The morphological ontogeny of Ctenobelba pilosella Jeleva, 1962 is described and illustrated. This species was investigated from its description mainly in ecological aspect. It was recorded from the forest and meadow soils, with rather low density, and small percent of juveniles.The adult is of medium size and has setiform bothridial seta, with 6–10 anterior spines. Notogaster is covered with asteriform granules and has 10 pairs of medium size, barbed setae. Seta d is absent from genua I–III and all tibia. Bothridial seta of juveniles is setiform and barbed, prodorsal and gastronotal setae are short, except of medium size ro and long, curved inwards lp. Nymphs are quadrideficient and eupheredermous i.e. they carry the exuvial scalps of previous instars. Anal valves of protonymph and deutonymph have two pairs of alveolar setae, which is rare in Brachypylina. The juveniles have seta d on all genua and tibiae.
Ctenobelba Balogh, 1943, with the type species Ctenobelba pectinigera (Berlese, 1908) (= Eremobelba pectinigera Berlese, 1908), comprises medium sized mites (adults: 340–577 µm). The diagnosis of Ctenobelba was given recently by Ahaniazad et al. (2017) as follows: prodorsum with one pair of long, parallel costulae, rostrum rounded or dentate, lamellar seta inserted apically on costula, bothridial seta bifurcate or setiform, with cilia or long branches unilaterally, notogaster with one pair of tubercles at anterior margin and 10 pairs of notogastral setae. Pedotecta I and II well developed, three to seven pairs of aggenital and three pairs of adanal setae present, lyrifissure iad located posterior to seta ad3, legs monodactylous. Subías (2004, 2020 update) included 22 species in Ctenobelba sensu stricto, and one of them he treated as species inquirenda.
According to the catalogue of juvenile oribatid mites by Norton and Ermilov (2014) and further literature, the morphology of juveniles of only C. pectinigera (Berlese 1908) is partially known in Ctenobelba. Grandjean (1943) investigated the shape of gastronotal setae of the larva and nymphs of C. pectinigera (his Elapheremaeus pectinigera), and some formulae like anal and gastronotal setae (Grandjean 1949) and leg setae (Grandjean 1951), mainly in a phylogenetic context. He also briefly described the morphology of nymphs and illustrated the bothridium, bothridial seta, anal region, and opisthonotal gland opening (Grandjean 1965).
The aim of this paper is to describe and illustrate the morphological ontogeny of C. pilosella, and compare the morphology of the adult with congeners. The original description of the adult by Csiszár and Jeleva (1962) is brief, incomplete and was based on a single specimen, therefore the redescription is reasonable and important.
Material and methods
The juveniles and adults of C. pilosella used in this study were collected on 18 July 2011 by O. Ivan from (1) chestnut-beech forest in Baia Mare, with Castanea sativa (Mill.) and Fagus sylvatica L., affected by industrial pollution (extraction and processing of non-ferrous metals), with a maximum in the 70s and 80s years (currently, Natura 2000 site). For ecological comparison, we also selected eight other habitats from Romania (Table 1): (2) beech-hornbeam forest in Zlatna, with Fagus sylvatica and Carpinus betulus L., also affected by industrial pollution (extraction and processing of non-ferrous metals), (3) Ponto-Sarmatic steppe meadow (hayfield) in Ponoare, with Festuca valesiaca Schleich. ex Gaudin, F. rubra L., Poa angustifolia L., Stipa capillata L., Dichanthium ischaemum (L.) Roberty (Natura 2000), (4) xero-mesophilous meadow (hayfield) in Calafindeşti, with Agrostis capillaris L., Filipendula vulgaris Moench, Arrhenatherum elatius (L.) Beauv. ex J. & C. Presl, Dactylis glomerata L., protected area of national interest (IUCN IV), (5) Dacian oak-hornbeam forest in Dobrina with Quercus petraea (Matt.) Liebl., Q. dalechampii Ten., Carpinus betulus, Tilia tomentosa Moench (Natura 2000), (6) oak forest in Tismana, with Quercus robur L. and Carpinus betulus, (7) beech forest in Nămăeşti, with Fagus sylvatica, affected by pollution with cement dust, (8) saxicolous vegetation in Horia, with Festuca valesiaca, Asplenium trichomanes L. and bryophytes on limestone cliffs and (9) Ponto-Sarmatic steppe meadow (hayfield) in Valea lui David, with Festuca valesiaca, Dichanthium ischaemum, Stipa capillata and S. joannis Čelak (Natura 2000). In all habitats, we investigated the density and stage structure of mites, sex ratio, number of gravid females and eggs, and body length and width. In abundant populations, 30 randomly selected specimens were used. We measured total length of mites (from tip of rostrum to posterior edge of notogaster) in lateral aspect, body width (widest part of notogaster) in dorsal aspect, and the length of anal and genital openings and setae perpendicularly to their length in µm. In total 178 adults were examined. In statistic calculations, to differentiate the variables (female length and width, male length and width) among sites, the basic statistical descriptors included the minimum, maximum, mean and standard deviation values. The values were log-transformed ln (x+1) (Łomnicki 2010), and normality of the distribution was justified with the Kolmogorov-Smirnov test, while the equality of variance in different samples was verified with the Levene test. The assumption of normality or equality of variance was not met, and the number of replicates in compared groups was different, so the non-parametric ANOVA Kruskal-Wallis was used and then, in case of significant differences between averages, the multiple comparison test between average ranks was applied. The level of significance for all statistical tests was accepted at α= 0.05. Statistical calculations were carried out with STATISTICA 13.1 Software.
