The Tribe Hyrtanellini Allen, 1980 (Ephemeroptera: Ephemerellidae) of Western and Central Asia with Description of a New Species

Simple Summary The larvae of tribe Hyrtanellini Allen, 1980 are very important from the biomonitoring and bioindication point of view. Unfortunately, the immature stage of some Hyrtanellini is difficult to identify as many authors have stressed. Moreover, in the mountainous areas of western and central Asia there is still clearly a significant number of undescribed new species. The present study focuses on the immature stage of the species of Hyrtanellini from Western and Central Asia. A new species of Serratella leonidi Martynov & Palatov, sp. nov. from Tajikistan is described, and generic placement of Serratella elissa Jacobus, Zhou & McCafferty, 2009 from Iran is discussed. Additionally, the phylogenetic reconstruction of Hyrtanellini based on the COI gene is proposed and discussed. Finally, considering the importance of correct identification of larvae for biomonitoring and investigation of aquatic fauna diversity, the key for the immature stage is shaped. Abstract A new species, Serratella leonidi Martynov & Palatov, sp. nov., is described from Tajikistan based on immature stage. Based on larval material from Iran including the topotypes, Serratella elissa Jacobus, Zhou & McCafferty, 2009 is complementary described, and its generic placement is clarified. The delimitation of three genera that are members of the tribe Hyrtanellini Allen, 1980, namely Serratella Edmunds, 1959, Torleya Lestage, 1917 and Quatica Jacobus & McCafferty, 2008 is briefly discussed. The phylogenetic reconstruction of Hyrtanellini based on the COI gene showed the relations of representatives of these genera on the one hand, and distinct delimitation of Serratella leonidi sp. nov. and S. elissa on the other. A list of species from Western and Central Asia attributed to Hyrtanellini, their currently known distribution and a key for the determination of the larvae are proposed.


Introduction
Spiny crawler mayflies (Ephemerellidae) are not speciose family comprising about 160 extant species known from the Nearctic, Palearctic and Oriental realms [1]. Despite the low number of species within the family, its taxonomical structure remains dynamic. A review by Jacobus and McCafferty [2] significantly ordered the family, but borders between some of the genera, including some genera of the tribe Hyrtanellini Allen, 1980, are still diffused. Serratella Edmunds, 1959, Torleya Lestage, 1917 and Quatica Jacobus & McCafferty, 2008 can be treated as such genera. Within Western and Central Asia, these genera were known with six species [3][4][5][6][7][8][9]. Another genus of Hyrtanellini Allen, 1980 known from these territories is Teloganopsis Ulmer, 1939, presented here with four species [5,8,[10][11][12]. Recently, Ding et al. [13] described Chinese Serratella and reported six valid species, including one new species from Sichuan and Yunnan Provinces. The authors also briefly discussed the importance of the future systematic studies of the genera Serratella, Ephemerella and Torleya.
Despite such a small number of Asian species of the tribe Hyrtanellini, the data on the distribution, variability and ecological preferences of its representatives are fragmentary. Populations of only some species from these areas of Asia were the object of molecular investigations [14][15][16].
The current contribution to Hyrtanellini contains: a description of a new species from Tajikistan, Serratella leonidi Martynov & Palatov, sp. nov.; a complementary description and change of generic placement of Serratella elissa Jacobus, Zhou & McCafferty, 2009 from Iran, based on material from several sites including the type locality; notes on the morphological affinities of genera Serratella, Torleya and Quatica; phylogenetic reconstruction of Hyrtanellini based on the COI gene that shows a close relationship between the representatives of the three genera mentioned above; and a species list of Western and Central Asia representatives of Hyrtanellini and a key for the immature stage.

