Baetis (Baetis) cypronyx sp. n., a new species of the Baetis alpinus species-group (Insecta, Ephemeroptera, Baetidae) from Cyprus, with annotated checklist of Baetidae in the Mediterranean islands

Abstract A detailed description of the larvae of Baetis (Baetis) cypronyx sp. n., a representative of the Baetis alpinus species-group within the mayfly family Baetidae, is provided, including a differential diagnosis with regard to closely related species of the group, especially Baetis melanonyx (Pictet, 1843) and Baetis baroukianus Thomas & Dia, 1984. The new species is mainly distinguished by mouthparts (i.e. the shape and setation of labrum, maxillary and labial palps, details of paraglossae and mandibular incisors), setation of legs and abdominal terga, and length of paracercus. All available data on the biology of this putative endemic species of Cyprus are summarized. Annotated distributional data of the 33 species of Baetidae so far recorded from the Mediterranean islands are given, including new records and also including first data from Malta.


Introduction
The first contribution to the Baetidae of Cyprus (Soldán and Godunko 2008) included the description of two new species from Cyprus and neighbouring island of Rhodos in Greece. Baetis mirkae Soldán & Godunko, 2008 of the Baetis lutheri species-group was found on both islands, and later considered as East Mediterranean (Pontomediterranean) species (Bauernfeind and Soldán 2012: 124). B. irenkae Soldán & Godunko, 2008 of the Baetis buceratus species-group is so far only known from three Cypriote localities and probably is endemic to Cyprus (Soldán andGodunko 2008: 95, Bauernfeind andSoldán 2012: 167). Two of these localities are in Limassol District (Kryos River at Kalidonia waterfalls and Diplos River at Chantara waterfalls) and were sampled during an extensive survey of aquatic invertebrates in May-June of 2004 (Soldán and Godunko 2008). Both localities revealed a relatively high diversity of benthic insects, namely high abundances of the mayfly genera Epeorus (Ironopsis), Electrogena, and Baetis (Baetis s. str., Nigrobaetis Novikova & Kluge, 1987). One species belonging to the Baetis alpinus species-group is described below as Baetis (Baetis) cypronyx sp. n.
The Baetis alpinus species-group was established by Müller-Liebenau (1969: 46) [i.e. alpinus-Gruppe] for three species, namely B. (B.) alpinus (Pictet, 1843) B (B.) melanonyx (Pictet, 1843) and B. (B.) nubecularis Eaton, 1898. This species-group with Holarctic distribution includes 12 Western Palaearctic species from Europe, Mediterranean, Minor Asia, and North Africa. According to Müller-Liebenau (1969), Jacob (2003), Soldán and Godunko (2009), and Bauernfeind and Soldán (2012), the distinguishing characters for this species-group can be summarized as follows: Larvae: (i) body flattened ventrally, with shortened abdomen; (ii) segments of antennal flagellum each shortened in the distal two thirds of the antenna; (iii) labrum usually with more than 6-7 (up to 22) long, submarginal setae; (iv) outer mandibular incisor group roughly triangular and often fused; (v) segment 2 of maxillary palp with one or more (sometimes numerous) stout setae on conical protuberance; (vi) pronotum with conspicuous dark pattern; (vii) sternal protuberances on meso-and metathorax more or less developed, pointed or rounded apically; (viii) outer margin of femora with medium or long bristles, acutely pointed or obtuse apically, arranged in 1-3 rows centrally and proximally; (ix) tarsal claws with a pair of fine subapical setae; (x) abdominal terga generally light, with marked dark spots centrally; (xi) posterior margins of abdominal terga with a row of triangular, more or less pointed spines; (xii) surface of abdominal terga usually without distinct corrugations, and usually covered with numerous, tongue-shaped, triangular or spatulate scales and their sockets; (xiii) paracercus more or less reduced (occasionally strongly reduced).
Imagines: (xiv) hind wings with three longitudinal veins, cross veins present or absent; (xv) abdominal terga relatively dark and translucent; (xvi) basal segment of forceps roughly cylindrical or subcylindrical, with inner, more or less expanded, conspicuous apicomedial projection, often forming a distinct rim; (xvii) forceps segment 2 subcylindrical, more or less constricted near base; (xviii) forceps segment 3 variable, egg-shaped or subcylindrical, nearly 2-3 times longer than wide.
