New fossil data reveal evolutionary pathways within the genus Trichoneura Loew, 1850 (Diptera, Limoniidae)

New inclusions of Trichoneura preserved in Upper Cretaceous (Cenomanian) Kachin amber allow the description of a new subgenus, Burmania subgen. nov., and four new species: Trichoneura (Burmania) burmitensis subgen. et sp. nov., Trichoneura (Burmania) chungkuni subgen. et sp. nov., Trichoneura (Burmania) sevciki subgen. et sp. nov. and Trichoneura (Burmania) wangi subgen. et sp. nov. The species differ mainly by the morphology of the hypopygium or wing venation but also the construction of the antenna. Based on a comparison of the wing venation and the morphology of the hypopygium it was possible to describe features which are characteristic of the new subgenus, especially the presence of vein R3+4. Moreover, it was possible to elucidate the evolutionary pattern of Trichoneura with two distinct extant and extinct branches. Trichoneura (Trichoneura) canadensis from Upper Cretaceous Canadian amber is transferred to the new subgenus Burmania.

Table 1.A list of recent species of the genus Trichoneura and their biogeographical distributions.The subgenus marked with an asterisk (*) is also represented in the fossil record.
Comparison.In Cretalinea gonocoxite is elongate, over 3 × as long as wide with huge, spoon-shaped lobe at apex measuring approximately 0.5 × the length of gonocoxite; gonostylus measuring less than 0.5 × the length of gonocoxite in Burmania subgen.nov.this lobe does not occur, gonocoxite is differentiated in length, gonostylus measuring more than 0.5 × the length of gonocoxite.Moreover, in Burmania subgen.nov.vein R 4 separating from R 2+3+4 far beyond separation of vein R 2 (r-r), and with vein R 3 forming sector R 3+4 ; in Trichoneura, Cretolimnobia and Xipholimnobia vein R 4 separates from R 2+3+4 before or at the same point of separation of vein R 2 (r-r), and R 3+4 does not occur.In Ceratolimnobia occur corniculus on vertex and gonostylus is deeply bifid, in Burmania subgen.nov.vertex is smoth and gonostylus is undivided.
Remark: Such features as smooth vertex, without the cornicuus, presence of vein R 3+4 and morphology of hypygium without huge, spoon-shaped lobe on its tip allow to classify this species to the new subgenus.
Comparison.In Trichoneura (Burmania) burmitensis subgen.et sp.nov. the tip of Sc is situated just before the fork of Rs, and sc-r at two of its length from the tip of Sc, in T. (B.) wangi the tip of Sc is situated opposite approximately 0.8 × before the fork of Rs, and vein sc-r at one of its length from the tip of Sc.Moreover, vein R 1 in T. (B.) burmitensis terminates at C opposite approximately 0.8 × common length of R 2+3+4 and R 3+4 , tip of R 1 is curved, R 3+4 is slightly longer than R 2 (r-r), while in T. (B.) wangi vein R 1 terminates at C opposite approximately 0.9 × common length of R 2+3+4 and R 3+4 , tip of R 1 is straight, R 3+4 is slightly shorter than R 2 (r-r).In T. (B.) burmitensis R 5 is widely separated from Rs, basal section of R 5 is equal in length to r-m, in T. (B.) wangi R 5 is narrowly separated from Rs, basal section of R 5 is shorter in length to r-m.There are also some differences in the position of tips Cu, A 1 and A 2 .In T. (B.) burmitensis tip of Cu is situated beyond d-cell level, tip of A 1 beyond m-cu level and tip of A 2 opposite approximately half the length of Mb, in T. (B.) wangi tip of Cu is situated at d-cell level, tip of A 1 before m-cu level and tip of A 2 opposite approximately 0.3 × the length of Mb.But, the main differences are visible in the shape of outer gonostylus: in T. (B.) burmitensis subgen.et sp.nov.this structure is narrow at the basal part, widened just before apex, in T. (B.) wangi outer gonostylus is wide along its entire length, tiped at apex.In contrast to Diagnosis.Tip of Sc situated just before fork of Rs, sc-r at two of its length from the tip of Sc; vein R 1 terminates at C opposite at fork of R 3+4 , tip of R 1 almost straight; R 3+4 shorter than R 2 (r-r); R 5 widely separated from Rs, basal section of R 5 equal in length to r-m; m-cu at midlength of d-cell; d-cell approximately twice as long as wide; tip of Cu beyond d-cell level; tip of A 1 before m-cu level; tip of A 2 situated just before half the length of Mb, medial-basal vein; gonocoxite very elongate, at least 3.5 × as long as wide with numerous, dense, elongate and thick setae especially concentrated at apex; outer gonostylus tiny, pointed and curved at apex, twice longer than inner, inner gonostylus narrow, thick with brush of setae at apex, aedeagus tiny, not very elogate, 0.6 × the length of gonocoxite, not divided.
Etymology.The specific epithet is dedicated to Prof. Chungkun Shih (Key Lab of Insect Evolution and Environmental Changes, College of Life Sciences, Capital Normal University, Beijing 100,048, China), the eminent specialist on fossil and recent insects.
