Four new scale insect species (Hemiptera: Coccomorpha) associated with coffee roots in Colombia, South America, with identification keys for genera Newsteadia Green, 1902, Distichlicoccus Ferris, 1950, and Paraputo Laing, 1929

ABSTRACT Coffee (Rubiaceae: Coffea spp.) is the host-plant of at least 214 scale insect species (Hemiptera: Coccomorpha). In this study, scale insects were collected from the roots of Coffea arabica Linnaeus, 1753 in five provinces of Colombia, South America, and the adult females were prepared as microscope slide mounts for identification. Four new scale species are described, based on the external morphology of the adult females: Newsteadia andreae n. sp. (Ortheziidae Amyot & Serville, 1843) differs from other Newsteadia Green, 1902 species by antennal length, presence of two spines on the trochanter, and absence of tubular ducts and groups of quadrilocular pores posterior to the vulva. Distichlicoccus takumasae n. sp. (Pseudococcidae Cockerell, 1905) is distinguished by having a few oral rim tubular ducts on both dorsum and venter and absence of circulus. Paraputo nasai n. sp. (Pseudococcidae) is characterized by having 16 pairs of cerarii, uniformity of length of its dorsal setae on all segments, and the sizes of oral collar tubular ducts. Pseudococcus luciae n. sp. (Pseudococcidae) is diagnosed by having few oral collar and oral rim tubular ducts, few multilocular disc pores, a small circulus and the eye not being associated with any sclerotized area or discoidal pores. The list of scale insect species on coffee roots in Colombia is updated to 65 species. Taxonomic keys to the New World species of Newsteadia, Distichlicoccus Ferris, 1950 and Paraputo Laing, 1902 are provided. To identify Pseudococcus luciae n. sp., modifications are provided for use with the existing taxonomic keys to New World and Neotropical mealybugs.

The species described here correspond to four genera of two families: Distichlicoccus Ferris, 1950, Paraputo Laing, 1929, and Pseudococcus Westwood, 1840of Pseudococcidae Cockerell, 1905and Newsteadia Green, 1902of Ortheziidae Amyot & Serville, 1843. Distichlicoccus is composed of 12 species and its records are restricted to New World, except by Distichlicoccus oracelloides De Lotto, 1969 which is recorded in South Africa (McKenzie 1967;De Lotto 1969;Williams & Granara de Willink 1992;Ben-Dov 1994). Its morphology is variable, but presents similarities with Pseudococcus Westwood, 1840 (presence of oral rim tubular ducts), Humococcus Ferris, 1953 (anal ring without pores in some species) and Chorizococcus McKenzie, 1960 (few number of cesarii pairs) (McKenzie 1967;Williams & Granara de Willink 1992). The genus Paraputo has 92 species described so far, with a cosmopolitan distribution. Its systematic is still under discussion mainly because of morphological character as anal bar (absent/presence) and number of setae in the anal ring (six or more than six), affecting the definition of species between Formicococcus Takahashi, 1928 and Paraputo (Williams 2004;Danzing & Gavrilov-Zimin 2014;Zhang & Wu 2017). The number of species of Paraputo increased after the synonymy of Cataenococcus Ferris, 1955(Tang 1992. Pseudococcus and Newsteadia are the biggest genera of each family, with 169 and 58 discovered species, respectively. In both cases, the distributions cover all the continents, except Antarctica (García Morales et al. 2016).