The illustrations of C. pilosella are limited to the body regions that show substantial differences between instars, including the dorsal and lateral aspect and some leg segments of the larva, tritonymph and adult, ventral regions of all instars, and the palp and chelicera of the adult. Illustrations were prepared from individuals mounted temporarily on slides in lactic acid, using the open-mount technique. In the text, tables and figures, we used the following abbreviations: rostral (ro), lamellar (le), interlamellar (in) and exobothridial (ex) setae, bothridium (bo), bothridial seta (bs), notogaster (Ng), notogastral or gastronotal setae (c-, d-, l-, h-, p-series), exuvial scalps of larva (L), protonymph (Pn) and deutonymph (Dn), lamellar costula (La), pedotectum (Pd), cupules or lyrifissures (im, ip, ih, ips, iad), opisthonotal gland opening (gla), humeral tubercle (ht), discidium (Dis), ovipositor setae (k, ψ, τ), subcapitular setae (a, m, h), cheliceral setae (cha, chb), Trägårdh organ (Tg), palp setae (sup, inf, l, d, vt, lt, ul, su) and solenidion ω, epimeral setae (1a–c, 2a, 3a–c, 4a–c), adanal and anal setae (ad-, an-series), aggenital seta (ag), leg setae (bv, ev, d, l, ft, tc, it, p, u, a, s, pv, pl, v), solenidia (σ, φ, ω) and famulus (ε). Terminology used follows that of Grandjean (1943, 1949, 1951, 1953, 1961, 1965) and Norton and Behan-Pelletier (2009). The species nomenclature follows Subías (2004, 2020 update).
Stage structure and density of Ctenobelba pilosella in different regions of Romania, details on plant cover are given in Material and methods; L—larva, Pn—protonymph, Dn—deutonymph, Tn—tritonymph, Juv—juveniles, Ad—adult.
The transmitted-light photomicrographs of the adult, larva and tritonymph of C. pilosella were prepared using a Leica DM3000 microscope and Leica DFC420 camera. For scanning electron microscopy (SEM), the mites were air-dried and coated with Au/Pd in a Polaron SC502, sputter coated and placed on Al-stubs with double-sticky carbontape. Observations and micrographs were made with a ZEISS Supra 55VP scanning electron microscope.
Adults of medium size (440–566), prodorsum elongated, with rounded rostrum and two long lamellar costulae, and barbed setae le and in on their basal and apical parts. Bothridial seta setiform, with 6–10 anterior spines. Anterior border of notogaster straight to concave, with humeral tubercle, and other tubercle positioned opposite to basal part of bothridium. Notogaster with asteriform granules and 10 pairs of thickened, barbed setae, gla opening close to lyrifissure im. Seta d absent from genua I–III and all tibia.
Juveniles unpigmented, cuticle with tubercles. Bothridial seta setiform, barbed, prodorsal and gastronotal setae short, except for medium sized ro and long, curving inwards lp. Nymphs quadrideficient and eupheredermous i.e. they carry exuvial scalps of previous instars, with reticulate pattern. Anal valves of protonymph and deutonymph with two pairs of alveolar setae, which is rare in Brachypylina. Seta d on all genua and tibia present.
