Description of a new species of Mediotipula from Albania, with consideration of the eastern Mediterranean as a diversity hotspot (Diptera, Tipulidae)

Abstract A new species of the TipulasubgenusMediotipula is described from the south-eastern part of Albania, south-eastern Europe. Morphologically, the new species is most similar to T. (M.) stigmatella Schummel, 1833, but differs mainly with respect to males, having a distinctly shaped posterior margin of tergite 9–10, a widened outer gonostylus and a series of details of the inner gonostylus (anterior end of the anterior arm, shape of the posterior arm), as well as having more bulbous and rounded hypogynal valves in the females. Further morphological differences of the male terminalia between allopatric populations of T. (M.) stigmatella in the Carpathians and Balkans, south-eastern Europe, are discussed.


Introduction
The Mediterranean region of Europe is one of the most species-rich biomes in the world with a high level of endemism shaped by tectonic shifts (Hewitt 2011, Oosterbroek and Arntzen 1992, Markova et al. 2010, Pleistocene climatic oscillations (Previšić et al. 2014), environmental heterogeneity (Triantis et al. 2005, Pinkert et al. 2017, and/ or natural selection (Kraitsek et al. 2008, Rodriguez-Ramirez et al. 2017. Biodiversity hotspots from such areas as Iberia, the Apennines, the Balkans (south-eastern Europe), the Mediterranean islands, Northwest Africa, western Turkey and the Caucasus (at the border of Europe and Asia) all have a decisive role in the glacial survival of numerous endemic or range-restricted taxa (De Jong 1998). Furthermore, they also provided a continuous supply of European biodiversity during several postglacial periods (Zachos and Habel 2011).
The western Palaearctic Tipula (Mediotipula) Pierre, 1924 is a small subgenus of only eleven species of moderately sized craneflies (Oosterbroek 2018). According to Savchenko (1966Savchenko ( , 1979Savchenko ( , 1983, Theowald (1978) andDe Jong (1995), Mediotipula is the sister group of the subgenus Savtshenkia Alexander, 1965. However, all members of Mediotipula are unique among Tipula, by having a relatively small discoidal cell, while almost all the other Tipula have a relatively large discoidal cell (length-width ratio of approximately two or more). Other synapomorphies include the entirely fused, platelike posterior apodemes of the sperm pump, in addition with a laterally compressed projection on the posterolateral corner of the gonocoxite in males (De Jong 1995). Additionally, females have the hypogynal valves fused for approximately one-half to two thirds of the length, in comparison with the totally separated (up to their bases) hypogynal valves in females of other Tipula. Sternite VIII of the females has a conspicuous sclerotisation of the ventral wall of the genital chamber near the openings of the gonopore, together with a usually less distinct and smaller posteroventral sclerite. Sternite IX is present as a slender and well-sclerotised structure, sometimes with a membranous medial part (De Jong 1995).
The majority of Mediotipula taxa have an isolated distribution in the western Palearctic area, showing high levels of endemism corresponding with the major biodiversity hotspots around the Mediterranean Sea while four species have a distribution area that is limited to the Iberian Peninsula (Oosterbroek 2018). The Balkans contain only three species with their ranges also covering Central Europe or the Caucasus. Only one species, T. (M.) mikiana Bergroth, 1888 has an exclusively extra-Mediterranean distribution, and only three species are range-restricted, one in Anatolia, (Asian Turkey) (T. (M.) anatoliensis Theowald, 1978), one in the Caucasus (T. (M.) caucasiensis Theowald, 1978) and one in Algeria (North Africa), (T. (M.) fulvogrisea Pierre, 1924) (Oosterbroek 2018).
Species of Mediotipula are distributed mostly in colline-to montane-subalpine ecosystems with a high level of humidity, often in very steep oak woods, and are rarely associated with open woods and hedges exposed to the sun (pers. obs., but see also Dufour 1986 andDe Jong 1995). Larvae are considered to be bryobionts, and are frequently collected under moist moss in/or along brooks, or banks of rivers and even under dry moss in the case of T. (M.) stigmatella (Savchenko 1966, Theowald 1967, Höchstetter 1963. A comprehensive phylogenetic analyses of the species which belong to Mediotipula was published by De Jong (1995), who compared and analyzed 24 morphological characters of both sexes (with the exception of the female of T. (M.) fulvogrisea), using parsimony. According to his phylogeny, only the previous T. (M.) brolemanni group (sensu Theowald, 1978) proved to be monophyletic, and together with the species T. (M.) siebkei and T. (M.) caucasiensis constituted a well-defined group within the subgenus Mediotipula.
In this paper we provide a morphological description of a new species and discuss its relationship with the similar T. (M.) stigmatella, based upon morphological features of the male and female terminalia. The morphological differences of male genital structures of different populations of T. (M.) stigmatella from the Carpathians and the Balkans are also discussed.

