Phylogeny of Calvittacus Revealing a New Species from China (Acari: Eriophyidae)

Simple Summary Eriophyoid mites (Eriophyoidea) are strictly phytophagous, consisting of over 5000 extant species, in which many species are pests. The genus Calvittacus includes only four species and is endemic in the Oriental Region. In this study, we combined morphological characters and molecular approaches to delimitate the Calvittacus species and recovered one new species, Calvittacus spectabilus sp. nov. The new species is vagrant on lower leaf surface, causing no apparent symptom to the host plant. Abstract Eriophyoid mites (Eriophyoidea) are distributed worldwide and are the largest superfamily in the Acari. After over one and a half centuries of field surveys, regional fauna of eriophyoid mites remains unclear. The genus Calvittacus Xue, Song & Hong 2006 is endemic in the Oriental Region, including four species—C. chenius Xue, Wang, Song & Hong, 2009; C. mollissimus Han, Xue & Hong, 2017; C. regiae Xue, Song & Hong 2006; and C. swidanus Song, Xue & Hong, 2009. In this study, we describe one new species, Calvittacus spectabilus sp. nov., collected on Bougainvillea spectabilis (Nyctaginaceae) from China (the Oriental Region). Phylogenetic analysis based on mitochondrial COI barcode sequences confirmed the C. spectabilus sp. nov., coinciding with the morphological delimitation. We further discussed the potential distribution of the Calvittacus species and underlined the integrative approaches in eriophyoid mite delimitation.


Introduction
Eriophyoid mites (Eriophyoidea) comprise over 5000 described species [1], leading to the largest superfamily in the Acari. They are strictly phytophagous and can cause massive economic losses, e.g., Aceria tosichella Keifer and A. guerreronis Keifer [2]. Although eriophyoid mites have a worldwide distribution [3], they are supposed to be distributed mainly in temperate regions [4]. After over one and a half centuries of field surveys of eriophyoid mites, new species continue to be described in recent years. For instance, given more than 1200 eriophyoid mites described in China, an average of 12 new species per year were added in the last three years [5]. Furthermore, the Chinese fauna of eriophyoid mites was suggested, including over 2300 species [6]. It is likely that 1000 species could be recovered in China in future studies.
The genus Calvittacus was established by Xue et al. [7], based on the type species C. regiae Xue, . It is characterized by broad dorsal annuli, forming thickened bands and a furrow; scapular setae ahead of the rear shield margin, in a centrad direction; all coxal setae present; and legs and opisthosoma with the usual setae [7]. To date, only four species have been reported in this genus; all of them infest angiosperms from China.
To understand the diversity of eriophyoid mites in China, especially the Calvittacus species, Xiao-Feng Xue and colleagues conducted a long-term field survey since 2002.
In this study, we describe and illustrate Calvittacus spectabilus sp. nov. on Bougainvillea spectabilis (Nyctaginaceae), which was collected from south China. We further discuss the potential distribution of the Calvittacus species and underline the integrative approaches in describing new eriophyoid mite species.

Taxa Sampling and Morphological Identification
Samples were collected from Bougainvillea spectabilis (Nyctaginaceae) in the field (Figure 1), using a hand lens (30×), in China. Mite samples were stored in 95-96% ethanol at −20 • C prior to DNA extraction. Mite specimens were also slide-mounted using Keifer's Booster and a modified Berlese medium [8], but without adding additional fibers as was suggested by de Lillo et al. [9]. The morphological terminology used herein follows Lindquist [10] and Amrine et al. [11], internal female genitalia nomenclature follows Chetverikov [12], and the generic classification is made according to Amrine et al. [11], in combination with descriptions of all the published genera after 2003. Specimens were measured following de Lillo et al. [9]. They were examined with the aid of a Zeiss A2 (Germany) research microscope with phase contrast, and semi-schematic drawings were made. Microphotographs were taken with a Zeiss A2 (microphoto camera AxioCam MRc) research microscope with phase contrast or differential interference, using 10× eyepieces at 100× oil magnification, connected to a computer using Axiovision image analysis software. For each species, the holotype female measurements precede the corresponding range for paratypes (given in parentheses). For males, only ranges are given. If no variation was observed among measurements, it will be indicated with an "*". All measurements are in micrometres (µm) and represent lengths, when not otherwise specified. The holotype and seven paratypes are deposited in the Arthropod/Mite Collection of the Department of Entomology, Nanjing Agricultural University (NJAU), Jiangsu Province, China [13]. this study, we describe and illustrate Calvittacus spectabilus sp. nov. on Bougainvillea spectabilis (Nyctaginaceae), which was collected from south China. We further discuss the potential distribution of the Calvittacus species and underline the integrative approaches in describing new eriophyoid mite species.

Taxa Sampling and Morphological Identification
Samples were collected from Bougainvillea spectabilis (Nyctaginaceae) in the field (Figure 1), using a hand lens (30×), in China. Mite samples were stored in 95-96% ethanol at −20 °C prior to DNA extraction. Mite specimens were also slide-mounted using Keifer's Booster and a modified Berlese medium [8], but without adding additional fibers as was suggested by de Lillo et al. [9]. The morphological terminology used herein follows Lindquist [10] and Amrine et al. [11], internal female genitalia nomenclature follows Chetverikov [12], and the generic classification is made according to Amrine et al. [11], in combination with descriptions of all the published genera after 2003. Specimens were measured following de Lillo et al. [9]. They were examined with the aid of a Zeiss A2 (Germany) research microscope with phase contrast, and semi-schematic drawings were made. Microphotographs were taken with a Zeiss A2 (microphoto camera AxioCam MRc) research microscope with phase contrast or differential interference, using 10× eyepieces at 100× oil magnification, connected to a computer using Axiovision image analysis software. For each species, the holotype female measurements precede the corresponding range for paratypes (given in parentheses). For males, only ranges are given. If no variation was observed among measurements, it will be indicated with an "*". All measurements are in micrometres (μm) and represent lengths, when not otherwise specified. The holotype and seven paratypes are deposited in the Arthropod/Mite Collection of the Department of Entomology, Nanjing Agricultural University (NJAU), Jiangsu Province, China [13].

