Methodology
Collecting methods. Our collection methods are designed to make possible quantitative estimates of the abundance of armored scale insect species without actually being significantly more laborious than traditional unquantified sampling methods. At each locality, we sample a diversity of host plants along a transect. We identify each woody plant to species (Gleason and Cronquist 1991), and sample at least one individual of each. We use one of two alternative sampling methods: the search method or the clip method. For the search method, each collector conducts a timed search, spending 5 minutes carefully examining the leaves and bark of one plant, collecting any leaves or twigs that appear to be infested with scale insects and placing them in a quart-sized Ziploc bag. At the end of the 5 minutes, the collector measures and records the length of the portion of branch that was searched, and also takes a 20 cm twig sample and 20 cm2 bark sample from a part of the plant that was not searched. For the clip method, the collector clips off a small branchlet, one that will fit in a quart-sized Ziploc bag. The choice of method is determined by field logistics. To sample foliage that is not within easy reach, we carry a pole pruner.
Each sample is assigned a unique lot number. Within an hour of collection, collected plant material is put on ice and then refrigerated until it can be examined. Samples collected using the clip method are weighed using a hanging scale (in the field) or electronic balance (in the lab). Within a week, all collected plant material is examined under a dissecting microscope. All live scale insect specimens are removed and preserved. Locally collected armored scale insects are cryo-preserved in an ultralow freezer (-80C). Other specimens are preserved in 100% ethanol and stored in a flammable-materials freezer at -20C.
Of the 22 type localities, half are located in a few midwestern states (Ohio, Iowa, Kansas), and the other half are scattered across 10 states: New York, New Jersey, Indiana, Illinois, Missouri, Virginia, Florida, Texas, Colorado, California (García Morales et al. 2016). Spending a few days at each site will require a total of about 11 weeks of travel and collecting, 8 weeks in summer 2023 and 3 weeks in summer 2024.
For the local collecting, we will involve a crew of undergraduates. There will be time for some of this during Years 1 and 2 of the project (in April-May 2023 and October 2024), and more time for it in Years 3 and 4 (April-September 2025 and 2026).
Specimen preparation. Each specimen is given a unique prep number, consisting of its lot number plus one or more additional letters. Its total genomic DNA is isolated using the Qiagen DNeasy Blood & Tissue kit (Qiagen, Valencia, California, U.S.A.). To facilitate digestion, the insect is punctured with a 000 insect pin before being placed in the lysis buffer. After an overnight digestion, the cuticle is retrieved using a wide-mouthed micropipette tip and stored in water at 4?C for later slide mounting. Following the manufacturer's protocol, spin columns are used to wash and then elute the genomic DNA. Slide mounting: if not fully cleared, the cuticle is placed in 10% potassium hydroxide at room temperature overnight or until cleared, and gently squeezed with a spatula to remove any remaining tissue contents. The specimen is then passed through a staining and dehydration series consisting of: distilled water, 5 min; double stain (BioQuip, Rancho Dominguez, California, U.S.A.), 5 min or until adequately stained; 70% ethanol, 5 min; 100% isopropanol, 5 min (X3); clove oil, 5 min or longer. The cuticle is then mounted individually on a microscope slide using Canada balsam thinned with Histoclear II (National Diagnostics Corporation, Lake Geneva, Wisconsin, U.S.A.), covered with a coverslip, and cured for 2 months at 45–50?C.
PCR and sequencing. We will amplify and sequence diagnostic fragments of 3 loci: the large ribosomal subunit (28S rDNA), elongation factor 1 alpha (EF-1a), and a portion of the mitochondrial genome including parts of cytochrome oxidase I and II (COI-II). We have sequenced these markers from hundreds of species of armored scale insects and shown that they are useful for discriminating between closely related species (Schneider et al. 2018; Normark et al. 2019). Although it has been argued that a single marker can provide a "DNA barcode" indicating species identity, the use of multiple markers is more robust approach. Inevitably, a few sequences appear in the 'wrong' place in the tree. When that happens, the investigator must decide: is this a new discovery or an error? The ability to compare multiple markers provides independent tests of such anomalous results, allowing the investigator to discriminate between different sources of error (Normark et al. 2019), and to use the degree of congruence between loci to infer species boundaries (Andersen et al. 2010; Yang and Rannala 2010; Gwiazdowski et al. 2011).
We will use PCR with our standard primer pairs to amplify these loci (Normark et al. 2019), and send them to Psomagen for Sanger sequencing. Although we have designed an ultraconserved element baitset for Diaspidiotus ancylus that has enabled us to sequence about 2000 loci at a time, the necessary baits and library prep kits remain prohibitively expensive. Therefore we continue to use Sanger sequencing of legacy markers as a primary means of molecular characterization of our samples while we seek funding for a more comprehensive population-genomic approach.
Species delimitation. This is the culminating goal of the project. We will integrate information from Sanger sequencing, ultraconserved element sequencing [funded from other sources], morphological characters, host tissue use, host taxon use, and geography to infer species boundaries in the Diaspididotus ancylus complex.
The backbone of these efforts will be a molecular phylogeny, obtained using all available sequence data and all available samples from the D. ancylus species complex and close outgroups. The most crucial analysis will be the BPP species-delimitation analysis, which yields a hypothesis of species boundaries. As a Bayesian method, BPP relies on input of prior probabilities of various parameters. We will test a range of priors to asses the extent to which they affect the outcome. We will also estimate species trees using BPP. For the putative species identified by BPP, we will compare morphology (for instance, number and shape of sclerotized lobes; numbers of dorsal ducts on different abdominal segments; arrangement and fimbriation of membranous plates) to see if there are any subtle diagnostic characters. We will also compare their host tissue use (leaves vs. bark), host taxon use, and geography.
Some have argued that BPP is susceptible to oversplitting, mistaking boundaries between populations for boundaries between species (Sukumaran and Knowles 2017). This is most likely to happen when ecologically similar populations have been geographically separated for a long time. We think this problem is unlikely to affect armored scale insects, which are typically wind-dispersed and also frequently moved by human agency, as suggested by their status as the family of insects with the highest proportion of invasive species (Miller et al. 2005). Nonetheless, we will seek to corroborate putative species boundaries using either morphological characters or ecological ones (host tissue or taxon use).
After assessing the boundaries and characteristics of the species within the complex, we address the problem of assigning the correct name to each. To do this we consider the morphology of the type specimens of each of the currently synonymized species, almost all of which are in the national collection in Beltsville, MD. We will also consider the DNA sequences and morphololgy of specimens collected from the type hosts and type localities.
