MOLECULAR SYSTEMATICS OF ARMORED SCALE INSECTS (DIASPIDIDAE)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0193179
• Grant No.: 2002-35302-12479
• Proposal No.: 2002-02791
• Project Start Date: Sep 1, 2002
• Project End Date: Aug 31, 2005
• Grant Year: 2002
• Program Code: [51.2]- (N/A)

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
PLANT, SOIL & INSECT SCIENCE

Non Technical Summary
Armored scale insects are serious pests of many trees and shrubs. There are a large number of species (about 2,400) but they all have simplified anatomy and are hard to tell apart. In spite of this similar, simplified anatomy, there is a great diversity of many important biological features, including use of host plants and natural enemies. Most strikingly, there is an amazing diversity of genetic systems, including systems in which only females have fathers and systems in which there are no males at all. __________________________________ The goal of the project is to sequence DNA from 3 different genes from 95 different armored scale species, including 65 pest species. With this set of DNA sequences we will be able to infer an evolutionary tree for these armored scale species, which will allow us to improve upon existing classification systems for armored scales, and will allow us to study their evolution. The data set will also make it possible to identify armored scales based upon their DNA sequences, and even to identify eggs and immature stages that cannot be identified by anatomical characters.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1130	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1130 - Entomology and acarology;

Keywords

insect taxonomy

systematics

dna

mitochondrial dna

evolution

parthenogenesis

genomes

coccoidea

hemiptera

homoptera

diaspididae

phylogenetics

elongation

reference collection

specimens

dna sequences

host specificity

insect anatomy

insect identification

insect population

species diversity

genetic diversity

insect reproduction

Goals / Objectives
Objective 1. To obtain a reference collection of specimens representative of the family Diaspididae, with at least 95 species in total, including at least 65 species from Miller and Davidson's list of armored scale insect pests, and also the tribe not represented on that list, Rugaspidiotini. For each species, the reference collection is to include a series of stably preserved voucher specimens, e.g., held in 100 percent ethanol at -20 degrees C, and slide-mounted material for traditional morphological study. Objective 2. To compile a matrix consisting of DNA sequence data for each species in the reference collection, including mitochondrial genes, cytochrome oxidase 1 and 2, and elongation factor 1-alpha. Objective 3. To use the data matrix to construct a maximally resolved phylogenetic hypothesis for the species in the reference collection. Objective 4. To use the phylogenetic hypothesis to investigate systematic and evolutionary questions, starting with (a) evaluating the monophyly of traditionally recognized genera and tribes, (b) characterizing evolutionary patterns of host specialization, and (c) investigating the evolution of paternal genome elimination.

Project Methods
For practical reasons most specimens will be collected either into 100 percent ethanol, 100 percent acetone, or some non-flammable alternative preservative such as RNAlater (Ambion, Inc.). Whenever practical we will collect fresh material and store some specimens at -80 degrees C without a preservative medium. DNA Preparation, PCR, and Sequencing DNA will be prepared by the salting-out method or any of various alternatives and stored at -80 degrees C. PCR will follow standard protocols, with strict separation of pre-PCR and post-PCR work areas and equipment, and negative (no genomic DNA) controls to check for cross-contamination. PCR products will be directly cycle-sequenced. Targeted loci We will sequence two mitochondrial loci, cytochrome oxidase 1 and 2 (CO1 and CO2), and on the single-copy nuclear locus elongation-factor 1-alpha. Sequence data management and phylogenetic analysis DNA sequence data will be compiled and edited in Sequencher 4.1 (Gene Codes Corporation). Due to the large number of sequences expected, phylogenetic analyses will primarily be parsimony-based and will employ the parsimony ratchet heuristic search algorithm (implemented in the programs PAUPrat, NONA and TNT). For comparison, we will also analyze the final data set using Bayesian phylogenetic inference, implemented in MrBayes, and maximum likelihood, implemented in PAUP 4.0 and or FastDNAml. We may encounter sequences of variable length, making it necessary to postulate insertion and/or deletion events. This is unlikely for the protein-coding sequences of CO1, CO2, and EF-1a, but it is likely in the case of introns within EF-1a. For the analysis of such sequences we will use the optimization alignment program POY. For comparison we will also align sequences in the program Clustalx using a range of gap costs and then use the phylogenetic inference procedures listed above (parsimony, Bayesian, likelihood). Integration with morphological characters My laboratory is collaborating with morphological systematists of Diaspididae: Dr. Lucia Claps, Instituto Lillo, Tucuman, Argentina; and doctoral student Megumi Shobu, Kyushu University, Fukuoka, Japan. In parallel with the molecular project propose here, we will be constructing a matrix of morphological characters for all the taxa from which molecular characters are sampled; we will ultimately analyze that matrix together with the morphological matrix using the parsimony-based programs mentioned above (e.g., PAUP, NONA). Evolutionary inference After completing the phylogenetic analysis we will use our estimate of the phylogeny of Diaspididae to study the evolution of a range of interesting characters, including hostplant use, viviparity, and bizarre features of diaspidid genetic systems such as paternal genome elimination (in which males lose their fathers' genomes and transmit only genes from their mother). We will map characters onto inferred phylogenetic trees using the principle of parsimony as implemented in programs such as MacClade.

