Corrections and Additions to Descriptions of Some Species of the Subgenus Orthocladius s. str. (Diptera, Chironomidae, Orthocladiinae)

Simple Summary The biodiversity study and conservation are primary objectives in preventing loss of species consequent to global climatic change. The present contribution adds information to a key taxon, a holometabolous insect belonging to Order Diptera, family Chironomidae, whose larvae are members of freshwater macroinvertebrate fauna and are considered good indicators of water quality. Abstract The larvae of some species of the subgenus Orthocladius s. str. (Diptera, Chironomidae) are here described for the first time with corrections and additions to the descriptions of adult males and pupal exuviae. The identification of larvae is generally not possible without association with pupal exuviae and/or adult males, so the descriptions here are based only on reared material or on pupae with the associated larval exuviae. Usually, Chironomidae larvae can be separated on the basis of morphometric characters, the most discriminant ones are: (1) the ratio between the width of median tooth of mentum (Dm) and the width of the first lateral tooth (Dl) = mental ratio (DmDl), (2) the ratio between the length of the first antennal segment (A1) and the combined length of segments 2–5 (A2–5) = antennal ratio (AR). The shape of mandible, maxilla, and other body parts are almost identical in all the species considered in this study. The larva of Orthocladius (Symposiocladius) lignicola is very characteristic and can be separated by the shape of mentum and the larvae of all the known species of Symposiocladius are characterized by the presence of large Lauterborn organs on antennae and of tufts of setae on abdominal segments. The larvae of Orthocladius (Orthocladius) oblidens and Orthocladius (Orthocladius) rhyacobius can be distinguished from other species basing on their large Dm and to each other by AR. A principal component analysis was carried out using 5 characters: (1) Dm, (2) Dl, (3) length of A1, (4) width of A1 (A1W), (5) combined length of segments 2–5 (A2–5). The most discriminant characters were Dm and A1, confirming that DmDl and AR can be used to separate species at larval stage, but the large superposition of morphometric characters in different species confirms that association with pupal exuviae is in any case needed to identify larvae. In future perspective, the development of reference DNA barcodes from specimens identified by specialists is recommended since possibly the best tool for larvae identification, but association of barcodes with morphotypes is in any case fundamental.


Introduction
The genus Orthocladius Wulp, 1874 is one of the richest in species within the Chironomidae subfamily Orthocladiinae [1]. It is among the most difficult genera to morphologically identify, as larvae of many species are quite similar and almost impossible to be separated guaranteed with aeration. A photoperiod of 14 h of light and 10 h of dark was maintained using a fluorescent lamp OSRAM LUMILUX COMBI-N/P, 18W.
Specimens from the collection of the Institut royal des Sciences naturelles de Belgique (IRSNB) and from the Swedish Museum of Natural History (NHM) were examined, including type material. These specimens, previously mounted on slides, dry pinned, or stored between two celluloid layers in isinglass, were mounted on permanent slides. Pinned specimens were prepared by boiling in KOH 10%, except the wings, transferred in acetic acid, butanol and in a phenol: xylene mixture 3:1, then mounted in balsam on a microscope slide. Specimens in isinglass were also gently boiled in KOH to dissolve gelatin, and thereafter treated as described above.
Sampled and reared individuals were fixed in 75% ethanol for preparation and mounted on microscopic slides according to Saether [12] and Wirth & Marston [13], using balsam or Faure as mounting medium. In the case of successful rearing, adult, larval and pupal exuviae were mounted on the same slide. The adult abdomen (including genitalia) was mounted in a dorso-ventral position. To examine the virga at high magnification (1000-1250×), it was sometimes necessary to dissect the IX tergite and to mount it as a separate part.
Descriptions provided in this study are based only on reared adult males with associated pupal exuviae and larvae, when available. Body parts measurements were made at different magnifications (10-1000×) using a LEICA DM LS B2 optic microscope connected to a LEICA DFC320 camera and analysed with LEICA LAS software V4.8. Measurements were given in µm unless otherwise stated. Photos of characters of taxonomic interest were taken with the LEICA DFC320 camera.