MATERIAL AND METHODS
Scale insect specimens collected manually from the roots of coffee (Coffea arabica) in Colombia, South America (in the departments of Antioquia, Cauca, Cundinamarca, Quindío and Tolima) were preserved in 75% ethanol and labelled with field data. Later, adult females were mounted on microscope slides according to the protocol of Sirisena et al. ZOOSYSTEMA • 2021 • 43 (18) (2013), using a Nikon MSZ-1 stereomicroscope. A Zeiss Axion Lab A1 and Nikon Eclipse E600 phase contrast microscopes were used for species identification. Image analyses were conducted using a Lumenera Infinity 1-5C microscope camera and Image Pro Insight 8.0 software. The type materials of the new species described below are deposited in the Scale Insect Collection of the Entomological Museum at the Universidad Nacional Agronomía Bogotá, Bogotá, Colombia (UNAB) and the Muséum national d'Histoire naturelle, Paris, France (MNHN).
The species concept provided by de Queiroz (2007) is applied, so the operational criterion to delimit species in this work is based on the external morphology of the adult females. The taxonomic terminology for the descriptions of Pseudococcidae species follows Beardsley (1965) for the body segmentation, Williams & Granara de Willink (1992) for setal nomenclature on abdominal segments VIII + IX, and Gimpel & Miller (1996) for numbering the cerarii. The term "vestigial cerarius" refers to a cerarius without, or with only one conical seta, one or two auxiliary flagellate setae, and few trilocular pores. Terminology for the description of the Newsteadia species (Ortheziidae) follows Kozár (2004) and Gavrilov-Zimin (2018). The taxonomic illustrations follow the style of Ferris (1953) and were composed by all studied specimens.
The measurement data corresponds to mean and standard deviation calculated for all the specimens examined, with the measurement of the holotype [in brackets] and the range of variation for the entire evaluated population (in parenthesis). Data for each cerarius gives the cerarius number followed by the number of conical setae of the holotype; number of auxiliary setae [in brackets] and then the entire evaluated population's range of conical seta number; range of auxiliary seta number (in parenthesis). For example, C 14 [1-3;2-3] (1-3;0-3) means that in the holotype, each cerarius XIV contains between one and three conical setae, with two or three flagellate setae; while in the entire evaluated population, each cerarius XIV has one to three conical setae and zero to three flagellate auxiliary setae.
The body width is the largest transverse measurement perpendicular to the longitudinal axis, and body length is the longest longitudinal measurement, in mm. All other measurements are given in µm. Leg length is the sum of the lengths of trochanter + femur, tibia + tarsus, and claw. The standardized measurements of anatomical features (e.g. antennal segments, leg segments, anal ring, pores) are shown in each figure. In the taxonomic illustrations, dorsal and ventral morphology is shown on the left and right sides, respectively.
Wax plates. Structure like that of dorsal plates, with spines each 16.3 ± 2.4 (11-22) µm long and apically rounded, flagellate setae each 40.6 ± 9.6 (20-60) µm long, and quadrilocular pores of similar diameter to those on dorsum; plates in marginal region well developed, similar to those on dorsum; plates in central region less conspicuous, composed of flagellate setae and few spines; head with three wax plates, thorax with 19, wax plates absent from abdomen. Ovisac band (Fig. 1N) surrounding the second abdominal segment, external edge composed of a transverse cluster of quadrilocular pores of type II, like those on dorsum (Fig. 1F), each 4-5 µm in diameter, accompanied by simple pores and spicules; inner edge of band formed of clustered spines, each 21.1 ± 4.4 (14)(15)(16)(17)(18)(19)(20)(21)(22) µm long and apically rounded, most posterior row of spines longest, truncate; flagellate setae forming mesial cluster in anterior section of band, each seta 43.8 ± 6.9 (32-55) µm long; other setae scattered through the band; quadrilocular pores of type I and simple pores with diameter similar to those on dorsum, scattered in the cluster of spines; area enclosed by ovisac band with six transverse clusters of quadrilocular pores of type II; scattered simple pores and spicules clustered with flagellate setae, each seta 33.2 ± 4.7 (26-42) µm long; inter-cluster areas bare.

