Vol.:(0123456789)

Experimental and Applied Acarology (2019) 78:373–401https://doi.org/10.1007/s10493-019-00388-y

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Integrative taxonomy of Abacarus mites (Eriophyidae) associated with hybrid sugarcane plants, including description of a new species

Mércia Elias Duarte1 · Renata Santos de Mendonça2 · Anna Skoracka3 · Edmilson Santos Silva4,5 · Denise Navia1

Received: 13 March 2019 / Accepted: 30 May 2019 / Published online: 5 July 2019 © Springer Nature Switzerland AG 2019

AbstractPhytophagous mites belonging to the Eriophyoidea are extremely diverse and highly host-specific. Their accurate morphological identification is hampered by their reduced size and simplified bodies and by the existence of cryptic species complexes. Previous studies have demonstrated the urgency of applying multisource methods to accurate taxonomic iden-tification of eriophyoid mites, especially species belonging to the genus Abacarus. This genus comprises 65 species, of which 37 are associated with grasses and four with sugar-cane Saccharum (Poaceae). Recently, Abacarus specimens very similar to Abacarus sac-chari were collected from the sugarcane crop in Brazil; however, their taxonomic place-ment was uncertain. In this study, we used an integrative approach to determine whether A. aff. sacchari specimens belong to A. sacchari or constitute a cryptic species. Morpho-logical data were combined with molecular phylogeny based on the nucleotide sequences of three markers, one mitochondrial (COI) and two nuclear (D2 region of 28S and ITS). Morphological differences were observed between A. aff. sacchari, A. sacchari and A. doctus. The phylogenetic relationships among these three taxa and the genetic distances separating them revealed an interspecific divergence. The results of the morphological and molecular methods were congruent and supported the existence of a new species: Abac-arus neosacchari n. sp. Duarte and Navia, herein described. This species belongs to the Abacarus cryptic species complex associated with sugarcane in the Americas. The results of this study, presenting the occurrence of multiple Abacarus species associated with sug-arcane, contribute to the knowledge on plants and mites diversity by adding up one more clue highlighting that plant hybridization can be an important mechanism contributing to the speciation of plant-feeding arthropods.

Keywords Phytophagous mite · Eriophyoidea · Molecular phylogeny · Multivariate morphometry · Saccharum

Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1049 3-019-00388 -y) contains supplementary material, which is available to authorized users.

* Denise Navia denise.navia@embrapa.br

Extended author information available on the last page of the article

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Introduction

Systematics has largely benefitted from the ongoing development of molecular tools, in addition to clarifying the taxonomic classification of organisms and uncovering cryptic diversity (e.g., Kornilios et al. 2018; Tamar et al. 2018), the use of an integrative approach combining morphology and molecular phylogenetics helps reveal evolutionary relation-ships among taxa (Samadi and Barberousse 2009; Skoracka et al. 2015; Radenkovic et al. 2018), including mites belonging to the Eriophyoidea superfamily (Skoracka and Dabert 2010; Skoracka et al. 2012; Lewandowski et al. 2014; Chetverikov et al. 2015; Cvrkovic et al. 2016).

The Eriophyoidea are phytophagous mites that are of great agricultural importance due to the direct damage they cause to their host plants, their ability to transmit phytoviruses, and their potential as biological agents for weed control (Lindquist et al. 1996). Approxi-mately 4600 valid species of eriophyoid mites colonizing a vast array of host plants have been described (J.W. Amrine, pers. comm.). Accurate taxonomic identification and solid knowledge of the systematics of these pestiferous phytophagous mites is absolutely needed for defining optimal control and risk mitigation strategies. Identification of eriophyoid mites is traditionally based on their morphological traits and is challenging due to their small size (80–500  µm long) and simplified body structure (Chetverikov and Petanović 2016). Many of the body structures of these animals have regressed during evolution (e.g., the third and fourth pairs of legs and the setae on the opisthosoma and legs). In addi-tion, species diagnostic traits are basically restricted to the females, making identification impossible when other stages are sampled (Lindquist 1996; Amrine et al. 2003; de Lillo et al. 2010).

Among the plant-feeding mites, eriophyoids are the most diverse and ecologically spe-cialized. Most eriophyoid species (ca. 80–95%) are highly host-specific, colonizing only a single host plant species or a few hosts within a single genus (Oldfield 1996; Skoracka et al. 2010). Such a high level of their host specificity has been considered to be a result of the intimate obligate interactions and the long-term evolutionary history of species with their host plants (Oldfield 1996).

Interestingly, cryptic diversity in eriophyoid mites has often been detected within spe-cies found to inhabit many host plant species. Such host-generalists appeared to be com-plexes of several species with restricted host ranges, supporting the notion of a high level of host specialization in the Eriophyoidea (Skoracka et al. 2012, 2013, 2018; Miller et al. 2013; Navia et al. 2015; Cvrković et al. 2016). One such example is the cereal rust mite (CRM hereafter), Abacarus hystrix (Nalepa), which has long been regarded as a gener-alist species that can easily adapt to many grass hosts (Frost and Ridland 1996). How-ever, quantitative morphometric and molecular analyses of three populations of the CRM from quackgrass [Elymus repens (L.) P. Beauv.], ryegrass (Lolium perenne L.) and smooth brome (Bromus inermis Leyss.) revealed cryptic speciation among populations from these different hosts (Skoracka 2008; Skoracka and Dabert 2010).

Another grass-associated Abacarus species complex whose diversity certainly merits deeper investigation are species associated with sugarcane. Cultivated sugarcane plants (Saccharum spp.) are hybrid cultivars that were derived from crossings between plants of S. officinarum and S. spontaneum (Dillon et  al. 2007). These hybrid cultivars have been widely cultivated in tropical and subtropical areas on several continents (Daniels and Roach 1987; Fauconnier 1993). It is known that hybridization between plants may impact the evolution of host-dependent arthropod species (Floate and Whitham 1993; Evans et al.

375Experimental and Applied Acarology (2019) 78:373–401

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2008). For example, Floate and Whitham (1993) have argued that hybridization in plants could influence host shifts and speciation in arthropods and pathogens. Therefore, it is likely that the current composition of the eriophyoid community associated with cultivated sugarcane around the world might be related to the history of dissemination of their hosts among continents. Currently, there are four valid Abacarus species associated with Sac-charum spp.: Abacarus sacchari ChannaBasavanna, which is widely disseminated around the world; Abacarus delhiensis ChannaBasavanna, reported from India; Abacarus queens-landiensis Ozman-Sullivan et al., reported from Australia; and Abacarus doctus Navia and Flechtmann, reported from Costa Rica (Navia et al. 2011) and El Salvador (Guzzo et al. 2014).

Given that there is solid evidence of high level of cryptic diversity among all Acari, including eriophyoid mites (Skoracka et al. 2015) and that taxonomy, including synonymi-zation, of sugarcane-associated Abacarus species have been based solely on morphology (e.g., Ozman-Sullivan et al. 2006) a comprehensive investigation of this taxon is warranted to assess its real diversity and to avoid further taxonomic confusion. All the more, speci-mens very similar morphologically to A. sacchari were recently collected in the sugarcane crop in Brazil and identified as A. aff. sacchari; however, their taxonomic placement is still questionable. To clear up the status of sugarcane-associated Abacarus taxa, including specimens akin to A. sacchari newly found in Brazil, in this study we employed an integra-tive systematics approach to test hypotheses whether A. aff. sacchari is: (i) a synonym of A. sacchari with the morphological differences resulting from intraspecific variation (such as those established by Ozman-Sullivan et al. 2006) or (ii) a separate species belonging to the cryptic species complex. We investigated the cryptic diversity of Abacarus species associ-ated with sugarcane in the Americas through detailed morphological study, morphometric analysis and molecular phylogenetic analysis. On this basis, a new Abacarus species was described. Moreover, including DNA sequences of other Abacarus species associated with grasses in the phylogenetic analyses in our study allowed us to define the ranges of intra- and interspecific variability for mitochondrial and nuclear molecular markers in this genus. The hypotheses on the effect of sugarcane hybridization on Abacarus species radiation are also discussed.