Sampling, Type Series and Morphological Observation
The present contribution is based on the larval material collected by D. Palatov and J. Bojková in 2016 and 2019, in several regions of Tajikistan and Northern Iran. All specimens were collected by kick sampling using hand nets in different freshwater habitats and preserved in the field in 80-96% ethanol. For long-term preservation, 80-95% ethanol was used. Several specimens of each species discussed in the present contribution were mounted on slides using Canada balsam. Photographs of the specimens mounted on slides were made using a Canon Power Shot A 630 with a Ulab XY-B2T microscope at the National Museum of Natural History, National Academy of Sciences of Ukraine (Kyiv, Ukraine) [further NMNH NASU]; photographs of specimens preserved in alcohol were made using a Leica Z16 APO stereomicroscope, with a Leica DFC450 Digital Camera at the I.I. Schmalhausen Institute of Zoology, National Academy of Sciences of Ukraine. The pictures were subsequently processed with LAS Core 3.8 and the Helicon Focus program. The Scanning Electron Microscopy (SEM) images were made using a Vega3 Tescan. The specimens studied under the scanning electron microscope were first dehydrated using ethanol and dried by critical point drying. Material from the type locality for both species were used for SEM photography.
The type material Serratella leonidi sp. nov. is deposited in the NMNH NASU [A.V. Martynov's collection]. The material of Serratella elissa used in this contribution is housed in J. Bojková's collection (Masaryk University, Brno, Czech Republic) [further MUNI] and A.V. Martynov's collection at the NMNH NASU.

Molecular Study
For main part of the specimens, the total genomic DNA was extracted and mitochondrial cytochrome oxidase subunit I (COI) was sequenced according to Guglya [17]; the procedure was carried out at the Natural History Museum, University of Oslo (Norway). For five specimens, the total DNA was extracted using the Chelex procedure [18] and sequenced according to Trontelj and Utevsky [19] at the V.N. Karazin Kharkiv National University (Ukraine). The GenBank accession numbers for 39 new sequences (8 species from 4 genera) used in the molecular study are given in the Table 1; the nomenclature for the gene sequences follows Chakrabarty et al. [20]. A total of 144 COI sequences of other Hyrtanellini species from 6 genera and three outgroup species (two genera) used in our study were obtained from Cardoni et al. [21], Gattolliat et al. [22], Wakimura et al. [23], Corse et al. [24], Morinière et al. [25], Tenchini et al. [26], Suh et al. [14], Li et al. [15], Xu et al. [16], Behrens-Chapuis et al. [27], Roslin et al. [28] and GenBank (unpublished data). The alignment of the analyzed sequences were made in BioEdit 7.0.5.3. In studies of species delimitation based on single-locus data, we used both tree-and distance-based methods to make the results more persuasive.
Genetic distances within groups and between taxa were calculated in MEGA 11 [29]. We used IQ-Tree and FigTree v. 1.4.4 for constructing phylogenetic trees from sequence data based on a maximum likelihood (ML) analysis. We used two models of molecular evolution: the Kimura 2-parameter (K2) model [30] and Tamura 3-parameter (T92) model [29], with a gamma distribution (shape parameter = 0.16). This analysis involved 147 nucleotide sequences. The codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were 662 positions in the final dataset.
Mouthparts: Labrum wide, anterolateral angles rounded; anteromedian emargination wide and shallow ( Figure 2c). Dorsal surface of labrum with paired groups of long, thin, hair-like setae on lateral lobes; anterolateral angles and adjacent part of dorsal surface covered with long, stout, hair-like setae with smooth and feathered margins; anterior margin of labrum; dorsal surface along the labrum margin covered with numerous short, feathered setae and its anterior margin with six very short, stout setae feathered apically. Additionally, dorsal surface of labrum with densely scattered short, thin, hair-like setae and scale sockets. On both mandibles outer incisor with three teeth; inner incisor with two teeth (Figure 2a,b). Central part of outer margin of mandibles and adjacent area of dorsal surface with a group of long, thin, hair-like setae. Dorsal surface of mandibles also covered with scale sockets. Right mandible with a row of eight long, stout, hair-like setae, feathered marginally. Superlinguae of hypopharynx with rounded apices covered with mainly long, stout, hair-like setae ( Figure 2d). Outer margins of superlinguae without setae. Lingua densely covered with short, thin, hair-like setae. Rows of setae on lateral sides of lingua consist of 6-7 elongated, pointed, stout setae. Maxillary palp elongated, three-segmented, with distinct articulation (Figure 2e); segments I and II distinctly longer than segment III; distal segment III with margins convergent from middle, rounded at the tip, its inner margin covered with fine setae apically and distally. Galea-lacinia with a row of 6-7 long, pointed, stout setae with feathered margins, and two dentisetae on inner margin; a group of about 10 long setae near of bases of maxillary canines; one robust denticle rounded apically on inner margin of galea-lacinia above dentisetae. Base of galea-lacinia with a group of 5-6 long, hair-like, stout setae with feathered margins on inner margin; one elongated stout seta near base of maxillary palp. Dorsal surface of mentum with a group of 6-7 long, thin, hair-like setae near base of glossae ( Figure 2f). Ventral surface of mentum with densely scattered scale sockets. Dorsal and ventral surfaces of glossae and paraglossae tips covered with differently sized stout, hair-like setae; the same type of setae covers ventral surface of paraglossae along outer margin. Labial palp three-segmented; segments I and II subequal in length, flattened; segment III short, rounded apically, covered with numerous fine setae, with convergent margins; segment III length/width ratio at base: 1.3-1.5; segments I and II outer and inner margins (mainly in distal half of segment II) covered with numerous thin, hair-like setae; segments I and II outer margins and areas along of ventral surface covered with long, stout, hair-like setae and spine-like setae.
