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One step closer but still far from solving the puzzle – The phylogeny of marine associated mites (Acari, Oribatida, Ameronothroidea) inferred from morphological and molecular genetic data

In: Contributions to Zoology
Authors:
Tobias Pfingstl Institute of Biology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria

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Iris Bardel-Kahr Institute of Biology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria

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Klaus Schliep Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria

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Abstract

Marine associated oribatid mites belong mainly to the Ameronothroidea which represent a very small percentage of all Oribatida. Over the last decades the systematics and evolutionary history of this group has been discussed controversially and still there is no consensus concerning several issues. The extreme marine environment may have resulted in parallel morphologies complicating the classification and estimation of phylogeny based on discrete morphological traits. In the present study, we performed a molecular genetic study using a mitochondrial and two nuclear markers to infer the phylogeny of this group. Additionally, we reconstructed the phylogeny of Ameronothroidea based on morphological data using different algorithms. Both methods resulted in largely congruent topologies and highlight the following important points: the Ameronothroidea represent a paraphyletic assemblage; the Podacaridae are a distinct family and should be excluded from Ameronothridae; the Fortuyniidae, Selenoribatidae and Tegeocranellidae constitute a monophyletic lineage; and certain genera of Selenoribatidae need a revision. These results demonstrate that the classification of Ameronothroidea and certain positions within this group need to be thoroughly reconsidered and revised. The present study also shows that phylogenetic estimates based on coded morphological data can be a very helpful tool for verifying and supporting molecular phylogenies.

Introduction

Intertidal oribatid mites mostly belong to the superfamily of Ameronothroidea, a group with about 110 described species, representing only one percent of all known Oribatida. In contrast to the terrestrial habitats of other oribatid mites, these mites have managed to conquer the marine littoral, where they mainly graze on lichen or algae (e.g., Pfingstl, 2017). Although they still largely look like their terrestrial relatives, they have evolved specific adaptations allowing them to survive in this extreme environment. For example, an elaborate plastron respiration to breathe underwater (e.g., Pfingstl & Krisper, 2014), or enlarged hook-like claws to maintain attachment during strong wave action (e.g., Karasawa & Hijii, 2004). The Ameronothroidea consist presently of five families that show variable associations with the marine environment and different global distribution patterns. The Ameronothridae and Podacaridae possess strictly intertidal species (= living between the tides), transition species (= able to dwell in intertidal and terrestrial habitats) and a few typical terrestrial species, that may occur far inland (e.g., Pfingstl, 2017). Both families are restricted to polar and cold temperate zones. The Ameronothridae are confined to the northern hemisphere and the Podacaridae are contained in the southern hemisphere (e.g., Procheş & Marshall, 2001). The Fortuyniidae and Selenoribatidae represent exclusively intertidal taxa, that are confined to subtropical and tropical coasts (e.g., Pfingstl, 2017). The Tegeocranellidae are also restricted to warmer climates but live in temporary swamps and streams (Norton & Behan-Pelletier, 2009) and have no association with the marine environment.

The systematics of this group has been a matter of several controversial debates and still there is no consent about their phylogenetic history. Details of these long-lasting debates are summarized in Pfingstl (2017) and Norton & Franklin (2018), the most important points are shortly resumed as follows: The Podacaridae, originally established by Grandjean (1955), were included in Ameronothridae based on their very similar morphologies (Weigmann & Schulte, 1977) and they are still placed in this family according to the only existing and frequently used catalogue of Oribatid mites of the world (Subías, 2022). A few authors argued against the unification of these two families (e.g., Woas, 2002; Pfingstl, 2017; Norton & Franklin, 2018) and recent molecular genetic data point to an independent origin of Podacaridae (Schäffer et al., 2010; Pfingstl et al., 2022a). Within Selenoribatidae, morphological diagnoses of certain genera are overlapping which has led to several erroneous taxonomic classifications and consequently to yet unsolved problems at generic and species level (Pfingstl & Schuster, 2012a). After several taxonomic transfers, the limnic Tegeocranellidae were suggested to show reasonable morphological synapomorphies with the Ameronothroidea and thus were included in this superfamily (Behan-Pelletier, 1997). Nevertheless, they are still not accepted as a valid member of this group by most researchers (e.g., Subías, 2022). And finally, the monophyly of the whole superfamily Ameronothroidea was questioned by several researchers, arguing that the present members of this group have evolved their littoral lifestyle independently in three different latitudinal bands (Procheş, 2001; Marshall & Procheş, 2007; Pfingstl, 2017).

The reasons for all these issues and why they are still not resolved are manifold, but the main points are that I) morphological data show a mosaic distribution among certain taxa and are still incomplete (descriptions often lack important details about legs, mouthparts, reproductive organs etc., juveniles of many taxa are completely unknown), ii) the extreme littoral environment may have triggered parallel evolution leading to morphologically similar but convergent traits and iii) molecular genetic data exists for very few species impeding a comprehensive phylogenetic reconstruction using the same markers.

At present, it is not possible to resolve the phylogeny of this group by classifying them with the available discrete morphological data, therefore other means of analyses or data are necessary to take a step forward. Using molecular genetic markers to infer the phylogenetic history of Ameronothroidea is a great option to do so, but, as indicated above, some taxa are difficult to include as they are hardly available or there are only few specimens that are preserved in ways making the extraction of dna very difficult. So, resulting phylogenies will not cover all relevant species and may thus show only vague topologies.

Nevertheless, we performed molecular genetic investigations on available marine associated ameronothroid mites using three different markers to get deeper insights into the evolutionary history of this group of mites. To account for possible shortcomings of this molecular study, we additionally conducted comprehensive phylogenetic estimates based on morphology. By combining these two different approaches, we intended to (I) reveal erroneous classifications and paraphyletic positions, (ii) identify morphological convergences, (iii) check for congruence between results, and (iv) test the performance of morphology-based phylogenies in oribatid mites.

