The Oldest Evolutionary Lineage of Trichoneura Loew, 1850 (Diptera, Limoniidae) and the First Evidence of This Genus in Cretaceous Spanish Amber

Simple Summary A new established subgenus (genus Trichoneura, family Limoniidae) from Lower Cretaceous Spanish amber represents the oldest lineage of the genus and exhibits a unique morphology of hypopygium characterized by a huge lobe on the gonocoxite. The discovery of this new subgenus sheds new light on the chronostratigraphic distribution and diversity of the genus Trichoneura and the evolution of the Limoniidae. The is oldest known species of Trichoneura and is important for understanding the evolution of this group of insects. Abstract A new subgenus Cretalinea subgen. nov. of Trichoneura (Diptera, Limoniidae) is established with one new species: Trichoneura (Cretalinea) xavieri subgen. et sp. nov. This is the first report of the genus Trichoneura in Spanish amber and the first record of the genus from the Lower Cretaceous period. The oldest described species of Trichoneura is compared with other species of the genus with particular reference to those known species from the Upper Cretaceous. A list and key of fossil species of Trichoneura are given.


Introduction
With regard to extant fauna, the genus Trichoneura Loew, 1850 [1] is sparse in its number of species and is found in three zoogeographical regions, primarily in the southern hemisphere. The genus Trichoneura is most rich in species in the Oriental region, where eight species of the genus have been recorded. Less abundantly they occur in the Afrotropical region (four species), and rarely in Australia/Oceania where only one species has been reported [2,3].
We only know five species from the fossil record, four from Eocene Baltic amber. Only one species was previously known from the Cretaceous period, from Upper Cretaceous Canadian amber [6] (Table 1). A representative of the genus Trichoneura has been found recently for the first time in Spanish Cretaceous amber and is described herein. It is also the oldest representative of the genus Trichoneura. Thanks to this new discovery, it was possible to shift the stratigraphic range of the genus Trichoneura from the late Cretaceous to the early Cretaceous. This new, peculiar species from amber of Spain described herein, provides evidence of the existence of Trichoneura flies in the early Cretaceous. Table 1. List of species of Trichoneura previously known from fossil record.

Material and Methods
The study was based on material from Cretaceous Spanish amber. The specimen comes from the upper Albian amber-bearing deposit of Peñacerrada I (Basque-Cantabrian Basin, near the village of Moraza, Province of Burgos) ( Figure 1). The specimen is deposited at the Museo de Ciencias Naturales de Álava, (Vitoria, Spain).
The specimen was embedded in epoxy resin (EPO-TEK 301) [10,11] which allowed physical protection and optimal study in ventral, lateral and dorsal views.
The biological inclusion was examined with a Nikon (SMZ25) stereomicroscope, Nikon SMZ 1500 equipped with a Nikon DS-Fi1 camera. The measurements were taken with NIS-Elements D 3.0 software. 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 tip of the gonocoxite. The measurements were given only for undamaged structures, in millimeters (mm), and the length of scape, pedicel, flagellomeres and particular segments of palpus were given according to the pattern: antenna or palpus section number/length of this section, in millimeters. Drawings were made by tracing the photographs. The terminology, wing venation and male genitalia nomenclature followed that of [12][13][14].        Horizon and locality. The type specimen was found in amber from coal levels with abundant plant remains deposited in delta plain areas that correspond to the top of filling sequences of interdistributary bays. It is also found in filling deposits of abandoned fluvial channels or crevasse splay in the Utrillas Group [15], Lower Cretaceous, upper Albian. The outcrop of Peñacerrada I [21] is located in the Basque-Cantabrian Basin, municipality of Moraza (Province of Burgos, Castilla y León Autonomous Community, northern Spain).

Discussion
Description. Body 3.96 mm long (Figure 2A), brown. Head (Figure 2A , scape approximately 3× longer than wide, with a few long and relatively strong elongate setae, longer than width of segments bearing them, 3× longer than pedicel, pedicel only 1.5× longer than first flagellomere, last flagellomere shortest and tiny, 3× longer than wide; flagellomeres 1-15 with two elongate setae, setae on last flagellomere rather short, last flagellomere with two rather short setae arranged at the tip of this segment of antennae. Maxillary palp (Figure 2A,B and Figure 3B) elongate, first and third palpomeres equal in length, 2× longer than wide, widened in distal part; second palpomere shortest, 1.5× longer than wide; last palpomere elongated and narrow, approximately 7× longer than wide, widened in apical part, length of palpomeres 0.47 mm: 1/0.12; 2/0.09 mm; 3/0.14 mm; 4/0.12 mm.
Wing (Figure 2A,D and Figure 3C): Rs as long as R 2+3+4 and R 3+4 combined; fork of R 3+4 distal to R 1 insertion into C. Hypopygium ( Figure 2C,D and Figure 3D): elongate and relatively narrow, 0.63 mm long, gonocoxite 0.40 mm long, inner gonostylus 0.14 mm, outer gonostylus 0.12 mm, lobe on hypopygium 0.23 mm long with relatively short, strong, sparsely distributed setae; inner gonostylus approximately twice width of outer gonostylus, process structure with two short setae at apex 0.5x the length of inner gonostylus, measuring 0.6x width of inner gonostylus.
Remarks. The specimen is not well preserved. Wings of the specimen are not clearly visible, and are only partially preserved. The legs are almost completely destroyed. The hypopygium is well preserved and the most important taxonomic features of the species are visible.

