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Article

A New Aculodes Species (Prostigmata: Eriophyidae) Described from an Invasive Weed by Morphological, Morphometric and DNA Barcode Analyses †

by
Biljana Vidović
1,
Nikola Anđelković
1,
Vida Jojić
2,
Tatjana Cvrković
3,
Radmila Petanović
1,4,
Francesca Marini
5,
Massimo Cristofaro
5 and
Brian G. Rector
6,*
1
Department of Entomology and Agricultural Zoology, Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade, Serbia
2
Department of Genetic Research, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia
3
Institute for Plant Protection and Environment, Banatska 33, 11080 Belgrade, Serbia
4
Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11000 Belgrade, Serbia
5
Biotechnology and Biological Control Agency (BBCA), Via Angelo Signorelli 105, 00123 Rome, Italy
6
USDA-ARS, Great Basin Rangelands Research Unit, 920 Valley Road, Reno, NV 89512, USA
*
Author to whom correspondence should be addressed.
†
Zoobank link: urn:lsid:zoobank.org:pub:AD9C113F-5B5F-4BD6-B20C-E7FEF7B38568.
Insects 2022, 13(10), 877; https://doi.org/10.3390/insects13100877
Submission received: 19 August 2022 / Revised: 9 September 2022 / Accepted: 22 September 2022 / Published: 27 September 2022
(This article belongs to the Section Insect Systematics, Phylogeny and Evolution)
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Abstract

:

Simple Summary

Natural enemies of cheatgrass, an invasive plant in the western USA that is associated with recent increases in destructive wildfires, were sought as part of a classical biological control program targeting that plant. A population of mites was discovered infesting cheatgrass plants in central Bulgaria and determined to be a new species using morphological, morphometric analysis (i.e., measurement of specific body parts) and DNA barcoding (i.e., comparison of DNA sequences in genes shared by all mite species). Without these modern analytical tools, this mite would probably have been considered as the same species as another mite that infests other plants. This new mite species will now be tested to determine if it is suitable for importation and release in the USA as a biological control agent of cheatgrass.

Abstract

A new species of eriophyoid mite, Aculodes marcelli sp. nov., was discovered on cheatgrass, Anisantha tectorum (L.) Nevski (syn. Bromus tectorum L.), an annual grass that is native to Eurasia and Northern Africa. This grass was introduced to North America near the end of the 19th century and now is widespread and associated with the observed increases in the size, frequency, and intensity of wildfires in western N. America. In this paper, A. marcelli sp. nov., is morphologically described and illustrated. Compared with other Aculodes spp., it differs based on morphology and the sequence of the mitochondrial cytochrome oxidase gene, subunit I (MT-CO1). Results of morphometric analysis showed clear differentiation between A. marcelli sp. nov., and the most similar congener, A. altamurgiensis from Taeniatherum caput-medusae. Analysis of MT-CO1 sequence divergence revealed significant levels of genetic variation (17.7%) and supported the results from the morphometric analysis; therefore, it is determined that they are two different species. Aculodes marcelli sp. nov., is a new candidate agent for classical biological control of A. tectorum.

1. Introduction

Eriophyoid mites are obligate herbivores characterized by minute size and simplified body structure [1]. Traditional descriptions of new eriophyoid species only on the basis on morphological traits often provide inadequate separation between taxa, especially in cases where species are morphologically very similar. Most contemporary reports concerning alpha-taxonomy are prepared following traditional methods, but there are more and more examples of new species descriptions of eriophyoids, or splitting from the putative complex of taxa, using methods of traditional taxonomy combined with the support of DNA sequences of one (usually COI mitochondrial gene) or a few gene fragments. The potential for high host specialization, host-race formation, and cryptic speciation is particularly great in eriophyoid mites because they are often intimately associated with their hosts, lack long-range host seeking ability, and have high reproductive rates [2,3]. A contemporary approach in describing species diversity, so called integrative taxonomy, has recently revealed the occurrence of cryptic species in several eriophyoid mite genera [4,5,6,7]. This integrative approach to the taxonomy of Eriophyoidea has gained traction with contemporary authors and in many recent studies traditional descriptions are integrated with morphometric analyses, molecular tools, and other characteristics [8,9,10,11].
Searching for biological control agents of cheatgrass, Anisantha tectorum (L.) Nevski (syn. Bromus tectorum L.), an eriophyid mite belonging to the genus Aculodes, was found that was morphologically similar to Aculodes altamurgiensis de Lillo & Vidović, a recently described species from medusahead (Taeniatherum caput-medusae (L.) Nevski) [12]. In preliminary morphological investigations, some morphological differences were observed between the cheatgrass mite and A. altamurgiensis and detailed analyses were required to discriminate these morphologically similar mite entities. Moreover, comparative analysis of discriminatory characteristics of Aculodes spp., described to date was necessary to ensure that the mite from cheatgrass was a new species.
To date, about 30 species of eriophyid mites belonging to the genus Aculodes have been described worldwide. Most of them (approx. 26) have been recorded from grasses (Poaceae), while the others were found on hosts within the families Malvaceae, Salicaceae, Fabaceae, and Rosaceae [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41]. Until now, only two species of the genus Aculodes (A. multitricavus and A. janboczeki) have been described from Bromus spp., both from B. inermis Leyss. (syn. of Bromopsis inermis (Leyss.) Holub) [30,31] and by preliminary research of Kiedrowicz et al. [42], one new species belonging to the genus Aculodes is registered on Bromus armenus Boiss. (syn. of Bromopsis armena (Boiss.) Holub).
Cheatgrass is a typically winter annual grass that reproduces only by seed [43]. It is native in temperate regions throughout much of Eurasia and Northern Africa. The introduction of cheatgrass to North America probably occurred independently several times. It was first reported in the far western United States near the end of the 19th century and is now widespread throughout most of the U.S., Canada, Greenland, and northern Mexico [44,45,46].
The aim of this study is the description of the new eriophyid mite species, Aculodes marcelli sp. nov., from Anisantha tectorum using traditional morphological methods, morphometric analysis, and molecular tools.

