COMPARATIVE ANATOMY OF APHID REPRODUCTIVE SYSTEMS

Andrew Polaszek

A thesis submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of the Imperial College

Department of Entomology Department of Pure & Applied Biology British Museum (Natural History) Imperial College, London SW7 London, SW7

March 1987 ABSTRACT

Comparative studies of the gross anatomy of aphid reproductive systems are described. Aphids are seasonally polymorphic, and all of the various morphs which occur are considered. Aphid biology and classification are reviewed in the introduction, and comparisons are made with other Stemorrhyncha. Special features of aphid biology, including parthenogenesis, viviparity and polymorphism, are discussed, and literature concerning reproductive anatomy of female and male aphids is reviewed. Techniques of studying the external and internal genitalia of all morphs are described. These techniques include dissection and serial sectioning, as well as studies with stereoscan and light microscopy. A survey of the reproductive systems of 4-8 Western Palearctic species in 9 subfamilies is described. Ovariole numbers, embryo or egg numbers, and sizes of embryos were recorded. Results reveal that seasonal decreases in ovariole number and/or embryo/egg number occur in most species. The reproductive characteristics of the various morphs are then discussed, and comparisons are made between species and between different morphs of the same species. The implications of seasonal changes in ovariole number and embryo number, the occurrence of vestigial or undeveloped ovarioles, and variation in ovariole number within morphs, are all discussed. Experiments were conducted to determine the influence of environmental factors such as host-plants, photoperiod and parasitization, on the development of the female reproductive system, and the results are discussed in relation to the reproductive biology of the aphids. The appearance of the external genitalia of females of 4-8 genera in 13 subfamilies is recorded. Male external and internal genitalia are described and illustrated, and the results of a survey of genitalia in 55 species, representing all 15 subfamilies of Aphidoidea, are presented. Aphid male genitalia are compared with those of other Hemiptera, and the phylogenetic implications of these studies are discussed, taking into account other aspects of aphid biology and morphology. 3

ACKNOWLEDGEMENTS

I am grateful to Dr. Roger Blackman, my supervisor at the British Museum (Natural History), for his constant guidance, considered advice and constructive criticism during each stage of the work. I also thank my university supervisor, Dr. Valerie Brown, for her help. This study was made possible by the award of a research studentship by the trustees of the British Museum (Natural History), which is gratefully acknowledged. I would like to thank Dr L.A. Mound, Keeper of Entomology for the use of the collections and facilities. I would also like to thank Dr Victor Eastop (Natural History Museum) for giving invaluable information on many aspects of aphid biology. Dr Wilf Powell (Rothamsted Experimental Station) supplied aphid parasitoids, and Dr Natasha Pungerl helped in handling them. Dr Bas Ponsen (Agricultural University, Wageningen, The Netherlands) gave up much of his time to teach me techniques of dissecting and sectioning aphids. The members of staff of the British Museum (Natural History) who helped me in various ways are far too numerous to mention by name, but I am especially grateful to all of my colleagues in the Department of Entomology, and I thank the staff of the Library Services, Photographic Unit, Electron Microscope Unit, Engineering Workshop and Botany Department for their assistance. I would also like to thank the following: Clive Carter, Forestry Commision, Alice Holt Lodge, Farnham, Surrey; James Compton, Mary Gibby, Virginia Nightingale and Julie Westfold of Chelsea Physic Garden, London; the Cotmans of Oaker, Derbyshire; Professor R.G. Davies, Imperial College, London; Professor Tony Dixon, University of East Anglia, Norfolk; Malcolm Gillham, University College, Cardiff; The Godman Exploration Fund; Dinah Hales, Macquarie University, Sydney, Australia; Andrew Halstead, Royal Horticultural Society, Wisley, Surrey; Jim Hardie and Mike Cammell, Imperial College, Silwood Park, Berkshire; J. Keesing, the Arboretum, Royal Botanical Gardens, Kew, Surrey; Gijs von Kroonowicz; Martin Luff, University of Newcastle upon Tyne; Michael Iyth, East Mailing Research Station, Kent; N(y family, and Mrs. Swanson of Brookwood, Surrey. 4

CONTENTS

Page

Abstract 2 Acknowledgements 3 Contents 4 List of figures 7 List of tables 9

1 General introduction & literature survey 13 1.1 Aphidoidea within Stemorrhyncha 13 1.2 Classification 13 1.3 Reproductive systems 1$ 1.4 Polymorphism 18 1.5 Morph determination 20 1.6 Review of literature on the anatomy of reproductive systems 22 1.6.1 Female aphids 22 1.6.2 Male aphids 25

2 Aims and objectives of the project 26

3 Materials and methods 27 3*2 Collection of material 27 3.2.1 Field collected material 27 3.2.2 Insectary-reared samples - 30 3*3 Techniques of dissection and sectioning 33 3.3.1 Techniques of dissection 33 3.3.2 Technique of sectioning 35 3.4 Techniques of mounting and staining 36 3.5 Identification of aphids 37 3.6 Methods of processing data 38

4 Comparative anatomy of the female aphid reproductive systems 39 4*1 Systematic survey 39 4*1*1 Introduction 39 4*1.2 Results: systematic account 40 Lachninae 40 Pterocommatinae 49 Aphidinae 52 5

4. 1.2 Results: systematic account (Continued) Page

Chaitophorinae ' 68 Drepanosiphinae 71 Thelaxinae 73 Mindarinae 74 Anoeciinae 75 Pemphiginae 77 Adelgidae ' 81 Phylloxeridae 82 4.1.3 Morph by morph account 83 4.1.3.1 Apterae 83 Lachninae 83 Pterocommatinae 86 Aphidinae 86 Chaitophorinae 90 Drepanosiphinae, Thelaxinae, Mindarinae, Anoeciinae 93 Pemphiginae 93 Adelgidae 95 Phylloxeridae 96 Summary 96 4.1.3.2 Alatae 98 Lachninae, Pterocommatinae 98 Aphidinae 101 Chaitophorinae - 103 Drepanosiphinae 105 Thelaxinae, Mindarinae, Anoeciinae, Pemphiginae 106 Summary 107 4*1*3.3 Sexuparae 111 4*1.3.4 Oviparae 116 4-1.4 General discussion 119 4*2 Influence of the environment on reproduction 127 4*2.1 Host-plants and aphid reproduction 127 4*2.2 The effects of photoperiod on the reproductive system in a monoecious and a heteroecious aphid 134 4*2.3 The effects of parasitization on the female aphid reproductive system 148

5 External genitalia in female aphids 160 6

CONTENTS (Continued) Page

6 Comparative anatomy of the male aphid reproductive system 170 6.1 Introduction 170 6.2 Male genitalia 172 6.2.1 External genitalia 172 6.2.2 Internal genitalia 174- 6.3 Systematic survey 174 Lachninae ' 174 Pterocommatinae 185 Aphidinae 188 Anomalaphidinae, Greenideinae 197 Chaitophorinae 199 Drepanosiphinae 203 Phleomyzinae, Mindarinae, Thelaxinae 214 Anoeciinae 217 Hormaphidinae, Pemphiginae 220 Adelgidae 222 Phylloxeridae 224 6.4 Male internal genitalia in other Hemiptera 226 6.5 Discussion: The phylogenetic implications of this study 230 6.5.1 External genitalia 231 6.5.2 Internal genitalia 235 6.5.3 Summary 240

7 Phylogeny and higher classifications of Aphidoidea: general considerations 241 7.1 Host-alternation 241 7.2 Types of sexuparae 242 7.3 Host-plant 242 7.4 Alate sexual morphs 244 7.5 Dwarf sexual morphs 244 7.6 Reduction of mouthparts and digestive tract in sexuals 246 7.7 The three-lensed eye 246 7.8 Sub-siphuncular wax-gland plates in oviparae 246 7.9 Bilobed anal plate 247 7.10 Number of rudimentary gonapophyses 247 7.11 Presence of an ovipositor-like structure 247 Summary: Phylogeny 247 8 References 249 7

LIST OF FIGURES Page Frontispiece: Pterocomma salicis aptera on sallow 12 1 INTRODUCTION Fig. 1.1 Internal reproductive system of Amphorophora tuberculata virginopara 16 Fig. 1.2 Internal reproductive system of A. tuberculata ovipara 17

3 MATERIALS AND METHODS Fig. 3»1 A simple method of rearing aphids 31 Fig. 3.2 Ebccised-leaf cage 32 Fig. 3«3 A method of rearing very small aphids 32

4 FEMALE INTERNAL REPRODUCTIVE SYSTEMS Fig. 4-1 Reproductive system of Cinara pilicornis showing undeveloped ovarioles 42 Fig. 4-*2 Seasonal mean embryo and ovariole numbers in Cinara pini, C. pinea and Lachnus roboris 48 Fig. 4-3 Mean embryo/egg numbers in Pterocomma spp. 51 Fig. 4..4. Mean embryo/egg numbers in two Aphis spp. 54- Fig. 4.5 Mean embryo/egg and ovariole numbers in Anoecia comi 76 Fig. 4-.6 Ovarioles of Eiriosoma lanuginosum sexupara after deposition of embryos 78 Fig. 4-7 Ovarioles of successive instars of Acyrthosiphon pisum alatae 108 Fig. 4-8 Ovaries of successive instars of Eiriosoma ulnri alatae 109 Fig. 4-.9 Reproductive system of Euceraphis punctipennis sexupara showing absorption of advanced embryos 113 Fig. 4«10 Ovarioles of Drepanosiphum platanoidis sexupara showing unusual (?trophic) structures 113 Fig. 4-* 11 Ovary of Hyperomyzus lactucae gynopara showing undeveloped embryos 115 Fig. 4-12 Mean numbers of embryos in successive instars of A. pisum 138 Fig. 4.13 Mean sizes of largest embryos in successive instars of A. pisum 139 Fig. 4.14 Ovarioles of third instar presumptive gynopara of Myzus persicae 144 Fig* 4*15 One ovariole of an adult M. persicae gynopara 144 Fig. 4* 16 Changes in mean embryo number, maximum embryo size and body length in A. pisum parasitized at first instar 151 LIST OF FIGURES (Continued) Page

Fig. 4*17 Ovary of A. pisum nine days after parasitization at first instar by Aphidius ervi 152 Fig. 4..18 A-D Egg and larvae of Ephedrus plagiator and serosal cell of Aphidius ervi 152 Fig. 4-. 19 Changes in mean embryo number, maximum embryo size and body length in A. pisum parasitized at fourth instar 154- Fig. 4-*20 Ovary of adult A. pisum parasitized at fourth instar 154- Figs 4- .21-4-. 24- Scanning electron micrographs of parasitoid larva and serosal cells 156

5 FEMALE EXTERNAL GENITALIA Figs 5.1 -• 5.14- SEMs of the genital area of various aphid species 162/164 Fig. 5.15 SEM of the genital area in a Mindarus abietinus ovipara 169 Fig. 5.16 Genital area of a Paoliella harteni ovipara 169

6 MALE GENITALIA Figs 6.1, 6.2 SEMs of Megoura viciae external genitalia 173 Fig. 6.3 M. viciae internal genitalia 173 Fig. 6 .4. Lachnus roboris genitalia 175 Fig. 6.5 Serial sections through L. roboris reproductive system 176 Figs 6.6 -•6.29 Male genitalia in various aphid species: 6.6 Maculolachnus submacula 177 6.7 Stomaphis quercus, S. yannonis 179 6.8 Cinara pini, C. boerneri 180 6.9 C. stro.yani 182 6.10 Eulachnus rileyi, E. agilis 183 6.11 Pterocomma populeum 186 6.12 P. salicis 187 6.13 Aphis epilobiaria, 6.14- A. fabae, 6.15 A. farinosa 189 6.16 A. praeterita 189 6.17 Macrosiphoniella artemisiae 190 6.18 M. millefolii, 6.19 M. oblonga 190 6.20 Cavariella aegopodii, 6.21 Hvperomyzus lactucae 191 6.22 Macrosiphum albifrons, 6.23 M. rosae 191 6 .2 4 -6.29 Male genitalia of a further six Aphidinae 192 Fig. 6.30 Horizontal serial sections through testes of successive instars.of M. persicae 196 9

LIST OF FIGURES (Continued) Page

Figs 6,31-6*59 Male genitalia in various aphid species 6,31j 6,32 Scoutedenia lutea, 6.33 Greenidea anonae 198 6.34- Chaitophorus capreae 200 6.35 C. leucomelas 201 6 .36 Periphyllus hirticornis, 6.37 P. testudinaceus 202 6.38 Drepanosiphum platanoidis 205 6.39 Bucallipterus tiliae 206 6.4- 0 Euceraphis betulae 207 6.4- 1 Phyllaphis fagi 208 6.42 Neophyllaphis brimblecombei, N. ?podocarpi 210 6.4- 3 Israelaphis tavaresi, 6.44- Neuquenaphis edwardsi 212 6.4- 5 Lizerius ocoteae 212 6 .4.6 Tamalia coweni, 6.4-7 Paoliella harteni 213 6.4- 8 Phleomyzus passerinii 213 6.4.9 , Mindarus abietinus 215 6.50 Glyphina betulae 216 6.51 Thelaxes dryophila 218 6.52 Anoecia comi, 6.53 Hamamelistes spinosus 219 6.54- Pemphigus spyrothecae, 6.55 Erisoma lanigerum 221 6 .56 P. spyrothcae. E. lanigerum internal genitalia 221 6.57 Pineus orientalis 223 6.58 Adelges cooleyi 223 6.59 Phylloxera glabra 225 Fig. 6.60 External genitalia in other Sternorrhyncha 227 Fig. 6.61 Internal genitalia in other Stemorrhyncha 229

LIST OF TABLES

1 INTRODUCTION Table 1.1 Classification of Aphidoidea 14 Table 1.2 Types of aphid sexuparae 19-20

4 FEMALE INTERNAL REPRODUCTIVE SYSTEMS Table 4-.1 Numbers of species studied in each subfamily 4-0 Table 4-2 Numbers of functional and undeveloped ovarioles in Cinara pilicomis alatae 4.1 Table 4-. 3 Data concerning reproduction in Lachninae 46 10

LIST OF TABLES (Continued) Page

Table 4 .4 Data concerning reproduction in Cavariella fundatrices 63 Table 4*5 Summarized data concerning reproduction in Aphidinae 66 Table 4-.6 Frequency of ovariole numbers in C. leucomelas apterae 69 Table 4 .7 Correlation of pairs of variables concerning reproduction in apterae of Lachninae 84 Table 4*8 Mean body lengths and largest embryo sizes in apterae of Lachninae 85 Table 4*9 Mean embryo and ovariole numbers in Cinara 86 Table 4•'10 Correlation of pairs of variables concerning reproduction in apterae of Pterocommatinae 87 Table 4-11 Correlation of pairs of variables concerning reproduction in apterae of Aphidinae 88 Table 4*12 Correlation of pairs of variables concerning reproduction in apterae of Chaitophorinae 90 Table 4*13 Mean embryo and ovariole numbers in apterae of Chaitophorinae 91 Table 4»14 Correlation of pairs of variables concerning reproduction in apterae of seven species of Aphididae 92 Table 4»15 Mean embryo and ovariole numbers in apterae of five aphid species 93 Table 4*16 Mean body lengths and sizes of largest embryos in five species of Pemphiginae 94 Table 4*17 Mean embryo and ovariole numbers in pemphigine apterae _ 95 Table 4»18 Mean ovariole and egg numbers in apterae of Adelgidae 95 Table 4*19 Correlation of pairs of variables concerning reproduction in alatae of Lachninae 99 Table 4*20 Correlation of pairs of variables concerning reproduction in alatae of Pterocommatinae 100 Table 4*21 Correlation of pairs of variables concerning reproduction in alatae of Aphidinae 102 Table 4*22 Mean ovariole and embryo numbers in alatae of Aphidinae 103 Table 4-23 Correlation of pairs of variables concerning reproduction in alatae of 10 species of Aphididae 104 Table 4«24 Distribution of ovariole numbers in alatae of Drepanosiphinae 105 Table 4.25 Mean ovariole and embryo numbers in alatae of Drepanosiphinae 106 11

LIST OF TABLES (Continued) Page

Table J+,26 Mean embryo and ovariole numbers and sizes of largest embryos in wingless sexuparae 111 Table 4*27 Mean embryo and ovariole numbers in winged sexuparae 112 Table 4-*28 Summary of modal ovariole numbers in aphid oviparae 116 Table 4.29 Distribution of ovariole numbers in virginoparae having a modal ovariole number of 10 123 Table 4.30 Reproduction in P. salicis fundatrices 128 Table 4-31 Mean body lengths, embryo numbers and ovariole numbers in M. rosae virginoparae 129 Table 4*32 Mean body lengths, embryo numbers and ovariole numbers in P, testudinacea alatae 129 Table 4*33 Mean body lengths, embryo numbers and ovariole numbers in A. corni alatae 130 Table 4*34 Mean numbers of embryos in apterae associated with different host-plant categories 133 Tables 4*35-4*38 Data concerning reproduction in successive instars of A. pisum under different environments 137 Tables 4*39-4*41 ANOVAs on body length, embryo number and size of largest embryo in A. pisum under different conditions 140 Table 4*42 Mean body lengths, embryo numbers and sizes of largest embryos in M. persicae gynoparae 142 Table 4*43 Comparison of mean body lengths, embryo numbers and sizes of largest embryos in field-collected and lab-reared M. persicae gynoparae 143 Table 4*44 Nuumbers of ovarioles and embryos in winged virginoparae and gynoparae of heteroecious Aphidinae 146 5 FEMALE EXTERNAL GENITALIA Table 5*1 Numbers of genital processes and rudimentary gonapophyses in 48 genera of Aphididae 166-167

6 MALE GENITALIA Table 6.1 Sample sizes, presence of wings and numbers of testis follicles in 17 species of Aphidinae 194 Table 6.2 Forms of claspers in subfamilies of Aphidoidea 231 Table 6 .3 Fusion and separation of the testes in Aphidoidea 237 Table 6 .4 Numbers of testis follicles in subfamilies of Aphidoidea 238 7 PHYLOGENI AND HIGHER CLASSIFICATION Table 7.1 Host-plant associations in Aphidoidea 243

1. GENERAL INTRODUCTION & LITERATURE REVIEW

1.1. Aphidoidea within Sternorryncha Aphidoidea is a superfamily of small, hemipteran insects, which feed exclusively on plants. The superfamily comprises Aphididae, Adelgidae and Phylloxeridae, and is one of four superfamilies in the series Stemorryncha, the others being Coccoidea (scale insects and mealybugs), Aleyrodoidea (whiteflies) and Psylloidea (psyllids). Evans (1963) suggested that aphids and coccids are more closely related to each other than to either of the other superfamilies, and according to Schlee (1969a, 1969b) they share a number of autapomorphies which suggest that Aphidoidea and Coccoidea are sister-groups, together known as "Aphidiformes11. Aphidoidea are notable for their exploitation of thelytokous parthenogenesis, which in the Aphididae is combined with viviparity. Thelytoky is the process by which female offspring are produced from unfertilized eggs with a diploid number of chromosomes. It is to be distinguished from arrhenotoky, which is the production of usually haploid male offspring from unfertilized eggs. Male haploidy occurs in coccoids and whiteflies, but not in aphids. Within the Sternorryncha all Aphidoidea, and some coccids and whiteflies, are able to reproduce by thelytoky (Soumalainen, 1962), only Psylloidea have no known case of parthenogenetic reproduction (Hodkinson, 1971). Viviparity is found in some families of Coccoidea and throughout the Aphididae, but not in Adelgidae, Phylloxeridae, Aleyrodoidea or Psylloidea.

