University of Massachusetts Amherst ScholarWorks@UMass Amherst

Masters Theses 1911 - February 2014

2003

Molecular systematics of a sexual and parthenogenetic species complex : nerii Bouchè (Insecta: Hemipthera: ).

Lisa M. Provencher University of Massachusetts Amherst

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Provencher, Lisa M., "Molecular systematics of a sexual and parthenogenetic species complex : Bouchè (Insecta: Hemipthera: Diaspididae)." (2003). Masters Theses 1911 - February 2014. 3090. Retrieved from https://scholarworks.umass.edu/theses/3090

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MOLECULAR SYSTEMATICS OF A SEXUAL AND PARTHENOGENETIC SPECIES

COMPLEX: Aspidiotus nerii BOUCHE (INSECTA: : DIASPIDIDAE).

A Thesis Presented

by

LISA M. PROVENCHER

Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

May 2003

Entomology MOLECULAR SYSTEMATICS OF A SEXUAL AND PARTHENOGENETIC SPECIES

COMPLEX: Aspidiotus nerii BOUCHE (INSECTA: HEMIPTERA: DIASPIDIDAE).

A Thesis Presented by Lisa M. Provencher

Roy G. V^n Driesche, Department Head Department of Entomology What is the opposite of A. nerii?

iuvf y :j3MSuy ACKNOWLEDGEMENTS

I thank Edward and Mari, for their patience and understanding while I worked on this master’s thesis. And a thank you also goes to Michael Sacco for the A. nerii jokes.

I would like to thank my advisor Benjamin Normark, and a special thank you to committee member Jason Cryan for all his generous guidance, assistance and time. I would like to recognize Stefano Colazza, Ezio Peri, and Paolo LoBue from the University of Palermo for their assistance with the sex pheromone analysis and Andrew Weeks at the University of Davis California for his work on the endosymbionts.

I also thank the following for their help in collecting: J. Daneel (Nelspruit, South

Africa), L. Erkilic (Adana-Gar, Turkey), A. Garonna (Napoli, Italy), U. Gerson (Rehovot,

Israel), R. Gill (Sacramento, California), H. Glenn (Homestead, Florida), R. Henderson

(New Zealand), F. Kozar (Budapest, Hungary), D. Miller (USDA), G. Stathas (Athens,

Greece), G. M. Orphanides (Cyprus), P. Gullan (Davis, California). And J. Hare, P. Fanti,

M. Hoddle and H. Schmutterer for their contacts.

And special thanks to Ifedayo Nicholson for all her offerings to the PCR god.

This work was supported by funds from the Massachusetts Agricultural

Experiment Station (Hatch 839).

IV ABSTRACT

MOLECULAR SYSTEMATICS OF A SEXUAL AND PARTHENOGENETIC SPECIES

COMPLEX: Aspidiotus nerii BOUCHE (INSECTA: HEMIPTERA: DIASPIDIDAE).

MAY 2003

LISA M. PROVENCHER, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST

M.A., UNIVERSITY OF MASSACHUSETTS AMHERST

Directed by: Benjamin B. Normark

Aspidiotus nerii Bouche, the oleander scale, is a worldwide pest of woody plants, including many of agricultural importance such as , olive and ornamentals. There is a great amount of variation within this species with respect to genetic systems (a sexual system of haplodiploidy vs. apomictic parthenogenesis), host plant use (> 100 plant families), development time and fecundity. Aspidiotus nerii were obtained from 18 different world locations were tested for phylogenetic analysis using molecular markers.

Using molecular analysis of a 774 bp fragment of mtDNA (including portions of the COI and COII genes; the Leucine tRNA was not found between the COI and COII as expected). Six 6 most parsimonious trees were recovered. The consensus tree was made up of three highly divergent clades with an uncorrected pairwise difference of 5.8-6.7%.

Colleagues from Sicily tested sex pheromone emissions in sexual and parthenogenetic forms of A. nerii. They found the parthenogens did not emit sex pheromones. Here individuals were screened for endosymbiotic bacteria in the phylum Cytophaga-

Flavobacterium-Bacteriods (CFB). These bacteria have been found to cause parthenogenesis in some mites. The findings revealed the parthenogens harbored the CFB

v bacteria and the sexuals did not. Compiling all this evidence one clade was found to be made up of parthenogenetic lineages with two remaining clades made up of seven sexual haplotypes. This evidence suggests that A. nerii is a complex of at least two different species as Gerson and Hazan proposed in 1979 when they described a new parthenogenetic species, Aspidiotus paranerii Gerson. TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...i\

ABSTRACT.....v

LIST OF TABLES.viii

LIST OF FIGURES.ix

CHAPTER

1. INTRODUCTION.1

2. METHODS.5

2.1 Taxon Sampling ..5 2.2 Nucleotide Sampling and Laboratory Procedures.5 2.3 Sequence alignment and Phylogenetic Analysis.6 2.4 Bacterial Screen.7 2.5 Wolbachia Screen...7