2. Materials and Methods

2.1. Collection and Morphological Measurements

Due to the morphological similarity of the above mentioned Aculodes mites registered on cheatgrass and medusahead, mites collected from Anisantha tectorum, specimens of A. altamurgiensis from medusahead were also collected as comparative material.
Plant samples of Anisantha tectorum were collected in Starosel, Bulgaria (42.485°; 24.5522°) and Surduk, Serbia (45.0925°; 20.2975°); Taeniatherum caput-medusae plants were collected in Starosel, Bulgaria (42.485°; 24.5522°) and Andria, Italy (41.1172°; 16.4183°).
Mite specimens from each plant sample were mounted in Keifer’s F medium [47] and then examined using a Leica DMLS research microscope with phase-contrast and the software package IM 1000 (Leica, Wetzlar, Germany).
Material for morphological study included specimens of Aculodes altamurgiensis from T. caput-medusae collected in Italy and A. marcelli sp. nov., from A. tectorum collected in Bulgaria and Serbia. For the morphometric analyses, 25–27 females from each sample were examined in the dorso-ventral position. Among the total of 23 morphological characters, two (number of dorsal and ventral rings) were meristic. All analyzed variables were distributed normally according to Kolmogorov–Smirnov tests. A one-way multivariate analysis of variance (MANOVA) was used to examine the differences in morphological variation among the three mite populations. Canonical variate analysis (CVA) was performed to visualize differences and to determine the relative importance of characters as discriminators among the studied mites. All statistical analyses were conducted using the Statistica 6 software package [48].
Material for species description included only the population of A. marcelli sp. nov., collected from A. tectorum in Bulgaria. For the species description of A. marcelli sp. nov., measurements are given in micrometers (µm) and, unless stated otherwise, refer to the length of the structure. Each measurement of the holotype precedes the corresponding range for paratypes (given in parentheses). The morphology and nomenclature follow [1] and genus classification is based on [49]. Measurements and illustrations were made according to Amrine & Manson [47] and de Lillo et al. [50].
Host plant names and their synonyms are in accordance with the on-line database: Euro+Med PlantBase [51]. The holotype and paratype slides as well as all material examined were deposited in the Acarology Collection, Department of Entomology and Agricultural Zoology, Faculty of Agriculture, University of Belgrade, Serbia.

2.2. Scanning Electronic Microscopy

Scanning electron micrographs (SEM) were made according to Alberti & Nuzzaci [52] using a scanning electron microscope (JEOL-JSM 6390, JEOL GmbH, Munich, Germany) at the Faculty of Agriculture, University of Belgrade. Live mites were collected individually with a fine entomological needle from fresh plant parts under a stereomicroscope (Leica M60 Leica, Wetzlar, Germany) and placed in the SEM sample holder. Prior to the observation, holder mounted mites were gold-coated (BAL-TEC SC-RD005, Balzers, Liechtenstein) by a Sputter coater (BAL-TEC AG, Balzers, Liechtenstein) for 100 s under a 30 mA ion current.

