Chamobates borealis (Trägårdh 1902) has been considered by some authors as a junior synonym of Chamobates pusillus (Berlese 1895). In this study we used an integrated taxonomy approach, comparing mitochondrial coding gene COI and morphological ontogeny of these species to clarify their systematic status. The Bayesian inference tree based on COI sequences of C. borealis and C. pusillus, as well as C. birulai (Kulczyński 1902), C. bispinosusMahunka, 1987, C. cuspidatus (Michael 1884) and C. rastratus (Hull 1914) separated all these species. In terms of the morphology, the adults of C. borealis and C. pusillus have similar body size and shape, thin aggenital setae and two lateral teeth on the rostrum, but C. borealis has the medial incision between these teeth, which is absent in C. pusillus. The adults of these species differ also from each other by the shape of bothridial setae, size of area porose Aa, location of seta lm and lyrifissure im, and the shape of most setae on the hysterosoma. The morphological ontogeny of these species is similar, but the larva and nymphs of C. borealis differ from those of C. pusillus by the length of some prodorsal and gastronotal setae, and the nymphs of C. borealis have a humeral organ, which is absent in C. pusillus. The presence of a humeral organ in some Chamobates species supports a clade inferred by COI sequence data.
Introduction
Chamobates borealis (Trägårdh 1902) was firstly described as Notaspis cuspidatus borealis Trägårdh, 1902, but this description, as well as a later redescription (Trägårdh 1910) were brief and concerned mainly the anterior part of the body, whereas the location of setae and porose areas on the notogaster were not indicated. Later, however, most authors (Shaldybina 1975; Karppinen & Krivolutsky 1982; Golosova et al. 1983; Schatz 1983; Marshall et al. 1987; Pavlichenko 1994; Bernini et al. 1995; Olszanowski et al. 1996; Niemi et al. 1997; Weigmann 2006; Siepel et al. 2009; Miko 2016; Murvanidze & Mumladze 2016) considered C. borealis as a separate species, whereas some (Mahunka and Mahunka-Papp 1995; Subías 2004, 2019; Bayartogtokh 2010; Weigmann et al. 2015; Arroyo et al. 2017) treated it as a junior synonym of Chamobates pusillus (Berlese 1895). According to Weigmann (2006), C. borealis has a medial incision between the two lateral teeth, while this incision is absent in C. pusillus. The adult of C. borealis has thin aggenital setae, and Subías (2004, 2019) included it in Chamobates sensu stricto, with 21 nominative species.
The morphological ontogeny—an often useful systematic and taxonomic data—of C. borealis has not been investigated, yet. Based on the catalogue of Norton and Ermilov (2014) and papers of Seniczak and Seniczak (2014) and Seniczak et al. (2018), the full ontogeny of six species of Chamobates is known, which constitutes a third of all species of this genus. These species are: C. cuspidatus, C. pusillus, C. rastratus (Hull 1914), C. schuetzi (Oudemans 1902), C. subglobulus (Oudemans 1900) and C. voigtsi (Oudemans 1902).
The aim of this paper is to clarify the systematic status of C. borealis, based on mitochondrial COI gene sequences and morphological ontogeny of this species, which was investigated in detail for the first time.
Materials and Methods
Sampling
Chamobates borealis was collected in broadleaf forests in Norway, C. pusillus in a peatland in Ireland, from which the juveniles of this species were described previously (see Seniczak et al. 2018), while other species included in the phylogeny analyses originated from Norway and Greece (Table 1). Sampling was carried out during 2012–2018, in mainland Norway by Anna Seniczak, Steffen Roth and Per Djursvoll, in Svalbard by Steffen Roth, in Ireland by Anna Seniczak and Thomas Bolger, and in Greece by Stanislaw Seniczak and Stefanos Sgardelis. Samples of mosses, each of a volume of 500 cm3, were collected by hand from the ground. Additionally, in order to study the ecology of C. borealis, several microhabitats (mosses from tree bark at ground level and 1.5 m above it, from stumps and dead tree trunks and dead wood) were sampled in one forest (Norway, Hordaland, Kvam: Mundheim, 60.155°, 5.896°, 97 m a. s. l., 8 June 2017). This was a low-herb deciduous forest dominated by gray alder [Alnus incana (L.) Moench], ash (Fraxinus excelsior L.), hazel (Corylus avellana L.), wych elm (Ulmus glabra Hudson) and birch (Betula pendula Roth), while the forest floor was mostly overgrown by mosses. The detailed habitat characteristics were presented earlier (Seniczak et al. 2019). Mites were extracted in Tullgren funnels for 14 days, because the samples were relatively large and originated from wet forest (Seniczak et al. 2019), and preserved in 90% ethanol.