Material and methods

Material

Morphological data was mainly taken from literature and molecular genetic data from GenBank. Additional mite material used for molecular genetic and morphological analyses originated from the collection of marine associated mites (curated by tp) housed in the Institute of Biology of the University of Graz.

dna extraction and sequencing

Whole genome dna was extracted from various ethanol fixed mite specimens (see table A1 in Appendix) using Chelex resin according to Pfingstl et al. (2022b). We sequenced three gene fragments: the mitochondrial cytochrome c oxidase subunit 1, region 2 (coi-2), the nuclear 18S rRNA (18S) and the nuclear 28S gene fragment D3 (D3). For coi and 18S, the protocol described in Pfingstl et al. (2022b) was used. Annealing for the pcr was adjusted between 45°C and 52°C. For D3 the same protocol and conditions as 18S were used, with 52°C as annealing temperature for pcr amplification. All respective primers can be found in table 1. All generated sequences were deposited at GenBank and are accessible under the numbers listed in table A1 in Appendix.

T1

Morphological data

Morphological data was basically taken from literature (descriptions, redescriptions). If information on specific morphological features, e.g., legs, gnathosoma, was not given in the text, we checked the figures. If the information was still lacking in the publication, we studied microscopic slides of the respective species (if available in our collections) to complement this information. If the species was not contained in our collection, we included the info about the specific character in this species as missing to our analyses. A total of 66 (61 adult and five immature) characters or character states, respectively, were recorded for 102 oribatid mite species in total. Characters of immatures included only developmental formulas and discrete characters that were stable throughout ontogeny. Intraspecific variation was not accounted for and excluded from the analyses. Only discrete characters were included in the analyses, except for body size, whereas here only the mean body size was used as reference. Character coding was based on unordered multistate characters (table A2 in Appendix), no character weighting or polarization was applied. The created matrix can be found in the (table A3 in Appendix).

Phylogenetic analyses

In addition to herein generated sequence data, selected sequences were downloaded from GenBank (table A1 in Appendix). For the concatenated alignment, only the 38 species with all three gene sequences available were used. Additionally, single gene trees for coi, 18S and D3 were conducted with 53, 54 and 41 species, respectively.

All genetic phylogenies were done in PhyloSuite (Zhang et al., 2020). For the concatenated phylogeny, the three respective sequences were aligned in batches with mafft (Katoh & Standley, 2013) using ‘ – auto’ strategy and normal alignment mode. Gap sites were removed with trimAl (Capella‐Gutiérrez et al., 2009) using “-automated1” command. They were then concatenated in PhyloSuite (Zhang et al., 2020). ModelFinder (Kalyaanamoorthy et al., 2017) was used to select the best-fit model for maximum likelihood (ml) and Bayesian inference (bi) using the corrected Akaike Information Criterion (aic c). ml phylogenies were inferred using iq-tree (Nguyen et al., 2015) under the gtr+R5+F model for 5000 ultrafast (Minh et al., 2013) bootstraps, as well as the Shimodaira – Hasegawa – like approximate likelihood-ratio test (Guindon et al., 2010). Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al., 2012) under gtr+I+G+F model (2 parallel runs, 2 million generations), in which the initial 25% of sampled data were discarded as burn-in. The same pipeline was used for the single-gene trees. The model for coi and 18S was gtr+F+I+G4 for all calculations, for D3 the model tvm+F+I+G4 was used for ml and sym+I+G4 for bi.

For estimating phylogenies based on morphological data, we performed maximum parsimony (mp) and ml analyses using the phangorn (Schliep, 2011) package in R (R Core Team, 2022), and we conducted a Bayesian inference analysis using Beast2 (Drummond et al., 2012; Bouckaert et al., 2019). Maximum parsimony was done using the parsimony ratchet (Nixon, 1999) and maximum likelihood was conducted with an unrooted (Felsenstein, 1981) and with a strict clock (Felsenstein, 2004) model with ultrafast bootstrapping (Minh et al., 2013). For bi the morph-models package (Lewis, 2001) for a strict clock (Zuckerkandl & Pauling, 1965) and a relaxed clock (Drummond et al., 2006) model were used. For detailed information, please see https://iris-bk.github.io/methods_morpho/.

Photographs

For photographic documentation, specimens were air-dried and photographed with a Keyence vhx-5000 digital microscope using automated image stacking.

Taxonomy

In this paper, we refrain from changing the existing taxonomy because important molecular genetic reference data is missing for certain type species and because taxonomic changes should always be accompanied by a comprehensive fully traceable evaluation of all morphological characters of each single taxon, which is clearly beyond the scope of the present publication.

Results

Phylogenies based on molecular genetic data

Maximum Likelihood and Bayesian Inference tree searches result in almost identical tree topologies; therefore, only bi trees are presented here, ml trees can be found in the supplementary data (supplementary fig. S1). Phylogenetic inference based on 18S results in mostly distinct clusters for each ameronothroid family with high statistical support. The Selenoribatidae are monophyletic, with Arotrobates granulatus being distinctly separated from all other members of this family (fig. 1), this position is possibly a result of long branch attraction.

Figure 1
Figure 1

Bayesian inference tree of marine associated Ameronothroidea and terrestrial outgroups based on 18S sequences. Posterior probabilities >0.9 are shown near nodes; abbreviations: prt – Portugal, de – Germany, dr – Dominican Republic, jp – Japan, tw – Taiwan, my – Malaysia; families are given in different colours. Photographs of selected species are given to provide an insight into the basic habitus of each larger group.

