First record of the family Ameronothridae (Acari: Oribatida) from Japan – new species, juvenile morphology, ecology and biogeographic remarks

ABSTRACT The Ameronothridae are recorded for the first time from Japanese coasts with the new species Ameronothrus yoichi sp. n. from Hokkaido. The report of this species represents the most southern occurrence of an Ameronothrus species in the Asian Pacific region. Ameronothrus yoichi sp. n. can be easily distinguished from its congeners by the conspicuously pusticulate body surface and the loss of dorsal companion setae d on all genua in the adult stage. Based on adult and juvenile morphology, a close relation to Ameronothrus maculatus and A. schneideri is suggested. Ameronothrus yoichi sp. n. is classified as a lichenivorous inhabitant of sediment-free rocky coastal substrates. Due to a lack of genetic sequence data of nearly all ameronothrid species a molecular genetic comparison is yet unfeasible, but a Bayesian inference tree based on the 18S rRNA gene shows a paraphyletic clustering of the ameronothrid A. yoichi sp. n. and Paraquanothrus grahami. http://www.zoobank.org/urn:lsid:zoobank.org:pub:5B772E2C-7D5E-4C86-9955-AB84A84C50DA


Introduction
The Ameronothridae are a group of several genera of freshwater and marine associated oribatid mites that either live in ephemeral rock pools of terrestrial environments or in various habitats of coastal regions. The taxonomic history of this family was subject to many controversies and confusing changes so that differing concepts still can be found. Berlese early proposed the genus Ameronothrus Berlese, 1896 and included it in Carabodidae (Berlese 1896), but later Willmann (1931) noticed that this genus represents an independent taxon. Though the latter author is often given as family author in literature (e.g. Weigmann 2006), it was Vitzthum (1942) who first mentioned the name Ameronothridae and hence is the true author of this taxon (e.g. Pfingstl 2017). Decades later, Schulte (1975) noticed remarkable morphological similarities between the northern hemispheric marine associated Ameronothridae, till then only consisting of the single genus Ameronothrus, and the southern hemispheric marine associated Podacaridae, containing the genera Podacarus, Halozetes, Alaskozetes, Antarcticola and Pseudantarcticola, therefore he suggested merging the two families. Based on reasonable argumentation, Weigmann and Schulte (1977) supported his suggestion and hence included all the former Podacaridae but also the freshwater associated South African Aquanothridae in the family Ameronothridae and this concept was followed by most subsequent literatures (e.g. Subías 2004;Norton and Behan-Pelletier 2009). Nevertheless, Pfingstl (2017) questioned this taxonomic rearrangement highlighting molecular genetic and biogeographic studies that indicated an independent evolutionary origin of Ameronothridae and Podacaridae. Consequently, the latest concept of Ameronothridae (Norton and Franklin 2018) again excludes the Podacaridae, but retains the freshwater associated genera, Aquanothrus, Paraquanothrus and Chudalupia as subfamily Aquanothrinae besides the marine associated Ameronothrus. Due to this confusing background, we will mainly focus in this paper on the genus Ameronothrus, which represents the northern hemispheric littoral members of the Ameronothridae.
Ameronothrus presently contains 13 species (Subías 2004) that inhabit various coastal habitats, as, for example, rocky intertidal shores, salt marshes, limnic estuary zones, but also terrestrial habitats on further inlands . They are known to feed on a wide variety of food, including lichen, algae and fungi (Schulte 1976). Some species are stenotopic, others are holeurytopic inhabitants of marine-associated environments, whereas species at lower latitudes show greater affinity for littoral habitats and species found at higher latitudes are mainly restricted to terrestrial habitats Marshall and Convey 2004).
Apart from single exceptional records in the Caribbean (Willmann 1936) and on the South African coast (Weigmann 1975), Ameronothrus species are distributed in the Holarctic (Schulte 1975), where they predominantly occur within cold-temperate regions (e.g. Procheş and Marshall 2001). Most records are from European coasts of the Northern and Baltic Sea, but there are also numerous reports from European Atlantic shores, as well as from polar coasts of Greenland and other arctic islands. Further comprehensive findings stretch along the Pacific North American coast from Alaska to California (Schulte 1975), whereas few records are known from the Atlantic North American coast and from the Mediterranean Sea (Schulte 1975;Pfingstl 2017). For a long time, the Asian Pacific shorelines seemed to be devoid of Ameronothrus, until two species Ameronothrus nidicola Sitnikova, 1975 and Ameronothrus oblongus Sitnikova, 1975 were discovered in the Far East of Russia, on Kamchatka (Sitnikova 1975), and later they were also reported from the Kuril Islands (e.g. Klimov 1998). However, although there is important subsequent literature on the systematics of Ameronothridae (Krause et al. 2016;Pfingstl 2017;Norton and Franklin 2018) and on further records from various Holarctic locations (e.g. Bücking et al. 1998;Søvik 2003), no further Ameronothrus species have been discovered and published for the last four decades.
In the course of an ongoing project investigating the intertidal mite fauna of Japanese coasts, we found Ameronothrus individuals on the shore of Hokkaido. Therefore, aim of the present paper is, first, to assess the taxonomic status of the found individuals; second, provide a detailed description; third, give information on juvenile morphology and fourth, discuss the biogeographic aspects of this record.