Materials and methods
Adults were collected using sweep nets and then stored in 96% ethanol or dry-pinned. Altogether, 54 male and four female individuals of Mediotipula belonging to four species were examined (see Table 1). All collection data are available in the TransDiptera Online Database (Kolcsár et al. 2018). Male and female genitalia were examined after being cleared in 10% KOH. Layer photos were taken using an Olympus SZ61 stereomicroscope equipped with a Canon 650D camera and LM Digital SLR Adapter; photos were combined using the Zerene Stacker software. Male and female genitalia terminology is in accordance with De Jong (1995). All specimens used in the present study were collected by the authors and deposited in the Diptera Collection of the Faculty of Biology and Geology, University Babes-Bolyai, Cluj Napoca, Romania (DCFBG) ( Figure 1, Table 1).  Type specimens are deposited in the Diptera Collection of the Faculty of Biology and Geology (DCFBG), University Babes-Bolyai, Cluj Napoca, Romania.

Description of the new species
Diagnosis. Males: Tergite IX-X in males with the posterior margin having a medial spinous extension with a wide base and gradually narrowed tip. Lateral corner of the posterior margin of the tergite IX-X is mostly rounded. Outer gonostylus widened gradually to tip, ending oblique at dorsal margin. The anterior end of the anterior arm of the inner gonostylus has a long beak-like elongation. The posterior arm of the inner gonostylus has in its dorsal margin a concentration of strong stout setae directed anteriorly, and the anterior corner ending with a thorn-like process. On the middle of the posterior part of the inner gonostylus a small triangular posterolateral extension is present. Females have the base of the hypogynal valves bulbous and rounded.
Description. Medium sized species, Body length: holotype male 10 mm, paratype female 12 mm; wing length: holotype male 15 mm, paratype female 16 mm. Adult habitus: General colour yellowish brown. First two segments of antennae yellowish, third light brown, remainder brown. Nasus yellowish with stout black setae. Dorsal part of head, near antennae, with two whitish patches; rest of head grey-peach coloured with dark setae, except whitish yellow occipital area. Thorax -brownish grey, with four shiny brown stripes on dorsal surface. Scutellum yellowish. Wings transparent, Mediotipula-like, discoidal cell present. Coxae and trochanters yellowish; rest of leg segments brown. Abdomen yellowish, with dark brown patches to continuous bands on the posterior edge and lateral margin of tergites I-VII, tergite VIII entirely brown.
Male terminalia (Figures 2, 3). Tergite IX-X with medial part bearing a narrow medial longitudinal suture with reduced whitish area close to its posterior margin (Figure 2C) and a relatively small medial spiny extension with a wide base and gradually narrowed tip on its posterior margin ( Figure 2C, E). Ventral surface of posterior margin of tergite IX-X flat, lacking ventrally produced extension ( Figure 2E). Lateral corner of posterior margin of tergite IX-X rounded ( Figure 2C). Posterior margin of sternite 8 V-shaped, with posterior margin ending straight ( Figure 2D). Gonocoxite has a   Figure 3F-H). Parameres small and triangular ( Figure 2B). Female terminalia ( Figure 4A). Cercus slightly curved downward and terminating in a rounded apex ( Figure 4A). Hypogynal valves only moderately sclerotised and fused for approx. two thirds of their length. End of membranous area of sternite VIII at base of hypogynal valves distinctly acute. Base of hypogynal valves bulbous. Ventral wall of genital chamber, near opening of gonopore is distinctly sclerotised. Vestigial sternite IX present as a slender, but well-sclerotised structure.
Etymology. The species epithet gjipeensis translates to "from the Gjipe Gorge" and was formed by appending the Latin suffix -ensis to the name of the gorge where the new species was collected.
Ecological notes and distribution. During our investigation in the south-western part of Albania, the new species described here was only detected in the highly-isolated humid habitat in the Gjipe Gorge, near the shore of the Adriatic Sea and very close to Gjipe Beach. The gorge is cut by a small stenotherm brook, fed by a spring ca. 2-3 km upstream. The river bed is filled with large limestone boulders and rocks, and has abundant mossy cover. The brook spring is surrounded by dense riparian vegetation, where the adults flew during daytime or were sometimes seen to rest on trees near the river. The microclimate of the river valley was roughly 10 °C cooler than in the surrounding macchia (L Keresztes pers. obs.). Specimens were collected in early May. The river was completely dry in June and no additional flying adults were collected, suggesting a short phenology between April and May (LP Kolcsár pers. obs).

