”Unmasking the Villain”: Integrative Taxonomy Reveals the Real Identity of the Key Pest (Thysanoptera: Thripidae) of Peanuts (Arachis hypogaea L.) in South America

Simple Summary In this work, we aimed to resolve the identification of the peanut thrips, the key pest of Arachis hypogaea in South America. Based on morphological, biological, and molecular data, we conclude that the name historically applied to this pest, Enneothrips flavens, constitutes a misidentification and that the peanut thrips is actually an undescribed species, Enneothrips enigmaticus sp. n. Abstract The peanut thrips, Enneothrips enigmaticus sp. n., is the key pest of Arachis hypogaea L. in South America, where it can cause yield losses of up to 85%. This species has historically been identified as Enneothrips flavens, but access to the holotype of this species and freshly collected material from southeastern and northern Brazil revealed that specimens commonly collected on peanut crops are not conspecific with E. flavens. Biological, molecular, and morphological assessments were carried out and led to the conclusion that the key pest of A. hypogaea belongs to a previously undescribed species: Enneothrips enigmaticus sp. n.


Introduction
The peanut thrips is the key pest of peanuts crops, Arachis hypogaea L. (Fabaceae), in South America. This pest is found in Argentina, Brazil, and Paraguay and can cause losses of up to 85% [1,2]. The species is commonly found on leaflets, where it feeds on the tissue, causing bronze markings and deformation [1,3].
The first record of a name applied to the peanut thrips was Frankliniella fusca (Hinds), which was a misidentification subsequently corrected by Gallego de Sureda, who identified the species as Enneothrips (Enneothripiella) flavens Moulton [4]. This name was previously proposed by Moulton [5] to describe a species based on a unique female specimen collected in Minas Gerais state, Brazil, on "Indian tea" plants. Due to the identification by Gallego de Sureda [4], peanut thrips has been referred to as E. flavens and host-specific of A. hypogaea [6].
However, specimens of Enneothrips recently collected on plants other than A. hypogaea were identified as E. flavens. This fact suggests that either E. flavens is not host-specific or Enneothrips specimens previously collected from A. hypogaea were misidentified. Both hypotheses have a serious impact because if the species is not host-specific to A. hypogaea, the control tactics must take into account that alternative hosts may harbor the pest when

Molecular Data Analysis
The chromatogram of each COI sequence was verified, aligned, and edited in a consensus sequence using Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI). Afterwards, COI sequences were aligned, and the presence of nuclear paralogs of mitochondrial origin (NumtS) [11] was observed following steps described in [12]: (i) insertions/deletions (indels), (ii) stop codons leading to premature protein termination, and (iii) increased rates of non-synonymous mutations. The presence of signatures (i) and (ii) would be enough to consider a sequence as a NumtS. The presence of signature (iii) would be used to confirm the NumtS status of the sequence. The genetic distance among haplotypes was estimated using the Kimura-two-parameters (K2P) model with 5000 bootstrap replications in MEGA X [13]. A Bayesian phylogenetic tree was estimated using the best substitution model of evolution GTR+I selected using the software MRMODELTEST v2.3 [14]. The Bayesian phylogenetic analyses were carried out in MRBAYES v3.1.2 [15], using two simultaneous runs of 25 million generations with one cold and three heated chains in each run. At the end of the runs, the first 25% of the trees were discarded as burn-in samples. The consensus tree of the two independent runs was obtained with posterior probabilities > 0.50. The COI sequence of Frankliniella occidentalis (NCBI accession number: HQ930545) was used as outgroup. No sequences of other Enneothrips spp. are available in NCBI; thus, sequences of Insects 2022, 13, 120 4 of 13 the common species F. occidentalis were chosen exclusively to estimate the genetic distance, not phylogenetic inferences.