Material and methods
Samples of littoral algae and lichens were scraped off rocks and concrete walls with a knife or a small shovel and then put in Berlese-Tullgren funnels for about 24 h to extract mites. Afterwards, collected specimens were stored in ethanol (100%) for morphological and molecular genetic investigation.

Molecular genetic analyses
For inferring phylogenetic relationships and providing reference sequences, whole genomic DNA was extracted from 11 ethanolfixed adult specimens of the family Ameronothridae using Chelex resin according to the adjusted protocols in . A~566 bp long fragment of the mitochondrial DNA cytochrome oxidase subunit I (COI) was amplified as stated in  using the primer pair Mite COI-2F and Mite COI-2R (for primer composition see Otto and Wilson 2001). Furthermore, the complete 18S rRNA locus (~1733 bp) was amplified including primers shown in Dabert et al. (2010) along with the protocols in Skoracka and Dabert (2010). Subsequent DNA purification steps included enzymatic ExoSAP-IT (Affymetrix) and Sephadex G-50 resin (GE Healthcare). Cycle sequencing, using BigDye Sequence Terminator v3.1 kit (Applied Biosystems), was conducted according to Schäffer et al. (2008). Automatic capillary sequencing and sequence visualization was operated on an ABI 3130xl (Applied Biosystems) device. All generated sequences are deposited in GenBank under accession numbers MK883430 -MK883440 (COI) and MK880170 -MK880177 (18S) (see Table 1). MUSCLE (Edgar 2004), integrated in the software MEGA 7.0 (Kumar et al. 2016), was used to align sequences. Additionally, already published 18S fragment sequences were downloaded from GenBank for subsequent phylogenetic analysis (for Accession numbers see ed.ac.uk/software/figtree). Both approaches revealed the same overall topologies yielding in high node supports. However, only the BI tree is shown in this study. We only used the 18S locus for species delimitation and phylogenetic inference because relevant COI or 28S sequences were not available in GenBank.

Drawings and photographs
Preserved animals were embedded in Berlese mountant for microscopic investigation in transmitted light. Drawings were made with an Olympus BH-2 Microscope equipped with a drawing attachment. These drawings were first scanned, then processed and digitized with the free and open-source vector graphics editor Inkscape (https://inkscape.org). The map of Hokkaido Island was made by GMT5.2.1 (http://gmt. soest.hawaii.edu/) and further modified with Adobe Photoshop 7.0.
For photographic documentation, specimens were air-dried and photographed with a Keyence VHX-5000 digital microscope.
Etymology. The specific name 'yoichi" is given as noun in apposition and refers to its type locality.
Gastronotic region. (Figure 1A, 2A) Oval in dorsal view, convex in lateral view; no distinct border between anterior median notogastral and prodorsal region. Ascleritic incision pgn visible along the lateral humeral area. Fifteen pairs of short, thickened and blunt notogastral setae (12-15 µm), c 1-3 , da, dm, dp, la, lm, lp, h 1-3 , p 1-3 ; setae p 1 and h 1 conspicuously longer than others, the latter unilaterally barbed. Five pairs of notogastral lyrifissures present but difficult to trace due to the rough cuticular surface; ia between seta c 2 and c 3 , but closer to the latter; im between seta lm and lp; ih laterad and anterior to h 3 ; lyrifissures ip and ips laterally of seta p 3 and p 2 respectively. Orifice of the opisthonotal gland (gla) not traceable due to heavy ornamention.
Lateral aspect. (Figure 2C) Pedotectum I and II absent. Discidium dis between acetabulum III and IV, developed as conspicuous rounded ridge.
Legs. (Figure 4) Ambulacrum tridactylous, median claw broad and strong, lateral claws weaker developed and dorsally slightly dentate. Extensive brachytracheae with slit-like stigmata on the dorsal paraxial face of all femora and tracheal sacculi ventrally on all tibiae and dorsally on trochanter III and IV. Dorsal companion seta d (usually associated with solenidia) on genu I, II and III and all tibiae reduced to alveolus. Primilateral setae of tarsus I absent. Tectal (tc) and iteral (it) setae as well as most other terminal tarsal setae with spoon-shaped or nodular tips (these are sometimes difficult to observe). Famulus ε on tarsus I rod-like, blunt and next to solenidion ω 1 , solenidion ω 2 shorter and in slight paraxial First development of setae characterized by letters, () = pairs of setae, -= no change with regard to preceding stage, d lost = dorsal companion seta reduced.