2.3. DNA Extraction, PCR Amplification and Sequencing

Material subjected to molecular analysis included populations inhabiting A. tectorum from Bulgaria and Serbia and the population associated with T. caput-medusae from Bulgaria. Mite specimens collected from fresh leaves were preserved in 96% ethanol and stored at 4 °C until DNA extraction. Total DNA was extracted from pools of one to three whole specimens using QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions, with modifications based on the work of Dabert and colleagues [53]. The barcode region of the mitochondrial cytochrome oxidase (MT-CO1) gene was amplified using LCO1490 and HCOd primers [54,55]. The polymerase chain reactions (PCR) contained High Yield Reaction Buffer A with MgCl2 (1x), additional 2.5 mM MgCl2, 0.6 mM of each dNTP, 0.6 μM of each primer, 1U of FastGene Taq DNA polymerase (NIPPON Genetics Europe) and 5 μL of diluted template DNA in a 25 μL final volume. PCR was carried out in a Mastercycler ep gradient S thermal cycler (Eppendorf, Hamburg, Germany) following the protocol and amplification conditions of [56]. PCR amplicons were visualized in a 1% agarose gel and purified using the QIAquick PCR purification Kit (QIAGEN) according to the manufacturer’s instructions. Sequencing was performed in both directions with the same primer pairs as in the initial PCR procedure by Macrogen Europe (Amsterdam, Netherlands).

2.4. Alignments, Molecular Analyses and Genetic Distances

Both strands of each barcode MT-CO1 amplicon were assembled into contigs using FinchTV v.1.4.0 (www.geospiza.com; accessed on 21 September 2022), translated into amino acids to check for the absence of stop codons and aligned by CLUSTAL W integrated in MEGA11 software [57]. Sequences of A. marcelli sp. nov., from Anisantha tectorum populations collected in Serbia and Bulgaria and A. altamurgiensis associated with T. caput-medusae collected from Bulgaria were trimmed and compared with corresponding sequences of A. altamurgiensis (GenBank accession numbers MH352403 and MH352404), A. mckenziei (Keifer) (FJ387561 and FJ387562), and A. holcusi Skoracka (MW439276). Uncorrected pairwise genetic distances were used to calculate the average genetic distance between the sequences.

3. Results

3.1. Morphometric Analysis

Means and standard deviations for the total of 23 morphological traits are given in Table 1. The one-way MANOVA for 23 morphological characters revealed statistically significant differences among the analyzed mite populations (Wilks’ Lambda = 0.0832, F46, 104 = 5.57, p < 0.0001). As illustrated by a CVA scatter plot of the first two canonical variates (CV) (Figure 1), the CV1 axis (accounting for 77.6% of the total morphological differences) separated mites from T. caput-medusae from those hosted by A. tectorum collected in Serbia, while a segregation of mites from A. tectorum collected in Bulgaria and those hosted by A. tectorum collected in Serbia is visible along the CV2 axis (accounting for 22.4% of the total variation). The total percentage of correctly classified mite specimens from each of the three plant samples was 94.8% (100% for mites hosted by A. tectorum collected in Serbia; 96.3% for mites hosted by T. caput-medusae; 88% for mites hosted by A. tectorum collected in Bulgaria).
The morphological characters that contributed most to the separation of the analyzed mite populations along the CV1 axis were the lengths of setae sc, d, and c2, distance between 1a tubercles, and the number of dorsal annuli (Table 2). In comparison to the mites from T. caput-medusae (negative section of the CV1 axis in Figure 1), those hosted by A. tectorum collected in Serbia (positive section of the CV1 axis in Figure 1) were characterized by higher mean values for these characters (Table 1). Morphological characters that contributed most to the segregation of the analyzed mite populations along the CV2 axis were the lengths of setae sc, c2, and 3a, distance between 2a tubercles, length of tibia II, and width of genitalia. In comparison to the mites from A. tectorum collected in Bulgaria (negative section of the CV2 axis in Figure 1), those hosted by A. tectorum collected in Serbia (positive section of the CV2 axis in Figure 1) showed higher mean values for these characters, except for the distance between 2a tubercles and the length of tibia II (Table 1).

3.2. Molecular Analyses

A mitochondrial CO1 fragment of 606 bp in length was generated from all Aculodes specimens. Nucleotide sequence data were deposited in the GenBank database under the accession numbers indicated in Table 3. The translation of the nucleotide sequences resulted in 202 amino acid positions. Sequence comparison showed that populations of A. marcelli sp. nov., collected on A. tectorum in Bulgaria and Serbia, are 100% identical.
Pairwise comparison of trimmed MT-CO1 fragments (531 bp) between A. marcelli sp. nov., and A. altamurgiensis averaged 17.6% genetic divergence. Similar p-distances were estimated between the new species and A. holcusi (17.2%), while average pairwise distance estimated between A. marcelli sp. nov., and A. mckenziei was 21.8% (Table 3).
Translation into amino acid sequences revealed 1.1% divergence between populations of A. marcelli sp. nov., with both A. altamurgiensis and A. holcusi, which is approximately one-quarter the amino acid divergence between the new species and A. mckenziei (4%) (Table 3).