Species Distribution Modelling
As the distribution of E. enigmaticus sp. n. is not well studied, the potential distribution in the Americas was modelled based on taxonomic validated species records on peanuts from Thysanoptera collections from USNM, CHNUFPI MLP, and ESALQ (Table 1). For non-georeferenced records, we used the label information to retrieve the most probable coordinates. A total of 19 environmental variables were used to estimate the potential distribution, which included precipitation, temperature, moisture index, and radiation, at a 10 min spatial resolution. These were obtained from the WorldClim dataset and cover climate information in the period 1970-2000. Analysis for E. flavens was not performed because there are very few records of this species to make the analysis possible. Modelling was performed through multivariate analysis and maximum entropy using MaxEnt 3.4.4 [16]. MaxEnt estimates the probability of occurrence based on environmental parameters and has predictive power even with small datasets [17]. Model settings were defined after a bias analysis using the software R 4.0.2. The chosen model, with the least delta AIC, included linear features with a regularization multiplier of 1.0.
This information is relevant not only because of the economic importance of E. enigmaticus sp. n., but also because the model results provide indication of a possible shared distribution with E. flavens, which can help understand the evolutionary pathways of both species.

Results
Based on morphological, biological and molecular evidence detailed below, we describe the new species E. enigmaticus sp. n. as the pest on peanuts (Figure 1). We re-evaluate the morphological description of E. flavens while also describing the adult female and male and larvae II of both species. This information is essential for the recognition of both

Results
Based on morphological, biological and molecular evidence detailed below, we describe the new species E. enigmaticus sp. n. as the pest on peanuts (Figure 1). We re-evaluate the morphological description of E. flavens while also describing the adult female and male and larvae II of both species. This information is essential for the recognition of both species and constitutes the first step towards the adoption of management tactics for the key peanut pest. Enneothrips enigmaticus sp. n. http://zoobank.org/urn:lsid:zoobank.org:act:E2CBCE8C-7508-4C46-B380-5AF2602FFE9D Female macroptera. Color orange to reddish brown ( Figure 2A). Antennal segments I and II light brown, III brown with base pale, IV and V brown with extreme base pale, VI-IX brown ( Figure 2C). Head, thorax, and abdomen reddish brown, somewhat darker laterally ( Figure 2D,F); fore wing reddish brown with pale basal fifth ( Figure 2H); legs yellow; abdominal tergites uniformly reddish brown. Head with ocellar area without sculpture; ocellar setae III within the ocellar triangle ( Figure 2D,E); pronotum and mesonotum transversely striate, around 20 lines of sculpture on mesonotum ( Figure 2F,G); metanotum longitudinally sculptured on lateral thirds, transversely sculptured on anterior third, and reticulate on the median two-thirds, without internal lines inside reticles ( Figure 2F,G Male brachyptera. Similar to female ( Figure 2B), but smaller, brachypterous ( Figure  2I,J), with a broad pore plate between the abdominal sternites II and III ( Figure 2K Larva II. Color, pale ( Figure 3A) with brown areas anteromedially on head ( Figure  3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases of femora and tibiae. Antennal segment I pale, II brown with pale apex, III brown with pale base and apex, Enneothrips enigmaticus sp. n. http://zoobank.org/urn:lsid:zoobank.org:act:E2CBCE8C-7508-4C46-B380-5AF2602FFE9D Female macroptera. Color orange to reddish brown ( Figure 2A). Antennal segments I and II light brown, III brown with base pale, IV and V brown with extreme base pale, VI-IX brown ( Figure 2C). Head, thorax, and abdomen reddish brown, somewhat darker laterally ( Figure 2D,F); fore wing reddish brown with pale basal fifth ( Figure 2H); legs yellow; abdominal tergites uniformly reddish brown. Head with ocellar area without sculpture; ocellar setae III within the ocellar triangle ( Figure 2D,E); pronotum and mesonotum transversely striate, around 20 lines of sculpture on mesonotum ( Figure 2F,G); metanotum longitudinally sculptured on lateral thirds, transversely sculptured on anterior third, and reticulate on the median two-thirds, without internal lines inside reticles ( Figure 2F,G Male brachyptera. Similar to female ( Figure 2B), but smaller, brachypterous ( Figure 2I,J), with a broad pore plate between the abdominal sternites II and III ( Figure 2K Larva II. Color, pale ( Figure 3A) with brown areas anteromedially on head ( Figure 3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases of femora and tibiae. Antennal segment I pale, II brown with pale apex, III brown with pale base and apex, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body dorsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. Body densely covered with sclerotized plates, absent on head, less numerous on pronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and abdominal tergites II    Larva II. Color, pale ( Figure 3A) with brown areas antero 3C), posteriorly on abdominal segment X ( Figure 3F) and on tibiae. Antennal segment I pale, II brown with pale apex, III b apex, IV-VII brown. Antennal segment IV with rows of micr dorsal setae expanded, except for cephalic pair D3 acute (Figur Body densely covered with sclerotized plates, absent on pronotum and abdominal tergites IX and X ( Figure 3F) Figure 3F) and on the bases of femora and gment I pale, II brown