Citation: Contributions to Zoology 92, 4 (2023) ; 10.1163/18759866-bja10043

The genus Indopacifica is the only monophyletic genus within this cluster. The Fortuyniidae consist of two groups: One group contains all Fortuynia species, and the second group comprises Alismobates and Litoribates species – the latter are nested in between the Caribbean Alismobates and the Indo- and West-Pacific Alismobates species (fig. 1). Species of the genus Ameronothrus are monophyletic. Aquanothrus, another supposed member of Ameronothridae, is placed outside as sister taxon to Scutovertex sculptus. The Podacaridae are only represented by Halozetes capensis in this tree and are shown as sister taxon to the terrestrial Tectocepheus velatus, Scutovertex sculptus and members of Ameronothridae (fig. 1).

The phylogenetic tree based on sequences of the D3 gene fragment results in similar clusters but with very weak overall resolution (see supplementary fig. S2). Two clusters of Fortuyniidae and two groups of Selenoribatidae are given all as sister taxa in this tree. The Ameronothrus species are represented as monophyletic with Carabodes labyrinthicus as sister taxon. Halozetes capensis and Podacarus auberti form a distinct podacarid lineage outside Ameronothridae.

Phylogenetic analyses based on the concatenated dataset (coi, 18S and D3) have the best statistical support and show a similar pattern to the 18S single gene tree (fig. 2). Within Fortuyniidae, there are again two groups, one consisting only of Fortuynia species and the other comprising Alismobates and Litoribates. Within Selenoribatidae, Arotrobates granulatus is placed distinctly separated from all other members. Only the genus Indopacifica is monophyletic, whereas all the other genera are not (fig. 2).

Figure 2
Figure 2

Bayesian topology based on the combined data set of coi, D3 and 18S sequences. Posterior probabilities >0.9 are shown near nodes; abbreviations: prt – Portugal, de – Germany, dr – Dominican Republic, tw – Taiwan.

Citation: Contributions to Zoology 92, 4 (2023) ; 10.1163/18759866-bja10043

Phylogenies based on discrete morphological data

The phylogenetic reconstruction based on morphological data using parsimony renders most of the ameronothroid families as monophyletic lineages (fig. 3). The Podacaridae, Tegeocranellidae, Selenoribatidae and Fortuyniidae form distinct clusters, only the fortuyniid Circellobates venustus is placed outside the respective family (fig. 3). The Ameronothridae are given as paraphyletic taxon with Aquanothrus montanus and both Paraquanothrus species placed outside the Ameronothrus cluster. The genera Ameronothrus, Tegeocranellus, Indopacifica and Fortuynia are monophyletic. Halozetes also forms a distinct cluster but Podacarus auberti is placed within this group. Species of the genus Litoribates are nested within Alismobates and members of Schusteria are scattered on different branches of the Selenoribatidae.

Figure 3
Figure 3

One of 14 most parsimonious trees based on 66 characters or character states of 98 ameronothroid and four terrestrial oribatid mite species. Bootstrap values are shown near nodes. Colours refer to different families and are the same as in preceding figures.

Citation: Contributions to Zoology 92, 4 (2023) ; 10.1163/18759866-bja10043

The maximum likelihood algorithm shows a very similar topology and is thus only given in supplementary fig. S3.

The phylogenetic tree using Bayesian inference highlights monophyletic families. In Podacaridae, Podacarus auberti is nested within the genus Halozetes and in Fortuyniidae Litoribates is again placed within the Alismobates lineage (fig. 4). The genera Ameronothrus, Fortuynia, Tegeocranellus and Indopacifica form monophyletic groups, the genus Schusteria is distributed on different branches of the Selenoribatidae.

Figure 4
Figure 4

Bayesian inference topology based on 66 morphological traits of 102 oribatid mite species. Posterior probability values are shown near nodes. Photographs of selected species are given to provide an insight into the basic morphology of each larger group. *Photograph shows Tegeocranellus knysnaensis, this species was not used for the analyses but is given here to visualize the typical habitus of Tegeocranellus species.

Citation: Contributions to Zoology 92, 4 (2023) ; 10.1163/18759866-bja10043

Discussion

Higher level systematics (family and above)

Several authors (Marshall & Procheş, 2007; Pfingstl, 2017) questioned the monophyly of the superfamily Ameronothroidea, arguing that the land to sea transition has happened at least three times independently in different latitudinal bands. Preliminary molecular genetic studies (Schäffer et al., 2010; Krause et al., 2016; Pfingstl et al., 2022a) partially supported these theories but included very few ameronothroid members. The present data also do not include enough possible terrestrial relatives or enough molecular genetic sequences of Ameronothridae and Podacaridae to reliably answer that question. Nevertheless, both genetic and morphological phylogenies place the Ameronothridae and Podacaridae in paraphyletic positions and thus further corroborate an independent origin of the marine associated lifestyle. Apart from the question of different terrestrial ancestries, the present data clearly contradict the merger of Podacaridae and Ameronothridae, as recommended by Schubart (1975) and Weigmann & Schulte (1977). Each conducted analysis in this study renders the Podacaridae a distinct lineage without a close relation to Ameronothridae and thus justifies the distinctness of this family. Norton & Franklin (2018) already proposed a new diagnosis of Ameronothridae in which they explicitly excluded the Podacaridae, and Travé (2021) recently reclassified the Podacaridae as a family containing the genera Podacarus, Alaskozetes, Halozetes and Antarcticola. In the former publication, Norton & Franklin (2018) also proposed the Aquanothrinae, including Aquanothrus and Paraquanothrus, as a subfamily of Ameronothridae. Although there is not enough molecular genetic data to verify this taxonomic act, phylogenetic trees based on morphology place the Aquanothrinae close to the rest of Ameronothridae but not as direct sister group or branch within the Ameronothridae. Future molecular genetic studies, including all members of Aquanothrinae, are necessary to assess the true systematic position of this subfamily.