Progress 09/01/02 to 08/31/05

Outputs
OBJECTIVE 1: We have exceeded our numerical goals for collecting. We have approximately 150 identified, described species in 100% ethanol (over 160% of our goal). Of these, fully half (75) appear on Miller and Davidson's 1990 list of armored scale insect pests (115% of our goal). In addition we have about 20 identified, described species in 70% ethanol (from which DNA may be recoverable), and about 20 apparently undescribed species (mostly of New Zealand Leucaspis, supplied by Rosa Henderson, and various genera from Argentina, supplied by Lucia Claps). In addition we have hundreds of samples from around the world that have not yet been identified. OBJECTIVE 2: We completed and published a data matrix consisting of portions of 2 genes (28S rDNA + elongation factor 1-alpha) for each of 75 described species + at least 8 undescribed species (Morse & Normark 2006). This published matrix represents 92% of our objective in terms of taxa, and 67% of our objective in terms of genes. Since submitting this publication, we have expanded our matrix to 122 described species (135%) for 28S , and about 85 described species for elongation factor 1-alpha. We also have about 40 species sequenced for cytochrome oxidase 1 and 2, and about 20 for the new marker CAD. We also have 28S sequences for a few dozen undescribed and identifed additional species. On the whole, our published data matrix is about 60% of the size of the one proposed; and our latest (not yet completed or published) matrix is about the size of the one we proposed but contains more taxa with less complete coverage of each taxon. OBJECTIVE 3. We have completed this objective (constructing a phylogenetic tree) for our published matrix of 83 species X 2 genes and published the result (Morse and Normark 2006). OBJECTIVE 4 (a). Evaluation of the monophyly of traditionally recognized genera and tribes. We have completed this objective for the most species-rich tribes (Aspidiotini, Lepdosaphedini, Diaspidini) and some of the most species-rich genera (Morse & Normark 2006). None of the traditionally recognized tribes was recovered as perfectly monophyletic. However, on the whole there were few big surprises, and our tree reflected traditional taxonomy to a large extent. For extensive discussion and illustrations, see Morse & Normark (2006). Most of the genera for which multiple species were available were reconstructed as monophyletic (Lepidosaphes, Diaspis, Carulaspis, Pseudaulacaspis, Chrysomphalus, Pinnaspis) or nearly monophyletic (Fiorinia includes Ichythaspis; Melanaspis includes Mycetaspis). A few seem to have deeper problems: Aspidiotus, Parlatoria, Dynaspidiotus, Chionaspis. 4(b) Characterizing evolutionary patterns of host specialization. Our first publication does not address this complex issue. More work on this is planned. 4(c) Investigating the evolution of paternal genome elimination. Contrary to Herrick & Seger's 1997 hypothesis that there were at least 4 origins of early paternal genome elimination in Diaspididae, we find only two origins (Morse & Normark 2006). This conclusion remains tenative pending further sampling.

Impacts
This project has resulted in the most complete and rigorous study of armoured scale insect phylogeny ever published (Morse & Normark 2006). It has also provided the preliminary data for a major NSF grant (DEB-0447880, "CAREER: Phylogeny and evolution of armored scale insects (Hemiptera: Diaspididae) and their bacterial endosymbionts," $651,937) that will fund the work of 3 graduate students studying armored scale insects through 2010. This work is already revolutionizing our understanding of the evolution and proper classification of armored scale insects, and lays the groundwork for implementing a system of molecular identifcation. All of these improvements in our understanding will in turn improve our ability to synthesize biological information across diverse species and to manage the many armored scale insect pests. This study has contributed to a number of other discoveries: most directly and strikingly, we have found that the primary endosymbiotic bacteria in armored scale insects is not Proteobacteria, as in almost all other Sternorrhynchan insects, but rather Bacteroidetes, as in cockroaches (ME Gruwell, GE Morse, & BB Normark, in prep.)

