Clunio boudouresquei sp . n . and ThalassosmiTTia ballesTai sp . n . , two tyrrhenian marine species occurring in scandola nature reserve , west corsica ( diptera : chironomidae )

Clunio boudouresquei sp. n. and Thalassosmittia ballestai sp. n. are diagnosed and described based on associated material of male adults, pharate male adults and pupal exuviae recently collected in the marine littoral zone of Scandola Nature Reserve (Cala Litizia, Punta Palazzu, Focolara Bay, West Corsica). While C. boudouresquei sp. n. is described as male and female adults and pupal exuviae, T. ballestai sp. n. is described only as male adult and pupal exuviae. On the basis of some atypical characters found in the male adult and pupal exuviae, both C. boudouresquei sp. n. and T. ballestai sp. n. appear to belong, to a local Tyrrhenian element. Biological cycles of both new species are closely related to alternation between marine and terrestrial ecological conditions, which are strongly reinforced during spring tides of lunar rhythms. Larval stages of both new species are typically marine shore dwellers of the intertidal zone along seacoasts of the Tyrrhenian sub-region, where the biological and ecological quality of marine coastal habitats are being seriously damaged by various anthropogenic activities. In the latter sites, the Lithophyllum byssoides (Lamarck) Foslie pavements (trottoirs, encorbellements) are actually threatened by the massive proliferation of Sea Mussels (Mytilidae), which predominate when changes in water quality and level of pollution become increasingly high. The two new species are considered as pertinent biological indicators of the marine coastal habitats around the Tyrrhenian Region, in particular, those delimited by the mid-littoral large bio-constructions of the red calcified alga L. byssoides, where the larvae of C. boudouresquei sp. n. are exclusively confined. Such threatened species are considered biogeographic Tyrrhenian representatives and indicators of global warming and local climate change in the region, particularly to a rising sea level. Comments on the taxonomic position, ecology and geographical distribution of the new species are given.


Introduction
Recent investigations of marine chironomids conducted in the protected area of Scandola Nature Reserve (West Corsica), allowed us to sample fully developed pharate, adults, pupae and pupal exuviae of two new species, which belong to the genera Clunio Haliday, 1855 and Thalassosmittia Strenzke & Remmert, 1957. The two new species (C. boudouresquei sp. n. and T. ballestai sp. n.) were previously reported by Moubayed-Breil et al. (2013) and Moubayed-Breil & Ashe (2012) as, Clunio sp. 1 and Thalassosmittia sp. 1. Worldwide the genus Clunio is known from all geographic regions, while Thalassosmittia is restricted the Neotropical, Nearctic, Palaearctic and Afrotropical regions.
In this paper, C. boudouresquei sp. n. and T. ballestai sp. n. are described and diagnosed based on associated material recently collected in the marine littoral zone of Scandola Nature Reserve (Focolara Bay) located in western Corsica. While C. boudouresquei sp. n. is described as male and female adults and pupal exuviae, T. ballestai sp. n. is described only as male adult and pupal exuviae. On the basis of some atypical characters found in the male adult (shape of head, palpomeres, apodemes, tergite IX, inferior volsella, gonostylus) and pupal exuviae (distribution pattern of armament on tergites and sternites), C. boudouresquei sp. n. and T. ballestai sp. n. appear to belong, each to a local Tyrrhenian marine element.
Larval stages of both new species are typically marine shore dwellers of the intertidal zone along seacoasts, where dense populations are often reported. In particular, those of C. boudouresquei sp. n. are exclusively confined to the intertidal habitats delimited by the mid-littoral large bio-constructions (pavements, 'trottoirs') of the 'long-living' red calcified alga Lithophyllum byssoides (Lamarck) Foslie, which are locally present in both western Corsica (Punta Palazzu) and southern France. In addition, the latter pristine habitats are considered to be microrefugia for a well-diversified biocoenosis including marine and semi-terrestrial taxa/species. The two new species are considered as pertinent indicators of the environmental quality of seacoasts around the Tyrrhenian Region (insular and continental Provinces), where changes in biological and ecological conditions of the intertidal zone are presumably being the result of human activities and global warming in this region.

