A new Aceria species ( Acari : Trombidiformes : Eriophyoidea ) from West Asia , a potential biological control agent for the invasive weed camelthorn , Alhagi maurorum Medik . ( Leguminosae )

A new species of eriophyoid mite Aceria alhagi n. sp. inhabiting the weed Alhagi maurorum Medik., is described from the type locality in Iran, but it was also collected from Uzbekistan, Turkey and Armenia. This mite causes changes of the leaves and inflorescence. Infested plants develop cauliflower-like galls on the inflorescence and leaves deforming the reproductive structures and inhibiting seed production. The potential reduction in seed set suggests that this mite could constitute a potential biological control agent against this noxious weed. To investigate intraspecific variability between A. alhagi n. sp. populations from Iran, Turkey and Armenia, we analysed molecular sequences of the mitochondrial cytochrome oxidase subunit I (mtCOI). These results indicated that there are no significant intraspecific divergences among populations of A.alhagi n. sp. from the five different localities in three West Asia countries. This finding can be used in the future research of certain mite populations as biological control agent.


Introduction
The plant genus Alhagi belongs to the tribe Hedysareae of the Leguminosae together with another eight genera: Corethrodendron, Ebens, Eversmannia, Greuteria, Hedysarum, Onobrychis, Sula and Taverniera (Duan et al. 2015). According to the Plant List database (2016), Alhagi has 9 plant species. Our target species, Alhagi maurorum Medik., is a shrub commonly known as camelthorn, camelthorn-bush, Caspian manna, or Persian manna (CABI 2015). The native range of this plant extends from Cyprus and Egypt in the West, to Mongolia and China in the East and South to India and Saudi Arabia (ILDIS 2002, Li et al. 2010. In its region of origin, camelthorn is used as a medicinal herb (Suthar et al. 2016). However no uses have been reported from the areas where it was introduced. Conversely in these areas it has become an invasive weed: in the USA it is listed as a noxious weed in seven states (USDA-NRCS 2002); Australia has declared it a state prohibited weed in Victoria (Munakamwe 2016) and in South Africa it is a declared Category 1 invasive species (AGIS-WIP 2006). No records of biological control agents are reported for this weed, although there is a potential in this field (Rassoul et al. 1988).
All Eriophyoidae mites are phytophagous, many of them can reach pests status on crops, whereas many others are associated with weeds. Eriophyoid mites have high potential as classical biological control agents of weeds due to their strict host plant specificity , Smith et al. 2010.
According to the world catalogue (Amrine & Stasny 1994), Fauna Europaea (De Lillo 2004) and published records (Denizhan et al. 2007, Xue et al. 2012, Lotfollahi et al. 2014, six eriophyoid mites have been reported on Hedysareae hosts. These include, on Hedysareae type hosts, Aculus hedysari (Liro 1941) from Hedysarum sibiricum Ledeb. (junior synonym of Hedysarum alpinum L.); Aculus longifilis (Canestrini 1891) from Onobrychis viciifolia Scop.; Aceria novellae from Hedysarum sp. (Denizhan et al. 2007) and Aculodes alhagis Xue, Sadeghi & Hong from A. maurorum (Xue et al. 2012).The final two species, Aceria medicaginis (Keifer 1941) and Aculops allotrichus (Nalepa 1894) were recorded on Hedysarum coronarium L. and A. maurorum respectively as alternate hosts. Besides these six species one more was recorded as nomen nudum, i.e. Aceria alhagi from A. maurorum (Kamali 2011, Doryanizadeh et al. 2013. Reliable identification is one of the key elements in the search for biological control agents of pests, including target weeds, in order to avoid unfavourable non-target effects. Phenotypic differences among eriophyoid mites belonging to the same genus associated with the closely related host plants are usually small. Intraspecific variability of morphological traits and the existence of cryptic species makes species determination difficult (Amrine et al. 1994, Skoracka et al. 2002, Navia et al. 2006. Recently, molecular analyses are widely accepted and applied as supplementary methods that help to avoid errors in systematics in such situations (Navajas & Navia 2010). The current study presents the morphological description of a new eriophyoid mite species, A. alhagi n. sp., found on A. maurorum as well as mt-COI nucleotide sequences of populations from different geographical areas.

Materials and methods
Plant samples of Alhagi maurorum Medik. (Leguminosae) were collected from Iran, Uzbekistan, Turkey and Armenia during 2012, 2013 and 2015 and examined at the laboratory under a dissection stereomicroscope. Since this mite species was recorded first in Iran (Kamali 2011), we decided to use this material as the type material for morphological description and measurements. Mite specimens from samples representing certain populations were used for morphological and molecular analyses and fixed in 75% and 96% ethanol respectively.