1.2 Classification of Aphidoidea. There are a number of higher classifications of aphids in common use at the present time, the main disagreements between them being limited to whether the major aphid groups constitute families or subfamilies, and where a number of small but distinct taxa should be allocated. Very little is known about the phylogenetic relationships between the major taxa, although most workers accept that Chaitophorinae are more closely related to Drepanosiphinae than they are to any other subfamily. Aphidinae and Pterocommatinae are also closely related. The main life-cycle characteristics of all the higher taxa in the Aphidoidea which have been described as either families or subfamilies by Bomer (1952), Bomer & Heinze (1957), Eastop (1966, 1977), Eastop & van Emden (1972) and Heie (1980) are summarized in Table 1.1, and this classification will be used for the purposes of this thesis. Aphid higher classification is discussed further in section 7. Table 1.1 Classification of Aphidoidea

SUBFAMILY LIFE-CYCLE PRIMARY HOST SECONDARY HOST DISTRIBUTION Aphididae Lachninae Monoecious Conifers, Mostly woody plants holarctic Pterocommatinae Monoecious Salicaceae Holarctic Aphidinae Monoecious/ Usually woody Usually World-wide heteroecious plants herbaceous Greenideinae Monoecious Woody plants Oriental Anomalaphidinae Biology- Australasian poorly known Chaitophorinae Monoecious Salix, Acer, Mostly Populus or holarctic grasses Drepanosiphinae Monoecious Woody plants, World-wide Leguminosae or monocotyledons Phleomyzinae Monoecious Populus Holarctic Mindarinae Monoecious Abies or Holarctic, Picea oriental Thelaxinae Monoecious Quercus & Holarctic, Betula oriental Anoeciinae Monoecious/ Cornus Roots of Holarctic, heteroecious Graminae oriental Hormaphidinae Heteroecious Various, inc. Woody plants Nearctic, biennial woody plants (where known) oriental Pemphiginae Mostly Woody plants Roots of Holarctic, heteroecious various plants oriental Annual or biennial Adelgidae Adelginae and Heteroecious, Coniferae Coniferae World-wide Pineinae biennial Phylloxeridae Phylloxerinae Mostly Fagaceae, World-wide monoecious Salicaceae Juglandaceae & Vitis 1.3 Reproductive systems The female reproductive system of parthenogenetic, viviparous Aphididae consists of two ovaries, each comprising 1-25 ovarioles (Fig.1.1). These ovarioles have germaria at their distal ends, and are joined at their proximal ends to a common oviduct which leads to the genital opening. Each ovariole may contain up to seventeen embryos at successive stages of development. The additional features of the reproductive system of a sexual, oviparous aphid are a pair of colleterial glands and a seminal receptacle or spermatheca (Fig.1.2). Sexual, oviparous aphids rarely contain more than ten ovarioles in each ovary or more than three eggs in each ovariole. The ovarioles of both viviparous and oviparous aphids are of the meroistic, telotrophic type, with nurse cells present but restricted to the germarium and connected to the first few developing embryos by nutritive cords (Huxley, 1859; Weber, 1930). The external genitalia of female aphids are reduced in most species studied, consisting simply of a genital opening situated between the genital and anal plates. Some species have up to four small, haired tubercles, the so-called "rudimentary gonapophyses", below the genital opening, on the side nearest the anal plate. The oviparae of some species have the last few abdominal segments drawn out. The external genitalia of female Adelgidae are more fully developed, and resemble those of Aleyrodidae. There are a pair of lateral gonapophyses and an unpaired dorsal gonapophysis containing the efferent duct of the cement gland (Borner,1908; Ossiannilsson et al, 1956). The internal reproductive systems of male aphids consist of a ..pair of testes, which may be fused and so appear to be a single organ, with vasa efferentia, becoming vasa deferentia, leading from them to the ejaculatory duct. A pair of accessory glands also opens into the duct, although these glands are absent in some taxa. The external genitalia of male aphids consist of a pair of claspers (occasionally referred to in the literature as operculae) which may be used to grip the female during mating, and a pair of valves which cover the genital opening when the aedeagus is withdrawn, and also serve as levers for its extension. The general structure of both the internal and external genitalia of male aphids is dealt with in full in section 6.2 and illustrated in figures 6.1-6.3 (p.173). Fig 1.1 Internal reproductive system of Amphorophora tuberculata virginopara

O) 17

Fig. 1.2 Internal reproductive system of Amphorophora tubercular* ovipara 1.4. Polymorphism Aphids are polymorphic, each species having a number of forms or morphs, which differ functionally as well as morphologically, associated with their seasonal changes in reproduction. The nomenclature used in this thesis for these morphs is outlined below. The sexual, egg-laying females are called oviparae, and usually occur, together with the males, in the autumn. These morphs are known collectively as sexuales or sexuals. The oviparae may be morphologically similar to the parthenogenetic females, or they may be specialised. The morph which hatches from the fertilized egg, usually in the spring, is called a stem-mother or fundatrix, the foundress of a clone. Her offspring, and the following parthenogenetic, viviparous generations are all known as virginoparae (i.e. "producers of virgins"), and may be either winged or wingless. The first generation of virginoparae produced by the fundatrix may also be called fundatrigeniae. The morphs which give rise to the sexuals are known collectively as sexuparae. Both the biology and the nomenclature of sexuparae is complex when Aphidoidea are considered as a whole. Sexuparae may be winged or wingless, they may give birth to males or oviparae or both, and they may give birth to other morphs in addition to the sexuals. Sexuparae which give birth to oviparae, but not to males, are usually known as gynoparae, and those which give birth to males and not to oviparae are known as androparae (although this term is rarely used). Many aphid species show host-alternation (heteroecy), and separate terms exist for those morphs flying to or returning from their secondary host, and for those which spend all their time on that host. Alatae leaving the primary host in spring are called emigrants, and any virginoparae bom on the secondary host are exules. Mating, and oviposition of the sexual egg, normally occurs on the primary host, and so heteroecious aphids must undertake a return journey from the secondary host. This may be accomplished by winged sexuparae (e.g. in Pemphiginae, Heteroecious Anoeciinae, Hormaphidinae), winged gynoparae and males (Heteroecious Aphidinae) or winged androparae and gynoparae (?some Adelgidae and Phylloxeridae). In order to interpret the different reproductive anatomies and reproductive strategies of the sexuparae which were studied in this survey it is necessary to understand the role of the sexupara in the particular species studied. The different kinds of sexuparae which occur are summarised in Table 1.2. Table 1.2 Types of aphid sexuparae

SUBFAMILY SEXUPARAE Lachninae Wingless (?also winged) sexuparae give birth to winged or wingless males, wingless oviparae, and possibly other morphs as well. Pterocommatinae As above. Aphidinae (Monoecious) As above. Aphidinae (Heteroecious) Winged gynoparae born on the secondary host migrate to the primary host and deposit the oviparae which develop into wingless adults. The males are bom of (usually) wingless androparae, which may also give birth to gynoparae and other morphs, but not normally to oviparae. Males are winged and migrate to the primary host. Greenideinae It seems to be unrecorded whether the winged sexuals are bom of winged or wingless sexuparae, or indeed whether both sexes are bom of the same morph. Anomalaphidinae Little is known about either the sexuparae or sexuals in this subfamily. Male Schoutedenia spp. are winged. Chaitophorinae Winged or wingless sexuparae give birth to winged or wingless males and wingless oviparae. Drepanosiphinae Winged (?also wingless) sexuparae give birth to both sexuales of which the males are winged in most species, and oviparae are only very occasionally winged. Fhleomyzinae Wingless sexuparae give birth to the winged sexuales. Mindarinae and Thelaxinae All alatae are sexuparae, but wingless morphs may also produce the dwarf, wingless sexuals. Anoeciinae (Monoecious) It is not recorded whether the wingless sexuals are bom of winged or wingless sexuparae. Oviparae are normal-sized, but males are dwarfish in some species. Anoeciinae (Heteroecious) Winged sexuparae, bom on the secondary host, migrate to the primary host and deposit the dwarfish wingless sexuals. Hormaphidinae As in heteroecious Anoeciinae. Pemphiginae As in heteroecious Anoeciinae. Sexuals are without mouthparts. Table 1.2 (Continued) Adelgidae As in heteroecious Pemphiginae, but according to Speyer (1923) separate gynoparae and androparae are involved. Sexuals with mouthparts. Phylloxeridae Sexuparae are winged, sexuals are dwarfed and wingless with reduced, non-functioning mouthparts. In Adelgidae and Phylloxeridae, the naming of the various morphs is more complicated. Adelgids are heteroecious, always with a Picea species as the primary host, and conifers of other genera as secondary hosts. They have sexuals, fundatrices and sexuparae, like some Aphididae, but the intervening generations are called gallicolae if they migrate from galls on the primary host and sistens or progrediens if they are wingless and found on the secondary host (Carter, 1971). Like some Aphididae and all Adelgidae, Phylloxeridae also have fundatrices, sexuparae and sexuals, but in the species Viteus vitifolii Fitch some morphs form galls on the leaves, the gallicolae, and some feed on the roots, the radicolae. Some races of aphids, and occasionally entire species, have lost the ability to produce either one or both of the sexual morphs. Such races or species are called anholocyclic, in contrast to those which have a sexual phase, which are called holocyclic. It seems to be more common for partially anholocyclic populations to have lost the ability to produce oviparae, so males produced by such populations may fertilise oviparae from other populations. A partially anholocyclic race may therefore still contribute to the genetic diversification of the species (Blackman, 1974a,1974b).

1.5 Morph determination There have been many investigations into the stimuli responsible for the production of particular morphs, and these stimuli have been found to include crowding, temperature and photoperiod. The observations of Garner & Allard (1920) on the photoperiodic responses of plants drew the attention of Marcovitch (1924), who went on to demonstrate photoperiodicity in aphids, this being the first account of such a phenomenon occurring in animals. He found that if Capitophorus hippophaes and Aphis forbesi were exposed to long nights soon after they hatched in the spring, sexuals were produced several months before their occurrence in the field. Lees (1959,1960,1964,1966) demonstrated that the brain of Megoura viciae is sensitive to light passing throught the cuticle. The amount of light received is somehow registered by the "group 1" cells of the brain, and induces appropriate changes in their neurosecretions. It has been suggested that these neurosecretions are transported directly to the gonads and not released into the haemolymph, thus affecting the progeny but not the parent (Steel & Lees, 1977). When embryos of Aphis fabae reach a particular stage of development, morph determination switches from maternal to autonomous. Some aphids produce their sexual morphs under long-day conditions, e.g. Aphis farinosa and Dysaphis devecta. In D. devecta production of sexuals is induced by changes in host-plant quality (Forrest, 1970). In some root-feeding aphids' which live in darkness e.g. Eriosoma pyricola, production of sexuals is thought to be controlled in a similar way (Sethi and Swenson, 1967), although crowding effects may be the stimulus for production of sexuals in Pemphigus bursarius (Judge, 1968). In the sycamore aphid Drepanosiphum platanoidis (Drepanosiphinae) male production is not under photoperiodic control, males appearing after a certain number of generations have elapsed, and this may occur in other monoecious species (Dixon, 1973)* Hales & Mittler (1983) found that male production in Myzus persicae could be induced by destroying the mother’s corpus all atm with precocene. The determination of the progeny of virginoparae as either apterae or alatae is thought to depend mainly on crowding and host-plant quality. If aphids are reared in isolation, they tend to produce more apterae than if they are reared in crowded conditions (Bonnemaison, 1951; Shaw, 1970). However, experiments using artificial diets (Mittler & Dadd, 1966; Mittler & Kleinjan, 1970) have shown that more apterae are produced on poor quality artificial diets than are produced on diets which are optimal for larval development. These results appear to contradict the generally accepted concept that alata-production is enhanced by poor nutrition. Harrewijn (1976) has demonstrated that derivatives of tyrosine and tryptophan can regulate wing-bud development in new-born M. persicae larvae. Environmental stimuli are thought to affect the level of juvenile hormone in the aphid, and it is this hormone which controls the production of apterae (Hales, 1976). Presumptive winged gynoparae of A. fabae which have had juvenile hormone applied to them topically, complete their development to become perfectly normal apterae (Hardie, 1980a). Hales and Mittler (1981) found that treating adult Hyzus persicae with precocene caused their offspring to become "adultoid” by the third instar, with fully developed embryos inside them. These could not however be deposited due to incomplete differentiation of the reproductive tract. Thus, some species studied appear to have life-cycles which consist of a fixed sequence of morphs, the type of morph appearing at- each stage in the life-cycle being pre-determined long before the aphid's birth. Other species have fewer constraints on the type of morph produced at a particular stage in the life-cycle, and morph determination in these species is often dependent on environmental stimuli received by the individual or by its parent. Most of these studies have been on species of Aphidinae, and less is known about the mechanisms behind morph determination in the other sub-families.

1.6 A review of literature on aphid reproductive anatomy,

1.6.1 Female aphids In 1695) van Leeuwenhoek dissected some aphids which he found curling the leaves of gooseberry, having seen them giving birth to live young. Inside each of those dissected he found between 20 and 60 embryos, some of which were fully developed. Later it was observed in a number of species that several successive generations could be bred, apparently without the necessity of having males present (Reaumur,1712a,1712b; Bonnet,1713; Curtis,1802). The discovery of males and oviparous females, and the observation that they were usually found only during autumn led to the idea of "continued impregnation" whereby the effects of fertilization by a male were somehow passed on to later generations until the following autumn, when the presence of a male would once again become necessary (Tremblay,1776). A commonly held opinion at this time was that viviparous aphids were hermaphrodite, but this idea was not accepted by Bonnet, who recognised both types of egg as true ova, and is thus credited with the discovery of parthenogenesis. Newport (1816,1818), using rose aphids, demonstrated that the eggs laid by oviparae did not contain fully formed embryos, as some previous workers had suspected, but were normal insect eggs. Mistaken observations concerning the method of reproduction in viviparous aphids have, in the past, led to much controversy. Steenstrup (1815) claimed that aphids showed "alternation of generations" as in other organisms which have sexual and asexual reproductive phases. Burnett (1851) distinguished viviparity in aphids from that of other insects, saying that in aphids there are no ovaries or oviducts, the embryos developing from buds on the inside of the body wall. Balbiani (1866a,1866b) re-established the notion that viviparous aphids were hermaphrodite, claiming that a male factor ("1* element matle") could be distinguished in the germanium. Simultaneous with these findings, Mecznikow (1866) published his theory that aphids were parthenogenetic ("agamo-genetic"), and this gained the 23 support of Claparbde (1867) who rejected Balbiani's theory. Lemoine (1893) also attempted to clear up this controversy by carefully comparing the embryology of viviparous and oviparous aphids. A number of detailed studies of the reproductive anatomy of aphids were carried out in Germany during the second half of the nineteenth century, the work of von Heyden (1857), Leuckart (on Adelgidae) (1859) > Brass (1882) and Witlaczil (1882) being particularly noteworthy. These authors observed that the colleterial glands and seminal receptacle are absent in viviparous aphid morphs, and that the latter organ is absent in parthenogenetic adelgid morphs. Numbers of eggs, embryos and ovarioles were recorded in some aphids and adelgids (Leuckart, 1859; Brass, 1882). The occurrence of trophic cords connecting the germarial lumen to the first few developing oocytes was recorded (Will, 1883,1888). The embryological studies of Witlaczil (1881), Tannreuther (1907) and von Baehr (1909,1920), continued the steady stream of research by German workers. At this time in England, Buckton (1875,1877,1880,1882) wrote and illustrated a four volume monograph on British aphids. This (for the most part) conscientiously researched work contains chapters devoted specifically to reproductive anatomy and previous ideas about aphid reproduction. The various dissecting and staining methods he used are described, as well as the reproductive anatomy of viviparous and oviparous females of a few aphid species, and their ovariole numbers. He looked specifically at virginoparae of Siphonophora (=Macrosiphum) rosae and Siphonophora pelargonii (=Ac.yrthosiphon malvae), the ovipara of Callipterus quercus (=Tuberculoides annulatus), a Cherme s (=Adelge s) and a Phylloxera species. A series of studies of the life-histories of three pest aphids was carried out in America (Baker, 1915; Baker & Turner, 1916a, 1916b), and these included descriptions of their embryology and internal genitalia. Baker (1915) observed that the oviparae of Pemphiginae develop only one ovariole, the others becoming atrophied and eventually undetectable. Uichanco (1924.) made a close study of Macrosiphum (=Uroleucon) tanaceti, recording the ovariole number, and describing the contents of the germarium, although he was unable to locate any trophic cords or distinguish the germarial lumen. Apart from the work of Shull (1930), Weber's (1930) book on hemipteran biology, and Hagan's (1951) comprehensive review of the embryology of viviparous insects (which includes an extensive account of previous work on aphid reproduction), very little was published about aphid reproductive systems until Lees (1959) 1 in a study of morph determination in the vetch aphid, recorded numbers of ovarioles and embryos at various developmental 24 stages. A number of papers on aphid cytogenetics (Orlando,1972a, 1972b; Pagliai,1965; Crema,1971,1973), a subject which lies outside the scope of this review, paid particular attention to ’’ambiphasic” aphids, i.e. those which have some ovarioles containing developing embryos and some containing eggs. A description of the reproductive system of a viviparous aphid can be found in the embryological study by Brusle (1962). Oseto & Helms (1971) studied the post-parturienic development of the reproductive system in Schizaphis graminum, describing the origins of the ovaries and lateral oviducts from mesodermal tissues. The number of ovarioles in Schoutedenia lutea, an unusual species belonging to the Anomalaphidinae, was recorded, and the reproductive system of the ovipara illustrated, by Hales & Carver, 1976. In the late 1970s a surge of interest in aphid reproductive systems took place, and detailed studies were made of ovarian development in Aphis fabae (Tsitsipis & Mittler,1976) and A. craccivora (Elliot & Macdonald,1976). Both include records of numbers of ovarioles, embryos and oocytes found at various stages of the aphids’ development. Professor A.F.G. Dixon and co-workers at the School of Biological Sciences, University of East Anglia, have made a large contribution to the study of aphid reproduction, particularly its ecological aspects. Seasonal changes in fecundity and state of gonads were found in Drepanosiphum platanoidis (Dixon,1975) and Rhopalosiphum pad! (Dixon,1976), although in this work the ovariole numbers of the various morphs were not recorded. In a subsequent study of A. fabae (Dixon & Dharma,1980) ovarioles numbers were recorded, and a distinct pattern of seasonal variation in ovariole number was found. Further studies, on six species from various subfamilies, showed that this phenomenon of seasonal variation in ovariole number was probably widespread among the Aphididae, and was a ’’programmed” feature of their life-cycles (Wellings et al, 1980). Blackman (1978) described the differentiation of germarial cells into oocytes and nurse cells. Elliott et al (1975), Couchman & King (1979,1980) and Buning (1985) described the ultrastructure of aphid germaria. The only broad survey of female aphid reproductive systems was by Tashev & Markova (1982) who recorded ovariole numbers of 86 species, often in two or more morphs of each species, and numbers of embryos with pigmented eyes in 52 species, although sample sizes were not included. Embryo counts have been suggested as a method of measuring potential fecundity in aphids (Banks,1965; Adams & van Emden,1972). Brown and Llewellyn (1985) counted embryos in a number of aphid species and related potential fecundity to both seasonality and plant growth form. 1.6.2 Male aphids There have been very few studies of the reproductive anatomy of male aphids. This is mainly because males are normally present for only a few weeks of the year. In 1836, Morren described the male of Aphis (=?Myzus) persicae as having between four and five ’’testicules", and recorded the presence of accessory glands, although he considered the latter to be seminal vesicles. Balbiani (1869) made a detailed study of the internal and external genitalia of the males of Siphonophora (=Macrosiphoniella) millefolii and Drepanosiphum platanoidis. The excellent illustrations are fully and unambiguously annotated, and observations on some immature males and on the species Aphis (=Myzus) persicae and Siphonophora .jaceae (-Uroleucon (Uromelan) jaceae) are included. It is unfortunate that Balbiani is chiefly remembered for his mistaken theory of hermaphroditism in aphids (see p.22, 1-34), while this carefully produced study is largely forgotten. In volume four of his monograph, Buckton (1882) included a chapter on the genitalia of male aphids, most of the information having been culled from Balbiani1 s 1869 paper, from which a lot of the illustrations were also copied. As this was the most widely read British work on aphids at the time and remained so until Theobald’s (1926,1927,1929) monograph, it is a pity that Buckton mislabelled the illustrations copied from Balbiani, misnaming the species and some of the structures depicted. Dbrbes (1871) illustrated the external genitalia of a male Pemphigus (=Forda) from Pistacia. Blochmann (1887) and Cholodowsky (1900), made some conflicting observations on the genitalia of two Chermes (=Adelges) species, and Tannreuther (1907) recorded the number of testis follicles in an aphid identified as Melanoxanthus (=Pterocomma) salicis, although cytological studies (Blackman, 198$) suggest this was probably a misidentification. Nusslin (1910) described and figured the internal genitalia of Mindarus abietinus and Prociphilus fraxini males. Baker (1915) described the internal and external genitalia of Eriosoma lanigerum, and Baker & Turner (1916a) the external genitalia of Aphis pomi. The only recent work on this subject is reported in a series of papers from Poland by KLimaszewski et al (1973), GZowacka et al (1974a, 1974b) and Bochen et al, (1975). These point out the differences between the male reproductive systems in various aphid taxa, and the phylogenetic implications of these differences are discussed. Blackman (in press) describes the general structure of the reproductive system of a male aphid. 2. THE AIMS AND OBJECTIVES OF THIS PROJECT