Materials and methods

Sampling

The study included 13 populations of Abacarus collected from sugarcane (Saccharum spp.) from Alagoas, Pernambuco and São Paulo states, Brazil, and from Sonsonate, El Sal-vador, and Provincia de Puntarenas, Costa Rica (Table  1). Samples of sugarcane leaves were collected in the field and transported to the laboratory for further examination. Subse-quently, the mites were collected by direct inspection under a dissecting stereomicroscope (40 ×). Multiple mite individuals obtained from a given locality originating from several sugarcane plants were regarded as a single population in the analyses. The collected mite specimens used for molecular and morphometric analyses were placed in Eppendorf tubes containing absolute or 70% ethyl alcohol, respectively. For morphometric analysis, mite specimens were mounted on microscope slides using modified Berlese medium (Amrine and Manson 1996). The sample codes of the Abacarus populations studied are listed in Table 1.

376 Experimental and Applied Acarology (2019) 78:373–401

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Tabl

e 1

Dat

a co

llect

ion

of A

baca

rus s

pp. o

btai

ned

in th

is st

udy,

on

suga

rcan

e (S

acch

arum

sp.)

from

Sou

th a

nd C

entra

l Am

eric

as

a Popu

latio

ns u

sed

for m

orph

omet

ric a

naly

sis

b Popu

latio

ns u

sed

for m

olec

ular

ana

lysi

sc Po

pula

tions

use

d fo

r mor

phom

etric

and

mol

ecul

ar a

naly

zes

Popu

latio

nAb

acar

us M

orph

o ID

Loca

lity

Geo

grap

hica

l coo

rdin

ates

Col

lect

or

Ab1

cA.

sacc

hari

and

A. a

ff. sa

ccha

riB

razi

l, A

lago

as, T

eotô

nio

Vile

la09

°54′

09″S

, 36°

23′4

3″W

Dua

rte, M

.E.

Ab3

aA.

sacc

hari

and

A. a

ff. sa

ccha

riB

razi

l, A

lago

as, S

ão S

ebas

tião

09°5

4′09″S

, 36°

23′4

3″W

Dua

rte, M

.E.

Ab9

cA.

sacc

hari

Bra

zil,

São

Paul

o, P

iraci

caba

22°4

2′15″S

, 47°

37′5

8″W

Nav

ia, D

.A

b10a

A. d

octu

sEl

Sav

ador

, Son

sona

te, H

acie

nda

San

Erne

sto–

Mar

tí, F

.A.C

Ab1

1aa

A. d

octu

sEl

Sal

vado

r, So

nson

ate,

Izal

co13

°39′

51″N

, 88°

40′0

9″W

Mar

tí, F

.A.C

Ab1

1bb

A. d

octu

sEl

Sal

vado

r, So

nson

ate,

Izal

co13

°42′

59″N

, 89°

42′1

9″W

Mar

tí, F

.A.C

Ab1

1cb

A. d

octu

sEl

Sal

vado

r, So

nson

ate,

Izal

co13

°43′

50″N

, 89°

42′0

9″W

Mar

tí, F

.A.C

Ab1

2cA.

sacc

hari

and

A. a

ff. sa

ccha

riB

razi

l, Pe

rnam

buco

, Rec

ife08

°01′

04″S

, 34°

56′4

7″W

Nav

ia, D

.A

b14c

A. sa

ccha

ri a

nd A

. aff.

sacc

hari

Bra

zil,

Ala

goas

, Teo

tôni

o V

ilela

09°5

1′53″S

, 36°

19′4

5″W

Dua

rte, M

.E.

Ab1

5bA.

aff.

Sac

char

iB

rasi

l, A

lago

as, C

ampo

Ale

gre

10°0

1′04″S

, 36°

15′2

1″W

Dua

rte, M

.E.

Ab2

1cA.

sacc

hari

and

A. a

ff. sa

ccha

riB

rasi

l, Pe

rnam

buco

, Rec

ife08

°01′

04″S

, 34°

56′4

7″W

Nav

ia, D

.A

b29a

A. sa

ccha

ri a

nd A

. aff.

sacc

hari

Bra

zil,

Ala

goas

, Teo

tôni

o V

ilela

09°5

1′53″S

, 36°

19′4

5″W

Dua

rte, M

.E.

Ab3

3aA.

doc

tus

Cos

ta R

ica,

Pro

vinc

ia d

e Pu

ntar

enas

, Dist

rict o

f Pita

haya

–Sa

nabr

ia, C

.

377Experimental and Applied Acarology (2019) 78:373–401

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Morphological study and description

Slide-mounted specimens were studied using a phase-contrast and differential interference contrast microscope (Eclipse 80i Nikon, Tokyo, Japan). Structures relevant to taxonomic classification were measured using a graded eyepiece. Measurements are given in micro-metres (µm). All characters were measured or counted using a 100 × objective. To the mor-phometric study, 10–20 females (depending on the availability) from 10 populations were examined in the dorso-ventral position. Forty-two morphological traits of each individual were measured or counted. The terminology used follows that of Lindquist et al. (1996), and classification is based on Amrine et  al. (2003). The count of ventral opisthosomal annuli began with the first full annulus behind the genitalia. Dorsal opisthosomal annuli were counted from the first full annulus behind the middle of the prodorsal shield rear margin. Measurements were conducted according to de Lillo et al. (2010) except for the following: (1) body length was measured from the tip of the frontal lobe to the rear end of the anal lobe without considering the pedipalps; (2) the sc tubercles space was the distance between the tubercles rather than the distance between the sc setae; and (3) the empodium length included its basal portion inserted into the tarsus.

Univariate (ANOVA) and multivariate (principal components analysis, PCA) statisti-cal analyses were performed on the 42 quantitative variables using STATISTICA software v.10.0. Principal components analysis (PCA) was applied to reveal any discontinuities in morphological variation among the specimens originating from different regions.

To describe the new taxon discovered in this study, 72 morphological traits of each individual (10 females and five males) were measured or counted. The procedure used to describe the new species is the same as that described above in the morphometric study. In addition, the female and male were drawn using a camera lucida under the same micro-scope with a 100 × objective; the drawings were scanned, digitized and edited using Adobe Photoshop CS6. Micrographs were obtained using a digital system consisting of a phase and differential interference contrast microscope (Nikon Eclipse 80i) connected to a digital camera (Nikon DS-Ri 1, 12.7 megapixels) that was in turn connected to a computer with NIS Elements software (Nikon) and a camera control unit (NikonDS-Fil with DS-L2).

The terminology used to describe the elements of the female internal genitalia and the male external genitalia was according to Chetverikov et al. (2014), and Chetverikov (2014). The definition of epicoxal area was according to Chetverikov and Craemer (2015). In the description of the female, each measurement of the holotype precedes the corresponding range for the paratypes. Absence of variation among measurements is indicated by ‘*’.

Molecular studies

DNA extraction, amplification, and sequencing

DNA was isolated from specimens of eight Abacarus populations (Table  1). Genomic DNA was extracted from single specimens using the DNeasy Blood Tissue Kit (Qiagen, Brazil). Intact mites were individually transferred from absolute ethyl alcohol to 1.5 mL microcentrifuge tubes containing 90 μL of ATL buffer (Qiagen); 10 μL of Proteinase K (Qiagen) was then added to each sample. The mixture was incubated at 56 °C with shak-ing in a thermomixer for 18–22 h. Mites were not crushed. All other steps of the stand-ard Qiagen DNA extraction protocol ‘Purification of Total DNA from Animal Tissue’

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(SpinColumn Protocol) were modified for DNA extraction from tiny mites as described by Mendonça et al. (2011).

The specimens used in DNA extraction, when possible, had their exoskeleton recovered from the membrane of the extraction column and were mounted on microscope slides in Hoyer’s medium. These slides were deposited as voucher specimens in the mite collection at Embrapa Genetic Resources and Biotechnology, Brasília, Brazil.

Three target DNA fragments were PCR-amplified and sequenced per single mite: one mitochondrial gene—the cytochrome c oxidase subunit I (COI) (DNA barcode region cho-sen by the Consortium for the Barcode of Life (http://barco ding.si.edu)—and two nuclear DNA fragments—the subunit D2 region in 28S rDNA and the ITS nuclear region includ-ing ITS1 + 5.8S + ITS2 (Table S1).