Selected Species
A key for the identification of adult males and pupae of Orthocladius s. str. species and closest species is provided. The key does not include the subgenera Euorthocladius and Eudactylocladius since they can be easily separated from Orthocladius s. str. both as larvae, pupae and adults [8,9], whereas the species of the subgenera Mesorthocladius, Pogonocladius and Symposiocladius are included in the key, because their separation from Orthocladius s. str. species often is not straightforward. The list of the species included in the key is reported in Table 1, where asterisks (*) indicates that all the three life stages (male adults, pupal exuviae, larvae) belonging to the same specimen were examined, to guarantee membership to the same species.

Subgeneric Characters of Adult Male of Orthocladius s. str.
Body color. Head dark-yellow brown, antennal setae gold-dark brown, scapus black. Thorax: background thoracic color yellow-brown; mesonotal stripes, humeral region, metanotum and mesosternum darker than the background color. Abdomen, femur and tibiae dark, tarsomeres lighter, halteres from transparent to white (character not usable for mounted material, being observable only in pinned specimens). Body color is attenuated in specimens mounted in Canada balsam after 10% KOH clearing, but live or pinned specimens are generally not available for study, so only the color observable in slidemounted specimens is considered and is of low utility.
Thorax. Antepronotal lobes well developed, but narrowed medially, with a shallow median notch and a group of well developed lateral setae. Acrostichals begin near antepronotum. Dorsocentrals generally stout and long (ranging between 75-125 µm), in few cases (O. rubicundus) slender and shorter (less than 40 µm long), in one row, some setae not well lined up in some cases. Scutellars usually uniserial, not lined up or multiserial in Orthocladius (Orthocladius) wetterensis.
Wing. Wing membrane without setae, with fine punctation, microtrichia visible at 400×. Costa at most moderately extended beyond R 4+5 . R 2+3 ending from 1/3 of distance from R 1 to R 4+5 . R 4+5 generally ending distal to end of M 3+4 , FCu little distal to RM or at the same level (VR 1-1.2). Cu1 slightly bent, squama with setae. R 1 and R 4+5 generally without setae. Anal lobe rounded to strongly produced.
Legs. Pseudospurs generally present on tarsomeres 1 and 2 of mid leg and tarsomere 1 of hind leg, sensilla chaetica sometime present on Ta 1 of P 3 . Pulvilli absent, claw with 4 teeth apically.
Hypopygium. Anal point with lateral setae, usually triangular with pointed apex, with rounded apex only in subgenera Euorthocladius and Mesorthocladius. Virga present and well developed or very reduced to absent in subgenus Symposiocladius and in Orthocladius (Orthocladius) rhyacobius. Superior volsella hook-like, triangular or collar-like. Inferior volsella divided into a dorsal and a ventral lobe, dorsal lobe long and narrow, noselike, pinched, short and squared, short and rounded [7], differences have taxonomic value; IVr is here introduced as the ratio between the length of dorsal lobe and its width. Ventral lobe covered by dorsal lobe, well extended beyond dorsal lobe in some species. Gonostylus simple tapered to end or forming a right angle at outer margin, with a well developed megaseta, crista dorsalis present, but generally not developed, very developed only in a few species.