Thorax: Ridges and tubercles absent on thoracic surface. Anterolateral angels of pronotum without any distinct projections. Mesothorax without anterolateral projections. Dorsal surface of thorax covered with scattered short, hair-like setae and small bubble-like setae (Figure 3d-g).
Femora of all legs slightly flattened; length/width ratio of forefemur 2. Dorsal surface of forefemur covered with scattered long, thin, hair-like setae (occasionally in groups of 2-3 setae), several small bubble-like setae and area with spine-like microtrichia near basal edge; basal part of femoral surface along outer margin with several long, mainly rounded apically, stout setae (occasionally with slightly divergent margins); distal half of femoral surface with irregular, discontinuous, transverse band of long, rounded apically, stout setae with slightly divergent margins. Outer margin of forefemur with irregular row of the same type of stout setae and a few long, thin, hair-like setae (Figure 4a,d). Outer margin of foretibia and tarsus with a few long, thin, hair-like setae only (often grouped into 2-3 setae); their ventral surfaces with scattered mainly long, thin, hair-like setae, almost grouped into 2-4 setae; the setae most numerous on tarsus and distal margin of tibia. Dorsal surface of foretibia with longitudinal row of 4-6 long, rounded apically, stout setae with slightly divergent margins and a few thin, mainly long, hair-like setae ( Figure 4h). Inner margin of foretibia, its adjacent area of ventral surface, and inner margin of tarsus with a row of long, rounded, stout setae and a few thin, hair-like setae.
Dorsal surface of middle and hind femora with scattered, mainly long, thin, hair-like setae and small bubble-like setae; basal half with area of spine-like microtrichia; central part of surface with several long, apically rounded, stout setae with slightly divergent margins (4-5 setae on middle femur; 1-2 on hind femur) (Figure 4b,c). Outer margins of femora and adjacent areas of dorsal surfaces covered with above-mentioned type of stout setae and scattered long, thin, hair-like setae. Dorsal surfaces of middle and hind tibiae with irregular, discontinuous row of the same type of 7-17 stout setae, runs along patella-tibial suture and inner margin; scattered long, thin, hair-like setae also covered the dorsal surfaces. Inner margins of middle and hind tibiae, and its adjacent area on ventral surface with a row of mainly long, stout setae. Outer margins of the middle tibia with several stout setae; hind tibia with 10-15 stout setae ( Figure 4i); additionally, hind tibia margins with a few thin, hair-like setae (often grouped in pairs). Outer margins, dorsal and ventral surfaces of middle and hind tarsi with thin, mainly long, hair-like setae (mainly in groups by two setae); inner margins of tarsi with a row of long stout setae.