From the preceding review one can see that there is still a considerable amount to be learned from further studies of the reproductive systems of aphids. The only broad survey of reproductive systems in female aphids (Tashev & Markova,1982) is inadequate because few morphs of each species are included and sample sizes are not mentioned. A survey of the reproductive systems of females of over forty species was therefore carried out, including comparisons with adelgids and phylloxerids. The external genitalia of female aphids have been little investigated apart from some early records by van der Goot (1913) of numbers of "rudimentary gonapophyses" found in females of various species, which he attempted to use as a taxonomic character. A survey of female external genitalia in aphids was therefore undertaken. There has also been very little work on male aphid genitalia. Representative species from as many taxa as was practicable were therefore selected, and detailed studies were made of their external and internal genitalia. The genitalia of male Aphidoidea were also compared with those of Coccoidea, Aleyrodoidea and Psylloidea. The purpose of these two broad surveys was twofold: firstly to attempt to correlate seasonality with differences in the reproductive strategy between morphs of the same species and between corresponding morphs in different species, and secondly to try to deduce the phylogenetic relationships between the taxa studied, by comparing the anatomy of their reproductive systems. In connection with the survey of female aphids, a study was made of the changes in the reproductive systems of females during the transition from parthenogenetic to sexual reproduction. This study included the comparison of a monoecious species with a heteroecious species. Some aphids collected in the field were found to contain the larvae of parasitoid wasps, and the embryos of some parasitized aphids showed severe parasitogenic effects although their mothers had been active and had appeared to be healthy. A series of controlled experiments was therefore carried out to investigate the effects of parasitization on the reproductive systems of viviparous aphids at different stages in their development. 3. MATERIALS AND METHODS

3.1 Material available for study. Most of the information concerning male and female reproductive systems was obtained by dissection of freshly killed aphids. These aphids were either recently collected in the field or obtained from insectary-reared cultures. When necessary, samples were preserved in either 70-95% alcohol or cytological fixative for later examination and/or dissection. The BM(NH) spirit collection was a supplementary source of material, particularly of male Coccoidea. Slides in the BM(NH) collection were used for microscopical examination of the external male genitalia of some of the species I was unable to collect in the field or rear in the insectary. In a few cases data were supplemented by descriptions of aphid reproductive systems available in the literature.

3.2 Collection of material

3.2.1 Field collected samples. The majority of field collected samples were from London and the Home Counties, a few coming from elsewhere in England. Four samples were collected from Co. Kerry, Republic of Ireland, and one sample of Glyphina sp. came from Zabrzeg in Poland. Males of Schoutedenia and Neophyllaphis spp. were collected in New South Wales, Australia, and in Chile. The following abbreviations used in the text describe collecting sites:

AH Aldershot, Hampshire __ AHL Alice Holt Lodge, nr. Farnham, Surrey B Brookwood, Surrey BC Barnes Common, London SW13 BMC British Museum (Natural History) insectary culture BMG British Museum (Natural History) grounds, London, SW7 BTH The Bourne, nr. Tilford, Hampshire CB Chiswick Bridge, London SW14 CG Cranley Gardens, London SW7 CHN Close House, nr. Wylam, Northumberland CP Cassiobury Park, Hertfordshire CPG Chelsea Physic Garden, London SW3 CRP Chelverton Road, London SW15 CSA Cobham & Stoke D’Abemon, Surrey DD Dunstable Downs, Bedfordshire EM East Mailing Research Station, Kent F Fleet, Hampshire FPR Fulham Palace Road, London SW6 GP Gunnersbury Park, London W5 (21 Galpin’s Road, London SW16 H Heston, Middlesex HH Hampstead Heath, London N6 HMP Headstone Manor Park, Harrow, Middlesex HP Holland Park, London W14- IPRP Isabella Plantation, Richmond Park, Surrey K Kenwood House, London N6 KB Kew Bridge, Surrey KGS Kew Gardens Station, Kew, Surrey KK Killarney, County Kerry, Republic of Ireland M Montpelier Street, London SW7 MC Mitcham Common, Surrey MM Merthyr Mawr, Glamorgan, S. Wales MR Montserrat Road, London SW15 0 Oaker, nr. Matlock, Derbyshire OH Oxshott Heath, Surrey OWCH Ockham, Wisley & Chatley Heath, Surrey PCem Putney Cemetary, London SW15 PCom Putney Common, London SW15 PROK Public Records Office, Kew, Surrey RBGK Royal Botanical Gardens, Kew, Surrey RDIK Rossdohan Island, County Kerry, Republic of Ireland RHSW Royal Horticultural Society, Wisley, Surrey RIK - Ross Island, County Kerry, Republic of Ireland RL Rayner1s Lane, Middlesex ROT Radcliffe-on-Trent, Nottinghamshire RP Richmond Park, Surrey RS Redhill, Surrey SC Stanmore Common, Middlesex SP Silwood Park, Ascot, Berkshire SR Spencer Road, London SW18 TH Tilford, Hampshire TPK Towing Path, Kew, Surrey WC Wimbledon Common, London SW19 WC(CW) Caesar’s Well, Wimbledon Common, London SW19 W Wickford, Essex WWC Wandsworth Common, London SW18 ZKP Zabrzeg, Katowice, Poland The following abbreviations are used to describe the various morphs. \

UV(1) Wingless virginopara from the primary host WV(1) Winged virginopara from the primary host UV(2) Wingless virginopara from the secondary host WV(2) Winged virginopara from the secondary host US Wingless sexupara WS Winged sexupara G Gynopara 0 Ovipara

Aphids were collected using a number of techniques. In spring, eggs or fundatrices of species on trees or shrubs were collected by examining the newly bursting buds and removing the twig with secateurs in order not to disturb the young fundatrices. Later in the year, the undersides of leaves or the stems of host plants were examined, and any aphids were picked off individually with a fine paintbrush. Cryptic species, and those which drop off the plant when disturbed were collected by beating the plant over a beating tray. Cryptic species on hosts which could not be properly beaten, e.g. some species on grass blades, were collected with a sweep net. For root-feeding species, host plants were dug up without disturbing the roots, and the soil was carefully removed. White, waxy areas on the roots or in the soil were often evidence of the presence of root-feeding Pemphiginae. Ant-attended species could often be located by searching in or near ants' nests, or by following ant columns. This was found to be particularly useful for collecting Cinara, Thelaxes and Anoecia spp. The presence of large quantities of honeydew was also indicative of the presence of aphids. Where alatae were particularly abundant, e.g. Anoecia sexuparae migrating from grasses, they could be easily caught on the wing. Aphids were collected into polythene bags with a few leaves, or part of the stem, of their host-plant, and a piece of tissue paper was inserted to absorb any moisture released by the plant and so prevent the aphids' drowning. Predators such as syrphid, cecidomyid or neuropteran larvae, anthocorids or coccinellids, and any aphids that were obviously parasitized, were removed at the collection site. 3.2.2 Insectary-reared samples. Field-collected samples very often consisted mainly of immatures, or of a particular morph, and were therefore reared in captivity to attempt to produce other morphs or to bring immatures to adulthood before dissection. A number of rearing techniques were used. Where eggs, fundatrices or stem-feeding species were collected, the entire twig was cut off and the cut end bandaged with a damp cloth to prevent rapid desiccation. At the earliest opportunity the twigs were put into water-filled bottles (Fig. 3.1). A cork with a'hole through which the twig passed was used to prevent the aphids from drowning. The whole arrangement was then placed either under a perspex cover, or in a wooden cage with nylon and perspex sides. Where small numbers of aphids could be easily reared on single leaves, or on short sections of stems, excised-leaf cages were used (Fig. 3*2). These cages are divided into a small compartment containing a water-filled sponge, and a larger compartment containing the cut leaf or stem, with a hole connecting the two compartments through which the end of the stem or the leaf petiole passes. The cages are sealed with a sliding cover which prevents the aphids from escaping but allows water to enter the lower compartment, and the leaves are replaced every few days. It was often necessary to keep cultures going for one or more seasons at ambient temperature, but protected from parasites and predators. These were either reared on whole plants in pots within perspex and muslin cages, or caged onto the plant in the field using nylon sleeves constricted at each end with either elastic bands or wire. Certain morphs are very small, and easily lost, even in an excised leaf cage e.g. the sexuals of Thelaxes dryophila and Phylloxera glabra. For rearing these morphs to adulthood, a particular caging technique was developed. One section of a small gelatin capsule was placed over the immature aphid while it was feeding, and the edges were sealed onto the underside of the leaf with PVA glue (Fig. 3.3). This technique was not invariably successful as the aphids often became coated with the glue before it had dried, but for rearing very small morphs it was much better than using excised leaf cages. Aphid cultures were kept in a variety of environments. Two constant environment rooms were available for most of the duration of the project, one at 18°^.1* C and a "long-day" light regime (16L: 8D), the other at 1 0 C and a "short-day" light regime (12L: 12D). The long-day room was used for routine rearing of cultures under constant conditions, and the short-day room for inducing, or attempting to induce, the production of sexuals. Where it was not considered necessary to. keep cultures under constant 31 32

Fig. 3.2

Excised-leaf cage

CONFINING VERY SMALL APHIDS Fig. 3.3 conditions, they were kept on the window-sill of my study. Here, the number of hours of darkness fluctuated with the season, from about fourteen in mid-winter, to about six in mid-summer, and the temperature rarely fell below 20 #C. Two experiments were carried out for which it was necessary to raise large numbers of aphids with synchronised dates of birth. This was done by allowing an appropriate number (see below) of adult females to reproduce on uncolonised leaves for up to 2J+ hours. After this time the mother aphids were removed and their progeny divided into batches of twenty. Each batch was then placed on a fresh leaf in an excised leaf cage. The necessary number of individuals comprising the parent generation was arrived at by assuming each adult female will reproduce every four to six hours. This was an approximate calculation, and in most cases twice as many parent aphids as should have been necessary were used, in order to compensate both for below average birth-rates and mortality of immatures.

3«3 Dissecting and sectioning techniques

3.3.1 Dissecting techniques Fresh material was dissected in a saline solution which was considered approximately isotonic with aphid haemolymph, and had the following composition:

Sodium Chloride (NaCl)...... 6.4-5 g Potassium Chloride (KC1)...... 0.35 g Calcium Chloride (CaCl2)...... 0.76 g Magnesium Sulphate (MgSO^ ) .7EjO)..... 0.86 g 8.42 g /litre

Aphids were placed in clear glass cavity blocks, 2j" x 2^", which were filled with saline., Usually the aphid drowned within a few minutes, but where the aphid remained on the surface, a few drops of a very weak detergent solution were added to reduce the surface tension of the saline. When aphids had a dense covering of wax (particularly adelgids) this was first gently removed with a fine paintbrush. Dissections took place under dark-field microscopy, at magnifications which varied from 12.8x to 64x, depending on the size of the aphid. Where the specimen was too small or too delicate to grip with forceps, it was held in place in the bottom of the cavity block by a piece of double-sided sellotape. Aphids were dissected in the following way: i) The aphid was placed on its back in the bottom of the cavity block and the thorax was gripped by a pair of fine forceps in such a way that the limbs and antennae were prevented from getting in the way of the abdomen. Any large bubbles adhering to the aphid were removed with another pair of fine forceps. ii) A small fold of the integument of the ventral abdomen, just below the junction with the thorax, was gripped with the forceps, and the integument was carefully pulled downwards towards the cauda. iii) A mounted needle was then used to dislodge fat tissue, mycetome and any other matter obscuring the reproductive system, and such tissues were gently wafted away using a Pasteur pipette filled with saline. iv) The reproductive system was removed by gripping the common oviduct (or the ejaculatory duct in males) as close to its junction with the body wall as possible, and gently pulling. In many cases, removal of the reproductive system, without rupturing some ovarioles, was made difficult by the terminal filaments (see fig. 1.1) pulling the ovarioles in the opposite direction. Where this occurred, the terminal filaments were first carefully severed. With female aphids, ovarioles were separated from each other to facilitate counting and measuring of ovarioles and embryos. It was important that removal of the reproductive system resulted in the minimum amount of damage to the rest of the aphid, because it was necessary to mount the aphids on slides after dissection, so that they could be identified.

The following data were recorded from viviparous aphids: i) Length of body. ii) Length of one forewing, iii) Number of ovarioles. iv) Number of embryos, v) Number of embryos with pigmented eyes. vi) Length of the largest embryo, vii) Length of the smallest embryo.

The length of the body was measured from the base of the cauda to the ends of the frontal tubercules. Occasionally aphids contained some ovarioles which were either empty of embryos or appeared as though they had never developed fully. In such cases, the number of functional ovarioles was recorded, as well as the total number of ovarioles, and notes were made concerning the condition of any non-functional ovarioles. The embryo number included all embryos present in the reproductive system at the time of dissection. ’Oocytes and mature eggs which were clearly visible were also included. No presumptive oocytes visible inside the germarium, but not yet extruded, were included in the count. The size of the smallest embryo was an indication of whether ovulations were still occuring. Where the smallest embryo was three or four times the size of a recently extruded oocyte, ovulations had ceased to occur. The same data were recorded from oviparae, but numbers and sizes of eggs, rather than embryos, were recorded. The total number of eggs, and the number of eggs in which the chorion had formed, were both recorded. For preparations of the external genitalia of female aphids, alatae were used, where possible, as the alate morph is more primitive morphologically than the aptera, and the character in question is also essentially primitive. Individuals were gripped with forceps and pressure was applied to the abdomen, resulting in the partial expansion of the membranous genital opening and the extrusion of the external genitalia, where these were present. The aphid was then quickly placed into Dubosq-Brasil Bouin fixative for at least 21 hours. Aphids were then dehydrated through a series of increasingly concentrated acetone solutions and critical-point dried, before examination and photography under the scanning electron microscope. This technique was also used for small male aphids. Preparations of the external genitalia of male aphids were made by applying pressure gently • to the sides of the abdomen with a pair of forceps, which usually resulted in the extrusion of the aedeagus. The aphid was then quickly placed into alcohol or cytological fixative, thus preserving the external genitalia in their extruded position. The external and internal genitalia were drawn at the highest magnification which was practicable (usually 32x) and were then preserved in alcohol. Female internal genitalia. were drawn only in a few cases. The internal and external genitalia of males and the internal genitalia of females were preserved in 95% alcohol. The dissecting technique described above was also found to be appropriate for the reproductive systems of the other subfamilies of Sternorrhyncha.

3.3»2. Sectioning techniques In order to examine the reproductive systems of some species in greater detail than was possible using dissection, serial sections of some of the material were prepared using the microtome. Either whole aphids, or just 36 i the reproductive systems that had been dissected out, were used. Material for sectioning was first fixed for a mininum of 24 hours in Dubosq-Brasil Bouin, a cytolological fixative for which the recipe is given below:

Picric acid...... 1g Acetic acid (glacial)...... 15cm3 Formalin (40% HCHO)...... 60cm3 80% alcohol...... 150cm1

Material fixed in this way was then washed in several changes of 95% alcohol (to remove traces of picric acid) and brought to terpineol through a 1:1 solution of terpineol in 95% alcohol. Material was embedded into paraffin wax or "Paraplast" and the blocks trimmed, mounted and cut on the microtome in the way described in the standard histology textbooks (e.g. Gatenby & Painter,1937; Pantin,1960).

3-4 Mounting and staining techniques. In order to identify aphids correctly, after the removal of the reproductive system, it was necessary to mount them on slides, and the following technique was used (Martin, 1983): aphids were put in boiling alcohol for two minutes, and then macerated in 10% caustic potash (K0H) for 2-5 minutes. After washing in several changes of distilled water for at least half an hour, aphids were dehydrated by placing them in glacial acetic acid for a minute or two. They were then soaked in clove oil for at least 10 minutes, and mounted in Canada balsam. All stages of the preparation were carried out in a specimen tube in a dry-block heater maintained at 75°G. For the parasitoid and short-day experiments, entire ovaries or ovarioles were slide-mounted after dissection, and for this the following technique was used: Ovarioles were fixed for at least 24 hours in Dubosq-Brasil Bouin, after which the fixative was washed out using several changes of 95% alcohol. After passing through absolute alcohol, ovarioles were soaked in methyl-benzoate celloidin for two days, and then mounted in Canada balsam. Using this sequence of dehydrating and clearing agents, it was found that intact ovarioles could be mounted without undergoing any damage or distortion. Unstained slides prepared in this way were examined with phase-contrast microscopy. Some reproductive systems were stained in Erlich's acid haematoxylin before dehydration and mounting. Serial sections were stained using Heidenhain1s iron haematoxylin or haematoxylin and eosin. The latter combination of stains was found to be particularly useful for slide preparations of male internal genitalia. 3.5 Identification of aphids Material was identified to the species level wherever possible. Initially, most samples were identified by Dr. R.L. Blackman, Dr. V.F. Eastop and Mr. J.H. Martin of the BM(NH), and some conifer-feeding species were identified in the field by Mr. C.I. Carter. All such identifications were checked, and later I was able to carry out most identifications using keys to the various taxa, listed below, and by comparing material with slides in the BM(NH) collection.