Studies of type material
Type specimens of C. borealis (2 adults, slide label: “Oribata cuspidata var. borealis. Rör 3 Kårsonjuonje I.T-dh.”, 1 adult, slide label:“Oribata cuspidata var. borealis. N01, 07 I.T-dh.”), and one adult of C. pusillus collected by K.H. Forsslund in the type locality [label:“Chamobates pusillus (Berl.) ♀ K.-H.F. leg. det. Ital. Toscana, Vallombrosa 24.9.1961. mf. 1006”] were borrowed from the Swedish Museum of Natural History, Stockholm, Sweden. The type specimen from the Berlese collection (single specimen, slide 28/10) was not in a good condition to be studied, so instead the measurements and photographs of other material from the type locality from this collection (slides 68/11 and 12) were kindly provided by Dr. Roberto Nannelli (CREA-DC, Research Centre for Plant Protection and Certification, Florence, Italy).
Molecular analyses
Thirty-five specimens of six Chamobates species (Table 1) were analyzed. We used species of putatively close genera as outgroups, Ceratozetes parvulus Sellnick, 1922, Euzetes globulus (Nicolet 1855), Fuscozetes fuscipes (C.L. Koch 1844) and Mycobates sarekensis (Trägårdh 1910). Each specimen was photographed and the photos are the vouchers that are available at Barcode of Life Data System (BOLD, http://boldsystems.org/). The specimens were subsequently placed in a well containing 50 ml of 90% ethanol in a 96-well microplate, and send to the Canadian Centre for DNA Barcoding (CCDB). Mites were sequenced for the barcode region of the COI gene according to standard protocols at CCDB (CCDB 2019), using either LepF1/LepR1 (Hebert et al. 2003) or LCO1490/HCO2198 (Folmer et al. 1994) primer pairs. The DNA extracts were placed in archival storage at -80°C at the University Museum of Bergen (UiB). The sequences are available in GenBank (accessions numbers in Table 1).
TABLE 1.
Information about specimens used in this study; labels correspond to specimen numbers in BOLD database.
(Continued)
COI sequences (sequence length a ≥ 407 bp) were blasted against GenBank in order to detect and exclude possible contaminations. Sequence variation within Chamobates species and between-species was calculated in BOLD, using Kimura 2 Parameter distance model, pairwise deletion, and BOLD Aligner (Amino Acid based HMM).
The sequences were aligned by eye in BioEdit v7.0.5 sequence alignment editor (Hall 2011). The search for the best fitting substitution model was carried out in PAUP* 4.0 a164 (Swofford 2002) using ModelTest (Posada & Crandall 1998). Phylogenetic Bayesian inference (BI) analysis was conducted in MrBayes 3.2 (Ronquist et al. 2012). Posterior probabilities were generated from Markov chain Monte Carlo (MCMMC) sampling over 10 million generations in two independent runs using 4 chains, HKY+I+G model (Hasegawa et al.1985) and 25% burn-in. The trace files generated by Bayesian MCMC runs were analyzed in Tracer v.1.6. (Rambaut & Drummond 2007) in order to assess chain convergence. After this step a 50% majority rule consensus tree was summarized from post burn-in trees and BI topologies were visualized in FigTree 1.4.2 (available at http://tree.bio.ed.ac.uk/software/figtree).
