Phylogenetic Position of a New Trisetacus Mite Species (Nalepellidae) Destroying Seeds of North American Junipers and New Hypotheses on Basal Divergence of Eriophyoidea

Simple Summary Eriophyoid mites are microscopic herbivores associated with higher plants. Some of them are serious pests due to their ability to vector viruses and cause other damage to host plants. Mites of the genus Trisetacus are widespread parasites of conifers. They usually live in buds, cones, and rarely within needles of Pinaceae (pine family) and Cupressaceae (cypress family). We discovered a new species, Trisetacus indelis n. sp., severely damaging seeds of three North American junipers in the western USA. This species possesses two morphologically different forms of females and has two deletion mutations in the gene cytochrome oxidase subunit I (Cox1). Such mutations are rare in eriophyoids and were previously detected only in two pestiferous species from palms and hazelnut. Our molecular-phylogenetic analyses determine the closest known relatives of the new species and suggest that Old and New World Trisetacus independently transitioned to living in seeds of junipers. Additionally we show that reconstruction of the phylogeny of Eriophyoidea based on one gene, Cox1, produces a poorly-resolved but biologically consistent tree topology to hypothesize the evolution of Eriophyoidea. Overall, our study improves our understanding of the diversity of conifer-inhabiting mites and indicates further needs in investigating the phylogeny of Eriophyoidea. Abstract Eriophyoid mites of the genus Trisetacus Keifer are widespread parasites of conifers. A new oligophagous species, T. indelis n. sp., was discovered severely damaging seeds of North American junipers (Juniperus osteosperma, J. occidentalis, and J. californica) in the western USA. It has two codon deletions in the mitochondrial gene Cox1 rarely detected in Eriophyoidea and includes distinct morphological dimorphism of females. A phylogenetic analysis based on amino acid alignment of translated Cox1 sequences using a large set of out-groups (a) determined that two North American congeners, T. batonrougei and T. neoquadrisetus, were the closest known relatives of T. indelis n. sp., and (b) indicated that Old and New World seed-inhabiting Trisetacus from junipers do not form a distinct clade, suggesting a possible independent transition to living in seeds of junipers in America and Eurasia by Trisetacus spp. Our analysis produced a new topology consistent with a scenario assuming gradual reduction of prodorsal shield setation in Eriophyoidea and an ancient switch from gymnosperms to other hosts. Additionally, our analysis did not support monophyly of Trisetacus; recovered a new host-specific, moderately supported clade comprising Trisetacus and Nalepellinae (Nalepella + Setoptus) associated with Pinaceae; and questioned the monophyly of Trisetacus associated with Cupressaceae.


Introduction
Eriophyoid mites are an ancient group of phytoparasitic acariform mites exclusively associated with higher vascular plants and closely related to soil nematalycid mites [1][2][3]. Because of their microscopic size (about 200-300 µm), numerous undescribed species, and greatly simplified homogenous homoplastic morphology explained by miniaturization and adaptation to phytoparasitism, eriophyoids are among the most difficult groups of mites to classify taxonomically [4][5][6][7]. The identification of eriophyoids is usually based on laborious morphometrics and precise descriptions of morphology followed by a comparison with species descriptions scattered in different journals [8]. Since the beginning of the 21st century, DNA barcoding techniques typically complement morphological studies and have been proposed as a standard for taxonomic publications establishing new taxa in Eriophyoidea [9][10][11][12][13].
Eriophyoid mites of the genus Trisetacus Keifer (Acariformes, Eriophyoidea, Nalepellidae) are common and widespread arthropod parasites of conifers [21,22]. They have been recorded in all regions where conifers are abundant except Australasia, where no studies on eriophyoids from gymnosperms have been performed to date [23]. All known hosts of Trisetacus belong to the two most diverse gymnosperm families, Pinaceae and Cupressaceae. Currently this genus comprises approximately 60 species; however, this number is expected to increase as more conifer species are investigated. Damage to host plants by Trisetacus can occur in a variety of forms [21,24]. Bud deformation (Figure 2A,F) is the most common damage caused by Trisetacus on various pinacean (Abies, Picea, Larix, Cedrus, Pinus, Pseudotsuga) and cupressacean (Juniperus, Platycladus, Chamaecyparis, Cupressus) host genera. Along with living in buds, several cryptic species of the complex Trisetacus juniperinus s.l., are also capable of infesting male cones and causing swollen galls on junipers ( Figure 2D,E). Two endoparasitic species, T. abietis and T. neoabietis from Abies spp., live in air-cavities under the epidermis, causing necrosis of the parenchyma ( Figure 2C) and premature needle fall [25,26]. A unique lineage of Trisetacus, T. pini, has adapted to live in galls, which they induce on young twigs on pines in Europe ( Figure 2B). Damaging female cones and living inside seeds is typical only for Trisetacus associated with junipers ( Figure 3). Along with bud mites, e.g., T. juniperinus severely damaging cypress trees in Europe [27], seed mites are serious pests of junipers on different continents. Trisetacus kirghisorum causes serious problems in central Asia where it heavily damages cones and seeds and notably slows growth and reproduction of economically and culturally important Juniperus spp. [28,29]. Similarly a common European mite species, T. quadrisetus, and the North American species T. batonrougei and T. neoquadrisetus can destroy up to 95% of seeds of their juniper hosts [30]; (J. Amrine and P. Chetverikov, unpublished observations). This group of seed parasites is a potential serious threat to conifer nurseries and requires early detection and removal of infected plants.
During field surveys in mountainous areas of the western USA we observed groves of junipers heavily infested by a new Trisetacus species. In this paper we aim to describe this species and assess its phylogenetic position within the genus Trisetacus, based on sequence comparisons of the mitochondrial cytochrome oxidase c gene (Cox1), including a dataset from a previous study [23]. In addition, we investigated the phylogenetic structure of Eriophyoidea using a new set of outgroups and a translated Cox1 amino-acid alignment that was twice as long as that produced by the classical LCO-HCO fragment [31].

