Life Stages and Phylogenetic Position of the New Scale-Mite of the Genus Neopterygosoma (Acariformes: Pterygosomatidae) from Robert’s Tree Iguana

Simple Summary This research presents a description of a new ectoparasitic scale-mite species, Neopterygosoma robertmertensi sp. n., collected from a Robert’s tree iguana (Liolaemus robertmertensi) from Argentina. For the first time, the description of females was accompanied by the description of the male and juvenile stages. The morphology of all post-embryonic stages of this species was analyzed in detail using scanning electron microscopy. Additionally, we conducted a phylogenetic analysis to determine its position within the genus and created an updated identification key for all Neopterygosoma species. The findings show that N. robertmertensi sp. n. is a part of the chilensis group and is a sister taxon to all Neopterygosoma spp. collected from Liolaemus pictus and L. chiliensis. Abstract A new pterygosomatid mite species, Neopterygosoma robertmertensi sp. n. (Acariformes: Pterygosomatidae) was collected from two specimens of Liolaemus robertmertensi (Liolaemidae) from Argentina. This new species is described based on active stages: adults (female and male) and juveniles (deutonymphs, protonymphs, and larvae) and quiescent stages (nymphchrysalis, deutochrysalis and imagochrysalis). The changes in morphological characters that occur during the ontogeny of N. robertmertensi have been analyzed in detail. A difference in larval sex morphology was observed for the first time in the family Pterygosomatidae (female larvae differ from male larvae in terms of the shape and size of the idiosoma and the position of the genital area). This new mite species is most similar to N. cyanogasteri but can be distinguished by the presence of different leg chaetotaxy patterns of genua IV and femora IV, four to six genital setae, three to five dorsomedial setae, and two to three ventromedial setae. Phylogenetic analysis was conducted based on 120 morphological characters of all Neopterygosoma spp. and four outgroup species using the maximum parsimony approach. The results indicated that this species is nested within mites of the chilensis group of Neopterygosoma associated with host species of the section chiliensis of Liolaemus s. str. An updated diagnosis of the chilensis group of Neopterygosoma and an identification key for all species of this genus has been provided.


Introduction
Mites of the genus Neopterygosoma are permanent ectoparasites, with all life stages living on the hosts.They are associated with endemic South American iguanian lizards of the genus Liolaemus (Sauria: Liolaemidae), and until recently, they were placed in the genus Pterygosoma [1,2].The first species of this genus was described by Dittmar de la Cruz et al. [2] from tree lizards (Liolaemidae) in Argentina, exceeding the geographical range of the genus Pterygosoma.Later on, Fajfer and González-Acuña [1] described six new species from Chilean tree lizards and established a group ligare for mites associated with liolaemids.Nevertheless, the phylogenetic trees constructed by Fajfer [3] clearly showed that the genus Pterygosoma was paraphyletic; therefore, a new genus Neopterygosoma was erected for mites associated with liolaemids lizards [3].Since then, only one new species, N. schroederi Fajfer, 2020, has been described [4].
Currently, mites of the genus Neopterygosoma are represented by eight species associated with the lizards of the genus Liolaemus.They are divided into two groups: chilensis, represented by monoxenous species associated with lizards from Chile, and patagonica, represented by a single oligoxenous species, N. patagonica (Dittmar de la Cruz, Morando and Avila, 2004), recorded on several Liolaemus spp.from Argentina [2,5].
Although eight species have been described in the genus Neopterygosoma so far, most of these descriptions are based only on a few adult females.This was necessitated by the fact that most of the described mite material was accidentally collected by herpetologists during the investigation of lizards or was taken from museum-preserved specimens, which were often washed before being fixed in formalin or alcohol.It should be emphasized that to gain a complete understanding of the mite taxonomy, phylogeny, ecology, and biology, it is essential to study both immature instars and males.In Pterygosomatidae, as in other mites, the description of juvenile stages enabled the detection of homologous features and establishment of the nomenclature used during species description [6,7].So far, only immatures of one species, N. schroederi, and a male of N. patagonica have been described [2,4].Nonetheless, the original description of the male was insufficient, as it only presented the idiosoma's width and length, chaetotaxy of trochanter-tibiae I-IV, and a vague figure of the idiosoma dorsum without any details.Moreover, the type series (syntypes) consists of five males, all of which have been designated as holotypes (!), and five females.However, exact locality data were not provided; only the provinces and host species were listed separately.
In this paper, we describe a new species, Neopterygosoma robertmertensi sp.n., from Liolaemus robertmertensi from Argentina, including a first comprehensive description of the male within the genus.We extensively examine the post-embryonic stages using scanning electron microscopy, and we note differences between larval males and females for the first time within this family.Additionally, we infer the phylogenetic position of N. robertmertensi based on morphological data.Considering both morphology and phylogeny, this new species belongs to the chilensis group (the first record of Argentinian host species within the group) and is a sister taxon to Chilenian mite species associated with Liolaemus pictus and L. chiliensis.Additionally, we have revised the diagnosis of the chilensis group and provided an updated identification key for the genus (based on females).