Abdomen: Terga I-IX with paired distinct projections; those on terga IV-VIII strongest; those on tergum IX smallest among those on all other terga. All the projections and adjacent areas of terga covered with mainly elongated, apically rounded, stout setae with slightly divergent or subparallel margins; distant from projections stout setae distinctly shorter and tiny (Figure 5a-g). All terga covered with spine-like microtrichia and few thin hair-like setae; the microtrichia most numerous and distinct in medial areas of terga; in addition, few scattered small bubble-like setae covered terga. Lateral areas of terga IV-VIII additionally densely covered with round scale sockets. Segments IV-IX with posterolateral projections. Lateral margins of segments IV-IX covered with mainly long, apically rounded, stout setae with slightly divergent margins (Figure 5a). Posterior margins of terga I-VII with scattered thin, hair-like setae and spine-like microtrichia only; posterior margins of terga VIII-IX additionally with rows of above-mentioned kind of stout setae runs from paired projections to lateral margins ( Figure 5c); posterior margin of terga X also with stout setae, but they are shorter than on previous segments ( Figure 5e).
Caudal filaments subequal in length; posterior area of their segments bears discontinuous rings of long, rounded, unicolored apically stout setae, with subparallel or slightly divergent margins.
Winged stages: unknown. Distribution and biology. The new species is probably endemic to Central Asia. Based on current knowledge, it is distributed locally, in the reaches of the upper Amu Darya basin. Serratella leonidi sp. nov. inhabits the largest mountain rivers, e.g., the Kofarnihon River in central Tajikistan and the Pyanzh River as the natural border between Tajikistan and Afghanistan. The larvae of the new species inhabit sections with rocky substrates (Figure 11a,b), and were found within the river sections with pebbles, stones and roots, with current velocity about 0.3-0.7 m/s. Obviously, the size of the watercourse, the speed of the flow, and the nature of the bottom are key factors for this species distribution. Thus, Serratella leonidi sp. nov. was found both in a high mountain river with an average summer temperature of 10-13 • C, and in a foothill river that warms up to 20 • C or more in the summer. There is a high probability that Serratella leonidi sp. nov. will be found in other rivers with similar characteristics, such as the Vaksh, Vakhsu, Kunduz, Bartang and Surkhan. This species is not expected in lowland watercourses; we failed to find it during a special study of the lower flow of the Vaksh River where fine sand becomes the predominant substrate. Thus, the species can be found with high probability in the northern part of Afghanistan, south-eastern part of Uzbekistan and almost throughout Tajikistan.        The distinguishing characteristics of this species were provided in a brief but informative original description, which supported the figures of the general dorsal view of larva, maxilla and tarsal claw [6]. Herein is a complementary description of some characteristics including variable ones and illustrations useful for the species determination (Figures 6-10). All additional data are based on material collected in northern Iran in several areas, including the type locality of T. elissa.  Abdominal terga from dirty yellow to light brown; in some dark specimens, terga IV, V and VIII distinctly lighter (Figure 6a-c). Sterna I-VIII with paired dark submedian smudges; sometimes merged smudges looks like two longitudinal lines. Sternum IX distinctly darker than all other sterna (Figure 6d-f). Occasionally, several stout setae on femora, tibiae and posterior margins of terga are distinctly black.
Segments of the central part of caudal filaments fringed with long, mainly pointed, bluntly pointed or rounded stout setae ( Figure 10e); some of setae stout, hair-like, longer than corresponding segment. Several stout setae in basal part of filaments short and apically rounded.
Remark. Jacobus et al. [6] in the contribution with original description of S. elissa mentioned that they only provisionally consider this species to be part of the genus Serratella Edmunds, 1959. On our opinion, despite the absence of distinct borders in all stages, there are characteristics such as the absence of paired abdominal projections, which approximate S. elissa to the genus Torleya Lestage, 1917. Additionally, as showed on our ML tree (Figures 11-13), the relation of S. elissa with other sequenced Hyrtanellini are the reasons for its placing within Torleya.

Molecular Results
In this study, we used a series of COI sequences of Hyrtanellini species which were not in doubt. Based on position of some Operational Taxonomic Units' sequences (OTUs' sequences), vouchers that were not determined up to species level on our ML tree were assumed, in some cases, to represent new taxa that have not yet been described.
Our phylogenetic reconstruction based on the COI gene showed the absence of a distinct division between Serratella and Torleya ( Figure 12). Simultaneously, all Torleya OTUs analyzed by us were placed together ( Figure 13). This ML tree also clearly confirmed the suggestion of Jacobus et al. [6], that the genus Serratella is not a monophyletic taxon. The genus representatives did not place within one clade on the ML tree and were often significantly distant from each other ( Figure 12). This contradicts the results obtained by Ogden et al. [31] and Xu et al. [16] based on detailed studies of molecular and morphological data, but using only a few species belonging to two genera.