3. RESULTS. 8

3.1 Sequence Analysis.8 3.2 Phylogenetic Analysis.8 3.3 CFB Analysis.8 3.4 Wolbachia Analysis.9

4. DISSCUSSION.10

4.1 Phylogenetic Analysis. 10 4.2 Biogeographical Analysis.12 4.3 CFB Analysis.12 4.4 Conclusion.13

BIBLIOGRAPHY.33

Vll LIST OF TABLES

Table Page

1. Locations and information for the 18 specimens collected.15

2. Aspidiotus nerii haplotype sequences and out groups undet. sp., Aodinella sp. and sp., showing the COI, intergenic space and COIL Note the Luecine tRNA is not between the COI and COII as normally found in other mtDNA.17

3. Primers used in polymerase chain reaction amplification and sequencing of the mitochondrial (CO I and CO II) DNA regions studied.23

4. Uncorrected Pairwise Distance matrix.25

5. Descriptive Statistics .. 27

vm LIST OF FIGURES

Figure Page

1. Strict consensus tree of 11 haplotypes resolving into 3 main clades. Five haplotypes were placed in Clade 1, two haplotypes were placed in Clade 2, including Pomigliano Sicily Italy and San Juan California, four haplotypes were placed in Clade 3. Bootstrap (above) and Bremer support (below) are included...29

2. One of the six Maximum Parsimony analysis trees is shown here with character mapping. Mitochondrial DNA data results corroborate that the A. nerii complex is made up of sexual and parthenogenetic lineages. One clade is made up of parthenogenetic lineages. Confirmation for thelytoky in this clade is based on evidence that two of the tested populations were obtained from laboratory colonies that have produce no males in more than 10 years (Glenn 2001, Hare 2002 personal communication). CFB were found only in the parthenogenetic populations of A. nerii. The Sicilian individuals within the Group 1 tested negative for sex pheromone emissions and the Sicilian individuals within the Group 2 haplotype tested positive for sex pheromone emissions...31

IX CHAPTER 1

INTRODUCTION

Why sex? One of the enduring questions in evolutionary biology is the significance of sexual vs. asexual reproduction in eukaryotes (Ghiselin, 1974; Williams,

1975; Smith, 1978; Bell, 1982; Stearns, 1987; Michod and Levin, 1988; Barton and

Charlesworth, 1998; Hamilton, 2001; Kondrashov, 2001). In theory, a population with asexual reproduction is expected to increase at twice the rate of its sexual counterparts

(Smith, 1971). Yet, sexual reproduction in eukaryotes predominates. Therefore a strong counterbalance for the cost of sex must exist. Many mechanisms (>20) (Kondrashov,

1993; Hurst and Peck, 1996; Barton and Charlesworth, 1998) have been proposed to explain the persistence of sexual reproduction over asexual reproduction. Some intriguing possibilities are resistance to parasites (Lively et al., 1990), the evasion of mutation accumulation (Muller, 1964; Kondrashov, 1988) and most recently a pluralist approach which considers the involvement of multiple mechanisms, environmental and mutational

(West et al.,1999). A consensus, however, has not yet been established. An attempt to elucidate this question is to investigate the origin of thelytoky, the reproductive system in which females produce only daughters. Although, thelytoky is rare, it is one of the most widespread deviations from normal Mendelian sexual reproduction in eukaryotes

(Normark, 2002). It has been found that some groups that exhibit low mobility such as terrestrial tube worms (Reynolds, 1974) soil mites (Norton et al., 1993) and scale (Nur, 1980) have high frequencies of thelytokous reproduction. This suggests a common evolutionary response in taxa that have limited dispersal and female philopatry

1 (Williams, 1975; Bell, 1982). Through extant thelytokes a direct comparison of the differences in the amounts of genetic recombination per generation and their current levels of genetic variation with ancestral sexual taxa can be examined.

Insects have long played a critical role in the search for the significance of sex.

There is a barrage of this type of research surrounding aphids (Hales et al., 1977; Weeks and Hoffmann, 1998; Spence and Blackman, 2000), walking sticks (Marescalchi, 1993;

Law and Crespi, 2002), weevils (Normark et al., 1999) and scale insects (Nur, 1980), to mention a few. Additional advantages of using insects for investigating the hypotheses of sex include ease in rearing, short generation time, and a vast background in genetic manipulation.

To further research in this direction of sexual vs. asexual reproduction, preliminary analysis was carried out on the armored Aspidiotus nerii Bouche

(=hederae Vallot) (Hemiptera: Coccoidea: Diaspididae), commonly known as either the oleander scale or the ivy scale. Armored scale insects are a family of plant-feeding insects characterized by sexual dimorphism. The female is neotenic, legless, wingless and exhibits complete fusion of the head, thorax and abdomen into a flat, sac-like body. The male is has the appearance of a minute winged insect. The larva begin as mobile crawlers which settle and become sessile under a shield, or scale, formed out of wax filaments secreted mainly by the pygidial glands and mixed with exuviae. The females spend their entire life under the scale; the weak flying adult males emerge from under the scale and locate the females by sex pheromones (Pinto, 2002).

2 Aspidiotus nerii is particularly suitable for this study because it is a complex of sexual populations and parthenogenetic lineages. This complex of sexual and parthenogenetic forms has been explored through cross-breeding studies conducted by

Schmutterer (1952) and DeBach and Fisher (1956); the results of these experiments were repeatedly negative, compelling these researchers to state that there were two discrete taxa involved. Schmutterer proposed a new subspecies Aspidiotus hederae unisexualis. In

1969, Gerson and Hazan carried out host preference studies and recorded differences in reproduction, fecundity and lifecycle rates between the sexuals and parthenogens. They found parthenogens required more time to complete their development and produced fewer eggs; temperature also affected oviposition. They also found a slight difference in shield color. The parthenogens had a yellow-red shield color, whereas the sexuals had a yellow shield color. This prompted them to give the parthenogenetic form specific status by naming it Aspidiotus paranerii Gerson.