Mite Sampling
The mite specimens were collected from the geckos housed in the herpetological collection of HUJ (abbreviations of the institutions are presented below).All lizards were kept in separate jars with 75% ethyl alcohol and were examined for mites, which were then removed from the lizards under a stereomicroscope (Nikon SMZ745 (Nikon Corporation, Tokyo, Japan).Then, the mites were placed in small vials (2 mL) containing 75% ethyl alcohol.

Morphological Analysis
Before mounting in Hoyer's medium, mite specimens were cleared and softened in Nesbitt's solution at +45 • C for 8-48 h.All specimens were mounted as vouchers using Hoyer's medium on a glass slide following the standard method [8].
Specimens destined for scanning electron microscopy (SEM) were dehydrated in ethanol, covered with gold, and examined using a Carl Zeiss AG-EVO ® 40 electron microscope (Carl Zeiss Microscopy, Oberkochen, Germany) at the Institute of Plant Protection of the National Research Institute in Poznan (IPP NRI), Poland.Additionally, the mites were studied and measured using a Leica DMD108 microscope (Leica Microsystems, Mannheim, Germany).All measurements, including scale bars, are given in micrometers (µm).In species descriptions, measurements (ranges) of paratypes are given in parentheses, following the data of the holotype.

Terminology
In the species descriptions, names of the leg and idiosomal setae followed Grandjean [9,10], as described by Norton [6], whereas those of the palpal setae followed Grandjean [11].Grandjean's nomenclature [9,10] has been applied to the family Pterygosomatidae by Bochkov and O'Connor [7].The scientific names of the lizards follow the Reptile Database [12].All of the specimens were deposited in the arachnid collections of HUJ and CSWU.The type material of the Neopterygosoma spp. was loaned from the AMU.

Phylogeny Reconstructions Methods
For the analysis of phylogenetic relationships between Neopterygosoma species, all species from the genus were used.The outgroup taxa were selected based on the analyses of Reference [3].As a distant outgroup, Pimeliaphilus podapolipophagus Trägårdh, 1905 was designated, and as a close outgroup, the representatives of the genus Geckobia (3 spp.) of the family Pterygosomatidae were selected.We chose G. nitidus because it was a sister taxon to Neopterygosoma in the analyses of Fajfer [3], and G. gerrhopygus and G. hirsti because they were grouped separately in the analysis (see Figures 6 and 7 in Reference [3]).

