Updated Italian checklist of Tenuipalpidae with description of a new species and new worldwide records of the genus Cenopalpus (Pritchard et Baker)

ABSTRACT The family Tenuipalpidae includes agricultural pests that have garnered extensive attention from the global research community. Despite the economic importance of tenuipalpids, due to their ecology and global trade of the infested plants, there has not been any corresponding and comprehensive research on these mites in Italy in recent decades. This study aimed to determine the species composition of tenuipalpid mites in Italy, update the Italian checklist for the taxon, and include new records and host plant associations of the genera Brevipalpus Donnadieu and Cenopalpus Pritchard and Baker worldwide. The study found several new records of tenuipalpid species in Italy, including four Cenopalpus and two Brevipalpus species. Additionally, one new species Cenopalpus ulmifolius De Giosa, Ochoa et de Lillo was added to the list. The research also incorporated new records of Cenopalpus species in different countries and several new host plant associations and the description of novel taxonomic characters were present for the Cenopalpus species studied during this work, since they were the most abundant tenuipalpid collected in the sampled areas. The study’s findings are crucial for developing effective management for tenuipalpid mites, understanding the fauna composition, and constructing preparedness strategies for quarantine purposes.


Introduction
The family Tenuipalpidae in Italy has primarily been studied by Antonio Berlese, Riccardo and Giovanni Canestrini, Marisa Castagnoli, Filippo Fanzago, Giocondo Lombardini and Fausta Pegazzano (Prasad 1982).In recent years, the economic importance of the Tenuipalpidae stems from their global impact and their increased interceptions in international trade on plants (see De Giosa et al. 2021a).Despite their importance, no extensive research has been conducted on these mites in Italy.Consequently, a knowledge gap exists regarding the current status of tenuipalpid mites found in Italy.Furthermore, the economic importance of this family has increased significantly as recent studies have shown that some flat mites have the ability to transmit viruses (de Lillo et al. 2021).
The first Italian checklist of the family Tenuipalpidae was published by Bernini et al. in 1995, encompassing a total of 4 genera and 21 species.This checklist was comprised of eleven species from the genus Brevipalpus Donnadieu, six from the genus Cenopalpus Pritchard and Baker, three from the genus Tenuipalpus Donnadieu, and one species of the genus Pentamerismus McGregor.In addition to these records, a misidentification occurred in the checklist where Acarus mori (Rondani 1870) was inaccurately listed as Raoiella mori.Later, Castagnoli and Nannelli (2003) provided the first update to the checklist, adding one species each in the genera Cenopalpus and Pentamerismus, resulting in a modest increase of 23 tenuipalpid species recorded in Italy.Between 2021 and 2023, a series of studies contributed to an increased number of 31 tenuipalpids documented in Italy (Ueckermann and Ripka 2016;De Giosa et al. 2021a, 2021b, 2022).
The current study primarily aims to describe a new species of Cenopalpus from Italy, to update the Italian checklist of the family Tenuipalpidae reporting new species records.Cenopalpus mostly occurs in the Western Paleartic and Oriental zoogeographical regions (Mesa et al. 2009;Castro et al. 2023), but despite its distribution, this genus is not well documented in Italy.Therefore, we provide new Cenopalpus records and host plant associations based on the material studied, presenting new morphological traits, to update the taxonomically guided identification of this genus.

Material and methods
The new checklist was developed based on national and international revisions, verification of species intercepted from Italy at the ports of entry in the USA and recent surveys conducted in southern Italy.The material intercepted at the ports of entry in the USA was deposited at the United States National Insect and Mite Collection, US National Museum of Natural History, Smithsonian (housed at the Systematic Entomology Laboratory in Beltsville, Maryland, USA).
Surveys were conducted in 2019 in the Apulia District of southern Italy and consisted of randomly collected plant materials from various Mediterranean tree and plant families in different habitats.Each sample, consisting of small branches with leaves and flowers if present, was packaged individually in tightly sealed polyethylene bags with paper towels to control humidity.The samples were transferred to the laboratory the same day and stored at low temperature (about +4°C).The collection data for each sample includes the locality, date, host, and global positioning system Plant and Food Sciences, University of Bari Aldo Moro, Italy (UNIBA)