Illustrations and photomicrographs
Illustrations were prepared from individuals macerated in lactic acid, using the open-mount technique (Grandjean 1949). We measured total length (from tip of rostrum to posterior edge of notogaster) and width (widest part of notogaster without pteromorphs), and length of setae and some parts of the body of mites in µm. The illustrations of instars are limited to the body regions of mites that show substantial differences between instars, including the dorsal and lateral aspect of the larva, tritonymph and adult, some leg segments of these stages and ventral regions of all instars. Palp and chelicera of the adult are also illustrated. In the text and figures, we use the following abbreviations: rostral (ro), lamellar (le), interlamellar (in) and exobothridial (ex) setae, lamella (La), bothridium (bo), bothridial seta (bs), notogastral or gastronotal setae (c-, d-, l-, h-, p-series), lyrifissures or cupules (ia, im ip, ih, ips, iad), porose areas (Aa, A1–A3), opisthonotal gland opening (gla), pteromorph (Ptm), pedotecta (Pd1), tutorium (Tut), Claparède organ (Cl), subcapitular setae (a, m, h), genal tooth (gt), genal notch (gn), discidium (Dis), cheliceral setae (cha, chb), palp setae (sup, inf, l, d, cm, acm, lt, vt, 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 solenidia (σ, φ, ω), famulus (ε) and setae (bv, ev, d, l, ft, tc, it, p, u, a, s, pv, pl, v). Terminology used follows that of Grandjean (1953, 1962) and Norton and Behan-Pelletier (2009).
For scanning electron microscopy (SEM), mites were fixed in 90% ethanol and placed on Al-stubs with a double-sticky carbon tape and coated with Au/Pd in a Polaron SC502 Sputter coater. Observations and micrographs were made with a ZEISS Supra 55VP scanning electron microscope.
Results
Molecular analyses
The Bayesian inference tree based on COI sequences showed that C. borealis forms a separate clade from C. pusillus (Fig. 1). Each of the two taxa obtained maximum posterior probability and were separated from each other by other Chamobates species with high node support. The two species were separated by a minimum of 14.33% COI sequence divergence (Table 2), whereas intraspecific COI variation in any of the included Chamobates species did not exceed 3.77%.
TABLE 2.
COI-based maximum P-distances within Chamobates species (underlined) and minimum P-distances between species, na – only one specimen was available and it was not possible to calculate P-distance within species.
FIGURE 1.
Bayesian inference tree based on COI sequences (658 bp). Specimen numbers correspond to BOLD database ( http://boldsystems.org/) and the photographs are vouchers of the sequenced animals. Information about specimens used for sequencing with Gene Bank accession numbers are in Table 1. Node support values < 0.90 are not presented in the graph.
Systematics
Chamobates borealis (Trägårdh, 1902)
(Figs. 2a, 2b, 3–12)
Notaspis cuspidata var. borealis Trägårdh, 1902.
Oribata cuspidata var. borealis: Trägårdh 1910.
Chamobates borealis: Shaldybina 1975; Karppinen and Krivolutsky 1982; Golosova et al. 1983; Schatz 1983; Marshall et al. 1987; Pavlichenko 1994; Bernini et al. 1995; Olszanowski et al. 1996; Niemi et al. 1997; Weigmann 2006; Siepel et al. 2009; Miko 2016; Murvanidze and Mumladze 2016.
Chamobates schuetzi (Oudemans 1902): Willmann 1931.
Diagnosis
Adult rather small (length 325–384, width 202–247; n= 60). Rostrum with medial incision and two lateral teeth. Bothridial seta clavate, finely barbed. Prodorsal seta le long, ro and in of medium size and ex short. Lamella well developed, cusp with outer tooth, translamella absent. Notogastral setae minute. Porose area Aa distinctly larger than other porose areas. Seta lm located posterior-medially to porose area Aa at distance equal to diameter of Aa. Lyrifissure im placed midway between seta lm and porose area A1. Aggenital setae thin, adanal and anal setae short.
Juveniles unpigmented. Gastronotal shield absent in larva, but present in nymphs, with 10 pairs of setae (d-, l-, h-series and p1), setae p2, p3 and of c-series inserted on unsclerotized cuticle. In larva, seta in shorter than ro, seta lp almost two times longer than la, but of similar length as h1. Humeral organ present only in nymphs. Gastronotal setae short in nymphs, except for longer c3, dp and h1.