Because of the unusual genetic system of A. nerii, the definition of the terms

‘sexual’ and ‘parthenogenetic’ in regard to A. nerii need to be explained. In the sexual populations of A. nerii, fertilized eggs give rise to either males or females, but after fertilization if the paternal genome of the offspring is eliminated in the egg cytoplasm, the offspring will result will be a haploid male. As a result only the maternal genome is

passed to the progeny. This system is variously known as the “diaspidid chromosome

system” (Nur, 1980), embryonic paternal genome elimination (PGE) (Herrick and Seger

1999; Normark 2002), or pseudoarrhenotoky (Norton et al. 1993). The parthenogenetic

mode of reproduction in A. nerii is apomictic thelytoky, mitotic cloning (Schrader, 1929).

3 There is also a great amount of variation within this species with regard to host plants, with a list of more than 100 families of woody plants, including nut trees and ornamentals (Borchsenius, 1966). This species is well-documented on citrus

(Balachowsky, 1948) and olives and carob (Avidov & Harpaz, 1969), and was listed among the principal armored scale pests of the world by Beardsley and Gonzalez (1975).

Aspidiotus nerii feeds on nutrients from the plant tissue (Ben-Dov, 1990) causing general weakening of trees, discoloration of leaves and deterioration and discoloration of fruit

(Llorens, 1990).

To clarify this system of sexual vs. asexual reproduction, the examination of the molecular systematics is compared between the sexual and parthenogenetic lineages and the separate maternal lineages of parthenogens. A 774 bp fragment of mitochondrial

DNA (COI and COII) was tested from 42 individuals sampled from around the world

(Table 1), and compared to sex pheromone evidence and presence or absence of endosymbiotic bacteria from the phylum Cytophaga-Flavobacterium-Bacteriodes (CFB) and Wolbachia.

4 CHAPTER 2

METHODS

2.1 Taxon Sampling

Aspidiotus nerii specimens (sexual and parthenogenetic) from the Mediterranean area in Group 2, New Zealand and the United States were solicited from colleagues. I personally collected samples from two locales in Southern California. In addition, several specimens of a confirmed parthenogenetic lineage Aspidiotus paranerii Gerson from

Israel were collected to include in the phylogenetic study (Table 1). Hemiberlesia sp.,

Aodinella sp. sp., and undet. Aspidiotini were collected and used for outgroup analysis.

All specimens were preserved in 95-100% ethanol and stored at -80°C. Between one and four individuals from each locality were used for molecular analysis.

2.2 Nucleotide Sampling and Laboratory Procedures

Nucleic acids were extracted from whole insects using the salting out method of

Sunnucks and Hales (1996) as modified by Normark (1999). Universal and newly designed oligonucleotide primers (A. nerii specific; the Great American Gene Co.,

Ramona, CA) were used to amplify desired gene regions sequenced via standard polymerase chain reaction (PCR). Reactions were performed using a MJ Research, Inc., programmable thermocycler. The PCR protocol used was as follows: 95°C for 1 minute for initial denaturing; 95°C for 30 seconds, annealing 50°C for 1 minute, extension 72°C

5 for 2 minutes; repeat for 32 cycles with final extension at 72°C, for 10 minutes. A 900 bp fragment of mtDNA was amplified and sequenced in two to three contiguous regions, encompassing partial sequences of both strands of mtDNA cytochrome oxidase I (COI)

and cytochrome oxidase II (COII), See Table 3 for primer pairs. All PCRs included positive and negative controls to assure accuracy and detect possible contamination.

Double-stranded PCR amplification products were visualized on 1.5% agarose gels,

purified with Qiagen kits (MinElute or Qiaquick; Qiagen) and directly sequenced using

an ABI-377XL automated DNA sequencer at the UMass Sequencing Facility or with an

ABI-3700 automated high-throughput DNA analyzer at Kansas State University.

2.3 Sequence alignment and Phylogenetic Analysis

Alignment was done by hand. There were no insertions or deletions within A.

nerii (See table 4). Sequences were complied and edited using Sequencher 4.1 (Gene

Codes, 2002). Evolutionary trees were found using PAUP 4.0bl0 (Swofford 1993) with

unweighed heuristic searches under maximum parsimony (MP) criterion. Trees were

rooted using the armored scales Hemiberlesia sp., Aodinella sp. sp. and undetermined

Undet. Aspidiotini as outgroups. Neighbor joining (NJ) analysis was also carried out. The

data set included a total of 774 aligned nucleotides from fragments of the COI and COII

sequences (not including the leucine tRNA which was not in between the COI and COII

as expected) were included in the analysis. Non-parametric bootstrap and Bremer Support

were used to assess the degree of support for each clade.

6 2.4 Bacterial Screen

A polymerase chain reaction (PCR) assay for the presence of endosymbiotic bacteria (CFB) was carried out for 110 of the collected individuals (17 males and 96 females). This test was conducted at the University of California Riverside by Andrew

Weeks, see Table 1.