DNA Extraction and Sequencing
In total, 14 individuals collected from three locations (Baxianshan, Fujian; Wanlu, Hainan; Haikou, Hainan) were selected for molecular analysis. Genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen) following a modified protocol [14]. PCR was used to amplify the 658 bp barcode region of the mitochondrial COI gene using the primer pairs bcdF01/bcdR04 [14]. PCR reaction, purification, and sequencing followed Yin et al. [6].

DNA Extraction and Sequencing
In total, 14 individuals collected from three locations (Baxianshan, Fujian; Wanlu, Hainan; Haikou, Hainan) were selected for molecular analysis. Genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen) following a modified protocol [14]. PCR was used to amplify the 658 bp barcode region of the mitochondrial COI gene using the primer pairs bcdF01/bcdR04 [14]. PCR reaction, purification, and sequencing followed Yin et al. [6].

Species Delimitation
Fourteen COI sequences were aligned and trimmed in Geneious 8.1.9 [15]. All sequences were blasted in GenBank and checked for possible contaminants. All the sequences were deposited in GenBank under accession numbers: MZ482549-MZ482557, OM892490-OM892494. In addition, collection data, taxonomic information, sequences, and trace files were submitted to BOLD [16] under number: AEC8688. We constructed a Neighbor-Joining tree that employed the K2P distance metric and used the Taxon ID tree tool on BOLD. Genetic distances were calculated with MEGA 6.0 [17] employing the Kimura 2-parameter (K2P) distance parameter [18].
Etymology. The specific designation spectabilus is derived from the species name of the host plant, spectabilis, changing postfix -is to -us; masculine in gender.
Differential diagnosis. The new species is morphologically similar to four Calvittacus species (Table 1), but can be differentiated by median, admedian and submedian lines present on the prodorsal shield (simple prodorsal shield design with few lines in the other four Calvittacus species), 18 dorsal annuli (11 to 13 annuli in C. chenius, C. mollissimus, and C. regiae; 23 annuli in C. swidanus), and empodium 3-rayed (empodium 5-rayed in the other four species).

Genetic Distance and Phylogenetic Analysis
Fourteen COI sequences of C. spectabilus sp. nov. from three populations were nearly identical in composition, with an intraspecific distance (K2P) ranging from 0.000 to 0.001. We tried to sequence the COI sequences of C. chenius and C. swidanus, but failed, possibly due to DNA degradation of samples, which were collected for over 10 years and were kept in 75% ethanol at room temperature. We therefore included three Calvittacus species in the phylogenetic analyses.
The ML and BI analyses showed that C. spectabilus sp. nov. was nested within the Eriophyidae (Figures 5 and 6), which is consistent with the morphological assignment.

Discussion
In this study, we described and illustrated one new eriophyoid mite species belonging to the genus Calvittacus, based on the distinct morphological characters of dorsal annuli with thickened bands, forming a furrow [7]. Eriophyoid mites are characterized by ringed opisthosoma, in which dorsal annuli vary in number (ranged from 10 to 80) and shape (forming ridges, furrows, or evenly rounded) [11]. However, dorsal annuli formed by thickened bands were occasionally found in genus Achaetocoptes [29], Calvittacus [7], Johnella [30], Neovittacus [26], and Vittacus [31]; all these genera hold few species (less than ca. 10). Furthermore, this morphological character has not been phylogenetically tested at the generic level to determine whether it is a synapomorphy. Although four species have been described in the Calvittacus, all generic assignments were based on morphological characters. Our phylogenetic results showed a non-monophyly of Calvittacus; however, this result was based on a fragment of mitochondrial COI gene sequences. Multiple genes from mitochondria and nuclear, especially genome sequences, should be determined in future analyses to account for the position of C. spectabilus sp. nov. and to unveil the monophyly of Calvittacus.
The genus Calvittacus was established by Xue et al. [7] based on the type species C. regiae Xue, Song & Hong 2006, which was collected from the Oriental Region (Figure 7). After that, three more species, collected from the Oriental Region, were assigned to this genus ( Figure 7). Herein, the fifth species, C. spectabilus sp. nov., were also collected from the Oriental Region. Under these contexts, it is likely that the genus Calvittacus was endemic in the Oriental Region. However, more field surveys are warranted in future studies to understand the origin and biogeographical distribution of the Calvittacus species.  COI barcodes were recently explored in eriophyoid mite delimitation, showing a clear gap between interspecific divergence and intraspecific divergence, and thus enabling a discrimination of 99% of the eriophyoid mite species [6]. We provided 14 COI barcode sequences of C. spectabilus sp. nov., which were collected from three locations. Sequence analysis showed no intraspecific divergence. After comparing with the eriophyoid mite sequences in the Barcode of Life Data Systems database (DS-ERIYYDNA Barcodes for Eriophyoidea, available at doi: dx.doi.org/10.5883/DS-ERIYY.), all 14 sequences were assigned into one Barcode Index Number (BOLD: AEC8688), which reflects a single species; this molecular delimitation is in line with our morphological studies, confirming a new eriophyoid mite species of C. spectabilus sp. nov.

Data Availability Statement:
All data is available in this paper. All sequences were deposited in the GenBank under accession numbers of MZ482549-MZ482557, OM892490-OM892494.