Keys used for the identification of aphid species in this study:

Aphididae: Lachninae-Cinarini- Eastop, 1972; Carter & Maslen, 1982, Ptero commatinae- Stroyan, 1984. Aphidinae. Aphis spp.- Stroyan, 1984. Rhopalosiphum spp.- n ii Dysaphis spp- Stroyan, 1957, 1963. Brachycaudus spp.- Burger, 1975. Cavariella spp.- Hille Ris Lambers, 1947. Hyperomyzus spp.- Hille Ris Lambers, 1949. Macrosiphum spp. Hille Ris Lambers, 1939; Macrosiphoniella spp.- Hille Ris Lambers, 1938. Anomalaphidinae- Identified by Dr.Dinah Hales. Greenideinae- No species were studied. Chaitophorinae- Stroyan, 1977; Heie, 1982. Drepanosiphinae- Stroyan, 1977; Heie, 1982. Phleomyzinae- Examined from slides only. Mindarinae- Heie,1980. Thelaxinae-Thelaxes spp.- Remaudiere, 1982. Glyphina spp.- Heie, 1980. Anoeciinae- Heie, 1980. Pemphiginae-Eriosoma spp. -Danielsson, 1979>1982. Adelgidae- Carter, 1971. Phylloxeridae- Barson & Carter, 1972. 3.6 Methods of processing data For the purposes of the systematic survey, each sample collected was assigned a code number between 1 and 52 representing the week during which the sample was dissected. As well as giving each sample a code number, morphs were also assigned code numbers, as follows: 1=Fundatrix (F) 2=Wingless virginopara on primary host (UV(1) 3=Winged virginopara on primary host (WV(1)) 4-=Wingless virginopara on secondary host (UV(2)) 5=Winged virginopara on secondary host (W(2)) 6=Winged or wingless sexupara, or gynopara (US, WS, G) 7=0vipara (0) For each morph of each species, data from all samples were combined into one data set representing that particular morph of that species. The individual samples making up the set were still distinguishable by their different dissection date codes. Means, with 95% confidence intervals were calculated for body length, wing length, ovariole number, embryo or egg number and the length of the largest embryo for the data from the systematic survey, and for the data from the photoperiod and parasitoid experiments. In addition, the ranges and modal values of ovariole numbers were calculated for the data from the systematic survey. ’Where it was necessary to test for significant differences in body length, ovariole number etc. between two samples, means were compared using Student1 s-T tests. Where data from more than two samples were to be compared, differences between means were evaluated using two-way analysis of variance. For data from the sysematic survey, the extent to which each variable recorded was dependent on the others was quantified by calculating the correlation coefficients between each pair of variables. Since the various dissection dates and morphs had been numerically coded, it was thus also possible to examine the effects of both seasonality alone and the seasonal progression of morphs on the other variables. All data were stored on computer files and statistics were carried out using the Mini tab statistical computing system, developed by the staff of the Statistics Department at Pennsylvania State University (Ryan et al, 1981). 4 COMPARATIVE ANATOM! OF FEMALE APHID REPRODUCTIVE SYSTEMS

4-1 SYSTEMATIC SURVEY

4.1.1 INTRODUCTION References to the ovariole numbers of particular aphid species have occurred sporadically in the aphidological literature for about the last hundred years, but until recently there has been no attempt to compare ovariole numbers either between the morphs of the same species or between corresponding morphs of different species. There has been some controversy over the extent to which ovariole numbers are "programmed11 as an adaptation anticipating host plant seasonality. Wiktelius and Chiverton (1985) argued that the ovariole number is dependent on the weight of the aphid which is in turn dependent on the quality of the host plant. This to some extent conflicts with the findings of Dixon and co-workers (many papers; particularly important is Wellings et al, 1980). Kidd and Cleaver (1986) suggested that variation in ovariole number within alate morphs of Aphis fabae may not be as common a phenomenon as was thought previously, and so may not in fact be a deliberate reproductive strategy. Brown & Llewellyn (1985a) and Llewellyn & Brown (1985a, 1985b) counted embryo numbers in a large number of species, often in the same species from a number of different hosts. They used these data to compare the reproductive potential and weight of aphids on different host plants. However, they distinguished only between apterae and alatae, not taking other morphs into account, and ovariole numbers were not recorded. In the present study, as broad a survey as practicable was carried out, covering 48 species representing 9 sub-families of Aphididae, 2 adelgids and a phylloxerid (Table 4-1)• In addition, isolated samples of particular morphs of other species were studied. "Wherever possible, every morph was studied, i.e. fundatrix, winged and wingless virginoparae from the primary host -and in heteroecious species from the secondary host- plus sexuparae and oviparae. A total of 3,348 dissections were carried out during this survey. The data thus obtained are summarised and commented on below (4*1 • 2) by subfamily and by species within each subfamily. In section 4.1 *3 the same data are summarised, and discussed morph by morph. Table 4* 1 Numbers of species studied from more than one morph within the families and subfamilies of Aphidoidea.

Family Subfamily No. Species

Aphididae Lachninae 6 Pterocommatinae 3 Aphidinae 20 Chaitophorinae 4 Drepanosiphinae 4 Mindarinae 1 Thelaxinae 2 Anoeciinae 1 Pemphiginae 4 Adelgidae Adelginae 1 Pineinae 1 Phylloxeridae Phylloxerinae 1 Total 48

4«1«2 RESULTS: Systematic account

LACHNINAE

Host-alternation does not occur in this subfamily. Six species were studied in detail, four belonging to the conifer-feeding genus Cinara, and two species on deciduous hosts. In addition nine samples of single morphs of other lachnine species were dissected.

Cinara pilicomis

AHL; SR; Picea abies.

MORPH N MEM BODI OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/-' +/- +/- pm +/- pm Cl Cl Cl Cl UV 23 2.65 0.13 11.2 0.59 10-14 10 33 4.8 1083 111 1425 W 13 2.75 0.21 10.6 0.72 9-12 12 33 4.9 981 158 1425

Only two out of a possible five morphs were collected for this species. Wingles s virginoparae had a mean ovariole number of 11.2, a modal ovariole number of 10, and a range of 10-14, thus the frequency distribution of ovariole numbers was clearly strongly biassed towards the bottom of the range. Winged virginoparae had a lower mean ovariole number and a lower range of ovariole numbers than wingless virginoparae, but a 41 higher modal number. The two morphs had the same mean embryo number. Almost all individuals collected from the single sample from SR contained between one and five vestigial or undeveloped ovarioles (Fig. 4.1). These occur frequently in Lachninae, and are discussed further on p47, but were particularly common in the above sample. Both alatae and apterae had a range of total ovariole numbers, and in alatae, subtraction of the number of vestigial ovarioles from the total results in there being eight functional ovarioles in nearly every individual (table 4*2). In apterae however the number of vestigial ovarioles does not appear to bear any relation to the total number.

Table 4*2 Numbers of functional and undeveloped ovarioles in apterae and alatae of C. pilicomis (SR samples).

0V. NO. APTERAE 0V. NO. ALATAE TOTAL UNDEVELOPED FUNCTIONAL TOTAL UNDEVELOPED FUNCTIONAL 14 5 9 12 4 8 14 3 11 12 4 8 13 5 8 12 4 8 13 3 10 12 4 8 12 2 10 11 3 8 12 4 8 10 2 8 12 1 11 10 2 8 12 1 11 9 1 8 12 0 12 9 1 8 12 3 9 11 4 7 11 3 8 10 3 7 10 3 7 10 0 10

Cinara stroyani

AHL. Picea abies. This species is closely related to C. oilicomis and is found on the same host-plant. All morphs were reared from a single sample of eggs on a potted spruce.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- Vim ci ■■ CI CI CI F 7 3.10 0.23 21.6 0.73 20-22 22 108 19.2 1207 148 1425 UV 10 3.35 0.24 13.4 0.50 12-14 14 74 6.2 1392 156 1500 WV 10 2.65 0.14 10.0 10 46 3.2 700 44 850 0 10 3.16 0.17 8.3 0.35 8- 9 8 4 0.5 1335 433 1950 1mm

Fig. 4.1 reproductive system of C. pilicornis showing undeveloped ovarioles

to C. stroyani fundatrices had high numbers of both ovarioles and embryos. Wingless virginoparae also had high numbers of embryos, although they had far fewer ovarioles than fundatrices. Ovariole numbers and embryo numbers were higher in wingless virginoparae of this species than in those of C. pilicomis. However, C. stroyani wingless virginoparae were all fundatrigeniae, whereas those of G. pilicornis were collected later in the season. Although most oviparae had eight ovarioles, no more than four of these ever contained eggs at any one time.

Cinara pinea

AHL; OH; OWCH; PCem; RBGK; WC(CW). Pinus sylvestris

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl uv 52 3.33 0.20 12.5 0.50 8—14- U 58 3.4 1362 154 2300 wv 8 3.61 0.29 12.7 1.05 11-14 12 54 11.1 1182 264 1575

Fundatrices and sexual morphs of this species were searched for at each of the above localities at the appropriate times of year, without success. Thus, most samples of this species are thought to have come from anholocyclic populations. However, a single alata collected at OH had a reproductive system equipped with accessory glands, a receptaculum seminis and unusually large germaria, as in an ovipara, but the ovarioles contained apparently normal embryos. Winged and wingless virginoparae had similar ovariole and embryo numbers, as was the case with G. pilicomis. 44

Cinara pini

AHL; BTH; B; OH; RBGK. This species shares the same host as C. pinea, but is smaller. All fundatrices were reared from eggs collected at either BTH or OH. All samples from Pinus sylvestris.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 19 2.53 0.20 20.8 0.90 17-22 22 62 5.3 1095 155 1425 UV 15 2.30 0.18 11.7 1.24. 8-15 10/14 31 2.1 1103 123 1425 WV 1 2.30 10.0 10 38 — 1250 — 1250 US 10 3.07 0.19 8.6 1.58 8-12 8 58 6.3 1360 124. 1650 0 28 2.93 0.14- 13.6 0.33 12-14- 14 12 1.0 1224. 76 1450

Fundatrices had high ovariole numbers, with a similar mean and the same modal number as in those of C. stroyani. Wingless virginoparae had a large range of ovariole numbers, encompassing all those ovariole numbers found in wingless virginoparae of other Cinara species studied in this survey. However, the data for wingless virginoparae represent two samples collected eleven weeks apart, the earlier sample containing individuals with a range of 10-14- ovarioles and the later sample, a range of 8-10. The largest morphs were the sexuparae which had the fewest ovarioles and gave birth to the largest embryos. This was also found in other aphids, including two Lachninae: Lachnus roboris and Maculolachnus submacula. The seasonal progression of ovariole numbers and embryo numbers in the successive morphs of C. pini was similar to that of C. stroyani, with the exception of the oviparae, which had much higher ovariole numbers and embryo numbers in C. pini.

Lachnus roboris

AHL; B; BTH; WC(CW). Quercus robur.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl ; Cl Cl Cl F 9 2.34 0 .12 14.0 — --- 14 53 5.9 658 48 700 UV 22 3.48 0.13 14.0 — --- 14 55 3.6 1316 209 2000 WV 3 3.62 1.27 14.0 — --- 14 53 26.5 1667 — 2100 US 10 4.01 0.20 14.0 — --- 14 46 3.8 1703 554 2450 0 21 3.32 0.17 14 .0 — --- 14 12 1 .0 917 181 1850 L. roboris was unusual in having complete constancy of ovariole number. Fundatrices were laboratory reared from eggs on poor quality host material, which could explain the very low mean and maximum lengths of the largest embryos. Leather (unpublished observations) found that fundatrigeniae in Finland had 14 ovarioles, but that third generation individuals had only 8. It is not clear from his data whether these were apterae or alatae. Strumpel (1983) mistakenly refers to females (presumably oviparae) of this species as containing an average of 1200 eggs'.

Maculolachnus submacula.

SR; CPG; Rosa spp.

MORPH N MEAN BODY OVARIOLENUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 10 2.53 0.23 14.0 14 58 8.3 953 155 1200 UV 23 3.02 0.17 12.0 0.15 11-13 12 26 3.9 1339 78 1625 W 10 2.73 0.18 11.8 0.30 11-12 12 39 4.9 695 36 1050 US 13 3.08 0.14 11.4 0.53 10-12 12 7 2.1 1470 112 1775 0 19 3.08 0.11 11.8 0.20 11-12 12 7 1.5 988 143 1375

Most of the abovei data were obtained from a single culture ofthis species, originating from a sample of eggs. The pattern of seasonal changes in ovariole number was similar to that found in the Drepanosiphinae, with the fundatrix having two more ovarioles than the remaining morphs. Embryo numbers in sexuparae were low, and some ovarioles did not contain any embryos at all, while those that did only contained a single embryo. This was in contrast to the closely related L. roboris, the sexuparae of which contained three or four embryos per ovariole. Leather (unpub. obs.) found fundatrices had a range of 10-12 ovarioles, fundatrigeniae and oviparae had 10.

Other Lachninae A further nine species of Lachninae were studied from single samples only, and the data for these are summarised in Table 4*3. The range of ovariole and embryo numbers in virginoparae of these species was similar to that found in the six Lachninae studied in greater detail. Protrama radicis and Trama troglodytes are closely related species with SPECIES COLLECTION SITE/IIOST 1MORPI1 N BODY LENGTH OVARIOLE NO. EMBRYO NO. LARGEST EMBRYO mm (mean-/-(2) mean +/-CI range mode mean +/-C1 mean +/-C max jum jam

Cedrobium lapportei RBGK; Cedrus atlantica - UV 6 1.70 0 .1 2 1 2 .0 — — 12 53 4.4 830 61 975 i -Cinara costata AHL; Picea abies WV 3 2.9 6 1.19 14.0 —— 14 65 2 0 .1 842

C. cupressi HMP; Cupressus sp. UV 10 3.33 0 .1 1 14.0 — — 14 43 8.9 1225 120 1423

C. iunireri RBGK; Juniperus communis UV 10 2 .3 8 0.38 1 2 .1 O.38 1 1 - 1 3 12 50 5.8 1127 30 1172

Eulachnus rileYi RBGK; Pinus sylvestris UV 3 2.09 0 .0 8 8 .0 ____ 8 29 7.7 990 194 1094

Schizolachnus pineti RBGK, OV/CH; P. sylvestris UV 3 2.19 0 .0 6 1 0 .0 10 37 5.7 975 70 1023

Protrama radicis RHSW; Artemisia douglasiana UV 4 2.93 0 .2 1 1 0 .0 10 65 8 .8 1223 398 1230

Trama trop-lodytes F; Helianthus tuberosus UV 3 2 .1 1 1 .2 1 5.0 2 048 4- 6 V5/6 15 1 .2 739 1^33 1546

Tuberolachnus salifrnus CB; Salix viminalis UV 2 if. 80 0 .2 2 14.0 —— 14 98 1 8 .0 1688 350 1750

Table 4.3

Summarised data for Lachninae from single samples 05 similar biologies, both living on the subterranean parts of herbaceous plants. Their ovariole and embryo numbers were, however, very different. P. radicis had ovariole and embryo numbers similar to those of the other Lachninae, whereas ovariole and embryo numbers in T. troglodytes were very low. The maximum embryo size in T. troglodytes was, however, very large. The largest embryo of one individual was 56% as long as its mother. It is possible that being larger at birth gives the species a survival advantage which compensates for being able to produce very few offspring.

General comments on Lachninae Complete data for all wingless, viviparous morphs were obtained for three species of Lachninae (C. pini, L. roboris and M. submacula) and in all three the sexuparae develop the largest embryos. In these species, therefore, the oviparae are larger at birth than any other morph. This was also found to be the case in Pterocomma populeum, a species with a similar biology, but not in the closely related P. salicis, and neither was it true of the wingless, tree-dwelling sexuparae of three of the four Chaitophorinae studied. The embryo numbers of C. pinea, C. pini and L. roboris were all found to decrease as the season progressed. Although samples of C. pinea came from anholocyclic populations, ovariole numbers also decreased with week number, as in the holocyclic C._pini, but not in the similarly holocyclic L. roboris (Fig 4..2). Undeveloped ovarioles were found to occur in all four Ginara species studied from more than one sample, and also in Lachnus roboris. They have also been described by van Rensburg (1981) in Cinara cronartii, and from the accuracy of the description there appears to be little doubt that we were observing the same phenomenon. These structures, when encountered in adult morphs, take the form of minute thread-like ovarioles, usually very difficult to see even at magnifications of up to 30x, because they are so small and are tucked between the larger embryos of the functional ovarioles (Fig. 4-.1). Often the undeveloped ovarioles contain a string of minute structures of roughly equal size, which probably represent oocytes which have begun to regress very shortly after their extrusion from the germarium. Undeveloped ovarioles occurred in samples at various times of the year and often in just one or two individuals in a sample, and so the occurrence of these did not appear to follow any seasonal patterns. The significance of these structures, which I have also found in some of the other subfamilies, is discussed in section 4-.1.4-. embryo no. vroe o. +5Cs in: nos. (+95%Cls) ovariole i. . ma eby & embryo mean 4.2 Fig. 10

20

A C B C. pini C. C. pinea C. L. roboris 40

50 r25 -15 -5

ovariole no. PTEROCOMMATINAE

Three species were studied, two from Salix and one from Populus. P. salicis, however, belongs to the subgenus Clavigerus, and so the other two species are actually more closely related.

Pterocomma salicis

KGS; PC; RBGK; Salix cinerea,. S. viminalis

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 16 3.40 0.07 21.5 1.14 16-24 21 100 10.6 1154 62 1350 UV 16 3.17 0.21 18.7 0.59 17-20 19 71 9.0 816 171 1250 W 6 2.91 0.12 19.0 0.87 18-20 18/20 64 4.1 661 74 703 US 10 3.25 0.28 18.3 1.23 16-20 18/19 58 12.5 1120182 1300 0 10 3.41 0.16 19.2 1.06 17-21 20 24 1.7 965 89 1125

All morphs consistently had very high ovariole numbers, with both the sexuparae and the oviparae having the highest ovariole numbers for those morphs in the Aphididae.

Pterocomma pilosum

CSA; IPRP; KGS; OH; SR; RBGK; TPK. Salix cinerea, Salix viminalis, Salix sp.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl , Cl Cl Cl UV 49 3.01 0.09 14.9 0.41 12-18 14 95 7.5 995 33 1225 W 40 2.66 0.08 14.7 0.34 13-17 14 76 6.0 731 48 1075

Sexual morphs were searched for at each of the above localities at the appropriate time of year, but only parthenogenetic morphs were ever found. These probably anholocyclic populations gradually diminished with the onset of winter, becoming heavily parasitized and often infected with disease. 50

Pterocomma populeum

BMG; CPG; RBGK; RBGK(RBM). Populus nigra var. betulifolia; P» yunnanensis; P. alba var. pyrami.dalis; P. "oxford”.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 9 3.U 0.15 33.8 0.84 32-36 34 212 15.8 975 104 1125 UV 26 2.85 0.12 15.3 0.53 13-18 14/15 82 6.6 1029 48 1225 W 18 3.19 0.09 16.8 1.46 14-22 14 95 17.4 889 54 1050 US 4 3.01 0.28 14.0 1.30 13-15 14 36 5.1 1113 154 1250 0 7 3.07 0.20 14.1 0.49 14-15 14 15 1.0 975 251 1350

Fundatrices of this species were extremely fecund; they had the highest modal ovariole number encountered in the Aphididae, and their embryo numbers were exceeded only by the fundatrices of Pemphigus and Eriosoma spp., which have a much more specialized morphology.

General comments on Pterocommatinae In the Pterocommatinae studied, embryo numbers were rather high in all morphs, compared with the rest of the Aphididae. A more marked seasonal reduction in ovariole number occurred in P. populeum than in P. salicis, although ovariole numbers remained consistently high in the later morphs of P.salicis (Fig. 4*3)• All three species studied showed seasonal reductions in both ovariole and embryo number, despite most samples of P. pilosum having come from (probably) anholocyclic populations. Ovariole numbers were also found to be very variable, odd numbers of ovarioles occurring very frequently, which would suggest lack of development or absorption of one or more ovarioles at some stage during development. embryo/egg no. trcma spp, Pterocomma Fig.4*3mean embryo embryo Fig.4*3mean 0 U WV S O US V W UV F j eg ubr in numbers egg o y r b m e OVARIOLE NO NO OVARIOLE / . salicis P. g g e populeum R

o n — --- -

•10 ■20 ■30 •40 40 20 10 30 52

APHIDINAE

Twenty species were studied in detail, of which twelve were host-alternating species. In addition a further eight species were studied from single samples. Where possible, pairs or groups of species were selected which contained closely related species having different life-cycles, to examine whether there was any association between life-cycle and reproductive biology.

Aphis epilobiaria

IPRP; M. Epilobium sp., E. hirsutum.

MORPH N MEAN BODY 0VARI0LE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- urn +/- pm Cl Cl Cl Cl UV 21 1.61 0.06 10.0 0.15 9-11 10 32 2.8 716 28 875 WV 11 1.69 0.10 9.9 0.36 9-11 10 28 1.5 676 50 766

Winged and wingless virginoparae had similar ovariole and embryo numbers in this species. Some winged virginoparae from a sample taken at IPRP in August contained undeveloped ovarioles, as discussed above for Cinara pilicomis.

Aphis epilobii

BC; BMG; CPG. Epilobium sp. This species shares the same host as A. epilobiara, but belongs to a different subgenus.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 14 1.52 0.11 10 10 33 5.5 676 70 875 UV 24 1.48 0.06' 10 0.09 10-11 10 34 3.6 571 33 671 WV 22 1.40 0.09 10 0.17 9-11 10 27 2.9 475 27 656

The single sample of fundatrices contained individuals of typical fundatrix appearance, with five-segmented antennae. However, a number of these fundatrices contained degenerate oviparous ovarioles in addition to their viviparous ones, suggesting that this may have been an abnormal population. The low embryo numbers of these fundatrices may not, therefore, be typical of this species. 53

Aphis grosaulariae

CHN; AHL; W. Ribes grossularia, Epilobium sp. This species is very closely related to A. epilobii, but is heteroecious. Unfortunately it was not possible to collect those morphs of this species which occur on the primary host, apart from a single fundatrix.