Spicules. Present on mesothorax and posterior body segments.
identificAtion key to new world species of Newsteadia green, 1902 (adapted from the descriptions by Morrison [1925,1952]  : no groups of quadrilocular pores posterior to the vulva (quadrilocular pores posterior to vulva in groups); a ratio of S ant III/S ant II of 3.7 (2.4); two spine-like setae on the trochanter (trochanter without spine-like setae), and each thoracic spiracle peritreme with quadrilocular pores inside (without quadrilocular pores in the peritremes).
Another species similar to N. andreae n. sp. is N. monikae Kozár & Konczné Benedicty, 2000, which also has three antennal segments, the second antennal segment longer that first one, distal fleshy setae on the antenna, flagellate setae with rounded apices on all antennal segments, and five pairs of abdominal spiracles. Newsteadia andreae n. sp. differs from N. monikae (characteristics given in parenthesis) by: spine-like setae absent from the coxa (present) and present on the trochanter (absent); apical antennal segment with fleshy longer setae and subapical setae, and apical shorter setae present (with flagellate longer setae and subapical setae, without apical shorter setae); tubular ducts absent from wax plates (present); and peritreme of each thoracic spiracle with 15-23 quadrilocular pores (pores absent).
In addition, N. andreae n. sp. is similar to N. milleri Kozár & Konczné Benedicty, 2000 in having antenna with three antennal segments, spine-like setae on the bases of trochanter and femur, an ovisac band without gaps, and five pairs of abdominal spiracles. Newsteadia andreae n. sp. differs from N. milleri (characteristics given in parenthesis) by having: all antennal segments with flagellate setae with rounded apices (all antennal segments with spine-like setae); apex of third antennal segment with subapical fleshy seta and apical short setae (apex of third antennal segment without either subapical setae or apical short setae); trochanter with two spine-like setae (one spine-like seta); and tubular ducts absent from both dorsum and venter (tubular ducts present in dorsal wax plates). etymology. -The specific epithet "takumasae" is assigned in honor of Dr Takumasa Kondo, mentor of the author.
Multilocular disc pores. Absent, except for one paratype, which has three pores on S abd VII, each pore about 9 µm in diameter (Figs 3I; 4G).
Oral collar tubular ducts. Each 8 ± 3.6 (5-11) µm long ( etymology. -The specific epithet is in recognition of the aboriginal community Nasa, who inhabit the region where specimens were collected.
diAgnosis. -Cerarii numbering 16 pairs; dorsal setae short, each 7-19 µm long; oral collar tubular ducts of two sizes, the smaller ones each with a diameter similar to a trilocular pore and the larger ones each with a diameter wider than that of a trilocular pore; anal bar absent; anal ring with 6 setae.

Oral collar tubular ducts. Absent.
Discoidal pores. Of two sizes: I) shorter type, each 2-3 µm in diameter, similar diameter to or smaller than a trilocular pore (Fig. 5C), distributed from head to S abd VI; II) longer type, each 4-5 µm in diameter, wider than a trilocular pore ( Fig. 5D) with border stout and sclerotized, distributed in S abd VII and S abd VIII + IX.