Results
In this study, we reported four Cenopalpus and two Brevipalpus species which are new records and one new species of the genus Cenopalpus.This increases to 37 the total number of tenuipalpid species present in Italy.Moreover, we reported new records of Cenopalpus species in seven countries and several new worldwide host plant associations.We did not provide updated identification keys to the world species of Cenopalpus, including the new species, since a revision of the genus is needed.Several types of Cenopalpus species have been lost (Mesa et al. 2009;Castro et al. 2023), descriptions in some cases are incomplete, and redescriptions of some species are based on specimens not collected in the local types.The current taxonomical information of the genus Cenopalpus present in the old species descriptions lacks important information in other tenuipalpid genera about morphological characteristics, as highlighted by Beard et al. (2015).
Based on the authors' personal observations, morphological characters such as spermathecal apparatus, microplates, and different dorsal and ventral reticulation patterns are also relevant in separating the species in the genus Cenopalpus.

Remarks
Brevipalpus californicus was reported for the first time from Sicily (southern Italy) on lemon (Di Martino 1960).However, the validity of its presence in Italy is questionable since it belongs to species complex (Tassi et al. in prep.).

Remarks
According to Vacante and Nucifora (2022), the presence of B. cuneatus on lemon trees in the Sicily region of southern Italy has been documented by Cavara and Mollica (1903).This species has been known to cause greyish patches on both lemon fruits and mandarin oranges in the same region (Tardo 1960).The family Rutaceae is a new host plant record for B. cuneatus.

Remarks
Brevipalpus lewisi has been reported for the first time in Italy by Tshikhudo et al. (2022).

Remarks
Brevipalpus oleae has been reported for the first time from Italy by Castagnoli and Pegazzano (1979) after surveys in olive orchard areas.

Remarks
Brevipalpus olearius has been reported for the first time from Italy by Castagnoli and Pegazzano (1979) during surveys conducted in olive orchard areas.

Remarks
Brevipalpus rotai has been described from Italy (Castagnoli and Pegazzano 1979).The redescription of the holotype and paratypes is in progress (De Giosa et al. in prep.).

Remarks
Brevipalpus recki has been reported for the first time from Italy by Pegazzano (1975) andDe Giosa et al. (2022) during surveys conducted on Quercus spp.

Remarks
Brevipalpus hondurani has been reported for the first time from Italy by Tshikhudo et al. (2022) due to an interception in South Africa.Arabia; South Africa; Spain; Sudan; Syria; Tahiti; Taiwan; Thailand; Tonga; Trinidad; Turkey; Uganda; Ukraine, USA.

Remarks
Brevipalpus phoenicis was reported for the first time from Calabria and Sicily regions (southern Italy) on mandarin orange (Di Martino 1960).This species has been found to cause greyish scabby patches and cracks on mandarin oranges (Di Martino 1985;Vacante 2010).However, the validity of its presence in Italy is questionable since it belongs to the Brevipalpus phoenicis species complex.As reported on the Tenuipalpidae Database (Castro et al. 2023) "All data pertaining to the taxon name B. phoenicis prior to the publication of Beard et al. (2015) should be considered questionable, due to synonymies and historic misidentifications (see Beard et al. (2015) for detailed information).Any data pertaining to this taxon name after 2015 that have not referenced Beard et al. (2015) for identifications should be assessed with care." 16. Brevipalpus yothersi Baker, 1949 (SM Figures 4(a-b))  Femora I-II with four setae (d, l′, v′, bv″); femur III with two setae (d, ev′); femur IV with one seta (ev′).Genua I-II with three setae (d, l′, l″); genu III with one seta (l′); genu IV without setae.Tibia I-II with five setae (d, v′, v″, l′, l″); tibia III-IV with three setae (d, v′, v″).Tarsi I-II with eight setae and each with one long, slender, and tapering solenidion; tarsi III-IV with five setae (ft′, tc′, tc″, u′, u″).7(a,b))*.*Mites were collected in southern Italy from two different host plant species, C. monogyna and P. domestica.The analysed samples presented three different dorsal cuticular patterns occurring simultaneously on both hosts.The females cannot be separated by several similar characteristics: 3 rounded lobes with the same reticulation; propodosoma with 3 bumps: large and regular cells in the middle and smaller in the lateral.Opisthosoma with the same reticulation and orientation of cells.Venter with regular reticulation between legs IV; anal plate with same shape and reticulation.The shape of the setae on the subcapitulum, palpus and legs is the same.The microplates confirm a match.Based on the current taxonomical data they are considered to belong to the same species.However, studies involving an integrative approach with morphological, morphometric, and molecular markers should be conducted to confirm the absence of cryptic species.