Material and methods
Material composed of adults, pharate adults and pupal exuviae belonging to both C. boudouresquei sp. n. and T. ballestai sp. n. was collected using standard methods: troubleau net for the benthos (larvae and pupae) and individuals floating on the surface of the water; Brundin drift nets towed behind a boat for pharates, pupae and drifted pupal exuviae; sweep net for flying adults. Additional inorganic material composed of plastics (micro-+ macroplastics) and pellet tar (0.5-5 to 10-15 mm), was also collected during the fieldwork, which were especially of great interest for useful comments on both biological and ecological quality of environment. Male adults were preserved in 80-85% ethanol, then cleared of musculature in 90% lactic acid (head, thorax, abdomen and anal segment) for 60 to 80 minutes; this 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 of the type and paratype material in dorsal view, the hypopygium including tergite IX and anal point, the gonocoxite and the gonostylus, were viewed ventrally and laterally to examine and draw from both sides all the necessary details of the species. In particular, the ventral view of hypopygium was illustrated when the anal point and tergite IX were removed. For a better examination of the specific features and more accurate description of the various taxonomic details of the pupa, the pupal abdomen was mounted not only in dorsal and ventral view, but separately in lateral view, which facilitates proper examination and illustration of all the relevant taxonomic characters. The proximal part of the abdomen and the halteres of the male adult were preserved in 85% ethanol for an eventual DNA analysis. Morphological terminology and measurements follow those of Saether (1980), Langton (1991) and Langton & Pinder (2007) for the imagines and pupal exuviae. Taxonomic remarks on some related known species from Europe with comments on the ecology and geographical distribution of the two new species are given. Paratypes (all leg J.M-B.): 2 male adults, 1 female adult, 6 pupal exuviae (4 males and 2 females), same locality as for holotype, 03.VI.2015.

Clunio boudouresquei
Holotype (mounted on 1 slide) and 2 pupal exuviae (1 male and 1 female) are deposited in the collections of the Zoologische Staatssammlung München (ZSM), Munich, Germany. Additional paratypes are deposited in the senior author's collection.

Diagnostic characters
Based on some characters found in the male adult (vertex with lateral projections, typical morphology of inferior volsella and both basal and caudal apodemes, presence of megaseta on gonostylus), C. boudouresquei sp. n. appears to belong to a local Tyrrhenian marine element. However, this new species can be distinguished from other European Clunio species by the blow listed characters.
Male adult: Vertex with two lateral triangular projections; antenna 10-segmented, last flagellomere longer than the 3 preceding segments; sensilla chaetica present on tibia and ta1 of PI-PIII; tergite VIII with a distinct elongate ellipse-like ridge located antero-medially, midline area with 6 short setae; apical expansion of tergite IX distinctly convex at apex; caudal apodeme with 5-6 curved claw-like tubercles; inferior volsella wider at base and narrowing distally; gonostylus unusually bearing a black fingernail-like megaseta, apex ending with a single finger-like tubercle.
Pupal exuviae: Antero-median area of frontal apotome and thorax with wrinkles; frontal setae present on distal part of frontal apotome; dorsocentrals Dc 1 -Dc 2 and Dc 3 -Dc 4 located close together; anterior transverse rows of spines interrupted on tergite II; posterior transverse rows of hooks present on sternites V-VII.
Etymology: the new species is named 'boudouresquei' in honour of our colleague Ch-Fr. Boudouresque (University of Sciences, Luminy, Mar-seille), who is still active in studying the biology and ecology of the Mediterranean marine flora and fauna including those of the protected area of Scandola Nature Reserve. As he always did in past, he keeps working on developing projects to preserve the marine protected area of Scandola Nature Reserve, which represents a precious and valuable inheritance area. (n = 5,2 pharates; Total length 2.70-2.90 mm. Wing length 1.35-1.40 mm, TL/WL = 2-2.10. General colouration contrasting brown to dark brown. Head and antennae dark brown; thorax contrasting light brown to brown with dark brown mesonotal stripes; wing pale translucent; legs brown to dark brown; tergites I-VII brownish, tergite VIII and anal distinctly contrasting light brown to dark brown.