Subgeneric Characters of Pupal Exuvia of Orthocladius s. str.
Color in species mounted on slides variable from yellow-gold to brown-black. Pigmentation with different distribution on abdominal segments, posterior margin of abdominal segments much darker in some species. Frontal setae on small cephalic tubercles, frontal warts [14,15]  horn usually with small spines, elongated (200-500 µm long), very long, slender and unarmed in Orthocladius (Mesorthocladius) frigidus and in Orthocladius (Mesorthocladius) vaillanti [18,19], enlarged in the middle in O. wetterensis and more or less rounded or pointed to apex in other species. The thoracic horn is sometimes bent at apex, but its shape is influenced by mounting position. Rows of hooks always present on posterior margin of tergite II (T II ) , width of hooks area variable in different species and of some taxonomic value: e.g., it is narrower in O. oblidens than in O. rhyacobius. Adhesion muscle-marks present on tergites: two oblique marks in the postero-median area, 2-5 less visible marks in the antero-lateral area on each side. Five dorsal setae (D I-V ) are present on tergites, three ventral setae (S I-III ) on sternites. Few setae on sternites may be branched as in Orthocladius (Orthocladius) rivinus. T II-V with very small points forming an area (= point patch), which may be divided into an anterior, median, posterior and apical field, continuous (O. rhyacobius) or well separated from each other (O. rubicundus). T VI with only an anterior, median and posterior field, T VII-VIII without point fields. The fields of points are more or less developed and extended laterally, often very small points may be observed extending to the antero-lateral corner of abdominal tergites, beyond the adhesion marks. Point patches are present also on sternites (especially S II-III ) with points often joined into groups of 2-3 ("Gruppen-shagreen"). The small points on the antero-lateral corner of tergites are variable in extension, similar points are present on sternites and in specimens mounted in dorso-ventral position dorsal and ventral points may be confounded. The number of antero-lateral adhesion marks and the position of seta D I are variable and have no taxonomic value. The different size of anterior, median, posterior and apical field of points on tergites is emphasised as an easily observable character without substantial variation within species, and thus a good candidate to be used in taxonomic keys for separating species.
Usually, very small blotches are present at the base of apical points on tergites, but in one species (O. pedestris) there are large brown blotches extending laterally to the apical point area of tergites and postero-medially on sternites. Pedes spurii B are present but reduced on segment II in O. frigidus and absent in Orthocladius (Symposiocladius) ruffoi. Pedes spurii A are present on S IV-VII , reduced on S VII in some species. Five, or more rarely four, lateral setae are present on abdominal segment VIII, with variation within species, very well developed in some species (e.g., O. ruffoi), sometime the anterior and/or the posterior one are bifid, with variation within species. Anal lobes with 3 anal macrosetae, usually strongly hooked at tip, but straight in O. ruffoi, straight or gently curved in O. dentifer. Tips of anal lobes with or without taeniate extensions of the cuticle. When present, they have different consistency and a taxonomic value, varying from very small, colourless light extensions (O. excavatus) to 'broken' setae (O. rubicundus), teeth [10], well sclerotized chitinous spurs (O. rhyacobius, O. pedestris) [6] or spiniferous processes (subgenus Symposiocladius) [4,20]. Additional small taeniate extensions, sometime appearing as a weak fringe of short setae, may be present (O. excavatus, O. pedestris). A well developed fringe of setae or hair-like teeth [10] is present only in O. vaillanti [6,19], a less developed one in O. ruffoi [6]. The ratio between the lengths of anal macrosetae and anal lobes varies around 1 and has taxonomic value.

Subgeneric Characters of Larva of Orthocladius s. str.
This description is based only on species reared to adults and included in the Keys to adult males and pupal exuviae. Medium sized larvae, up to 10 mm long.
Antenna with 5 segments, decreasing in size from 1 to 3, 4th segment equal or longer than 3rd, 5th segment small. Ring organ near the basis of 1st segment, blade shorter than flagellum, style long as 3rd segment. AR or the ratio between the length of the first segment and the other 2-5 has taxonomic value. Lauterborn organ developed only in larvae of the subgenus Symposiocladius (Figures 66-70 in [4]).
Mandible with a long apical tooth and 3 inner teeth increasing in size, the distal one (3rd) is the largest, a distal sclerotization of mola may be mistaken as a 4th inner tooth; seta subdentalis apically pointed, seta interna consisting of about 5-8 branches, some branch bifid, outer margin smooth, mola without spines.
Mentum. With a single median tooth and 6 lateral teeth. Ventromental plates reduced. Setae submenti well developed, inserted just a little more distal to the end of ventromental plates, about 50-70 µm long. Median tooth (Dm) of variable extension, from 1 to 4 times larger than the first lateral tooth (Dl1); the ratio between the width of median tooth and the 1st lateral one (DmDl) has taxonomic values.
Abdominal segments without robust setae, a tuft of setae present only in larvae of Symposiocladius (Figure 5l in [20]. Procerci only a little longer than wide, with 5-7 apical setae 350-400 µm long.