2.5 Wolbachia Screen

Wolbachia-specific primers 99F/994R, ftsZF/R (O’Neill et al., 1992) and wsp81F/691R (Holden et al., 1993) were used on the existing DNA extractions to determine any presence of Wolbachia in A. nerii.

7 CHAPTER 3

RESULTS

3.1 Sequence Analysis

A total of 774 aligned sites for the nineteen COI and COII sequences were used for the analysis. There are no insertions or deletions. Complete nucleotide sequences are available in GenBank under accession numbers xxxOOOO. The Leucine tRNA normally found between the COI and COII was not found and has most likely been translocated to a different position in the mitochondrial genome. This translocation appears to be in the norm in the family Diaspididae (Normark, personal corresp.). The sequence shows a small number of bases forming an intergenic spacer (See Table 2).

3.2 Phylogenetic Analysis

MP analyses resulted in six trees, each suggesting three distinct clades within the

A. nerii complex, and with a single origin of parthenogenesis (See Figure 2 for an

example). Neighbor joining analysis resulted in a tree that was identical with one

MP tree.

3.3 CFB analysis

All adult male specimens tested were negative for the eubacteria. All individuals

believed to be thelytokous tested positive, with the exception of one specimen from the

Homestead, Florida, colony that tested negative. Specimens from Pomigliano Italy

8 appeared to be a mix of two sexual haplotypes (Italy. 1 and Italy.3) and one thelytokous haplotype (Italy.2) that was identical to the New Zealand and California.2 haplotypes.

3.4 Wolbachia analysis

All Wolbachia analyses were negative. CHAPTER 4

DISCUSSION

4.1 Phylogenetic Analysis

The analysis of the molecular systematics of the sexual and parthenogenetic lineages and maternal-lineage genealogies of A. nerii populations based on mitochondrial

DNA show eleven haplotypes that resolve into 3 distinct clades. These clades have wide geographic distributions (see Table 1). There is high sequence divergence between the three clades (5.8-6.7%) (Table 4). Clade 3 is made up of parthenogenetic haplotypes and clade 1 and 2 are made up of sexual haplotypes (for supporting evidence see Figure 2).

The parthenogenetic clade apparently has one point of origin. This single point of origin suggest that all the parthenogens arose from one sexual haplotype and these diverged over time due to reproductive isolation and mutation (Figure 2). It is possible, however, that there were multiple origins of parthenogenesis as was found in the parasitoid wasp

Venturia canescens Gravenhorst, which is also made up of sexual and parthenogenetic forms (Schneider et al., 2002). Analysis of the genetic structure of this wasp showed the parthenogenic haplotypes mostly clustered together in one clade with low divergence between them, but several parthenogenic haplotypes appeared elsewhere in the tree among the sexual forms. Analyzing more A. nerii individuals, may lead to similar results.

It has long been suspected that the armored scale insect A. nerii is a species complex (Felt 1901, cited by Schrader 1929; DeBach and Fisher, 1956; Gerson and

10 Hazan, 1979). This is due to the large host range, slight morphological differences (color of shield) (Schmutterer, 1952; Gerson and Hazan, 1979) and different ecological requirements found in different A. nerii populations (DeBach and Fisher, 1956; Gerson and Hazan, 1979). Based on the presence of males, thelytoky confirmed by rearing, presence or absence of CFB bacteria and sex pheromone emission, this supports the

Gerson and Hazan hypothesis that there is a strictly sexual species and a strictly parthenogenetic species that includes all the haplotypes in the parthenogenetic clade. It does not support the hypothesis that geographically separate parthenogenetic lineages represent different species. There is, however, slim evidence for the support of a second sexual species, in the high sequence divergence and non-synonymous substitutions found in the California.3 and Italy.2 haplotypes that form a sister clade to the sexuals (See

Table 2). More data need to be compiled to investigate this possibility.

Within the parthenogenetic clade is the haplotype Group 1, which includes

confirmed parthenogenetic populations from Israel, Sicily and Florida. It was found that

the synthesis of the sex pheromones has been halted in the parthenogenetic Sicilian

lineages (Colazza personal communication). All individuals tested from clade 3 where

found to harbor the CFB bacteria. Using the standard mtDNA calculation of 2.0%

sequence divergence per one million years (Brower, 1995), estimates that clades 1 and 3

diverged approximately 3.0 my a and clades 2 and 3 diverged approximately 2.5 mya

from clade 1 and 2. The sequence divergence within clade 3 is low, yet the calculated

divergence from the Florida haplotype and the Group 1 is lmy. If ancient as defined in

11 insects is lmy old (Law and Crespi, 2002; Normark, unpub.), this implicates A. paranerii as a parthenogenetic lineage which is a candidate for an ancient asexual insect lineage.

4.2 Biogeographical analysis

Aspidiotus nerii is an aggressive pest insect with global distribution (Gerson and

Hazan, 1979). This wide-reaching distribution of A. nerii implies recent movement between continents. The spread of this insect appears to have taken place on plants translocated by humans, it is interesting that the pattern of distribution for this insect has been brought about through ancient and present agricultural trade. The California and

Italy haplotypes are all distantly related to each other yet are closely related to non-

California, non-Italian haplotypes (See table 1), illustrating a great deal of movement of these insects since the haplotypes have diversified. More specifically, a single lemon in

Pomigliano Sicily was found to be host to three haplotypes, two sexual and one parthenogenetic, that were all distantly related.