Amplification and sequencing of the COI (mtDNA) was performed using the degenerate primers bcd F01 and bcd R04 (Table S1). PCR was conducted in 25-µL reaction volumes containing 2.5 µL of 10 × reaction buffer supplied by the manufacturer (Qiagen), 1.5 µL MgCl2 (25 mM), 1.5 µL dNTP mix (10 mM), 1.25 µL of each primer (10 µM), 0.25-µL U µL−1 (5 units) of Taq DNA polymerase for standard and specialized PCR applications (Qiagen), 12.75 µL of sterile water and 4 µL of DNA template; using a thermocycling pro-file of one cycle of 3 min at 96 °C followed by 45 cycles of 10 s at 95 °C, 30 s at 50 °C, and 1 min at 72 °C, and a final step of 5 min at 72 °C.

Amplification of the D2 region in 28S rDNA was performed using the primers f1230 and D1D2 rev 04 (Table S1). PCR was conducted in 25-µL volumes enclosing 2.5 µL of 10 × reaction buffer (Qiagen), 1.5 µL MgCl2 (25 mM), 1 µL dNTP mix (2.5 mM of each base), 0.625 µL of each primer (10 µM), 0.25-µL U µL−1 (5 units) of Taq polymerase (Qia-gen), 15.5 µL of sterile water and 3 µL of DNA template; using a thermocycling profile of one cycle of 3 min at 96 °C followed by 35 cycles of 10 s at 95 °C, 1.15 min at 50 °C, and 2 min at 72 °C, and a final step of 5 min at 72 °C. For sequencing of the D2 region, the primers fw2 and D1D2 rev 04 were used.

The ITS nuclear region was amplified and sequenced using the forward and reverse primers 18S and 28SC, respectively (Table S1). PCR was conducted in 25-µL reaction vol-umes with 2.5 µL of 10 × buffer (Qiagen), 1 µL MgCl2 (25 mM), 0.3 µL BSA (10 mg mL−1, Biolabs), 1 µL dNTP mix (2.5 mM of each base), 0.5 µL of each primer (10 µM), 0.25-µL U µL−1 (5 units) of Taq polymerase (Qiagen), 14.95 µL water and 2 µL of DNA template; using a thermocycling profile of one cycle of 4 min at 94 °C followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C, and a final step of 10 min at 72 °C.

After amplification, 5 μL of the PCR reaction was analyzed by electrophoresis on a 1% agarose gel and visualized by GelRed staining (Biotum, Fremont, CA, USA) to assess the product size and concentration. Both strands directions of the amplified fragments (COI, D2 and ITS) containing visible and single bands were directly sequenced using an ABI 3730 XLs automated DNA sequencer (Applied Biosystems, Korea).

Sequences and phylogenetic analyses

The software Staden Package v.1.6.0 (Staden et  al. 2000) was used for checking, editing and assembling the raw data into sequence contigs. The COI, D2 and ITS sequences were aligned using the ClustalW multiple alignment procedure (Thompson et al. 1994) implemented in BIOEDIT v.7.0.4 (Hall 1999) with default gap-weighting parameters. No manual adjustments were made to the CLUSTAL alignment. The dis-tributions and frequencies of haplotypes (COI) and sequences variant (D2 and ITS) of

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the Abacarus species included in this study were inferred using the option ‘Haplotype Collapse’ in the program ALTER (Alignment Transformation Environment) available at http://sing.ei.uvigo .es/ALTER / (Glez-Peña et al. 2010).

Analyses of the pairwise genetic distances between and within nucleotide sequences among lineages were performed using MEGA v.6 (Tamura et  al. 2013) and the most appropriate evolutionary model for estimation of inter- and intra-lineage genetic var-iation was also selected using MEGA v.6. The Tamura-Nei (TN93) model (Tamura et al. 2007) was applied to the COI dataset, and the Kimura 2-Parameters (K2P) model (Kimura 1980) was applied to the D2 and ITS datasets. Standard error estimates were obtained by a bootstrap procedure (1000 replicates).

The phylogenetic analyses for studied fragments were conducted using the maxi-mum likelihood (ML) optimality criterion. The best-fit models of nucleotide substitu-tion for the three fragments were selected using the jModeltest v.2.1.1 program (Dar-riba et  al. 2012) based on the likelihood scores for 88 different models. The Akaike information criterion corrected (AICc) and the Bayesian information criterion (BIC) were calculated (Table S2). The ML models were tested in PhyML v.3.0 (Guindon and Gascuel 2003; Guindon et al. 2010), NJ in MEGA v.7 (Kumar et al. 2016), and Bayes-ian inference (BI) was tested in MrBayes v.3.2.6 (Ronquist et al. 2012). This software and these procedures were used to test NJ, ML and BI phylogenies in analyses of COI, D2 and ITS sequences. The estimated frequencies of nucleotide bases are summarized in Table S2.

The sequences of Abacarus acutatus Sukhareva, Abacarus lolii Skoracka, A. hystrix, Abacarus longilobus Skoracka, Abacarus plumiger Laska et al. and Abacarus sp. available in GenBank were included in the analyses as an internal group, and the sequences of Ace-ria tosichella Keifer and Aceria eximia Sukhareva were included as an outgroup.

Sequences were also checked against Abacarus sequences retrieved from GenBank (Skoracka and Dabert 2010; Skoracka et al. 2012; Laska et al. 2018) and used to edit a neighbor joining tree to verify their reliability. The alignment of COI sequences was confirmed by translating the aligned DNA into amino acids using MEGA v.6. To ini-tially identify candidate protein coding regions in DNA sequences searching start and stop codons, an open reading frame was determined using a graphical analysis tool (ORF FINDER) available at http://www.ncbi.nlm.nih.gov/proje cts/gorf/. The complete dataset is available upon request.

Combined analysis—COI, D2 and ITS

To perform the combined analysis of COI, D2 and ITS fragments, the sequence files were individually organized using MEGA v.6. Alignment of the three fragments was performed separately by the CLUSTAL W multiple alignment method (Thompson et al. 1994) implemented in the BioEdit program. The files were then concatenated in a single matrix in Mesquite v.3.0.4 (a modular system for evolutionary analysis) (Mad-dison and Maddison 2016), and the BI combined analysis was performed in MrBayes v.3.2. The number of categories used to approximate the gamma distribution was set at four, and four Markov chains were run for 10,000,000 generations; the final aver-age standard deviation of split frequencies was less than 0.01, and the stabilization of model parameters (burn-in = 0.25) occurred at approximately 250 generations. The edition of the phylogenetic tree was performed using TreeView v.0.5.0.

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Results

Morphological and morphometric analyses

The means, standard deviations and ranges of the measured values of the 42 analysed mor-phological characters of three species (10 populations, see Table 1) of Abacarus females associated with sugarcane are presented in Table S3. The main morphometric differences between the specimens of A. neosacchari n. sp., A. sacchari and A. doctus observed in the present study were associated with the following characters: length of the scapular seta (sc); length and width of the frontal lobe (Fig. 1a, b, c); length of leg setae (l” and ft”) and tibia I and II; number of ventral annuli; and number of empodium rays. In addition to morphometric differences, our detailed morphological observation allowed us to observe differences that were constant in the whole morphological group: (i) the shape of the fron-tal lobe, which is subtriangular and apically pointed in A. neosacchari n. sp. (Figure 1a), subelliptical and pointed apically in A. sacchari (Fig. 1b), and subelliptical and apically rounded in A. doctus (Fig. 1c); and (ii) the prodorsal shield ornamentation, which presents submedian lines that are slightly curved in the anterior region of the shield in A. neosac-chari n. sp. morphotype (Fig. 1a), markedly curved in A. sacchari (Fig. 1b), and barely visible in A. doctus (Fig. 1c).