The position of the type species of the genus Quatica (i.e., Q. ikonomovi (Puthz, 1971)) is defined on the ML tree in one clade with representatives of Torleya (Figures 12 and 13). Together with the absence of a significant division of these genera, and morphological notes on Quatica mentioned below, should initiate a further detail morphological and molecular study of this genus' representatives. We cannot exclude that there is a high possibility that Quatica will be considered a junior synonym of Torleya (Figures 12 and 13).  Table 2). The genetic distance within the species is 0.003 (K2/T92+G, n = 3); this small value can be partially caused by the collection of all the specimens in one locality. Table 2. Genetic distances (COI) between sequenced Hyrtanellini species, which occur in Western and Central Asia, and within the species calculated using the Kimura 2-parameter (K2) model and Based on the ML tree, T. elissa is closely related to the Torleya representatives (Figures 12 and 13), and it is partially reasoned change of the generic position of this species. The genetic distances within the species are 0.013/0.014 (K2/T92+G, n = 4).
Gattoliat et al. [22] registered a single haplotype (from southern France) of S. ignita which significantly diverged from Corsican specimens (main territory of their study). They mentioned that the genus is supposed to be monospecific in Europe and no morphological differences were observed, suggesting continental cryptic species in Serratella. On our ML tree, S. ignita (n = 109) was also presented by distinct two lineages ( Figure 14) and support the presence of cryptic species within southeastern France, and South and West Germany. In addition, the clade of samples from Western Asia (Turkey, Iran, Armenia and Russian Krasnodar krai) and the clade of samples from Ukraine (Donetsk Region), Finland, Russia (Murmansk and Tula Regions, Republic of Karelia), China (Liaoning) and South Korea, were distinct but not significantly diverged. Notably, samples from the Donetsk Region (Ukraine) where the species presented with a geographically isolated population and considered a glacial relict [32], are the most closely relate to samples from Finland and Russia (Tula and Murmansk Regions, Republic of Karelia), in contrast to samples from the Zakarpattia Region (Ukraine) that are situated in another clade with specimens from Germany. The presence of the clade of samples that belong to the territories of China (Liaoning) and South Korea cannot be currently explained by zoogeographical consideration. Another large clade joined samples from Bulgaria, Germany, Finland, Ukraine (Zakarpattia Region), Italy (continental and peninsular, Sardinia), France (Corsica); they are divided into three smaller groups, but their distance and bootstrap support are low. Generally, in our opinion, it is doubtful whether the presence of the mayfly species with such a large distribution that S. ignita has, and the series of cryptic species will be described from different parts of Eurasia.  Within the north-eastern Black Sea coastal area (Krasnodar krai of Russia), we found larvae of Torleya species (Torleya sp. Cauc1) which are morphologically and genetically closely related to T. elissa and Torleya major (Klapálek, 1905). The genetic distance (K2/T92+G) between this OTU and T. elissa (0.148/0.349) and T. major (0.216/0.755) are large (Figures 12 and 13, Table 2). Currently, there are not enough data for morphological description of the Torleya sp. Cauc1 as a new species, but this information will be useful for the study of Caucasian freshwaters diversity. The registered Caucasian taxon, as well as Centroptilum volodymyri Martynov, Godunko & Palatov, 2022 that showed large genetic distances between Caucasian and Iranian populations (K2-0.13) which can be a separate cryptic species, are new evidences that will help to understand the origin and relationship of Caucasian and Iranian mayfly faunas [33].

List of Species, Currently Known Distribution and Key for Larvae of Hyrtanellini Allen, 1980 of Western and Central Asia
The composition and boundaries of Serratella, Torleya and Quatica are debatable [2,16,[34][35][36]; the generic position of some of their representatives will be changed in the future, but herein (see in species list, key, etc.) they are provided according to the current systematic position.
For now, eleven species of the tribe Hyrtanellini are known from Western and Central Asia (see also  (Allen, 1973) Furthermore, the one species mentioned above as the undescribed Torleya sp. Cauc1, can be found in some West Asian countries.