Spicules. Present from metathorax to S abd VIII + IX.
Body setae. Flagellate, each 20 ± 5 (12-40) µm long, longest setae distributed in submesial and mesial areas, forming clusters in interantennal region, S abd VI to S abd VIII + IX and a few adjacent to cerarii; shortest setae evenly distributed over the entire surface.
identificAtion key to new world species of ParaPuto lAing, 1929 [adapted from descriptions and taxonomic keys in Balachowsky (1959)  There is a debate about the generic character state that defines Paraputo and Formicocccus. As Paraputo nasai n. sp. has six setae in the anal ring and no anal bar, there is no conflict to put it into the Paraputo genus. Paraputo nasai n. sp. comes closest to Paraputo colombiensis (Williams & Granara de Willink, 1992), another species with translucent pores on the hind femur and tibia and with a circulus present. Paraputo nasai n. sp. differs from P. colombiensis (character states in parenthesis) in having oral collar tubular ducts of two distinct sizes (oral collar ducts of one size only); multilocular disc pores on S abd IV and posterior segments (present on S abd VI and posterior segments); dorsal setae of uniform length over the entire surface (dorsal setae of two sizes, the shorter ones present from head to S abd III, the longer ones present on S abd VI and posterior segments); number of conical cerarian setae progressively reduced anteriorly, from 6-12 setae in each of C 1 to C 6 , to 2-3 setae in C 7 to C 18 (conical setae number constant amongst all the cerarii, with 4-6 setae per cerarius); and conical setae absent from marginal intercerarian spaces (  Paratypes. 8 adult ♀ on 5 slides • 1 ♀ (marked as "Para" shares a slide with the holotype); same data as for holotype; UNAB 4635 • 1 ♀ (marked as "Para" shares a slide with an immature specimen); same data as for holotype; UNAB 4635 • 4 ♀ (on 2 slides); same data as for holotype; UNAB 4635 • 2 ♀ (on 1 slide); same data as for holotype; MNHN.
diAgnosis. -Combination of only a few oral collar tubular ducts (< 75 ducts), a few oral rim tubular ducts (< 15 over entire body), and few multilocular disc pores (< 12), a small circulus (< 61 µm wide) and eyes not associated with either a sclerotized area nor with discoidal pores.
description Pre-mounting specimens Appearance in situ: not observed. Specimens preserved in 75% ethanol with milky appearence; color unchanged in 10% potassium hydroxide.
In addition to having a small circulus, these species have only a few oral rim tubular ducts. Morphological differences between them include absence of discoidal pores associated with eyespot (P. neomicrocirculus and P. spanocera have discoidal pores associated with eyespots), translucent pores restricted to femur and tibia (present in all segments of the hind leg in P. spanocera; restricted to the hind tibia in P. neomicrocirculus), and less than five multilocular disc pores on each abdominal segment from S abd V and posterior segments (P. neomicrocirculus and P. spanocera have more than 10 multilocular disc pores on each abdominal segment) [character data of P. neomicrocirculus and P. spanocera taken from Gimpel & Miller (1996)].

vAriAtions
The distribution and number of oral rim tubular ducts are a recurrent character used to separate species of Pseudococcus. In P. luciae n. sp. these are highly variable and overlap the ranges found in several other species. Some specimens lack ducts on the dorsum of the abdomen (see Table 2, Ptypes 2-4 and 7) like P. dendrobiorum; other specimens lack ducts on the venter (see Table 2, Ptypes 4-7) like P. dasyliriae Gimpel &Miller, 1996 andP. mandoi Williams, 1985.

DISCUSSION
The family Pseudococcidae has 256 genera and Ortheziidae has 24 (García Morales et al. 2016). Some of those genera have similar morphologic characters and their phylogenetic relationship is yet unclear. The species described here provide new information to discuss the generic character states, particularly of Distichlicoccous and Paraputo. Distichlicoccous takumasae n. sp. shows morphological variations that should be considered to define the genus. The last described species for Distichlicoccous present highly variable taxonomic characters such as the presence and number of circulus and the number of cerarii. Until the species described by Williams & Granara de Willink (1992), the genus had only one species with circulus and the cerarii numbers were less than 14, but those new discoveries make necessary to review the generic diagnosis. Those variations fit with character states from species of genera Chorizococcus, Spilococcus Ferris, 1950, Pseudococcus, and to a lesser degree with Humococcus Ferris, 1953. Distichlicoccous takumasae n. sp. belongs to the nocirculus group only with most of the species of the genus but also increased the list of species with more than 14 cerarii pairs. Paraputo nasai n. sp. and Neotropical Paraputo species (previously placed in the synonymized genus Cataenococcus) should be consider into the discussion of taxonomic definition between In this paper, four new species are described, and their taxonomic locations must be confirmed by other tools besides the morphological one. Furthermore, the morphological variations found in these species could contribute to a more accurate taxonomic delimitation, especially in the cases of genera Distichlicoccus and Paraputo.