The distinctness of the families Fortuyniidae and Selenoribatidae, on the other hand, has never been seriously challenged, only Schuster (1977) questioned the separation of both families based on found intermediate specimens from the Maldives, but these were apparently lost somehow and consequently never described (Pfingstl & Schuster, 2012a). In addition to this doubt, a preliminary molecular genetic study (Iseki & Karasawa, 2014) indicated that one species of Fortuyniidae might be closer related to Selenoribatidae than to confamilial taxa. Present phylogenetic estimates based on morphology speak a clear language presenting both families as distinct monophyletic and closely related lineages. Only in the Parsimony tree the fortuyniid Circellobates venustus is placed outside other Fortuyniidae. But this monotypic species was described in an insufficient manner (Luxton, 1992) which led to many gaps in the matrix possibly resulting in its separate placement. Phylogenies based on gene sequences, show a slightly different picture. The 18S and the concatenated tree indicate that Fortuyniidae and Selenoribatidae together form a clear monophyletic lineage, but the latter possibly evolved from a fortuyniid ancestor and the families are consequently not the product of a dichotomous split of a common ancestor. However, this does not affect their present classification as both families are morphologically well defined (Pfingstl & Schuster, 2012a; b) and newly described species can still be easily assigned to the respective family so far. Considering the evolution of certain morphological features, some interesting interpretations are possible. The Fortuyniidae are characterized by the possession of the so called ‘Van der Hammen’s organ’, a unique system of lateral cuticular canals, which is part of the plastron respiration (e.g., Pfingstl & Schuster, 2012b). According to herein inferred molecular genetic phylogenies, this trait either evolved twice within Fortuyniidae or it was completely lost in Selenoribatidae. Although the specific configuration of these lateral channels slightly differs between fortuyniid genera (Pfingstl & Krisper, 2014) and given clusters (Fortuynia vs. Alismobates/Litoribates), it is rather unlikely that such a complex system has evolved independently in closely related taxa. Therefore, it is more likely that this organ was lost in one fortuyniid species, which then diversified into the Selenoribatidae. To clarify the real evolutionary relationship between those two groups it will be necessary to include the limnic Tegeocranellidae, which are supposed to be closely related to these families (Behan-Pelletier, 1997), in molecular genetic analyses.

Historically, the Tegeocranellidae were placed in different superfamilies (summarized in Pfingstl, 2017) before they were suggested to be members of Ameronothroidea, closely related to Fortuyniidae and Selenoribatidae (Behan-Pelletier, 1997). Some researchers did not accept this suggestion, possibly because this family is not marine associated. Consequently, they are still included in Tectocepheoidea in certain current classifications (e.g., Subías, 2022), leaving some doubt as to their real systematic placement. Although no gene sequence of any tegeocranellid species could be included in the present study, they form a well-supported monophyletic clade with Fortuyniidae and Selenoribatidae in the phylogenetic trees based on morphology and thus clearly support the assumption of Behan-Pelletier (1997). Of course, this relationship still must be confirmed by molecular genetic data, but any other result than a monophyletic group would be surprising. Norton & Franklin (2018) emphasized this relationship by referring to them as the ‘fortuynioid’ families without implying formal taxonomic rank and the present data clearly support this grouping which should be accepted until the contrary is proven.

Generic and species level

The genera Ameronothrus, Fortuynia and Indopacifica are monophyletic in all trees and thus unquestionable systematic and phylogenetic groups. Their morphology and molecular genetic sequences are well separated from the other taxa and their classification is apparently based on solid ground.

The few known and investigated members of the fortuyniid genus Litoribates also form a monophyletic clade but are nested within the genus Alismobates in all trees. This specific configuration was first shown by Pfingstl et al. (2022b) using only 18S sequence data. The authors suggested that Litoribates has evolved from Caribbean Alismobates stocks and has diverged rapidly from its ancestors because of their specific adaptation to mangrove habitats. The phylogenetic trees based on morphology show the exact same results indicating that there are strong morphological divergences classifying Litoribates as a distinct morpho-group within the genus Alismobates and corroborate the above-given assumption. From a strict phylogenetic point of view, Litoribates is nested within Alismobates and thus could be synonymized, but the genus is morphologically well-delimited and therefore should be retained or at least be given as a subgenus of Alismobates, as long as the generic diagnoses work out well.

Within Podacaridae, the phylogenetic estimates based on morphological characters only, place the two species of Alaskozetes as sister group to Halozetes, whereas the latter clade includes the monotypic Podacarus auberti. Interestingly, an earlier study, using mitochondrial coi and nuclear histone-3 (H3) gene sequences, showed the opposite topology, with Podacarus being sister taxon and Alaskozetes antarcticus being part of the Halozetes cluster (Mortimer et al., 2011). The authors of this study concluded that A. antarcticus should be transferred to Halozetes. Due to the absence of genetic data for most podacarid species, we are not able to confirm or reject this proposed transfer, but our morphological data at least indicate that we should not act too quickly. From a morphological point of view, the genera Alaskozetes and Podacarus are well separated from Halozetes (Travé, 2021), but the latter is quite diverse with 16 species and five subspecies (Subías, 2022). Moreover, juvenile morphology strongly diverges within this genus, with most species having juvenile porose body sclerites, like the immatures of Alaskozetes and Podacarus, and the other species lacking such sclerites completely (e.g., Ermilov et al., 2012). Consequently, the true problematic taxon could be the diverse Halozetes, not the species poor Alaskozetes and Podacarus. The inclusion of more species and more morphological data of Halozetes and Alaskozetes, as well as using more than a single nuclear marker should clarify possible synonymies. The study of Mortimer et al. (2011) also indicated that Halozetes belgicae and H. intermedius each represent more than a single species. This is possible, as cryptic diversity seems to be a common phenomenon in marine associated oribatid mite species (Pfingstl et al., 2021). Moreover, cryptic diversity is also revealed in the present dataset: the Ameronothrus maculatus specimens from Germany and Portugal are always placed apart in genetic phylogenies, indicating strong molecular divergence. Future comprehensive studies on Ameronothrus species from various areas may reveal further cryptic taxa.

