Five new species of Aspidiotini (Hemiptera, Diaspididae, Aspidiotinae) from Argentina, with a key to Argentine species

Abstract Five new species of armored scale insect from Argentina are described and illustrated based upon morphological and molecular evidence from adult females: Chortinaspisjujuyensissp. nov., Clavaspispatagonensissp. nov., Hemiberlesiaozolitasp. nov., Melanaspislilloisp. nov., and Melanaspistargionoidessp. nov. The genera Chortinaspis and Melanaspis are recorded for the first time from this country. An identification key to all recorded species from tribe Aspidiotini occurring in Argentina is provided.


Introduction
Armored scale insects are ubiquitous, highly invasive herbivores that often become pests on trees, shrubs, and ornamentals (Miller and Davidson 1990;Normark et al. 2019). They comprise the largest family of scale insects (Hemiptera, Coccomorpha, Diaspididae) with over 2600 species in 422 genera (García Morales et al. 2016). About onequarter of armored scales belong to the tribe Aspidiotini Westwood, a particularly pestiferous group containing numerous cosmopolitan species (Miller and Davidson 1990;Schneider et al. 2019). At present the tribe comprises over 720 species in 88 genera; however, recent phylogenetic evidence has revealed rampant artificiality among these genera (Schneider et al. 2018), and their classification is in need of extensive revision.
This article describes five new species of Aspidiotini from Argentina. Generic designations are made following morphology and the best available evidence from molecular studies (Schneider et al. 2018;Normark et al. 2019), keeping in mind the ultimate goal to identify genera of Aspidiotini that delimit natural groups of species. This article also provides an identification key to the species of Aspidiotini recorded from Argentina (García Morales et al. 2016Morales et al. , last accessed 21.V.2020. This work adds to our knowledge of armored scale diversity in this region, for which a foundation has largely been established by Claps, Wolff, and colleagues (Claps and Terán 2001;Claps et al. 2001a;b;Claps and Wolff 2003;Granara de Willink and Claps 2003;and additional works).

Materials and methods
Specimens were preserved in 100% ethanol and stored at -20 °C before being slidemounted. Specimens were slide-mounted according to the joint DNA-morphology preparation protocol described in Normark et al. (2019), except that some specimens were prepared according to an earlier set of protocols. In the earlier protocols some specimens were ground to powder for DNA preparation and others from the same series were transferred directly to 10% KOH for mounting on microscope slides.
Morphological terminology conforms to descriptions and illustrations provided by Schneider et al. (2019). Measurements were made on a Zeiss Axio Imager.M2 (Carl Zeiss Microscopy, LLC, White Plains, NY, USA) microscope with the aid of an Axi-oCam and AxioVision software. Illustrations were made with the aid of a camera lucida. Slide-mounted specimens were examined by the authors under phase contrast and Differential Interference Contrast microscopy.  segments I-III. Small clusters of ventral submarginal microducts present on abdominal segments II-VI. Anal opening: Small and slightly oval, 11-17 (median 14) μm in diameter, positioned 2.2-3.3 (median 2.3) anal lengths from base of L1, located in posterior third of pygidium. Perivulvar pores: Absent.
Remarks. Chortinaspis jujuyensis sp. nov. shares similarities with C. graminella (Cockerell) and C. frankliniana Ferris. The median lobes of C. jujuyensis sp. nov. are apically truncate or nearly rectangular in shape like those of C. graminella and have rough apical edges like those of C. frankliniana. But C. jujuyensis sp. nov. can be distinguished from both species by its narrow, smooth second lobes, in contrast to the broadly truncate and notched second lobes seen in the other two species. It differs from C. chortina (Ferris) in that it lacks any plates anterior to the position of the third lobes.
Host plant. Not recorded. Etymology. The specific epithet is an adjective formed from the name Jujuy, the province in which it was found + the suffix -ensis, meaning of or from a place.