Redescription of morphology of adult
Measurements. Mean length (range) of females—503.5±26.0 (458–566, N= 124) and males—475.2±21.9 (440–518, N= 54), and mean width (range) of females 281.4±13.2 (253–313) and males —260.7±11.8 (241–283).
Prodorsum. Subtriangular, longer (187–191) than wide (129–133). Rostrum rounded, with pair of medium sized (45–47) and smooth seta ro (Figs. 1a, 2, 3a, 4a, 4b, 5d). Two long (85–88) prodorsal lamellar costulae present, separated from bothridium, with medium sized, barbed setae le (44–47) and in (48–51) on their basal and apical parts (Figs. 1a, 3a, 4a, 4b, 6a, 6b). Bothridium rounded, bothridial seta setiform (107–111), with 7–8 anterior spines, basal spine of medium size (35), length decreasing to apical part (Figs. 1a, 3a, 4a–c, 5a, 6c). Seta ex short (12) and smooth, located lateroventral to bothridium (Fig. 3a).
Notogaster. Longer (311–315) than wide (295–299). Anterior border of notogaster straight to concave, humeral tubercle present, other tubercle opposite to basal part of bothridium (Figs. 1a, 4a–c). Notogastral setae (10 pairs, including c2) of medium size (Table 2), barbed and of different shapes (Figs. 1a, 1b, 3a, 4, 5b, 5c). Lyrifissures ia and ip not observed, im close to opisthonotal gland opening, ih and ips in normal positions (Figs. 1a, 3a).
Gnathosoma. Subcapitular setae h and m slightly longer (24–26) than a (16), all smooth (Figs. 2, 5d). Palp relatively short (98), most palp setae smooth, except barbed seta l″ on tibia (Fig. 3b), solenidion ω separated from seta acm, seta lt″ on tarsus absent. Formula of palp setae (and solenidion): 0-2-1-3-8(1). Chelicera elongated (154 × 62), chelate (Fig. 3c), chb shorter than cha, both smooth. Axillary saccule of subcapitulum long (24).
Ventral and lateral regions. Epimeral seta 1b, 1c, longer (30) than other setae (15–21, Fig. 2). Genital setae (5 pairs) short (15–17) and smooth, g1 and g3 in inner row, other setae in lateral row. All setae on ovipositor short and smooth (Fig. 3a). Aggenital setae (3 pairs), adanal setae (3 pairs) and anal setae (2 pairs) short (17–23) and smooth. Lyrifissure iad short, located lateral to medial part of anal plates. Pedotectum I large, oval (48 × 32), Pedotectum II smaller (36 × 19), discidium well-formed, distally rounded (Figs. 3a, 4).
Legs. Leg femora relatively slim, most leg setae finely barbed, seta d from genua I–III and all tibiae absent (Figs. 7a, 8). Most leg solenidia of medium size, except long φ1 on tibia I and φ on tibia III and IV. Formulae of leg setae (and solenidia, from trochanter to tarsus): I – 1-5-3(1)-4(2)-20(2); II – 1-5-3(1)-4(1)-15(2); III – 2-3-1(1)-3(1)-15; IV – 1-2-2-3(1)-12. Legs monodactylous.
Description of juvenile stages
Larva oval in dorsal aspect, unpigmented, cuticle with tubercles (Figs. 6e, 6f, 9, 10a, 12a). Prodorsum subtriangular, prodorsal seta ro of medium size, setae le, in and ex short, all smooth (Table 2). Mutual distance between setal pair le as between setal pair ro, and between setal pair in nearly four times longer than between pair ro. Pair le inserted approximately midway between pairs ro and in. Seta in inserted close to bothridium. Bothridium rounded, bothridial seta setiform, barbed.
Gastronotum of larva with 12 pairs of setae, including h3 inserted laterally to medial part of anal valves (Figs. 10a, 11a). All gastronotal setae short, except slightly longer h2 and long lp (Figs. 9a, 10a, 11a), all smooth. Setae da, dm and dp on large apophyses. Anal valves (segment P) with two pairs of small setae. Cupules ia and im not observed in plicate cuticle, cupule ip between setae h1 and h2, cupule ih lateral to anterior part of anal valves. Tube-like opisthonotal gland opening anterior to seta lp. Most leg setae barbed, seta d coupled to proper solenidia present (Fig. 13), most of medium size, except longer d on tibia I. Distal part of tarsus I relatively thin and long, with setae u and p.
