Rheocricotopus ( s . str . ) costai sp . n . AND R . ( s . str . ) pyrenaeus sp . n . , TWO RELICT SPECIES FROM GLACIAL RHEOCRENES AND STREAMS IN CORSICA AND THE EASTERN PYRENEES ( DIPTERA : CHIRONOMIDAE , ORTHOCLADIINAE )

Two new species of the genus Rheocricotopus subgenus Rheocricotopus (R. costai sp. n. and R. pyrenaeus sp. n.) are diagnosed and described, based on material collected in some glacial rheocrenes and streams located in the high mountains of Corsica and the Eastern Pyrenees. Rheocricotopus costai sp. n. is described as male and pupal exuviae, while R. pyrenaeus sp. n. is described as male and female adults and pupal exuviae. Rheocricotopus costai sp. n. is known from both western Corsica and the Eastern Pyrenees, while the geographical distribution of R. pyrenaeus sp. n. is restricted to the protected area of the Mantet Nature Reserve (Eastern Pyrenees). Larvae of both R. costai sp. n. and R. pyrenaeus sp. n. are exclusively rheophilic being confined to lotic habitats located at high altitude (crenal and rhithral). Apart from the presence of an additional median circular small patch of spinules on tergite III of the exuviae, R. costai sp. n. directly keys into the effusus-group on the basis of several specific characters found in the male adult. Nevertheless, R. pyrenaeus sp. n. keys near both of R. reduncus Sæther & Schnell, 1988 (known from Finland, Norway and Russian Far East) and R. tchernovskii Makarchenko & Makarchenko, 2005 (known from Russian Far East), based in particular, on the unusual shape of the superior volsella which is inwardly markedly turned over distally. The genus Rheocricotopus is currently represented by 10 species in continental France and by 8 species in Corsica (Moubayed-Breil 2016). Consequently, the description of R. costai sp. n. and R. pyrenaeus sp. n. increases the total number in the genus to 12 for continental France and to 9 for Corsica. Taxonomic remarks, discussion and comments on the ecology and geographical distribution of the two new species are given.


Introduction
Data on the taxonomy and geographical distribution of the known Rheocricotopus species from Europe and some neighbouring areas (Brundin 1956 The genus Rheocricotopus has been revised and divided into two subgenera (Psilocricotopus and Rheocricotopus) by Saether (1985), who recognised and created three different groups within each subgenus: godavarius-group, chalybeatus-group and atripes-group for Psilocricotopus subgenus; effusus-group, fuscipes-group and tuberculatusgroup for Rheocricotopus subgenus.
Rheoccricotopus costai sp.n. and R. pyrenaeus are diagnosed and described based on material collected in some glacial rheocrenes and streams located in the high mountains of Corsica and the Pyrenees.Rheoccricotopus costai sp.n. is described as male adult and pupal exuviae, while R. pyrenaeus sp.n. is described as male and female adults and pupal exuviae.
The two new described species directly key into the effusus-group created by Saether (1986) for known Rheocricotopus species (Nearctic, Palaearctic and Oriental Regions), on the basis of the following characters found in the male adult and pupal exuviae: humeral pit reduced or ellipsoid with smaller separate ellipsoid pit below; superior volsella rounded or right-angled with distal short or long pronounced projection; gonostylus with or without long crista dorsalis; frontal setae present on frontal apotome; median circular patch of spines/or strong spinules present on tergites IV-VI.Meanwhile the male adult and pupal exuviae of R. costai sp.n. show some morphological affinities with some members of the effusus-group, those of R. pyrenaeus sp.n. keys near both R. reduncus Saether & Schnell, 1988 and R. tchernovskii Makarchenko & Makarchenko, 2005 on the basis, in particular, of the unusual shape of the superior volsella which is markedly turned over inwards distally.Moreover, R. costai sp.n. seems to belong to a local 'Pyreneo-corsican element', based on the presence of an additional small median patch of spinules on tergite III of the exuviae.Consequently, a fourth additional group within the subgenus Rheocricotopus, the 'reduncusgroup', could be created which would include, R. reduncus, R. tchernovskii and R. pyrenaeus sp.n.
In this paper, R. costai sp.n. (known from western Corsica and France) and R. pyrenaeus sp.n. (known from south western France) are diagnosed and described based on material collected in glacial springs and streams at high altitude.Rheocricotopus costai sp.n. is identical with 'R.(Rh.) sp.1' in Moubayed-Breil & Ashe (2012), while R. (Rh.) pyrenaeus sp.n. is the same as 'R.(Rheocricotopus) sp.1' in Moubayed-Breil & Ashe (2016).The first new species (R. costai ) is described as male adult and pupal exuviae, the second (R. pyrenaeus ) as male and female adults and pupal exuviae.