3.3. Taxonomy

Aculodes marcelli Vidović sp. nov., (Figure 2, Figure 3 and Figure 4)
Description. FEMALE (n = 10). Body wormlike 277 (217–293), 60 (57–65) wide, whitish in color. Gnathosoma 19 (18–20) curved downwards, chelicerae 16 (14–16), setae ep 3, setae d 10 (8–10) unbranched. Prodorsal shield 34 (34–37) including the frontal lobe, 38 (33–38) wide; triangular with a pointed frontal lobe over the gnathosoma; median line present on rear half of the shield; admedian lines complete, from anterior lobe diverging to lateral margin of shield; I pair of submedian lines present on rear half, incomplete, parallel to admedian; II pair of submedian lines present, incomplete, arched. I and II submedian lines connecting in the 1/4 anterior part of the shield; conical microtubercles and dashes present between the lines on rear surface of the shield. Tubercles sc on rear shield margin 16 (16–20) apart, scapular setae sc 39 (38–54). Leg I 38 (37–42); femur 12 (9–12), setae bv 13 (13–15); genu 6 (5–7), setae l″ 22 (22–27); tibia 8 (8–9), setae l′ 9 (9–13); tarsus 8 (8–10), setae ft′ 17 (17– 21), setae ft″ 27 (26–28); tarsal solenidion ω 8 (8–10) with a thinner and rounded end; tarsal empodium 9 (8–10), 8-rayed. Leg II 34 (34–38); femur 12 (10–12), setae bv 18 (18–26); genu 6 (5–6), setae l″ 14 (13–16); tibia 7 (6–7); tarsus 9 (8–9), setae ft′ 9 (9–11), setae ft″ 26 (24–28); tarsal solenidion ω 8 (8–10) similar to that on leg I; tarsal empodium 9 (8–10), 8-rayed. Coxae with a pattern of wavy lines and dashes; sternal line 11 (11–13); setae 1b 9 (9–11), tubercles 1b 10 (9–11) apart; setae 1a 21 (19–28), tubercles 1a 7 (6–9) apart, setae 2a 43 (27–44), tubercles 2a 23 (21–25) apart. Genital coverflap 12 (11–15), 23 (20–23) wide, with 9 (9–12) longitudinal striae in a single row; setae 3a 19 (18–23), 17 (14–17) apart. Internal genitalia with anterior apodeme trapezoidal, longitudinal bridge relatively long, the post-spermathecal part of the longitudinal bridge is reduced; spermathecal tubes directed latero-posterad, composed of two parts: basal part egg-shaped, distal more tubulose; spermathecae globose. Opisthosoma with subequal annuli: 63 (63–69) dorsal and 66 (61–68) ventral annuli; 5 (5–6) coxigenital annuli. Dorsal and ventral opisthosoma with pointed microtubercles close to the rear margins of annuli. Setae c2 29 (29–40), 51 (46–56) apart, on annulus 9 (8–10); setae d 38 (34–49), 35 (30–38) apart, on annulus 21 (21–23); setae e 34 (31–35), 16 (13–18) apart, on annulus 37 (35–41); setae f 24 (24–27), 18 (16–19) apart, on annulus 60 (57–62); seta h2 66 (55–79), 9 (7–9) apart; setae h1 7 (6–9), 5 (4–5) apart.
MALE (n = 2). Body wormlike, 197–200, 53–54 wide, whitish in color. Gnathosoma 17 curved down, cheliceral stylets 14–15. Prodorsal shield 33, 33–34 wide. Prodorsal shield tubercles on the rear shield margin 18–19 apart, setae sc 34–39, projecting posteriorly. Shield design similar to female. Leg I 30–33; femur 9, setae bv 9–10; genu 5, setae l″ 21–24, tibia 6–7, tibial setae l′ 10; tarsus 8, setae ft′ 16, setae ft″ 21–23; solenidion ω 8, empodium em 6, and 8-rayed. Leg II 28–29; femur 8–9, setae bv 17–18; genu 4–5, genual setae l″ 13–15, tibia 5; tarsus 7–8; setae ft′ 8–11, setae ft″ 21–22; solenidion ω 9–10, empodium em 6–7. Coxae granulated; sternal line 10–11; setae 1b 10–12, 1b tubercles 9–10 apart; setae 1a 22–27, 1a tubercles 5 apart; setae 2a 29–34, 2a tubercles 18 apart. Genitalia 19–20 wide; setae 3a 20, 3a tubercles 14–15 apart. Opisthosoma with subequal annuli: 61–66 dorsal and 61–63 ventral annuli; 4–5 coxigenital annuli. Setae c2 35–37, 41–44 apart, on annulus 8–10; setae d 42–44, 31–33 apart, on annulus 19–21; setae e 26–30, 15–16 apart, on annulus 36; setae f 16–24, 18 apart, on annulus 57–59; setae h2 50, 7–8 apart, setae h1 5, 4 apart.
Type host plant.Anisantha tectorum (L.) Nevski (Poaceae), cheatgrass.
Type locality. Starosel, Bulgaria 42.485°; 24.5522°, 396 m elev.