Thorax (Fig. 4A): wing 3.80 mm long, 0.94 mm wide (Figs.4A,D, 5C); vein R 1 elongate, ending opposite fork of R 3+4 on R 3 and R 4 ; tip of R 3 beyond half the length of R 4 ; R 4 approximately twice the length of d-cell; d-cell 0.46 mm long, approximately twice as long as wide; crossvein r-m rather elongate, equal in length to basal section of R 5 ; M 3 0.81 mm long, shorter than M 1+2 , longer than M 4 ; A 1 and A 2 elongate, A 1 almost straight, A 2 curved at the tip.
Comparison.The main difference between the Trichoneura (Burmania) chungkuni subgen.et sp.nov.and other species of Burmania subgen.nov.known from Cretaceous is the morphology of hypopygium.Hypopygium of T. (B.) chungkuni is narrow, with elongate, at least 3.5 × as long as wide.Gonocoxite with dense, elongated setae, especially at the tip of gonocoxite with aedeagus not very elongate, reaching at most 0.6 × the length of gonocoxite and lobe shaped inner gonostylus.In other species of Burmania the hypopygium is wide with not very elongate gonocoxite, similarly to these species known from Baltic amber, aedeagus is almost as long as gonocoxite or longer and inner gonostylus is narrow and tipped.Some differences are well visible in wing venation.R 1 in T. Description.Body (Fig. 6A,F) brown, 3.08-3.72(holotype) mm long.Head (Fig. 6A,B) with antenna (Figs.6A,B,E, 7A) 0.64 mm long, shorter than head and thorax combined; scape elongate, narrow, cylindrical, longer than pedicel, wider than other segments of antenna; pedicel elongate, longer than wide, widened, approximately as long as first flagellomere; flagellomeres wide, approximately as wide as long, but becoming progressively slender toward antennal tip; first flagellomere elongate, longer than the rest, approximately 4 × as long as wide; last flagellomere longer than penultimate one.Antenna with two moderately elongate setae on each flagellomeres, one on one side and one on the opposite side of each member; palpus (Figs.6A,B, 7B) four-segmented, first, second and fourth palpomeres narrow, sleder, third palpomere widened distally, last palpomere longer than rest, all palpomeres with few, rather not very elongate setae, shorter than segments bearing them.Key to species of Burmania subgen.nov.
-Gonocoxite at least 3.5 × as long as wide, very elongate setae on apex of gonocoxite longer than half of its length; R 1 ending opposite fork of R 3+4 on R    www.nature.com/scientificreports/positioned beyond this fork.The hypopygium in the Eocene species of subgenus Trichoneura is rather wide (this feature is well visible).A huge lobe which occurred on the gonocoxite of upper Albian (Lower Cretaceous) T. (C.) xavieri was probably subsequently reduced and the apex of the gonocoxite was shortened, as seen in Eocene and recent species of this genus.In Burmania subgen.nov. the huge lobe on the gonocoxite is not presented.The Cretaceous line with a huge lobe on the gonocoxite or elongate gonocoxite is completely extinct (Fig. 10).Analysis of morphological features of craneflies of the genus Trichoneura shows that two evolutionary branches were probably separated at the early stage of evolution of these insects.In the recent fauna this group is almost relict, represented by only 13 species within three subgenera.In the evolution of this group the evolutionary tendencies are visible, especially in the morphology of the wing venation (Fig. 10), whereby the radial vein R 1 was gradually shortened.In Cretaceous representatives we can observe an elongate R 1 which terminates far beyond half the length of Rs.Also, in Cretaceous representatives, such as T. (C.) xavieri from Spanish amber and in those described herein from Burmese amber under the new subgenus Burmania occurs a short vein R 3+4 , whereas in Eocene species vein R 3+4 doesn't occur.
The Trichoneura genus, dynamically developing in the Mesozoic, evolved mainly in what was then Laurasia.Evidence of its widespread occurrence in this subcontinent is found in the fossil resins of Europe 15,17,19,41 , Asia (the species described herein) and North America 40 (Table 2).Unfortunately, we have no fossil evidence of the presence of representatives of Trichoneura from Gondwana in the Mesozoic.In the modern fauna only in Africa we find four representatives of the Trichoneura genus and several species in the Australian/Oceanian Region (Fig. 11).In modern fauna, representatives of the subgenus Xipholimnobia are more numerous in species than  Limoniidae are highly variable regarding their ecology and biology, their larvae are found in a wide spectrum of habitats, ranging from running waters, through still and stagnant ones, bottom sediments, to terrestrial habitats such as soils, litter, and detritus [44][45][46][47] .Unlike larvae, imagines of Limoniidae are more habitats restricted and usually present in shady and moist places, often near the shores and banks of waters, feeding on nectar and plant juices exuded on their surface [48][49][50][51] .
Palaeoentomological investigations on fossil Limoniinae demonstrated the existence of obstacles and needs to reinterpretation of biogeographic opinions concerning these flies.A better knowledge of fossil Limoniinae had enabled to provide palaeohabitats reconstructions and ecological interpretations of the past environments, in which these insects existed.

