Juvenile morphology of seven intertidal mite species (Acari, Oribatida, Ameronothroidea) from the East Asian region

ABSTRACT The present paper provides information on juvenile stages of seven intertidal oribatid mite species from the East Asian region. Larva and nymphs of the fortuyniid species Fortuynia churaumi, F. shibai, F. rotunda and Alismobates reticulatus are described and details about juvenile morphology of the selenoribatid species, Arotrobates granulatus, Indopacifica taiyo and I. tyida are given. Juveniles of the genus Arotrobates are reported for the first time and strong ventral femoral carinae are supposed to be diagnostic characters. Basically, the conservative morphology of immature stages could be confirmed in every studied taxon and its importance for phylogenetic considerations is emphasized.


Introduction
Much of the life of oribatid mites is spent as a juvenile and therefore knowledge about these active developmental stages is important for reasons relating to ecology and systematics (Travé 1964). Grandjean (1954) argued that juvenile morphology of oribatid mites is more conservative than that of the adults and thus better suited to infer phylogenetic relationships. Zhang (2018) also emphasized that immature instars show a diversity of traits and other information that could be potentially useful for understanding mite taxonomy, phylogeny and biology. Despite these facts, juveniles of most species of mites are still unknown. For example, in oribatid mites (Astigmata excluded) juvenile instars were studied in only about 8% of all known species and as a consequence, immature morphology is completely unknown for many families (Norton and Ermilov 2014). Reasons for this tremendous lack of knowledge are that resources for making proper adult-juvenile associations are scarce (Norton and Ermilov 2014), and that rearing experiments in the laboratory require living specimens, considerable efforts, time and luck.
The intertidal oribatid mite families Fortuyniidae and Selenoribatidae represent two of the few exceptions, as juvenile morphology played an important role in early systematic considerations. Moreover, numerous species descriptions provided additional information on immatures resulting in a considerable amount of knowledge about juvenile morphology. Van der Hammen (1963) erected the Fortuyniidae based on juvenile morphology, and when Schuster (1963) proposed the Selenoribatidae, he also gave information on the juveniles of Thalassozetes riparius Schuster, 1963, which he described in this work. Shortly afterwards, Grandjean (1966Grandjean ( , 1968 provided diagnoses for the latter family and these included juvenile characteristics, which he also used for a thorough comparison with the Fortuyniidae. Another important study using the juvenile morphology of these families for phylogenetic considerations, was that of Behan-Pelletier (1997), where she provided strong arguments supporting a relationship among Tegeocranellidae, Fortuyniidae and Selenoribatidae. This relationship was further corroborated by Pfingstl and Schuster (2012) using new data on fortuyniid juveniles. With the knowledge of these important studies, it was easier for experts to identify juveniles contained in a sample and to make the correct adult-juvenile association, which resulted in a relatively high number of described juveniles in Fortuyniidae and Selenoribatidae. Both families contain 63 species together and juvenile stages are known from 25 of these, which accounts for approx. 40%.
At the moment, 14 species of Fortuyniidae and Selenoribatidae have been described from Chinese and Japanese coastlines but immatures are known in none of them, except for Circellobates venustus, where Luxton (1992) only provided four sketchy figures of tritonymphal features that he described in two short sentences in his text. Apart from these insufficient data, most of the species were only found once in a single location with very few specimens (Luxton 1992;Karasawa and Aoki 2005), i.e. Arotrobates lanceolatus Luxton, 1992, Psednobates uncunguis Luxton, 1992, Fortuynia sinensis Luxton, 1992, Circellobates venustus Luxton, 1992 and Alismobates rotundus Luxton, 1992, Rhizophobates shimojanai Karasawa & Aoki, 2005, Schusteria nagisa Karasawa & Aoki, 2005 and Schusteria saxea Karasawa & Aoki, 2005, which could explain the lack of juveniles and thus the lack of knowledge. Other species were reported on several occasions and in different areas indicating high abundances and wider distributions for example, Arotrobates granulatus Luxton, 1992 and Alismobates reticulatus Luxton, 1992 which were both discovered in Hong Kong (Luxton 1992) and later found on several Japanese islands (Karasawa and Aoki 2005;Pfingstl et al. 2019bPfingstl et al. , 2021, or Fortuynia rotunda which was described from Mozambique (Marshall and Pugh 2002) and later reported from the Japanese Ryukyu islands (Karasawa and Aoki 2005), and Fortuynia shibai Aoki, 1974 which was originally found in a single location on the island of Shikoku (Aoki 1974) and later it was reported from various Japanese islands (Pfingstl et al. 2019b). In some of these cases, juveniles may have been present in the samples but were either not recognized or simply ignored and consequently they were discarded or stowed unnoticed in a collection. Fortuynia churaumi Pfingstl, Shimano & Hiruta, 2019, Indpacifica taiyo Pfingstl, Shimano & Hiruta, 2021and Indopacifica tyida Pfingstl, Shimano & Hiruta, 2021, on the other hand, were described very recently (Pfingstl et al. 2019b(Pfingstl et al. , 2021, whereas these descriptions were accompanied by comprehensive morphometric and molecular genetic investigations that prevented the inclusion of detailed juvenile data in the first place.
Nevertheless, samples used for the latter two studies contained numerous juvenile specimens from these and several aforementioned species. Therefore, it is the aim of the present paper to provide detailed descriptions of the juvenile stages of these species, which are F. churaumi, F. shibai, F. rotunda, Alismobates reticulatus, Arotrobates granulatus, I. taiyo and I. tyida. Providing this important data is supposed to further contribute to the knowledge about ameronothroid juvenile morphology, which should represent a comprehensive basis for future phylogenetic considerations and reconstructions.