Univariate analysis revealed significant differences among all of the characters analysed in the studied Abacarus species (Table  S3). The traits that most influenced the first two principal components (PCs) and showed higher weights in the covari-ance matrix are listed in Table  S4. Of the total morphological variability, 56.3% was explained by two principal components (PC) (Fig.  2). No overlap was observed between plotted individuals of the different species in the space of the first two prin-cipal components, showing complete morphometric discontinuity among them. The first PC, which explained 42.2% of the total variation, clearly separated A. doctus from A. neosacchari n. sp. and A. sacchari. Abacarus doctus individuals were plot-ted in the positive section of the PC1 axis, whereas A. sacchari and A. neosacchari n. sp. appeared mostly along the negative section of this axis. The main PC1 traits loading were: length of the frontal lobe (longer in A. doctus than in A. sacchari and

Fig. 1 Details of the prodorsal shield of Abacarus neosacchari n. sp. (a), Abacarus sacchari (b) and Abac-arus doctus (c) presenting differences in the shape of the frontal lobe (FL), shield ornamentation and length of the scapular seta (sc)

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A. neosacchari n. sp.), length of tibia I and II, scapular seta, antaxial genual seta II, antaxial seta II and seta c2, distance between ventral seta d and f (shorter in A. doc-tus than in A. sacchari and A. neosacchari sp. n.), and number of ventral annuli (less numerous in A. doctus than in A. sacchari and A. neosacchari n. sp.) (Figure 2, Tables S3 and S4). The second PC, which explained 14.1% of the variation, mainly separated A. neosacchari n. sp. from A. doctus and A. sacchari. Abacarus neosacchari n. sp. individuals were plotted along the PC2 positive section, A. sacchari individuals were plotted along the negative axis, and A. doctus individuals were plotted in both the posi-tive and the negative sections. The main PC2 traits loading were: number of empodium rays I–II, number of dorsal annuli (more numerous in A. neosacchari n. sp. than in A. doctus and A. sacchari), and shape of the frontal lobe base (narrower in A. neosacchari n. sp. than in A. doctus and A. sacchari) (Fig. 2, Table S4).

Molecular analyses

The general topologies of the phylogenetic trees inferred by the NJ, ML analyses and BI for the COI, D2 and ITS datasets were similar and consistently revealed the same structure for Abacarus and external group populations, thus, only the BI model is shown.

Fig. 2 First two principal components for morphometric data of Abacarus mites. 1, 3 Abacarus neosac-chari sp. n., 2 Abacarus sacchari (Teotônio Vilela, Brazil), 4 Abacarus sacchari (São Sebastião, Brazil), 5 Abacarus neosacchari n. sp. (Recife, Brazil), 6 Abacarus sacchari (Recife, Brazil), 9 Abacarus sacchari (Piracicaba, Brazil), 7–8 Abacarus doctus (El Salvador) and 10 Abacarus doctus (Costa Rica)

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Sequence diversity

The translation of the mtDNA COI nucleotide sequences resulted in 195 amino acid sequences, with 15 (7.7%) variable amino acid positions. The final COI dataset consisted of 22 aligned sequences of 604 bps, representing seven populations of Abacarus associated with sugarcane obtained in this study, 23 sequences of Abacarus available in the GenBank and two species of Aceria mites as an outgroup. No insertions or deletions were found. In the alignment, 162 (26.9%) sites were parsimony informative, and 166 (27.5%) sites were variable. Twenty-nine haplotypes were identified from 45 COI sequences of Abacarus (Table S5). The average mean divergence over all the sequence pairs (including the out-group taxa) was 21.5% (SE = 2.8) and ranged from 5.0 to 30.4%. The average mean diver-gence over the Abacarus sequences was 21.1% (SE = 2.4) and ranged from 5.0 to 29.4%. The mean divergence among sugarcane-associated Abacarus sequences was 22.47% and ranged from 19.5 to 24.7% (Table 2).

The nuclear sequence data of the D2 region of the 28S rDNA comprised 47 aligned sequences of 512 bps (23 obtained in this study from seven populations of Abacarus and 22 retrieved from GenBank) and two outgroup species. In the alignment, 115 (22.0%) sites were parsimony informative, and 128 (24.4%) sites were variable. Twenty-three sequence variants were identified from 47 D2 sequences (Table S5). The average mean divergence over all sequence pairs (including the outgroup taxa) was 12.5% (SE = 1.6%) and ranged from 0.4 to 25.0%. The average mean divergence over the Abacarus spp. sequences was 12.0% (SE = 1.4) and ranged from 0.4 to 21.3% (Table 3).

The final ITS dataset consisted of 40 sequences of 900 bps, including 38 sequences of eight populations of Abacarus obtained in this study and two outgroups. The ITS sequences obtained during this study are the first sequences available in the Genbank for this genus; therefore, only sequences of the ‘Saccharum group’ could be used in the analy-ses. No insertions or deletions were found. In the alignment, 230 (23.4%) sites were parsi-mony informative, and 264 (27.0%) sites were variable. Fourteen sequence variants were identified from 38 ITS sequences of Abacarus species (Table S5). The average mean diver-gence over all sequence pairs, including the outgroup taxa, was 10.3% (SE = 1.8%) and ranged from 4.0 to 35.3%. The average mean divergence over the Abacarus spp. sequences was 7.9% (SE = 1.0%) and ranged from 4.0 to 17.9% (Table 4).

Phylogenetic analyses

The Bayesian inference tree of nucleotide sequences of COI, 28S D2, ITS as well as combined analysis that included all sequence variants supported the monophyly of the Abacarus genus (Figs.  3, 4, 5, 6). In COI analysis, Abacarus species were separated into two main well-supported clades, one of which contains all Abacarus associated with sugarcane: A. sacchari, A. doctus and A. neosacchari n. sp. (this clade is hereafter referred to as the ‘Saccharum group’); the other clade comprises species associated with other grass species (hereafter referred to as the ‘other grasses group’) whose sequences were obtained from GenBank. The ‘Saccharum group’ was supported by a posterior probability score of 100%, and within this clade three species clustered distinctly, each with a high posterior probability score (99 to 100%) (Fig. 3). The ‘other grasses group’ was supported by a posterior probability of 69% and contained the four previously estab-lished groups A. acutatus, A. lolii, A. hystrix, and A. longilobus with its sister species A.

383Experimental and Applied Acarology (2019) 78:373–401

1 3

Tabl

e 2

Esti

mat

es o

f av

erag

e di

verg

ence

(sh

own

as p

erce

ntag

es w

ith s

tand

ard

erro

r es

timat

es in

par

enth

eses

) fo

r CO

I re

gion

seq

uenc

e pa

irs w

ithin

(on

the

diag

onal

) an

d be

twee

n Ab

acar

us sp

ecie

s and

the

Acer

ia o

utgr

oup

spec

ies

Ana

lyse

s wer

e co

nduc

ted

usin

g th

e Ta

mur

a-N

ei (T

N93

) mod

el

A. sa

ccha

riA.

neo

sacc

hari

n. s

p.A.

doc

tus

A. a

cuta

tus

A. lo

liiA.

hys

trix

A. sp

.A.

plu

mig

erA.

long

ilobu

sAc

. tos

iche

llaAc

. exi

mia

A. sa

ccha

ri0.

0 (0

.0)

A. n

eosa

ccha

ri n

. sp.

19.5

(2.3

)0.

1 (0

.1)

A. d

octu

s24

.7 (2

.7)

22.2

(2.5

)0.

0 (0

.0)

A. a

cuta

tus

26.3

(2.6

)23

.6 (2

.3)

25.0

(2.5

)5.

1 (0

.8)

A. lo

lii25

.0 (2

.8)

24.9

(2.7

)24

.0 (2

.6)

22.3

(2.5

)0.

2 (0

.1)

A. h

ystr

ix25

.0 (2

.3)

26.8

(2.5

)28

.3 (2

.6)

21.9

(2.1

)20

.6 (2

.0)

13.3

(1.3

)A.

sp.

24.1

(2.6

)26

.8 (2

.8)

24.6

(2.5

)23

.2 (2

.5)

20.3

(2.4

)23

.4 (2

.3)

0.7

(0.3

)A.

plu

mig

er27

.0 (2

.8)

24.3

(2.5

)23

.1 (2

.5)

23.1

(2.5

)21

.2 (2

.6)

22.0

(2.2

)21

.0 (2

.5)

0.0

(0.0

)A.

long

ilobu

s29

.4 (2

.9)

25.4

(2.7

)24

.2 (2

.5)

24.1

(2.6

)22

.9 (2

.7)

23.4

(2.2

)22

.5 (2

.6)

5.0

(1.0

)0.