Cladistic Analysis
All of the characters were unordered and unweighted.In total, 13 species and 120 morphological characters of adult females were included in the analysis (List S1, Table S1).Preparing and editing of the data matix were completed using NEXUS Data Editor 0.5.0 [13].The missing states were designated as "?" and inapplicable characters as "-".The reconstruction of phylogenetic relationships was performed in PAUP 4.0.a147 for Microsoft Windows [14].The branch-and-bound option was used for maximum parsimony analysis.Nodal support was evaluated using the Bremer indices calculated using PRAP2 (http://bioinfweb.info/Software/PRAP2)[15].Analysis of the charactersdistributions and the drawing and editing of the trees were performed using FigTree v1.4.3 [16], and the final illustrations were made in Adobe Illustrator CS6.
Etymology The species name is derived from the species name of the host.

Differential diagnosis
This species is most similar to Neopterygosoma cyanogasteri from Liolaemus cyanogaster (Duméril and Bibron) from Chile [1].In females of both species, the setation of tarsi I-IV, tibiae I-IV, genua I-III, femora I and III, and trochanters I-IV is the same, fixed cheliceral digit is spinous, palp seta dF is longer than dG, subcapitular seta n is smooth or with barely discernible serration, the antero-mid cluster of dorsal setae is represented by about 60 setae, and five pseudanal setae ps are present.In Neopterygosoma robertmertensi setae lv'GIV, lv'GII and ld'FIV are absent, coxal setae 3a are smooth, 4-6 pairs of serrate genital setae are present, 3-5 pairs of dorsomedial setae, and 2 or 3 pairs of ventromedial setae are present.In N. cyanogasteri setae lv'GIV, lv'GII and ld'FIV are present, coxal setae 3a are serrate, one smooth genital seta, 17-21 dorsomedial setae, and 14-18 ventromedial setae are present.
Remarks Our research used scanning electron microscopy to enhance taxonomic descriptions of the new Neopterygosoma species.As a result, we noticed that in the original description of Neopterygosoma spp.[1], some inaccuracies are mentioned.The detailed photographs revealed that a smooth and weakly sclerotized propodonotal shield is present in all Neopterygosoma mites (Figure 4b) (it appears in protonymph).

Phylogeny Unweighted Parsimony Analysis
The analysis of the data matrix (Table S1) showed that out of 120 characters (List S1), 85 were informative.The analysis with all characters treated as unordered and unweighted was performed with Paup and produced one parsimonious tree (Figure 15).The tree is 219 steps long and has a consistency index (CI) of 0.64; retention index (RI) of 0.56, and rescaled consistency index (RC) of 0.36.The monophyly of the genus Neopterygosoma is supported by four synapomorphies (Bremer index 3), of which two are unique (length of coxae I, absence of coxal setae 2a and 4a).As expected, the resulting topology in this analysis is very similar to that in Fajfer [3]; in that hypothesis, the P. patagonica was the sister taxon to three species of Chilean species (P.chilensis, P. ligare, and P. formosus) included in the analysis.Our analysis confirms that N. patagonica from Argentina, considered less specialized (it has a circular body shape that is unable to hide under the host's scales), is the sister group to all the other species of the genus from Chile, considered more specialized (their idiosoma is wider than long, therefore, they live completely hidden beneath the scales).Its position is also supported by five common synapomorphies (Bremer index 2), of which three are unique (e.g., the presence of much longer setae in the postero-lateral part and peripheral part of idiosoma than in the anterior half of the dorsum).
The new species, L. robertmertensi, is a sister taxon to all species collected from Liolaemus pictus (N.formosus, N. ovata, N. ligare, and N. levissima) and L. chiliensis (N.chilensis) and is supported by the presence of five non-unique synapomorphies (Bremer index 1).The node uniting all of the above-mentioned mite species collected from the two host species is supported by five non-unique synapomorphies (Bremer index 2).Within the clade, the relationship within the species is weakly supported: N. formosus is a sister taxon to N. ovata + N. ligare (Bremer index 1), and the three species are a sister group to N. levissima + N. chilensis (Bremer index 1).Notably, the positions of both N. schroederi and N. cyanogasteri, are weakly supported by several non-unique synapomorphies (Bremer index of 1).
The only differences between the tree presented in Reference [3] and this tree lay in the position of the outgroup species.In the analysis [3], the genus Geckobia was paraphyletic with G. nitidus as a sister taxon to representatives of species of the genus Neopterygosoma, while Geckobia gerrhopygus + G. hirsti were as a sister taxon to the genera: Gerrhosaurobia + Zanurobia + Ixodiderma + Scaphotrix + Pterygosoma.In our analysis, all the outgroup Geckobia spp.are grouped in a common clade.