Materials and methods
Samples of littoral algae were scraped off rocks, concrete walls and mangrove roots with a small shovel and then put in Berlese-Tullgren funnels for 12 to 24 hours to extract mites. Afterwards, specimens were preserved in ethanol (100%) for morphological investigation. Most samples were collected by the authors, and different collectors are given next to the respective sample. Juveniles were assigned to a specific species only if adults of a single species were contained in the respective sample.

Preparation and visual documentation
Preserved specimens were embedded in Berlese mountant for microscopic analysis in transmitted light. Drawings were performed with an Olympus BH-2 Microscope equipped with a drawing attachment, then they were scanned and afterwards processed and digitized with the free and open-source vector graphics editor Inkscape (https://inkscape.org) and finally they were further modified with Adobe Photoshop CS. Morphological terminology used in this paper follows that of Grandjean (1954Grandjean ( , 1966Grandjean ( , 1968 and Norton and Behan-Pelletier (2009).
Legs ( Figure 2). Femora I and II with large triangular porous areas on paraxial side. Two slender paraxial longitudinal porous areas on femur III, one dorsally the other ventrally. Setation and solenidia see Table 1.
Legs. Setation and solenidia are shown in Table 1.

Remarks
The barbulation of notogastral setae shown in the adults of F. shibai is already present in the larval stage and remains constant throughout the development. Fortuynia shibai adults also show porous areas on all femora and trochanters III and IV. The femoral porous areas are already present in all juvenile stages but porous areas on trochanters could not be confirmed in any ontogenetic stage (either they are difficult to trace or they are only developed in the adult stage). Pfingstl, Shimano & Hiruta, 2019 Larva and protonymph unknown.
Legs. Setation and solenidia are shown in Table 2.

Remarks
Fortuynia churaumi adults show a specific reticulate pattern (fine dense granulation interrupted by non-granular lines) on their femora (see Pfingstl et al. 2019b, Figure 3), this pattern is already observable in the tritonymph.
Fortuynia shibai and F. churaumi may occur syntopically in some locations on the Japanese islands (Pfingstl et al. 2019b). Adults can be easily distinguished and the same applies to juveniles allowing to easily separate them if contained in a single sample. Fortuynia shibai immatures always show distally barbed notogastral setae (vs. smooth in F. churaumi) and these setae are considerably longer than in F. churaumi. Marshall & Pugh, 2002 Larva (N = 1): length 262 µm.
Legs. Setation and solenidia are shown in Table 3.
Podosoma and venter. Epimeral setation 3-1-3-2, seta 3 c added in this stage, close to trochanter III. Four pairs of genital setae. Three pairs of adanal setae. Two pairs of anal setae, fully developed in this stage.
Legs. Large porous areas on paraxial side of each femur. Porous areas on trochanters not traceable. Setation and solenidia see Table 3.

Remarks
When Marshall and Pugh (2002) described Fortuynia rotunda from African coasts, they gave short information on the legs, e.g. colour and length of segments, and only details about the setae of genu I and II (p. 179, Figure 4(b)). Karasawa and Aoki (2005) provided a redescription of F. rotunda based on their specimens found on the Japanese Ryukyu islands but also gave very few information about the legs, e.g. lengths of segments, smoothness of claws. Accordingly, we present herein the setation and solenidia for the legs of this species for the first time in detail and although these formulas may be the same for all Japanese F. rotunda, they may differ in the African F. rotunda. Therefore, the development of leg setation given in the present paper is so far only valid for Japanese F. rotunda.
Fortuynia rotunda is characterized by its very rounded notogaster and the juveniles already show a similar body shape with the gastronotic region being considerably broad and rounded. However, this trait should not be considered diagnostic, neither for adults nor for juveniles because it is also present in all developmental stages of F. smiti (Pfingstl 2015). It is further assumable that other species with similarly rounded body shapes of adults, like Fortuynia maculata, show similar rounded larva and nymphs.
Legs. Setation and solenidia are shown in Table 4.