Apart from the description of the new species, additional objectives of this contribution are to discuss its differential diagnosis and its difference to other representatives of the B. alpinus species-group, to summarise available data on the biology and distribution of the new species, and to present an annotated checklist of the Baetidae in the Mediterranean islands.

Material
Most specimens of the new species were collected in the Kryos River at Kalidonian Waterfalls; additional material was collected in Diplos River at Chantara Waterfalls (for numbers of specimens, their proper localities, and deposition see below). Holotype and 45 paratypes of the new species are housed in the Institute of Entomology, BC CAS (České Budějovice, Czech Republic), 22 paratypes in the collection of State Museum of Natural History NASU (Lviv, Ukraine), and 22 paratypes are stored in the Staatliches Museum für Naturkunde (Stuttgart, Germany). Additional paratypes are deposited in the collection of CNR-IRSA Water Research Institute (Brugherio, Italy).

Morphological study
The specimens were preserved in 70-80% ethanol. Eight paratypes were mounted on slides with Euparal liquid. Drawings were made using a Zeiss Axioplan microscope with a camera lucida. Photographs of larvae were taken using a Leica Z16 APO macroscope and processed with Leica Application Suite™ Version 3.1.8 to obtain combined photographs with enlarged depth of field. Photographs were subsequently enhanced with Adobe Photoshop™ CS3.
Specimens used for SEM were dissected and dehydrated through a stepwise immersion in ethanol and then dried by critical point drying (Leica EM CPD300). The mounted material was coated with a 5 nm Au/Pd layer (Leica EM ACE200) and subsequently examined and photographed with a Zeiss EVO LS 15 scanning electron microscope. SEMs were subsequently enhanced with Adobe Photoshop™ CS3.
Diagnosis. Baetis cypronyx sp. n. differs from all other representatives of the Baetis alpinus species-group by the following combination of larval characters (see Table 1): (i) labrum of distinctly oblong shape, nearly rectangular ( Fig. 6a-c), (іі) outer mandibular incisor group distinctly fused, narrow and triangular (Fig. 8); (iii) segment 2 of maxillary palps usually with single seta, exceptionally with two stout apical setae Table 1.

Cuticular coloration
General colour yellowish brown to brown. Head light brown with paler genae; clypeus light brown; frons with several small, isolated brown spots. Antennae light brown, flagellum paler than scape and pedicel.
Pronotum yellowish brown with two pairs of oblique brownish bands; mesonotum yellowish brown to brown, with longitudinal brown bands centrally, and several spots of the same colour centrally and laterally; metanotum brown with darker smudge centrally (Figs 1, 4). Lateral sides of thorax with brown pleurites (Fig. 2). Ventral side of thorax paler than dorsal side; sterna yellowish ( Fig. 3). Legs pale. Femora yellowish brown with two darker, usually isolated longitudinal spots along outer margin; tibia light brown; base and apex of tarsi brown, darker than middle part; tarsal claw brown (Figs 2, 3). Abdominal terga (Figs 1, 4) yellowish brown to brown; terga I-III (IV) and VI-VIII darker. Terga I-III (IV) brownish centrally, with broad pale area laterally; median brown spot on terga III and IV occasionally divided into two longitudinal spots; all terga with more or less well visible brownish band along anterior margin of segment; a pair of diffuse brownish maculae near posterior margin of terga V-VIII; a pair of brownish paramedian dots on terga II-X, terga III-VI occasionally with additional oblique streaks fused with paramedian dots and forming a diffuse brownish U-shaped pattern in anterior half of segment. Abdominal sterna with a pair of sublateral elongated spots. Cerci yellowish brown to brown, 3-5 first segments slightly darker.
Hypodermal coloration. Hypoderm without contrasting markings. Head. Surface of clypeus and frons covered with solitary FT, B, and Hr setae. Larval turbinate eyes brown to intensively brown apically. Antennae slightly longer than 1/2 of body length. Scape and pedicel with solitary FT and Hr, and more abundant B setae only, without any particular cuticular ornamentation (e.g. corrugation/chagrin; see Bauernfeind and Soldán 2012), which is present in some representatives of the B. alpinus species-group and in the closely related B. lutheri and B. pavidus species-groups.