4.3 CFB analysis

All of the parthenogenetic lineages we collected tested positive for endosymbiotic bacteria from the phylum Cytophaga-Flavobaterium-Bacteriods (CFB). Closely related bacteria were also found in the parthenogenetic females of the false spider mite

Brevipalpus phoenicis Geijskes (Weeks, 2001). This is evidence that horizontal transmission of the bacteria may be taking place. Because the CFB bacterial endosymbionts are mostly found in parthenogenetic individuals, and in this case only parthenogenetic individuals, they may at least be one of the mechanisms involved in the

12 cause of thelytoky in A. nerii. Unfortunately, the usual test to check if bacteria are the cause of parthenogenesis is to treat the insect with tetracycline, but in the case of scale insects that harbor many bacteria for nutritional support (Buchner, 1965), such as A. nerii, antibiotics would destroy all the bacteria and the host as well. Hopefully future research on B. phoenicis will elucidate the function of these bacteria and their role in the mites and in turn reveal the reason of their presence in A. nerii. CFB were also found in the parasitic wasps in the genus Aphytis that parasitizes A. nerii (Weeks unpublished data). Pinto et al., (2002) found that this parasitoid uses the sex pheromone of third instar immature female A. nerii as a chemical stimulus to locate its host. Since A. paranerii does not emit the sex pheromone, this adds a small advantage that may assist the asexual lineages to persist longer. Aphytis chilensis Howard, attacks the third instar female scales, laying eggs under the scale cover. The parasitoid larvae eventually kill the host and emerge as adults. But it has been observed that Aphytis uses A. nerii for direct feeding

(Pinto et al., 2002), allowing for the possibility of infection of the eggs with the bacteria.

These infected eggs would in turn produce parthenogenetic offspring. This suggests that direct evidence for horizontal transmission of the CFB by these wasps should be sought.

4.4 Conclusion

The evidence gathered in this preliminary research has outlined the importance of studying sexual and parthenogenetic clades to investigate the origins of thelytoky and evolution of ancient asexual lineages. Aspidiotus paranerii is a clonal lineage that attacks a mosaic of sexual and clonal Citrus and is in turn attacked by a clonal wasp, Aphytis.

Both A. paranerii and Aphytis have been found to harbor clonal endosymbiotic bacteria.

13 Research initiated further in this direction will have great impact on supporting the hypotheses for the evolution of sexual and asexual reproduction.

14 Table 1. Locations and information for the 18 specimens collected.

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16 /

Table 2. Aspidiotus nerii haplotype sequences and outgroups undet. Aspidiotini, Aodinella sp. and Hemiberlesia sp., showing the COI, intergenic space and COIL Note the leucine tRNA is not between the COI and COII as normally found in other insect mtDNA.

17 ..-—COI-- Undet. Aspidiotini ATT TCA AGA TTA ATT TTT TGA TTT CCA TTA TTA ATT AAT TTC AAT TTT Aodinella sp. ATT TCA AGA CTA ATT TTC TGA TTA CCA TTA TTA ACT AGA TTT AAT TTT Hemiberlesia ATT TCA AGA TTA ATT TTT TGG TTA CCT TTA TTG ACA AAT TTT AAT TTC Italy.2 ATT TCA AGA CTA ATT TTC TGA CTT CCT CTA CTA ACT AAC TTT AAT TTT New Zealand .T. . . .C . .T.T. . T.T. Florida .T. . ..C ..T.T. . T.T. Group 1 .T. . . .C . .T.T. . T.T. California.5 fji *P T T T Greece ip fp fp ip rp California.2 rp fp T T T Group 3 Group 2 California.3 T.. ..C ..T ... T.A ... T.. A Italy.1

..COI-- Undet. Aspidiotini AAT AAT AAT TGA ATA ATA ATT AAT TTT ATT AAT ATT ATT TCA TCA ACA Aodinella sp. AAT AAT AAT TGA ATA ATA ATT AAT TTT ATT AAT ATC ATT ACT TCA ACA Hemiberlesia sp. AAC AAT AAT TGA ATA ATA ATT AAT TTC ATT AAT ATC ATT ACT TCA ACA Italy.2 AAT AAT AAT TGA ATA ATA ATT AAT TTT ATT AAT ATT ATT ACA TCA ACT New Zealand ...... C.C. Florida ...... T.C. Group 1 .T.C ... . California.5 . .T.C. Greece .T.C. California.2 .T...... Group 3 .T. Group 2 .T.. . California.3 .T ... .G. Italy.1 .T.