2 (0

.0)

Acer

ia to

sich

ella

28.4

(2.9

)30

.4 (3

.1)

25.1

(2.7

)25

.7 (2

.6)

24.3

(2.7

)25

.0 (2

.3)

25.7

(2.7

)27

.4 (2

.9)

25.4

(2.6

)n/

cAc

eria

exi

mia

25.8

(2.7

)25

.6 (2

.7)

22.8

(2.5

)21

.5 (2

.3)

20.1

(2.3

)25

.1 (2

.4)

26.1

(2.9

)24

.5 (2

.8)

24.3

(2.8

)18

.2 (2

.2)

n/c

384 Experimental and Applied Acarology (2019) 78:373–401

1 3

Tabl

e 3

Esti

mat

es o

f ave

rage

div

erge

nce

(sho

wn

as p

erce

ntag

es w

ith st

anda

rd e

rror

esti

mat

es in

par

enth

eses

) for

D2

regi

on o

f 28S

rDN

A se

quen

ce p

airs

with

in (o

n th

e di

ago-

nal)

and

betw

een

Abac

arus

spec

ies a

nd th

e Ac

eria

out

grou

p sp

ecie

s

Ana

lyse

s wer

e co

nduc

ted

usin

g th

e K

imur

a 2-

para

met

er (K

2P) m

odel

A. sa

ccha

riA.

neo

sacc

hari

sp. n

.A.

doc

tus

A. a

cuta

tus

A. lo

liiA.

hys

trix

A. sp

.A.

plu

mig

erA.

long

ilobu

sAc

. exi

mia

Ac. t

osic

hella

A. sa

ccha

ri0.

3 (0

.2)

A. n

eosa

ccha

ri sp

. n.

2.2

(0.7

)0.

0 (0

.0)

A. d

octu

s15

.5 (2

.1)

14.4

(1.9

)0.

0 (0

.0)

A. a

cuta

tus

17.8

(2.1

)16

.9 (1

.9)

17.1

(2.2

)0.

3 (0

.2)

A. lo

lii18

.1 (2

.0)

17.5

(2.0

)19

.5 (2

.4)

5.8

(1.2

)0.

0 (0

.0)

A. h

ystr

ix17

.9 (2

.0)

17.4

(2.0

)19

.9 (2

.4)

5.9

(1.2

)0.

4 (0

.2)

0.2

(0.1

)A.

sp.

18.5

(2.1

)18

.2 (2

.1)

18.6

(2.3

)3.

6 (1

.0)

3.6

(0.9

)4.

0 (0

.9)

0.0

(0.0

)A.

plu

mig

er17

.8 (1

.9)

17.2

(1.9

)19

.2 (2

.3)

5.2

(1.1

)1.

2 (0

.4)

1.5

(0.5

)4.

1 (0

.8)

0.0

(0.0

)A.

long

ilobu

s19

.9 (2

.2)

19.3

(2.2

)21

.3 (2

.5)

7.0

(1.4

)2.

9 (0

.8)

3.2

(0.9

)5.

9 (1

.1)

1.7

(0.6

)n/

cAc

eria

exi

mia

24.5

(2.8

)23

.6 (2

.7)

25.0

(3.1

)15

.2 (2

.2)

11.2

(2.0

)11

.6 (2

.0)

13.0

(2.0

)12

.6 (1

.9)

14.6

(2.0

)n/

cAc

eria

tosi

chel

la23

.5 (2

.6)

22.6

(2.6

)24

.0 (3

.1)

13.4

(2.1

)9.

8 (1

.7)

10.2

(1.8

)11

.2 (1

.8)

10.7

(1.7

)12

.6 (1

.9)

2.4

(0.8

)n/

c

385Experimental and Applied Acarology (2019) 78:373–401

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plumiger, one unidentified group represented by specimens of Abacarus sp., and an iso-lated branch formed by a specimen identified as A. hystrix (KC412843) collected from Elymus hispidus (Opiz) Melderis subsp. barbulatus in Turkey. These five groups pre-sented posterior probability values equal to or greater than 99%. In 28S D2 analysis the ‘Saccharum group’ formed a separated and well-supported (97%) cluster within a clade (60% support) including A. acutatus and undefined Abacarus sp. (FJ392671, FJ392670). Other Abacarus species from the ‘other grasses group’ showed polytomy with two distinct clades: A. hystrix, A. plumiger with its species A. longilobus, and the branch formed by A. lolii (Fig. 4). Finally, in ITS analysis ‘Saccharum group’ was divided into two major branches, one containing A. sacchari and A. neosacchari n. sp. specimens and the other including A. doctus. This result was similar to that observed for COI and D2 phylogenies. The presence of these three species in the ‘Saccharum group’ was sup-ported by a robust posterior probability value (100%).

Description of a new Abacarus species in the ‘Saccharum group’

Our results based on uni- and multivariate morphometric analyses (ANOVA and PCA) showed that there is a discontinuous variation among the three studied sugarcane-associated Abacarus morphotypes (Fig. 2, Tables S3–S4), suggesting the occurrence of three Abac-arus species inhabiting sugarcane in Brazil and Central America. The genetic distances and phylogenetic analyses based on the sequences of mtDNA COI and nuclear D2 and ITS (Tables 2, 3, 4, Figs. 3, 4, 5, 6) also supported the occurrence of three genetic lineages of sugarcane-associated Abacarus. Thus, the A. aff. sacchari morphotype is considered a new species and is named Abacarus neosacchari Duarte and Navia n. sp. (ZooBank registration details: urn:lsid:zoobank.org:act:999F392E-90FC-4857-98D5-3ED6D1455416).

Taxonomy

Family Eriophyidae Nalepa, 1898.Subfamily Phyllocoptinae Nalepa, 1892.Tribe Anthocoptini Amrine and Stasny, 1994.Genus Abacarus Keifer, 1944.Abacarus neosacchari n. sp. Duarte and Navia (Figs. 7, 8, 9).

Table 4 Estimates of average divergence (shown as percentages with standard error estimates in parenthe-ses) for ITS region sequence pairs within (on the diagonal) and between Abacarus species and the Aceria outgroup species

Analyses were conducted using the Kimura 2-parameter (K2P) model

A. sacchari A. neosacchari sp. n. A. doctus Ac. tosichella Ac. eximia

A. sacchari 0.1 (0.1)A. neosacchari sp. n. 4.0 (0.7) 0.0 (0.0)A. doctus 17.9 (1.6) 17.8 (1.6) 0.3 (0.1)Aceria tosichella 34.3 (2.7) 34.2 (2.6) 33.1 (2.6) n/cAceria eximia 34.5 (2.7) 35.3 (2.7) 34.6 (2.8) 6.6 (0.9) n/c

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Fig. 3 Bayesian inference (BI) tree performed using HKY+I+G model on data from the COI sequences of Abacarus eriophyoid mites and the outgroup species. The number of times that a haplotype was found in the dataset is indicated in parentheses after the GenBank accession numbers. Concordant trees were obtained by Maximum-likelihood (ML) and Neighbour-Joining (NJ) analyses, which produced the same topology in defining groups. Statistical supports indicate Bayesian posterior probabilities values as a per-centage. Only statistical supports greater than 50% are indicated above branches

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Holotype Adult female, collected on 01 June 2012, by M.E. Duarte and E.S. Silva, deposited at the reference collection at the Laboratory of Acarology, Embrapa Recursos Geneticos e Biotecnologia, Brasilia, DF, Brazil.

Type locality Brazil: TeotônioVilela, Alagoas State on hybrid sugarcane, Saccharum sp. (Poaceae) (9º51′54″S, 36º20′01″W).