Mouthparts. Labrum (Figs 6a-c) distinctly wider than long, nearly rectangular, with width/length ratio 1.80-1.88; dorsal surface with 1 + 11-18 long submarginal setae, arranged in a single irregular row (occasionally 1-4 bristles form an additional weekly defined row); 6-8 smaller setae laterally on both margins; dorsal surface of labrum covered with sparsely arranged B and only a few FT setae grouped mainly posterolaterally; ventral side with 2-5 small pointed setae anterolaterally. Median incurvation of middle part of anterior margin of labrum clearly shallow and wide.
Outer mandibular incisor group narrow and triangular, distinctly fused; inner incisor group not prominent, with 3-4 small teeth (of which most distal tooth is the biggest), both groups separated by a shallow incision. Right and left prostheca of same size, nearly symmetrical, with 8-10 apical teeth (Fig. 8).
Hypopharynx relatively slender, anterior side laterodistally covered with fine, elongated setae along outer margins of lingua and superlinguae, lingua with prominent central lobe, superlinguae with marked hump (Fig. 7).
Outer margin of femora with 2-3 rows of long bristles with obtuse to bluntly pointed tips proximally and centrally (Figs 13,20,21), and one row of shorter and stouter obtuse bristles distally; central part of outer margin of femora occasionally with long bristles arranged in 1-2 rows. Long marginal bristles alternating with submarginal STSm-bp setae and elongated Hr setae. Inner margin with 2-6 STSs-bp setae near to proximal end. Surface of femora with STSs-bp and STSs-ov setae and tiny setae [Hr and more abundant FT setae]. Outer and inner margins of tibiae with STSm-p and STSmbp setae and short Hr setae; surface of tibia with STSs-bp to nearly STSs-ov setae; a group of long Hr setae near distal end of outer margin of tibia. Tarsi with 6-10 middle to elongated STS-p setae along the inner margin, and several STSm-p and/or STSmbp setae on outer margin; both margins of tarsi covered with tiny Hr setae; surface of tarsi with a few FT and more abundant Hr setae, and small STS-bp setae. Tarsal claws not elongated, moderately hooked; with 10-11 teeth arranged in single row and two subapical tiny Hr setae (Figs 13, 14).
Abdomen. Posterior margin of terga with broad triangular spines of different size, bluntly pointed or occasionally pointed apically; broader spines along posterior margin of terga III-VIII; spines alternating with 1-3 tiny B and a single Hr setae. Irregular row of smaller submarginal spines on terga III-VIII (IX) (Fig. 22). Surface of terga with few, not elongated, tongue-shaped [SC-tg] to triangular [SC-it; bluntly pointed to rounded apically] scales, and their few sockets (mainly lacking on tergum X), concentrated on central part of segment (Figs 23, 24); solitary Hr and more abundant FT setae stretched over the whole surface of terga I-X. Posterior margin and surface of sterna without spines, stout setae or scales, with B and Hr setae only.
Paraproct plate as in Figs 16-18. Inner margin of paraproct with 8-12 spines of different size along apical half, alternating with tiny setae [solitary FT and more abundant B setae], and 2-8 (mainly 4-7) submarginal STSm-bp setae (Figs 16, 17); a single row of relatively small and stout spines along inner margin of cercotractor (for definition of cercotractor see Kluge 2004) (Fig. 18). Surface of paraproct covered with sparse FT, B and Hr setae and their bases only.
Tracheal gills whitish yellow to light brown, not elongated, broadly rounded apically (Fig.15, I-VII); gills I and VII nearly symmetrical; gills II-VI asymmetrical; serrated margins of gills more or less well marked, with tiny Hr setae inserting in small, articulated bases; tracheation poorly visible.