The state of our knowledge of the specific differences the adults and larvae of Hyrtanellini is not the same. The imaginal stage is only described for three species from target territories, namely in S. ignita, T. major and Q. euphratica. However, all twelve species are known at larval stage. Thus, there is no useful key for adults of Hyrtanellini, but a determination key for larvae is necessary and it is provided below. Part of the key on Teloganopsis is based on Marie et al. [11]: 1 Claw with one median row of denticles, where preapical denticle stout and larger than all other species-2 (genus Teloganopsis Ulmer, 1939).
-Claw with one median row of denticles, where preapical denticle not stouter and not larger than all other-5.
-Antennae at least two times longer than fore tibia length-3.

3(2)
Apex of maxilla is wide, spoon-like, with concavity; head, pronotum and mesonotum with distinct, narrow median line as wide as median suture; outer margin and dorsal surface of femora with short and widely rounded setae-Teloganopsis bauernfeindi (Thomas, Marie and Dia, 1999 [in Marie, Dia and Thomas]).
-Apex of maxilla narrow; head, pronotum and mesonotum with whitish median line distinctly wider than median suture; outer margin and dorsal surface of femora with long and narrow setae-4.
-Prothorax without anterolateral projections; transverse irregular row of stout setae on forefemur consists of a few elongated and slender stout setae-Serratella karia (Kazanci, 1990). 8(6) Dorsal surface of head, thorax, middle and hind femora covered with small bubblelike setae. Each of abdominal terga I-VIII with a pair of distinct submedian tubercles; only on tergum I the smallest, knob-like, all submedian tubercles bear numerous, mainly elongated, apically rounded, stout setae; caudal filaments and legs are unicolored yellow; coloration of terga III-IX similar, yellow, with brown transverse elongated spots that do not touch each other; terga II and III with transverse brown band; tergum X with indistinct pair of smudged submedian spots-Serratella leonidi Martynov & Palatov, sp. nov.
-Dorsal surface of head, thorax and femora without bubble-like setae. Distinct elongated submedian tubercles present on terga IV-VII (VIII), terga II, III and VIII with knob-like tubercles; tergum VIII with occasionally well-developed tubercles; within submedian tubercles only those of terga IV-VIII had more than several short, apically rounded, stout setae; legs with a series of brown slings; apices of caudal filaments brown, basal part with alternating pairs of brown and yellow segments, yellow centrally; abdominal terga dark, without distinct coloration pattern, terga IV-VI and X lighter than other terga-Serratella ignita (Poda, 1761). 9(5) Head with a pair of occipital protuberances, sometimes indistinct; prothorax with anterolateral projections-Quatica ikonomovi (Puthz, 1971).
-Dorsal surface of forefemur without distinct transverse row; several elongated, stout setae rounded or pointed apically, irregularly scattered on dorsal surface. Gill III not narrowed-11. 11(10) Lateral areas of posterior margin of tergum VIII with long stout setae pointed or apically rounded. Pronotum and occasionally dorsal surface of femora with small, robust, rounded stout setae (these small setae often almost absent)-Torleya elissa (Jacobus, Zhou & McCafferty, 2009) comb. nov.