Material and methods
The study was based on 10 inclusions of the genus Trichoneura (Limoniidae: Limoniinae) preserved in Cretaceous Kachin amber, aged on 98.79 ± 0.62 Ma, (Upper Cretaceous, Cenomanian) 2,52,53 .The specimens were found as inclusions in the deposits located at the Hukawng Valley in the northern Myanmar, Myitkyina and Upper Chindwin districts (Myanmar) 27,54 2).The specimens were examined using a Nikon SMZ 1500 stereomicroscope equipped with a Nikon DS-Fi1 camera, and the measurements were taken with NIS-Elements D 3.0 software in University of Rzeszów.Measurements of individual parts of the body were given only when the measured morphological structures were not distorted.The length of the vein M 3 was given from the point of its connection with the crossvein m-m to the margin of wing, the length of the discal cell was given from its posterior edge to the point of connection of vein m-m with vein M 3 .The length of hypopygium was measured from the posterior margin of tergite IX to the apex of gonocoxite.Drawings were made based on specimens and the photographs.Drawings and photographs were made by Iwona Kania-Kłosok.The wing venation nomenclature and the designation of the hypopygium is followed by Kania 41 .Maps were built using the map Maps-For-Free (https:// maps-for-free.com) and modified with the software programs Corel Draw and Corel Photopaint X7.

Figure 1 .
Figure 1.Maps of location of recent amber mining area in the Hukawng Valley, Myitkina Province, Burma.(A) Map of the world with location of Hukawng Valley; (B) Enlarged view of location of Hukawng Valley.(C).Geological setting of the Kachin amber deposits, after Kania41 , modified.Maps were built using the map Maps-For-Free (https:// maps-for-free.com) and modified with the software programs Corel Draw and Corel Photopaint X7.