MORPH N MEAN BODY OVARIOLE NUMBER emb/bgg no LGST EMB/BGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 1 1.97 14.0 14 103 766 — 766 UV(2) 21 1.27 0.06 10.0 0.15 9-11 10 35 5.4 562 21 641 WV(2) 10 1.29 0.05 9.4 0.76 7-10 10 25 3.6 473 12 500

The single fundatrix dissected not only had many more embryos than the Epilobium-feeding generations, but also had a higher ovariole number. The ovariole numbers of the Epilobium-feeding generations of this species were the same as those of both A.epilobiara and A. epilobii.

Aphis salicariae (=A. comiella)

AHL; GPG; SP. Comus sanguineum, Comus sp., Chamaenerion (=Epilobium) angustifolium. This is another heteroecious species with a member of the Onagraceae, very close to Epilobium, as its secondary host.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 13 1.43 0.07 10.0 10 63 3.9 523 26 578 UV(1) 11 1.35 1.01 10.0 10 41 4.2 547 29 609 WV(1) 13 1.50 0.09 10.0 10 38 4.8 453 31 500 WV(2) 10 1.60 0.08 10.0 10 38 6.9 592 4 687 G 4 1.19 0.11 10.0 10 36 21.3 512 183 594 0 10 1.09 0.10 10.0 10 7 0.8 328 67 453

Ovariole numbers were completely constant in eleven samples from at least four populations of this species (Fig. 4.4)» although embryo/egg number still showed a seasonal decline. In Finland, Leather (unpublished observations) found fundatrices of A. salicariae had a range of ovariole numbers from 16-20, but that winged virginoparae on the primary host had 10 ovarioles. All the Epilobium-feeding morphs of Aphis species had ten ovarioles, and very similar numbers of embryos. A. salicariae also had ten ovarioles in all morphs, although the summer generations on Chaemaenerion, closely related to Epilobium, contained more embryos than Epilobium-feeding Aphis> Fig.4.4 mean embryo/egg numbers in two

Aphis spp. e m b r y o/ e g g n o . ------

OVARIOIE NO. ------ovariole ovariole no. Aphis euonymi

SP. Euonymus europaeus. This species is monoecious on Euonymus, and closely related to A. fabae, which often uses Euonymus as its primary host.

MORPH N MEAN BODY 0VARI0LE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- p m +/- pm Cl Cl Cl Cl UV 16 1.^6 0.08 11.7 0.29 11-12 12 40 3.3 594 64 766 WV 12 1.4-3 0.10 11.5 0.81 8-12 12 34 3.4 520 95 609

All individuals dissected came from a single sample, and all had either eleven or twelve ovarioles, except for a single alata with eight. The differences between winged and wingless virginoparae, although not statistically significant, followed the general trend found throughout the Aphidinae, with apterae having the same modal number as alatae, but a slightly higher mean number of ovarioles ; apterae had higher embryo numbers, and their largest embryos were larger than those of apterae.

Aphis fabae

BMC; H; 0D; RBGK; SP. Euonymus europaeus, Philadelphus sp., Viburnum opulus, Phaseolus sp., Vicia faba. This is a species complex with a number of subspecies showing different host preferences. The subspecies cirsiiacanthoidis is apparently the only one able to complete its holocycle on Philadelphus (Stroyan, 1984)• Other closely related species also use Euonymus and Viburnum as primary hosts.

MORPH N MEAN BODY 0VARI0LE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 17 1.69 0.14 19.2 0.95 17-24 20 100 14.2 562 70 844 UV(1) 21 1.71 0.11 13.8 0.28 12-15 14 80 7.5 589 45 765 WV(1) 10 1.59 0.11 12.3 0.48 12-14 12 45 9.7 559 62 750 UV(2) 39 2.15 0.08 12.5 0.32 10-15 12 .78 5.4 756 34 969 WV(2) 15 2.11 0.12 11.9 0.14 11-13 12 51 3.9 503 86 797 G 20 1.73 0.10 11.5 0.32 10-12 12 — .. * 566 26 688 0 10 1.56 0.06 11.7 0.48 10-12 12 11 0.6 619 16 641 ^Embryo numbers were not recorded in this sample

Ovariole number varied in all morphs, especially in the fundatrix. Two

samples of fundatrices were collected, one from Euonymus and one from Philadelphus. In both samples the ovariole number ranged from 17 to 21. A single fundatrix from Viburnum had 24- ovarioles, and may have belonged to a different species. The samples of UV(2) and WV(2) with the highest embryo numbers were from long-standing cultures belonging to the BM(NH), which are continually provisioned with fresh excised Vicia leaves. Other workers have studied ovariole and embryo numbers in this species. Tashev & Markova (1982) found fundatrices on Viburnum opulus with 16-20 ovarioles, WV(1) morphs with 13-18, and oviparae with 12. Dixon & Dharma (1980) obtained ovariole numbers of 18.47 +/-0.27 (range: 14-22) for fundatrices, and 13-5 +/-0.13 (range: 12-14 ) for wingless fundatrigeniae, values which compare reasonably well with my own results. However, they found that their W(2) morph had ovariole numbers ranging from 6-12. Kidd & Cleaver (1986) found that their WV(2) morphs had a constant ovariole number of 12, and suggested that the differences may be due to variation between clones. All morphs from the third generation onwards had the same ovariole number as A. euonymi virginoparae. The ovariole number of A. euonymi fundatrices would be of further value in comparing reproduction in these two species.

Aphis farinosa

B; KGS. Salix caprea, S. cinerea. This monoecious species has a shortened life-cycle, fundatrices appearing in early May, and the first oviparae being produced in the first week of July.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/E3GG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAI +/- +/- +/- p m +/- pm Cl Cl Cl Cl UV 19 1.99 0.07 12.4 0.36 11-14 12 69 5.9 674 43 797 WV 6 1.58 0.19 10.3 0.54 10-11 10 36 14.7 537 25 563 US 11 1.55 0.10 10.6 0.92 8-12 10/12 46 6.3 632 106 844 0 1$ 1.41 0.09 8.2 0.48 6-10 8 4 1.4 628 60 734

Male embryos were orange, and thus distinguishable from other embryonic morphs. One or two male embryos were found among the embryos of wingless virginoparae, although in many cases these seemed to be undergoing absorption. Because of the short life-cycle of this species, the wingless virginoparae and wingless sexuparae probably do not represent two discreet morphs, but overlap considerably in the kinds of progeny they produce. Tashev and Markova (1982) found wingless virginoparae with 10-12 ovarioles, and winged virginoparae with 10. The following 8 species all belong to genera which are primitively heteroecious, with Rosaceae as primary hosts, and herbaceous secondary hosts. The first belongs to the tribe Aphidini, and the remainder to the Macrosiphini.

Rhopalosiphum insertum

BMG; EMRS; GR; GP; GR; PROK; SR. Crataegus monogyna, Malus domestica. This species alternates between apple and grass, but unfortunately no grass-feeding morphs were collected.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 12 1.95 0.28 11.7 0.95 10-14 10 48 12.1 730 80 938 W(1) 12 1.26 0.06 9.8 0.37 8-10 10 31 4.0 404 33 $16 G 7 1.57 0.10 9.7 0.86 8-10 10 — — 0 30 1.38 0.06 6.0 0.07 6- 7 6 4 0.7 $82 28 688

The fundatrices of this species were unusual among the heteroecious Aphidinae in having the same modal ovariole number as other virginoparae, although the mean and range were much higher. This was also found in A. salicariae, and the following species, Brachycaudus helichrysi. In other heteroecious Aphidinae, fundatrices had at least two more ovarioles than fundatrigeniae and subsequent generations.

Brachycaudus helichrysi

BMG; CHN; MRS. Prunus insititia, Prunus sp., Chysanthemum sp., Myosotis sp. Heteroecious, from plum or damson to various herbaceous secondary hosts.

MORPH N MEAN BODY' OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 31 1.97 0.09 12.$ 0.34 11-14 12 8$ 6.8 600 28 719 UV(1) 13 2.20 0.08 11.8 0.46 11-13 12 82 13.0 6$ 6 2$ 703 WV(1) 11 1.35 0.12 11.1 0.93 8-12 12 29 $.4 490 16 $31 UV(2) 22 1.6 4 0.09 8.0 8 44 3.0 $70 23 672 W(2) 10 1.1$ 0.06 7.9 0.23 7- 8 8 18 0.7 434 42 $16 G 7 1.09 0.08 7.9 0.3$ 7- 8 8 11 2.6 431 14 4$3

Fundatrices, represented by two different samples, resembled those of R.

insertum in having the same modal ovariole number as the following two generations, although the mean and range were slightly higher. Morphs produced on secondary host plants all had eight ovarioles but very different embryo numbers.

Brachycaudus klugkisti

CP; HP; K. Silene dioica, Silene sp. Monoecious

MORPH N MEAN BODY OVARIOLE NUMBER EMB/BGG NO LOST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl uv 11 2.07 0.09 11.3 0.77 9-13 12 62 6.5 733 35 813 wv 10 1.67 0.08 10.1 0.10 9-11 10 23 2.1 553 17 591 0 18 1.53 0.05 9.1 0.36 8-10 9 7 0.8 590 11 656

Both embryo numbers and ovariole numbers of B. klugkisti wingless virginoparae were lower than those of B. helichrysi on its primary host, but higher than those of B. helichrysi on its secondary host. Oviparae of this species were unusual in having an odd number of ovarioles in 9 out of 18 specimens.

Dysaphis crataegi

BMG; GR; PROK; ROT; SR. Crataegus monogyna, Aethusa cynapium, Daucus carota. This species is heteroecious between hawthorn and Umbelliferae. On the primary host, it lives in pseudo-galls of curled leaves, and is very often attended by ants.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 15 2.31 0.10 13.6 0.35 12-11 11 101 16.1 819 61 1078 WV(1) 19 1.87 0.06 9.6 0 .10 7-10 10 10 2.9 665 31 813 UV(2) 20 1.83 0.06 9.9 0.22 8-10 10 11 7.3 618 30 796 WV(2) 9 1.81 0.05 10.0 10 31 2.8 518 61 796 G 27 1.71 0.05 10.1 0.13 8-10 10 25 1.0 577 27 703 0 11 1.11 0.05 8.5 0.85 8-10 8 3 0.8 508 79 593

Fundatrices 'had very high embryo numbers, and four more ovarioles than the subsequent generations of viviparae. Leather (unpub. obs.) found gynoparae with 6-10 ovarioles. 59

Dysaphis plantaginea

SP. Malus domestica, Plantago major. Heteroecious between apple and plantain, generations on apple living in pseudo-galls of curled leaves.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 19 2.12 0.11 21.6 0.63 20-24 22 185 11.6 629 57 797 UV(1) 31 2.34 0.07 16.3 1.07 12-20 18 120 8.4 815 33 953 WV(1) 22 1.91 0.11 12.6 0.46 11-14 12 35 4.2 512 31 625 UV(2) 10 1.38 0.06 10.0 10 34 2.0 616 33 656 G 11 1.21 0.12 8.0 8 16 2.7 450 10 578 0 8 1.38 0.10 8.8 0.83 8-10 8 8 0.8 598 22 641

Fundatrices and fundatrigeniae had both the highest mean ovariole and embryo numbers for those morphs encountered in the Aphidinae. Tashev & -Markova (1982) found fundatrices of D, mali had 24. ovarioles. D. plantaginea belongs to a different subgenus from the previous species, D. crataegi. The seasonal decline in both ovariole and embryo numbers was striking in these two species, oviparae of both having very low numbers of eggs. Winged virginoparae were not found on plantain (c.f. Longicaudus trirhodus). In both species of Dysaphis studied, fundatrices were readily distinguishable from other morphs. They have large globular bodies, short legs, and are rather sedentary. The early generations of both species inhabit pseudo-galls formed from leaves which have been curled due .to the feeding of the young fundatrix. This pair of species, with their highly fecund fundatrices specialized for reproduction, are in some ways comparable to fundatrices of Pemphiginae (q.v.), also living in galls and with very high fecundities.

Longicaudus trirhodus

CPG; OD; RL; SR. . Rosa spp., Aquilegia vulgaris. This species is heteroecious, migrating betwen rose and columbine.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 22 2.21 0.09 20.0 0.39 18-22 20 117 6.3 649 24 797 UV(1) 10 2.52 0.12 11.5 0.69 10-13 12 58 10.7 753 29 813 WV(1) 11 1.63 0.09 11.1 0.59 10-12 12 39 3.3 513 15 547 UV(2) 21 1.26 0.05 10.0 0.10 9-10 10 32 2.8 563 40 906 G 21 1.88 0.07 9.91 0.25 9-11 10 15 2.1 540 22 641 0 10 1.23 0.11 10.0 10 8 0.9 484 81 563 Ovariole numbers were high in fundatrices of this species, 15 out of 22 individuals dissected having 20 ovarioles. A marked decline in both ovariole number and embryo number occurred between the fundatrices and fundatrigeniae. Tashev & Markova (1982) found fundatrices on rose had 18 ovarioles, and winged and wingless virginopara on columbine had 10. No other species closely related to L. trirhodus was studied for comparative purposes, although both ovariole and embryo numbers were lower in virginoparae of this species than they were in Macrosiphum rosae, which is also heteroecious with rose as its primary host. Winged virginoparae were not found on columbine, although records in the EM(NH) collection suggest that this morph is common on columbine throughout the summer. Some species of Aphidinae however, do not produce winged virginoparae on the secondary host, e.g Fhorodon humili (Campbell, 1984).

Macrosiphum rosae

CHN; CPG; 0D; RHSW; Rosa sp., Dipsacus fullonum, Scabiosa sp. This species is heteroecious between rose and Dipsacaceae.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl UV(1) 26 2.69 0.10 13.9 0.39 12-16 U 92 4.9 776 34 891 WV(1) 21 2.64 0.10 13.1 0.50 10-14 14 72 7.5 775 67 969 UV(2) 29 2.17 0.10 12.5 0.37 10-14 12 53 2.9 749 46 906 WV(2)* 2 2.41 12.5 12-13 12/13 58 — 797 — 844 0 10 2.49 0.06 11.8 0.30 11-12 12 12 1.1 706 58 828 ^Confidence limits are not included for this very small sample.

Wingless virginoparae of M. rosae were collected from six different localities between the first week of April and the first week of May. None of these virginoparae completely fitted the description of M. rosae fundatrices. (Hille Ris Lambers,1939)• Some of them may, however, have been true fundatrices, others may have been wingless virginoparae which had overwintered, or their progeny, and some were probably second or third generation virginoparae. Ovariole and embryo numbers of virginoparae on Rosa were higher than in those on the secondary host. 61

Myzus persicae

BMC; EMRS; RHSW. Primus persica, Solanum tuberosum*

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 11 1.76 0.11 15.6 0.46 15-17 15 77 7.5 580 46 672 UV(1 10 1.80 0.12 12.3 0.83 10-14 13 77 6.2 661 27 734 WV(1) 10 1.85 0.10 10.9 1.04 9-13 10 ' 46 2.4 598 20 672 UV(2) 22 2.06 0.05 10.0 10 75 4.4 738 18 812 W(2) 10 2.05 0.04 9.9 0.23 9-10 10 44 3.9 684 30 766 G 10 1.98 0.06 9.9 0.23 9-10 10 37 2.0 655 29 734 0 10 1.89 0.08 10.0 10 13 1.0 642 64 719

This species was rather unusual in that fundatrices and wingless fundatrigeniae frequently had odd numbers of ovarioles (15 and 13 respectively). Individuals of later generations almost invariably had 10 ovarioles, Takada (1984) dissected 50 M, persicae fundatrices, arising from four different crossing experiments. Fundatrices were obtained with ovariole numbers of 10, 12, 14, 16, and 18. The mean ovariole number of fundatrices arising from three of the four crosses was 15, as was the mean ovariole number overall. However, rather suprisingly, no fundatrices with odd numbers of ovarioles were obtained at all. 49 out of 50 third generation individuals dissected had 10 ovarioles, the remaining individual had 9. Tashev & Markova (1982) found fundatrices had 18 ovarioles and remaining generation 9-10 ovarioles. Leather (unpub. obs.) found that winged virginoparae on their secondary host had a range of 4-10 ovarioles in a sample of 99 individuals.

Hyperomyzus laetucae

AH; BMG; CHN; CPG; HP; MR; ROT; SR. Ribes alpinum, R. nigrum, R. rubrum, R. sanguineum, R. uva-crispa, Sonchus oleraceus. This species is heteroecious between various species of currants and sowthistle or lettuce.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 17 1.90 0.27 14 .0 14 79 7.2 632 68 891 UV(1) 15 1.96 0.15 10.1 0.20 10-11 10 59 10.5 635 62 797 WV(1) 17 1.6 4 0.09 10.0 0.18 9-11 10 29 1-5 563 37 688 UV(2) 20 2.43 0.10 10.0 — 10 65 6.2 752 21 828 WV(2) 11 2.03 0.07 10.0 — 10 49 3.7 602 31 656 G 22 1.95 0.18 9 .9 0.29 8-10 10 35 4.1 652 39 813 0 26 1.54 0.07 9 .9 0.16 8-10 10 9 0.6 669 49 781 H. lactucae had remarkable stability of both ovariole and embryo number in its various morphs, despite the large number of samples (15) examined. Both Tashev & Markova (1982) and Leather (unpub. obs.) found all virginoparae had 10 ovarioles.

Cavariella aegopodii

AHL; BC; BMG; CB; CPG; PCom; TH; WC. Salix fragilis, S. viminalis, Salix sp., Anthriscus sylvestris, Heracleum sp. This is a heteroecious species, migrating from various Salix spp. to various species of Umbelliferae.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/BGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 14 2.37 0.22 14.0 — 14 92 7.5 656 68 812 UV(1) 24 2.06 0.14 10.0 0.02 9-11 10 52 1.7 670 52 828 WV(1) 13 2.15 0.07 9.9 0.17 9-10 10 42 2.5 678 40 796 UV(2) 31 2.26 0.05 10.0 — 10 60 5.5 746 39 1000 W(2) 10 2.15 0.08 9.9 0.22 9-10 10 39 3.8 561 27 609 G 25 1.96 0.20 9.8 0.34 6-10 10 28 3.8 600 41 859 0 13 1.68 0.15 10.0 10 8 3.0 612 132 797

The seasonal changes in ovariole and embryo number were very similar to those found in the previous species, Hyperomyzus lactucae, with fundatrices having 14- ovarioles and the remaining generations having 10. Three of the seven morphs studied (fundatrices, wingless virginoparae on the secondary host and oviparae) showed complete constancy of ovariole number. Of the remaining morphs, gynoparae had the most variable ovariole numbers. This was one of the few species in which wingless virginoparae on the secondary host had significantly more embryos than those on the primary host. Oviparae of this species contained few eggs.

Cavariella theobaldi

BMG: CB. Salix fragilis

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 16 2.58 0.11 18.7 0.72 16-20 19 121 4.0 912 31 922

Fundatrices of the two species of Cavariella had differences in ovariole number. These differences were discovered when three samples were collected at different times and were found to contain individuals, original 1 y thought to be all C. aegopodii, with different numbers of ovarioles. One sample (CB 6.III.84.) contained individuals which all had 14 ovarioles, a second sample (BM 12.IV.85) showed a continuous variation in ovariole number from 16 to 20, and a third (CB 23.IV.85) contained four individuals with 14, four with 20, and one with 19* Individuals belonging to the third sample were kept individually isolated and allowed to reproduce for a few days before they were dissected. After dissection, all fundatrices and the second generation progeny from the third sample were mounted on slides. Examination under the microscope revealed that fundatrices with 14 ovarioles, and their progeny, were C. aegopodii, whereas those with 16 or more ovarioles, and their progeny, belonged to the C._theobaldi group. Chromosome preparations by Dr. R.L. Blackman confirmed the identity of the aphids.