Description of morphological ontogeny
Adult
Morphology of adult (Figs. 2a, 2b, 3–6) similar to that investigated by Weigmann (2006), with triangular prodorsum and almost spherical, convex notogaster. Mean length of females 368.1 (range 356–384, n=30) and width 238.5 (228–247), and mean length of males 346.7 (range 325–358, n= 30) and width 209.4 (202–215). All notogastral setae minute. Porose area Aa rounded and larger than other porose areas. Seta lm located closer to Aa than seta la, lyrifissure im located midway between seta lm and gla opening. Cheliceral setae cha longer than chb, both barbed (Fig. 5b). Most palp setae barbed, except for smooth tarsal setae (Fig. 5c). Anteroventral apophysis on genua I and II absent, tibia I with anterodorsal apophysis (Fig. 6). Most leg setae with short barbs, setae pv and s on all tarsi with longer barbs. Formulae of leg setae [trochanter to tarsus (+ solenidia)]: I—1-5-3(1)-4(2)-20(2); II—1-5-3(1)-4(1)-15(2); III—2-2-1(1)-3(1)-15; IV—1-2-2-3(1)-12. Tarsi heterotridactylous.
Juvenile stages
Larva oval (Fig. 7a), unpigmented. Prodorsum subtriangular, prodorsal seta ro longer than in and le (Table 3), all barbed; seta ex short and smooth. Mutual distance between pair le about two times longer, and between pair in about three times longer than between pair ro. Setal pair le inserted closer to in than ro. Opening of bothridium rounded, bothridial seta clavate, with barbed head.
FIGURE 5.
Chamobates borealis, female. (a) Lateral aspect, legs partially drawn, scale bar 50 µm; mouthparts, right side, scale bars 20 µm, (b) chelicera, (c) palp.
Gastronotum of larva with 12 pairs of setae, including h3 inserted laterally to posterior part of anal valves (Figs. 8a, 9a); most of medium size (Table 3) and barbed, except for short and smooth h3, length of setae increasing from anterior to posterior. Longer gastronotal setae darkly pigmented, except for light basal part (Fig. 7b). Gastronotal shield absent. Cupule ia located posterior to seta c3, cupule im between setae lm and lp, cupule ip between setae h1 and h2, and gland opening anterolateral to seta lp. Anal valves (segment PS) glabrous (Figs. 8a, 9a).
Nymphs with relatively shorter prodorsum and slimmer bothridial seta than in larva, length of prodorsal setae increasing during ontogeny (Table 3). Gastronotum of protonymph with 15 pairs of setae because setae of p-series appear in protonymph (Fig. 8b), and remain in other nymphs (Figs. 10a, 10b). Gastronotal shield present, with 10 pairs of setae (d-, l-, h-series and p1), setae p2, p3 and of c-series inserted on unsclerotized cuticle. Seta c3 of medium size and barbed, other gastronotal setae short, except for slightly longer dp and h1 (Table 3); longer setae with short barbs, other setae smooth (Fig. 11). Longer gastronotal setae dark pigmented, except for light basal part. In protonymph, one pair of setae appears on genital valves (Fig. 8b), and two pairs are added in deutonymph and tritonymph each (Figs. 10a, 10b); all short and smooth. In deutonymph, one pair of aggenital setae and three pairs of adanal setae appear and remain in tritonymph; all short and smooth. Anal valves of protonymph (segment AD) and deutonymph (segment AN) glabrous, but in tritonymph two pairs of small, smooth setae present (Fig. 10b). In nymphs cupules ia and im placed as in larva, cupule ip located between seta h2 and p2 (protonymph) or between p1 and h2 (other nymphs), cupule iad located lateral to anterior part of anal valves, cupules ips and ih displaced posterolateral and lateral to iad (Figs. 8a, 8b, 10a, 10b). Gland opening as in larva. Leg setae of tritonymph with short barbs or smooth (Fig. 12).
