Systematic status of Hydrozetes octosetosus Willmann, 1932 (Acari: Oribatida: Hydrozetidae) in the light of ontogenetic and ecological studies

The systematic status of Hydrozetes octosetosus was investigated by comparing this species to H. lacustris, which is the type species for the genus Hydrozetes Berlese, 1902. These species are similar, but probably not synonyms as recently proposed. They differ mainly by the position of notogastral seta lm in the adult and position and length of this seta and total number of long setae in the posterior part of nymphs. In the adult of H. octosetosus, this seta inserts behind the opisthosomal gland opening (gla), but in front of it in H. lacustris. In the nymphs of H. octosetosus seta lm inserts behind gla opening, but medially to it in H. lacustris. In H. octosetosus this seta is very long and the total number of long setae in nymphs is four pairs, while in H. lacustris seta lm is short and the total number of long setae in nymphs is three pairs.


Introduction
Most oribatid mites live in terrestrial ecosystems, where they transform organic matter and release mineral elements for plant growth. But overall, this group of mites inhabits various ecosystems, from extremely dry to wet and flooded. Some species, like those of the genus Hydrozetes, live in freshwater habitats, such as lakes, ponds, and rivers (Krantz and Baker 1982). They occur mainly on plants, but also burrow inside stalks and in damaged and decaying leaves (Kranz 1978;Norton 1994).
Species of Hydrozetes are well adapted to fresh water, both by their physiology and morphology, and can live submersed in water indefinitely. The adults have microtubercles near the respiratory stigmata, which hold a thin layer of air (Krantz and Baker 1982); this plastron allows the mites to exchange respiratory gases with water. Some morphological adaptations, such as the boat shape of nymphs, long setae on the posterior part of the body, and thick and stiff setae on distal parts of the legs also seem important. considered as a separate species. According to Subìas (2004) H. octosetosus is recorded in central Europe, while H. lacustris is a boreal species.
The aim of this paper is to revise the systematic status of H. octosetosus Willmann, 1932, which, contrary to Deichsel (2004, we consider to be a separate species. We compare the morphological characters of juvenile and adult stages of H. octosetosus with those of H. lacustris (Michael, 1882), which is the type species for the genus Hydrozetes Berlese, 1902, and also the distribution of both species.

Material and techniques
For the morphological study on the ontogeny of H. lacustris and H. octosetosus we chose lakes where these species did not occur together. Hydrozetes lacustris was collected in lake U1 (Ulriken, Bergen, Norway), where it occurred alone and in high density (Table I).
Hydrozetes octosetosus was collected in lake Martwe (Tuchola Forest, Poland), where it was distinctly more abundant than the co-occurring Hydrozetes sp. 1 and H. lemnae. The morphological description and illustrations of H. lacustris include the dorsal aspect of larva and tritonymph, anal region of larva and anogenital region of all nymphal stages, where new segments and setae appear during ontogeny. As the ontogeny of both species is similar, the drawings of H. octosetosus are limited to the dorsal aspect of larva and tritonymph, and the anal region of the larva and the anogenital region of the tritonymph. The adults of investigated species are also considered, to document the full development of setation during ontogeny. Terminology used follows that of Grandjean (see Travè and Vachon 1975 for many references).
The density of H. octosetosus and H. lacustris was investigated in Sphagnum mosses on the edges of some lakes in Bergen, Norway (lakes U1 and U2, Ulriken; lake Blåmansvannet and lakes F1 and F2, Fløyen; in each lake one sample of 20 cm620 cm65 cm was taken in June 2005) and in the Tuchola Forest, Poland (lakes Martwe and Wielkie Gacno; in each lake 10 samples of 10 cm610 cm65 cm were taken in September 2005).