Type material. Female holotype (slides # 862/1) and paratypes 42 females, 4 males; 3 May 2018, collected by Brian Rector.
Additional studied material. Starosel, Bulgaria (42.485°; 24.5522°), 8 May 2017, 12 slides; 20 May 2018, 10 slides collected by F. Di Cristina, F. Marini, and M. Cristofaro. Surduk, Serbia (45.0925°; 20.2975°), 15 May 2018, 46 slides, collected by D. Smiljanić and B. Vidović.
Relation to the host. The mites are vagrants on the leaf surfaces and in spikes, sometimes in large colonies. No visible damage was observed although effects on the host’s life cycle have yet to be tested.
Etymology. The species name, marcelli, is derived from the name of Marcello Barlattani, a member of our technical support crew who died during the course of this work, in honor and memory of him.
Differential diagnosis and remarks. All Aculodes spp., from Poaceae can split out in the three groups based on the presence and length of the median line on the prodorsal shield and based on the number of empodial rays (Table 4). Aculodes marcelli sp. nov., has an incomplete median line in the posterior half of the shield, which is different from the species that lack a median line (A. capillarisi Skoracka, A. dubius (Nalepa), A. janboczeki Skoracka, A. koeleriae Sukhareva, A. kransnovi Sukhareva, A. stoloniferae Skoracka, A. sylvatici Skoracka, Labrzycka & Rector) and species with a complete median line (A. bambusae Kuang, A. levis Huang, A. ponticus Sukhareva).
Aculodes marcellisp. nov., has an incomplete median line in the posterior half of the shield, which is different from the species that lack a median line (A. capillarisi Skoracka, A. dubius (Nalepa), A. janboczeki Skoracka, A. koeleriae Sukhareva, A. kransnovi Sukhareva, A. stoloniferae Skoracka, A. sylvatici Skoracka, Labrzycka & Rector) and species with a complete median line (A. bambusae Kuang, A. levis Huang, A. ponticus Sukhareva).
Aculodes marcellisp. nov., can be distinguished from other Aculodes spp., that also have an incomplete median line on the basis of the number of empoidal rays (Aculodes marcelli sp. nov., 8-rayed; A. multitricavus Skoracka, A. mckenziei (Keifer), and A. stipacolus Alemandri & Navia 9-rayed; A. altamurgiensis de Lillo & Vidović, A. festucae Skoracka, Labrzycka & Rector, A. fuller (Keifer), A. tsukushiensis Xue, Song & Hong, A. caespiticolus Ripka & Szabo, A. tinaniensis Elhalawany & Amrine, and A. skorackae Elhalawany & Amrine 7-rayed).
Among the Aculodes spp., with incomplete median lines and eight empodial rays, Aculodes marcelli sp. nov., may differ on the basis on presence and position of their submedian lines, the presence of microtubercles on the prodorsal shield and the shape of the microtubercles on the dorsal and ventral side of the opisthosoma.
Differential characteristics of Aculodes marcelli sp. nov., in comparison with the most similar species are the following: dashes are present between the lines of prodorsal shield, two submedian lines which touch each other at their anterior part are present, of which the second is longer. A. agropyronis differs by the absence of dashes between the lines, A. calamaabditus and A. deschampisae differ by the presence of only I submedian line, while A. mongolicus has a longer I submedian line, and in A. neglectivagrans submedian lines I and II do not touch each other. The dorsal and ventral opisthosoma in Aculodes marcelli sp. nov., have pointed microtubercles, unlike the A. holcusi opisthosoma, which has subrounded dorsal and slightly pointed ventral microtubercles.
With regard to the prodorsal shield design Aculodes marcelli sp. nov., is most similar to A. altamurgiensis, in that they have the same design of prodorsal shield. The only obvious characteristics that distinguish these two species are the number of empodial rays (A. marcelli sp. nov., 8-rayed; A. altamurgiensis, 7-rayed) and the number of dorsal (A. marcelli sp. nov., 63–69; A. altamurgiensis, 51–58) and ventral annuli (A. marcelli sp. nov., 61–68; A. altamurgiensis, 55–66).