Description of Species
The species considered in the keys to adult males and pupal exuviae are in Table 1. In Supplementary material there is a legend of additional information given (Table S1). Hereafter are reported additional notes for some species. References to contributions to description of life stages present in previous publications and species distribution are given in Supplementary material (Tables S2 and S3), with additional taxonomic notes. Morphometric measures of adult males of some species are summarized in Table 2. Morphometric measures of larvae are in Table 3. A detailed list of all larval measures is in Supplementary material (Table S4). Measures from larval exuviae reared to adults were preferred, when available (see * in Table 1), otherwise measures are from larvae only tentatively assigned to a species. Ranges of measures are given when available. When a value is given before range, it is the value considered more accurate, because it comes from the best mounted specimen, because some measures are affected by error bound to the different position of the mounted parts [21].

Orthocladius (Orthocladius) decoratus (Holmgren, 1869)
The species is described as adult male and pupal exuviae [6,8,23]. The larva is still undescribed, probably cannot be separated from the larva of O. excavatus.  Brundin, 1947 Only the adult male [6,22] and pupal exuviae [6,8] are described, the larva is unknown. Brundin, 1947 The species is described in several papers [6,24,25]. The adult male of O. excavatus is separated from other species by the presence of a developed virga (Figure 1), the triangular superior volsella and the digitiform dorsal lobe of inferior volsella. The adult male may be confounded with O. marchettii, but this species has a larger dorsal lobe of inferior volsella.

Orthocladius (Orthocladius) excavatus
The pupal exuviae are characterized by reduced taeniate extensions and extensive cover of points on abdominal tergites III to VI.

Orthocladius (Orthocladius) marchettii
The species can be separated from O. excavatus by the larger dorsal lobe of the inferior volsella (Figure 2c), and the more prominent superior volsella ( Figure 2b).
The adult male is separated by the large dorsal lobe of the inferior volsella (Figure 3d), the pupal exuviae are characterized by short anal macrosetae (Figure 3h). The larva by a large median mental tooth (Figure 3j).  Orthocladius (Orthocladius) obumbratus (Johannsen, 1905). This species was firstly described from Nearctic, its presence in the Palaearctic region was recently questioned [6]; the reason is that the lectotype of O. obumbratus differs from the specimens collected in other Nearctic stations and identified as O. obumbratus [7]. An accurate examination of dorsal and ventral lobe of inferior volsella from type material of O. obumbratus shows that the dorsal lobe is short and squared, near to the one of O. oblidens, with a length/width ratio (IVr) below 2 (Figure 4d). Other North American Orthocladius (Orthocladius) obumbratus (Johannsen, 1905) This species was firstly described from Nearctic, its presence in the Palaearctic region was recently questioned [6]; the reason is that the lectotype of O. obumbratus differs from the specimens collected in other Nearctic stations and identified as O. obumbratus [7]. An accurate examination of dorsal and ventral lobe of inferior volsella from type material of O. obumbratus shows that the dorsal lobe is short and squared, near to the one of O. oblidens, with a length/width ratio (IVr) below 2 ( Figure 4d). Other North American specimens, gently furnished from Dr. Caldwell and identified as O. obumbratus, have a digitiform inferior volsella (Figure 4h) with an IVr near to 3, a value observable in O. excavatus, which has always an IVr well above 2 ( Kieffer, 1909.

Orthocladius (Orthocladius) pedestris
The adult male of the species was described in [6,9], type material are pupal exuviae [10].   Kieffer, 1909 The adult male of the species was described in [6,9], type material are pupal exuviae [10]. The adult is characterized by the hooked superior volsella (Figure 5b), and can be confounded with O. decoratus. The pupa is characterized by brown blotches in the intersegmental area of abdominal segments. The larva is very similar to those of O. excavatus and O. marchettii and is characterized by a low mental ratio DmDl and high AR (Figure 5i-l (11.7-13).