Results
Morphology of juvenile stages of Hydrozetes lacustris (Michael, 1882) Larva. Larva with three pairs of legs, shape oval, body flesh-coloured ( Figure 1). Prodorsum triangular, porose, lateral part slightly wrinkled. Setae ro, in, and ex smooth, seta le barbed; seta le longest, seta ex shortest. Bothridium (bo) weakly developed, sensillus (ss) long, setiform. Gastronotal region with 12 pairs of setae, including seta h 3 positioned near anal opening ( Figure 2A). Setae of c-series, da, dm, and la shorter than other setae and smooth. Seta lm also smooth and situated medially to opisthosomal gland opening (gla), setae dp, lp, h 1 , and h 2 on small apophyses and barbed; seta h 1 thicker and shorter than h 2 , stiff and pointed. Length of setae of d-and l-series increases from anterior to posterior part of gastronotal region. Seta h 2 longest, seta h 3 shortest. Cupule pair ih positioned lateral to anterior part of anal valves. Cuticle with small pits.
Nymphal stages. All nymphs with four pairs of legs, slimmer than larva, boat-shaped and flesh-coloured. Number and shape of setae on the prodorsum as in larva, but more setae (15 pairs) present in the gastronotal region.
The successive juvenile stages differ distinctly in the body size (Table I) and the number of setae in the anogenital region. Genital valves of protonymph with two pairs of setae  Figure 2B); three pairs of pseudanal setae (p 1 2p 3 ) on paraproctal valves; setae p 2 and p 3 thin, seta p 1 thick and stiff; all setae smooth. Pairs of cupules ips positioned lateral to anterior part of anal valves. Genital valves of deutonymph with four pairs of setae ( Figure 3A). A pair of aggenital setae (ag) between genital and anal valves; three pairs of pseudanal setae present; three pairs of adanal setae (ad 1 2ad 3 ) on paraproctal valves; all setae short, thin, and smooth. Anogenital region rarely with pits. Genital valves of tritonymph usually with six pairs of setae ( Figure 3B), seta ag present. Setae p 2 and p 3 and three pairs of adanal setae small. Anal valves with two pairs of small setae. Pairs of cupules iad positioned lateral to anterior part of anal valves of deuto-and tritonymph. Anogenital region of nymphs rarely with pits.
The shape of setae on dorsal and lateral part of gastronotal region is similar to those in the tritonymph (Figure 4). Setae of c-series, d-series, la, and lm short and smooth, seta lm situated medially to gla. Other setae longer and thicker and positioned on small apophyses in posterior part of body. Setae lp, h 2 , and h 1 as long as body, thick at basal part, pointed and smooth; setae h 3 and p 1 distinctly shorter, but thicker, stiff, pointed, and smooth. Central part of gastronotal region with small pits, frontal and lateral parts slightly wrinkled.
Morphology of juvenile stages of Hydrozetes octosetosus Willmann, 1932 The juvenile stages of H. octosetosus are similar to those of H. lacustris in the body shape and the number of setae (Figures 527), but differ from them in several important morphological characters, as follows: 1. The position of seta lm in relation to opening gla: in H. octosetosus this seta inserts posteriorly to gla, but medially to it in H. lacustris; consequently in the former species the distance between seta lm and lp is distinctly shorter than between seta lm and la, while in the latter species the distance between setae of l-series is similar. 2. The length of seta lm: in H. octosetosus this seta is rather long in the larva and very long in the nymphal stages, while in H. lacustris it is short.  3. The length of setae le and ex in all juvenile stages: in H. octosetosus seta le is longer but seta ex is shorter than in H. lacustris. 4. The length of seta in in the larva: in H. octosetosus this seta is longer than in H. lacustris. 5. The number of genital setae: the nymphs of H. octosetosus usually have more of these setae than those of H. lacustris. 6. The shape of some setae on tarsus I, which is considered an important morphological character of the genus Hydrozetes (Willmann 1931;Grandjean 1948;Ghilarov and Krivoluckij 1975;Weigmann 2006): in the tritonymph of H. octosetosus seta pv0 is thinner, while seta s is thicker than in H. lacustris ( Figure 8); H. octosetosus has also thinner seta pv0 on tarsus II than H. lacustris.