Materials and Methods
Morphology. Live mites of T. indelis n. sp. were collected in 2021 in the USA from plants using a fine minuten pin and a dissecting microscope, then placed in Eppendorf tubes filled with 96% ethanol. The mites were mounted in modified Berlese medium with iodine [32] and cleared on a heating block at 90 • C for 3-5 h. Slide-mounted specimens were examined with differential interference contrast light microscopy (DIC LM) using a Leica DM2500 (Leica Microsystems GmbH, Wetzlar, Germany) and photographed with a ToupCam UCMOS09000KPB digital camera (Hangzhou ToupTek Photonics Co., Hangzhou 310030, China). Images and specimens were analyzed and measured using ToupTek ToupView software (Hangzhou ToupTek Photonics Co., Hangzhou 310030, China). In the description of the new species, measurements of a holotype (female) are presented followed by measurement ranges for paratype females. All measurements are given in micrometers (µm) except when specified otherwise. Classification and terminology of morphology follow [4,14,33]. The scientific names of host plants are given according to [34]. Drawings of mites were sketched by pencil using a video projector [35], scanned, and finalized in Adobe Illustrator CC 2014 (Adobe Systems, San Jose, CA, USA) using a Wacom Intuos S CTL-4100K-N (Wacom Co., Ltd, Kazo, Saitama, Japan) graphics tablet.
DNA extraction and sequencing. For DNA extraction, 1-3 mite specimens of each species were crushed with a fine pin in a 3 µL drop of distilled water on a cavity well microscope slide. The drop was pipetted into a thin-walled PCR tube with 30 µL of 5% solution of Chelex ® 100 Resin (Bio-Rad Laboratories, Inc., Hercules, California, USA) before being heated three times (5 min at 95 • C). The solution above the settled Chelex ® granules was used as the DNA template for PCR to amplify a fragment of subunit I of Cox1. Thermal cycling profiles and primers (for PCR and for sequencing) used were as specified by [36]. After amplification, 4 µL of each reaction product was mixed with 0.5 µL of SYBR Green I (Lumiprobe, Hannover, Germany) and analyzed by electrophoresis in a 1% agarose gel to assess the product size and concentration. Sequences were obtained using BigDye Terminator v.3.1 chemistry in a 3500xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Trace files were checked and edited using GeneStudioTM Professional 2.2.0.0. (www.genestudio.com, accessed on 10 February 2022).
Type material. Holotype female from slide E4679, paratype females and males from slide series E4680-E4683 collected on 24 February 2021 near Madeline, California (41.129000, −120.501000), from inside enlarged seeds of Juniperus occidentalis Hook. (Cupressaceae). Type material is deposited in the Acarological Collection of the Zoological Institute of the Russian Academy of Science (ZIN RAS) in Saint-Petersburg and in the collection of eriophyoid mites of J. Amrine (West Virginia University, USA).
Relation to host. Mites penetrate immature female cones, feed on seed endopsperm, and cause seed deformation; infested seeds may protrude from the cones up to 1-2 mm ( Figure 3D-F).
Additional material. Females and males of Trisetacus indelis n. sp. from slide series E4684-E4688 collected on 10 February 2021 in the Sweetwater Range, Nevada (38.607000, −119.240000) from inside enlarged seeds of Juniperus osteosperma Hook.; and from slide series E4689-E4693 collected on 9 February 2021, near Dales, California (40.291000, −122.112111) from inside enlarged seeds of J. californica Carr. (Cupressaceae). This material has been deposited in the Acarological Collection of ZIN RAS, Saint-Petersburg, Russia.
Etymology. The specific epithet, indelis, is an adjective, gender masculine, derived from "indel" and corresponding to the codon deletion mutations present in the Cox1 sequence of this species in comparison to all currently known members of the genus Trisetacus [23].
Differential diagnosis. T. indelis n. sp., is morphologically close to T. kirghisorum and T. batonrougei. Main differences between them concerns prodorsal shield ornamentation and shapes of the spermathecal apparatus. In T. batonrougei median and admedian lines are absent [30], whereas in T. kirghisorum three short lines (median and two admedian) [42] and in T. indelis n. sp. one long median line are present ( Figure 4E; Figure 5A,B). In T. indelis n. sp. the spermatheca is small, tear drop-shaped, and the spermathecal tube is long and narrow ( Figure 3F). In T. kirghisorum and T. batonrougei the spermatheca is notably larger and cucumber-shaped, and the spermathecal tube in T. batonrougei has an additional expanded segment [23].