Remarks
Adults of A. reticulatus are characterized by the name giving reticulate cuticular notogastral pattern, whereas this pattern is best observed around the medial notogastral setae (e.g. Luxton 1992, own observation). The reticulation results from slightly elevated cuticular lines that are connected at various points. Juveniles also show a distinct reticulate pattern on their centrodorsal plate, but the pattern is clearly visible all over this plate (except for the inverted Y on the posterior half) and it results from irregular scalelike elevations (a negative shape from the structure shown in the adults, so to speak). Juveniles of Alismobates pseudoreticulatus show the exact same pattern (Pfingstl 2015) but the adults of this species possess only remnants of the reticulate pattern framing the bases of centrodorsal notogastral setae. So, adults can be distinguished based on this trait, while juveniles cannot be. Moreover, we could not find any difference between juveniles of both species that makes an identification on species level impossible.
Legs. Setation and solenidia are shown in Table 5.

Remarks
When Luxton (1992) erected the genus Arotrobates, he provided sketchy drawings of the legs of A. lanceolatus and mentioned at least in the key that members of this genus show conspicuous crests on all femora. Apart from this info, he provided very few further details on leg features whereas he mostly reported appearances of solenidia, for both known species. Later, Karasawa and Aoki (2005) redescribed A. granulatus from the southern and central Ryukyus and also gave only vague information on the legs, e.g. they provided length of each segment and wrote a sentence about pigmentation, but in contrast to Luxton (1992) they did not mention ventral carinae on femora at all. The latter trait, however, is present in the Japanese specimens and represents a prominent feature that is already developed in the juvenile stages. The setation and solenidia, presented herein for the first time in detail, are only based on Japanese A. granulatus material. No porous areas could be  -bv´´, l´, d (l), σ v´, (l), φ 1-2 (pl), (pv), s, (a), (u), (p), (tc), (ft), ω 1-2 , ε 0-3-2-3-16 detected on any leg segment in any stage. Another characteristic trait of Arotrobates are the plough-like ventral epimeral carinae and although this feature is not developed in any juvenile stage, the epimeral integument of the nymphs appears to be more folded and less tight than in any other selenoribatid juvenile. This extension of integumental surface could already be an indication of the later developing ventral epimeral carinae.       Tarsus  Chaetome  Solenidia  Leg I deutonymph -d, bv´´, (l) (l), σ (l), v´, φ 1 , φ 2 (pl), (pv), s, (a), (u), (p), (tc), (ft), ε, ω 1 , ω 2 0-4-2-3-16    Grandjean (1954) organized the Brachypylina using the conservatism of juvenile morphology. Indeed, juveniles show more conservative morphologies that may allow to infer high-level relationships (e.g. Norton and Ermilov 2014). On the other hand, this conservatism may mask relationships between species so that it may be impossible to differentiate two closely related species based on their juveniles (e.g. Pfingstl and Krisper 2011) and therefore juvenile morphology is less suitable for species determination. Ermilov et al. (2012) provided diagnostic keys for ameronothrid juveniles but only to generic level for the reason given above. Herein, we even refrain from giving any diagnostic key because of strongly overlapping characteristics in selenoribatid juvenile instars and because of missing data from several genera, e.g. Psednobates, Rhizophobates, Thasecazetes. Nevertheless, certain taxonomic and phylogenetic statements can be drawn from present data. First of all, juveniles of Alismobates reticulatus and A. pseudoreticulatus show a distinct reticulate pattern on their centrodorsal plate and this pattern is also present in the adults. All other known Alismobates juveniles show a clear foveate pattern that is absent in the adult stage. Consequently, A. reticulatus and A. pseudoreticulatus may share a common ancestor and the reticulate pattern may represent a synapomorphy of these two species. Alismobates reticulatus is distributed from Hong Kong (Luxton 1992) to the southern Japanese islands and A. pseudoreticulatus was reported from Thailand and the Malayan peninsula (Pfingstl 2015;Pfingstl et al. 2019a) and together with A. rotundus they are the only species of this genus reported from Southeast and East Asia. The latter species was reported from Hong Kong but unfortunately juveniles are unknown and thus it remains unclear if they also show a reticulate centrodorsal plate and if this trait relates to a phylogeographic lineage.