Development and variability of setation
The number of setae on the prodorsum remains similar during the ontogeny of H. lacustris and H. octosetosus, but these species differ in the length of some setae (Table I). Compared to those of H. lacustris, all juvenile stages of H. octosetosus have seta le nearly twice as long and seta ex distinctly shorter, while seta in is longer only in the larva. The bothridium is weakly developed in all juvenile stages, with long, setiform sensillus. In the adults of these species the sensillus is short, setiform or absent, rarely clavate. From among 535 adults of H. lacustris from lake U1 (Ulriken, Norway) only seven specimens had a clavate sensillus on one or both sides. The number of gastronotal setae changes similarly during the ontogeny of these species, because new segments and setae appear in the anal region. There are 12 pairs of gastronotal setae in the larva, while in the protonymph three pairs of pseudanal setae (p 1 2p 3 ) first appear on segment PS, and the number of gastronotal setae is 15 pairs; p-series setae are present in the deutonymph, tritonymph, and adults (Figures 9, 10). In the protonymph some setae (lp, h 1 , and h 2 in H. lacustris and lm, lp, h 1 , and h 2 in H. octosetosus) grow very long in the posterior part of the body, and remain similar in the deutonymph and tritonymph. In the deutonymph three pairs of adanal setae (ad 1 2ad 3 ) first appear on segment AD, and are present in the tritonymph and adult. In the tritonymph two pairs of setae appear on the anal valves and are present in the adult. In both species the coxisternal setal formula is 32122 (larva), 3212221 (protonymph), 3212222 (deutonymph), and 3212223 (tritonymph and adult). In the larva seta 1c is difficult to observe, because it is scaliform and covers Claparède's organ, which is well presented by Grandjean (1963). In the investigated populations the formula of the genital setae was 2242(627)2(627) (protonymph to adult) in H. lacustris and (223)-(526)2(728)2(728) in H. octosetosus. Some nymphs and adults have one genital seta more on one side than on the other.
In the juvenile stages of H. octosetosus the migration of seta lm is observed from medial to posterior position in relation to opening gla, compared to H. lacustris. In all juvenile stages of H. lacustris this seta is small, thin, and smooth and placed medially to gla, rarely behind it, while in the adult it is usually in front of gla. In H. octosetosus this seta is rather long and barbed in the larva, and is placed slightly behind gla opening, while in the protonymph, Figure 10. Hydrozetes octosetosus, adult, dorsal aspect. deutonymph, and tritonymph it is placed behind gla and is very long. In the adult seta lm is similar in shape to the other notogastral setae, and is positioned behind gla, near seta lp.
In the juvenile stages of both species some variability in the gastronotal setae also occurred. In H. octosetosus one seta lm was sometimes thin and, when it was broken near the basal part, it looked like a short seta. In some individuals one seta lm grew closer to gla than the other, and some other gastronotal setae were positioned at different distances from the anterior border.
In the adults of H. octosetosus and H. lacustris the loss of some setae of the c-series was observed, compared to the tritonymph. Based on the position of these setae in the tritonymph, the first seta or pair of setae c 1 was lost, and also the next seta or pair of setae c 3 , and only setae c 2 remained. Among 165 adults of H. octosetosus from lake Martwe (Tuchola Forest, Poland) one had all setae of the c-series, five adults had five setae, 61 adults had four setae, 35 adults had three setae and 63 adults had a pair of setae c 2 . Among 57 adults of H. lacustris from this lake eight had five setae, 33 adults had four setae, three adults had three setae, and 13 adults had a pair of setae c 2 . Interestingly, the Norwegian adults of H. lacustris had more c-series setae than the Polish adults. Among 535 adults of this species from lake U1 (Ulriken) nine had all setae of the c-series, 11 adults had five setae, and the others had four setae (pairs c 2 and c 1 ).
The adult of H. octosetosus differs from that of H. lacustris in several important morphological characters, as follows: 1. The position of seta lm relative to opening gla: in H. octosetosus this seta inserts behind this opening, while in H. lacustris it is in front of it; consequently in the former species seta lm inserts far from seta la and near seta lp, while in the latter species the distance between setae of l-series is similar. 2. The length of dorsal setae, mainly the d-series: in H. octosetosus these setae are longer than in H. lacustris. 3. The length of prodorsal setae le and in: in H. octosetosus these setae are longer than in H.
lacustris. 4. The number of genital setae: H. octosetosus usually has more of these setae than H. lacustris. 5. The length of seta ft9 and shape of seta s on tarsus I: in H. octosetosus seta ft9 is shorter than in H. lacustris and seta s is barbed, while in the latter species is smooth ( Figure 11).