Publications

• Provencher. L. M., G. E. Morse, A. R. Weeks, and B. B. Normark. 2005. Parthenogenesis in the Aspidiotus nerii complex (Hemiptera: Diaspididae): a single origin of a worldwide, polyphagous lineage associated with Cardinium bacteria. Annals of the Entomological Society of America 98: 629-635.
• Normark, B. B. 2004. Haplodiploidy as an outcome of coevolution between male-killing cytoplasmic elements and their hosts. Evolution 58: 790-798.
• Normark, B. B. 2005. Using flow cytometry to assess ploidy in armored scale insects (Diaspididae). Pp. 49-57 in L. B. Erkilic and M. B. Kaydan (Eds.), Proceedings of the Tenth International Symposium on Scale Insect Studies, Adana, Turkey, 19-23 April 2004. Scientific and Technical Research Council of Turkey, Ankara.
• Morse, G. E., M. E. Gruwell, L. E. Claps, and B. B. Normark. 2005. Towards a combined molecular and morphological phylogenetic analysis of Diaspididae. Pp. 79-94 in L. B. Erkilic and M. B. Kaydan (Eds.), Proceedings of the Tenth International Symposium on Scale Insect Studies, Adana, Turkey, 19-23 April 2004. Scientific and Technical Research Council of Turkey, Ankara.
• Gruwell, M. E., C. D. von Dohlen, K. Patch, and B. B. Normark. 2005. Preliminary PCR survey of bacteria associated with scale insects (Hemiptera: Coccoidea). Pp. 49-57 in L. B. Erkilic and M. B. Kaydan (Eds.), Proceedings of the Tenth International Symposium on Scale Insect Studies, Adana, Turkey, 19-23 April 2004. Scientific and Technical Research Council of Turkey, Ankara.
• Normark, B. B. 2004. The sex lives of scales. Natural History 113: 38-44.

Progress 10/01/03 to 09/30/04

Outputs
OBJECTIVE 1: We have exceeded our numerical goals for collecting, with 107 species in total, and many more remaining to be identified. OBJECTIVE 2: As of 9/30/2004 the data matrix was about 80% complete, with 2 gene fragments complete for 94 taxa (elongation factor 1-alpha and 28S ribosomal DNA) and the third gene fragment complete for 48 species.

Impacts
Armored scale insects have relatively few anatomical characters on which a classification can be based. By sequencing some of their genes we provide a new source of info. that can be used to classify them. We are finding that certain aspects of the old classification of armored scale insects are supported by the molecular characters and others are not. It is looking like some of the most commonly recognized tribes and subtribes are not natural groups and will need to be completely revised. It is much too early to say what the revised system will look like. But our results (which we hope to submit in March) will show that there are some groups well-supported by DNA evidence that have never been recognized by morphologists and that some groups recognized by morphologists are unsupported by the DNA evidence. Another result of our work is that it will become possible in principle to use molecular methods to identify the common armored scale insects. Neither of these effects will be immediate -- it will take time for a new classification system to take shape, and for new identification methods to be developed. And since we have no intention of slackening the pace of our molecular studies our laboratory is likely to be instrumental in developing the new classification and identification systems. Our preliminary results represent the most detailed studies ever made of the evolutionary relationships between armored scale insects.They will provide a solid basis for a stable classification when we publish them next year.

Publications

• No publications reported this period

Progress 10/01/02 to 09/30/03

Outputs
OBJECTIVE 1: The numerical goals for collecting are nearly complete, with about 90 identified species total (out of 95) and about 55 pest species (out of 65). OBJECTIVE 2: As of 09/30/2003 the data matrix was about 25% complete. Postdoc Geoff Morse has developed excellent primers for amplifying the gene elongation factor 1-alpha. Progress has been very rapid and we are planning to submit our first two publications, reporting our initial phylogenetic results, in early 2004.

Impacts
Armored scale insects have relatively few anatomical characters on which a classification can be based. By sequencing some of their genes we provide a new source of info. that can be used to classify them. We are finding that certain aspects of the old classification of armored scale insects are supported by the molecular characters and others are not. It is looking like some of the most commonly recognized tribes and subtribes are not natural groups and will need to be completely revised. It is much too early to say what the revised system will look like. But our results (which we hope to submit in March) will show that there are some groups well-supported by DNA evidence that have never been recognized by morphologists and that some groups recognized by morphologists are unsupported by the DNA evidence. Another result of our work is that it will become possible in principle to use molecular methods to identify the common armored scale insects. Neither of these effects will be immediate -- it will take time for a new classification system to take shape, and for new identification methods to be developed. And since we have no intention of slackening the pace of our molecular studies our laboratory is likely to be instrumental in developing the new classification and identification systems. Our preliminary results represent the most detailed studies ever made of the evolutionary relationships between armored scale insects.They will provide a solid basis for a stable classification when we publish them next year.

Publications

• No publications reported this period

Progress 10/01/01 to 09/30/02

Outputs
Brand new project no report necessary.

Impacts
(N/A)

Publications

• No publications reported this period