Ontogenetic transformation
The relative length of prodorsal setae ro, in and le changes during the ontogeny. In the larva, the longest of these three setae is ro, in the nymphs in, and in the adult le. In all instars, seta ex remains short. The opening of bothridium is rounded in all instars, but in the adult it is larger and has lateral and medial scales. In all instars, the bothridial seta is clavate and barbed, but in the nymphs its head is slimmer than in the larva and adult. The larva has 12 pairs of gastronotal setae, the nymphs have 15 pairs (p-series appears), whereas the notogaster of the adults loses setae c1, c3 and d-series, such that 10 pairs of setae (c2, l- and h- and p-series) remain. The formula of gastronotal setae of C. borealis is 12-15-15-15-10 (from larva to adult). Formulae of epimeral, genital, aggenital setae and segments PS–AN are as in C. pusillus (Seniczak et al. 2018). The ontogeny of leg setae and solenidia of C. borealis is similar to that of C. pusillus (Seniczak et al. 2018).
Distribution and ecology of Chamobates borealis
Chamobates borealis is considered a Holarctic species (Weigmann 2006), typically found in soils in forests of different humidity (Weigmann 2006). It is a silvicolous, microphytophagous (Schatz 2016) and secondary decomposer (Schneider et al. 2004). It was also reported on feathers of birds (Lebedeva & Krivolutsky 2003).
FIGURE. 7–8.
Chamobates borealis, legs partially drawn, scale bar 50 µm. 7. Larva, (a) dorsal aspect, (b) shape of seta lp (enlarged). 8. Ventral part of hysterosoma, (a) larva, (b) protonymph.
TABLE 3.
Measurements of some morphological characters of juvenile stages and adults of Chamobates borealis (mean measurements of 2–10 individuals per instar in µm); Nd – not developed.
FIGURE 9.
Chamobates borealis, lateral aspect, legs partially drawn, scale bars 50 µm. (a) Larva, (b) tritonymph.
FIGURE 10–11.
Chamobates borealis, legs partially drawn, scale bars 50 µm. 10. Ventral part of hysterosoma, (a) deutonymph, (b) tritonymph. 11. Tritonymph, (a) dorsal aspect, (b) shape of seta in (enlarged).
Chamobates borealis was the second most abundant oribatid species in a rich broadleaf forest in western Norway (dominance index, 17%; average density 83 individuals per 500 cm3). It was found in all microhabitats studied (mosses from soil, tree bark, stump, dead wood, and in dead wood) but was most abundant in mosses growing on dead wood (Seniczak et al. 2019). The juveniles made on average 10% of the population, but were most abundant in mosses on ground and absent from tree bark and dead wood. In mosses on the ground (total of five samples, sampled 8 June), the stage structure of C. borealis was the following: 17 larvae (3%), seven protonymphs (1%), 22 deutonymphs (4%), 19 tritonymphs (4%) and 457 adults (88%). The sex ratio (females: males) calculated for one sample (120 adults) was 1:1.3 and 50% of females were gravid, carrying one to four large eggs (160 × 77), which made about 43% of their total body length.
Note on Chamobates pusillus (Berlese, 1895)
Seniczak et al. (2018) described morphological ontogeny of Chamobates pusillus (Berlese, 1895) but did not provide diagnosis of this species. To facilitated future identification, we supplementary present it as follows: Diagnosis of adult: Adult of similar size (length 345–390, width 234–267 (n= 35), rostrum with two teeth, medial incision absent (Figs. 2c, 2d). Most notogastral setae alveolar, except minute c2 and p-series. Porose area Aa slightly larger than other porose areas. Seta lm located medially from porose area Aa at distance of two times diameter of Aa; lyrifissure im placed closer to porose area A1 than to seta lm; aggenital setae thin, adanal and anal setae alveolar. Diagnosis of juveniles: Gastronotal shield absent in larva, present in nymphs, with 10 pairs of setae (d-, l-, h-series and p1), setae p2, p3 and of c-series inserted on unsclerotized cuticle. In larva, seta in longer than ro, in nymphs humeral organ absent and gastronotal setae fine and short, except for distinctly longer and thicker c3.