Remarks
Cenopalpus halperini has been observed in Sardinia (Italy) on Pinus pinaster (see Castagnoli 1974), associated with large populations of C. wainsteini (Livshitz and Mitrofanov 1967).In addition, C. halperini has also been found in the Apulian District on Pinus halepensis (Mill), near needles and scaly leaves, always associated with C. wainsteini.No alteration has been observed in the presence of C. halperini from our study and Castagnoli (1974).The biology of C. halperini is poorly known and requires detailed study.

Diagnosis (adult female)
As per C. spinosus species group, in addition to the following.Body ovate, flat, and completely reticulated.Developed rostral shield and it can vary: the first rostral shape is with 4 short medial and submedial lobes (i.e.medial lobes more acute), 2 short lateral lobes, the submedial and lateral lobes distal end more rounded than medially; the second rostral shape can be with 2 medial lobes strong and developed, 4 short (strongly reduced) submedial and lateral lobes.Hood reticulation is irregular with elongate to rounded cells and oblique to vertical folds.Propodosoma with one bump medially (slightly flat) on which there are large and polygonal cells medially and posteriorly; dorsolateral cells smaller and polygonal to elongate.A strong sejugal furrow between propodosoma and opisthosoma.Dorsal opisthosoma completely reticulated with one bump medially (between c1-c1 and e1-e1) that becomes narrow on posterior margin of opisthosoma; cuticle on bump with largest cells (some fused creating folds) than dorsolateral and lateral ones.Dorsolateral and lateral cells Spermathecal apparatus.(Figure 23(e)) A long, narrow, and convoluted duct ending in a spherical bulb or in a small and rounded vesicle.Vesicle may be undeveloped, with duct ending blindly or in small, membranous bulb.24(a-d)) Leg chaetotaxy formula as C. spinosus species group, in addition to the following.Femur I with setae d spatulate and bv,' l, v' smooth; genu I with spatulate and finely serrate setae (d, l') and smooth seta (l''); all tibia I setae moderately barbed and lanceolate (l,' l',' v,' v''), except d (smooth).Shape of setae on leg II, as following leg I, except the setae on femur: bv'' and l' spatulate and lanceolate, such as d.Femur III with finely barbedlanceolate seta d and smooth seta ev'; genu III with smooth setae (l') and tibia III (d, v,' v'').Shape of setae on leg IV, as following leg III, except v' and v'' on tibia moderately barbed.Measurements of legs (coxae to tarsi): I 170-180, II 145-150, III 140-145, IV 145-150.

Remarks
Cenopalpus lanceolatisetae is a new record for England, Italy, and Jordan.Therefore, new host plant associations include C. lacteus, C. nobilis, E. angustifolia, and P. spinosa (amygdaliformis).We retain inappropriate to design M. sylvestris as a new host plant association since only 1 specimen of C. lanceolatisetae has been found on this host plant species.(Livshitz et Mitrofanov, 1967) (Figures 29(a-36b))

Type depository
Unknown.

Remarks
Cenopalpus mespili has been reported in the updated Tenuipalpidae checklist (Castagnoli and Nannelli 2003).The effective presence of this species in Italy remains questionable due to the absence of information in both national and international scientific journals.

Remarks
Currently C. pegazzanoae has been described and reported only from Italy.

Type depository
Faculté des Sciences de Lyon, France.