4. Discussion

Variability of phenotypic traits among populations of mites inhabiting different host plants may occur as different mite species (i.e., total separation of gene pools); host races (i.e., partial differentiation of gene pools); or intraspecific phenotypic plasticity (i.e., no separation of the gene pool) [58]. As a result of these multiple possible causes of phenotypic variability, taxonomic assessment based on traditional, strictly morphological methods may fail to adequately separate species, especially in the case of cryptic species. Morphometric analysis may indicate that there is variability but without molecular analysis, it is impossible to declare two morphologically different eriophyoid populations collected from different hosts to be different species, short of elaborate and time-consuming cross-breeding and host-preference bioassays [4,6,8,59,60,61]. Molecular tools and methods, including sequencing and publishing barcode genes in publicly available databases, are useful in discrimination of cryptic species and should be included with alpha taxonomy whenever it is possible [4,5,7,9,62,63,64].
Analysis of MT-CO1 sequence divergence in the present study supported the morphometric analyses, revealing significant levels of genetic variation and host-associated genetic structure between Aculodes spp., mites collected on A. tectorum and on T. caput-medusae. Although A. marcelli sp. nov., is morphologically very similar to A. altamurgiensis, interspecific nucleotide sequence divergence of MT-CO1 was 17.7%, which corresponds to p-distance values between MT-CO1 sequences available in databases of other morphologically different congeneric mite species that are also associated with closely related host plants (e.g., A. holcusi and A. mckenziei). Similarly, clear differentiation was shown in other eriophyoid genera inhabiting different grass hosts such as Aceria tosichella s.l., 13.9% [8] and Abacarus hystrix, 22.6% [65].
Because of the great morphological similarity between already described Aculodes altamurgiensis and recently discovered Aculodes marcelli sp. nov., there was an early suspicion that they were the same species, but from different host plants (BV, pers. comm.). However, the integrative approach for description of the new species of eriophyoid mites presented herein, i.e., results of both phenotypic and genotypic variability did not confirm this hypothesis. According to the results of our morphometric analysis, A. altamurgiensis is clearly separated from two populations of A. marcelli sp. nov., and the analysis of MT-CO1 sequence divergence revealed significant levels of genetic variation (17.7%), indicating that they are indeed two different species. However, while some morphometric differences were observed between two populations of A. marcelli sp. nov., collected from Bulgaria and Serbia, no genetic variability was identified between them, which leads us to the conclusion that it is a single species exhibiting phenotypic plasticity in two reproductively isolated populations.
Like T. caput-medusae, A. tectorum is an important weed in western N. America [66]. The dried, senesced plants of both species persist in arid environments and provide highly flammable tinder to ignite and carry fire to larger fuel sources; this contributes significantly to the recent increases in the frequency, size, and intensity of wildfires in that region [67,68]. The United States Department of Agriculture, Agricultural Research Service leads an international classical biological control program targeting both A. tectorum and T. caput-medusae. In order to determine its suitability as a classical biological control agent of A. tectorum, Aculodes marcelli sp. nov., will undergo similar biological testing as is being performed to evaluate A. altamurgiensis as a candidate agent for control of T. caput-medusae [69]. The fact that A. marcelli sp. nov., was never described prior to the discovery reported herein provides a strong indication that it does not cause damage to cereal species or other economically important plants.

Author Contributions

Conceptualization, B.V., M.C. and B.G.R.; methodology, B.V., V.J., T.C., R.P. and B.G.R.; software, N.A. and V.J.; validation, B.V., T.C., R.P. and B.G.R.; formal analysis, B.V., N.A., V.J. and T.C.; investigation, B.V., F.M., M.C. and B.G.R.; resources, B.V., M.C. and B.G.R.; data curation, B.V.; writing—original draft preparation, B.V., V.J. and T.C.; writing—review and editing, B.V., T.C., V.J., R.P., F.M. and B.G.R.; visualization, B.V. and N.A.; supervision, B.V., M.C. and B.G.R.; project administration, B.V. and B.G.R.; funding acquisition, B.V. and B.G.R. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by: Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant No. 451-03-68/2022-14/200116, No. 451-03-68/2022-14/200007, No. 451-03-68/2022-14/200010; Serbian Academy of Sciences and Arts, Grant No. F-195; United States Department of the Interior, Interagency Agreement No. L16PG00228.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data produced in this study are reported herein.

Acknowledgments

The authors are grateful to Franca Di Cristina and Marcello Barlattani, BBCA ONLUS, Italy, for mite collections; Dragica Smiljanić Faculty of Agriculture, Serbia, for collecting mites and creating the technical illustrations. Special thanks to Dean Vili Harizanova and Atanaska Stoeva of Agrarian University, Plovdiv, Bulgaria for directing the searches for A. tectorum populations in that country.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