Although the family Selenoribatidae represents a well-defined monophyletic lineage, the classification within this group is supposed to be partly erroneous and therefore questionable (Pfingstl & Schuster, 2012a), and it is indeed a considerable mess. Apart from the monotypic taxa, i.e., Psednobates, Rhizophobates, Thasecazetes, and the genus Indopacifica, members of each genus are given in paraphyletic positions. Carinozetes species nicely cluster together in the morphology trees, but in the trees based on gene fragments they are mostly given in closely related but separate positions. Earlier studies focused on Carinozetes bermudensis and C. mangrovi (Pfingstl et al., 2014, 2019) and demonstrated a close relationship based on their almost identical phenotype and genetic similarity. By contrast, the type species Carinozetes trifoveatus shares the prominent and name giving ventral carinae (= keels) (Pfingstl & Schuster, 2012a), but none of the genetic markers places it as sister taxon to the other Carinozetes species. Although fairly different, ventral keels have also evolved in the two Arotrobates species and Selenoribates arotroventer, therefore, we have to consider that the characteristic ventral keels of C. trifoveatus are also just a product of convergence. In this case, C. bermudensis and C. mangrovi should be given a new genus name and generic diagnoses should be amended. Nevertheless, as all Carinozetes species are contained in the Caribbean area and as they still cluster together in the ‘morpho’ trees, a detailed morphological reassessment of the three Carinozetes species and phylogenetic analysis with another genetic marker are necessary to verify a possible convergence.

Apart from these uncertainties in Carinozetes, all our phylogenetic reconstructions show a very close relationship between Carinozetes and the Brazilian type species Schusteria littorea and the Caribbean Schusteria marina. Based on discrete morphological traits, such a relationship was already suggested and it was assumed that an ancestor of the Western Atlantic Schusteria clade, which includes the above-mentioned species, also gave birth to the keel bearing Carinozetes (Pfingstl & Lienhard, 2017). Our results clearly support this assumption, but question at the same time the taxonomic identity of all other Schusteria species. The African Schusteria melanomerus and Schusteria ugraseni do not cluster with the above mentioned Schusteria species at all and can be found either close to Rhizophobates, Indopacifica or Thalassozetes. Interestingly, both African species were first transferred to Rhizophobates by Karasawa & Aoki (2005) and then they were placed in the genus Thalassozetes (Subías 2004, 2022), whereas Pfingstl & Schuster (2012a) argued that both transfers were not justified as the morphology of both species is in accordance with the original genus diagnosis for Schusteria (Grandjean, 1968). The Japanese Schusteria nagisa and Schusteria saxea, on the other hand, were originally described (Karasawa & Aoki, 2005) based on inaccurate premises (Pfingstl & Schuster, 2012a), therefore it is not surprising that they show no close relation to any of the other Schusteria species in the present trees. According to our data, neither the African nor the Japanese species are ‘true’ Schusteria, and although they are apparently closely related to Rhizophobates and Indopacifica, they cannot be unambiguously assigned to any of these selenoribatid taxa.

The genus Thalassozetes and its members have not been questioned so far but present results demonstrate that at least two species represent problematic taxa. First, Thalassozetes tenuisetosus does not cluster with the other Thalassozetes species in the morphological phylogenetic trees. This species is characterized by the genus specific prodorsal ridges (Bayartogtokh & Chatterjee, 2010) but deviates in many other non-diagnostic characters, as for example cuticular pattern, number of epimeral setae, shape of tarsal famulus etc. (Pfingstl et al., 2020), which already points to a position as an outsider in this group. Unfortunately, we do not have molecular genetic data to verify the real phylogenetic position of this species. Second, in the phylogenetic estimates using gene fragments, Thalassozetes canariensis is given as sister taxon of Selenoribates divergens, distinctly separated from the main Thalassozetes clade. From a morphological point of view, this is very surprising because T. canariensis strongly corresponds to the genus specific morphology and is even very similar to the type species Thalassozetes riparius (Pfingstl et al., 2020). Phylogenetic estimates based on morphological characters place T. canariensis in the Thalassozetes clade, but they also support Selenoribates divergens as being the sister species. Here we hold the wolf by the ears, because S. divergens may be a result of an incorrect classification and thus could be transferred to Thalassozetes due to its close relationship to T. canariensis, but genetic data implies that the latter species may not belong to Thalassozetes, which leaves us empty-handed for the moment. The name ‘divergens’ was chosen for this Selenoribates species as it diverges the most from the type species Selenoribates foveiventris (Pfingstl, 2015) and this divergence apparently already indicated a possible different systematic position.