Materials and Methods
The examined material was collected using some standard methods: Surber net for the benthos (larvae and pupae); Brundin drift nets for pharates, pupae and drifted pupal exuviae; troubleau net for individuals floating on the surface of the water and a sweep net for flying adults.Male adults were preserved in 80% Ethanol, then cleared of musculature in 90% lactic acid (head, thorax, abdomen and anal segment) for about 60 to 80 minutes, but can be left overnight at room temperature without any detrimental effect or damage.The specimens were checked under a binocular microscope after 20 minutes in lactic acid to determine how the clearing was progressing.When clearing was complete the specimens were washed in two changes of 70% Ethanol to ensure that all traces of lactic acid were removed.
The studied material was mounted in polyvinyl lactophenol.Before the final slide mountings (dorsally) of the male holotype and paratype material, the hypopygium including the IX th tergum, the anal point, the gonocoxite and the gonostylus, were viewed ventrally and laterally to examine and draw from both sides all the necessary details of each species.In particular, the ventral view of the hypopygium was illustrated when the anal point and tergite IX were removed.
Part of the abdomen and the halters of the male adults were preserved in 85% ethanol for an eventual DNA analysis.Morphological terminology and measurements follow that of Saether (1980Saether ( , 1985) ) and Langton & Pinder (2007) for the adults and Saether (1980) and Langton (1991) for the pupal exuviae.Taxonomic remarks on some related species from Europe and neighbouring geographical areas, as well as discussion and comments on the ecology and geographical distribution of the two new species are provided.

Material examined
Holotype.Corsica. 1 male adult, leg.J. Moubayed-Breil, glacial stream, upper basin of the Asco River at the locality of 'High-Asco', (42° 27 ' 13 The Holotype (male adult, on one slide) is deposited in the collections of the National Museum of Ireland, Kildare Street, Dublin 2, Ireland.Remaining paratypes are deposited in the senior author's collection.
Type material was preserved in 80% ethanol, and later mounted in polyvinyl lactophenol.For each adult, the head, thorax and abdomen were cleared in 90% lactic acid, then washed in 70% ethanol before mounting on slides.
Etymology: The new species is named "costai" in honour of Jacques Costa (president of the Nature Regional Park of Corsica), who is actively contributing to preserving the environment and species associated with all aquatic habitats occurring in the protected areas of Corsica.

Diagnostic characters
Apart from the presence of an additional median small patch of spinules on tergite III of exuviae, R. costai sp.n. belongs to the effusus-group based on some other characters found in the male adult: humeral pit elongate ellipse-like, superior volsella with a pronounced distal projection.However, this new species can be distinguished from other related members of the effusus-group in having: -male adult.Coronal area with 2 coronals (1 seta on each side); lobes of antepronotum not gaping and thinner medially; tarsomere ta5 of PI, PII and PIII entirely blackish; sensilla chaetica present on tarsomeres ta4-ta5 of PI, PII and PIII; humeral pit extremely elongate ellipsoid, narrowing distally, with a distinct smaller separate oval pit below; tergite IX with a weak hump medially, which is clearly visible in lateral view; anal point with 12-14 lateral setae, apex unusually rounded; superior volsella broadly bent downwards, nose-like apically, distal part short and distinctly projecting downwards; inferior volsella long, finger-like in shape, dorsal side large and lobe-like, ventral side triangular; posterior side of gonostylus swollen medially and abruptly narrowing apically, anterior side with 2 rows of setae at obtuse angle; crista dorsalis short, tooth-like, only visible when viewed laterally and at right-angle, located distally and occupying 20 to 30% of the total length of the gonostylus.
-pupal exuviae.Frontal apotome weakly covered with wrinkles; thoracic horn finger-like and strongly toothed on one side.Circular median patch of spines and spinules present on tergites III-VI; patch on tergite III smaller and armed with short spinules; patches on tergites IV-VI are subequal, more extensive and armed with much bigger and longer spines.
Holotype (male adult + its pupal exuvie, on 1 slide) is deposited in the collections of the National Museum of Ireland, Kildare Street, Dublin 2, Ireland.Paratypes are deposited in the senior author's collection.
Type material was preserved in 80% ethanol, and later mounted in polyvinyl lactophenol.For each adult, the head, thorax and abdomen were cleared in 90% lactic acid, then washed in 70% ethanol before mounting on slides.

Etymology:
The new species is named 'pyrenaeus' after the Pyrenees mountains where its geographical distribution is currently restricted to the protected area of the 'Mantet Nature Reserve', (located in the Eastern Pyrenees of France), which covers glacial springs, peat bogs and pristine cold streams.