Table 4«4« Supplementary data concerning reproduction in Cavariella fundatrices. C. aegopodii C. theobaldi

Mean body length 2.37 mm 2.58 mm Max. length largest embryo 0.81 mm 0.92 mm Max. length lgst embryo/Body length X100 34 36 Mean ovariole no. 14 19 Mean embryo no. 92 121 No. of embryos/ovariole 6.6 6.5 Mean no. pigmented embryos 18 24

There is virtually no difference in number of embryos per ovariole between the two species, so the greater number of ovarioles in C, theobaldi has resulted in a proportionate increase in embryo number (table 4*4)> C. theobaldi fundatrices thus appear to have a reproductive advantage over those of C. aegopodii, although later generations of Cavariel1 a species all have ten ovarioles, almost without exception. C. theobaldi perhaps needs to produce more alatae to find its secondary host. These two species are an ideal pair for further research into the causes of variation in ovariole number in aphids. A similar situation is found in fundatrices of two Periphyllus species (p.91 ). 64

The following three species are all monoecious on Umbelli ferae. Macrosiphoniella millefolii and M._artemisiae are closely related, M. oblonga belongs to a different subgenus.

Macrosiphoniella millefolii

PC; RBGK; TH; WC. Achillea millefolium

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/BGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl UV 18 2.43 0.23 12.1 0.16 12-13 12 58 8.8 878 113 1200 WV 20 2.71 0.13 13.4 0.41 12-14 14 46 4.3 955 49 1075 US 20 2.53 0.10 12.0 0.26 10-13 12 49 3.9 936 40 1100 0 6 2.25 0.37 12.0 — 12 12 2.1 608 92 825

This species was unusual in having winged virginoparae with a greater ovariole number than wingless virginoparae. However, the sample of wingless virginoparae was collected five days later from the same locality as the winged virginoparae, and it is possible that there may be an earlier generation of wingless virginoparae with 14 ovarioles. Leather (unpub. obs.) found wingless virginoparae with 10 ovarioles and winged virginoparae with 14-16.

Macrosiphoniella artemisiae

CP; DD; HP; PCom; TPK; WC. Artemisia vulgaris.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl UV 52 2.28 0.11 13.9 0.06 13-14 14 84 5.3 822 62 1141 WV 12 1.36 0.21 14.0 14 69 6.7 607 112 969 US 10 1.74 0.11 13.9 0.23 13-14 14 39 2.8 686 97 906 0 13 2.44 0.23 13.8 0.23 13-14 14 18 1.8 662 102 781

Six samples of wingless virginoparae were collected from various localities between the last week of April and the first week of August. Those collected earlier in the year had shorter antennae, a characteristic of fundatrices, but did not completely fulfill the criteria for fundatrices as described by Hille Ris Lambers (1938). Almost every individual of this species dissected had fourteen ovarioles. Macrosiphoniella oblonga

HP; PCom; PROK; TPK; WC. Artemisia vulgaris

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- urn +/- pm Cl Cl Cl Cl UV 20 2.75 0.11 12.0 ------12 75 4.1 1003 40 1109 WV 19 2.15 0.16 12.0 12 44 4*2 798 101 1125 0 12 2.37 0.17 12.0 ------12 22 2.4 563 & 594

Fundatrices were searched for at all of the above localities, but without success. This species was similar to M. artemisiae in having constancy of ovariole number, and was often found on the same plant. However, despite being a much larger species it had both fewer ovarioles and embryos.

Other Aphidinae A further seven species of Aphidinae were studied from single samples. The data for these are summarised in Table 4»5 and their life-cycles are summarized below.

SPECIES TRIBE LIFE-CYCLE PRIMARY SECONDARY HOST HOST Aphis praeterita Aphidini Monoecious Epilobium Anuraphis subterranea Macrosiphini Heteroecious Pyrus Pastinaca Myzus cerasi u Heteroecious Prunus Galium Myzus ligustri it Monoecious Ligustrum Nasonovia ribis-nigri n Heteroecious Ribes Compositae Macrosiphum Mainly euphorbiae tt anholocyclic various Macrosiphoniella usquertensis tt Monoecious Achillea

Aphis praeterita, monoecious on Epilobium had the same mean and modal ovariole numbers as A. epilobiara and A. epilobii, both also monoecious on Epilobium, and the same mean and modal ovariole numbers as the summer morphs of A. grossulariae and A. salicariae, which feed on Epilobium and Chamaenerion respectively. SPECIES COLLECTION SITE/ HOST MORPH N BODY LENGTH OVARIOLE NO. EMBRYO NO| L/VRGEST EMBRYO mm mean+/-d mean 7-Cl range mode mean-*/* Cl mean +/-QI max pm urn Anuraphis SR. Pastinaca sativa UV( 2) 9 2 . 0 6 0 . 0 7 11.9 0 . 2 ^ 1 1 - 1 2 12 60 1.6 692 82 ~ ^ k k subterranea

Aphis MM, Epilobium hirsutum 0 9 1 .3 9 0 . 0 9 9 .8 0 . 3 2 9 - 1 0 10 9 1 .9 5 8 9 98 625 praeterita

Macrosiphoniella WC. Achillea millefolium u v 9 2 .2 3 0 . 2 5 10.0 ——- 10 99 9 .9 906 150 1031 usquertensis

Macrosiphum CHN Lactuca sp. uv 2 k 2 . 9 8 0.11 11.0 0 . 3 6 1 0 - 1 2 10 69 6 .3 8 5 ^ ‘ 33 1 0^7 euphorbiae

Myzus BMG. Prunus cerasi u v 11 2 . 8 6 0 . 1 3 1*f.O 1.15 12-18 1*f 127 9 .6 788 2 k 891 cerasi

M. ligustri SR, Ligustrum vulgare F 9 l.9*f 0 . 0 7 10.2 O.k? 9-12 10 k o 1.2 600 kk 6*H

Nasonovia ribis-nigri CPG. Ribes nigrum F 9 2 mkk 0 .1 3 1*f.O — — I k 96 k . 6 838 39 891

Table 4.5 Summarized data for Aphidinae from single samples Wingless virginoparae of Myzus cerasi had high numbers of both ovarioles and embryos. M. ligustri fundatrices had 10 ovarioles, which is rather low for fundatrices of Myzus and its related genera, but the same as that of summer generations of M, persicae. It is probable that M. ligustri has become secondarily monoecious on Ligustrum, original 1 y having had a rosaceous primary host. The low ovariole and embryo numbers of the fundatrix, more typical of a virginopara, suggest that the expression of a typical Myzus fundatrix, as found in heteroecious species, has been modified by the loss of the primary host. There is a par all el between this phenomenon and that which occurs in fundatrices of Pemphigus spyrothecae, except that the latter species has become secondarily monoecious on its primary host. Nasonovia is closely related to Hyperomyzus (p.6l ), and fundatrices of N. ribis-nigri had the same number of ovarioles as H. lactucae. M. usquertensis wingless virginoparae had the lowest mean and modal ovariole number of the four species of Macrosiphoniella studied, but like many of the other Macro siphoniella samples, their ovariole numbers were completely constant.

General comments on Aphidinae In the Aphidinae the commonest ovariole number was 10, occurring in 4&% of all the morphs of all species studied. This is in contrast to the Lachninae and Pterocommatinae in both of which 14 was the most common modal ovariole number. Of a total of 103 morphs of Aphidinae studied, 47 had a modal ovariole number of 10, 27 had the mode at 12, and 14 had the mode at 14- Thus these three ovariole numbers account for 85% of all the morphs studied. Seasonal decreases in ovariole number occurred in 10 of the 12 species for which data on a full range of morphs, including fundatrices, were obtained. The two exceptions were Aphis epilobii, in which the single sample of fundatrices may have been abnormal, and A. salicariae. In most species a seasonal decrease in ovariole number was coupled with a decrease in embryo number, in A. salicariae only embryo number showed a seasonal decrease (fig. 4*4)• 68

CHAITOPHORINAE Four species, two from each of the main genera were studied, all are monoecious on their respective host plants.

Chaitophorus capreae

BMG; CG; FPR; KGS; OH; WC; VJWC. Salix caprea, S. cinerea.

MORPH N MEAN BODY OVARIOLE NUMBER ' EMB/EGG NO LGST EMB/BGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 28 1.54- 0.08 8.0 8 55 6.0 583 28 719 UV 14 1.59 0.08 8.0 8 4-7 3.7 4-86 31 594 WV 3 1.4-8 0.26 8.0 8 26 10.0 4-53 — 453 US 5 1.31 0.10 8.0 8 25 4*4 519 32 547 0 15 1.55 0.09 8.0 8 8 1.2 549 48 625

Ovariole numbers were completely constant in this small, willow-feeding species. The number of embryos, however, still declined with the seasonal progression of morphs. One ovipara (not included in the above data) had ambiphasic ovaries, i.e. containing both eggs and embryos.

Chaitophorus leucomelas

BMG; CPG; EMRS. Populus nigra, P. tacamahaca.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODEMEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 24 2.06 0.09 13.8 0.30 12-14 14 84 5.0 637 41 844 UV (early) 30 1.98 0.07 10.8 0.25 10-13 11 65 2.3 657 32 844 UV (late) 29 1.90 0.08 8.1 0.10 8- 9 8 49 6.0 672 28 797 W 13 2.16 0.08 8.2 0.23 8- 9 8 41 2.3 639 39 797 US 10 1.74 0.19 7.7 0.48 6- 8 8 30 13.8 625 94 766 0 14 2.11 0.22 8.0 8 10 1.3 602 54 703

In apterous virginoparae of this species there was a marked difference in ovariole number between early and later collections, and variation occurred in all samples (cf. Cinara pini, p,44). Samples collected on different dates had different ovariole numbers. Those collected earlier in the season were probably fundatrigeniae and had mostly 10 or 11 ovarioles, whereas in later samples nearly all individuals had 8. Table 4* 6 shows the frequency of the various ovariole numbers at different collection dates. 69

Table J+.6. Chaitophorus leucomelas collected at different dates and at different sites.

OV. NO. DATE & PLACE OF COLLECTION & FREQUENCY 22.V.83 27.V.83 27.V.84 2.VI.83 14-. VI. 83 (BMG) (CPG) (BMG) (BMG) (CPG)

8 1 —— 4 10

9 1 - 1 -

10 - 6 1 3 -

11 6 4 4 5 -

12 1 - 1 -- 13 _ — 1 ——

Tashev & Markova (1982) looked at ovariole numbers of Chaitophorus albus and C. populialbae on poplar, and a Chaitophorus sp. on Salix, collected between July and November. Winged and wingless virginoparae and oviparae invariably had eight ovarioles in all three species.

Periphyllus hirticomis

CPG; WWC. Acer campestre

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F u 2.29 0.22 2 2 . A 2.51 23-26 26 138 22.3 674 95 922 UV 21 2.18 0.15 12.1 0.64- 10-14 12 53 8.4 695 62 984 US 15 2.31 0.13 11.4 0.68 10-13 12 53 9.7 817 59 969 0 15 2.29 0.25 10.9 0.54 10-12 10 12 2.3 550 142 812

No alatae of this species were found. Fundatrices had high ovariole numbers, about twice the ovariole number of their* progeny, the wingless virginoparae. Both this species and the next species produce specialized first instar nymphs, known as dimorphs, at the end of spring. These aestivate throughout the summer, resuming their development at the beginning of autumn, when it is probable that they develop into sexuparae. Given the differences in development and reproductive functions between wingless virginoparae and wingless sexuparae their ovariole and embryo numbers were surprisingly similar. Periphyllus testudinacea

BMG; CPG; OD; PCom; RDIK; ROT. Acer campestre, A. pseudoplatanus.

MORPH N MEAN BODY OVARIOLE NUMBER emb/egg no LGST MB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 26 2.34 0.09 13.9 0.11 13-14 U x 107 5.2 694 51 875 UV 12 2.58 0.25 14 .1 0.18 14-15 14 111 16.3 738 66 875 W 31 2.31 0.10 14.0 0.09 13-15 14 68 8.0 640 22 797 WS 17 2.16 0.20 7.9 0.12 7- 8 8 17 3.5 775 13 922 0 22 2.25 0.08 10.0 0.36 8-11 10 15 1.5 602 39 750

The marked differences between this species and P._hirticomis were suprising. Fundatrices had far fewer ovarioles than those of P. hirticomis, but contained large numbers of embryos. The wingless virginoparae, which were all fundatrigeniae, were also highly fecund, and had the same ovariole number as their mothers, and similar numbers of embryos• Data for the two winged morphs contrasted strongly, with spring alatae (giving rise to aestivating dimorphs) having six more ovarioles than the winged sexuparae, in oviparae the ovariole number went up again to 10, as in P. hirticomis. Differences between the fundatrices of this and the previous species are further discussed on p. 91 .

General comments on Chaitophorinae

Seasonal changes in ovariole numbers were found to be different in the two pairs of species in this subfamily. In the genus Chaitophorus, C. capreae had a constant ovariole number in all of its morphs, whereas C. leucomelas showed definite seasonality, with the wingless virginoparae switching to a different ovariole number as the season progressed. Both Periphyllus species showed seasonality in this character, but with a big difference in the ovariole number of the fundatrix between the two species. All four species, including C. capreae with its constant ovariole number, underwent a seasonal decrease in embryo number. 71

DREPANOSIPHINAE Four species were studied, all monoecious on their respective woody host plants. D. platanoidis belongs to the tribe Drepanosiphini, and the remainder belong to the Phyll aphidini.

Drepanosiphum platanoidis BMG; CRP; OD; WC. Acer pseudoplatanus MORPH N MEAN BODY OVARIOLE NUMBER , EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 27 2.94 0.11 11.9 0.21 10-13 12 79 3.5 811 46 1025 WV 11 2.54 0.26 10.0 10 40 4.1 723 181 1025 WS 5 3.06 0.48 9.8 0.55 9-10 10 37 12.7 1245 146 1450 0 14 3.04 0.15 10.0 10 26 1.9 667 28 750

Two samples of winged virginoparae were collected, one in the second week of July and the other in the first week of August. In both samples many individuals were found to be absorbing their most advanced embryos, i.e. those nearest to the genital opening. During the summer, D. platanoidis adults enter a state of reproductive diapause, and only resume the development of their gonads in the autumn when they give rise to the sexual generation (Dixon, 1985). It seems likely, therefore, that both samples of winged virginoparae were about to enter such a state, and were in fact young adult sexuparae. Embryos within sexuparae collected during the last two weeks of November were very large compared with the largest embryos of other morphs. These embryos would have been oviparae, developed after the absorption of the embryos (presumably virginoparae) which had previously occupied their position in each ovariole. The seasonal pattern of changing ovariole numbers obtained was the same as that found by Wellings et al (1980) for this species.

Eucallipterus tiliae

EMG; CPG; RL. Tilia europaea

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 13 1.94 0.27 10.0 0.49 10-12 10 94 7.7 692 73 922 W 23 2.13 0.08 8.0 8 59 6.3 631 40 781 WS 11 2.03 0.09 8.0 8 61 9.1 604 72 719 0 10 1.82 0.13 8.0 8 17 3.6 603 28 656 Unlike D. platanoidis and E. punctipennis, E. tiliae does not aestivate during the summer, but continues actively reproducing (Dixon, 1985) and only one of the 23 virginoparae collected between mid-July and mid-August showed any signs of absorbing its largest embryos. Wellings et al (1980) found the same seasonal patterns of changing ovariole number in this species.

Euceraphis punctipennis

CPG; HP; KK; SC; WC. Betula pubescens

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 12 3-44. 0.15 10.0 10 57 7.3 985 68 1250 WV (early) 10 3.02 0.13 8.0 8 34. 6.1 1043 49 1150 WV (late) 8 2.51 0.21 8.0 8 26 7.6 603 219 850 0 16 3.60 0.24 8.0 8 24 3.3 813 78 925

Virginoparae collected earlier in the summer (15.VI.84) had normal embryos, whereas those collected later (6.VII.83) were absorbing their oldest embryos, as described above for D. platanoidis. A comparison of the mean and maximum lengths of the largest embryos illustrates this. It seems likely that the earlier sample consisted of actively reproducing virginoparae, whereas the later sample consisted of individuals entering reproductive diapause for the summer, and destined to produce sexuals when they resumed reproduction in the autumn. The appearance of the ovarioles and embryos of such an individual is illustrated in fig. 4-.9.

Phyllaphis fagi AHL; CPG; IPRP; RIK; RS; WC. Fagus sylvatica

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 24 2.35 0.10 12.1 0.21 11-14 12 115 4.6 647 29 766 UV 14 1.40 0.20 9.5 0.49 8-10 10 50 12.0 483 38 563 WV 18 1.86 0.11 10.0 10 52 7.1 500 49 578 0 15 2.00 0.10 9.8 0.23 8-10 10 21 3.2 559 35 594

Fundatrices of this species had high numbers of embryos, and differed from those of the other three species of Drepanosiphinae examined in being wingless. Additionally, this species produces wingless virginoparae, whereas the other three species examined do not. General comments on Drepanosiphinae Changes in ovariole numbers in this subfamily followed the same seasonal pattern in all four species studied, with all fundatrices having two more ovarioles than the remaining morphs. Numbers of embryos per ovariole were particularly high in E. tiliae and P. fagi funda trices, reaching 9»k and 9.6 .respectively. All species underwent a seasonal reduction in embryo number. Absorption of the oldest embryos by summer morphs was found in both D. platanoidis and E. punctipennis, this phenomenon is discussed further in the section dealing with sexuparae (k- 1.3-3) •

THELAXINAE Two species, representative of the two British genera, were examined.

Thelaxes dryophila

B; GP; RP; WC; WWC; Quercus castanifolia, Q. cerris, Q. robur. Monoecious on oaks, always ant-attended, producing alatae from the beginning of June onwards. These are all sexuparae, and deposit the sexuals which aestivate throughout the summer (Polaszek, 1986b).

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 19 2.19 0.1 2 9 .8 0.20 9-10 10 133 9 .7 665 21 73k UV 19 1 .6 k 0.1 9 10.0 10 77 20.1 567 kk 766 WS 22 1.16 0.05 9 .3 0 .k6 6-10 10 18 3 .0 3k2 k8 500 0 8 0.82 0.05 k .o — k 2 1 .2 272 83 391

Fundatrices had very high numbers of embryos, despite having the same modal ovariole number as the remaining parthenogenetic morphs. The oviparae all, had four ovarioles and very few eggs. Glyphina betulae

RBGK;ZKP. Betula pendula, B. populifolia. Monoecious on birch, ant-attended. The life-cycle is abbreviateded, with sexuals appearing in early- to mid-summer.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl GI Cl W?S 10 1.42 0.04 10.0 10 40 3.4 420 24 4.69 US u 1.45 0.03 9.9 0.15 9-10 10 44 6.8 581 28 641 0 10 1.17 0.07 6.7 0.89 6- 8 6/7/8 5 1.3 431 89 563

Fundatrices of this species were not obtained. Winged and wingless morphs were found together with the sexuals, and may have both been sexuparae. Viviparous morphs had the same- ovariole number as those of T. dryophila, but the oviparae, which are not dwarfish like those of T. dryophila, had more ovarioles.

MINDARINAE

Mindarus abietinus

RBGK. Abies cephalonica. Monoecious on fir; life-cycle abbreviated with only three generations a year (Heie, 1980).