The genus Selenoribates itself shows unusually high morphological diversity (Pfingstl, 2015) and generic concepts were subsequently adjusted to accommodate the newly described species (Pfingstl, 2013, 2015). All Selenoribates species, except for S. divergens, cluster together in the phylogenetic estimates based on morphology, but according to used gene markers, they are placed in far distant positions on the selenoribatid branch, indicating paraphyly. Pfingstl (2015) highlighted three distinct morphological groups in Selenoribates, the ‘foveiventris’ group with no obvious morphological structures, the ‘satanicus’ group with strong modifications of the anterior part of the notogaster and the ‘divergens’ group showing branched notogastral setae. None of these groups is reflected in any of our phylogenies indicating convergent evolution of the characters used for grouping. Considering these points, a comprehensive re-evaluation of the morphology and revision of the genus diagnosis of Selenoribates is necessary.

Classifying the Selenoribatidae based on discrete morphological characters presently seems to be a very tricky endeavour as they show highly diverse features and apparently numerous convergent traits. Correcting the present classification will require (I) identifying all convergent structures to elucidate real synapomorphies and redefine genus diagnoses, (ii) collecting more molecular genetic data, especially from type species, i.e., Thalassozetes riparius, Selenoribates foveiventris, Rhizophobates shimojanai and Psednobates uncunguis (these were not available for the present study) for clear reference points, and (iii) creating well delimited new genera for non-assignable taxa.

Conclusions

The present classification of Ameronothroidea suffers from major problems and thus should be thoroughly revised. Molecular genetic analyses provide us with important clues about the evolutionary phylogeny of this group, but data from many taxa are still missing and only a few genetic markers were used for the present analyses which could have resulted in partly erroneous or vague tree topologies. Therefore, it is always important to have morphology-based phylogenies to check the validity of molecular genetic results (e.g., Hillis & Wiens, 2000). Here we used morphological data and different algorithms to reconstruct the phylogenetic history of ameronothroid mites and the results strongly support most molecular genetic data. Congruent tree topologies created by the different methods allow us to identify the most probable evolutionary relationships, which are the following: I) the Ameronothroidea represent a paraphyletic assemblage, ii) the Podacaridae are a distinct family, its inclusion to Ameronothridae can no longer be justified; iii) the Fortuyniidae and Selenoribatidae are a clear monophyletic group; iv) Ameronothrus, Fortuynia and Indopacifica are well-supported monophyletic genera; v) the genus Litoribates evolved from Alismobates but represents a distinct genetic and morphological group within this taxon; vi) most of the genera of Selenoribatidae are clearly paraphyletic taxa, especially Schusteria and Selenoribates; and vii) the Tegeocranellidae are closely related to Fortuyniidae and Selenoribatidae, with all three families forming a monophyletic lineage (only supported by morphological data, molecular data is lacking).

Morphological data is rarely used to perform phylogenetic estimates, as the hierarchical interdependence of many morphological traits complicates phylogenetic analyses (Brazeau et al., 2019). There is also disagreement about the criteria for character selection and coding (e.g., Poe & Wiens, 2000), and about which algorithm performs best and shows the most reliable results (e.g., Schrago et al., 2018). The present study nicely demonstrates that using phylogenetic estimates based on morphology can be an important additional tool to verify and complement molecular phylogenies, and that such analyses can succeed in cases where traditional morphological cladistics fail due to preconceived ideas of relationships or trait significance.

Editor: R. Vonk

Acknowledgments

The authors would like to thank two anonymous reviewers for their valuable comments and corrections helping to improve the quality of this paper. This investigation was funded by the Austrian Science Fund (fwf): [I 3815]. No potential conflict of interest was reported by the authors.

Supplementary material

Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.22592194

References

  • Bayartogtokh, B. & Chatterjee, T. (2010). Oribatid mites from marine littoral and freshwater habitats in India with remarks on world species of Thalassozetes (Acari: Oribatida). Zool. Stud., 49, 839854s.

    • Search Google Scholar
    • Export Citation
  • Behan-Pelletier, V.M. (1997). The semiaquatic genus Tegeocranellus (Acari: Oribatida: Ameronothroidea) of North and Central America. Can. Entomol., 129, 537577.

    • Search Google Scholar
    • Export Citation
  • Bouckaert, R., Vaughan, T.G, Barido-Sottani, J., Duchêne, S., Fourment, M., Gavryushkina, A., Heled, J., et al. (2019). beast 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Comput. Biol., 15, e1006650.

    • Search Google Scholar
    • Export Citation
  • Brazeau, M.D., Guillerme, T. & Smith, M.R. (2019). An algorithm for morphological phylogenetic analysis with inapplicable data. Syst. Biol., 68, 619631.

    • Search Google Scholar
    • Export Citation
  • Capella-Gutierrez, S., Silla-Martinez, J.M. & Gabaldon, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 25, 19721973.

    • Search Google Scholar
    • Export Citation
  • Dabert, M., Witalinski, W., Kazmierski, A., Olszanowski, Z. & Dabert, J. (2010). Molecular phylogeny of acariform mites (Acari, Arachnida): strong conflict between phylogenetic signal and long-branch attraction artifacts. Mol. Phylogen. Evol., 56, 222241.

    • Search Google Scholar
    • Export Citation
  • Drummond, A.J, Ho, S.Y.J, Phillips, M.J. & Rambaut, A. (2006). Relaxed Phylogenetics and Dating with Confidence. PLoS Biol., 4: e88.

  • Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A. (2012). Bayesian Phylogenetics with BEAUti and the beast 1.7. Mol. Biol. Evol., 29, 19691973.

    • Search Google Scholar
    • Export Citation
  • Ermilov, S.G., Stary, J. & Block, W. (2012). Morphology of juvenile instars of Ameronothridae (Acari: Oribatida). Zootaxa 3224, 140.

  • Felsenstein, J. (1981). Evolutionary trees from dna sequences: A maximum likelihood approach. J. Mol. Evol., 17, 368376.