Diagnostic characters
Rheocricotopus pyrenaeus sp.n. directly keys near both R. reduncus (Finland, Norway and Russian Far East) and R. tchernovskii (Russian Far East) in the effususgroup based, on the following two main imaginal and pupal characters: distal part of superior volsella markedly turned over and curved inwards; median circular patch of spinules on tergite IV much smaller than those on tergites V-VI.However, this new species can be separated from other related members of the effusus-group in having: -male adult.Coronal area with 4 coronals (2 setae on each side); lobes of antepronotum thick and widely gaping medially; distal half of tarsomere ta5 of PI, PII and PIII blackish; humeral pit ellipsoidal, moderately elongate, larger in proximal part with a distinct smaller half ovallike pit below; tergite IX bearing a distinct hump medially, which is clearly visible in lateral view; apex of anal point sharply pointed; superior volsella broad, noselike apically, distal part distinctly projecting and turned over inwards; dorsal side of inferior volsella triangular with large thumb-like apex, ventral side triangular; posterior side of gonostylus swollen medially in both lateral view and at right-angles, distal part constricted near the apex; crista dorsalis clearly visible in lateral and at right-angles, widely extended distally from middistance till apex, semicircular and occupying 70 to 80 % of the total length of gonostylus.
-pupal exuviae.Frontal apotome nearly semicircular, anterior half covered with faint and fine wrinkles; thoracic horn markedly clubbed and toothed on one side.Circular median patch of spines present on tergites IV-VI; patch on tergite IV distinctly smaller and armed with short spinules; patches on tergites V-VI are subequal, more extensive and armed with much longer and larger spines.

Pupal exuviae
(n = 4: 2 males and 2 females; Figs.60-66) Total length 3.65-3.75mm.General colouration brownish.Frontal apotome brown, anterior half covered with fine wrinkles.Anterio-median area of cephalothorax and suture of thorax markedly rugulose and wrinkled; base of thoracic horn and wing sheath with blackish shading; outer and inner margin of antennal sheath brownish.Abdomen brown yellowish, lateral margin of segments I-VIII brownish.Anal lobe and genital sac

Larva
Known but not described.

Taxonomic position
Though R. costai sp.n. apparently shows a closely phylogenetic relationships with the effusus-group (shape of the humeral pit and gonostylus), some other relevant specific characters found in the male adult and exuviae (lobes of antepronotum thin; apex of anal point rounded; distal part of superior volsella short and widely projecting downwards; tergite III of exuviae bearing a distinct median circular patch of spinules) allowed us to consider this new species as a separate local 'Pyreneo-corsican element'.Nevertheless, due to some specific characters found in the male adult and exuviae of R. pyrenaeus sp.n. (distal part of superior volsella markedly inwardly turned over distally; circular median patch of spines on tergite IV distinctly smaller than those on tergites V-VI), this new species keys near both R. reduncus (known from Norway) and R. tchernovskii (known from Far East Russia), based in particular, on the elongate distal part of the superior volsella which is markedly turned over inwards.Therefore, a fourth additional group within the subgenus Rheocricotopus, the 'reduncus-group', could be created to include the three following species: R. reduncus, R. tchernovskii and R. pyrenaeus sp.n.However, the specific characters found in the male and female adults and pupal exuviae will separate the two new described species from other related known members of the effusus-group, on the basis of the following combination of characters.

Ecology and geographical distribution
Rheocricotopus costai sp.n. and R. pyrenaeus sp.n. are both rheophilic species exclusively encountered in lotic habitats (peat bogs, springs, upper streams) with siliceous water and low water conductivity.Localities where larvae and pharate adults were collected consist of moderately to weakly shaded ruisselets and cold mountain streams.Bryocolous and hygropetric habitats including waterfalls probably represent the most common aquatic areas for larval populations.Environmental data of habitat recorded along the crenal and upper rhithral of the Rivers Asco (Corsica) and Mantet (Pyrenees) are: siliceous water, low value of conductivity (20-40) µS/cm; pH 5.5-5.7;temperature 6-12°C.Such pristine habitats, which are endangered by pastoralism and both natural and accidental flooding, deserve much greater consideration, protection and preservation.Material was collected in some glacial helocrenes and streams delimited by well preserved and protected areas covered by local Nature Reserves located in both Corsica and the Eastern Pyrenees.
The two new species are regarded as typical relict representatives of glacial helocrenes and cold stenothermic streams.They belong to the crenobiontic and crenophilous community of species as documented by Lindegaard (1995).The discovery of R. costai sp.n. and R. pyrenaeus sp.n. in such preserved lotic habitats highlights the importance of glacial springs and streams, which are considered to be microrefugia and hotspots of diversity.
Rheocricotopus costai sp.n. is known from both western Corsica and the Eastern Pyrenees, while the geographical distribution of R. pyrenaeus sp.n. is currently restricted to springs and streams located in the Eastern Pyrenees.In particular, R. costai sp.n. can be expected to occur in other similar areas all around both the continental and insular Tyrrhenian Provinces (Italy, Spain).This indicates and reinforces the importance of headwaters and cold enclaves in the preservation and persistence of autochthonous glacial relict species, which can be considered as biological indicators of the global warming and climate change in the Mediterranean biogeographical region.

Table 2 .
Abdomen.Hypopygium in dorsal and ventral view (Figs.40-41).Tergite IX semicircular, with a distinct rounded median hump, which is clearly visible in lateral view.Anal point about 45-50 µm long, triangular and slightly curved downwards, large at base and uniformly narrowed distally with a sharp pointed apex; 12 setae are present laterally (6 on each side).Latero-sternite IX with 6 setae.Transverse sternapodeme and phallapodeme as in