Occurrence of investigated species in lakes
In Norwegian lakes H. lacustris and H. octosetosus occurred together, but in different proportions (Table II)

Discussion and remarks
Juvenile stages of oribatid mites are generally poorly known, and keys to species concern mainly the adults. In the higher Oribatida (Brachypylina or Circumdehiscentiae), to which the genus Hydrozetes belongs, the juvenile stages differ distinctly from the adults, mainly in the body shape, cuticular sclerotization, and the shape and numbers of setae on the dorsal part of the hysterosoma. In the case of H. lacustris and H. octosetosus, the nymphs have some long setae on the posterior part of the body and their determination seems to be easier than for the adults. Besides, the morphological characters of juveniles are very helpful in a more precise determination of species. Our conclusions regarding H. octosetosus and H. lacustris are inconsistent with the results of Deichsel (2004), who considered H. octosetosus a junior synonym of H. lacustris, with a position intermediate between the latter and mites that would usually be identified as H.  parisiensis. In the adults he considered such morphological characters as the body length, distance between insertions of setal pairs la, da, and bothridia, and the number of notogastral setae in l-and h-series. In the juvenile stages he observed no morphological differences except the number of long setae on the posterior part of nymphs. Among 186 studied adults from six sites, Deichsel (2004) observed a gradual transition of the total number of l-and h-series setae between five and ten pairs. Similarly, among 56 studied nymphs the number of long setae varied between two and eight pairs. The author quantified the systematic value of these characters, using the Wards method with Euclidean distances in Statistica 6.0 (StatSoft, Tulsa, OK, USA). Cluster analysis did not confirm the diagnostic value of the total number of l-and h-series setae in the adult. Consequently, Deichsel (2004) assumed that the morphological variability he observed was intraspecific, and considered H. parisiensis Grandjean, 1948 andH. octosetosus Willmann, 1932 to be junior synonyms of H. lacustris (Michael, 1882). He also proposed two morphotypes of H. lacustris: H. lacustris lacustris (forma typica), with a total number of 13 pairs of notogastral setae in the adult, and at most four pairs of long setae in the posterior part of nymphs, and H. lacustris parisiensis, with more than 13 pairs of notogastral setae in the adult and more than four pairs of long setae in the nymphs. These morphotypes are also considered in Weigmann's (2006) key to the adults of German species of Hydrozetes. In Weigmann and Deichsel (2006), the description of H. lacustris included an illustration of H. lacustris f. parisiensis, but in the key to the nymphs only four species are included: H. confervae, H. lacustris, H. lemnae, and H. thienemanni.
In the light of our results Deichsel's (2004) synonymies can be questioned. Hydrozetes octosetosus differs from H. lacustris in several morphological characters that he did not study: namely the position of seta lm relative to the opening gla; the length of seta in in the larva and setae le and ex in all juvenile stages; the number of genital setae; and the shape of some setae on tarsus I and II. While he counted the total number of setae of l-and h-series in the adults, he did not separate the series, which is made necessary by the presence of H. parisiensis in the population. In the latter species, neotrichy occurs in the h-series (Grandjean 1948), where usually more than three pairs of setae are present. As H. lacustris has only three pairs of h-series setae and the same number of l-series setae, which variability did Deichsel (2004) really observe: l-series, h-series, or both series? We believe he was observing variability of h-series setae in H. parisiensis. Since he did not take note of the relative positions of seta lm to opening gla, he could not recognize the adults of H. octosetosus; if they were present, they were included in H. lacustris. Deichsel (2004) did not name the c setae in his drawing, so we can only conclude that German populations of H. lacustris had one pair of setae c 2 , if the total number of notogastral setae was 13 pairs. However, adults of H. lacustris from Norway have two to three pairs of setae of c-series, and those from Poland have one to three pairs, so the total number of notogastral setae is higher than that reported by Deichsel. This problem applies also to adults of H. octosetosus from lake Martwe in the Tuchola Forest, which have one to three pairs of c-series setae. Taking this into consideration, it is useless to compare total numbers of notogastral setae when adults of H. lacustris or H. octosetosus are mixed with those of H. parisiensis, because two series of setae (c-and h-series) vary. For example, adults of H. lacustris and H. octosetosus with two pairs in the c-series have the same total number of notogastral setae as adults of H. parisiensis, which have one pair in the c-series and four pairs in the h-series.