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 10 1.56 0.09 11.7 1.88 6-14 12/14 21 3.5 597 39 656 WS 8 1.86 0.12 10.6 2.53 7-14 14 12 2.5 602 39 656 0 10 0.91 0.07 5.0 1.54 1- 8 6 4 1.3 352 22 391

All morphs of this species showed a great deal of variation in ovariole number, with adult oviparae having between one and eight ovarioles. Three immature oviparae of this species were dissected and found to contain seven, ten and ten ovarioles respectively. Although this was a rather small sample on which to base any conclusions, it may be that ovarioles degenerate or are absorbed during development. Baker (1915) described embryonic regression of ovarioles in Briosoma lanigerum, but disappearance of ovarioles during larval development has not been noted previously, and is worthy of further investigation. ANOECIINAE This family contains two genera, one • of which, Anoecia, occurs throughout the world. The most common species of Anoecia are heteroecious between Comus and the roots of Gramineae, although some are monoecious on roots of Gramineae or Cyperaceae. A complete set of data was obtained for one species.

Anoecia corn!

BMG; CPG; PC; RBGK; SR. Cornus alba; C. hemsleyi; Comus sp., roots of various Gramineae.

MORPH N MEAN BODY OVARIOIE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 18 1.73 0.08 7.9 0.12 7- 8 8 63 5.6 760 49 953 UV(1) 13 1.80 0.10 9.9 0.17 9-10 10 63 4.2 716 37 828 W(1) 20 1.39 0.03 9.8 0.19 9-10 10 45 2.9 616 24 688 UV(2) 20 1.78 0.10 9.5 0.39 8-10 10 49 5.7 767 37 875 W(2) 4 1.66 0.10 10.0 — 10 41 5.3 660 31 688 WS 20 1.82 0.06 9.3 0.37 8-10 10 21 5.1 657 36 797 0 22 1.12 0.05 3.8 0.23 2- 4 4 3 0.2 457 33 563

These data are summarized in fig. 4*5 A. comi differed from all other species so far reported in having fundatrices with fewer ovarioles than the other viviparous morphs, although they contained a similar number of embryos to the succeeding generation In the sexuparae, it was possible to distinguish male embryos from those of oviparae by their appearance and size. Male and ovipara embryos were never found together in the same ovariole. Out of a total of 221 embryos of which the sex was recorded, 122 were found to be males, approximately a 1:1 sex ratio. However, males were found to be far less common in the field than females, and indeed, Paul (1977) records the sex ratio of this species as 5:1 in favour of oviparae. Male embryos appeared to be being selectively absorbed in the ovaries of some sexuparae, and if this is a widespread phenomenon it may explain the difference between the sex ratio encountered in the field and that within the sexupara reproductive system. Compared with spring alatae, sexuparae were larger and had fewer, larger embryos. Oviparae, which are dwarfish in this species, had only four ovarioles, and in this respect resembled the oviparae of T. dryophila. 76

6 c ovariole ovariole no. E CD

morph

Fig. 4.5 mean embryo/egg nos. & ovariole nos.

in Anoecia corni

OVARIOLE NO- -

e m b r y o / e g g NO. PEMPHIGINAE Four species were studied, two from the Pemphigini and two Eriosomatini, both tribes containing mostly heteroecious species. The secondary hosts of heteroecious species are usually the roots of plants.

Eriosoma lamiginosum

MC; PCom; Ulmus procera, Pyrus communis (roots). Spring generations on elm develop in large closed galls.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGST EMB/EGG LENGTH ram MEAN RMGE MODE MEAN MEAN MAX +/- +/- +/- j m +/- pm Cl Cl Cl Cl F 18 2.4-7 0.14 30.3 1.07 26-34 30/32155 25.7 806 21 891 UV(1) 20 2.00 0.08 8.0 8 86 6.0 682 25 766 W(1) 20 2.24 0.11 8.0 — 8 26 5.0 519 24 625 UV(2) 1 8.0 — 8 — — - ---- — WS 3 1.31 0.30 8.0 — 8 —— - ---- —

Fundatrices had a very high but rather variable number of ovarioles, and the remaining morphs all had eight. Embryo numbers were very high in fundatrices, but their progeny (UV(1)) had a much greater number of embryos per ovariole. Expensive searches were made at many localities for wingless virginoparae on pear roots. Similarly, sexuparae and sexuals were searched for, but with little success, during the whole of autumn, in two successive years. The single sample of a wingless virginopara on the secondary host was obtained by confining the previous (winged) generation with pear roots. No progeny survived beyond the first instar however, although the opportunity was taken to record the ovariole number in one first instar. The three sexuparae collected had already deposited all of their progeny (which were not found) before they were collected. The appearance of the empty ovarioles was as shown in Fig.4-.6. 78

FIG.-4.6 OVARIOLES OF E . lanuqinosum SEXUPARA, AFTER DEPOSITION OF EMBRYOS Eriosoma ulmi

CPG; HP; MC; RBGK; WWC. Ulmus procerat Ribes sanguineum (roots). Spring generations on elm develop in open galls formed from rolled leaves. Unlike the previous species, all fundatrigeniae are winged.

MORPH N MEAN BODY 0VARI0LE NUMBER EMB/EGG NO LGST MB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pn +/- pm Cl Cl v Cl Cl F 20 2.56 0.12 22.7 0.88 18-25 24 284 37.9 893 35 1016 WV(1) 13 2.15 0.18 8.0 -- 8 19 7.0 517 46 578 UV(2) 10 1.48 0.14 8.0 --- 8 67 21.0 592 53 703

Numbers of ovarioles in the fundatrices of this species were also high, but lower than those of E. lanuginosum. Embryo numbers in fundatrices were, however, much higher than in E. lanuginosum, due to the far greater numbers of embryos per ovariole. In addition, the largest embryos of this species reached a greater size before birth.

Pemphigus bursarius

BMG; SR; TPK. Populus nigra, Sonchus oleraceus (roots), Taraxacum officinale (roots). Heteroecious between poplar, on which it lives in closed galls, and roots of various Compositae.

MORPH N MEAN BODY OVARIOLE NUMBER EMB/EGG NO LGSTEMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 9 2.16 0.18 22.7 1.27 20-24 24 359 56.0 762 71 891 WV(1) 10 1.96 0.08 12.1 0.23 12-13 12 22 1.4 491 21 563 UV(2) 12 1.37 0.10 8.0 8 84 16.1 533 30 578

Fundatrices of this species had the highest embryo number encountered in the Aphididae, and this was due partly to their having a high ovariole number, but more particularly to having, on average, almost 15 embryos per ovariole. Winged emigrants had four more ovarioles than those of the two Eriosoma species. The summer apterae on roots had a constant ovariole number of eight. Leather (unpub. obs.) also examined ovariole numbers in this species in Finland, and found fundatrices had between 18 and 28 ovarioles, and winged fundatrigeniae 10—14* Tashev & Markova (1982) found winged fundatrigeniae had 10 or 12 ovarioles. Pemphigus spyrothecae

BMG; PCom; TPK; WWC. Populus nigra. Monoecious on poplar, in contrast to P. bursarius which is heteroecious.

MORPH N MEAN BODY OVARIOLENUMBER EMB/EGG NO LGST EMB/EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 27 1.53 0.07 8.2 0.52 6-10 8 56 7.6 668 31 813 UV 20 1.30 0.05 9.9 0.27 8-11 10 36 1.4 658 19 734 WS 29 2.01 0.08 7.5 0.24- 6- 8 8 8 0.2 751 18 891 0 20 0.87 0.03 1.0 1 1 — 519 13 563

Fundatrices of this species were, like those of A. comi, unusual in having fewer ovarioles than the fundatrigeniae. Moreover, their ovariole numbers were very low and untypical of fundatrices of Pemphiginae. This species has a shortened life-cycle and all alatae, which appear in the second or the third generation, are sexuparae. The possibilty that this species has lost the ability to produce a true fundatrix, due to having become secondarily monoecious, is further discussed on p.120. It was possible to distinguish male embryos from ovipara embryos in P. sp/rothecae sexuparae. Out of a total of 221 embryos of which the sex was recorded, 57 were males and 164 oviparae, a sex ratio of approximately 3:1 in favour of oviparae.

General comments on Pemphiginae Marked seasonal changes in embryo number were found in all four species studied. Wingless virginoparae of E. ulmi and P._bursarius from their secondary hosts had higher numbers of embryos than winged virginoparae had. With the exception of the monoecious P spyrothecae, ovariole numbers decreased as the season progressed, with great differences in ovariole numbers between fundatrices and oviparae. The contrasting ovariole numbers of fundatrices, summer morphs and oviparae are associated with the high degree of specialization of the external morphology of these morphs, discussed further in section 4*1 *4. In alate Pemphiginae, the development of the embryos within each ovariole is synchronized, unlike the other subfamilies, in which embryos are in a progressively advanced stage of development from the germarium to the common oviduct. ADELGIDAE The life-cycles of Adelgidae are rather complex. Most species are heteroecious, with a biennial life-cycle. The • primary host is always a Picea sp., and the secondary hosts are conifers of other genera. All morphs are oviparous. Many species produce closed galls on the primary host, which do not open until their occupants have become adult.

Adelges cooleyi

AHL; RIK. Picea sitchensis, Pseudotsuga douglasiana.

MORPH N MEAN BODY OVARIOLE NUMBER EGG NO LGST EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- - +/- pm +/- pm Cl Cl Cl Cl F 10 1.03 0.10 48.2 4.26 40-57 44/48 81 11.7 306 16 344 UV(2) 19 0.66 0.04 7.5 0.88 5-10 10 11 2.7 236 33 344

In both morphs the number of eggs per ovariole was similar to that found in oviparae of Aphididae, and rarely exceeded two. High numbers of eggs in the fundatrices of this species were achieved by their having high numbers of ovarioles.

Adelges viridis

AHL. Picea orientalis

MORPH N MEAN BODY OVARIOLE NUMBER EGG NO LGST EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- p m +/- p m Cl Cl Cl Cl F 4 1.58 0.13 84.5 23.1 57-110 — 106 27.1 394 117 500

Fundatrices were much larger than in A. cooleyi, with very high and very variable numbers of ovarioles.

Pineus orientalis/P. pini (It is impossible to distinguish the anholocyclic species P. pini from P. orientalis when it is found on Pinus sylvestris.

AHL; HH. Picea orientalis, Pinus sylvestris Pineus orientalis/P* pini (continued) MORPH N MEAN BODY OVARIOLE NUMBER EGG NO. LGST EGG LENGTH mm MEAN RANGE MODE , MEAN MEAN MAX +/- +/- +/- urn +/- pm Cl Cl Cl Cl F 6 1.74 0.07 51.2 5.19 45-60 — 67 10.8 339 26 375 UV(2) 11 0.90 0.08 10.5 1.22 8-14 9/10/12 13 4.8 210 78 391

Both ovariole numbers and embryo numbers in the two morphs of this species examined were similar to those found in A. cooleyi Leuckart (1859) counted counted as many as 200 eggs deposited by the "fir-louse" (=?A. viridis) and recorded alatae of (?) A. laricis as having four ovarioles.

General comments on Adelgidae The above data on Adelgidae are rather sparse, but there appears to be a seasonal decline in both ovariole and egg numbers, as in the Aphididae. Ovariole numbers were very high in the fundatrices of Adelgidae, higher than any encountered in the Aphididae, and varied a great deal. No previous studies on ovariole or embryo numbers in the Adelgidae have been carried out.

PHXLLOXERIDAE All morphs of phylloxerids are oviparous, as in Adelgidae. Some species are heteroecious (Stoetzel, 1985)> and others monoecious.

Phylloxera glabra SR. Quercus robur Monoecious on oaks. MORPH N MEAN BODY OVARIOLE NUMBER EGG NO. LGST EGG LENGTH mm MEAN RANGE MODE MEAN MEAN MAX +/- +/- +/- pm +/- pm Cl Cl Cl Cl F 10 1.14 0.06 27.6 3.14 26-36 26 51 5.5 272 18 313 UV 10 1.01 0.10 6.0 0.47 5- 7 6 10 2.9 320 46 375 WS 13 0.96 0.07 4.2 0.43 3- 6 4 8 1.5 328 18 375

Fundatrices had high numbers of ovarioles, and a seasonal decline in ovariole number occurred. As in Adelgidae, each ovariole contained only one or two eggs. Although a sample of P. glabra oviparae belonging to the sexual generation was not examined during the course of the systematic survey, the reproductive system of this morph was examined during a study of the sexual morphs, and found to contain a single ovariole. 4.1.3 MORPH BY MORPH ACCOUNT

Polymorphism in Aphidoidea was discussed briefly in section 1.4> and the data from section 4«1«2 show that for most species each morph has a characteristic number or range of ovarioles, embryos or eggs. Most species are therefore seasonally polymorphic regarding their reproductive strategies. For this reason, the data from section 4.. 1.2 are further summarized below in a morph by morph account. The morphs discussed are: apterae (from both the primary and secondary hosts, including fundatrices), alatae, sexuparae and oviparae.

4-.1.3.1 APTERAE APHIDIDAE LACHNINAE The five species of Lachninae studied in detail showed a number of correlations between the variables studied (Table 4«7). The length of the body and the length of the largest embryo were significantly correlated in all species, and length of the largest embryo and number of pigmented-eyed embryos were correlated in four species. The apparent relationship between length of largest embryo and number of pigmented embryos may have been due to an age effect; individuals with fewer pigmented embryos were probably slightly younger, so that their largest embryos, although pigmented, would have been smaller than those of older adults with a greater number of pigmented-eyed embryos. The correlation between adult body length and number of pigmented-eyed embryos may have been due to the fact that larger females are able to contain more large embryos, and since they have greater nutritional resources at the disposal of their embryos than do smaller aphids, their embryos may be able to develop more quickly. This would result in there being, for any given species, a greater number of pigmented-eyed embryos within larger aphids than within smaller ones of the same age. The potential fecundity would remain unaffected. to U • o o £ b o u <0 X> • 10 >> jb •- b B W to X> b <1) E ft o E o (I) ft ft o • 0) b i <0 \ to b v\ 0) \ +> \ -P o \ to to >> b p b • 0 0 hG no. t xf B o no. b XJ cl TJ (D o O O H o o o s o 0 b b h0 ft rQ hO x> x> > a a 0 -p O

with pign 6 t-i Hi Hi > s Longest e 5

* * * * * * ♦ * * * * * * * Cinara pinea ♦ * * * * N.A. N.A. N.A. 1 7

* * * * * + * ** * * *+* * * * * * * * * * * * *' 3 C. pini 6

* * * ♦ * * * + * * * * * * * * # * C. stroyani * * * 2 3 * * * * * * * * * * * * * Lachnus roboris * * * * 3 5 * * * * * * * * * * * * * * * Maculolachnus submacula * * * * * * ** * * 3 6

Direction of correlation + ve +ve +ve +ve + ve +ve -ve -ve -ve -ve

Table 4.7 Correlation of pairs of variables relating to reproduction in the apterae of five species of Lachninae

*** P<0.001 N.A.= Not Applicable (C. pinea was studied from only one morph) ^ ** P<0.01 ^ * P<0.05 Table 4-.8 Mean lengths of adult and largest embryo in Lachninae

SPECIES LENGTH LENGTH •: LARGEST EMBRYO/ BODY (mm) LARGEST BODY LENGTH X100 EMBRYO (mm) Cedrobium lapportei 1.70 0.85 50 Cinara cupressi 3.35 1.20 36 G. juniperi 2.60 1.15 44 C. pilicomis 2.65 1.10 42 C. pinea 3.35 1.35 40 C. pini 2.30 1.10 48 C. stroyani 3.25 1.20 37

Lachnus roboris 3.50 1.30 37 Maculolachnus submacula 3.00 1.35 45

Compared with most other aphid taxa, the embryos of Lachninae reach a particularly large size before being born, growing to an average length of 48% of that of the mother aphid in C. pini (Table 4*8). Overall, the size of the largest embryo ranged from 36% to 50% of the mother’s body length. In four of the five species a seasonal decrease in ovariole number was associated with a decrease in embryo number. In Lachnus roboris the number of ovarioles remained constant although embryo number underwent a seasonal decrease. The seasonality of a number of the characters described above, including bodylength, ovariole number and embryo number, is associated with the correlation between morph and week number, which is characteristic of holocyclic species. Although the fundatrix may be morphologically indistinct from the aptera in most of the Lachninae, her reproductive potential is greater. However, most samples of Cinara pinea came from anholocyclic populations, yet this species also showed significant negative correlations between week number and both embryo number and ovariole number, despite the absence of a true fundatrix. The maximum ovariole number in this species was 1 4 > lower than that of fundatrices of holocyclic Cinara species. Funda trices of only two Cinara spp. were collected, C. pini and C. stroyani. The modal number of ovarioles was 22 in both species (Table 4*9) • Fundatrices of C. stroyani contained an average of 108 embryos, whereas those of C. pini had only 62 embryos, fewer even than apterae of either C. stroyani or C. pinea which had considerably fewer ovarioles. However, embryos of C. pini reached a far greater size (relative to the size of the mother) than did those of the other species (Table 4.8). Table 4 . 9 Summary of mean numbers of ovarioles, embryos and embryos per ovariole for the genus Cinara.

SPECIES MORPH MEAN MEAN EMB. NO./ 0V.N0. EMB.N0. 0V. NO.

C. cupressi Aptera U 43 3.07 C. juniperi Aptera 12 50 4.17 C. pilicomis Aptera 11 33 3.00 C. pinea Aptera 14 63 4.50 C. pini Fundatrix 21 62 2.95 C. pini Aptera 12 32 2.66 C. stroyani Fundatrix 22 108 4.91 C. stroyani Aptera 13 74 5.69

Of the remaining Lachninae studied, apterae of the large willow aphid Tuberolachnus salignus, with adult body length up to 4*8 mm, contained only fourteen ovarioles, although embryo number was high, exceeding 100 in some cases.

PTEROCOMMATINAE Three species were studied and some of the correlations obtained between the various variables are shown in Table 4*10. In P. salicis and P. populeum, as in Lachninae, the length of the body was correlated with both the length of the largest embryo, and with the number of pigmented embryos. In all three species, those individuals with the most ovarioles had the greatest number of developing embryos. Embryo number decreased as the season progressed, except in P._pilosum, many samples of which appear to have come from anholocyclic populations. The largest embryos were smaller in relation to the mother’s body length than in Lachninae, ranging from 26% in P. salicis apterae to 34% in both P. populeum apterae and P. salicis fundatrices.

APHIDINAE In all species both the number of pigmented embryos and the length of the largest embryo increased with body length of the parent (table 4*11 )• The average length of the largest embryo varied between 0.52 mm for embryos inside funda trices of Aphis salicariae and 1.00 mm for those of Macrosiphoniella oblonga apterae. In both of these examples however, the f .