Distribution. Argentina (Jujuy). Derm membranous throughout at maturity except for light pygidial sclerotization. Antennae simple, each with one spine-like seta. Distance between antennae 150-185 μm. Without disc pores associated with anterior or posterior spiracles. Lobes: Only L1 well developed and sclerotized, slightly wider than long, inner margins parallel or slightly converging, with 0-1 medial notch and 1-2 lateral notches; median lobes separated by space 1/5 their width; L2 and L3 absent in typical form, one specimen with single poorly formed L2 present in type series. Paraphyses: With 1 pair of paraphysis-like pyriform sclerotizations between L1; interlobular spaces between L1 and L2 and between L2 and L3 each with 2 clavate paraphyses, inner paraphysis slightly larger than outer paraphysis of each pair; paraphyses arising from lateral angle of L1 only slightly swollen at anterior end and directed away from meson. Plates: Difficult to observe; apparently 1 or 2 present between L1 and L2, 2 present between L2 and L3, 0-3 beyond L3, all roughly rectangular with minor fringing at apex, about as long as L1. Ducts: Dorsal pygidial macroducts of 1-barred type; one macroduct present between median lobes with duct exceeding beyond posterior margin of anal opening; with 2-3 macroducts arising from first interlobular space; roughly single-file row of 7-8 macroducts arising from second interlobular space; 8-13 in marginal and submarginal areas of abdominal segment V, arising from third interlobular space. Few pre-pygidial macroducts on marginal line from mesothorax to abdominal segment III, 1-3 per segment on each side, shorter than pygidial macroducts; 1-2 submarginal macroducts present on each side of abdominal segment IV; small sets of 1-4 short submedial macroducts present on each side of abdominal segments I-IV. Ventral marginal or submarginal microducts present in small groups on each segment from prothorax to abdominal segment VI. Informal synonyms. A specimen from the type series (D0274B) has appeared in published molecular-phylogenetic analyses, designated as "Clavaspis undescr" (Schneider et al. 2018) and "Clavaspis ud0274" .

Clavaspis patagonensis
Remarks. The traditional morphology-based assignment for this species would be in the genus Diaspidiotus, but recent molecular-phylogenetic studies have shown that Diaspidiotus is radically non-monophyletic and that the true affinities of this species lie with the genus Clavaspis (Schneider et al. 2018). The morphological character traditionally used to distinguish between these genera is the shape of paraphyses arising from the lateral angles of median lobes. In typical Clavaspis species, these paraphyses are swollen at the anterior end and directed toward the midline of the body or they have a detached knob giving them a mushroom-like appearance (Ferris 1938). In C. patagonensis sp. nov., the paraphyses are slightly swollen at the anterior end but they are pointing away from the midline, similar in appearance to those found in species of Diaspidiotus and other near relatives, like Hemiberlesia.
Adult females of C. patagonensis sp. nov. are nearly identical in appearance to C. covilleae (Ferris), but the species are separated on the phylogeny by several other members of Clavaspis. The two can be distinguished based on the shape of paraphyses arising from the lateral angles of median lobes and the distribution of macroducts. Clavaspis patagonensis sp. nov. has fairly narrow paraphyses and possesses one or two dorsal submarginal macroducts on abdominal segment IV. Clavaspis covilleae has broadly swollen paraphyses, typical of Clavaspis, and lacks any submarginal macroducts on abdominal segment IV. The new species could also be easily confused with Diaspidiotus osborni (Newell & Cockerell). In this case, C. patagonensis sp. nov. can be distinguished by possessing submarginal macroducts on IV, having more than one marginal macroduct on at least one pre-pygidial segment, lacking dorsal submarginal microducts on pre-pygidial segments, and having a prosoma that remains membranous in mature adult females. In contrast, D. osborni lacks submarginal macroducts on IV, typically has one marginal macroduct per pre-pygidial segment, has small groups of dorsal submarginal microducts on pre-pygidial segments, and the prosoma becomes sclerotized in mature adult females.
Host plant. Embothrium coccineum J. R. Forst. & G. Forst. (Proteaceae) Etymology. The specific epithet is an adjective formed from the name Patagonia, the region in which it was found + the suffix -ensis, meaning of or from a place.
DNA sequences. DNA sequences of several loci of Hemiberlesia ozolita sp. nov. have been published from one paratype (D0288C) and one other individual from the type series that was ground to powder during the preparation of DNA (D0288A): 28S, Informal synonyms. Specimens from the type series and their endosymbionts have appeared in several published phylogenetic trees, and have been referred to variously as "Diaspidiotus sp undesc #1" (Morse and Normark 2006;Rugman-Jones et al. 2010), "Diaspidiotus sp nov 1" (Andersen et al. 2010), and "Hemiberlesia ud0288" (Schneider et al. 2018;Normark et al. 2019).
Remarks. Hemiberlesia ozolita sp. nov. is most similar to H. nothofagi Williams, but H. ozolita sp. nov. is distinctive in having plates in the first space shorter than L1, 3 plates beyond L2, anal opening relatively small (< 25 μm in diameter), 31-42 macroducts on each side of the pygidium, and groups of ventral submarginal microducts running from the pygidium to the thorax. In contrast, H. nothofagi has plates in the first space exceeding L1 in length, 6-7 plates beyond L2, a large anal opening (30 μm in diameter), about 26 pygidial macroducts per side, and few microducts present on the venter, not arranged in submarginal groups on thoracic and pre-pygidial segments. The new species is also similar to H. rapax except it has a much smaller anal opening and the pre-pygidial macroducts are longer, about as long as the pygidial macroducts.