Paratype The same locality as for holotype. 14 February 2012, 01 June 2012, 14 August 2012, 18 October 2013 and 10 April 2014. Collected by M.E. Duarte and E.S. Silva (57 females, 28 males, and 6 immatures) were deposited at the reference collection at the Laboratory of Acarology, Embrapa Genetic Resources and Biotechnology, Brasilia, Distrito Federal, Brazil. Other paratypes (10 females and 5 males) were deposited in the

Fig. 4 Bayesian inference (BI) tree performed using TPM3+G model on data from the 28S r-RNA subunit D2 sequences of Abacarus eriophyoid mites and the outgroup species. The number of times that a sequence variant was found in the dataset is indicated in parentheses after the GenBank accession numbers. Concord-ant trees were obtained by Maximum-likelihood (ML) and Neighbour-Joining (NJ) analyses, which pro-duced the same topology in defining groups. Statistical supports indicate Bayesian posterior probabilities values as a percentage. Only statistical supports greater than 50% are indicated above branches

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Mite Reference Collection at Department of Entomology and Acarology, ESALQ/USP, Piracicaba, São Paulo, Brazil.

Other material All material collected from Brazil. 19 specimens from Junqueiro (9°51′53″S, 36°19′45″W), 165 from São Sebastião (09°54′09″S, 36°23′43″W), 12 from Campo Alegre (10°01′04″S, 36°15′21″W), 124 from Coruripe (09°55′40″S, 36°16′45″W), 23 from São Miguel dos Campos (09°52′09″S, 36°23′43″W), 28 from Barra de São Miguel (09°52′55″S, 36°19′45″W), 9 from Barra de Santo Antônio (09°52′08″S, 36°23′43″W) and 34 from Rio Largo (9°50′53″S, 36°19′45″W)—Alagoas State; 14 from Sergipe State, coll. Silva, E.S.;160 from Recife–Pernambuco (08°01′04″S, 34°56′47″W), coll. Navia, D., 16 from Saltinho (22°42′15″S, 47°37′58″W), 8 from Rio das Pedras (22°42′15″S, 47°37′58″W), 36 from Piracicaba (22°42′15″S, 47°37′58″W)—São Paulo State, coll. Duarte, M.E. and Navia, D.; 1 from Teresina–Piauí (05º02′21″S, 42º47′22″W), coll. Querino-da-Silva, R.B. and 6 from Boa Vista–Roraima (02°43′28″N, 60°48′08″W), coll. Santos, E.

Fig. 5 Bayesian inference (BI) tree performed using GTR+G model on data from the ribosomal region ITS of Abacarus eriophyoid mites and the outgroup species. The number of times that a sequence variant was found in the dataset is indicated in parentheses after the GenBank accession numbers. Concordant trees were obtained by Maximum-likelihood (ML) and Neighbour-Joining (NJ) analyses, which produced the same topology indefining groups. Statistical supports indicate Bayesian posterior probabilities values as a percentage. Only statistical supports greater than 50% are indicated above branches

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Fig. 6 Combined Bayesian inference (BI) analysis tree for Abacarus eriophyoid mites calculated from the concatenated cytochrome c oxidase subunit I sequences (COI), 28S r-RNA subunit D2 sequences and ribo-somal region ITS. The number of times that a sequence variant was found in the dataset is indicated in parentheses after the species name. Statistical supports indicate Bayesian posterior probabilities values as a percentage. Only statistical supports greater than 0.5 are indicated above branches

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DNA GeneBank accession numbers DNA sequences of additional material were obtained for mitochondrial and nuclear markers and deposited in GeneBank with the following accession numbers: COI (KX892623, KX892629-KX892631, KX892635, KX892639-

Fig. 7 Abacarus neosacchari n. sp.—a dorsal view, female; b ventral view, female

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KX892642), D2 (KX855692, KX855694, KX855698-KX855702, KX855706-KX855707) and ITS (KX855715, KX855726, KX855728, KX855731, KX855733, KX855734, KX855737, KX855742-KX855746, and KX855751-KX855752). See Table S4 for collec-tion data of specimens from each sequence were obtained.

Fig. 8 Abacarus neosacchari n. sp.—a lateral view, male; b empodium; c internal genitalia, female; d coxi-genital region, male; e Leg I, female; f leg II, female

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Etymology The specific designation ‘neosacchari’ is derived from the Greek word ‘neos’ (neutral) meaning ‘new’ and ‘sacchari’ referring to specific name of Abacarus sacchari, which is very similar to the new species, both associated with sugarcane in the Americas.

Relation to host This new species is a vagrant on the inner surface of the leaves. Pre-sumably associated with reddish spots, rust. Since sugarcane plants presenting reddish spots were infested by both A. neosacchari n. sp. and A. sacchari it is not possible to state if just one or if both of them were responsible for these rust symptoms. Such symptoms could be confused with those caused by rust fungi, which are commonly associated with sugarcane.

Fig. 9 Micrographs of Abacarus neosacchari sp. n. a, b Prodorsal shield, female; c coxigenital region, female; d coxigenital region, male. Scale bar: 20 μm

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Differential diagnosis The new species A. neosacchari n. sp. is similar to A. sacchari, A. queenslandiensis and A. doctus. It can be distinguished by: the prodorsal shield ornamen-tation with submedian lines slightly curved in the anterior region of the shield (markedly curved in A. sacchari and barely visible in A. doctus); the shape of the frontal lobe, which is subtriangular and apically pointed (subeliptical and pointed apically in A. sacchari and subeliptical and rounded apically in A. doctus); the length of sc seta which can reach the 5th dorsal annulus (in A. doctus reach the 3rd dorsal annulus, in A. queenslandiensis the 4th and in A. sacchari the 7th); the coverflap ornamentation with two or three transversal lines (in A. doctus two lines, in A. queenslandiensis and A. sacchari missing) and the 7–8 empodium rays (6 in A. queenslandiensis, 7 in A. sacchari and 6–7 in A. doctus). Genetic divergences clearly supported distinction among the three sugarcane-associated species in the Americas (Tables 2, 3 and 4).

Description

Female (n = 10) Body fusiform, 201 (173–207), 64 (56–64) wide, yellowish. Gnathosoma (Fig.  7a) 19 (18–22), projecting slightly downwards; pedipalp coxal seta (ep) 3*, dorsal pedipalp genual seta (d) 10*; chelicerae 16 (14–16), auxiliary stylet 13 (10–13). Prodorsal shield (Figs. 1a, 7a, 8a, b) subtriagular 53 (48–53), 57 (53–60) wide, frontal lobe, acuminate apically, 8 (7–9), 14 (11–14) wide; scapular tubercles and seta on the posterior rear shield margin 35 (29–36) apart, scapular seta (sc) 14 (11–14), directed backward, reaching the 5th dorsal annuli. Shield ornamentation (Figs. 1a, 7a, 9a, b) with median line missing; admedian lines extending along entire shield, diverging in near rear shield margin; irregular sinuous submedian lines anteriorly; epicoxal area with dashes and granulations. Coxigenital region (Figs. 7b, 9c) with 2 (2–3) complete annuli, 5 (4–5) incomplete annuli, microtuber-culated. Coxisternal plates (Figs. 7b, 9c) granulated, prosternal apodeme 8 (8–10), antero lateral seta on coxisternum I (1b) 10 (8–10), 15 (13–15) apart; proximal seta on coxister-num I (1a) 29 (25–32), 8 (6–8) apart; proximal seta on coxisternum II (2a) 39 (39–52), 27 (21–29) apart. Legs (Figs. 7a, 8e, f) with all segments and seta. Leg I (Fig. 8e) 35 (36–37), femur 12 (10–12–), ventral basifemoral seta (bv) 13 (10–13); genu 6 (5–6), antaxialgenual seta (l”) 30 (26–30); tibia 8 (7–9); paraxial tibial seta (l’)7 (6–7); tarsus 9 (8–9), antaxial fastigial tarsal seta (ft”) 26 (21–26), paraxial fastigial tarsal seta (ft’) 22 (20–22), paraxial unguinal tarsal seta (u’) 7 (6–7), tarsal empodium 9 (9–10), entire, 8 (7–8)-rayed, tarsal solenidion (ω) 9 (9–10), slightly curved. Leg II (Fig. 8f) 33 (32–34); femur 10 (10–11), (bv) 24 (17–25); genu 6 (5–6), (l”) 10*; tibia 7 (6–7); tarsus 9 (8–9), (ft”) 25 (24–25), (ft’) 8 (7–8), (u’) 6 (5–6), tarsal empodium10 (9–10), entire, 8 (7–8)-rayed, tarsal solenidion (ω) 9 (9–10), slightly curved. Opisthosoma (Fig. 7a, b) with 43 (40–47) dorsal annuli, smooth; 53 (50–59) ventral annuli microtuberculated. Seta c2 39 (35–45), on ventral annulus 1–2 (1–2); seta d 72 (64–73), 38 (35–40) apart, 36 (36–44) microtubercles between them, on ventral annulus 14 (12–16); seta e 13 (10–13), 16 (12–16) apart, 13 (10–15) microtubercles between them, on ventral annulus 31–32 (29–35); seta f 27 (25–29), 24 (21–24) apart, 23 (18–29) microtubercles between them, on ventral annulus 52 (46–52). Seta h1 3*, h2 65 (65–86). External genitalia (Figs. 7b, 9c) 18 (16–19), 26 (20–26) wide, coverflap with 12 (12–15) longitudinal lines, basally with two or three transversal lines, granulated; setae 3a 27 (23–29). Internal genitalia (Fig. 8c) with anterior genital apodeme trapezoidal, presenting two distinct parts (anterior- apex and posterior part) and distinct oblique apodeme present under the anterior genital apodeme, associated with anterior portion of pre-spermathecal