.-.COI.-.. Undet. Aspidiotini AAT TTA ACA TTT TTT CCT CAA CAT TTT ATA GGA TTA AAT GGA ATG CCC Aodinella sp. AAT TTA ACA TTT TTT CCT CAG CAT TTT CTT GGA TTA AAT GGA ATA CCA Hemiberlesia sp. AAT TTA ACT TTC TTT CCA CAA CAC TTN TTA GGA TTA AAT GGG ATA CCT Italy.2 AAT TTA ACA TTT TTC CCT CAA CAT TTT TTA GGA TTA AAT GGT ATA CCA New Zealand C . . T. A Florida C . .T.A Group 1 C . .T.A California.5 C ..T. A Greece C ..T.A California.2 . . .T...'. .A Group 3 Group 2 California.3 A Italy.1

.-COI- Undet. Aspidiotini CGA CGA TAC ATT ATT TAT TCA GAT TTT ATT ATT ATA TGA AAC AAT CTT Aodinella sp. CGA CGA TAC ATC ATT TAT TCT GAT TTC CTA ATT TTC TGA AAT AAT TTA Hemiberlesia sp. CGA CGT TAT ATT ATT TAT TCA GAT TTT GTA ATT TTC TGA AAT AAT TTA Italy. 2 CGT CGT TAC ATT ATT TAC TCA GAT TTT ATT ATT TTT TGA AAT AAT TTA New Zealand .T. Florida .T.A. Group 1 .T.A. California.5 Greece California.2 Group 3 Group 2 California.3 T Italy.1

Continued, next page

18 -coi- Undet. Aspidiotini TCA ACT TTA GGT TCA TTA ATA ACA ATC TTA TTT ATT TTT ATA TTT TTT Aodinella sp. TCA ACC TTA GGA TCA ATA ATA ACA ATT ATA TTT ATT TTC ATA TTT ATT Hemiberlesia sp. TCA ACA TTA GGA TCA ATA NCC ACT ATC TTA TTT ATT TTC ATA TTC ATT Italy.2 TCA ACT TTA GGA TCA ATA ATA ACA ATT ATA TTT ATT TTT ATA TTT TTT New Zealand . Florida ...... Group 1 ...... California.5 . Greece . California.2 ... Group 3 . Group 2 ..... California.3 ...... Italy.1 .

--COI.-.. Undet. Aspidiotini TAT TCT ATT ATT GAT TTA ATC AAT TCA AAA CGA AAA ATT ATT TTC ATT Aodinella sp. TAT TCA ATT ATN GAT TTA ATT AAC TCA AAA CGA AAA ATT ATT TTT ATT Hemiberlesia sp. TAC TCA ATT ATT GAC TTA ATT AAT TCA AAA CGA AAA ATT ATT TTC TTA Italy.2 TAT TCA ATT ATT GAA CTT TTA ATT TCA AAA CGA AAA ATT ATT ATT ATT New Zealand Florida Group 1 California.5 Greece California.2 Group 3 Group 2 California.3 C Italy.1

--COI-- Undet. Aspidiotini ATT AAA TCT AAT AAT AAT GAA TGA AAA AAT AAT ATT CCA ATT ATT AAT Aodinella sp. ATT AAA ACT AAT AAT AAT GAA TGA AAA AAT AAT TAC CCA ATT TTA TCA Hemiberlesia sp. ATC AAA TCA AAT AAT AAT GAA TGA AAA AAT AAC TCA CCT ATT CTC TCA Italy.2 ATT AAA TCT AAT AAT AAT GAA TGA AAA AAT AAT TTA CCA ACT ATT TCA New Zealand ...... C.N Florida ...C .C. ... T.T Group 1 ...C .C. . .G. California.5 Greece California.2 Group 3 Group 2 C California.3 Italy.1

Continued, next page.

19 -CU1COI Undet. Aspidiotini CAT TCA AAT ATT GAA AAT AAC TTC ATA TTT AAA AAA AAA TAA ACA TAA Aodinella sp. CAT CCA AAT AAA GAA TCA ATA TTT TTA TTT AAT AAA ATT AAA ATA TTA Hemiberlesia sp. CAT ACA AAT AAA GAA ATA GTA TTC TTA TTT AAT AAA AAC TAA AAA TAA Italy.2 CAT TCA AAT ATT GAA AAT AAT TTT ATA TTC AAT AAA AAC TAA AT New Zealand NNN NN. . .C . .C Florida Group 1 California.5 Greece .NN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNT California.2 . .C . .C Group 3 Group 2 California.3 . .N NNN NNN NNN NNN NNN NNN NNN NNN N. . . .N N Italy.1 . .C . .T

—V^Wilcon Undet. Aspidiotini ATC ATA TCA TGA ATA AAT YTA AAT TTT CAA AAT CCA AAT TCA ATT AAT Aodinella sp. ATT ATA TCA TGA ATA AAT ATA AAT TTC CAA AAT CCA AAT TCT ATT AAT Hemiberlesia sp. ATT ATA TCA TGA ATA AGA ATA AAT TTC CAA AAT CCT AAT TCT ATT AAT Italy.2 T ATA TCA TGA ATA AAC TTA AAT TTT CAA TAT CCA AAC TCA ATT AGT New Zealand . .C .A. Florida Group 1 California.5 C. . A. C . .T . .T . .C .A. Greece c.. A. C . .T . .T . .C .A. California.2 c.. A. C . .T . .T . .C .A. Group 3 c.. A. C . .T . .T . .c .A. Group 2 c.. A. C . .T . .c .A. California. 3 : N. NNN NNN NNG . .C AG...... T . . • . .c .A. Italy.1 . .G . .C AG. • • « . .T • • • . .c .A.