Discussion
The genus Liolaemus is the most ecologically diverse and species-rich genus distributed in South America from the high Andes of central Perú to the shores of Tierra del Fuego, and it spans an altitudinal range from sea level to over 5000 m [17].The liolaemid lizards cover various climatic regimes and inhabit a great diversity of habitats (e.g., arid Atacama desert or humid rainforests).Moreover, the lizards exhibit a wide range of reproductive modes, types of diets, coloration patterns, and body sizes [18].They also have a long evolutionary history dating back to 18-22 million years ago [19,20].
Currently, the genus includes over 280 species [12], but new species are being discovered at a rapid rate every year, e.g., [21,22]; therefore, it is estimated that the actual number of the species may be much higher.The genus is subdivided into two subgenera-Liolaemus (sensu stricto) and Eulaemus [23]-which appear to have separated at least 12.6 million years ago and are currently each divided into several groups.The presence of Neopterygosoma mites has been detected in 12 different species of hosts belonging to Liolaemus s. str.living on both sides of the Andes at different elevations, having different types of scales, coloration patterns, etc. [18,24].
As a rule, mites from different pterygosomatid genera are strictly specific with respect to lizard hosts, and cospeciation has a strong influence on the architecture of host-parasite relationships within the family Pterygosomatidae [3].All representatives of the genus Neopterygosoma are monoxenous parasites (the chilensis group) except for N. patagonica collected from several Liolaemus spp.(oligoxenous parasite).Since host species from the same communities (these host taxa distributions partially overlap [17]) do not carry the same set of parasite species, we can expect to observe at least partially parallel evolution of Neopterygosoma mites of the chilensis group and Liolaemus hosts.
Nonetheless, the co-phylogenetic studies require phylogenetic hypotheses or data matrices for both lineages involved in the coevolutionary process.So far, the relationships between Liolaemus lizards at the species level are still questionable, e.g., [19,25].Recently, Troncoso-Palacios et al. [26] conducted a phylogenetic study of the relationship of species of Liolaemus s. str.(based on three fragments of the mitochondrial genome); as a result, the species were divided into two main clades named: chiliensis and nigromaculatus sections.These findings were congruent with the phylogenetical tree (Figure 3 in Reference [17]) based on previous works [19,25,27].Until now, all Neopterygosoma spp.are associated with closely related hosts belonging to the chiliensis section, whereas representatives of another pterygosomatid genus, i.e., Geckobia nitidus and G. zapallarensis, were collected from lizards of the nigromaculatus section [28] (marked on Figure 15).
However, not all of the host species groups were recovered monophyletic in the work of Troncoso-Palacios et al. [26]; therefore, Parenza et al. [29] infer a robust phylogeny (based on 541 ultra-conserved elements and 44 protein-coding genes) for a Chilenian clade of Liolaemus s. str.using representatives of all thirteen groups.As a result, only the relationship among the major Chilean clade of Liolaemus was resolved, as in previous studies [26] (Figure 15).All mites of the chilensis group (i.e., monoxenous 'more specialized' mite species) have been associated with closely related hosts belonging to three host groups of [26], i.e., robertmertensi, gravenhorsti, and pictus.The pterygosomatids have been found on all representatives within the two former groups except for L. sanjuanensis (robertmertensi group) and L. gravenhorsti (gravenhorsti group), which suggests that checking numerous host specimens of the two species for mites might lead to new species descriptions.
The highest number of Neopterygosoma spp. was described from a single host species-L.pictus (4 spp.)-belonging to the pictus group, including 11 host species.However, the number of species in this group is debatable because a few species have been treated as subspecies of L. pictus [30,31] or synonymized with L. pictus [32].This host species has a wide distribution and forms a local population at low elevations (0-1600 a.s.l.) on both sides of the Andes, whereas the remaining Liolaemus spp.live either in the eastern or western slopes of the mountains [19].It is unknown if the mite species occupy the full geographical range of their main host because so far, they have been found only in Isla Mocha (Arauco Province, Chile), although attempts to collect the mites from different localities were made (by M. Fajfer in ZSM and NHM).This could be interpreted as a consequence of the recent evolution of new mite species which are competing on the same host; therefore, further studies may prove that this group of parasites undergoes rapid adaptive radiation.