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part of the longitudinal bridge and additional perpendicular apodeme also present. Sper-mathecal tube directed posterad, almost funnel-like with the thorn-like process, situated in the area where the spermathecal tube joins the spermatheca, directed posterad. Spermatheca ellipsoid, directed posterad.

Male (n = 5) Smaller than females and generally similar to them, 157–168, 48–52 wide. Gnathosoma (Fig. 8a) 18–19; (ep) 3*, (d) 8–10; chelicerae 14–16; auxiliary stylet 11–13. Prodorsal shield 44–47, 46–50 wide, frontal lobe 7–9, 11–14 wide; scapular tubercles 19–21 apart; scapular setae (sc) 10–12, reaching the fifth dorsal annuli. Coxigenital region (Fig. 8d, 9d) with seven incomplete annuli, microtuberculated. Coxisternal plates (Figs. 8d, 9d) granulated; prosternal apodeme 8–10, (1b) 8–10, 11–12 apart; (1a) 23–27, 6–7 apart; (2a) 35–41, 18–21 apart. Leg I 36–37, femur 10*, (bv) 10*; genu 5–6, (l”) 23–25; tibia 6–7, paraxial tibial seta (l’) 6*; tarsus 8*, (ft”) 20–22, (ft’) 16–18, (u’) 5–7, tarsal empodium 8–9, entire, 7–8-rayed, (ω) 8–9. Leg II 29–31, femur 10*, (bv) 14–18; genu 5–6, (l”) 9–10; tibia 6–7; tarsus 7–8, (ft”) 19–21, (ft’) 4–6, (u’) 5*, tarsal empodium 8–9, entire, 7–8-rayed, (ω) 8–9. Opisthosoma (Fig. 8a) with 34–42 dorsal annuli and 46–49 ventral annuli. Seta c2 29–39, on annulus 1–2; seta d 38–54, 29–31 apart, 25–38 microtubercles between them, on ventral annulus 10–13; seta e 10–11, 11–13 apart, 8–14 microtubercles between them, on ventral annulus 25–27; seta f 22–26, 19–22 apart, 18–25 microtubercles apart, on ventral annulus 42–46. Seta h1 3*, h2 67–71. External genitalia (Figs. 8d, 9d) subtriangular, 19–20 wide, setae 3a 19–26. Posterior part of the epiandrium (external genitalia), between and behind genital setae 3a, with microtubercles aligned irregularly.

Discussion

Divergence within grass‑associated Abacarus complex

The integrative approach combining morphometric and molecular data allowed us to explain the observed morphological variation in mites associated with sugarcane in the Americas. The pattern of clustering of morphometric traits together with the genetic diver-gence of three gene fragments revealed the existence of three Abacarus species on sugar-cane in the Americas, one of which is a newly recognized and described species.

The divergence in DNA sequences among these species was comparable to or greater than estimates of interspecific variation in other invertebrate taxa (e.g., Skoracka et  al. 2012; Arribas et  al. 2013; Miller et  al. 2013; Lewandowski et  al. 2014; Li et  al. 2014; Cvrković et  al. 2016; Oca et  al. 2016; Xue et  al. 2017; Richter et  al. 2018). The high-est divergences among Abacarus species associated with sugarcane were detected for the mitochondrial gene fragment COI; they ranged from 19.5 to 24.7%. These values are almost as great as the distances between other Abacarus phylogenetic lineages and the out-group species (Table 2). Similar distance values (ca. 20%) have been detected between two Abacarus species collected from quackgrass and ryegrass (Skoracka and Dabert 2010) for which reproductive barriers has been shown in crossing experiments (Skoracka 2008).

The divergence in nuclear markers also supports the existence of three sugarcane-asso-ciated species (Tables 3 and 4). The ITS distances among them range from 4.0 to 17.9%, suggesting the interspecific level by comparison to the distance between the well-defined Ac. tosichella and Ac. eximia species (6.6%), 1.4% distance between two separate species

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of Ac. tosichella complex (Carew et al. 2009), 2% for tetranychid mites (e.g., Navajas et al. 1994, 1998; Ben-David et al. 2007), 1.2% for Anopheles albitarsis complex (Li and Wilk-erson 2007), or 3.5% for cryptic species of the monogean ectoparasite Gyrodactylus sp. (Bueno-Silva et al. 2011). The variation in the D2 sequences between A. sacchari and A. neosacchari n. sp.was lower (2.2%) than the observed variation for the ITS marker but comparable as between the outgroup species Ac. eximia and Ac. tosichella (2.4%) (Table 3). It was also higher than the variation between A. hystrix and A. lolii (0.4%), for which repro-ductive barriers have been demonstrated experimentally (Skoracka 2008). Low D2 varia-tion between species can be expected since nuclear ribosomal sequences are much more conserved than COI sequences, and closely related species often possess 28S rDNA that is identical or very similar (Lee and O’Foighil 2004).

Interestingly, in our analyses A. hystrix clustered in two clades (Fig. 3), and the COI intraspecific distance between them was high (ca. 13%) and comparable to the interspecific distances in many animal taxa (Hebert et  al. 2003). This may indicate that they in fact consist of different species. These results demonstrate that a vast amount of undescribed diversity may be hidden within grass-associated Abacarus complex, what has been also indicated by Laska et al. (2018). Certainly, the relationships and species boundaries within the Abacarus complex still require further attention.

In summary, our results confirmed the monophyly of Abacarus species associated with grasses as well as the monophyly of three sugarcane-associated species. In addition, our findings showed the association of three phylogenetically related taxa with the same sugar-cane host. This finding is interesting from the ecological point of view and raises questions about their possible evolution, which is discussed below.

Abacarus species associated with sugarcane

Five valid Abacarus species associated with Saccharum spp. have been reported around the world up to date, i.e., A. sacchari, A. delhiensis, A. queenslandiensis, A. doctus, and herein described A. neosacchari n. sp. Two species, Abacarus officinari Keifer and Abacarus fujianensis Xin and Ding, which were reported from sugarcane, were synonymized as A. sacchari (Ozman-Sullivan et al. 2006). However, based on the original descriptions, some morphometric traits of A. sacchari clearly differ from those of A. officinari and A. fujian-ensis. Therefore, it would be worthwhile to apply molecular and morphometric approaches to verify whether these synonimized species actually constitute the same taxa or, as in the case of A. sacchari and A. neosacchari n. sp., constitute a complex of cryptic species.