GenBank Data and Cox1 Sequence Diversity
Seven nearly identical Cox1 sequences of T. indelis n. sp. were obtained (K2P distances = 0.000-0.001, Table 1). Two sequences (isolates ost6 and ost10) have one synonymous substitution (C/Y vs T in all other sequences) in the position 852 of our alignment in a codon corresponding to asparagine. All sequences of T. indelis n. sp. are lacking two nucleotide triplets (alignment positions 484-489) in comparison to all other known Trisetacus spp. [23]. Among all other (approximately 1500) Cox1 sequences of eriophyoid mites that are currently present in GenBank, codon deletions are present only in the 21 sequences of Phytoptus avellanae (1 codon deletion) and four sequences of Retracrus (2 codon deletions). They are situated in the same region of the Cox1 gene (alignment positions 469-474) as in T. indelis n. sp.

Molecular Phylogenetic Analysis
The molecular phylogenetic analysis revealed a monophyletic Eriophyoidea, as expected, comprising three large clades (Phytoptidae s.str., Eriophyidae s.l, and Nalepellidae) with poorly resolved relations between them (Figure 7). Two archaic pentasetacid genera (Loboquintus and Pentasetacus) were situated in the unresolved basal part of the tree and clustered with different large eriophyoid clades. The genus Trisetacus was polyphyletic. All members of Trisetacus associated with Pinaceae formed a well-supported clade sister to Nalepellinae (Setoptus + Nalepella), which are also associated with Pinaceae. Trisetacus from Cupressaceae appeared as polyphyletic and comprised several well-supported clades, each of them including mite species associated with different species of the plant genus Juniperus. The mite genus Boczekella was sister to one of the clades of Trisetacus from Cupressaceae. Trisetacus spp., living in seeds of Juniperus spp., did not cluster together. However, all analyzed species (T. batonrougei, T. quadrisetus, and T. indelis n. sp.) from seeds of American junipers (Juniperus californica, J. occidentalis, J. osteosperma, and J. virginiana) formed a highly supported clade sister to a clade comprising a complex of cryptic species, viz. Trisetacus juniperinus s.l. + T. bioti s.l. associated with various cupressacean genera (Cupressus, Juniperus, Platycladus). Only one population from this complex (Trisetacus sp. F20 from Crimea) lives in seeds. The species T. quadrisetus, T. kirghisorum, and Trisetacus sp. F20 associated with seeds of the Eurasian junipers J. excelsa, J. foetidissima, and J. communis, were not clustered together although monophyly of the lineage T. quadrisetus + T. kirghisorum was strongly supported.