Our phylogenetic analysis shows that N. patagonica is a sister taxon to all monoxenous mites of the chilensis group.It agrees with the findings of Fajfer [3].P. patagonica inhabits various host species of three different groups (see Figure 15) [17,26], which might suggest that this mite species' association is not fully recovered, and we can expect even more multihost associations.P. patagonica, due to its circular shape of idiosoma, is morphologically unable to take shelter under the scales; therefore, most of its idiosoma protrudes beyond the scales.This probably allows the mite, by virtue of its effective dispersal abilities, to switch off quickly from a host when the opportunity arises, and then locate and colonize another host.This is especially probable if the host species, as in this case, share the same diet and occur at least partially in the same habitat [17].
The phylogenetical analysis indicates that the newly described species, N. robertmertensi, is nested within the mites of the chilensis group of Neopterygosoma associated with species of the section chiliensis of Liolaemus s. str.Its placement is also confirmed by a set of morphological features (see Figure 15), although the Bremmer support is only 1.The reason for this may be that N. robertmertensi has many unique features (e.g., the number of dorsomedial, ventromedial, or genital setae, i.e., characters 36, 40, and 49-52 in Figure 15, respectively), which do not match the description of the chilensis group provided in Reference [4].Therefore, a revised description of the species group is presented here.
For the first time, we collected all mites from the host specimens that were preserved directly after collection.As a result, we collected hundreds of mites which were completely hidden beneath the lizard's scales.We found 1-12 specimens under a single scale, and the mites inhabited each body part of the host specimens.This lack of topical (habitat) specificity is quite surprising because in pterygosomatids living under the scales (such as Pterygosoma or Geckobia), a high preference towards a microhabitat on the host body is observed [33,34].
This large number of mites allowed us to observe morphological diversity among juveniles and adults and to illustrate for the first time the complete morphological ontogeny of these mites.For the first time in the family Pterygosomatidae, we were able to determine differences between the sexes of larvae.In male larvae of N. robertmertensi, the idiosoma is smaller and almost as long as wide (155-200 long and 170-215 wide), the genital region is situated ventrally, and the male develops directly in chrysalis inside the larval integument.In female larvae, the idiosoma is bigger and wider than long (170-250 long and 290-360 wide), the genital region is situated terminally, and the life cycle of the larva consists of both: active stages that feed on blood (protonymph, deutonymph, and adult) and legless inactive stages (nymphchrysalis, deutochrysalis, and imagochrysalis).
Our study shows that a female larva forms a chrysalis that resembles those found in other pterygosomatids (e.g., see Figure 8C in Reference [35]).Inside the chrysalis, a coiled protonymph develops.After molting, the newly emerged protonymph is larger than larva, and we observe the appearance of four pair of legs with the full set of setae on femora-tarsi IV, numerous idiosomal setae arranged similarly to subsequent stages, subcapitular setae n, weakly sclerotized small propodonotal shield, additional setae ps3 in the genital region, leg setae on coxae II-III (2b, 3b, 3c, 3d), genua-trochanter I-III (v GI-III, v G-III, l GI, vFI, vTrI-III), and tarsi I, i.e., it (ζ), tc (ζ) and tc (ζ).
In the protonymph integument, we observed a deutochrysalis with a completely formed coiled deutonymph.This stage differs from a protonymph by the presence of much smaller gnathosoma and longer palpal setae (dF, dG), fewer setae on the mid-dorsal cluster, and the arrangement of setae (fewer in number) that resembles that in females.An adult female develops in the imagochrysalis (tritonymph).It differs from a deutonymph by the size of the idiosoma, the presence of additional two or three genital setae (g4-g6) and pseudanal setae (ps4-ps5), and ventral setae on trochanter IV.The males develop directly in the chrysalis inside the exoskeleton of larvae.
At this point, it is unclear whether the presence of both male and female larvae is unique for the genus Neopterygosoma.In Pterygosomatidae, as a rule, the description of juvenile morphology is often neglected.This could be due to several factors, such as (i) the difficulty of associating juveniles with an adult if the adults are missing in the sample, (ii) a small number of specimens found on hosts in museum collections (the mites might fall off the host during its preservation), (iii) the presence of only female mites on hosts, which may be explained by the short duration of their juvenile stages or (iv) the small size and transparency of the juvenile stages which make them difficult to notice on the hosts.
It is interesting to note that the larvae of Neopterygosoma differ from those of other genera, such as Pterygosoma or Geckobia, due to the absence of setae on tarsi I, specifically it (ζ), tc (ζ) and tc (ζ).In other pterygosomatid larvae, only one fan-like proral setae p , one simple tectal seta tc , and paired iterals it and it in the form of eupathidia are present.Additionally, Norton's description of leg chaetotaxy [6], based on Grandjean's work [10,11], referred to the iterals as "post-larval setae" that are added in the protonymph stage.Yet, in Neopterygosoma spp.larvae, there is only one euphatidial setae it' while in contrast, the larvae of Pterygosoma have a pair of iterals (it' and it").