---coil-- Undet. Aspidiotini ATA ATT AAA ATT AAT ATA TTT CAT AAT TTT ATA TTA ATT ATT TTA ATT Aodinella sp. ATA ATT AAA ATC ATT ATA TTT CAT AAT TTC TTA ATG ATT TTG CTA ATT Hemiberlesia sp. ATA ATA AAA ATC NCT CTA TTT AAT AAT TTT TTA ATA ATT ATC CTA ATT Italy. 2 ATA ATT AAA ATT AGA ATA TTT CAT AAT TTC ATA CTA ATT ATT TTA ATT New Zealand Florida Group 1 California.5 T Greece T California.2 T Group 3 T Group 2 California.3 C c ... c Italy.1 C c ... c

Continued, next page.

20 COII -

Undet. Aspidiotini NAT ATN ATT ATA ATT TTA ATT TTA ATA ATT AAT TTT TCT TTA *-3 H! AAT Aodinella sp. TAC ATT ATT ATT ATT TTA ATC CTA ATA ATA AAA TTT TCA TTA AAA AAC Hemiberlesia sp. AAC ATC ATC ATT ATT TTA ATT ATA ATA ATA AAA TTC TCA CTA AAA AAT Italy.2 AAT ATT ATT ATA ATT TTA ATT TTA ATA ATT AAC TTT TCA CTA ATT AAT New Zealand Florida Group 1 California.5 T. .

Greece • o e T. . California.2 T. . Group 3 . .T T. . Group 2 T. .

California.3 . .c T. . G. . • • • Italy.1 . .c T. . G. . ...

COII—- Undet. Aspidiotini AAA TTN AAT NAT AAA ATA GTC TTA CAA NAT CAA ACA TTA GAA ATT TTA Aodinella sp. AAA TTT AAT AAC AAA ATT ATT TTA CAN AAT CAA AAA TTA GAA ATT ATT Hemiberlesia sp. AAA TTT AAT AAT AAA ATT ATT CTT CAA CAC CAA AAA TTA GAA ATT ATT Italy.2 AAA TTT AAT AAT AAA ATA ATT TTA CAA AAT CAA AAA TTA GAA ATT TTA New Zealand Florida Group 1 California.5 . .C Greece . .C California.2 . .c Group 3 . .c Group 2 . .c California.3 TTT . .C Italy.1 . . . TTT . .C

COII-- Undet. Aspidiotini TGA ACA ATT ATT CCT ATA TTT GTA ATT ATT ATN NNC AGA NCA ATT TCA Aodinella sp. TGA ACA ATA ATT CCA ATT ATT ATA ATT ATA ATC ATT AGA ATA TTA TCA Hemiberlesia sp. TGA ACA TTA ATG CCC ATC ATA ATC ATT ATA ATT ATT AGA AAT ATT TCA Italy.2 TGA ACA ATT ATT CCT ATA ATT ATT ATT ATT ATA ATT AGA ATA ATT TCT New Zealand Florida . Group 1 ...... California. 5 .C...... C.C.•. .A Greece .C.C.C ...... A California.2 C.C.A Group 3 C.C.A Group 2 C.C.A California.3 . .. Italy. 1 ......

Continued, next page.

21 COII Undet. Aspidiotini NNT AAT TTA TGA TGT GCA AAT AAT GAA ATA AAA GAT GAT GTT TTA AAT Aodinella sp. ATT AAT CTT TTA TAT TCA AAT AAT GAA ATA AAA AAC AAT ATT ATC AAT Hemiberlesia sp. ATT AAT TTA TTA TAT TCA AAT AAT GAA TTT AAA AAT AAT TTA ATT AAT Italy.2 ATT AAT TTA TTA TTT TCA AAT AAT GAA ATA AAA AAT AAT ATT CTT AAT New Zealand ATT AAT TTA TTA TTT TCA AAT AAT GAA ATA AAA . .C AAT ATT CTT AAT Florida Group 1 California.5 Greece California.2 Group 3 Group 2 California.3 Italy.1

COII—- Undet. Aspidiotini ATT AAA ATT ATT GGT AAT CAN CGA TTT TGA AAT TAC GAA TAT TCA ATA Aodinella sp. ATT AAA ATT ATT GGA AAT CAA TGA TTT TGA AAT TAT GAA TAT CCA ATA Hemiberlesia sp. ATT AAA ATT ACT GGT AAT CAA TGA TTT TGA AAT TAT GAA TAT CCA TTA Italy.2 ATT AAG ATT ATT GGT AAT CAA TGA TTT TGA AAT TAT GAA TAT TCA TTA New Zealand . .A A. . Florida Group 1 California.5 . .A . .C . .C

Greece • • • . .A . .C . .C California.2 . .A . .C . .c Group 3 . .A . .C . .c Group 2 . .C . .c California.3 . .A . .A . .A . .C Italy.1 . . : . .A . .A . .C

---con- Undet. Aspidiotini TTT AAT AAA AAN TTT AAT TCT TAT CTA TTA ATT AAG AAT AAT TTT ACT Aodinella sp. TTT AAC AAA AAT TTT AAT TCC TA: TCT TTA ATA AAC AAC AAT TTT AAC Hemiberlesia sp. TTT AAT AAA AAT TTT AAT TCT TAC TTA ATC ATA AAT AAT AAT TTC AAT Italy.2 TTT AAT AAA AAT TTC AAT TCA TAT TTA TTA ATA AAT AAT AAT TTT AAC New Zealand . Florida ...... Group 1 . California.5 ... . .C.C ..T.. ..T Greece .C.C . .T...... T California.2 C ... . .C ..T. T Group 3 C.C..T.T Group 2 C.C..T. T California.3 . .C.T.. .C.T Italy.1 . .C.T.T

-COII- Undet. Aspidiotini NNT TTC Aodinella sp. TTT TCA Hemiberlesia sp. TTT ATA Italy.2 TTT TTT New Zealand . Florida . Group 1 . California.5 . Greece . California.2 . Group 3 . Group 2 . California.3 . Italy.1 .