Orthocladius (Orthocladius) rhyacobius Kieffer, 1911
The species was described in [6,27] as adult male and its pupal exuviae in [6]. The adult is characterized by the absence or the strong reduction of virga ( Figure 6). The lectotype was established based on a pupal exuvia [10]. O. rhyacobius and O. excavatus were considered junior synonyms of O. obumbratus in [10], but the pupal exuvia of O. rhyacobius has very strong taeniate extensions (Figure 6g), whereas O. excavatus has small taeniate extensions (Figure 1g). The associations of reared pupal exuviae with adult males support the evidence that O. rhyacobius and O. excavatus are different species, the former lacking virga (Figure 6b), the latter with a well developed virga (Figure 1b). The larva of O. rhyacobius is characterized by a large median mental tooth and a high AR (Figure 6i-l). Kieffer, 1909. The adult male of the species was described in [6,9], type material are pupal exuviae [10]. The adult is characterized by the hooked superior volsella (Figure 5b), and can be confounded with O. decoratus. The pupa is characterized by brown blotches in the intersegmental area of abdominal segments. The larva is very similar to those of O. excavatus and O. marchettii and is characterized by a low mental ratio DmDl and high AR (Figure 5i-l).  Orthocladius (Orthocladius) rhyacobius Kieffer, 1911.

Orthocladius (Orthocladius) pedestris
The species was described in [6,27] as adult male and its pupal exuviae in [6]. The adult is characterized by the absence or the strong reduction of virga ( Figure 6). The lectotype was established based on a pupal exuvia [10]. O. rhyacobius and O. excavatus were considered junior synonyms of O. obumbratus in [10], but the pupal exuvia of O. rhyacobius has very strong taeniate extensions (Figure 6g), whereas O. excavatus has small taeniate extensions (Figure 1g). The associations of reared pupal exuviae with adult males support the evidence that O. rhyacobius and O. excavatus are different species, the former lacking virga (Figure 6b), the latter with a well developed virga (Figure 1b). The larva of O. rhyacobius is characterized by a large median mental tooth and a high AR (Figure 6i-l).  Potthast, 1914.

Orthocladius (Orthocladius) rivinus
The lectotype was established on pupal exuviae [10]. The species was described in [6,9] as adult male. The adult is characterized by a triangular superior volsella (Figure 7c) and a squared dorsal lobe of inferior volsella (Figure 7d). Pupal exuviae are characterized by the splitting of some setae on sternites III (Figure 7g).

Orthocladius (Orthocladius) rivinus Potthast, 1914
The lectotype was established on pupal exuviae [10]. The species was described in [6,9] as adult male. The adult is characterized by a triangular superior volsella ( Figure 7c) and a squared dorsal lobe of inferior volsella (Figure 7d). Pupal exuviae are characterized by the splitting of some setae on sternites III (Figure 7g).
The adult male ( Figure 8) is characterized by the small and thin dorsocentral setae (see Section 3.6 Key to adult males).
The pupal exuviae are characterized by the presence of a postero-lateral patch of granules (chitinized rings) on apical bands of Tergites II-V extending ventrally, the patch of granules may be complete ventrally across the sternites or medially broken ( Figure  8f,g).
The larva has a variable AR and a median mental tooth with intermediate values (Figure 8i-l
The adult male ( Figure 8) is characterized by the small and thin dorsocentral setae (see Section 3.6 Key to adult males).
The pupal exuviae are characterized by the presence of a postero-lateral patch of granules (chitinized rings) on apical bands of Tergites II-V extending ventrally, the patch of granules may be complete ventrally across the sternites or medially broken (Figure 8f,g).
The larva has a variable AR and a median mental tooth with intermediate values (Figure 8i-l Brundin, 1956 The adult male was described in [9,24], the pupal exuviae in [8] and redescribed in [6] from adult male and pupal exuviae. Inferior volsella is very characteristic (Figure 9d), scutellar setae are arranged in multiple rows (Figure 10d), this character separates O. wetterensis from other species. The pupal exuviae are characterized by an enlargement in the middle of the thoracic horn (Figure 9e). The larva is reported has having a high AR [30], but the AR is low in reared larvae, lower in comparison with other species, so this character cannot be used to identify the species. More useful is the width of median tooth of mentum (Figure 9j).  Brundin, 1956.