Morphological analysis
Mites were extracted from the plants using a fine pin with the aid of direct examination under a dissection stereomicroscope and/or using extraction methods described by de Lillo (2001) and Monfreda et al. (2007). The mites were mounted in Keifer's F medium (Amrine & Manson 1996) and then examined using a Leica DMLS research microscope with phase-contrast. The morphology and nomenclature follows Lindquist (1996) and genus classification is based on Amrine et al. (2003). Measurements and illustrations were made according to Amrine & Manson (1996) and de Lillo et al. (2010). Morphometry was performed using the software package IM 1000 (Leica, Wetzlar, Germany) and for drawings a camera lucida was used. All measurements are given in micrometers (μm) and, unless stated otherwise, are the lengths of the structures. Morphometric data of A. medicaginis which were used for comparison with A. alhagi n. sp., were obtained from the literature (Keifer 1941). Plant names are in accordance with The Plant List (2016) on-line database.
The holotype and the paratype slides are deposited in the collections of the Acarology Laboratory, Department of Entomology and Agricultural Zoology, Faculty of Agriculture, University of Belgrade, Serbia; one paratype slide is deposited in the Department of Plant Protection, Razavi Agricultural and Natural Resources Research Centre, Mashhad, Iran.
Scanning electron micrographs (SEM) were taken according to Alberti & Nuzzaci (1996). Live mites were collected individually using a fine pin from fresh plant material under a stereomicroscope. For sample preparation before being placed on the SEM holder, mites were sputter-coated with gold for 100 s under 30 mA ion current.
The samples were then studied in the vacuum chamber of a JEOL Scanning Electron Microscope (SEM, JEOL-JSM6390) at the Laboratory of Electron Microscopy, Faculty of Agriculture, University of Belgrade, Serbia.

Morphometric characters
Aceria alhagi n. sp.  (Denizhan et al. 2007) on the Hedysarum sp.. A. alhagi n. sp. is close to A. medicaginis, but it can be distinguished by the following characters: number of rays on the tarsal empodium (A. alhagi n. sp.= 6; A. medicaginis =5); number of striae on the female genital coverflap (A. alhagi n. sp. with 15 -17; A. medicaginis with 10 -12); prodorsal shield design (A. alhagi n. sp. with almost smooth prodorsal shield, with short admedian and submedian lines on the rear margin of the shield, while A. medicaginis with completely smooth prodorsal shield without any lines). The differing morphometric characters between A. alhagi n. sp. and A. medicaginis are presented in Table 2.

Molecular Analyses
The final alignment included the whole mt-COI barcoding region. No insertions or deletions were found between the sequences. In total, 4/658 (0.6%) nucleotides were polymorphic, of which two were parsimony informative. Base pair frequencies show that the region is AT-rich (A: 0.228, C: 0.153-0.160, G: 0.157-0.158, T: 0.456-0.460). The translation of the nucleotide sequence resulted in a 219 amino acid positions, of which one was variable. The average mean divergence over all the sequence pairs was 0.3% and ranged from 0% to 0.6% (Table 3). Similar level of divergence was obtained within six populations of Aceria tulipae (Keifer), range 0.0 -0.5% (Kiedrowicz et al., 2017), as well as within Phytoptus avellanae (Nal.) range 0.0 -0.4% (Cvrković et al., 2016). In Tegolophus celtis Guo, Li, Wong, Xue & Hong, 2015, divergence within the same gene, among protogyne and deutogyne females was 0% to 0.9% (Guo et al. 2015), while intraspecific nucleotide sequence divergence within each host strain of Abacarus hystrix ranged from 0.2% -13.9% (Skoracka & Dabert 2010). Results obtained in this study indicate that there are no significant intraspecific divergences among populations of A. alhagi n. sp. from the five different localities in western Asia.
Four different haplotypes were obtained for populations of A. alhagi n. sp. collected on A. maurorum from different geographic localities, and the sequences are available from GenBank under accession numbers MF150169 -MF150173.

Discussion
Alhagi maurorum is considered a weed of significance in many regions of the world where it has been introduced (AGIS-WIP 2006, Munakamwe 2016; USDA-NRCS 2002) and potential control options need to be considered. Biological control offers a safe and sustainable method, which can be used to control a weeds density and reduce spread (McFadyen 1998). The main mode of spread of camelthorn is through seed dispersal, where each self-fertile plant can produce up to 6000 seeds (Ambasht 1963). The eriophyoid, A. alhagi n. sp. has been observed causing serious damage on the reproductive output of A. maurorum in western Asia (five regions of the current study). The inflorescences of the infested plants are replaced by cauliflower-like galls and seed production is massively reduced. Although A. alhagi n. sp. is unlikely to reduce densities of the weed in the short term, a reduction in seed set of plants in the introduced range would limit the long distance dispersal of this weed through various abiotic (wind and water) (Ambasht 1963) and biotic (cattle, sheep, horses) (Kerr et al. 1965) factors.
The finding from this study allows us to conclude that only one species of Aceria is present on A. maurorum, with no doubt about its taxonomic status, A. alhagi n. sp.. This is an important finding for the development of a biological control programme since eriophyoid mites are usually highly host specific and are gaining popularity in classical weed biological control , Smith et al. 2010. Future classical biological control studies targeting the reduction in reproductive output of A. maurorum should consider A. alhagi n. sp. a potential biological control agent with a high impact on inflorescence production, ultimately reducing seed set.