Nymphs slimmer and with relatively shorter prodorsum than in larva, but shape of prodorsal setae as in larva. Prodorsum with longitudinal ridges between bothridia and setae le, posterior part porose. Gastronotum of protonymph with 11 pairs of setae due to appearance of p-series setae (Fig. 10b), and loss of setae c3 and d-series, remaining absent in deutonymph and tritonymph (Figs. 11b, 14a, 14b). All nymphs unpigmented, cuticle with tubercles, gastronotum relatively flat, and setae of l- and h-series on marginal part of gastronotum, all short except long and inward curving lp, positioned anteroventral to tube-like gla opening (Figs. 7b, 10b, 12d, 14a, 14b, 16a–c, Table 2). All nymphs carry exuvial scalps of previous instars (Fig. 15), easily lost in alcohol samples (Figs. 7b, 7c, 11b, 12b–d). In protonymph, one pair of setae appearing on genital valves, and two pairs added in deutonymph and tritonymph each (Figs. 10b, 14a, 14b), all short and smooth. In deutonymph, one pair of aggenital setae and three pairs of adanal setae appearing; next pair of aggenital setae added in tritonymph; all short and smooth. Anal valves of protonymph (segment AD) and deutonymph (segment AN) with two pairs of alveolar setae, in tritonymph two pairs of anal setae present (Figs. 10b, 11b, 14a, 14b); all short and smooth. In all nymphs, cupules ia and im not observed in plicate cuticle, cupule ip observed in ventral aspect posterolateral to seta p2, cupules ih, ips and iad in normal positions. Most setae on legs barbed, tarsi I and II with uniformly narrow distal stalk of medium length, with one claw and short setae u and p, whereas in other tarsi distal part relatively shorter (Fig. 17). Seta d on all genua and tibiae of medium size, barbed and coupled with proper solenidia, except longer and smooth d on tibia I. Solenidion φ1 on tibia I longest, solenidia ω1 and ω2 on tarsi I and II shorter, other solenidia short.
Summary of ontogenetic transformations
In all juvenile instars of C. pilosella, the prodorsal seta ro is of medium size, and setae in, le and ex are short, whereas in the adult all setae are of medium size. In all instars, the opening of bothridium is rounded and the bothridial seta is setiform; in the juveniles it is barbed, whereas in the adult it has 7–8 long anterior cilia. The larva has 12 pairs of gastronotal setae, the nymphs have 11 pairs (p-series appear, c3 and d-series lost), whereas the notogaster of adult loses seta c1 such that 10 pairs of notogastral setae remain. The formula of gastronotal setae in C. pilosella is 12-11-11-11-10 (from larva to adult). In deutonymph, one pair of aggenital setae appears, and one pair is added in the tritonymph and adult each. Formulae of epimeral setae is 3-1-2 (larva, including scaliform 1c), 3-1-3-1 (protonymph), 3-1-3-2 (deutonymph) and 3-1-3-3 (tritonymph and adult). Formula of genital setae is 1-3-5-6 (protonymph to adult), aggenital setae is 1-2-3 (deutonymph to adult), and segments PS–AN is 23333-2333-222 (including alveolar setae). Ontogeny of leg setae and solenidia is shown in Table 3.
Distribution, ecology and biology
According to Subías (2004, 2020) and Mahunka and Mahunka-Papp (2004), C. pilosella has Mediterranean and Ponto-Mediterranean distribution, respectively, but Shtanchaeva and Subías (2010) extended this distribution to South Europe. This species was recorded from Bulgaria (Csiszár & Jeleva 1962), Slovenia and Croatia (Tarman 1983), Slovakia (Miko 1990), Romania (Vasiliu et al. 1993), Hungary (Mahunka & Mahunka-Papp 2004) and Caucasus (Shtanchaeva & Subías 2010; Murvanidze & Mumladze 2016).
Mahunka and Mahunka-Papp (2004) and Murvanidze and Mumladze (2016) recorded C. pilosella from forest soils. In Romania C. pilosella was recorded both from forests (Ivan & Vasiliu 2000) and open habitats, such as meso-xerophilous meadows (Ivan 2007) and saxicolous habitats (Ivan & Călugăr 2004), which indicates its ecological plasticity. Moreover, C. pilosella is tolerant to the presence of heavy metals (Vasiliu & Vasiliu 1989) and cement dust (Ivan & Vasiliu 2012) in soil.