Hemiberlesia ozolita sp. nov. constitutes the sister-lineage of a clade that includes all other sampled Hemiberlesia species and Palinaspis sordidata, according to the phylogenetic estimate of Schneider et al. (2018). The relatively small anal opening in this species is a trait shared in common with several other species formerly placed in Abgrallaspis that have since been transferred to Hemiberlesia (Normark et al. 2014), a decision supported by molecular evidence.
Host plant. Not recorded. Etymology. The specific epithet is an adjective formed from the Greek terms ozotos, meaning branching, and litos, meaning simple, and is used to describe the distinctly simple pygidial plates of this species.
Distribution. Argentina (Jujuy). broadest near mesothorax. Body outline turbinate. Derm membranous throughout at maturity except for pygidium, which has characteristic dorsal sclerotized areas; sclerotization of these areas unusually heavy, such that paraphyses and basal scleroses of lobes difficult to discern clearly on some specimens. Antennae, simple, each with 1 long seta, distance between antennae 200-330 μm (median 260). Without disc pores near spiracles. Lobes: With 4 pairs of well-developed pygidial lobes, L1-L3 apically rounded and L4 truncate or pointed, notches absent from lobes; L1 slightly wider than long, median lobes separated by narrow space 0.15 times width of L1, with basal sclerosis about 1/2 width of L1 arising from mesal edge; L2 and L3 similar in size and shape, shorter and broader than L1; L4 somewhat variable in shape, truncate or with sloping edges. Paraphyses: Short and clavate, scarcely longer than L1; absent between L1, paraphysis formula 2-2-3 or 2-2-4; 1 interlobular paraphysis near outer corner of L1, 1 attached to inner corner of L2, 1 in interlobular space between L2 and L3, 1 attached to inner and outer corners of L3, 1 narrow paraphysis attached to inner corner of L4 and 2-3 narrow paraphyses in interlobular space between L3 and L4, these often fused into a single complex mass and difficult to count; several paraphysis-like sclerotizations surrounding macroduct orifices present beyond L4. Plates: Apparently absent. Ducts: Dorsal pygidial macroducts of 1-barred type, nearly uniform in size, with minute orifices and long slender ducts, most arranged in distinct furrows between sclerotized areas arising from interlobular spaces; 1 submarginal macroduct orifice immediately anterior to each L1, with ducts extending beyond posterior margin of anal opening; 10-21 (median 16) duct orifices in furrow of first space, originating between L1 and L2 and extending in elongate cluster anteriorly 60-85% of distance to anus, each duct about 120-130 μm in length; 18-40 (median 29) in furrow of second space, originating between L2 and L3 and extending anteriorly to a point laterad or anterolaterad of anus; 3-9 (median 6) on sclerotized area arising from L3; 2-24 (median 15) in furrow of third space, originating between L3 and L4 and extending anteriorly to a point anterolaterad of anus; duct orifices in furrows of second and third spaces membranous, especially towards anterolateral corner of furrow, or surrounded by partial or complete sclerotized ring, especially near posterior end and along medial margin of furrow; submedial clusters of dorsal macroducts present on each pre-pygidial abdominal segment, shorter and narrower than pygidial ducts. Ventral pygidial microducts similar to dorsal macroducts in size and shape and similarly arranged in rows on segments V-VII, 21-44 (median 33) on each side; ventral duct orifices on segment V each surrounded by conspicuous Informal synonyms. Specimens from the type series have appeared in several published phylogenetic trees, and have been referred to variously as "Melanaspis sp. nov." (Gruwell et al. 2005) "Melanaspis sp undesc #2" and "Melanaspis sp undesc #3" (Gruwell et al. 2007;Morse and Normark 2006;Rugman-Jones et al. 2010), "Melanaspis sp. undesc." (Gruwell et al. 2009), "Melanaspis sp nov 1" and "Melanaspis sp nov 2" (Andersen et al. 2010), and "Melanaspis ud0276" (Schneider et al. 2018;Normark et al. 2019).

Melanaspis lilloi
Remarks. This species is very similar to M. targionoides sp. nov. The diagnosis and affinities of M. lilloi sp. nov. are discussed below under the remarks for M. targionoides sp. nov.
Host plant. Not recorded. Etymology. The specific epithet is a noun in the genitive case, meaning "of Lillo". It honors the Instituto Miguel Lillo, academic home of Lucia Claps and the other scientists who first collected the species described in this manuscript.