Systematic position and affinities of M. gjipeensis sp. n.
The new species belongs to the subgenus Mediotipula, a cranefly group restricted to the western Palearctic area, with the majority of taxa (eight out of twelve) having a rangerestricted distribution in the Mediterranean area and the Caucasus. Mediotipula has a rather isolated phylogenetic position among the western Palearctic Tipulidae, sharing a combination of synapomorphies that is unique for this subgenus (De Jong 1995), and present also in T. (M.) gjippensis, such as the relatively small discoidal cell, the entirely fused, plate-like posterior apodemes of the sperm pump and a laterally compressed projection on the posterolateral corner of the gonocoxite in males. Additionally, females have the hypogynal valves fused for approx. one-half to two thirds of the length, in comparison with the totally separated (up to their bases) hypogynal valves in females of other Tipula (for more details see De Jong 1995).
The new species is most similar to T. (M.) stigmatella, having the inner gonostylus of the male terminalia approx. two times as high as inner gonostyli of other species of Mediotipula (Figs 3D, 6B, G). However, the new species can be easily distinguished from the latter species, because in the males the posterior margin of tergite IX-X has a medial spiny extension with a wide base that gradually narrows towards the tip ( Figure 2C), instead of the narrowed and rounded medial spinous extensions seen in T. (M.) stigmatella ( Figure  5A, C). The medial spiny extension is relatively small ( Figure 2E Figure 4B).

Morphological variability of M. stigmatella in the Carpathian-Balkan area
During our investigation, morphological differences were detected between allopatric populations of T. (M.) stigmatella in the Carpathian-Balkan area. Most important divergences were detected in the shape of the posterior margin of the tergite IX-X between populations from the Carpathians ( Figure 5C) and the Balkans ( Figure 5A), but no important differences in the general shape of the outer and inner gonostylus were found between populations from Lunca Bradului (the Carpathians) ( Figure 6D, E) and Kavala (Greece)( Figs 5F, 6A-C). However, a series of important structures were detected between more proximal populations from Kavala (Greece) together with individuals from Vevchani (FY of Macedonia) and Kulata (Bulgaria), mostly on the width of the outer gonostylus ( Figure 5F, G), and on the apex of the posterior arm of the inner gonostylus ( Figure 6A-C, F-H). The triangular-like apex of the posterior side of the posterior arm of the inner gonostylus of the specimens from Kavala (Greece) and Vevchani (FY of Macedonia) is shorter, with a broader base ( Figure 6B), while in specimens collected from Kulata (Bulgaria) such similar structure is longer, with a narrowed base ( Figure 6G). Without having a clear geographic pattern of the detected morphological divergences in the studied population, and with only a limited number of individuals, more data is required, covering the whole of the species distribution area, in order to evaluate the taxonomic importance of the detected differences.