Discussion
Deposits of Spanish amber were formed in the Lower Cretaceous period in upper Albian later than the deposits of Lower Cretaceous Lebanese amber (lower Barremian [22,23]) and earlier than the Burmese amber deposits (Upper Cretaceous, Cenomanian [23]. Among the inclusions originating from this period there are many significant transformations of fauna and flora. Some of the oldest members of the subfamily Limoniinae e.g., the oldest representatives of the genus Helius Lepeletiere et Serville, 1928 [24] [29]. Added to these the newly described species-T. (Cretalinea) xavieri subgen. et sp. nov.-the oldest representative of the genus and new subgenus (Figure 4). The genus Trichoneura has so far been represented in the fossil record only by one subgenus, Trichoneura. In recent fauna, the subgenus Trichoneura is represented by only by one species, while the subgenus Xipholimnobia is the richest in species but is not present in the fossil record. There are more extant than fossil species, although the subgenus Ceratolimnobia is represented by only one species -Trichoneura (Ceratolimnobia) munroi (Alexander, 1920) [4]. Subgenera differ mainly in the morphology of the hypopygium and wing venation. The diversity of wings of fossil species belonging to the family and representatives of each subgenus are shown in Figure 5. There are clearly visible differences in the presence or reduction of the terminal sector of vein R 1 , and development of R 3+4 as in T. (T.) canadensis ( Figure 5). Five species from the fossil record, including a new species, T. (Cretalinea) xavieri subgen. et sp. nov., have obvious R 1 . Due to the fact that there is a tendency to reduce the end of R 1 , e.g. in Tipulidae Latreille, 1802 [30], it can be supposed that the well-developed R 1 end is a plesiomorphic feature, and is more often characteristic of fossil species than modern ones. However, in Trichoneura this section of venation is variable, R 1 is sometimes obsolete, appearing as short spur.
It is also worth noting that all fossil species of the genus Trichoneura, both those known from the Cretaceous and the Eocene, have been found in the northern hemisphere, while most of the modern species classified to the genus occur in the southern hemisphere. This may be related to the climate, because deposits of resins such as Lebanese were formed in tropical or subtropical, moderate to hot and very wet, dense forests in the north-east of Gondwana [31], while Baltic amber deposits were formed during warming episode, in the Middle Eocene Climatic Optimum (MECO), the wormest period in Earth's history [32]. Today, representatives of the genus Trichoneura are species associated with warmer areas on Earth including parts of Africa and Southeast Asia [2] (Figure 6). the presence or reduction of the terminal sector of vein R1, and development of R3+4 as in T. (T.) canadensis ( Figure 5). Five species from the fossil record, including a new species, T. (Cretalinea) xavieri subgen. et sp. nov., have obvious R1. Due to the fact that there is a tendency to reduce the end of R1, e.g. in Tipulidae Latreille, 1802 [30], it can be supposed that the well-developed R1 end is a plesiomorphic feature, and is more often characteristic of fossil species than modern ones. However, in Trichoneura this section of venation is variable, R1 is sometimes obsolete, appearing as short spur.  It is also worth noting that all fossil species of the genus Trichoneura, both those known from the Cretaceous and the Eocene, have been found in the northern hemisphere, while most of the modern species classified to the genus occur in the southern hemisphere. This may be related to the climate, because deposits of resins such as Lebanese were formed in tropical or subtropical, moderate to hot and very wet, dense forests in the northeast of Gondwana [31], while Baltic amber deposits were formed during warming epi- known from the Cretaceous and the Eocene, have been found in the northern hemisphere, while most of the modern species classified to the genus occur in the southern hemisphere. This may be related to the climate, because deposits of resins such as Lebanese were formed in tropical or subtropical, moderate to hot and very wet, dense forests in the northeast of Gondwana [31], while Baltic amber deposits were formed during warming episode, in the Middle Eocene Climatic Optimum (MECO), the wormest period in Earth's history [32]. Today, representatives of the genus Trichoneura are species associated with warmer areas on Earth including parts of Africa and Southeast Asia [2] (Figure 6).

Conclusions
Cretalinea subgen. nov. shows a unique morphology of hypopygium in which a relatively large, spoon-shaped lobe occurs measuring almost half the length of the gonocoxite. This particular structure of hypopygium is currently known only from one place and time, the Cretaceous Spanish amber period, and probably did not survive to recent times. This Cretaceous evolutionary lineage of Trichoneura provides the possibility to change the chronostratigraphic distribution of the genus from the late Cretaceous to the early Cretaceous. The newly discovered species is the oldest representative of Trichoneura and sheds new light on the evolution of this genus.

Conclusions
Cretalinea subgen. nov. shows a unique morphology of hypopygium in which a relatively large, spoon-shaped lobe occurs measuring almost half the length of the gonocoxite. This particular structure of hypopygium is currently known only from one place and time, the Cretaceous Spanish amber period, and probably did not survive to recent times. This Cretaceous evolutionary lineage of Trichoneura provides the possibility to change the chronostratigraphic distribution of the genus from the late Cretaceous to the early Cretaceous. The newly discovered species is the oldest representative of Trichoneura and sheds new light on the evolution of this genus.