Distribution. Argentina (Jujuy). broadest near mesothorax. Body outline turbinate. Prosoma becoming sclerotized at full maturity (length > 1400 μm); derm otherwise membranous except for pygidium, which has characteristic dorsal sclerotized areas; sclerotization of these areas unusually heavy, such that paraphyses and basal scleroses of lobes difficult to discern clearly on some specimens. Antennae simple, each with 1 long seta, distance between antennae 130-410 (median 230) μm. Without disc pores near spiracles. Lobes: With 4 pairs of well-developed pygidial lobes, L1-L3 apically rounded and L4 truncate or pointed, notches absent from lobes; L1 slightly wider than long, median lobes separated by narrow space 0.15 times width of L1, with basal sclerosis about 1/2 width of L1 arising from mesal edge; L2 and L3 similar in size and shape, shorter and broader than L1; L4 somewhat variable in shape, truncate or with sloping edges. Paraphyses: Short and clavate, scarcely longer than L1; absent between L1, paraphysis formula 2-2-3 or 2-2-4; 1 interlobular paraphysis near outer corner of L1, 1 attached to inner corner of L2, 1 in interlobular space between L2 and L3, 1 attached to inner and outer corners of L3, 1 narrow paraphysis attached to inner corner of L4 and 2-3 narrow paraphyses in interlobular space between L3 and L4, these often fused into a single complex mass and difficult to count; several paraphysis-like sclerotizations surrounding macroduct orifices present beyond L4. Plates: Apparently absent. Ducts: Dorsal pygidial macroducts of 1-barred type, nearly uniform in size, with minute orifices and long slender ducts, most arranged in distinct furrows between sclerotized areas arising from interlobular spaces; 1 submarginal macroduct orifice immediately anterior to each L1, with ducts extending beyond posterior margin of anal opening; 17-36 (median 29) duct orifices in furrow of first space, originating between L1 and L2 and extending in elongate cluster anteriorly 90% of distance to anus or farther, anterior end of cluster usually directly laterad of anus, each duct about 120-130 μm in length; 18-53 (median 35) in furrow of second space, originating between L2 and L3 and extending anteriorly to a point laterad or anterolaterad of anus; 7-14 (median 9) on sclerotized area arising from L3; 15-30 (median 19) in furrow of third space, originating between L3 and L4 and extending anteriorly to a point anterolaterad of anus; duct orifices in furrows of second and third spaces each surrounded by sclerotized ring; submedial clusters of dorsal macroducts present on each pre-pygidial abdominal segment, shorter and narrower than pygidial ducts. Ventral pygidial microducts similar to dorsal macroducts in size and shape and similarly arranged in rows on segments V-VII, 23-51 (median 35) on each side; ventral duct orifices on segment V each surrounded by conspicuous sclerotized ring, degree of sclerotization decreasing towards anterolateral corner of segment; microducts also distributed along head, thorax, and pre-pygidial margins, as well as rows extending from marginal area toward each spiracle. Anal opening: Oval, 14-31 (median 20) μm long, positioned 4-9 (median 6) anal lengths (102-144 μm, median 129 μm) from base of L1, near midpoint of pygidium. Perivulvar pores: Absent or present; 0-11 (median 0, holotype 1) pores in total, distributed as one loose cluster on only one side of the body (5 of 7 individuals with pores present) or as one loose cluster on each side of the body. ing DNA preparation; morphological vouchers were not preserved, but DNA sequences were published. These include sequences of 28S (D0264A, DQ145361.2; D0264C, KY218983.1; D0291A, KY219004.1 and DQ145395.2), EF-1α (D0264A, DQ145473.1), and cytochrome oxidase I and II (COI-II, D0276G, MH919222.2). An additional sequence of COI-II purporting to be from a member of the type series of this species, D0264A, was available on GenBank from 2010-2020 under accession number GQ424989.1. This was actually a sequence of a different species, Aonidomytilus espinosai Porter, GQ424988.1, erroneously assigned due to contamination or mislabeling; it has been retracted from GenBank. DNA sequences of the primary bacterial endosymbiont, Uzinura diaspidicola, of M. targionoides sp. nov. have also been published, including a fragment of the small ribosomal subunit (16S) of a paratype (D0264C, KY220091.1) and 2 ground-up specimens of the type series (D0264A, GQ424852.1; D0291A, DQ868844.1), and a fragment of the large ribosomal subunit (23S of D0291B, DQ873256.1).