Discussion
Chamobates borealis has been considered a junior synonym of C. pusillus by some authors, but in the light of our investigations these species differ clearly from each other, both on the molecular and morphological level (Tables 2, 4). Although not so common in the field of acarology, yet, morphology-based species identification should be accompanied by DNA characters. There is an increasing number of studies using COI sequence data to assess species boundaries in Oribatida (Schäffer et al. 2010; Lienhard et al. 2014; Kreipe 2015; Pfingstl et al. 2019a, 2019b). The level of sequence divergence in the Chamobates species analyzed here is generally similar to species in those studies. Although the threshold between intra- and interspecific variance for many groups can be much higher than 2% (Hebert et al. 2003; Cognato 2006), the more than 14% observed here between C. borealis and C. pusillus is far beyond what can be accepted within a species. More importantly, the phylogenetic analysis clearly set apart C. borealis from C. pusillus, with several taxa separating the two with a strong support.
The morphology of adult C. borealis differs from C. pusillus by several distinct characters (Weigmann 2006; Seniczak et al. 2018). Chamobates borealis has the medial incision between the rostral teeth, whereas C. pusillus has no such incision. The former species has more slender bothridial seta, and larger porose area Aa than the latter species. In C. borealis, the seta lm is located closer to Aa than in C. pusillus. In the latter species, lyrifissure im is placed closer to the gla opening than in the former species. Moreover, in C. borealis all setae of the hysterosoma are short, whereas in C. pusillus most notogastral setae and setae of ad- and an-series are alveolar.
Also the juveniles of C. borealis differ from those of C. pusillus. The larva of the former species has longer prodorsal setae ro than in, while in the latter species it is the opposite. The nymphs of C. borealis have longer setae dp and h1 than C. pusillus and possess a humeral organ, which is absent in the latter species. This organ is also present in the nymphs of C. cuspidatus, C. rastratus and C. subglobulus, whereas it is absent in C. schuetzi and C. voigtsi (Seniczak & Solhøy 1988; Seniczak & Żelazna 1994; Seniczak & Seniczak 2014; Seniczak et al. 2018). It is interesting to note that the presence or absence of a humeral organ in these taxa at least partly fit with the clades inferred by COI sequence data (Fig. 1). The gastronotal shield is on the other hand present in the nymphs of both C. borealis and C. pusillus, and additionally in C. rastratus and C. schuetzi, whereas absent in other species. In the nymphs of C. borealis, C. pusillus and C. schuetzi, most gastronotal setae are short (Seniczak & Solhøy 1988; Seniczak et al. 2018), in C. rastratus and C. subglobulus they are long (Seniczak & Żelazna 1994; Seniczak & Seniczak 2014), whereas in C. cuspidatus they are of medium size (Seniczak & Solhøy 1988). In the larvae of most species, most gastronotal setae are of medium size, except for C. subglobulus, in which these setae are very long. Our molecular data have indicated that these characters are of limited value in phylogenetic studies, but can be of great taxonomic value.
Willmann (1931) considered C. borealis a synonym of C. schuetzi, but in the former species the porose area Aa is larger than in the latter species, and seta in is shorter than in C. schuetzi. Therefore, C. schuetzi needs more studies, also on the molecular level to confirm the systematic status.
Acknowledgements
We thank two anonymous reviewers for helpful suggestions and Dr. Marut Fuangarworn, a section Editor of Systematic & Applied Acarology for editorial suggestions that improved the scientific value of this paper. We are grateful to Ms. Gunvi Lindberg (Swedish Museum of Natural History, Stockholm, Sweden) for the loan of the material used in this study, and to Dr. Roberto Nannelli (CREA-DC, Research Centre for Plant Protection and Certification, Florence, Italy) for the photographs and measurements of C. pusillus from the Berlese Collection, and to Dr. Steffen Roth (University Museum of Bergen, Bergen, Norway) for collecting samples in Svalbard. This study was supported by grant No. 811030 from The Norwegian Taxonomy Initiative – KR 35-16 (Norwegian Forest Oribatida (NFO) - highly diverse, but poorly known). DNA barcode data in this publication was generated in collaboration with the Norwegian Barcode of Life Network (NorBOL) funded by the Research Council of Norway and the Norwegian Biodiversity Information Centre. Part of the study was done within the program of Polish Minister of Science and Higher Education “Regional Initiative of Excellence” in 2019–2022 (Grant No. 008/RID/2018/19).