Discussion
Adults of the genus Fortuynia show a consistently homogeneous morphology with very few distinguishing characters (Pfingstl 2015) and this homogeneity is even more pronounced in the immature stages. In 10 out of 16 described species, immatures share the same habitus, colouration and developmental formulas of notogastral, epimeral and anogenital setae. Variation between juveniles of different species can mainly be found in the length of setae and the presence or absence of barbs on notogastral setae.  (Bayartogtokh et al. 2009). The Table 6. Indopacifica taiyo, chaetome and solenidia from protonymph to adult (tritonymph excluded due to missing data). *information for adult taken from Pfingstl et al. (2021).

Instars
Trochanter Femur Genu Tibia Tarsus  Chaetome  Solenidia  Leg I  protonymph d, bv´´, l´(l), σ l´, v´, φ 1 (pl), (pv), s, (a), (u), (p), (tc), (ft), ε, ω 1 , ω 2 0-3-2-2-16 bv´´, l´(l), σ l´, v´, φ (pv), s, (a), (u), (p), (tc), (ft), ω 0-3-2-2-13 deutonymph of F. arabica exhibits only 10 gastronotic setae whereas the tritonymph shows the full set with 15 setae (Bayartoghtokh et al. 2009), all other Fortuynia juveniles already show the full set of gastronotic setae from the protonymphal stage and never show any reduction during development. The authors based their observation on a single deutonymphal specimen that is absolutely okay if there is only one specimen available but it could be that this specimen represents an aberrant individual. This individual shows another striking deviation as it possesses three genital setae but lacks the aggenital seta (Bayartogtokh et al. 2009), whereas all other Fortuynia deutonymphs show only two genital setae and the presence of one aggenital seta. We think that this deviation is based on a simple mistake because the third genital seta of the F. arabica deutonymph could be the aggenital seta that usually appears in the deutonymph. The protonymph of F. taiwanica shows another developmental abnormality because it already shows one pair of adanal setae (Bayartogtokh et al. 2009). These setae usually appear not before the deutonymphal stage. However, this adanal seta is absent in the ventral depiction of the protonymph of F. taiwanica (Bayartogtokh et al. 2009, p. 249, Figure 4(b)) so it is possible that the authors just made a little slip in the text causing this unusual divergence. The present description of the juvenile stages of Arotrobates granulatus represents the first report of immature stages in the genus and thus we cannot compare it to larvae and nymphs of congeneric species. However, nymphal characteristics of A. granulatus are in accordance with juvenile traits of Selenoribatidae (Grandjean 1966) confirming its systematic position and nymphs can hardly be distinguished from any other selenoribatid juvenile at first glance. Possible diagnostic characters for juveniles of Arotrobates are only the more folded and less tight anterior epimeral integument and the very prominent ventral femoral carinae. The former is supposed to be an indication of the later developing adult ventral epimeral carinae. The genus Carinozetes shows similar epimeral carinae in the adult stage but nymphs lack any similarity with Arotrobates nymphs in this respect. The other trait, femoral ventral carinae are a widespread feature among Selenoribatidae and certain species, e.g. Schusteria littorea, Thalassozetes canariensis, are known to show this feature early in their development (Grandjean 1968;Pfingstl et al. 2020) but adults as well as nymphs of Arotrobates granulatus possess more prominent carinae than any other known selenoribatid taxon. Nonetheless, these characters are difficult to assess without direct comparison and it is unclear if they are also present in Arotobates lanceolatus juveniles; therefore, they should be viewed with caution.
Similar to Fortuynia, adults of the genus Indopacifica show a remarkable homogeneous morphology with very few characters allowing determination (Pfingstl et al. 2021) and the same applies to the juveniles. Immature stages of I. iohanna, I. taiyo and I. tyida show conformity in all characters except for the legs. Indodpacifica iohanna juveniles can be distinguished by the lack of proximoventral teeth on the claws (Resch et al. 2019), a character that is present in the juveniles of the two other species. Indopacifica taiyo and I. tyida show slight differences in the development of leg setation (details are outlined in the respective remarks section), which would allow distinction at least in the deutonymphal stage. Basically, all Indopacifica juveniles show a strongly granular cerotegument, a feature that is only similarly shown in A. granulatus nymphs, and they all possess exobothridial setae that are shaped like a minute lamella. The latter trait may be diagnostic for the juveniles of this genus but such tiny structures are often misinterpreted in light microscopic view and thus need further confirmation with other means of observation. Table 7. Indopacifica tyida, chaetome and solenidia from deutonymph to adult. *information for adult taken from Pfingstl et al. (2021).