Cerci as long as 1.20-1.32 of body length. Paracercus reduced to 2-16 segments (Fig. 24). Posterior margin of cercal and paracercal segments with row of broad, triangular spines, and uneven submarginal row of smaller spines. Length of paracercus of mature larvae apparently variable in different populations, as well as in specimens within each population. Paracercus in larvae from Cryos River (type locality) vestigial (evidently shorter than abdominal tergum X, consisting of approximately up to 5-7 segments, some segments at least partially fused, Fig. 1); paracercus in paratype larvae from Diplos River strongly reduced (but evidently longer than abdominal tergum X), only consisting of about 10 or more apparently separated or distinguishable segments; Fig. 4).
Male and female adults. Unknown. Etymology. The specific epithet is a combination of the name of Cyprus, where the new species was found, and the specific epithet of the closely related species B. melanonyx.

Affinities
Baetis cypronyx sp. n. can be undoubtedly attributed to the B. alpinus species-group as defined above based on larval body shape and presence of (i) numerous submarginal long setae on dorsal surface of labrum, (ii) triangular outer mandibular incisor group, (iii) 1-2 stout setae at tip of maxillary palp segment 2, (iv) conspicuous brownish pattern on pronotum (similar to that in B. alpinus (Pictet, 1843)) and well visible pair of dark spots on abdominal terga, (v) numerous long bristles on outer margin of femora, The new species appears to be closely related to B. melanonyx known throughout Europe and to B. baroukianus  described from Lebanon. For the latter two species a separate subgenus Patites Thomas & Dia, 1999 was established based on larval and imaginal characters (Thomas and Dia 1999: 107; type species Baetis (Patites) baroukianus . On the other hand, Bauernfeind and Soldán (2012: 101) consider that the delimitation of taxa of B. alpinus species-group is rather difficult due to the high level of (probably clinal) variability combined with disjunctive area of many species. A separation of the B. alpinus species-group on genus or subgenus level is recently not considered to reflect phylogenetic lineages under the concept used for genera within Baetinae by these and other authors (e.g. Jacob 2003: 89; Bauernfeind and Soldán 2012: 101).
Differences between three above listed species can be observed in the arrangement of long setae on the dorsal surface of the labrum, i.e. B. cypronyx sp. n. with 1 + 11-18 long submarginal setae, in contrast to 1 + 14-21 long submarginal setae in B. melanonyx, and mainly 1 + 19-21 in B. baroukianus (Fig. 6a- fig. 27a; Thomas and Dia 1984: 8, fig. 2b). Additionally, in contrast to B. melanonyx with proximally narrowed labrum B. cypronyx sp. n. and B. baroukianus can also be characterized by a nearly rectangular labrum that is distinctly wider than long.
Two irregular rows of long, stout bristles can be observed on the tips of paraglossae in the new species, in contrast to 3 rows in B. melanonyx and 4-5 rows in B. baroukianus (see Table 1 and Fig. 9; Müller-Liebenau 1969: 62, fig. 27i; Thomas and Dia 1984: 8, fig. 6b).
Other differences concern the shape of labial palp segment 3, which is nearly symmetrical in B. cypronyx sp. n. and in B. melanonyx (quotient q from 0.76 to 0.94), in contrast to a markedly asymmetrical segment 3 in B. baroukianus, with q = 0.52-0.56 (Figs 12a-c; Thomas and Dia 1984: 9, figs 7a-c). Thomas and Dia (1984: 7, 8, figs 1b, 1m) depicted the heads of the female larva of B. baroukianus and B. melanonyx in dorsal view, discussing the head width ratio for both species. For B. baroukianus was noted that its head is widest below the eyes between the genae, while in B. melanonyx the widest part was determined at eye level. According to  the head width ratio for B. baroukianus / B. melanonyx below the eyes is 1.59 (with maximal value 1.46). However, in larvae of B. baroukianus from Iran that we examined, the width of head both at eye level and below eyes is nearly equal in both sexes, respectively (Figs 31, 32).
In contrast, female larvae of B. cypronyx sp. n. and B. melanonyx both have their maximal width of head at the level of eyes; the head width however is only slightly smaller at genal level below the eyes in both species (Fig. 5C, D). Similar proportions also apply for male larvae of the latter two species (Fig. 5A, B). The larval head width ratio for B. cypronyx sp. n. / B. melanonyx below the eyes at genal level is 1.05-1.15 in females, and 1.15-1.20 in males; at eye level the ratio is 1.00-1.05 in females, and 1.14-1.20 in males.