Common features of juvenile stages
Apheredermous. Colour dark brown. Integument soft and strongly plicate ("nymphes plissées" in the sense of Grandjean 1953). Prodorsum triangular, rostrum rounded, cerotegument overall finely granular. Rostral seta (ro) thin, long and smooth, lamellar (le) and interlamellar setae (in) thickened, blunt and barbed. Exobothridial seta (ex) small and smooth. Bothridia small cup-like. Sensillum short, clavate, head with inconspicuous linear elevations. Hysterosoma oval in dorsal view, slightly convex in lateral view. Notogastral setae thickened and barbed, posterior setae longest, median dorsal setae shortest. Orifice of opisthonotal gland gla located in latero-ventral folds on a level with anterior third of anal valves. Legs monodactylous, with slightly invaginated porose areas on the same leg segments and position as brachytracheae in adults. Solenidia on genu and tibia always associated with dorsal companion setae (both approx. the same size).
Ventral region of idiosoma. Epimeral setation 2(3)-1-2, third seta on epimeron 1(given in parentheses) developed as protective seta (number in parentheses includes this seta) covering Claparède's organ. No anogenital setae present in this stage. Posterior third of anal orifice framed by smooth and short seta h 3 and smooth and long seta h 2 . Legs. Chaetome and solenidia see Table 2.

Ecological data
The specimen from Muroran (Figure 8) was found in black algae growing on a large rock in the upper eulittoral zone of a rough coastal area with strong wave action. The sea at this location belongs to the North Pacific Ocean but still is influenced by the warm Tsugaru current which flows eastwards through the strait of Tsugaru (between Hokkaido and Honshu connecting the Sea of Japan with the Pacific Ocean). The air temperature at Muroran ranges from −2.6°C in February to 20.1°C in August, while the sea surface temperature ranges from approximately 2°C in the coldest month to 23°C in the warmest month (Table 3). The population from Yoichi was collected from large layers of grey lichen growing on a massive quay wall. The sample was taken in the supralittoral area, which is only exposed to spray from the seawater. This location lies at the Sea of Japan (Figure 8) in an area that is influenced by the warm northwards flowing Tsushima current. The air temperature in Yoichi ranges from −5°C in the winter to 20°C in the summer, whereas sea surface temperature is the lowest in March with approx. 4.8°C and the highest in September with 23.4°C (Table 3).

Genetic data
COI as well as 18S rRNA sequence data confirm all investigated specimens as members of A. yoichi. The Bayesian inference tree based on 18S rRNA further shows a well-separated A. yoichi clade but places it in paraphyly to Paraquanothrus grahami Norton & Franklin, 2018, a supposed member of the Ameronothridae (Figure 9). Additionally, A. yoichi as well as P. grahami are placed outside other members of the superfamily Ameronothroidea, namely the Selenoribatidae and Fortuyniidae that are represented as a monophyletic group in the tree.