with pale apex, III brown with pale base and n. Antennal segment IV with rows of microtrichia ( Figure 3B). Body ded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. vered with sclerotized plates, absent on head, less numerous on dominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E  Larva II. Color, pale ( Figure 3A) with brown areas anteromedially on head ( Figure  3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases of femora and tibiae. Antennal segment I pale, II brown with pale apex, III brown with pale base and apex, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body dorsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. Body densely covered with sclerotized plates, absent on head, less numerous on pronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and abdominal tergites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D Figure 3A) with brown areas anteromedially on 3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases o tibiae. Antennal segment I pale, II brown with pale apex, III brown with p apex, IV-VII brown. Antennal segment IV with rows of microtrichia (Figu dorsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventr Body densely covered with sclerotized plates, absent on head, less n pronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on me abdominal tergites II and VIII; spiracle facets variably with zero or one pore Abdominal tergites IX and X with one pair of campaniform sensilla.
Measurements (in microns   Color, pale ( Figure 3A) with brown areas anteromedially on head (Figure ly on abdominal segment X ( Figure 3F) and on the bases of femora and al segment I pale, II brown with pale apex, III brown with pale base and rown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body panded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. covered with sclerotized plates, absent on head, less numerous on abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and gites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E   Larva II. Color, pale ( Figure 3A) with brown areas anteromedially on head ( Figure  3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases of femora and tibiae. Antennal segment I pale, II brown with pale apex, III brown with pale base and apex, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body dorsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. Body densely covered with sclerotized plates, absent on head, less numerous on pronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and abdominal tergites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E)   Larva II. Color, pale ( Figure 3A) with brown areas anteromedially on head ( Figure  3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases of femora and tibiae. Antennal segment I pale, II brown with pale apex, III brown with pale base and apex, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body dorsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. Body densely covered with sclerotized plates, absent on head, less numerous on pronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and abdominal tergites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E) Figure 3A) with brown areas anteromedially on head ( Figure  3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases of femora and tibiae. Antennal segment I pale, II brown with pale apex, III brown with pale base and apex, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body dorsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. Body densely covered with sclerotized plates, absent on head, less numerous on pronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and abdominal tergites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E) Figure 3F) and on the bases of femora and nal segment I pale, II brown with pale apex, III brown with pale base and brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body xpanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. y covered with sclerotized plates, absent on head, less numerous on d abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and rgites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E) Figure 3F) and on the bases of femora and al segment I pale, II brown with pale apex, III brown with pale base and brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body xpanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. covered with sclerotized plates, absent on head, less numerous on d abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and gites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E) Figure 3A) with brown areas anteromedially on head 3C), posteriorly on abdominal segment X ( Figure 3F) and on the bases of fem tibiae. Antennal segment I pale, II brown with pale apex, III brown with pale apex, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3 dorsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral set Body densely covered with sclerotized plates, absent on head, less nume pronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesono abdominal tergites II and VIII; spiracle facets variably with zero or one pore (Figu Abdominal tergites IX and X with one pair of campaniform sensilla. Measurements ( Figure 3F) and on the bases of femora and ibiae. Antennal segment I pale, II brown with pale apex, III brown with pale base and pex, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body orsal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. ody densely covered with sclerotized plates, absent on head, less numerous on ronotum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and bdominal tergites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E). bdominal tergites IX and X with one pair of campaniform sensilla.