3 and R 4 …
Trichoneura (Burmania) chungkuni subgen.et sp.nov.(Figs.4C, 5D). 3. Vein sc-r at two of its length from the tip of Sc; vein R 1 terminates at C opposite approximately 0.8 × common length of R 2+3+4 and R 3+4 ; R 3+4 slightly longer than R 2 (r-r); R 5 widely separated from Rs; m-cu just before midlength of d-cell; tip of Cu beyond d-cell level; tip of A 1 beyond m-cu level; tip of A 2 situated opposite approximately half the length of Mb; inner gonostylus narrow, lobe shaped, rounded at apex, slightly folded … Trichoneura (Burmania) burmitensis subgen.et sp.nov.(Figs.2A,C, 3C).Vein sc-r at one of its length from the tip of Sc; vein R 1 terminates at C opposite approximately 0.9 × common length of R 2+3+4 and R 3+4 ; R 3+4 shorter than R 2 (r-r); R 5 narrowly separated from Rs; m-cu in midlength of d-cell; tip of Cu at d-cell level; tip of A 1 before m-cu level; tip of A 2 situated opposite approximately 0.3 × the length of Mb; gonocoxite with few, not very elongate setae at apex; outer gonostylus broad in distal part and strongly curved at apex, pointed, strongly sclerotized, inner gonostylus narrow, lobe shaped, pointed at apex; … Trichoneura (Burmania) wangi subgen.et sp.nov.(Figs.8C,D, 9C).

Figure 10 .
Figure 10.Diagram of potential lines of evolution within the genus Trichoneura in chronostratigraphical view with examples of wing venation and morphology of gonocoxites and gonostyles of chosen species of the genus.
Ceratolimnobia, species belonging to both subgenera occur at similar latitiudes, e.g.T. (C.) ishigakiensis is found in Japan, as is T. (X.) japonica 21 , and T. (C.) munroi occur in Madagascar similarly to T. (X.) madagascariensis14 .The presence of these modern, relict species may indicate the occurrence of representatives of the Trichoneurana genus also in Gondwana in past geological epochs.
(Fig. 1) and are housed in Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków (ISEA PAS) (eight specimens) and in State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, China, coll.B. Wang (one specimen) (Table

Figure 11 .
Figure 11.Geographical distribution of fossil and recent species of the genus Trichoneura.Points indicate fossil localities of Trichoneura, colour shadow-widespread of recent species of the genus.Map was built using the map Maps-For-Free (https:// maps-for-free.com) and modified with the software programs Corel Draw and Corel Photopaint X7.

Table 2 .
List of fossils known belonging to the genus Trichoneura, with their ages, localities and information about the holotypes.

cell approximately twice as long as wide; tip of Cu beyond d-cell; tip of A 1 beyond m-cu; tip of
A 2 situated opposite approximately half the length of Mb, medial-basal vein; gonocoxite not elongate, at most 2.5 × as long as wide with few, not very elongate setae at apex; outer gonostylus strongly curved, narrow in basal part, widened and sclerotized just before apex, apex of outer gonostylus narrow, pointed, with a brush of very short and coarse bristles at the end; inner gonostylus narrow, slightly sclerotized with narrow, pointed apex, inner gonostylus only approximately 0.3 × longer than outer; aedeagus thick, almost as long as gonocoxite, curved at apex.Etymology.The specific epithet is derived from the Burmite.
T. (B.) chungkuni, in T. (B.) burmitensis gonocoxite is not elongate, at most 2.5 × as long as wide with only few, not very elongate setae at apex, aedeagus is thick, almost as long as gonocoxite, curved at apex, in T. (B.) chungkuni gonocoxite is elongate, at least 3.5 × as long as wide, aedeagus is distinctly shorter than gonocoxite, approximately 0.6 × of its length.In T. (B.) sevciki, aedeagus is longer than gonocoxite, outer gonostylus is rather narrow and inner gonostylus is elongate and lobe shaped, widened at apex, in T. (B.) burmitensis outer gonostylus is strongly curved, widened and sclerotized just before apex, apex of outer gonostylus is narrow, pointed, inner gonostylus is narrow, slightly sclerotized with narrow, pointed apex.From Canadian amber T. (B.) canadensis comb.nov.inner gonostylus is long, twice longer than outer gonostylus, in T. (B.) burmitensis inner gonostylus is shorter.