Discussion
A new mite species destroying seeds of North American junipers. In this study, we report on a new Trisetacus species, T. indelis n. sp., associated with junipers native to the western USA. This species is remarkable in several aspects. First, this is the first member of Nalepellidae possessing codon-deletion mutations in the Cox1 gene. Such mutations in Cox1 are rare in Eriophyoidea, currently known in only two other eriophyoid genera (both from Phytoptidae s.str.): Phytoptus and Retracrus [36,43]. In all cases, the codon deletions occur in approximately the same region, about 500 bp in the 3 direction from the Cox1 start codon, which should be noted in future when designing primers.
Second, the new species is a new distinct example of deuterogeny, with two morphologically different forms of females present in the life cycle that possess identical Cox1 barcodes [44,45]. Among other members of Trisetacus, distinct deuterogeny has been reported for T. kirghisorum and T. piceae. However, contrary to all known examples of deuterogeny in Eriophyoidea, in these latter two species both sexes (females and males) are subjected to seasonal dimorphism [29,46,47]. This fact possibly correlates with the closer relation of T. kirghisorum to the clade comprising Trisetacus from Pinaceae (which includes T. piceae) than to the clade comprising Trisetacus from seeds of American junipers revealed in the molecular phylogenetic analysis presented here (Figure 7). Although a deep investigation of the problem of deuterogeny is out of the scope of this study, we hypothesize that deuterogeny may be common for many Trisetacus spp., and that possibly all eriophyoids may have seasonally adapted female forms that differ in their morphologies and physiological functions to varying extents in different phylogenetic lineages.
Third, Trisetacus indelis n. sp., provides a new documented case of oligophagy in Eriophyoidea, a superfamily in which most members are considered highly host-specific monophages [48]. This species inhabits seeds of three partially sympatric species of Juniperus (J. californica, J. occidentalis, and J. osteosperma) growing in mountainous areas of the western USA [49]. Our analyses suggest that this mite species is closely related to the two other presumed monophagous North American Trisetacus species, T. batonrougei and T. quadrisetus, associated with Juniperus scopulorum and J. virginiana, respectively, which have a more eastern distribution in USA than the known hosts of T. indelis n. sp. Because Cox1 is a relatively quickly evolving gene often used for delimiting host races and cryptic species [50][51][52][53][54][55], we expected to see variation, particularly in 3d codon positions, between populations of T. indelis n. sp., from different hosts and localities. The observed striking pairwise sequence similarity and presence of the indicative indels, which are very rare in Cox1 genes of Eriophyoidea, suggest that T. indelis n. sp., may be a recently evolved species, quickly expanding its host range and area of distribution. The single synonymous nucleotide substitution observed in a population of J. osteosperma (isolates ost6 and ost 10) may be a signature of early host-dependent species divergence in the form of a point mutation. Future studies are needed to trace the evolutionary history of T. indelis n. sp., and assess its impact on the seed ecology of its hosts, two of which (J. occidentalis and J. osteosperma) are considered weedy in parts of their native ranges [56].
Implications to the phylogeny of Eriophyoidea. No morphological or multigene molecular phylogenetic analyses to date have resolved the basal phylogeny of Eriophyoidea [3,6,7,15,16,36,57]. One general conclusion from prior studies was that genes usually used in molecular phylogenetics by acarologists (rDNA, Cox1, 16S) are suboptimal for resolving the phylogeny of Eriophyoidea. However, it was noted that in many cases when it was possible to obtain well-resolved multigene phylogenies of different acarine groups, it has often largely reproduced the topology of trees based only on the Cox1 gene [58]. Our analyses based on translated Cox1 amino acid alignments produced an incompletely resolved tree of Eriophyoidea but revealed several new groupings marked by host associations and common morphology.
The analysis presented here unambiguously placed Trisetacus indelis n. sp., within a clade of American seed-infesting mites from junipers. Similar to a previous phylogeny of Nalepellidae based on the D1D2 28S fragment [23], our current analysis (a) questions the monophyly of Trisetacus, (b) produces a new moderately supported clade from pinacean hosts (Nalepellinae + Trisetacus from Pinaceae), (c) rejects monophyly of Trisetacus from Cupressaceae, (d) suggests a possible independent transition of Trisetacus to living in seeds of New and Old World junipers, and (e) again indicates an uncertain position of Boczekella associated with Laricoideae (Pinaceae) in the basal part of the nalepellid clade. Additionally, our analysis offers a new hypothesis on the basal divergence of Eriophyoidea suggesting (i) gradual loss of prodorsal shield setae (vi, ve, sc), and opisthosomal setae c1 in major eriophyoid lineages-Nalepellidae, Phytoptidae s.str., and Eriophyidae s.l., (ii) gymnosperms as the most likely ancestral hosts of Eriophyoidea, and (iii) pentasetacids as an archaic polyphyletic group comprising "living fossils." While these hypotheses have been widely discussed in the relevant literature [7,16,[18][19][20]36,59,60], the presented topology is the first case in which all of them are combined together. Future analyses based on larger mitogenomic and genomic datasets may produce a breakthrough in our knowledge of the early steps of the evolution of Eriophyoidea and generate a phylogenetically consistent classification of this important taxon.

Conflicts of Interest:
The authors declare no conflict of interest.