The new species found on sugarcane in Brazil is the second Abacarus species described from the Americas. We hypothesize that, as was suggested for A. doctus (Navia et  al. 2011), A. neosacchari n. sp. is not native to the Neotropical region but was introduced (e.g., through the exchange of plant material) from the region of origin of its sugarcane host plant, namely, from the tropical areas of Asia. Another possibility is that this new species was associated with an American native grass species and has recently shifted and adapted to sugarcane. Such a scenario was suspected for the coconut mite Aceria guer-reronis Keifer adaptation event to coconut in the Americas (Navia et  al. 2005), which, however, appeared to originate in the Americas (Navia et al. 2005). Host shifts and subse-quent adaptation to novel host plants are important drivers of speciation in phytophagous invertebrates (e.g., Berlocher and Feder 2002; Janz et al. 2006), with the apple maggot fly, Rhagoletis pomonella, the most classic example of a shift between its native host hawthorn (Crataegus spp.) to the introduced and domesticated apple Malus domestica (Bush 1969;

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Feder et al. 1988). To determine whether A. neosacchari n. sp. was associated with another grass host in Brazil or whether its occurrence on sugarcane in America is a result of inter-continental shipment, more extensive sampling and the use of population genetic tools is warranted.

Both A. sacchari and A. neosacchari n. sp. are widely distributed in Brazil, and mixed infestations of these species occur on cultivated sugarcane (Duarte 2016, unpubl. thesis). Assemblages consisting of several cryptic species on cultivated plants appear to be com-mon in eriophyoid mites. For example, mixed infestations of the MT-1, MT-3 and MT-8 genotypes of the wheat curl mite complex have frequently been found on wheat (Skoracka et  al. 2017). Such infestations are challenging to management decision-making because different (even closely related) species may respond differently to plant resistance and may have different abilities to transmit plant pathogens (Bickford et al. 2007).

Surveys and detailed taxonomic studies should be conducted to determine whether the new taxon, A. neosacchari sp. n., is present in other countries where sugarcane is culti-vated in Asia, Americas, and Oceania, just as A. sacchari is widespread around the world (Ozman-Sullivan et al. 2006; J.W. Amrine, pers. comm.). Increased knowledge on the dis-tribution of Abacarus species associated with sugarcane around the world is crucial to the establishment of phytosanitary barriers to avoid the dissemination of phytophagous mites that can reach pest status through the exchange of plant material.

Host‑plant hybridization and cryptic diversity of eriophyoid mites

Hybridization is an important mechanism contributing to speciation and responsible for the evolution of many plant taxa (Goulet et al. 2017). This process also influences the dis-tribution, performance, and level of parasitism of herbivores associated with hybrid versus parental plant species (Whitham 1989; Preszler and Boecken 1994; Whitham et al. 1999; Hochwender and Fritz 2004). It has also been suggested that hybridization may have evo-lutionary consequences not only for hybridizing plants but also for host-dependent plant-feeding arthropods by influencing their host shifts, host-race formation and diversification (Floate and Whitham 1993; Evans et al. 2008). Evans et al. (2008) observed that popula-tions of the bud-galling eriophyoid mite Aceria parapopuli (Keifer) found on pure Populus angustifolia and P. angustifolia × P. fremontii hybrids are phylogenetically separated, indi-cating that these populations represent morphologically cryptic species.

Actually, many eriophyoid mites that have been classified as single species have been found, upon closer inspection, to represent complexes of cryptic species (Skoracka and Dabert 2010; Skoracka et  al. 2012, 2014; Miller et  al. 2013; Lewandowski et  al. 2014; Navia et al. 2015). The most noticeable examples include: grass-associated wheat curl mite (Ac. tosichella) which consists of at least 29 genetic lineages that differ in their host speci-ficity (Skoracka et al. 2012, 2018; Miller et al. 2013); Retracrus johnstoni Keifer, which constituted at least two cryptic species, one associated with Heliconiaceae and the second associated with Arecaceae (Navia et  al. 2015); and Phytoptus avellanae Nalepa, associ-ated with hazel species which consists of two distinct lineages: gall-making and vagrant (Cvrković et al. 2016). Our study also contributes to the knowledge of cryptic diversity in eriophyoid mites by providing evidence of three cryptic species within sugarcane-associ-ated Abacarus mites. They can be discriminated based on morphometric and molecular tools. Two of them, A. sacchari and A. neosacchari sp. n., are genetically closer to each other than to A. doctus what indicates a recent speciation event. All three sugarcane-asso-ciated species are genetically more closely related to each other than to other Abacarus

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species associated with other grasses. The finding that closely related species are associ-ated with the same host-plant is intriguing because theory suggests that related species seg-regate their niches as a result of competition (McArthur and Levins 1967). Indeed, cryptic lineages of Ac. tosichella complex inhabiting the same plant species are distinctly related, and their current coexistence was interpreted as an effect of secondary contact while their speciation occurred in allopatry (Skoracka et al. 2017). Sharing of plant species by geneti-cally related A. sacchari, A. neosacchari n. sp. and A. doctus could be explained by the scenario in which each of the mite species evolved from a common sugarcane ancestor but on different cultivars of sugarcane that have been subsequently used for crosses to obtain commercial sugarcane hybrids. The phenomenon observed nowadays may be an effect of secondary contact between mite species and some competition between them is expected. To reveal whether this scenario is true requires further genetic and ecological observations.

The diversity of Abacarus mites associated with sugarcane may be related to the his-tory and dissemination of this crop in the world. As a cultivated plant, sugarcane spread along human migration routes to Southeast Asia, India and the Pacific, where it hybrid-ized with wild canes. It reached the Mediterranean in approximately 500 B.C. (Fauconnier 1993) and spread to many southern European countries, followed by introduction to West Africa, Central and South America, the West Indies (Fauconnier 1993), and Australia (Bull and Glasziou 1979). The fact that sugarcane originated from crosses between species of Saccharum and other grasses (Daniels and Roach 1987) may have influenced the genetic differentiation and emergence of different Abacarus species. Further studies focusing on eriophyoid mite communities associated with sugarcane progenitors around the world are necessary to enhance our understanding of the effect of plant hybridization on speciation in this specialized group of phytophagous mites. Our study, which reveals cryptic diversity within sugarcane-associated Abacarus mites, is the first step in determining whether the hybridization of Saccharum species has played an important role in the evolutionary diver-sification of Abacarus mites.

Acknowledgements We sincerely thank Dr. Elisângela Gomes Fidelis, Embrapa Roraima, Roraima; Dra. Ranyse Barbosa Querino da Silva, Embrapa Meio Norte, Piauí; Dr. Elio Cesar Guzzo, Embrapa Tabuleiros Costeiros, Alagoas, Brazil; Mr. Felipe Alfredo Cerón Martí, Ingênio Central Izalco, Sonsonate, El Salvador; Dr. Hugo Aguilar Piedra, Universidad de Costa Rica, Costa Rica, for their help with sampling. To ‘Coorde-nadoria de Aperfeiçoamento de Pessoal do Ensino Superior’ (CAPES) for the financial support to R.S. Men-donça (PNPD/Agronomia Proc. No. 88882.305808/2018-1), to Fundação de Amparo à Pesquisa do Estado de Alagoas (FAPEAL) and to Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF) for the fellowship to the first author, to ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)’, Brazil, for granting the fellowship to the first and last authors.

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Affiliations

Mércia Elias Duarte1 · Renata Santos de Mendonça2 · Anna Skoracka3 · Edmilson Santos Silva4,5 · Denise Navia1

Mércia Elias Duarte mercia_elias@hotmail.com

Renata Santos de Mendonça mendonca.rsm@gmail.com

Anna Skoracka skoracka@amu.edu.pl

Edmilson Santos Silva silva_es@yahoo.com.br

1 Embrapa Recursos Genéticos e Biotecnologia, Brasilia, Distrito Federal 70770-900, Brazil2 Faculdade de Agronomia e Medicina Veterinária, Universidade de Brasília, Campus Darcy

Ribeiro, Brasília, DF 70297-400, Brazil3 Population Ecology Lab, Faculty of Biology, Institute of Environmental Biology, Adam

Mickiewicz University, Uniwersytetu Poznańskiego 6, Poznań 61-614, Poland4 Universidade Federal de Alagoas, Campus Arapiraca, Arapiraca 57309-005, Brazil5 Universidade Federal de Alagoas, Centro de Ciências Agrárias (CECA), Alagoas 57100-000,

Brazil