyohei Saigusa (Fukuoka, Japan) for information on occurrence of C. itoae in Honshu Island, Japan. We are grateful for the helpful comments and suggestions provided by Peter Chandler and Jan Ševčík in their reviews of this manuscript.
Order of the 37 genes on the Mitochondrial genome of Coelophthinia loraasi sp. nov., their affiliation to the forward (+) or reverese (-) strands, start and end positions (begin/end), size in base pairs (size), start codon (start cd) and number of intergeneric nucleotides (inc). A negative intergeneric nucleotide number indicates overlap between the genes.
Barplot of the relative frequencies of codons used in the mitochondrial genome of C. loraasi sp. nov., with their encoded amino acids.