Remarks. This species is very similar to the previous one, Melanaspis lilloi sp. nov. The two were considered to belong to a single undescribed species, ("Melanaspis ud0276") by Schneider et al. (2018) and Normark et al. (2019). The diversity of informal designations assigned to members of the two species prior to 2018 were recordkeeping artifacts and did not reflect any diversity of evidence-based hypotheses. Careful study of the type series during the preparation of this manuscript revealed slight but consistent morphological differences between what are here regarded as separate species. The two species are also distinguishable by DNA, and appear as separate clusters in published phylogenies (Morse and Normark 2006;Normark et al. 2019). The pattern is seen most clearly in figure S2 of Normark et al. (2019), and is seen in each of the loci sampled from both species in that study: 2.2% divergence at 28S (compared to 0.14% within M. lilloi sp. nov.), 2.2-2.3% divergence at EF-1α (compared to 0.12-0.35% within each species), and 1.45% divergence at the primary endosymbiont's 16S locus (compared to 0.18-0.19% within each species). Although the species are morphologically very similar, M. targionoides sp. nov. has more numerous and more sclerotized dorsal ducts, along with sclerotization of the prosoma at full maturity. Specifically, the two species may be distinguished by the following characters. In M. targionoides sp. nov., the elongate cluster of dorsal ducts in the furrow of the first space (arising between L1 and L2) extends anteriorly at least 90% of the distance to the anus, its anterior end usually being directly lateral to the anus; in M. lilloi sp. nov., this cluster of dorsal ducts extends only 60-85% of the distance to the anus, its anterior end always lying posterolateral to the anus. In M. targionoides sp. nov. the furrow of the third space (arising between L3 and L4) has a single or double line of conspicuous subcircular sclerotized duct orifices extending from near the posterior margin to the anterior third of the pygidium; in M. lilloi sp. nov., the furrow of the third space has sclerotized duct openings only in the posterior third of the pygidium, with sometimes a few present further anteriorly along the medial edge of the furrow (lateral edge of the sclerotized area arising from L3) -these are often only partially sclerotized and anteroposteriorly compressed, thus appearing as partial ellipses rather than complete circles. Melanaspis targionoides sp. nov. has the prosoma sclerotized at full maturity (body length greater than 1.4 mm); M. lilloi sp. nov. has the prosoma membranous at full maturity. Melanaspis targionoides sp. nov. sometimes has perivulvar pores; M. lilloi sp. nov. lacks perivulvar pores.
Melanaspis targionoides sp. nov. and M. lilloi sp. nov. are referrable to Melanaspis based upon the characteristic sclerotization pattern of the dorsal pygidium, and their placement in Melanaspis is supported by molecular evidence (Morse and Normark 2006;Andersen et al. 2010;Rugman-Jones et al. 2010;Schneider et al. 2018;Normark et al. 2019;). However, they possess a combination of traits often seen in species of Targionia, including the absence of plates, presence of numerous small, slender macroducts arranged in distinct furrows, and simple, rounded pygidial lobes lacking notches. Plates are typically present in species of Melanaspis but can be highly reduced and difficult to view. The simple lobes and short paraphyses found in M. targionoides sp. nov. and M. lilloi sp. nov. are similar in appearance to those of M. enceliae (Ferris), but the number and distribution of macroducts is quite distinct from any other species observed for this genus.
Host plant. Not recorded. Etymology. The specific epithet is an adjective describing the resemblance this species bears to others placed in Targionia by adding the suffix -oides to indicate likeness in form.
Distribution. Argentina (Jujuy). Dorsum of pygidium with several sclerotized areas divided by furrows, one of which isolates L2 from remaining sclerotized areas; anterior submarginal region of dorsal pygidium appears reticulated or striped with variously branching furrows; longest paraphyses attached to lobes, never arising from interlobular spaces (Crenulaspidiotus Minimally fringed plates present between L1-L2 and L2-L3, absent or present anterior to position of L3, broad-based with 2 or 3 fringes when present; Additional online resources aiding in the identification of Aspidiotini are provided by Schneider et al. (2019) and Dooley (2006). N. Vajda, B. Peters, R. Carlton, M. Fizdale, M. Nichols, A. Krewinski, and E. Stangle. JW's participation was supported by the China Scholarship Council. This research was supported in part by the National Science Foundation (DEB-0447880, DEB-1258001). It was also supported in part by the U. S. Department of Agriculture's National Institute of Food and Agriculture, and by the University of Massachusetts Amherst Biology Department and Center for Agriculture, Food, and the Environment, under project number MAS00535. It was also supported in part by the U.S. Department of Agriculture's Agricultural Research Service. The authors have declared that no competing interests exist. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the USDA or NIFA. USDA is an equal opportunity provider and employer.