-Lateral areas of posterior margin of tergum VIII with extremely long, stout setae pointed apically and long, thin, stout hair-like setae. Pronotum and dorsal surface of femora with numerous relatively large, robust, rounded, stout setae-Torleya sp. Cauc1. It should be noted that almost all of the species, excluding S. ignita, Q. ikonomovi, T. major, T. mesoleuca and T. maculocaudata, are locally distributed and their distributions do not overlap (Table 3). Torleya elissa is relatively widely distributed within the Iranian Plateau, within central and southern parts (Zagros Range) and the northern part (Alborz Range). There is a high probability of finding this species within the Talish Mountains. Serratella leonidi Martynov & Palatov sp. nov. was found only within the upper part of the Amu Darya river basin in Central Asia. Serratella karia is locally distributed within southwestern Anatolia, and Quatica euphratica (Kazanci, 1987) in the eastern part of Anatolia, where it is found within the upper part of the Euphrates River basin. If there are thorough investigations of target regions, similar local distribution can be evidence of relict nature and the gradual extinction of the group [46]. The unusually stretched (more than 1600 km) but obviously fragmented distribution of Teloganopsis subsolana (Allen, 1973)  Serratella and Quatica are related and are possibly polyphyletic genera. Some of their representatives are morphologically similar at the larval stage. The most recent detailed combined molecular and morphological analysis by Ogden et al. [31] did not include any species of Quatica. Ubero-Pascal & Sartori [35] included Quatica species in their phylogenetic analysis, and they considered Q. paradinasi and Q. ikonomovi as closely related forms characterized by three characteristics related to larval and egg morphologies: the presence of tubercles and star-like setae in the head and furrows demarcating the mesh units of chorion sculpturing. They did not include Q. euphratica in their analysis. The strong support of this clade (Figure 1 in [35]) led them to confirm that Q. paradinasi and Q. ikonomovi form a monophyletic group belonging to the genus Quatica. It is notable that these characteristics are a part of the main distinguishing characteristics of larval section of Quatica classification (see [2]). However, at least some larvae of Q. ikonomovi lack the star-like setae on head protuberances, based on specimens from Bosnia and Herzegovina which were examined. In these specimens only several short setae with divergent margins and feathered apices are present on the protuberances. Moreover, some of Serratella species, e.g., S. zapekinae Bajkova, 1967 also have head protuberances that bear similar types of setae. At the same time, S. zapekinae distinctly differs from Quatica by the shape of the imago male genitalia ( Figure 5 in [47]) and structure of the egg chorion ( Figures 30-32 in [48]). Therefore, there is currently no set of only larval unique modality of morphological characters that are invariably characteristic for Quatica and absent in Serratella species. For now, only one character modality of Quatica does not occur in all known Serratella representatives, namely the presence of anterolateral projections on the pronotum. This characteristic is mentioned in Quatica classification by Jacobus & McCafferty [2]. Quatica appears to be restricted to the Mediterranean areas [49]; Serratella representatives also occur within this region.
The genus Quatica requires further study, especially considering the fact that Jacobus & McCafferty ([2]: Figure 99) united a polytomy to create its present composition. According to our phylogenetic reconstruction based on the COI gene, Quatica (sequences of the type species Q. ikonomovi, were used) is closely related to Torleya (including the type species T. major), and any significant division of these genera was not observed (Figures 12 and 13). Notably, some of the examined larvae of Q. ikonomovi (from Bosnia and Herzegovina) lack the distinct paired median projections on abdominal terga and star-like setae, and only because of prominent anterolateral projections of pronotum, it cannot be placed in the Torleya genus. At the same time, some species of Torleya, e.g., T. major and Torleya sp. Cauc1, bear numerous large, feathered, rounded, stout setae that looks like bulbous star-like setae (see Figure 36 in Jacobus et al. [6]).
The genus Serratella is also related to Torleya. Both genera have no distinct differences at all stages, and were therefore considered as one taxon by some specialists, e.g., as taxon Torleya/g2 in Kluge [34]. We noted that the type species of Serratella has well-developed, paired abdominal projections, and non-operculate gills; the type species of Torleya has abdominal terga without projections, and operculate gills; the genera or subgenera of Torleya and Serratella are often separated based on one or both of these characteristics, but such taxa appear to be artificial. It should be noted that the Torleya type species T. major has indistinct operculate gills, so there is only one characters for separation left. At the same time, according to molecular and combined (based on molecular and morphological data) studies, Torleya and Serratella are spaced apart (see [16,31]). Unfortunately, only several species of these taxa were used in the studies. Our ML tree (Figures 12 and 13) shows the close relation of sequenced Torleya species and dispersion of Serratella representatives.
On our opinion, the main goal of the future investigations of the genera is to review them based on molecular and morphological data using as many species as possible. Torleya representatives with and without distal palisade of denticles on tarsal claw, with and without maxillary palp mandatory, and with and without projections on abdominal terga should be used in future studies.
The winged stages for a significant number of species from the target regions and the presence of cryptic species are still undescribed, together with a lack of data on regional species distribution, and uncertain composition and boundaries of the genera Serratella, Torleya and Quatica show a perspectivity and necessity for the further study of the tribe Hyrtanellini in general, and especially within Western and Central Asia.