Corroborating molecular species discovery: Four new pine-feeding species of Chionaspis (Hemiptera, Diaspididae)

Abstract The genus Chionaspis (Hemiptera, Diaspididae) includes two North American species of armored scale insects feeding on Pinaceae: Chionaspis heterophyllae Cooley, and Chionaspis pinifoliae (Fitch). Despite the economic impact of conifer-feeding Chionaspis on horticulture, the species diversity in this group has only recently been systematically investigated using samples from across the group’s geographic and host range. This paper provides morphological recognition characters for four new species that were recently hypothesized to exist on the basis of molecular evidence. The new species, here described, are Chionaspis brachycephalon Vea sp. n., Chionaspis caudata Vea sp. n., Chionaspis sonorae Vea sp. n. and Chionaspis torreyanae Vea sp. n. One of the new species, Chionaspis caudata Vea, has a gland spine at the apex of the pygidium, between the median lobes, unlike any other species of Chionaspis. An identification key to the species of Chionaspis feeding on pine in North America is provided.


introduction
The armored scales insects (Hemiptera, Diaspididae) are a group of over 2500 described species of plant parasites . Adult females are characterized by a reduced morphology and a sessile habit on plant surfaces (Miller and Davidson 2005). Because armored scale insects are often cryptic in the field and are not susceptible to mass-collecting techniques, they are often overlooked in faunal surveys, and so new species tend to be discovered as agricultural pests (Evans et al. 2009, Wolff and Claps 2010, Dones and Evans 2011. Conventionally, new species of armored scales are discovered based on unique combinations of morphological characters. However, some shortcomings of conventional morphological species descriptions are their reliance on specimens sampled from a limited number of locations, or hosts, as is often the case when identifying agricultural pests. Such limited sampling may fail to observe a range of intraspecific morphological variation across hosts and geography. More importantly, conventional species descriptions of armored scales are not often corroborated with genetic measures of species boundaries (but see Evans et al. (2009) and Rugman-Jones et al. (2009)). This reliance on morphology alone may obscure the nature of species diversity in armored scales. Some putative species, identified only by molecular markers, may be cryptic species (Bickford et al. 2007) -which show no interspecific morphological variation (Andersen et al. 2010. Because conventional, morphological criteria for species differences may fail to distinguish new species, Gwiazdowski et al. (2011) undertook a molecular study of species diversity of Chionaspis collected across North America from 54 species of Pinus. Here we report a parallel morphological study of species diversity in this same sample.
Current taxonomy of the genus Chionaspis Signoret recognizes two pine-feeding species, C. heterophyllae Cooley and C. pinifoliae (Fitch). These species are native to North America (Watson 2005), and are considered pests on Pinus in forests and ornamental settings (Miller 1996, Miller andDavidson 2005). Chionaspis pinifoliae has been recognized as a pest for over 150 years and has been a subject of at least 189 publications. Chionaspis heterophyllae has been a subject of at least 55 publications (Veilleux et al. 2011), and together, the two species have been the focus of three PhD dissertations (Nielsen 1970, Shour 1986, Gwiazdowski 2011. Aspidiotus pinifoliae was first described by Fitch as a pest of pines, "which fixes itself upon the leaves, exhausting them of their juices and then causing them to perish and fall, and the end of the limbs to die when thus defoliated" (Fitch 1956:488). Fitch described the species based on specimens on pine needles that were sent to him from Robert W. Kennicott who collected them in the "yard of S. Francis, Esq. in the city of Springfield" in Illinois. Fitch describes the arrangement of the scale insects on the pine needle as well as their general shape and color, but the pine species was not identified and the scale insects were never prepared and mounted on a slide. Nevertheless, the series of specimens on needles used for the description were found at the New York State Agricultural Society (New York State Museum). This should allow the designation of a lectotype for C. pinifoliae (not treated here). The species was subsequently placed in five different genera (Mytilaspis pinifoliae, LeBaron 1872: 83;Chionaspis pinifoliae, Comstock 1881: 318;Leucaspis pinifoliae, García Mercet 1912: 215;Chionaspis (Phenacaspis) pinifoliae, Balachowsky 1930d: 266;Polyaspis pinifolii, Lindinger 1935: 140;Phenacaspis pinifoliae, Ferris 1937: SI-93.) Chionaspis pinifoliae heterophyllae was first described by Cooley in 1897Cooley in (1897 from specimens collected in Florida (Cooley 1899). Cooley's original description distinguished this subspecies (which he called a variety) from Chionaspis pinifoliae by its smaller body size and more rounded and less conspicuous median lobes (Cooley 1897, Andresen 1957. The differences in these characters were later illustrated, on plate 7, of Cooley's monograph of Chionaspis (Cooley 1899). Subsequently, C. pinifoliae heterophyllae has undergone two taxonomic changes; the first in rank to full species by the name Phenacaspis heterophyllae, (MacGillivray 1921:347), and the second by reassignment to Chionaspis as Chionaspis heterophyllae, (Borchsenius 1966: 122).