  • Felsenstein, J. (2004). Inferring Phylogenies. Sinauer Associates, Sunderland.

  • Grandjean, F. (1955). Sur un acarien des iles Kerguelen. Podacarus auberti (Oribates). Mém. Mus. Nat. Hist. Natur. Paris, 8, 109150.

    • Search Google Scholar
    • Export Citation
  • Grandjean, F. (1968). Schusteria littorea n.g., n.sp. et les Selenoribatidae (Oribates). Acarologia, 10, 116150.

  • Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307321.

    • Search Google Scholar
    • Export Citation
  • Hillis, D.M. & Wiens, J.J. (2000). Molecules versus morphology in systematics: Conflicts, artifacts, and misconceptions. In: J.J. Wiens (Ed), Phylogenetic analysis of morphological data, pp. 119. Smithsonian Institution Press, Washington, DC.

    • Search Google Scholar
    • Export Citation
  • Iseki, A. & Karasawa, S. (2014). First record of Maculobates (Acari: Oribatida: Liebstadiidae) from Japan, with a Redescription Based on Specimens from the Ryukyu Archipelago. Spec. Divers., 19, 5969.

    • Search Google Scholar
    • Export Citation
  • Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A. & Jermiin, L.S. (2017). ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods, 14, 587589.

    • Search Google Scholar
    • Export Citation
  • Karasawa, S. & Hijii, N. (2004). Morphological modifications among oribatid mites (Acari: Oribatida) in relation to habitat differentiation in mangrove forests. Pedobiologia, 48, 383394.

    • Search Google Scholar
    • Export Citation
  • Karasawa, S. & Aoki, J. (2005). Oribatid Mites (Arachnida: Acari: Oribatida) from the Marine Littoral of the Ryukyu Archipelago, Southwestern Japan. Spec. Divers., 10, 209233.

    • Search Google Scholar
    • Export Citation
  • Katoh, K. & Standley, D.M. (2013). mafft multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol., 30, 772780.

    • Search Google Scholar
    • Export Citation
  • Krause, A., Pachl, P., Schulz, G., Lehmitz, R., Seniczak, A., Schaefer, I., Scheu, S. & Maraun, M. (2016). Convergent evolution of aquatic life by sexual and parthenogenetic oribatid mites. Exp. Appl. Acarol., 70, 439453.

    • Search Google Scholar
    • Export Citation
  • Lewis, P.O. (2001). A Likelihood Approach to Estimating Phylogeny from Discrete Morphological Character Data. Syst. Biol., 50, 913925.

    • Search Google Scholar
    • Export Citation
  • Litvaitis, M.K., Nunn, G., Thomas, W.K. & Kocher T.D. (1994). A molecular approach for the identification of meiofaunal turbellarians (Platyhelminthes, Turbellaria). Mar. Biol., 120, 437442

    • Search Google Scholar
    • Export Citation
  • Luxton, M. (1992). Oribatid mites from the marine littoral of Hong Kong (Acari: Cryptostigmata). In: B. Morton (Ed) The Marine Flora and Fauna of Hong Kong and Southern China iii. Proceedings of the Fourth International Marine Biological Workshop, pp. 211227. Hong Kong, Hong Kong University Press.

    • Search Google Scholar
    • Export Citation
  • Marshall, D.J. & Procheş S. (2007). The origins of marine mites: interpreting geographical and ecological patterns. In: Morales-Malacara J.B., Behan-Pelletier V., Ueckermann E., Pérez T.M., Estrada-Venegas E.G. & Badii M. (Eds.), Acarology xi: Proceedings of the International Congress, pp. 97103. Sociedad Latinoamericana de Acarología, Mexico.

    • Search Google Scholar
    • Export Citation
  • Minh, B.Q., Nguyen, M.A.& von Haeseler, A. (2013). Ultrafast approximation for phylogenetic bootstrap. Mol. Biol. Evol., 30, 11881195.

    • Search Google Scholar
    • Export Citation
  • Mortimer, E., Jansen van Vuuren B., Lee, J.E., Marshall, D.J., Convey, P. & Chown S.L. (2011). Mite dispersal among the Southern Ocean Islands and Antarctica before the last glacial maximum. Proc. Royal Soc. B, 278, 12471255.

    • Search Google Scholar
    • Export Citation
  • Nguyen, L.T., Schmidt, H.A., von Haeseler, A. & Minh, B.Q. (2015). iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol., 32, 268274.

    • Search Google Scholar
    • Export Citation
  • Nixon, K.C. (1999) The Parsimony Ratchet, a New Method for Rapid Parsimony Analysis. Cladistics, 15, 407-14.

  • Norton, R.A. & Behan-Pelletier, V.M. (2009). Suborder Oribatida. In: G.W Krantz. & D.E. Walter (Eds), A Manual of Acarology – Third Edition, pp. 430564. Texas Tech University Press, Texas.

    • Search Google Scholar
    • Export Citation
  • Norton, R.A. & Franklin, E. (2018). Paraquanothrus n. gen. from freshwater rock pools in the USA, with new diagnoses of Aquanothrus, Aquanothrinae, and Ameronothridae (Acari, Oribatida). Acarologia, 58, 557627.