Female adult
(n = 2, 1 pharate; Figs 1i-j, 4a-e) Small sized species. Total length 1.65-1.70 mm. General shape is Clunio female-type. Colouration as in the male adult except for the thorax, which is less dark. Antennae light brown; legs brownish with blackish claws. Abdominal tergites and anal segment contrasting brown to dark brown. Head.
Abdomen. Anal segment (dorsal, Fig. 4a    Abdomen. Armament, chaetotaxy, distribution pattern of shagreen and details of armament on tergites and sternites II-VII as in Figs 5a, 5c, 5e-f. Tergite I and sternites I-III bare, sternite IV occasionally with 1-2 rows of small spines (Fig. 5a). Conjunctives of tergites III-VII and sternites V-VII with one transverse row of hooks, which are smaller on sternites, conjunctive on segment VIII composed only of short posteriorly directed spines. Antero-median transverse rows of spines present on tergites II-VII, sparsely present and interrupted medially on tergite II, becoming denser and more extensive on tergites III-VI. Pedes spurii A and B absent; apophyses on tergites and sternites absent. Number and distribution pattern of lateral setae on segments I-VIII: 2 on segment I; 2/3 on II-VII; 3 on VIII. Anal segment is Clunio-type, genital sac 490-500 µm long, 70 µm maximum width, ending each with 1 pointed tubercle.

Larva
Known but not described.

Differential diagnosis
Male adult and pupal exuviae of C. boudouresquei sp. n. are compared to those of known Clunio species from seacoasts of Europe and neighbouring areas, based on material collected by the senior author in Corsica, continental France, Italy, Spain except for Bulgaria (Varna seashores, leg. P. Michailova). Some relevant specific features found in the male adult and pupal exuviae will easily separate the new species from other members of Clunio by the following combination of characters: Male adult: Frontal area of head bearing 2 apical projections (Figs 1c-d), is differently shaped in C. marinus (Figs 1a-b); last flagellomere narrowed apically (Fig. 1f), is widely clubbed in C. mediterraneus (Fig. 1k) and linearly curved in C. sp. 1 (Fig. 1l); typical long finger-like expansion of left palp (Fig. 1g), is absent in both C. sp. 2 (Fig.  1m) and C. marinus (Fig. 1n); caudal apodeme composed of basal brush-like and 5-6 apical claws ( Fig. 2b), is lacking basal brush and less branched apically in C. mediterraneus (Fig. 2e); megaseta present on gonostylus (Fig. 2c), is absent in C. mediterraneus (Fig. 2f) and C. marinus (Fig. 2h); apex of gonostylus with only one single finger-like tubercle (Figs 2c-d), while consists of several unequal tubercles in both C. sp. 1 (Fig. 2g) and C. marinus (Figs 2h-i); basal and apical parts of axial apodeme (Figs 3a-b), are differently shaped in C. mediterraneus (Figs 3c-d and C. sp. 1 (Figs 3e-f).

Ecology and remarks
The immature stages of Clunio spp. are typically marine dwellers of the intertidal zone along the littoral and mid-littoral zones of rocky shores, sometimes in association with populations of Mytilus spp. In some species (in particular those associated with Lithophyllum beds) the emergence of the adults is synchronized with the lunar cycle (Neumann 1976, Neumann et al. 1997, Kaiser & Heckel 2012. The biological cycle (reproduction and emergence) of C. boudouresquei sp. n. is closely related to the typology of the intertidal zone including alternation between submerged marine habitats and terrestrial ecological conditions, which are strongly reinforced during spring tides of lunar rhythms (new and full moon).
The pavements, 'trottoirs' of L. byssoides represent a combination of habitats that typically characterize the intertidal zone of the protected area of Scandola Nature Reserve. They mainly consist of a pristine combination of habitats considered to be microrefugia for a dense and diversified community of marine, semi-aquatic and semi-terrestrial species, including members of several closely integrated dipteran families (Chironomidae, Ceratopogonidae and Dolichopdidae). The newly described species is encountered in the marine mid-littoral zone of Punta Palazzu (Fig. 6), where larval stages occur exclusively within the large bio-constructions of the 'long-living' red calcified alga L. byssoides, which clearly delimit alternate cycles of both submerged and terrestrial habitats. In addition, the bio-concretions of Punta Palazzu are currently considered as the largest Lithophyllum beds in Europe, where valuable knowledge on the biology (growth rate) and ecology of the algal communities are documented by Verlaque (2010).  While the biological and ecological quality of L. byssoides rims are still well-preserved at Punta Palazzu and Port-Cros Island (Figs 6-7), other similar marine sites located along the coastal Mediterranean ecosystem of continental France are becoming extinct, or have been deeply damaged and degraded (Figs 8-9) during the last four decades by human activities, including ecotourism and release of toxic chemical pollutants (e.g., HAP, PCB, abundance of macro-and microplastics). In addition, the L. byssoides beds delimited by the latter endangered sites, are heavily threatened by a massive proliferation of an invasive Mytilidae species (Mytilus galloprovincialis Lamarck, 1819). This sea mussel significantly predominates when changes in water quality and level of pollution become increasingly high ( Consequently, in some of the Tyrrhenian mid-littoral coastlines (Punta Palazzu, Port-Cros, Banyuls), some relevant and vulnerable Clunio species are closely confined to the Lithophyllum beds, and therefore their loss would be clearly indicative of a combination of anthropogenic impacts and global warming in this geographical region. Such relict Tyrrhenian species are considered as potentially biogeographic representatives and biological indicators of local climate change (in particular, the rise of sea level), which strongly affect both sustainability and viability of the Clunio populations.