22 Table 3. Primers used in polymerase chain reaction amplification and sequencing of the mitochondrial (CO I and CO II) DNA regions studied.

23 Name of primer Sequence region Position

S2442 CCA ACA GGA ATT AAA ATT TTT AGA TGA TTA GC CO I C2-n-3662 CCA CAA ATT TCT GAA CAT TGA CC CO II S497t CTC TTT CTC ATT CAA ATA TTG CO I A497t CAA TAT TTG AAT GTG AAA TAG CO I S2543nerii GGA GGA TTA ACT GGA ATT ATT TTA TC CO I S3399nerii CAT TGG TAA TCA ATG ATT TTG CO II A3546nerii GGT TCT TGA ATG AAT AAC ATC TAA AGA TG CO I S2513neriib GAT CCA TAG GAT TTT TAT TAA TAT TTA C CO I

24 Table 4. Uncorrected Pairwise Distance Matrix. i ON CC cn o o

i o Cd VO Cd o o d o

i l> (N NO o M in OOO odd

1 >n m NO ■d 0 O NO *—< 10 r-H O O 0 O d d d d

1 00 00 00 m t" f—« in 0 cn ON 0 O O 0 O d d d d d

1 d- d- ON 00 r- 0 t-H »-H 0 m r-H in OO O O 0 0 0 0

d o' d d d d

1 00 r-~ 00 cn r- O n d- d- NO NO NO O 0 O O O O O

d O O O O d O

0 00 in in OO 00 d- • in in d- d- NO in NO NO s 0 O O 0 0 O O O d O d 0 0 O O O

. t- t- >n d- >n Cd 0 in 00 i-H r—H •d in d- d- NO m in >n O O O 0 O O O 0 0 d d d d d d d d d

1 ON d- Cd r~ in m in *—1 00 0 d-h Cd Cd Cd d- cn in >n NO m d- N O O O 0 O 0 0 O 0 0 d d d d d d d d d d

1 0 00 O r-H in d- ON m d- d- m cd r-H cd CN O r-H 0 r-H cd r-H Cd >—1 r—I r—H r-H r-H ^H r-H d d d d d d d d d d d

r-H 1 NO r- On r- 00 r- 0 00 d" cd ON c- NO NO so NO r- NO c- t-- 00 ^H r—H r-H r-H r-H 1—1 r-H O O O O O O d d d d d d

1 NO OO NO O in ■d 00 00 cd in ON 0 cn r-H d- NO d- in in in d- d- in d- d- m in r-H r-H r-H r-H r-H T—1 r-H ^H

d d 0 O O O d d d d d d d

.£ & o *- £2 "O s<3 c Cd co «o Xl 1/3 _cd d d cd d < Id Cd cd ^ e .«

26 Table 5. Descriptive Statistics. Statistics COI /con

Alignment Length (bp) 774 %A 42.72 %C 9.37 %G 4.06 %T 43.86 %Pairwise Distance (uncorrected) 0.6-6.8%

28 Figure 1. Strict consensus tree of 11 haplotypes resolving into 3 main clades. Five haplotypes were placed in Clade 1, two haplotypes were placed in Clade 2, including Pomigliano Sicily Italy and San Juan California, four haplotypes were placed in Clade 3. Bootstrap (above) and Bremer support (below) are included.

29 undet. Aspidiotini Hemifoerlesia sp. 100 26 Aodinelfa sp.

California.2

83 Group 3 84 3 Group 2 - Clade 1

94 California.4 100 Greece - 30 99 California.5 - - Clade 2 13 Italy .1 -

Italy .2 - 88 New Zealand 75 — Clade 3 97 Florida Group 1 -

30 Figure 2. One of the six Maximum Parsimony analysis trees is shown here with character mapping. Mitochondrial DNA data results corroborate that the A. nerii complex is made up of sexual and parthenogenetic lineages. One clade is made up of parthenogenetic lineages. Confirmation for thelytoky in this clade is based on evidence that two of the tested populations were obtained from laboratory colonies that have produce no males in more than 10 years (Glenn 2001, Hare 2002 personal communication). CFB were found only in the parthenogenetic populations of A. nerii. The Sicilian individuals within the Group 1 tested negative for sex pheromone emissions and the Sicilian individuals within the Group 2 haplotype tested positive for sex pheromone emissions.

31 Hemiberlesia sp.

Legend i- i apparent parthenogenetic sexuals

d’d' Males present O Thelytoke T confirmed by rearing -J- Tested positive for CFB

_ Tested negative for CFB TUI Tested positive for sex pheromone

I_I Tested negative for sex pheromone

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37