Since 1921 (MacGillivray 1921) taxonomists have only recognized two pine-feeding Chionaspis (previously Phenacaspis) in North America, but the recent reanalysis of species diversity within this group by Gwiazdowski et al. (2011) indicated the presence of at least 10 closely related species feeding on pine. The methods of Gwiazdowski et al. (2011) used multi-locus genealogical concordance to delimit species. This method is expected to be conservative because it should only detect species boundaries that are old and impermeable enough for monophyly to have evolved at a majority of loci (Neigel andAvise 1986, Hudson andCoyne 2002).
Most of the specimens collected by Gwiazdowski et al. (2011) are morphologically indistinguishable from C. pinifoliae or C. heterophyllae. The few specimens that could be distinguished from both C. pinifoliae and C. heterophyllae could be placed in five morphological groups, and these groups would be recognized as species by conventional morphological criteria (Miller andDavidson 2005, Watson 2005). Only one of these four morphological groups was recognized as a species by Gwiazdowski et al. (2011), whereas the other four morphological groups were subsumed within more inclusive undescribed species. All morphological groups are tightly correlated with geography, host affiliation or both (see Gwiazdowski et al. (2011) and species descriptions below).
These results highlight the possibility that even species that are well known, as in the case of pest species, may be more diverse than previously thought (i.e. contain cryptic species). It is not immediately clear how best to assign taxonomic status to cryptic species (but see Cook et al. (2010)), and so here we provide morphological descriptions for four new species of Chionaspis which are distinguished in the analyses of Gwiazdowski et al. (2011) as belonging to clearly diverged lineages, and possessing a unique combination of morphological characters distinct from both C. pinifoliae and C. heterophyllae.

Materials and methods
Field collection and slide mounting of all specimens were accomplished using the protocols described by Gwiazdowski et al. (2011). While some species of Chionaspis can show a tissue-specific morphology --where broodmates developing on different plant tissues (e.g. leaves or bark) have very different morphology (Takagi 1985, Liu et al. 1989) --specimens from Gwiazdowski et al. (2011) were all collected from the same host tissue: pine needles. Illustrations were made by hand using a camera lucida on a Zeiss 47 46 20-9900 microscope and an Olympus CHBS, and digitally edited with Photoshop CS4 14.0.0. They follow the convention used in scale insect illustration, with each figure displaying the dorsal body surface on the left side and the ventral body surface on the right side. Enlargements of significant features are located around the body. The morphological terminology and measurements in the descriptions below follows the conventions of Miller and Davidson (2005). In brief, abbreviations in the text refer to different pygidial lobes (trullae of Takagi): L1 for the median lobes, L2 for the second pair of lobes, L3 for the third pair of lobes and L4 for the fourth pair of lobes. Formulas are provided for the number of gland spines and microducts present between the pygidial lobes. For example, 1-1-1 indicates 1 gland spine in the first space (between L1 and L2); one in the 2nd space (between L2 and L3); and one in the 3rd space (between L3 and the position where L4 would be). Occasionally, the number of microducts subtending gland spines differs from the number of gland spines, and the microduct formula (microducts subtending gland spines in the 1st-2nd-3rd spaces) is indicated in parentheses. Length measurements are given as ranges with the median value in parentheses. The distance between the median lobes is measured from the medial margins, at the midpoint between base and apex. The species described here correspond to the morphological groups indicated by the letters B, C, D, and E in Figures 2 through 4 from Gwiazdowski et al. (2011). Here, each species name is followed by the corresponding morphogroup letter from Gwiazdowski et al. (2011).