Geographical distribution
Geographical distribution of known Clunio species from European seacoasts (Ashe & O'Connor 2012) and the Tyrrhenian sub-region is given in in Figure 10.

Diagnostic characters
Though the pupal exuviae of T. ballestai sp. n. apparently shows a close morphological resemblance with that of T. thalassophila (distribution pattern of armament on tergites and shape of anal lobe), some relevant specific characters found in the male adult (shape of tergite IX, anal point and inferior volsella) will sufficiently separate the species described here from other related members of genus Thalassosmittia by the below listed characters.
Male adult: Temporals with 3 inner and 3 verticals; last flagellomere of antenna distinctly clubbed, abruptly narrowing distally and bearing a brush of curved sensilla chaetica; antennal groove reaching segments 2, AR 0.72; lobes of antepronotum widely opened; antepronotals absent. Brachiolum with 1 seta, veins and squama bare. Tarsomere ta 5 of PI-PII wider and rounded apically; spurs present on tarsomeres ta 1 -ta 4 ; sensilla chaetica present on tibia and tarsomeres ta 1 -ta 4 of PI-PIII. Tergite anal band absent on tergite IX; anal point drop-like shaped and bearing a rounded setiferous lobe at base; virga with 2 closely grouped spines; inferior volsella rounded lobe-like shaped with bifid basal margin; gonostylus distinctly swollen at base and thinner distally when viewed laterally.
Pupal exuviae: Frontal apotome with sub-cylindrical tubercles; dorsocentral Dc 1 distances between dorsocentral Dc 1 vestigial about 5-7 µm long; Dc 1 and Dc 2 separated by 25 µm, Dc 2 and Dc 3 by 70 µm; transverse row of hooks and orally directed pins present on conjunctives of sternites IV/V-VII/VIII is occasionally absent on IV/V; anal lobe sub-trapezoidal, genital sac distinctly swollen distally and bearing an apical finger-like tubercle. Pedes spurii A and Pedes spurii B absent. Anal lobe sub-trapezoidal to sub-triangular, bearing 2 subequal macrosetae on dorsal side; genital sac swollen distally, bearing a projecting outwards tubercle.
Etymology: the new species is named 'ballestai' in honour of our colleague Laurent Ballesta (Andromède Océanologie, Carnon, South France) who is still an active marine biologist studying and preserving the Mediterranean marine fauna and flora including the protected area of Scandola Nature Reserve, which represents a precious and valuable inheritance area.
Gonocoxite about 265 µm long, 75 µm maximum width, much wider at base, narrowing distally to a rounded apex, inner margin with 10-11 stout setae. Inferior volsella large lobe-like shaped, rounded apically with posterior margin distinctly bi-lobed, apical and caudal parts contrasting and hyaline. Gonostylus in dorsal (Fig. 11e) and lateral view (Fig. 11h) 105 µm long, 30 µm maximum width; swollen medially and less wide distally when view laterally; posterior margin sinuous and swollen medially; crista dorsalis low and widely extended Abdomen. Armament, chaetotaxy and distribution pattern of shagreen with details of armament on tergites and sternites: III-VIII (Fig. 12e); V-VII (Figs 12f-k). Tergite I and sternites I-IV bare. Anterior transverse rows of spines present on tergites II-VIII, those on tergites VII-VIII are smaller and less extensive; posterior transverse rows of spines present on tergites III-VIII, becoming gradually more extensive on VI-VIII. Conjunctives of tergites III/IV-VII/VIII and sternites IV/V-VII/VIII with rows of hooks and orally directed pin-shaped setae (Figs 12e, 12h-k), those on sternite IV/V are occasionally absent (Fig. 12g). Caudo-lateral area of tergites and sternites II-VII with a group of short spines (Figs 12e-k). Pedes spurii A and PSB absent. Number and distribution pattern of lateral setae on segments I-VII, 2, postero-lateral seta on segments V-VI forked; segment VIII with 3 setae located distally. Anal segment in dorsal and ventral view as in Fig. 13e; anal lobe sub-trapezoidal to sub-triangular, 130-135 µm long, 135-140 µm minimum width at base, 185-190 µm maximum width at apex, a rounded patch of short spine present medially, apex bearing a finger-like tubercle which is projecting outwards; genital sac 220 µm long, distinctly swollen distally and overreaching apical margin of anal lobe by 70-75 µm; macrosetae consist of 2 subequal setae, about 90-95 µm long, separated by about 35-40 µm.