Baetis melanonyx and B. baroukianus markedly differ from the new species by their arrangement of setation at the outer margin of femora. There is only a single row of acutely pointed long bristles proximally and centrally, alternating with STSe-bp (in B. melanonyx) and STSm-bp (in B. baroukianus) submarginal setae, in contrast to Baetis cypronyx sp. n. that features 2-3 rows of bluntly pointed long bristles centrally and a group of STSm-bp submarginal setae (Figs 13, 20, 21). This character has been recently used for delimitation of two distinct evolutionary units of B. alpinus within the Central Alps (Leys et al. 2016), and much earlier for delimitation of B. alpinus and B. melanonyx (Figs 25, 26;Müller-Liebenau 1969;Godunko 1999: 26, fig. 3C).
The new species also clearly differs from B. melanonyx and B. baroukianus in the sternal protuberances near the coxae on meso-and metathorax that are pointed apically in the former species, in contrast to rounded apically protuberances in both latter species (Table 1).
Abdominal terga of B. melanonyx and B. baroukianus (including tergum X) are covered by numerous scale sockets, in contrast to only a few scales on terga of B. cypronyx sp. n., where scales and their sockets are missing on tergum X (see Figs 23, 24 for B. cypronyx sp. n.;Godunko 1999: 26, fig. 3D, and our Fig. 28 for B. melanonyx [the same for B. baroukianus]; Table 1); the shape of scales is similar in all three discussed species. The shape of marginal spines along the posterior margin of abdominal terga in all three species is generally similar, but the new species can be markedly recognized by the presence of not shortened stout spines and additional, submarginal, irregular row of smaller spines on terga III-VIII (IX) (see Fig. 22 for the new species in contrast to Fig. 27 for B. melanonyx); in B. baroukianus the single row of shortened stout spines is figured by Thomas and Dia (1984: 9, fig. 9). Marginal large spines alternating with Hr setae and with 1-3 setae of sensillum basiconicum type can be recognised in the new species (similarly to B. alpinus), in contrast to B. melanonyx and B. baroukianus showing a group of 1-5 setae.
Inner margin of paraproct plate of B. cypronyx sp. n. and B. melanonyx with more or less similar number of marginal spines (see Table 1), in contrast to B. baroukianus with not more than four spines only. Other differences between species discussed above can be recognized in the number and shape of submarginal stout setae, i.e. 2-8 STSsbp and STSm-bp setae in B. cypronyx sp. n., in contrast to 8-12 in B. melanonyx and up to 7 STSs-ov and STSm-ov setae in B. baroukianus.
Other differences between the closely related species B. cypronyx sp. n. B, baroukianus and B. melanonyx are summarized in Table 1. Thomas and Gazagnes (1984) described B. cyrneus Thomas & Gazagnes, 1984 from Corsica and placed this species in the B. alpinus species-group. Baetis cyrneus most probably also is an endemite of the Mediterranean islands (see below). It differs from B. cypronyx sp. n. by the arrangement of mouthparts, especially by the shape and setation of mandible with both groups of incisors well developed, segment 2 of maxillary palps with two regular stout setae apically, and by the elongated shape of labial palp segment 3. Additional differences can be observed in the paraproct plate, with numerous scale sockets on its surface, and in the length of paracercus with 10-25 segments.
2 Described by Thomas and Gazagnes (1984: 199) from Corsica. According to Bauernfeind and Soldán (2012: 105) only known from a few localities in terra typica (see also OPIE-benthos data). Nevertheless, Belfiore (1988), , and Buffagni et al. (2003) report B. cyrneus also from the Toscana Region and some Mediterranean islands, i.e. Sicily and Sardinia. DNA barcoding (Gattolliat et al. 2015) however revealed that specimens determined as B. cyrneus represent four different cryptic species occurring in Corsica and Sardinia. So far no morphological differences have been determined for these putative species. The high intra-specific genetic distance in B. cyrneus recently detected by Cardoni et al. (2015) for populations from Corsica and Sardinia also point to cryptic variation. 3 Recorded by  and  for the first time. 4 Recorded by Belfiore and Gaino (1988: 77) for the first time in Sardinia and later also listed from Sicily Buffagni et al. 2003); for Corsica based on OPIE-benthos data.