Diagnosis (adult female)
As per C. lineola species group, in addition to the following.The body is oval and very developed, in some cases contracted at the junction of hysterosoma and propodosoma.Further, C. wainsteini is completely flat without any bumps.Anterior margin of propodosoma is not developed and appears like a narrow border with a shallow oval depression in the middle.The posterior margin of opisthosoma gradually becoming narrow at its base.Cuticle    completely plicate, covered with mostly transverse striae, except some longitudinal ones.In particular, dorsal propodosoma with longitudinal and transversal wrinkles: dorsomedial striae minutesmall, transverse and oblique, laterally long and longitudinal.Anterior margin of opisthosoma, between c3-c3 and d3-d3, with generally long-short, transverse, oblique and few longitudinal striae; from e3-e3 to h2-h2 becoming elongate strong and longitudinal.Three to five distinct transversal folds (between d3-d3 and e3-e3) on the border of the anterior and posterior of opisthosoma.Between e1-e1 and h1-h1 almost smooth, with fine and weak longitudinal striae.The propodosomal and opisthosomal setae, except c1, c2, d1, e1, palmate.Otherwise c1, c2, d1, e1 slightly serrate.Cuticular microplates: separate individual, rounded to irregularly rounded plates, with multiple short irregular ridges over dorsal surface (Figures 66(a-b)).Metapodosoma almost smooth between coxae III and coxae IV, with light and fine striae.Beyond setae 4a, cuticle completely covered with narrow to developed transverse bands (11 or more folds).Ventral, genital and anal plates are well developed but not sclerotized, with fine transverse.

Remarks
Cenopalpus wainsteini has been first reported from Italy by Pegazzano (1971).Cenopalpus wainsteini by could be responsible for causing alterations on Pinus sp., as reported by Huanca et al. (2021).This species needs future detailed study concerning the biology, ecology, and its interactions with trees of the family Pinaceae.

Remarks
Cenopalpus adventicius was collected and described from Rosmarinus officinalis Linnaeus imported from Italy.This species is morphologically close to C. officinalis.Future morphological comparisons between the types and molecular analyses are needed to exclude possible synonyms.

Remarks
Pentamerismus taxi has been reported in the first Tenuipalpidae checklist for Italy (Bernini et al. 1995).The effective presence of this species in Italy remains questionable due to the absence of information in both national and international scientific journals.

Remarks
Tenuipalpus caudatus has been reported for the first time from Italy by Castagnoli and Pegazzano (1979).

Remarks
Tenuipalpus granati has been found on different varieties of grapes in Italy (Peverieri et al. 2009).

Remarks
Tenuipalpus pacificus has been reported in the first Tenuipalpidae checklist for Italy (Bernini et al. 1995).The effective presence of this species in Italy remains questionable due to the absence of information in both national and international scientific journals.