In the nymphal stages Deichsel (2004) observed the number of long setae to vary between two and eight pairs, but as noted above the reader does not know the source of the variability: it may be in the l-series, the h-series or both series. In the nymphal stages this problem is more complicated than in the adults, because in a population of H. lacustris mixed with H. parisiensis the number of long setae varies in both l-series and h-series. Hydrozetes lacustris has one long pair in the l-series (lp), while H. parisiensis (after Grandjean 1948) has two pairs (lm and lp), just as in H. octosetosus. Additionally, the number of long hseries setae varies in H. parisiensis because of neotrichy, so the total number of long setae has no systematic value. If nymphs of H. octosetosus were present in Deichsel's (2004) study, they would have been included in H. parisiensis. It would be atypical of adults of H. lacustris to have a total of five pairs of setae in the l-and h-series, and nymphs with a total of two pairs of long setae in the posterior part of body, which Deichsel observed. The adults of both H. lacustris and H. octosetosus have six pairs of setae in the combined l-and h-series, while H. parisiensis has more setae because of neotrichy. In the nymphs of H. lacustris three pairs of long setae (lp, h 1 , h 2 ) are present, while in H. octosetosus there are four pairs (lm, lp, h 1 , h 2 ) and in H. parisiensis more than four pairs because of neotrichy (lm, lp, and all long setae of the h-series).
Hydrozetes lacustris and H. octosetosus are interesting in that some setae of the c-series have been lost, relative to the tritonymph, and such patterns are thought to reflect phylogeny. During this phylogeny two pairs of setae (c 1 , c 3 ) are subject to loss, after which only setae c 2 remained. However, it is still not clear which pair is lost first, c 3 or c 1 ; Grandjean (1951Grandjean ( , 1968 believed it was pair c 3 , while Shaldybina (1972) thought it was pair c 1 . The order of loss of setae of the c-series in this study is c 1 then c 3 , consistent with Shaldybina (1972). However, Grandjean's (1951Grandjean's ( , 1968 pattern of loss of setae of this series also appears in the higher oribatid mites, for example, in the family Ceratozetidae, where pair c 3 is lost before pair c 1 (Seniczak et al. 1990).
In studied adults of H. octosetosus and H. lacustris the sensillus was short, setiform, or was absent, rarely clavate. Willmann (1932) observed only a clavate sensillus in H. octosetosus, while Michael (1898) mentioned both forms of sensillus in H. lacustris.
In European species of Hydrozetes the shape of some setae on tarsus I of adults may have a diagnostic value (Grandjean 1948), but is probably relevant to the juvenile stages too and varies geographically. For example, in the Norwegian adults of H. lacustris seta s was smooth, while in those studied by Willmann (1931) it was barbed; the Norwegian adults also had thinner seta pv0 and thicker seta u compared to those studied by Grandjean (1948). Therefore the leg setation of Hydrozetes should be investigated on wider geographical material.
The distribution of H. octosetosus differs from that of H. lacustris. In some lakes these species occur together, but in different proportions. Moreover, H. lacustris occurred alone in lake U1 (Ulriken, Norway), while H. octosetosus occurred in lake Martwe (Tuchola Forest, Poland) without H. lacustris. This kind of distribution would not be expected from a single highly variable species of the type proposed by Deichsel (2004). Hydrozetes octosetosus is new to the fauna of Poland, based on the list published by Olszanowski et al. (1996), and to the Norwegian fauna (Mehl 1979).