11
The species can be considered endemic to Corsica. This conclusion is confirmed by recent DNA barcoding (Gattolliat et al. 2015;Cardoni et al. 2015 Sartori and Thomas (1991: 224) used the specimens from the type series of B. albinatii also to specify distinguishing characters of representatives of the B. muticus species-group. 13 Two records of this species from the islands Kos (Belfiore 1990: 266) and Rhodos (Soldán and Godunko 2009: 9) belong to hitherto undescribed species. 22 Numerous records from Mediterranean islands. Russev (1959: 272)  The record from the Balearic Islands needs to be confirmed (Alba-Tercedor and Jáimez-Cuéllar 2003). Species inquirenda according to Bauernfeind and Soldán (2012: 191 Bauernfeind and Soldán (2012: 194). Taxonomical status and presence in Corsica needs to be clarified. 25 The record from the Balearic Islands needs to be confirmed (Alba-Tercedor and Jáimez-Cuéllar 2003). Most probably part of material belongs to C. simile. Species inquirenda according to Bauernfeind and Soldán (2012: 199). 26 The record from the Balearic Islands needs to be confirmed (Alba-Tercedor and Jáimez-Cuéllar 2003). Most probably part of material belongs to C. simile. Species inquirenda according to Bauernfeind and Soldán (2012: 199). 27 Several records from the Western and Eastern Mediterranean Region. The record from Corsica is based on OPIE-benthos data. The information published by Belfiore and D'Antonio (1991: 260) [sub. Cloeon gr. simile] needs to be clarified. Recently DNA barcoded by Cardoni et al. (2015). 28 So far known from several localities in Sicily and Sardinia Buffagni et al. 2003;Bauernfeind and Soldán 2012). Reported from Elba and Sardinia by Cardoni et al. (2015), with remarks on its possible presence in the Corse-Sardinian biogeographic region; based on DNA barcoding the specimens from Elba and Sardinia may however represent a cryptic endemic species as they differ significantly from specimens of Continental Europe (Cardoni et al. 2015). 29 So far only known from several localities in Rhodos (see Sowa 1985;Sroka et al. 2010); probably endemic to the island (Bauernfeind and Soldán 2012). 30 The record from Sicily by Buffagni et al. (2003) refers to an earlier record by Belfiore et al. (1991: 32) [sub. Pseudocentroptilum sp. gr. pulchrum).
According to a tabular summary by . P. pulchrum was considered absent from the island. Most probably recorded by Grandi (1966: 327) [sub. Centroptilum pennulatum Etn.] from Sicily for the first time. The problem with proper identification of material previously assigned to the P. pennulatum species-group is briefly discussed by Belfiore (1988),  and . The respective taxonomical status of this material needs to be clarified. 31 The single record from Lesbos [sub. Pseudocentroptilum motasi Bogoescu, 1947] by Keffermüller and Sowa (1984:  Flight period probably from May and during first half of summer months, since several nymphs ready to emerge were collected together with younger larvae.

Notes on distribution
As well as B. irenkae, a new species so far known only from several localities in Cyprus (type locality at Kryos River within Kalidonian Waterfalls, and another one locality at Diplos River within Chantara Waterfalls), and thus might be considered presently as endemic to this island (Table 2).

Annotated checklist of Baetidae in the Mediterranean islands
The history on the mayfly fauna of the Mediterranean islands dates back to the first published observations by Hagen (1864). In this contribution, seven mayfly species were reported from Corsica, including three species of Baetidae. Significant early publications dealing with the Corsican mayfly fauna and also including the description of new species were contributed by Esben Petersen (1912;1913). All other publications in the early 20 th century (Jakobson and Bianki 1905;Lestage 1922;Kimmins 1930) in fact were just compilations and summaries of H.A. Hagen's and M. Esben-Petersen's earlier investigations. The first records of the mayfly fauna of the Balearic Islands was published by Navás (1914). Literature on the distribution of Baetidae in the Mediterranean Islands however is scattered. The annotated checklist presented here (Table 2) provides the first comprehensive compilation of records of Baetidae in the Mediterranean islands incorporating also most recent records and findings along with detailed critical comments on previous records.