Slide mounted type specimens have been deposited at the National Insect Collection, Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City (CNIN), the United States National Entomological Collection (Coccoidea collection) at the U.S. National Museum of Natural History (USNM), USA and the University of Massachusetts Insect Collection, Amherst, MA, USA (UMAM). Genomic DNA from all types, as well as lots supplying the type material (additional specimens in-situ on host tissue) from the study of Gwiazdowski et al. (2011), has been deposited at the American Museum of Natural History's Ambrose Monell Cryo Collection (AMCC). The DNA sequences used by Gwiazdowski et al. (2011) comprise three loci: the D2-D3 portion of the large ribosomal subunit rDNA (28S), elongation factor 1-alpha (EF-1a), and a mitochondrial fragment spanning parts of cytochrome C oxidase subunits I and II (COI-COII). Sequences for these three loci, for all type specimens, have been deposited in GenBank (Benson et al. 2012). The repository, associated accession numbers, and locality information for all type material and material with published DNA sequences is provided in the species description and Appendix 1and 2. The USNM and CNIN do not assign accession numbers to type specimens; types are incorporated within the general Coccoidea collections, and these are arranged alphabetically by family, genus, species. Holotype specimens are prominently marked in red, additionally CNIN paratypes are marked in green. Diagnosis. Chionaspis brachycephalon Vea differs from other Chionaspis by the following combination of characters (Table 1): small head, gland spine formula variable from 1-1-1 to 2-2-2 (median: 2-2-2), microduct formula also variable from 2-2-2 to 3-3-4 (median: 3-2-2); numerous marginal gland spines on abdominal segments 3 to 5, absent from abdominal segment 1 and 2; variable number of notches present on all pygidial lobes.
Description. Field characters: All pine-feeding Chionaspis discussed here, including C. heterophyllae and C. pinifoliae, are indistinguishable by eye in the field. The adult female for all species possesses a white oystershell-shaped and slightly convex cover, with the amount of posterior expansion varying according to the diameter of host needles. Body elongate, color varying from yellow when immature to reddish brownish in specimens containing eggs, with lateral protrusion on the anterior abdominal segments. Found on needles.
Prepygidium: Gland spines. Near each body margin on segments 3 and 4, absent from segment 1 and 2; with 2 -6 (4) on segment 3 and 2-5 (4) gland spines on segment 4, all protruding from margin..Ducts. Macroducts of 2 sizes; large macroducts in submedian and submarginal areas of abdominal segments 3 and 4. Small macroducts in submedian area of any or all of segments 2 to 4, and in marginal areas from meso-or metathorax to segment 3. Prepygidial microducts present on venter and dorsum from segment 1 to 4, sparsely distributed.
Etymology. The epithet brachycephalon is a noun, derived from Greek, meaning "short head", from brachy-short + cephalon head. The epithet refers to the head shape of this species, which appears smaller than that of other pine-feeding Chionaspis.
Description. Field characters: All pine-feeding Chionaspis reported here, including C. heterophyllae and C. pinifoliae are indistinguishable by eye in the field. See the description above for Chionaspis brachycephalon Vea.
Prepygidium: Gland spines. Near each body margin from segment 1 or 2 to 4, with 0 -4 on segment 1, 0 -5 (4) on segment 2, 1 -7 (4) on segment 3 and 1 -2 (1) gland spines on segment 4, which are short and protrude from the margin. Gland spines from segment 1 to 3 are the smallest, and never protrude from the margin. Ducts. Macroducts of 2 sizes; largest macroducts in submedian areas of abdominal segments 4 and 3. Small macroducts in submedian area of segments 3 and 4, and in submarginal areas of segments 1 to 4. Prepygidial microducts present on venter from segment 1 to segment 3, in marginal or submarginal areas from head to segments 2 to 3. Prepygidial microducts on dorsum on segments 1 to 4, often in conspicuous clusters submedially.
Etymology. Chionaspis caudata Vea possesses an unusual median gland spine between the median lobes. The epithet caudata is a Latin adjective meaning tailed (caudate), derived from cauda, tail, and referring to this peculiar feature.