Discussion
Several microscopy techniques have been proven to be necessary for identifying tenuipalpid species: Low Temperature Scanning Electron Microscopy (LT-SEM) and cryomicroscopy are invaluable and fundamental for morphological studies.Indeed LT-SEM techniques provide to observe delicate structures that are commonly destroyed or seriously altered during the slide mounting process, resulting in some artefacts (Beard et al. 2015;Castro et al. 2016).Following Beard et al. (2013), phase contrast microscopy is almost inadequate to investigate the ornamentations of a tenuipalpid specimen, but it is great for studying the shape of the setae and spermathecal apparatus.Differential interference contrast (DIC) is recommended whenever possible for tenuipalpid diagnostics.For more information, follow "Flat mites in the world -microscopy for mites" (http://idtools.org/id/mites/flatmites/). Since the family Tenuipalpidae present species-complex groups and the number of morphological characters used at the beginning of the taxonomical studies were scarce, morphometric studies and new morphological traits such as microplates (Welbourn et al. 2003), spermathecal apparatus, leg chaetotaxy, and plant associations are enhancing our understanding of the systematics of Cenopalpus, while allowing the identification of new significant and critical characters for species separation in this group.
The paper aims not to redescribe Cenopalpus species since we have not compared types or designed neotypes when the types were lost but to introduce novel morphological characters for this genus, such as microplates.We have provided morphological data for specimens collected in Italy, and we suggest that the information should be used as a base for future research on the genus Cenopalpus.Although we have made comprehensive morphological studies of some Cenopalpus species, we still miss relevant molecular and morphological information.Most original descriptions and redescriptions do not meet the current expectations for identifying and describing new species, resulting in misidentification and confusion of the Cenopalpus species.For instance, the specimens of Cenopalpus from Iran (Khanjani et al. 2012) and Turkey (Çobanoğlu et al. 2016) were identified as bakeri exhibit two different chaetotaxies of the legs, each differing from the specimens of bakeri collected in Italy.
Defining the taxonomic identity of organisms is a prerequisite for their study and, in the case of economically important species, misidentification and unprecise host plant associations may lead to the application of inappropriate prevention and control strategies.The diagnostic certainty coupled with the phylogenetic signal as a predictor of the host range of plant pests and pathogens is an evolutionarily based tool for phytosanitary risk analysis (Gilbert et al. 2012).Knowing which local plant species are vulnerable is necessary for calculating the risk posed by a novel pest or pathogen (Robles-Fernández and Lira-Noriega 2017).Empirical data on the local host range of novel pests are usually lacking, but we know that some pests are more likely to attack closely related plant species than species separated by greater evolutionary distance.
Brevipalpus are known to be vectors of plant viruses.Therefore, the presence in Italy of B. papayensis, B. yothersi, and B. obovatus should alert the phytosanitary system to the introduction of these vectors in the country and Europe since contact with the viruses they transmit can cause outbreaks.Moreover, it is still unclear if some Cenopalpus species (e.g. C. waisteini-see Huanca et al. 2021) can be vectors of plant pathogens or cause severe mechanical damage to the plants.
We encourage researchers to contribute to the expansion of our understanding of the family Tenuipalpidae by comparison with type specimens or vouchers and better documenting species.micrographs, Andrew Ulsamer, former Debra Creel and Lucrecia Rodriguez (USDA), Armando Rosario-Lebron (APHIS, USDA), for their help with information, references, and suggestions.To Smithsonian Natural History Museum, National Agricultural Library (NAL-USDA), USDA National Program-Forest Service and APHIS for support and assistance with references, permits and funding for this study.This work would not have been possible without the help of Pasquale Trotti, Franca Todisco and Giuseppe Bari (Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro).Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA; USDA is an equal opportunity provider and employer.
setae smooth and lanceolate.Subcapitulum extending to the distal end of femur I. Subcapitulum with subcapitular setae m 12-15; distance between setae m -m 14.Spermathecal apparatus.(Figure 1(e)) Very long and narrow, distal end not visible.

Figure 7 .
Figure 7. Microplates of adult female C. bakeri collected on C. monogyna, characterized by variations in the reticulation of the dorsal pattern (scales 50 µm).
genua I-IV (l,' l'') and tibia I-IV (l') of the deutonymph.Also, the setae on the genua I (l') and tibia I (d, l''), and on the trochanter (d), genua (l'), and tibia (d) III of the larvae differ in shapes in both redescriptions.A detailed study involving and integrative approach with molecular markers and comprehensive morphological review of the holotypes and paratypes is needed to understand the taxonomical status of this species.The mouthparts were studied byNuzzaci and de Lillo (1989, 1991)  and de Lillo et al. (2002) on
), 42(a-b), 43(b)) Setal formula for palps as in diagnosis of C. spinosus species group.Solenidion 6-7 and eupathidia 4-6.Femur seta short and strongly barbed in the end; genu-tibia setae smooth and slightly barbed in the distal end.Palps extending to end of femur I.The distal end of subcapitulum always reaching the posterior margin of genu I. Subcapitulum with lanceolate, and slightly barbed subcapitular setae m 34-35; distance between setae m-m 32.Spermathecal apparatus.(Figure 37(e)) A long narrow, convoluted duct is visible, ending in a small, rounded vesicle.
Measurements of legs (coxae to tarsi): I 136-145, II 105-108, III 93-110, IV 107-111.Protonymph.LARVA.EGGS.Not examined.Distribution (Castro et al. 2023) Afghanistan; Algeria; Armenia; Austria; China; Cyprus; Denmark; Egypt; England; France; Georgia; Germany; Greece; Hungary; India; Iran; Iraq; Israel; Italy; Jordan; Lebanon; Libya; Morocco; Netherlands; Pakistan; Portugal; Syria; Tunisia; Turkey; Ukraine; USA: Oregon.Remarks.Cenopalpus pulcher has been described from Italy (Canestrini and Fanzago 1876).Cenopalpus pulcher is a new record for Argentina, England, Mauritius, North Ireland and the former Yugoslavia.Moreover, Buxus sp., C. lacteus, Ligustrum sp., and M. scions are new host plant associations.Furthermore, the adult female of C. pulcher is morphologically close to the one of C. bakeri.For this reason, both species are difficult to separate if the nymphs are unavailable, resulting in misidentification.The current worldwide distribution of C. pulcher should be re-evaluated and confirmed.