Differential diagnosis
Only the pupal exuviae of T. ballestai sp. n. directly key close to those of T. thalassophila, while the male adult is quite different and likely belongs to a local 'Tyrrhenian element'. On the basis of some relevant specific characters found in the male adult and pupal exuviae, T. ballestai sp. n. is compared, as male adult, to that of T. thalassophila and, as pupal exuviae, to other undescribed morphotypes collected in Corsica, continental France, Italy and Spain. However, T. ballestai sp. n. is easily distinguished from other related species or taxa/species of Thalassosmittia by the following combination of characters.

Ecology and remarks
The examined material of T. ballestai sp. n. (male adults, male pharate adults and pupal were collected in the type locality of Focolara Bay (Fig.  14) located in Scandola Natural Reserve, western Corsica. Additional material including associated larval stages is needed to determine and confirm the ecology of the new described species. While the marine intertidal zone at Focolara Bay is better preserved during winter and spring periods, it still heavily degraded, as other seacoasts around the Mediterranean Basin, by the impact of pollution and ecotourism activities, which highly increase each year between June and September.
The increasing abundance of plastics along the coastal ecosystem, estuarine zones and the littoral marine environment of Corsica (Bastia, Ajaccio, Porto, etc.) including those of Scandola Nature Reserve, has been observed since the last three decades. Inorganic matter, composed of pellet tar and plastics, collected in both drift and troubleau nets (as shown and detailed in Figs 15-16), has become more dominant major threat to all types of marine organisms. Small particles of plastics, consisting of several forms of both macro-and micro-particles of 0.5-5 to 10-15 mm size, are systematically found from the surface and water column to the seabed sediment and beach, where marine organisms (especially deposit feeders and detritivores) ingest them. Therefore, these pollutants may adversely impact species that inhabit intertidal zones that cannot adapt to changing conditions via behavioural plasticity; T. ballestai may be among these species. Consequently, a major challenge in marine environmental disciplines (evolutionary biology, ecology, conservation) is to better understand and predict how these sensitive species will respond to the impact of human activities and the rise of sea level, which is directly related to the global warming.

Geographical distribution
Geographical distribution of known Thalassosmittia species from European seacoasts (Ashe & O'Connor 2012) and the Tyrrhenian sub-region is given in figure 10. Thalasosmittia atlantica '✩' is known only from the western Atlantic seacoasts including the Canary Islands (type-locality), Madeira, Spain and Portugal. Thalasosmittia ballestai sp. n. '❄︎ ' is common and abundant in the seashores of Focolara Bay (West Corsica). The records of T. ballestai sp. n. (pupal exuviae) from some seashores in southern France need to be confirmed by the presence of associated material composed of adults and pupae. Thalasosmittia thalassophila '✤' is present along the Atlantic and some Mediterranean seacoasts in Europe including: France, England, Germany, Greece, Ireland, Italy, Netherland, Romania and Spain.