Notes. Chionaspis caudata Vea differs from the other species by the rather squareshaped head and noticeably longer body, the presence of a single gland spine subtended by one microduct between the median lobes, and the gland spine formula. The presence of the median gland spine is striking as this feature prevents this species from keying to the genus Chionaspis (or indeed any related genus) in available keys to genera; however, the phylogenetic analyses of Gwiazdowski et al. (2011) unambiguously place C. caudata Vea within Chionaspis. Diagnosis. Chionaspis sonorae Vea is distinguishable from other Chionaspis by the combination of the following characters (Table 1): median lobe shape unusual, broad, medial margins parallel or slightly convergent in basal half, abruptly angled near midpoint, with distal half divergent, serrated; yoke horseshoe-shaped; microducts sparse.

Chionaspis sonorae
Description. Field characters: All pine-feeding Chionaspis reported here, including C. heterophyllae and C. pinifoliae are indistinguishable by eye in the field. See the description above for Chionaspis brachycephalon Vea.
Prepygidium: Gland spines. Near each body margin from segment 2 to 4, absent from mesothorax, metathorax and segment 1; 4 -8 (6) on segment 2, 4 -7 (5) on segment 3 and 1 -4 (2) on segment 4. Gland spines on segments 3 and 4 protruding from margin and about same size as those on segment 5. Gland spines on segment 2 the smallest and never protruding from the margin. Ducts. Macroducts of 2 sizes; larger macroducts in submedian areas of abdominal segments 4 and 3. Small macroducts in submedian area of any or all of segments 3 and 4, in marginal areas from meso-or metathorax to segment 3. Prepygidial microducts almost absent on both surfaces, with a few on segment 2.
Etymology. The epithet sonorae is a Latin noun, the genitive form of Sonora, meaning "of Sonora".  Paratypes: Adult females on separate slides, D2238D, E and G, same information as D2238A, deposited at USNM.
Description. Field characters: All pine-feeding Chionaspis reported here, including C. heterophyllae and C. pinifoliae are indistinguishable by eye in the field. See the description above for Chionaspis brachycephalon Vea.
Etymology. The epithet torreyanae is a Latin noun, genitive case, meaning "of torreyana", referring to the pine species Pinus torreyana, on which Chionaspis torreyanae Vea was collected.
Key to the species of pine feeding Chionaspis in North America: 1 Gland spine present between median lobes; zygosis absent between medial lobes (Figure 2 Head reduced, with margins converging rapidly toward anterior end of cephalothorax; gland spine cluster in 1st, 2nd, and 3rd spaces each with 2 or more microducts (Figure 1)  Medial margins of median lobes curving abruptly outward near midpoint between base and apex: parallel near base and becoming suddenly divergent (and slightly notched) in apical half (Figure 3)

Chionaspis caudata Vea and a modified diagnosis of the genus Chionaspis
The gland spine located between the median lobes of Chionaspis caudata Vea is unique among the species of Chionaspis. Takagi (1985), in his description of the genus, describes the median lobes as "united together in a basal zygosis". If considering this character, conventional taxonomy would not place C. caudata Vea in Chionaspis, even though this species also possesses a suite of morphological features consistent with the genus, such as the relative position of perivulvar pores, macroducts and gland spines on the pygidium and other abdominal segments. Ferris's (1937) key to North American diaspidids, still the most useful resource for the Mexican fauna, is utterly confounded by C. caudata Vea (it comes closest to the genus Pseudoparlatoria Cockerell, which has pair of conjoined gland spines between the median lobes). The phylogenetic results from Gwiazdowski et al. (2011) unambiguously place C. caudata Vea within the pine-feeding Chionaspis species complex, and the genus Chionaspis. Mexico is an undersampled region where specimens have only been collected recently, and these recent collections indicate that the genus Chionaspis is more variable than previously thought, especially regarding variation concerning key characters involving the pygidial median lobes.
Morphogroup A and other yet-undescribed species Gwiazdowski et al. (2011) mentioned a fifth novel morphogroup, Morphogroup A, which we have not described here. Although this group of specimens (from Pinus cembroides in the state of Queretaro) at first appeared to have a distinctive morphology, we found it challenging to write a key that could consistently discriminate it from Chionaspis pinifoliae, so we have conservatively omitted to describe it here. The molecular evidence of Gwiazdowski et al. (2011) suggests that several additional species of pinefeeding Chionaspis remain undescribed.