Figure 53 .
Figure 53.Differential interference contrast micrographs of adult females C. ulmifolius: (a-c) Dorsal and ventral habitus of specimens collected on R. canina; (b-d) Dorsal and ventral habitus of specimens collected on R. ulmifolius (scales 50 μm).

.
The deutonymph of C. ulmifolius nov.sp.morphologically resembles several other species, including C. brachypalpus Hatzinikolis et al., C. pseudospinosus Livshitz et Mitrofanov, C. quadricornis Livshitz et Mitrofanov, C. rubusi Khanjani et al., C. spinosus Donnadieu, and C. taygeticus Hatzinikolis et al.However, there are also notable differences between the deutonymphs of these species.Deutonymph of C. ulmifolius with dorsal setae c1, h2, h1 long (whereas these setae are short in C. brachypalpus and c1 are short in C. pseudospinosus).Deutonymph of C. ulmifolius with setae e1 and f2 long (whereas these setae are short in C. quadricornis).Deutonymph of C. ulmifolius with c1, c3, e3 long (whereas shorter in C. spinosus).Deutonymph of C. ulmifolius with d1 and h2 short (whereas longer in C. taygeticus).Deutonymph of C. ulmifolius with f3 long (whereas shorter in C. rubusi).Morphological differences in the subcapitulum length and cell shape on the rostral shield are also present between the females of the mentioned species.Female of C. ulmifolius with subcapitulum reaching beyond the distal end of the femur (whereas rostrum not reaching the end of the femur in C. quadricornis and C. taygeticus).Female of C. ulmifolius with rugose-transverse cells on the rostral shield (whereas with areolate cells in C. brachypalpus, C. pseudospinosus and C. quadricornis).
Spermathecal apparatus.(Figure 61(e)) Only the beginning of duct is visible at genital opening.
Welbourn et Beard, 2017 Type depository Florida State Collection of Arthropods, Florida, USA.Distribution (Castro et al. 2023) Guatemala; Italy; USA.Remarks Tenuipalpus sarcophilus has been reported for the first time from Italy by De Giosa et al. (2021) due to an interception in the United States.

Distribution (Castro et al. 2023) India
: West Bengal; Italy.RemarksBrevipalpus mitrofanovi has been described from Italy on Quercus cerris Linnaeus(Pegazzano 1975).The redescription of the holotype and paratypes is in progress(De Giosa et al. in prep.).

Diagnosis (adult female)
As per C. spinosus species group, in addition to the following.In the current description, two females with different morphology are reported.Rostral shield with regular reticulation: longitudinal cells between medial and submedial lobes; vertical reticulation on lateral lobes.Coxisternal area between coxae I-II with weak transverse striae, becoming almost smooth medially.Metapodosoma smooth between coxae III and IV, with transverse folds on each coxa.The region posterior to coxae IV with uniform reticulation.

Type depository USNM Distribution (Castro et al. 2023)
RemarksPentamerismus oregonensis has been reported for the first time from Italy by De Giosa et al. (2021) due to an interception in the United States.