Conclusions
In this research, we meticulously described and illustrated the morphology of the new species of pterygosomatid mite, Neopterygosoma robertmertensi, using scanning electron microphotography.As a result, we found new morphological features which were not recognized in previous studies of Neopterygosoma spp., such as the presence of a weakly sclerotized propodonotal shield.We observed the species morphological ontogeny and analyzed the main morphological differences between juvenile stages.For the first time in Pterygosomatidae, we observed both male and female larvae that differ mainly by the size and shape of idiosoma and from other pterygosomatid larvae by chaetotaxy of tarsi I. Additionally, the phylogenetic analysis showed that this species is nested within the chilensis group of Neopterygosoma, which was consistent with the morphological analysis.Neopterygosoma mites occur only on hosts belonging to three groups of the chiliensis section of the subgenus Liolaemus s. str., whose distributions partially overlap.Nonetheless, the hosts do not carry the same sets of parasite species.This suggests that mites of the chilensis group might be a good fit for cophylogenetic studies, especially if we take into account the fact that some studies conducted on pterygosomatid mites revealed a cophylogenetic pattern [3].

Figure 15 .
Figure 15.The most parsimonious tree (tree length 219, CI of 0.64, RI of 0.56, RC of 0.36) found using the branch-and-bound search option for the unordered and unweighted dataset.Numbers at nodes are Bremer indices.Numbers below branches are common synapomorphies (character numbers refer to List S1).Distribution of the mite species within host groups and section are marked in different colours.
AMU-Department of Animal Morphology, Adam Mickiewicz University, Poznan, Poland; CSWU-Department of Molecular Biology and Genetics, Institute of Biological Sciences, Cardinal Stefan Wyszynski University in Warsaw, Poland; HUJ-National Natural History Collections of the Hebrew University of Jerusalem, Israel; NHM-Natural History Museum, London, the United Kingdom; ZSM-Bavarian State Collection of Zoology, Munich, Germany.