In this study, C. pilosella was most abundant in soils of beech-hornbeam forest (density 21 individuals per 500 cm3), and also abundant in chestnut-beech forest (19.6 individuals per 500 cm3), where the juveniles were most abundant (Table 1). This species was relatively abundant in Ponto-Sarmatic steppe meadow and xero-mesophilous meadow, whereas in other habitats it had low densities, illustrating its ecological plasticity.
In all habitats, adults dominated, and only in four juveniles were present, comprising 12–16% of all individuals. In the chestnut-beech forest, where the juveniles were most abundant, the stage structure of C. pilosella was the following: one larva, two protonymphs, three deutonymphs, 10 tritonymphs and 82 adults. In all habitats, females were more abundant than males, the sex ratio (females: males) varied slightly (1:0.36–1:0.50) and 0–60% of females (mean 24%) were gravid. The gravid females carried 1–2 relatively large eggs, each 229 × 145, which constituted about 46% of the total body length of females.
Females with the longest body size occurred in the soil in Baia Mare (chestnut-beech forest) (see Table 4 for details). The widest females of C. pilosella occurred in Baia Mare and Dobrina (Dacian oak-hornbeam forest), while they were significantly narrower in the other habitats. The longest and widest males were found in Baia Mare and Dobrina, while they were significantly shorter in the other habitats. In most habitats, females were significantly larger than males (Table 4).
Measurements of some morphological characters of juvenile stages and adult of Ctenobelba pilosella (mean measurements of 1–10 individuals in µm); Nd — not developed.
Ontogeny of leg setae (Roman letters) and solenidia and famulus (Greek letters) of Ctenobelba pilosella.
Sex ratio, number of gravid females and mean body length and width (and range) of Ctenobelba pilosella in µm in different regions of Romania.
Comparison of morphology of Ctenobelba pilosella with congeners and remarks
Among Ctenobelba species, C. mikaeeli Ahaniazad et al. 2017 is the largest while C. csiszarae Mahunka, 1977 is the smallest (Table 5). The notogastral setae of most species are setiform, but they are flagelliform in C. pseudomahnerti Subías & Shtanchaeva, 2013 and leaf-like in C. foliata Hammer, 1961, C. heterosetosa Murvanidze & Weigmann, 2007, C. martyanensis Ermilov et al., 2012, C. mikaeeli, C. parafoliata Pérez-Íñigo, 1991, C. parapulchellula Subías & Shtanchaeva, 2013 and C. pulchellula Gil-Martín & Subías, 1997. Ctenobelba brevipilosa Mahunka, 1964 and C. ayyildizi Baran, 2012 have the largest number of cilia on the bothridial seta (13–15 cilia) while C. martyanensis and C. perezinigoi Moraza, 1985 have the lowest number (4–5 cilia). Most species have three pairs of aggenital setae, C. apatomorpha Iturrondobeitia et al., 1998 has two pairs, C. marcuzzii Mahunka, 1974 has 5–6 pairs and C. mahnerti Mahunka, 1974 – seven pairs (unknown in C. foliata and C. serrata Mahunka, 1964). These species differ also from one another by other morphological characters given in Table 5 (also see Ahaniazad et al. 2017 for comparison of Ctenobelba species). Ahaniazad et al. (2017) stated that C. mikaeeli is most similar to C. pilosella, differing mainly in having more branches on the bothridial setae, serrated prodorsal seta le (versus barbed in C. pilosella), most gastronotal setae leaf-like, including p1 (versus setiform in C. pilosella) and shape of epimeral setae. Here we also add a difference in the shape of the lamellar costulae, where it extends up to the bothridium in C. mikaeeli, whereas in C. pilosella they most often do not extend up to the bothridia.
Selected morphological characters of the adults of Ctenobelba species, abbreviations explained in Material and methods.