    • Search Google Scholar
    • Export Citation
  • Otto, J.C. & Wilson, K. (2001). Assessment of the usefulness of ribosomal 18S and mitochondrial coi sequences in Prostigmata phylogeny. Proceedings of the 10th International Congress (Vol. 100, No. 9). Melbourne: csiro Publishing.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T. (2013). Revealing the diversity of a once small taxon: the genus Selenoribates (Acari, Oribatida, Selenoribatidae). ZooKeys, 312, 3963.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T. (2015). Morphological diversity in Selenoribates (Acari, Oribatida): new species from coasts of the Red Sea and the Indo-Pacific. Int. J. Acarol., 41, 356370.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T. (2017). The marine-associated lifestyle of ameronothroid mites (Acari, Oribatida) and its evolutionary origin: A review. Acarologia, 57, 693721.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T. & Krisper, G. (2014). Plastron respiration in marine intertidal oribatid mites (Acari, Fortuyniidae and Selenoribatidae). Zoomorphology, 133, 359378.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T. & Lienhard, A. (2017). Schusteria marina sp. nov. (Acari, Oribatida, Selenoribatidae) an intertidal mite from Caribbean coasts, with remarks on taxonomy, biogeography, and ecology. Int. J. Acarol., 43, 462467.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T. & Schuster, R. (2012a). Carinozetes nov. gen. (Acari: Oribatida) from Bermuda and remarks on the present status of the Family Selenoribatidae. Acarologia, 52, 377409.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T. & Schuster, R. (2012b). First record of the littoral genus Alismobates (Acari: Oribatida) from the Atlantic Ocean, with a redefinition of the family Fortuyniidae based on adult and juvenile morphology. Zootaxa, 3301, 133.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T., Baumann, J. & Lienhard A. (2019). The Caribbean enigma: the presence of unusual cryptic diversity in intertidal mites (Arachnida, Acari, Oribatida). Org. Divers. Evol., 19, 609623.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T., De la Paz, J.C. & Hernández-Teixidor, D. (2020). First record of intertidal oribatid mites (Acari, Oribatida) from the Canaries – a new species and its complete ontogeny. Syst & Appl. Acarol., 25, 19011914.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T., Lienhard, A. & Jagersbacher-Baumann, J. (2014). Hidden in the mangrove forest: the cryptic intertidal mite Carinozetes mangrovi sp. nov. (Acari, Oribatida, Selenoribatidae). Exp. Appl. Acarol., 63, 481495.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T., Lienhard, A., Baumann, J. & Koblmüller, S. (2021). A taxonomist’s nightmare–cryptic diversity in Caribbean intertidal arthropods (Arachnida, Acari, Oribatida). Mol Phylogenet Evol., 163, 107240.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T., Hiruta, S.F., Bardel-Kahr, I., Obae, Y. & Shimano, S. (2022a). Another mite species discovered via social media – Ameronothrus retweet sp. nov. (Acari, Oribatida) from Japanese coasts, exhibiting an interesting sexual dimorphism. Int. J. Acarol., 48, 348358.

    • Search Google Scholar
    • Export Citation
  • Pfingstl, T., Schäffer, S., Bardel-Kahr, I., & Baumann, J. (2022b) A closer look reveals hidden diversity in the intertidal Caribbean Fortuyniidae (Acari, Oribatida). PloS one, 17, e0268964.

    • Search Google Scholar
    • Export Citation
  • Poe, S., & Wiens, J.J. (2000). Character selection and the methodology of morphological phylogenetics. In: J.J. Wiens (Ed), Phylogenetic analysis of morphological data, pp. 2036. Smithsonian Institution Press, Washington, DC.

    • Search Google Scholar
    • Export Citation
  • Procheş, S. (2001). Back to the sea: secondary marine organisms from a biogeographical perspective. Biol. J. Linn. Soc., 74, 197203.

    • Search Google Scholar
    • Export Citation
  • Procheş, S. & Marshall, D.J. (2001) Global distribution patterns of non-halacarid marine intertidal mites: implications for their origins in marine habitats. J. Biogeogr., 28, 4758.

    • Search Google Scholar
    • Export Citation
  • Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., et al. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol., 61, 539542.

    • Search Google Scholar
    • Export Citation
  • Schäffer, S., Koblmüller, S., Pfingstl, T., Sturmbauer, C. & Krisper, G. (2010). Ancestral state reconstructions reveal multiple independent evolution of diagnostic morphological characters in the “Higher Oribatida” (Acari), conflicting current classification schemes. bmc Evol. Biol., 10, 246.

    • Search Google Scholar
    • Export Citation
  • Schliep, K. (2011). Phangorn: Phylogenetic Analysis in R. Bioinformatics, 27, 592593.

  • Schrago, C.G., Aguiar, B.O. & Mello, B. (2018). Comparative evaluation of maximum parsimony and Bayesian phylogenetic reconstruction using empirical morphological data. J. Evol. Biol., 31, 14771484.

    • Search Google Scholar
    • Export Citation
  • Schubart, H. (1975). Morphologische Grundlagen für die Klärung der Verwandtschaftsbeziehungen innerhalb der Milbenfamilie Ameronothridae (Acari, Oribatei). Zoologica, 123, 2391.

    • Search Google Scholar
    • Export Citation
  • Schuster, R. (1977). Die Selenoribatidae, eine thalassobionte Familie der Hornmilben (Oribatei). Acarologia, 19, 155160.

  • Skoracka, A. & Dabert, M. (2010). The cereal rust mite Abacarus hystrix (Acari: Eriophyoidea) is a complex of species: evidence from mitochondrial and nuclear dna sequences. Bull. Entomol. Res., 100, 263272.

    • Search Google Scholar
    • Export Citation
  • Subías, L.S. (2004). Listado sistemático, sinonímico y biogeográphico de los ácaros oribátidos (Acariformes: Oribatida) del mundo. Graellsia, 60, 3305.

    • Search Google Scholar
    • Export Citation
  • Subías, L.S. (2022) Listado sistemático, sinonímico y biogeográfico de los ácaros oribátidos (Acariformes, Oribatida) del mundo (excepto fósiles). Monografías electrónicas, 12, 1538.