Orthocladius (Orthocladius) wetterensis
The adult male was described in [9,24], the pupal exuviae in [8] and redescribed in [6] from adult male and pupal exuviae. Inferior volsella is very characteristic (Figure 9d) scutellar setae are arranged in multiple rows (Figure 10d), this character separates O wetterensis from other species. The pupal exuviae are characterized by an enlargement in the middle of the thoracic horn (Figure 9e). The larva is reported has having a high AR [30], but the AR is low in reared larvae, lower in comparison with other species, so this character cannot be used to identify the species. More useful is the width of median tooth of mentum (Figure 9 j).   Brundin, 1956.

Orthocladius (Orthocladius) wetterensis
The adult male was described in [9,24], the pupal exuviae in [8] and redescribed in [6] from adult male and pupal exuviae. Inferior volsella is very characteristic (Figure 9d) scutellar setae are arranged in multiple rows (Figure 10d), this character separates O wetterensis from other species. The pupal exuviae are characterized by an enlargement in the middle of the thoracic horn (Figure 9e). The larva is reported has having a high AR [30], but the AR is low in reared larvae, lower in comparison with other species, so this character cannot be used to identify the species. More useful is the width of median tooth of mentum (Figure 9 j).

Orthocladius (Pogonocladius) consobrinus (Holmgren, 1869)
The adult male is described in [9,26], the pupal exuviae in [8]. Absence of sensilla chaetica in flagellomeres 2-5 cannot be used as a character separating the subgenus [3], because they are always observed in the specimens examined, including type material deposited at NHM. The larva is briefly described (Figure 9.53G in [22]).
Other characters as the length of each of the antennal segments 2-5, mandible teeth length, distance between setae submenti, size of head capsule [30] were no more considered because their measures are subjected to large errors.
A principal component analysis (PCA) was carried out using 5 selected characters to find the ones responsible of the largest variance (Tables 4 and 5, Figure 12). The width of the median tooth of mentum (Dm) and the length of the first antennal segment (A 1 ) were the most discriminating characters, while the width of the first antennal segment (A 1 w) was a little more discriminating than the length of antennal segments 2-5 combined (A 2-5 ). In PCA, the width of the first antennal segment (A 1 W) was a little more discriminating than the length of A 2-5 combined in the first axis, but the reverse was true in the second axis ( Table 4). As a practical measure to separate species, the ratio DmDl (ratio between the width of the median and first lateral mental tooth) and the antennal ratio AR (ratio between the length of the first and the 2-5 length of antennal segments) were selected (Table 3, Figure 13). In the impossibility to give a dichotomous key rough outlines to separate species are given (Table 6).  Table 6. DmDl and AR values useful to separate larvae.
A principal component analysis (PCA) was carried out using 5 selected characters to find the ones responsible of the largest variance (Tables 4 and 5, Figure 12). The width of the median tooth of mentum (Dm) and the length of the first antennal segment (A1) were the most discriminating characters, while the width of the first antennal segment (A1w) was a little more discriminating than the length of antennal segments 2-5 combined (A2-5). In PCA, the width of the first antennal segment (A1W) was a little more discriminating than the length of A2-5 combined in the first axis, but the reverse was true in the second axis (Table 4). As a practical measure to separate species, the ratio DmDl (ratio between the width of the median and first lateral mental tooth) and the antennal ratio AR (ratio between the length of the first and the 2-5 length of antennal segments) were selected (Table 3, Figure 13). In the impossibility to give a dichotomous key rough outlines to separate species are given (Table 6). Figure 12. plot of species scores and character loadings in principal component analysis in the first two axes, grey triangles refer to samples of larvae belonging to reared specimens, see Table 3 for abbreviations. Figure 12. Plot of species scores and character loadings in principal component analysis in the first two axes, grey triangles refer to samples of larvae belonging to reared specimens, see Table 3 for abbreviations.
Insects 2022, 12, x FOR PEER REVIEW 20 of 24 Figure 13. plot of the ratio DmDl against the antennal ratio (AR). Dashed lines separates species with different morphometry, see Table 3 for abbreviations.  Figure 13. Plot of the ratio DmDl against the antennal ratio (AR). Dashed lines separates species with different morphometry, see Table 3 for abbreviations.