Measurements    Figure 3F) and on the bases of femora and ae. Antennal segment I pale, II brown with pale apex, III brown with pale base and x, IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body sal setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. y densely covered with sclerotized plates, absent on head, less numerous on notum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and ominal tergites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E 14.xii.1993, ; 21.xii.1993, 19♀; 4.i.1994 Figure 3F) and on the bases of femora and al segment I pale, II brown with pale apex, III brown with pale base and rown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body panded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. covered with sclerotized plates, absent on head, less numerous on abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and gites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E Figure 4A,C-E,G) but with shadings, especially medially on abdominal tergites ( Figure 4A),  Figure 3F) and on the bases of femora and l segment I pale, II brown with pale apex, III brown with pale base and rown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body panded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. covered with sclerotized plates, absent on head, less numerous on abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and ites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E   Figure 3F) and on the bases of femora and e. Antennal segment I pale, II brown with pale apex, III brown with pale base and , IV-VII brown. Antennal segment IV with rows of microtrichia ( Figure 3B). Body al setae expanded, except for cephalic pair D3 acute ( Figure 3C). Ventral setae acute. y densely covered with sclerotized plates, absent on head, less numerous on otum and abdominal tergites IX and X ( Figure 3F). Spiracles on mesonotum and minal tergites II and VIII; spiracle facets variably with zero or one pore ( Figure 3D,E   single mutation step and distributed in both sample sites, Jaboticabal (H3 = 2 and H4 =1) and Rio Branco (H3 = 1 and H4 = 3) (NCBI accession number: H3 = MT947753-MT947755 and H4 = MT947756-MT947759) ( Figure 5). The intraspecific genetic distance of the COI gene fragment under each species was 0.0015. The interspecific genetic distance (K2P) of the COI gene fragment between E. flavens and E. enigmaticus sp. n. varied from 0.1872 to 0.1892. Enneothrips enigmaticus sp. n. and E. flavens are the only representatives of the genus occurring south of 20° S, and at present the only confirmed records are in South America, especially near the Tropic of Capricorn. Modelling, however, indicates that the species may find suitable conditions in other areas of the continent, especially in tropical countries ( Figure 6). Morphological comparison of specimens collected in peanuts (E. enigmaticus sp. n.) with the types of species currently classified in Enneothrips confirmed that E. flavens is the closest related species. However, the following morphological differences between E. enigmaticus sp. n. and E. flavens were noticed: (i) internal markings between sculpturing lines on metanotum present only in E. flavens (Figures 2F and 4E); (ii) sculpturing lines on mesonotum less numerous in E. enigmaticus sp. n. (Figure 2F,G); (iii) abdominal tergites lighter and more uniformly colored in E. enigmaticus sp. n. (Figure 2A,B) and shaded brown medially in E. flavens ( Figure 4A,B). In addition, individuals morphologically identical to the E. flavens holotype were collected in the states of São Paulo and Minas Gerais and were examined from mounted material deposited in USNM from Santa Catarina state. Among the individuals, males of E. enigmaticus sp. n. were brachypterous ( Figure 2B), while males of E. flavens were always macropterous ( Figure 4B).
Forward and reverse sequences were analyzed together, but as they generated different sizes, a consensus sequence was made, according to the usual procedure for estimating haplotypes. COI sequences were edited in a consensus size of 672 pb. Evidence of NumtS presence was not found in the sequences. The two sequences of E. flavens resulted in two haplotypes (H1 and H2) separated by a single mutation step ( Figure 5) (NCBI accession number: H1 = MT947751 and H2 = MT947752). The seven sequences of E. enigmaticus sp. n. resulted also in the presence of two haplotypes (H3 and H4) separated by a single mutation step and distributed in both sample sites, Jaboticabal (H3 = 2 and H4 = 1) and Rio Branco (H3 = 1 and H4 = 3) (NCBI accession number: H3 = MT947753-MT947755 and H4 = MT947756-MT947759) ( Figure 5). The intraspecific genetic distance of the COI gene fragment under each species was 0.0015. The interspecific genetic distance (K2P) of the COI gene fragment between E. flavens and E. enigmaticus sp. n. varied from 0.1872 to 0.1892.