The adults investigated herein are of similar length as in Csiszár and Jeleva (1962, length 516, width 287, sex not investigated) and Miko (1990, length 505–560, width 275–290, sex not investigated). In our adults, the lamellar costula has setae in and le on its basal and apical parts, respectively and is separated from the bothridium (versus it is connected with bothridium in figure 36 by Csiszár & Jeleva 1962), as in Miko (1990). Setae le and in are of medium size and barbed (versus le is smooth and in is broken in Csiszár & Jeleva 1962), and the tubercle is present on the anterior margin of the notogaster, opposite to basal part of bothridium (versus it is absent in Csiszár & Jeleva 1962), as in Miko (1990). In our individuals, the notogastral setae are barbed and barbs are of different shape (versus they are only barbed in figure by Csiszár & Jeleva 1962). The drawing of C. pilosella by Mahunka (1964) is similar to our specimen, except we observed thinner gastronotal setae. In the adult of C. pilosella drew by Csiszár and Jeleva (1962), the bothridial seta has seven cilia, in our individuals and those studies by Mahunka (1964) it has 7–8 cilia, whereas in those investigated by Miko (1990), this seta has 6–10 cilia.
The juvenile stages of C. pilosella are similar to those of C. pectinigera investigated by Grandjean (1965) in having the bothridial seta setiform and barbed, tube-like opisthonotal gland opening, long gastronotal seta lp and other setae short. In both species, two pairs of alveolar setae are present on the anal valves of protonymph and deutonymph, which are rare in Brachypylina. The juveniles of C. pectinigera were only partially described by Grandjean (1965), which limits more detailed comparison of morphology of juveniles the two species.
The nymphs of C. pilosella carry the exuvial scalps of previous instars on the gastronotum, which are easily lost in alcohol samples. Most gastronotal setae of nymphs are short, except for long and inward curving lp, which does not protect the exuvial scalps against loss, so the question appears how the exuvial scalps are fasten to the gastronotum of living nymphs. The nymphs of other species of the superfamily Ameroidea, to which Ctenobelba and Ctenobelbidae belong, use different methods of protection against loss of exuvial scalps. For example, the nymphs of Gymnodampia setata (Berlese 1916) from Ameridae retain setae of d-series on the gastronotum and are apheredermous, with a circular line of dehiscence (Chen et al. 2004), whereas the nymphs of Amerus polonicus Kulczynski, 1902 lost setae of d-series and are eupheredermous (Seniczak et al. 2020c), but the peripheral setae on the gastronotum are long and protect the exuvial scalps against lost. The nymphs of Hungarobelba pyrenaica Miko & Travé, 1996 (Hungarobelbidae) also have long dorsal peripheral setae on the gastronotum (Miko & Travé 1996), but Mongaillardia granjeani Călugăr & Vasiliu, 1984 (Amerobelbidae) and Damaeolus ornatissimus Csiszár, 1962 (Damaeolidae) have short marginal gastronotal setae, which do protect exuvial scalps against loss (Călugăr & Vasiliu 1984; Seniczak et al. 2020a), as in C. pilosella. By contrast, Basilobelba parmata Okayama, 1980 (Basilobelbidae) and Caleremaeus arboricolus Norton & Behan-Pelletier, 2020, C. monilipes (Michael 1882) and C. retractus (Banks 1947) (Caleremaeidae) use a cornicle (Seniczak et al. 2019, Norton & Behan-Pelletier 2020), typical for Damaeidae (Norton 1978, 1980; Seniczak & Seniczak 2011, 2013; Seniczak et al. 2013, 2016).
The percentage of juvenile C. pilosella was rather low or juveniles were absent in the soil samples of investigated habitats. For example, the highest abundance was found in the beech-hornbeam forest in Zlatna, but no juveniles were found. The absence of juveniles cannot be explained by the season (May) because in other samples from different localities, taken during this time, juveniles were present. The absence of juveniles in the Zlatna samples is probably a result of the small number of replicates (5) and the aggregated occurrence of C. pilosella in soil. Similar ecology was observed with Damaeolus ornatissimus Csiszár, 1962, Oribatella hungarica Balogh, 1943 and Tectoribates ornatus (Schuster 1958) (Seniczak et al. 2019, 2020a, b).
We thank two anonymous reviewers for helpful suggestions that improved the scientific value of this paper. This study was done under the program of the Polish Minister of Science and Higher Education “Regional Initiative of Excellence” in 2019–2022 (Grant No. 008/RID/2018/19). The ecological investigation was supported by a grant of Romanian Ministry of Education and Research through the Core Program, Contract no. 25N/2019, project 19-270301.