The eastern Mediterranean area as a diversity hotspot
Current systematic and phylogeographical studies of the Mediterranean terrestrial fauna (Oosterbroek and Arntzen 1992, De Jong 1998, Sloan et al. 2014) reveals complex origins and evolutionary histories of temperate taxa in relation with the complex paleoecological history of the area. High levels of species diversity are related to ancient events (Paleocene-Miocene) and also to more recent events (Pliocene-Pleistocene) followed by active speciation and even explosive radiations, leading finally to the emergence of the socalled "Mediterranean Sanctuary" of diversity, the second largest hotspot in the world and the largest of the world's five Mediterranean-climatic regions (Grabowski et al. 2017, Vargas et al. 2018. Albania belongs to the Eastern Mediterranean chorotype of the fauna, as proposed by Taglianti et al. (1999) including the NE-Mediterranean, Palestino-Cyprioto-Taurian, Palestino-Taurian, and Aegean distribution patterns of different animal species.
The eastern Mediterranean area, where the new member of the subgenus Mediotipula was collected, is recognised as an important centre of endemism, but no range-restricted Mediotipula species were detected in the area to date. Tipula (M.) gjipeensis sp. n. was identified by us only in a small, humid limestone gorge in Albania. We hypothesised a restricted distribution of the species, most probably as a result of the presently isolated distribution of humid ecosystems in the Mediterranean area. Several aquatic and semiaquatic organisms have similarly restricted or fragmented distributions in the Mediterranean area, which most probably followed the general decline of humid ecosystems during the late Cainozoic and simultaneous retreat in highly-fragmented local populations (sometimes highly-divergent evolutionary significant units, or in many cases distinct species) (Fernandez-Mazuecos et al. 2014, Godunko et al. 2017, Neubauer et al. 2016).
However, a number of case studies on plant species (Allegrucci et al. 2017, Crowl et al. 2015 hypothesise an evolutionary process in lineages that are adapted to pre-Mediterranean (pre-Pliocene) conditions in relatively small, xeric areas. Later, they became strongly competitive and expanded as the Mediterranean climate became dominant (Pliocene-Quaternary) across the Mediterranean Basin, and most probably also began to penetrate into the northern parts of Europe. Similar patterns were detected in the case of the Lacerta viridis complex (Cassel-Lundhagen 2010), with leading edge populations that colonised large parts of Europe, but several fringe populations became genetically divergent or morphologically different in highly isolated populations in the Balkan area. This pattern is also most likely true for the sibling pairs T. (M.) stigmatella / T. (M.) gjipeensis, with a larger distribution in the south-central part of Europe to Caucasus, but with morphologically deeply divergent T. (M.) gjipeensis from Albania, and less, but notable morphological differences between populations of T. (M.) stigmatella in the Balkan area, identified for example in Kavala (Greece) and Kulata (Bulgaria) ( Figure 6B, G). The presence of morphologically divergent allopatric populations of T. (M.) stigmatella (possibly cryptic species) is likely a result of more recent isolation, followed by repeated dispersions and area fragmentations during the Pleistocene climate change. This needs to be subjected to a more comprehensive analyses of the species over its whole range.

Faunistic and ecological notes
Albania is located in the western part of the Balkan Peninsula and is one of the most important Mediterranean biodiversity hotspots (Caković et al. 2018, Szabolcs et al. 2017, but is still considered to be the last "terra incognita" for European biodiversity. A series of recent studies, which focused on different groups of plants and animals, brings major contributions to the diversity and distribution of a number of organisms from Albania (Barina et al. 2013, Graf et al. 2018, Kaltsas et al. 2008, Stevanović et al. 2007). The description of T. Mediotipula gjipeensis sp. n. is an important addition to the faunal list of the Albanian Tipulidae and highlights the importance of similarly isolated pristine ecosystems to local and regional diversity of the Mediterranean Area.
Ecological demands of the majority of Mediotipula species are largely overlooked because of a high number of undescribed larvae. Based on adult distributions, species inhabiting open woods and hedges exposed to the sun seem to have generally larger distributions (e.g., T.  (Oosterbroek 2018). Such humid and sheltered "ecological islands" set in larger xeric areas could act as refuges for a number of rangerestricted or endemic species. For example, within the steep Gjipe Gorge (only a few kilometres in length), other than T. (M.) gjipeensis, the Mediterranean cranefly species Dolichopeza (D.) furiscipes Bergroth, 1889 was also detected (L Keresztes unpublished data), having a fragmented distribution in similar habitats in the southern part of continental Europe, but also on some Mediterranean islands (Mederos Lopez 2012, Oosterbroek 2018). The mayfly Electrogena hellenica Zurwerra & Tomka, 1986 has an even more restricted distribution in the Mediterranean area which is limited to western Greece, along rivulets in steep valleys at 300 m a.s.l. (Bauernfeind and Soldan 2012), but which was also collected by the first author of this paper from the Gjipe Gorge (L Keresztes unpublished data). Similar ecosystems in the Mediterranean area are rare and isolated but they are seriously affected by human activity (limestone and rock quarries, water extraction, habitat deterioration, discharge of sewage effluents, etc.) enforced by ongoing global climate change and therefore needing special attention and perhaps protection.