Systematics
The new species Ameronothrus yoichi can be easily distinguished from its congeners by its unique prodorsal ridge structure, the conspicuously pusticulate body surface showing densely packed large nodules and the loss of dorsal companion setae d on all genua in the adult stage. Most other characteristics do overlap with certain species and hence can only be used in specific combinations for determination. Schulte (1975) classified four groups of Ameronothrus species based on morphological correlations, (I) the Ameronothrus marinus group containing A. marinus  setae (pl), the reduction of the ascleritic incision pgn and the complete labiogenal articulation with the A. maculatus group, but shows a differing anal setation with two instead of only one pair of setae. Based on these conformities, A. yoichi seems to be closest related to A. maculatus and A. schneideri, though there is no complete match with the group. Unfortunately, the descriptions of A. dubinini, A. nidicola and A. oblongus (Sitnikova 1975(Sitnikova , 1977 are not detailed enough for a thorough comparison and classification into one evolutionary group in the sense of Schulte (1975); therefore, a clear assessment of phylogenetic relationships within Ameronothrus is not feasible yet.
The same applies to juvenile morphology as information on the above-mentioned three species and A. nigrofemoratus and A. schubarti is lacking. Nevertheless, a comparison with known Ameronothrus juveniles shows that A. yoichi exhibits the same developmental patterns and formulas, e.g. prodorsal, notogastral, epimeral and anogenital setation, but differs from other species in certain characteristics. Ameronothrus marinus, A. bilineatus and A. schneideri already lack a trichobothrium in the juvenile stages and A. lineatus and A. lapponicus show uniform shapes of notogastral setae (e.g. Schulte 1975;Ermilov et al. 2012) whereas A. yoichi juveniles show enlarged and broadened, posterior notogastral setae. A similar condition can only be found in the nymphs of A. maculatus, but here seta lp is not thickened and enlarged as it is in A. yoichi.
In a larger phylogenetic context, the present data places A. yoichi in a paraphyletic position to Paraquanothrus grahami, the second supposedly member of Ameronothridae. This contrasts with the recently suggested close relationship of Aquanothrinae and Ameronothridae (Norton and Franklin 2018) and questions its inclusion in the latter. The suggested independent evolutionary origin of the littoral Ameronothridae, Fortuyniidae and Selenoribatidae (Krause et al. 2016;Pfingstl 2017), all members of the superfamily Ameronothroidea, on the other hand, is supported as shown by their paraphyletic placement. The monophyletic origin of Fortuyniidae and Selenoribatidae was already confirmed by certain studies (Iseki and Karasawa 2014;Krause et al. 2016) and is further affirmed by  the present data. Nonetheless, comprehensive molecular genetic studies, including many more taxa, are necessary to solve found discrepancies and to verify indicated phylogenetic positions.
Ecology Schulte et al. (1975) postulated four ecological groups of ameronothrid mites: (I) stenotopic inhabitants of the marine littoral populating characteristic salinity ranges, (II) eurytopic inhabitants of the littoral living in saline and brackish waters, (III) holeurytopic inhabitants of the littoral living in limnic, brackish, marine and terrestrial habitats and (IV) stenotopic inhabitants of inland regions dwelling exclusively in terrestrial habitats. Based on the few present records of A. yoichi it is difficult to assess the ecology of this species but the populations were found in the upper marine eulittoral and supralittoral area hence a strictly terrestrial lifestyle (group IV) can be excluded. Additionally, they were found at relatively low latitudes and species occurring at lower latitudes usually tend to be stenotopic and restricted to marine environments Marshall and Convey 2004); therefore, A. yoichi most likely belongs to group I or II. Several Ameronothrus species, i.e. A. marinus, A. schusteri, A. lineatus, A. maculatus and A. schubarti are known to live mainly in the sediment-free rock littoral ) and the same may apply to A. yoichi as both investigated populations were found restricted to this habitat. Ameronothrus yoichi shows three claws on each leg, which further points to the rocky substrate as the main habitat because tridactylous Ameronothrus species are usually found on solid substrates while monodactylous species are mainly restricted to soft substrates (Schulte 1975).
All Ameronothrus species are microphytophagous and can be classified into three groups, namely, lichenivorous species feeding on lichen growing on hard substrates of the coast or inland, algivorous species subsisting on diverse algae from hard coastal substrates and fungivorous species feeding on fungal growth in the upper soil layer of salt marshes (Schulte 1976). Based on its predominant occurrence in lichen on a hard substrate, A. yoichi most likely belongs to the lichenivorous group. However, feeding experiments in the lab would be necessary to confirm such a food preference.
In all known cases, Ameronothrus species are larviparous (Bücking et al. 1998) or at least ovoviviparous (Søvik 2003), which means that larvae hatch immediately after deposition. The same is true for A. yoichi as we found fully developed larvae contained within eggshells inside the body of a female.

Biogeographic aspects
In a global context, the occurrence of A. yoichi at a latitude of approximately 42 degrees North is nothing unusual because distributions of most Ameronothrus species stretch further south (Schulte 1975). In the Asian Pacific region, on the other hand, represents the record of A. yoichi on Hokkaido the southernmost occurrence of an Ameronothrus species. The congeneric A. nidicola and A. oblongus were reported from Kunashir Island (Klimov 1998), which is very close to Hokkaido but lies approximately two degrees more northern (see Figure 8). The distribution of both species stretches further north with records from Sakhalin Island, Lovushki and the Peninsula of Kamchatka (Ryabinin 2015). This pattern indicates that A. nidicola and A. oblongus are adapted to colder climates than A. yoichi whereas it is not clear yet how far the distribution of the latter reaches into the South. Records from Honshu are lacking, but occurrences in the north and hence colder regions of this Japanese Island should be considered. Water temperatures seem to play an important role in shaping the distributions of Ameronothrus as shown in A. lineatus and A. nigrofemoratus. These species reach the latitude of 38°North at the Pacific American shore where consistently cold waters prevail while they only reach a latitude of 50°North at European coasts where comparable water temperatures are present (Schulte 1975). Japanese coasts are strongly influenced by the warm Kuroshio Current and its branch the Tsushima Current and these warm waters may limit the southern distribution of Asian Pacific Ameronothrus species.  Table 1.
However, biogeographic studies combined with local climate data would be necessary to prove such a correlation. Unfortunately, most studies on Ameronothrus species provide the locations of the species found, but no detailed information on the climate prevailing at these locations, therefore only vague conclusions might be drawn. In the light of global warming, local climates may change rapidly and hence distributions of Ameronothrus species may alter, therefore providing reference data is the first step in predicting these shifts.