Discussion
Species identification within the subgenus Orthocladius takes advantage from characters expressed in pupal exuviae. Most species can be distinguished also on the basis of differences in adult male genitalia or some other character as dorsocentral and scutellar setae, while others require the association of pupal exuviae with adult males for achieving identification (see Key). Absence of virga, collar-like superior volsella, ventral part of inferior volsella not extending below dorsal part were considered as characters supporting the separation of subgenus Symposiocladius from Orthocladius s. str. [4], but O. rhyacobius (Figure 6b) and O. dentifer (Figure 3 in [6]), not included in Symposiocladius, have characters compatible with this definition of the subgenus. The subgenus Symposiocladius include pupal exuviae with quite different morphological characters, i.e., both species with a large spur on anal lobes, such as O. (S). lignicola (Figures 10 and 12 in [2]), and species without spur, such as O. (S). holsatus (Figure 4 in [20]). The larva of O. (S). lignicola is quite different from the others of this subgenus, because of the peculiar mentum ( Figure 19 in [2], Figure 76 in [4]), but the presence of large Lauterbon's organs on antennae and of setal tufts on the abdominal segments of larvae of all the known species of Symposiocladius (Figures 13-19 in [2], Figures 60-70 in [4], Figure 5 in [20]) supports the separation of the subgenus. Contrasting evidence about the subgeneric limits of Orthocladius s. str. and Symposiocladius should be clarified.
After an accurate morphometric analysis of reared material, it is possible to conclude that in principle larvae cannot be identified without association with pupal exuviae. In some situation the larvae can be also confounded with species belonging to other related genera, as is the case of Symposiocladius larvae, which can be assigned to some species of Cricotopus (see point 104/103 at page 197 in [22]). Similarly, the subgenus Cricotopus (Paratrichocladius) has larvae which cannot be easily separated from species of Orthocladius s. str. (see point 112/113 at page 198 [22]). The morphometric characters suggested to separate larvae (AR and DmDl) are only indicative. Antennal ratio (AR) is useful to separate species, but the ratio between the width of median tooth and the 1st lateral tooth of mentum (DmDl) is probably the most useful character for separating larvae. In particular, O. oblidens and O. rhyacobius are separated from other species thanks to wider Dm (Figures 3j, 6j, 12 and 13). Unfortunately, morphometric measures of larvae have large superposition in different species, e.g., in O. rubicundus and O. excavatus, and for some species (O. marchettii, O. pedestris, O. glabripennis) the data available are very limited. The criteria given in previous papers [30,32] are not supported by the present evidence.
DNA-based taxonomy could be very useful to identify species at each life stage, overcoming difficulties related to morphological identification. However, the development of reference DNA sequences allowing the identification of the species of Orthocladius is still in an embryonic phase. In fact, DNA barcodes are available for a few species (see Supplementary Material, [23,33]). Future needs include collaboration between morphological and molecular taxonomists for the development of barcode libraries for the molecular identification including species not barcoded yet [34].
Orthocladius taxonomy would benefit from bringing to light the specialized literature about the species described in local or less known journals. Very similar species, and possibly some of them conspecific, were described from Nearctic [7]. In the present paper one of these cases is discussed, i.e., the problem of the relation between O. obumbratus with O. excavatus, but further cases of similarity between Nearctic and Palaearctic species are known. Just to give another example, the similarity of O. carlatus (Roback, 1957) and O. curtiseta Saether, 1969 with O. rubicundus would deserve further study.
Some Orthocladius s. str. species are known from the East Palaearctic [35][36][37], many of them described in all three stages (larva, pupa, adult male) with possible affinities with O. excavatus and O. rubicundus. Other Orthocladius species for which all the life stages have been described are known for Japan [38][39][40]; some are very similar to West Palaearctic ones. Here again, the problem of possible synonymy could be solved with the support of molecular taxonomy, even if finding specimens could be not easy due to very large number of species described and the distances among collection localities.
Supplementary Materials: The following supporting informations are available online at: http: //www.mdpi.com/xxx/s1. Table S1: Legend of abbreviations used in the paper and in the tables. Table S2: Geographical distribution of Orthocladius species, with related bibliographic references. Table S3: References cited in Table S2. Table S4: Detailed measures of different morphological parameters cited in the paper.  Data Availability Statement: All specimens analyzed and the related data are deposited at University of Milan, if not specified otherwise, and can be requested to the corresponding author.