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Figure 1. Canonical variate analysis (CVA) of 23 morphological traits. “T. caput-medusae” = Aculodes altamurgiensis from Taeniatherum caput-medusae collected in Italy, “A. tectorum SR” = Aculodes marcelli sp. nov., from Anisantha tectorum collected in Serbia, “A. tectorum BG” = Aculodes marcelli sp. nov., from A. tectorum collected in Bulgaria.
Figure 1. Canonical variate analysis (CVA) of 23 morphological traits. “T. caput-medusae” = Aculodes altamurgiensis from Taeniatherum caput-medusae collected in Italy, “A. tectorum SR” = Aculodes marcelli sp. nov., from Anisantha tectorum collected in Serbia, “A. tectorum BG” = Aculodes marcelli sp. nov., from A. tectorum collected in Bulgaria.
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Figure 2. Schematic drawings of female Aculodes marcelli sp. nov.: D=dorsal mite; V=ventral mite.
Figure 2. Schematic drawings of female Aculodes marcelli sp. nov.: D=dorsal mite; V=ventral mite.
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Figure 3. Schematic drawings of female Aculodes marcelli sp. nov.: AD=Prodorsal shield; AL=Lateral view of anterior body region; CG=Female coxigenital region; em=Empodium; GM=Male genital region; IG=Internal female genitalia; LO=Lateral view of annuli; L1=Leg I; PM=Lateral view of posterior opisthosoma.
Figure 3. Schematic drawings of female Aculodes marcelli sp. nov.: AD=Prodorsal shield; AL=Lateral view of anterior body region; CG=Female coxigenital region; em=Empodium; GM=Male genital region; IG=Internal female genitalia; LO=Lateral view of annuli; L1=Leg I; PM=Lateral view of posterior opisthosoma.
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Figure 4. Scanning electron micrographs of Aculodes marcelli sp. nov.: (A) Dorsal view; (B) Dorsal view with detail of prodorsal shield; (C) Ventral view; (D) Ventral view, anterior half; (E) Dorsal view with apparent wax-like secretion; (F) Detail of tarsus.
Figure 4. Scanning electron micrographs of Aculodes marcelli sp. nov.: (A) Dorsal view; (B) Dorsal view with detail of prodorsal shield; (C) Ventral view; (D) Ventral view, anterior half; (E) Dorsal view with apparent wax-like secretion; (F) Detail of tarsus.
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Table
Table 1. Means and standard deviations (SD) for 23 morphological traits of Aculodes altamurgiensis from Taeniatherum caput-medusae collected in Italy and Aculodes marcelli sp. nov., from Anisantha tectorum collected in Bulgaria and Serbia (n = number of specimens).
Table 1. Means and standard deviations (SD) for 23 morphological traits of Aculodes altamurgiensis from Taeniatherum caput-medusae collected in Italy and Aculodes marcelli sp. nov., from Anisantha tectorum collected in Bulgaria and Serbia (n = number of specimens).
TraitA. altamurgiensis Italy (n = 27)A. marcelli sp. nov.
(n = 25) Bulgaria		A. marcelli sp. nov.
(n = 25) Serbia
MeanSDMeanSDMeanSD
A: body length235.4124.04257.6726.70259.4618.50
B: prodorsal shield length35.681.5936.671.4837.491.58
C: prodorsal shield width35.191.9636.763.0737.622.51
D: setae sc length46.273.3844.205.4448.213.87
E: tubercles sc apart18.381.0318.831.3719.251.14
F: number of dorsal annuli61.812.7766.962.9468.963.69
G: number of ventral annuli62.003.1565.683.4669.283.99
H: setae c2 length32.392.9234.673.9636.233.86
I: setae d length37.883.5742.984.6847.964.47
J: setae e length30.134.2333.614.8837.425.07
K: setae f length25.012.2426.271.8028.522.56
L: genitalia length11.191.0511.530.9911.801.49
M: genitalia width20.220.7520.291.1820.930.83
N: setae 3a length19.772.0622.013.2922.032.81
O: tubercles 3a apart14.790.7915.251.2315.120.61
P: tubercles 1b apart8.340.779.671.029.660.95
Q: tubercles 1a apart5.710.646.250.966.040.68
R: tubercles 2a apart19.371.6021.641.7421.161.10
S: setae 2a length35.658.2440.575.2040.556.02
T: tibia I length7.990.578.120.718.030.93
U: tarsus I length8.060.628.390.528.910.61
V: tibia II length6.560.566.810.556.690.83
W: tarsus II length7.810.578.110.698.430.75
Table
Table 2. Standardized coefficients for canonical variables on the first two (CV1 and CV2) canonical axes (Cum. Prop. = cumulative proportion of described differences between mite populations. Morphological characters that contribute most to the separation of mite populations are marked in bold).
Table 2. Standardized coefficients for canonical variables on the first two (CV1 and CV2) canonical axes (Cum. Prop. = cumulative proportion of described differences between mite populations. Morphological characters that contribute most to the separation of mite populations are marked in bold).
TraitCV1CV2
A: body length0.066−0.280
B: prodorsal shield length−0.122−0.047
C: prodorsal shield width0.3160.127
D: seta sc length−0.7380.927
E: tubercles sc apart0.238−0.325
F: no. of dorsal annuli0.524−0.253
G: no. of ventral annuli−0.0000.287
H: setae c2 length0.474−0.624
I: setae d length0.5560.427
J: setae e length0.1210.105
K: setae f length0.1740.384
L: genitalia length0.0130.245
M: genitalia width0.0490.583
N: setae 3a length0.076−0.514
O: tubercles 3a apart−0.092−0.028
P: tubercles 1b apart0.350−0.240
Q: tubercles 1a apart−0.5500.470
R: tubercles 2a apart0.147−0.629
S: setae 2a length−0.129−0.429
T: tibia I length−0.0580.059
U: tarsus I length0.4070.401
V: tibia II length0.084−0.505
W: tarsus II length0.3030.098
Eigenvalue4.3361.251
Cum. Prop.0.7761.000
Table
Table 3. Pairwise distance matrix with uncorrected p-distances between Aculodes marcelli sp. nov., and three congeners (A. altamurgiensis, A. mckenziei, and A. holcusi) based on CO1 nucleotide (below the diagonal) and amino acid sequences (above the diagonal). Numbers in column headers refer to species and populations listed in their respective rows. Genbank accession numbers provided in parentheses.
Table 3. Pairwise distance matrix with uncorrected p-distances between Aculodes marcelli sp. nov., and three congeners (A. altamurgiensis, A. mckenziei, and A. holcusi) based on CO1 nucleotide (below the diagonal) and amino acid sequences (above the diagonal). Numbers in column headers refer to species and populations listed in their respective rows. Genbank accession numbers provided in parentheses.
12345678
1Aculodes marcellisp. nov.
Bulgaria (OP028122)		 0.0000.0110.0110.0110.0400.0400.011
2Aculodes marcellisp. nov.
Serbia (OP028123)		0.000 0.0110.0110.0110.0400.0400.011
3Aculodes altamurgiensis
Bulgaria (OP028124)		0.1770.177 0.0000.0000.0450.0450.000
4Aculodes altamurgiensis
(MH352403)		0.1770.1770.032 0.0000.0450.0450.000
5Aculodes altamurgiensis
(MH352404)		0.1750.1750.0210.011 0.0450.0450.000
6Aculodes mckenziei
(FJ387561)		0.2170.2170.2110.2150.213 0.0000.045
7Aculodes mckenziei
(FJ387562)		0.2190.2190.2000.2020.2020.045 0.045
8Aculodes holcusi
(MW439276)		0.1720.1720.1580.1620.1580.2060.208
Table
Table 4. Design of prodorsal shield and number of empodial rays of species in the genus Aculodes from grasses.
Table 4. Design of prodorsal shield and number of empodial rays of species in the genus Aculodes from grasses.
Without a Median LineComplete Median Line
A. capillarisiA. bambusae
A. dubiusA. levis
A. janboczekiA. ponticus
A. koeleriae
A. krasnovi
A. stoliniferae
A. sylvatici
Incomplete median line
7 rayed empodium7–8 rayed empodium8 rayed empodium9 rayed empodium
A. festucaeA. calamaabditusA. agropyronisA. mckenziei
A. fullerA. deschampisaeA. holcusiA. multitricavus
A. tsukushiensisA. mongolicusAculodes marcellisp. nov.A. stipacolus
A. altamurgiensisA. neglectivagrans
A.caespiticolus
A. tinaniensis
A. skorackae
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MDPI and ACS Style