Enneothrips enigmaticus sp. n. and E. flavens are the only representatives of the genus occurring south of 20 • S, and at present the only confirmed records are in South America, especially near the Tropic of Capricorn. Modelling, however, indicates that the species may find suitable conditions in other areas of the continent, especially in tropical countries ( Figure 6).

Discussion
The differences reported above are based on comparisons of more than 100 specimens of Enneothrips from peanuts either collected or examined from collections and almost 30 individuals identified here as E. flavens, including the species holotype. Thus, they constitute robust evidence that the name historically applied to the key peanut pest has been misidentified. Males of both Enneothrips enigmaticus sp. n. and E. flavens exhibit an internal pore plate between sternites II and III, a character shared with other thrips genera from the Neotropics, namely, Ameranathrips, Apterothrips, Baileyothrips, Charassothrips, Desartathrips, Enneothrips, Pseudothrips, Psydrothrips, and Xerothrips [18]. The easily found males of E. enigmaticus sp. n. might open possibilities to study their behavior and the significance of such pore plate character. It is also possible to develop studies aiming to understand the role of the reduced wings in E. enigmaticus sp. n. males, a character found only in this species in the genus.
Immatures of peanut thrips have been found only from that crop, although a few adults have been collected from other plants [6]. In our surveys, we found larvae of E. enigmaticus sp. n. on peanuts, thus confirming the host association with the A. hypogaea ( Figure 3K). A good number of adults have also been collected from Arachis pintoi¸ a possible alternative host in the same plant genus that could indicate Arachis spp. as hosts of E. enigmaticus sp. n. As for E. flavens, no specimens that match its holotype morphology have been collected from peanuts. On the other hand, several specimens of this thrips species were collected from at least four plant species, one of which (Campomanesia guazumifolia) ( Figure 3L) with immatures. The large number of adult specimens from several plants and the larvae found in one of the plant species suggests that, unlike E. enigmaticus

Discussion
The differences reported above are based on comparisons of more than 100 specimens of Enneothrips from peanuts either collected or examined from collections and almost 30 individuals identified here as E. flavens, including the species holotype. Thus, they constitute robust evidence that the name historically applied to the key peanut pest has been misidentified. Males of both Enneothrips enigmaticus sp. n. and E. flavens exhibit an internal pore plate between sternites II and III, a character shared with other thrips genera from the Neotropics, namely, Ameranathrips, Apterothrips, Baileyothrips, Charassothrips, Desartathrips, Enneothrips, Pseudothrips, Psydrothrips, and Xerothrips [18]. The easily found males of E. enigmaticus sp. n. might open possibilities to study their behavior and the significance of such pore plate character. It is also possible to develop studies aiming to understand the role of the reduced wings in E. enigmaticus sp. n. males, a character found only in this species in the genus.
Immatures of peanut thrips have been found only from that crop, although a few adults have been collected from other plants [6]. In our surveys, we found larvae of E. enigmaticus sp. n. on peanuts, thus confirming the host association with the A. hypogaea ( Figure 3K). A good number of adults have also been collected from Arachis pintoi¸a possible alternative host in the same plant genus that could indicate Arachis spp. as hosts of E. enigmaticus sp. n. As for E. flavens, no specimens that match its holotype morphology have been collected from peanuts. On the other hand, several specimens of this thrips species were collected from at least four plant species, one of which (Campomanesia guazumifolia) ( Figure 3L) with immatures. The large number of adult specimens from several plants and the larvae found in one of the plant species suggests that, unlike E. enigmaticus sp. n., E. flavens is probably