Vidović, B.; Anđelković, N.; Jojić, V.; Cvrković, T.; Petanović, R.; Marini, F.; Cristofaro, M.; Rector, B.G. A New Aculodes Species (Prostigmata: Eriophyidae) Described from an Invasive Weed by Morphological, Morphometric and DNA Barcode Analyses. Insects 2022, 13, 877. https://doi.org/10.3390/insects13100877

AMA Style

Vidović B, Anđelković N, Jojić V, Cvrković T, Petanović R, Marini F, Cristofaro M, Rector BG. A New Aculodes Species (Prostigmata: Eriophyidae) Described from an Invasive Weed by Morphological, Morphometric and DNA Barcode Analyses. Insects. 2022; 13(10):877. https://doi.org/10.3390/insects13100877

Chicago/Turabian Style

Vidović, Biljana, Nikola Anđelković, Vida Jojić, Tatjana Cvrković, Radmila Petanović, Francesca Marini, Massimo Cristofaro, and Brian G. Rector. 2022. "A New Aculodes Species (Prostigmata: Eriophyidae) Described from an Invasive Weed by Morphological, Morphometric and DNA Barcode Analyses" Insects 13, no. 10: 877. https://doi.org/10.3390/insects13100877

APA Style

Vidović, B., Anđelković, N., Jojić, V., Cvrković, T., Petanović, R., Marini, F., Cristofaro, M., & Rector, B. G. (2022). A New Aculodes Species (Prostigmata: Eriophyidae) Described from an Invasive Weed by Morphological, Morphometric and DNA Barcode Analyses. Insects, 13(10), 877. https://doi.org/10.3390/insects13100877

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