10 o . >> in G • x o o B G >7 0) o G O > 7 X . in jc u B W O d) G p . AJ X V P a : X u p X > 7 Ai K Al G P i P p G o p p. a : 0) • o u G p 10 o o <0 W o 10 (0 " jg . O X o p -P ’AS X 53 P P in d) > 7 O • i— 1 ’H rH \ G *P • • ttO 'Cl P. p \ -p G o o o B t o > 7 G >■> • G G T3 G d) G G a •H -P G X G X o X o o o o p o o P G G 2 w in X X LO P o a> X rH X U) •P -H H H H «M W •H O O o o o X XX Pi X X -P -P -p -P p •H G G P i bO bn A l bo bo bo a : G A3 • G » bO G -P G a A) o a G G -p G G > G G o o P P -H d) O -H > > M ;z a " x . SPECIES t> <*> > O o o W X M

Pterocomma pilosum ** * * * H.A. * * * * ★ N.A. N.A. 1 7 * * * * * P. populeum ♦ * * * * * * 3 7 * * * * * * * * * * * ** * * * * * * P.salicis *** 3 6

Direction of correlTtion +ve +ve +ve +ve +ve +ve -ve -ve -ve -ve

Correlation of pairs of variables relating to reproduction in the apterae of three species of Pterocommatinae *** PCO.OOl N.A. Not a p p lic a b le ** P> Q x i *>■>o ,0d (1) (1) X! O 0) s e P i Sd P-> xi 2 TJ s o *d r?3 ■a. ^a) H -H to to x> d d JO bO x) d >> bO d o to x s d XS Q) xs Tf B (1) d 1 d d o o o a o O

o o .Q bO rQ bO a) d: > to •H 4-> -H rH 4-> +j +> 4-> d) •H O Xi of d O v a r i o l 2 3 1-4 i-4 3 'S Ovariol' E m b r y o o % ; a W X

* * * * # Aphis epilobii 2 3 M * * * * * * # * * ♦ * * *** * * * * * * * * * ♦ ♦ * « # A. fabae 3 1 2 H * * * * 4t # * * * * # * ♦ ♦ * # * # ♦ ♦ * A. farinosa ♦ * 3 M * * * * # * * * * * ♦ * * * * * * # * # * * * # « # # A. grossulariae 2 3 H * * * ♦ * * * * * A. salicariae * # # ♦ # ♦ * * * 2 b H * * * Brachycaudus helichrysi ♦ * # ♦ * * ** * ♦ ♦♦ * *♦ * * ♦ 3 5 H #4i# Cavariella aagopodii ♦ * * # 4t * ♦ # * *♦ * # * * * * • # * 3 7 H ♦ # * ♦ * * * * * * * * * * * * ♦ # # # # * # # Dysaphis crataegi *** * * * 2 5 H * * * * ♦ ♦ * * * * * * * * * * * # ♦ * * # * * * * # # # # D. plantaginea 3 5 H * # # * * * # * * * * * ♦ * ♦ * * Hyperomyzus lactucae 1***1 3 8 H * * ** # #** * * * * # * ♦ * * * * ♦ Longicaudus trirhodus ♦ * ♦ * • * * 3 5 H * * * * *# * * * Macrociphoniella artemisiae 2 7 M ♦ * * * * * * * * * * * * # # M. millefolii 2 b M * ♦ ♦ * * * * M. o b l o n g a N.A. N.A. N.A. 1 2 M

* * * 4* * * * * 4> * * # « # # * * * * ♦ ♦ Macrosiphum rosae * * * * * * 2 7 H 1 * * + * * * * j" * * *■] ♦ * * ♦ * * __ 1 Myzus persicae I— 1 3 5 H Rhopalosiphura insertum * * * * * * * * * * * ♦ * * + 4> * * * ♦ * # # « * # # # • ♦ 2 b H

Direction of correlation +ve +ve -ve -ve +ve +ve -ve -ve -ve -ve

T a b le 4 .11 Correlation of variables relating to reproduction in the apterae of 17 species of Aphidinae (asterisks in square brackets indicate that the direction of the correlation is contrary to that for the rest of the samples) oo 0 0 length of the largest embryo was 36% of the adult bodylength. Over the whole subfamily, the length of the largest embryo ranged from 28% to 45% of adult body length, 30-36% for the majority of species. Those heteroecious Aphidinae which underwent a seasonal decrease in body length also showed a seasonal decrease in the length of the largest embryo. However, large decreases in bodylength resulted in only small decreases in the length of the largest embryo. For example, summer apterae of Dysaphis plantaginea on the secondary host were on average 4-0% smaller than spring apterae on apple, but the average length of the largest embryo was only 23% lower. Similarly, apterae of Longicaudus trirhodus on Aquilegia were on average half the size of those in spring populations on rose, although their largest embryos were on average only 25% shorter. In 10 species out of 17, adult bodylength decreased as the season progressed, and all but two of these species were heteroecious. However, four heteroecious species showed no such correlation, and two of these, Myzus persicae and Hyperomyzus lactucae showed a seasonal increase in adult body length. A seasonal decrease in ovariole number was found in 11 species out of 17, and a seasonal decrease in embryo number in 13 species. Those species which did not conform to these seasonal patterns included both monoecious and heteroecious species. In one such species, Aphis salicariae, all morphs had ten ovarioles. According to Leather (unpubl. obs.) however, the fundatrix of this species in Finland has between 16 and 20 ovarioles. Ovariole numbers in the apterae of Aphidinae varied a great deal between species, from eight in Brachycaudus helichrysi on the secondary host, to 22 for D. plantaginea fundatrices. However, particular morphs of particular species had characteristic ovariole numbers as summarised previously (Section J+A.2), Ovariole numbers in apterae of Aphidinae are compared with those of alatae in Table J+,22. The highest embryo numbers were found in those morphs with the highest ovariole numbers. The average number of embryos per ovariole varied from 3.2 to 8.4-. There are few further generalisations which can be made about the reproductive strategy of the Aphidinae as a group. It seems that the type of life-cycle involved is more important than any taxonomic factors in deteimning that strategy. Aphidinae appear to have greater potential fecundities than do the Lachninae, because they have higher numbers of embryos per ovariole. There may, however, be differences in the potential number of oocytes in the germarium between the two taxa. In addition, neither the developmental rate of the embryos nor the rate of extrusion of oocytes from the germarium were considered in this study, and if the rate of embryo development was slower

c wingless morphs of . o studied No. samples No. of N

Embryo Weeknumber/Embryo Embryo number/ Morph Embryo Ovariole Weeknumber/ Ovariole Ovariole number/Morph Ovariole Ovariole number/ Ovariole Embryo number Embryo Longest embryo/ No. embryo/ No. Longest embryos pigmented with eyes Length body/ Week body/ Length n Length body/ Morph body/Length with pigmented pigmented with eyes Length body/ No. body/No. Length embryos Length longest Length longest embryo Length Length body/ CO M M 8 c P 4 4 4 4 P CD p CD o c o p O z r H" p* p rt * * * * * * * * * * * * * # * * * * * * * * 50 01 01 3 M o Q O c M 0) rt" o o • * * * * ** * ** ** * * * * *# * * * * * * * * P H- CD o P rt" H- O P p P* H" CD H P cr H P p P CD P vo . v>i j «

1 1 * *

ct" H- O P o H* n> O I P

1 1 1 1

. r t j P CD 0 n H- C P 0 CD H- CD i—■ cn < p f t H- ►-> 0 CD f+ p CD t— CD f t H- 3 cr CD CD 0 13 0 Hi P ft CD P CD H* CT H* p CD I— H CD fO CD 0 Hi n U p CD Hi 0 c p o p L rt CD p* 0 o p H* P P UD P p [*] rpooitive correlations 06 in Lachninae, then at a given moment there would be fewer embryos present in the mother’s ovarioles, while the germarium still contained a number of potential oocytes.

CHAITOPHORINAE The correlations between variables studied in four species of Chaitophorinae (Table 4-12) followed the same pattern as for the monoecious species belonging to the other subfamilies. Adult body length and length of largest embryo were correlated in all species, as were body length and number of pigmented embryos. The relationships between these variables have already been discussed. The length of the largest embryo was 29%-38% of parental bodylength for the eight wingless morphs studied (average 32%), slightly less than in Aphidinae, and much less than in Lachninae.

Table 4-. 13 Summary of. mean numbers of ovarioles, embryos and embryos per ovariole for apterae in the subfamily Chaitophorinae.

SPECIES MORPH m e a n MEAN MB. NO, OV. NO. MB. NO. 0V. NO. C. capreae Fundatrix 8 55 6.9 n ii Aptera 8 47 5.8 C.leucomelas Fundatrix 14 84 6.0 it Fundatrigenia 11 65 5.9 it Aptera 8 49 6.1 P. hirticomis Fundatrix 22 138 6.3 ti it Aptera 12 53 4*4 P. testudinacea Fundatrix 14 107 7.6 i i Aptera 14 111 7.9

The number of embryos per ovariole in Chaitophorus was remarkably constant, despite seasonal differences in ovariole number in C. leucomelas (Table 4*13). The large difference in ovariole numbers between fundatrices of P. testudinacea and P. hirticornis, both species being often found side by side on the same host, is a similar phenomenon to that already described for Cavariella aegopodii and theobaldi (p.63 ). Despite having eight more ovarioles, P. hirticornis fundatrices are only 23% potentially more fecund than those of P. testudinacea, as they have fewer embryos per ovariole. Spring apterae of P. hirticomis contain on average only half the number of embryos of P. testudinacea apterae, and perhaps the fundatrices are more fecund to compensate for this. Overall, the potential fecundity of Chaitophorinae is similar to that of Aphidinae and Pterocommatinae, but greater than that of Lachninae which tend to have fewer embryos per ovariole.

studied No, No, of samples No, morphs wingless No, of Embryo number/ Week number/Embryo Embryo number/ Morph Embryo number/ Ovariole number/ Week Ovariole number/ Ovariole Morph Ovariole number/ Embryo number Embryo 0variole 0variole number/ embryos with with pigmented embryos eyes Length longest Length embryo/ longest No, Length body/ V/eek Length body/ LengthMorph body/ n> Length body/ No, Length body/No, embryos with pigmented eyes Length body Length longest Length embryo longest o w w M Ifi C * * * * * * * * * * * * * * -F- * * * * * * * * ** * * ** * # * ** ** ** * * ** r o r o * * * i * ** ** ** * ** * * * * * *** * VoJ ___ i * * ♦ * * * ** * * * * * * * * * * ** * »* * * * * * *** * * #*** ** *** *** * * ** * * * r o * ** • « * * * * * ♦ * * * «* * * ** ** ♦ ♦ ** ** *** * * * * i i * ♦ ** * * * * * * * * + * * ** * r o r u ** * * *** ** VJl 4 r* — i ______— i i— i i i i i 1 I i i <* CT> CD <* a > i < a> < i + o < + < a> a> < o < a> + < + s o < o

Table 4.14 Correlation of variables relating to reproduction in apterae of seven species of Aphididae 6 Z DREPANOSIPHINAE, THELAXINAE, MINDARINAE & ANOECIINAE Only one of the drepanosiphine species studied in this survey, Phyl1aphis fagi, has wingless viviparous morphs. Glyphina betulae and Mindarus abietinus were represented by single samples only, and because of the abbreviated life-cycles of these species, these small samples consisted of individuals containing the embryos of both parthenogenetic and sexual morphs, and so are a rather special case. Correlations for the remaining members of the above subfamilies are shown in table 4«14* The pattern of correlations followed that already discussed for other species, adult body length being correlated with both largest embryo length and number of pigmented embryos. The length of the largest embryo, as a proportion of adult body length, was similar in Phyllaphis, Thelaxes, Glyphina and Mindarus, but was greater in Anoecia where it approached that of Lachninae. The successive morphs of P. fagi and T. dryophila underwent a seasonal decrease in bodylength, since both species have large fecund fundatrices, and P. fagi produces dwarfish virginoparae during the summer. The range of ovariole numbers was quite low in all these species, from 12 in P. fagi fundatrices to eight in those of A. corni (Table 4*15), and there was little variation in ovariole number within each sample.The increased fecundity of fundatrices was achieved largely by developing more embryos in each ovariole rather than by increasing the number of ovarioles.

Table 4«15* Mean ovariole and embryo numbers and numbers of embryos per ovariole in Phyllaphis, Thelaxes, Glyphina, Mindarus and Anoecia apterae.

SPECIES MORPH MEAN MEAN EMB. NO, OV. NO. EMB. NO. 0V. NO. Phyllaphis fagi F 12 115 9.6 I! UV 10 50 5.0 Thelaxes dryophila F 10 133 13.3 UV 10 77 7.7 Glyphina betulae UV 10 44 4.4 Mindarus abietinus UV 11 22 2.0 Anoecia comi F 8 63 7.9 1! UV(I) 10 63 6.3 1! UV(2) 10 49 4.9

PEMPHIGINAE Because of the great differences in most of the characters studied between fundatrices and other morphs in Pemphiginae, significant correlations were found between almost every pair of variables (Table 4--14-) • The monoecious^ species P. spyrothecae was exceptional in having fundatrices with fewer ovarioles than the fundatrigeniae, and hence showing an increase in both embryo and ovariole number as the season progressed.

Table 4*16 Mean body lengths and lengths of largest embryos for five species of Pemphiginae, including largest embryo size as a percentage of adult body length.

SPECIES MORPH BODY LENGTH SIZE LGST. LGST. EMB./ (mm) EMBRYO (mm) BODY LENGTH X 100 P. spyrothecae F 1.55 0.65 42 !! UV 1.30 0.65 50 P. bursarius F 2.15 0.75 35 it UV(2) 1.40 0.55 39 E. ulmi F 2.55 0.90 35 ti UV(2) 1.50 0.60 40 E. lanuginosum F 2.45 0.80 33 it UV 2.00 0.70 35 E. lanigerum UV(2) 1.70 0.65 38

The ratios of lengths of adult to length of largest embryo are all within the range covered by the other aphid species (Table 4.16), although the embryonic sexuparae within the second generation apterae of P. spyrothecae are much longer than those of the apterae of the other Pemphiginae. With the exception of P. spyrothecae all post-fundatrix apterae contained eight ovarioles, with very little variation either within or between samples (table 4*17). In some samples, individuals were deliberately taken from different galls to avoid using the progeny of a single fundatrix, and other samples were taken from a single gall for an assessment of intraclonal variation. Ovariole numbers in fundatrices varied a lot between individuals, but virtually all individuals of later generations contained eight ovarioles. These results suggest that ovariole numbers in the post-fundatrix generations of Pemphiginae are under strict genetic control, whereas the ovariole numbers of fundatrices are not. The similarities between ovariole numbers of P. bursarius and E. ulmi apterae are striking, the main difference being the exceptionally high number of embryos per ovariole in P. bursarius, the highest I have encountered in the Aphidoidea. Table 4.17 Summary of mean ovariole and embryo numbers and numbers of embryos per ovariole for some pemphigine apterae.

SPECIES MORPH MEAN MEAN EMB. NO./ OV. NO. EMB. NO. 0V. NO. P. spyrothecae F 8 56 7.0 n UV 10 36 3.6 P. bursarius F 23 359 15.6 UV(2) 8 85 10.7 E. ulmi F 23 284- 12.3 n UV(2) 8 67 8.4- E. lanuginosum F 30 155 5.2 ii UV 8 86 10.8 E. lanigerum UV(2) 8 4-8 6.0 Of all the Aphidoidea, fecundities are highest in the fundatrices of heteroecious Pemphiginae, and Tashev and Markova (1982) found that fundatrices of Thecabius affinis had 50 ovarioles, which is the highest number recorded in Aphididae. The habit of gall formation and the protection which the gall affords the fundatrix and her progeny have enabled the fundatrices to adopt a sedentary existence, directing their energies into the production of vast numbers of offspring. However, the fundatrices of P. spyrothecae, also protected in a gall, have an untypically low fecundity, but this is connected with their having a monoecious life-cycle (see p.120).

ADELGIDAE

Samples of adelgidae were rather small, and few morphs were collected. Because Adelgidae, like Aphididae, are seasonally polymorphic, many similarities were found between the two families, despite the adelgids being exclusively oviparous. For both genera all individuals within each sample laid eggs of approximately the same size. Fundatrices were larger than apterae collected from secondary hosts.

Table 4-* 18 Mean ovariole and egg numbers in some adelgid apterae.

SPECIES N MORPH MEAN MEAN EGG NO./ 0V. NO. EGG NO. 0V. NO. A. cooleyi 10 F 48 81 1.7 n 10 UV(2) 9 16 1.7 n (RIK) 10 UV(2) 6 6 1.0 A. viridis 4 F 85 105 1.2 P. orientalis 6 F 51 68 1.3 P. pini 9 UV(2) 10 15 1.5 96

Like the fundatrices of Aphididae, those of Adelgidae and Phylloxeridae are more fecund than the later generations (table 4-•18). However, unlike aphids, the increased fecundity of adelgids and phylloxerids is only achieved by increasing the number of ovarioles, and not the number of eggs per ovariole, which is always low.

PHYLLOXERIDAE

Only two samples of Phylloxera glabra apterae were studied, correlations between the variables recorded were not, therefore calculated, but numbers of ovarioles, eggs and eggs per ovariole are shown below.

MORPH N MEAN 0V. NO. MEAN EGG NO. NO. EGGS/ 0V

F 10 27.6 51 1.85 UV 10 6.0 10 1.67

Although ovarioles in both oviparae and virginoparae throughout the Aphidoidea are of the telotrophic type (see section 1.3), the method by which more advanced embryos obtain their nutrition is not understood. In viviparous aphids, the oocytes and least developed embryos in any ovariole are connected to the germanium by trophic cords containing parallel arrangements of microtubules (Couchman & King, 1980) but no such structures are available to provision the more advanced embryos. Another source of nutrition must therefore be available to such embryos, such as absorption through the ovariole sheath. This may enable viviparae to develop greater numbers of progeny per ovariole than oviparae, whose growing oocytes are dependent on the trophic cord for their supply of nutrients.

APTERAE: SUMMARY 35 of the 4-1 species that were studied in detail showed significant correlations between adult body length and the length of the largest embryo. The size to which an embryo develops before birth is therefore related to the size of its mother. Embryos developed to between 30% and 40% of parental body length in over half of the wingless morphs of the species studied. Those species in which apterae developed their embryos to less than 30% of the adult body length did not share any particular taxonomic relationships or life-cycle characteristics. Similarly, species whose morphs developed relatively large embryos (i.e. over 4-0% of adult body length) came from a number of different subfamilies, although the Lachninae, the Epilobium-f eeding species of Aphis, and Anoecia comi all developed particularly large embryos. Large seasonal reductions in adult body length, such as those found in many of the heteroecious Aphidinae, resulted in much smaller reductions in the length of the largest embryo. Similar patterns of changing embryo and ovariole number were found throughout the Aphidoidea, and for most species, individuals collected earlier in the year had the greatest fecundites. This was also true of Adelgidae and Phylloxeridae, despite their different method of reproduction. Seasonal differences in potential fecundity were the result of changes in ovariole number, embryo number, or both, and although fecundity was dependent on ovariole number in many species, in others the number of embryos per ovariole was more important. The degree to which the fundatrix was morphologically distinct from the other apterae was directly related to the difference in fecundity between the fundatrix and other apterae. At one extreme, fundatrices of e.g. Lachnus roboris and Chaitophorus capreae are difficult to tell from apterae of the same species and have the same ovariole numbers, and at the other extreme are Pemphigus bursarius and Eriosoma lanuginosum with fundatrices that are very different from their daughters and granddaughters both in morphology and reproductive potential. These seasonal changes in fecundity are genetically programmed, and part of the polymorphic nature of the Aphidoidea, presumably anticipating changes in host-plant quality. The relationships between reproductive potential and host-plant type and quality are discussed in section 1.2.1. 4.1.3.2 ALATAE

APHIDIDAE

LACHNINAE Winged morphs are comparatively rare in the Lachninae, and were collected in sufficient numbers in only three species, Cinara pinea, C. pilicomis and Maculolachnus submacula. Fewer significant correlations between the variables studied were found than for the apterae, partly due to the smaller sample sizes (Table 4*19). In all three species individuals with a greater body length also had longer wings, which was to be expected. The number of pigmented embryos was correlated with the size of the largest embryo in all three species, as in the apterae (p. 84 ). The largest embryos of lachnine alatae were slightly shorter than those of the apterae, both in terms of actual length and as a proportion of adult length. Alatae showed less variation in ovariole number than apterae, with a range of eight to fourteen, and their embryo numbers were lower. Undeveloped ovarioles, as described in the apterae (Fig. 4»1)» were found frequently.

PTEROCOMMATINAE Significant correlations were found between wing length and body length in P._oilosum and P. populeum, the two species in which alatae were collected in sufficient numbers (Table 4*20). Alatae of P. salicis were only encountered early in the year. In alatae of al 1 species studied, average size of the largest embryo was less than in apterae, reaching only a quarter of the parental length, compared with a third for apterae. Ovariole numbers and embryo numbers were negatively correlated with week number for P. pilosum and P. populeum, showing a seasonal reduction in potential fecundity for these species. Ovariole number varied a lot within samples for all three species, 13-17 in P, pilosum, 14-22 in P. populeum and 19-24 in P. salicis. Compared with the other subfamiles, ovariole numbers and potential fecundities were very high in pterocommatine alatae; alatae of P. populeum, with a mean embryo number of 129, being particularly fecund. Casual observations on cultures of Pterocommatinae suggested that alatae began to reproduce almost immediately after reaching adulthood, without first having a flight period. It may be that in alatae in which the reproductive urge is greater than the urge to migrate or disperse, the nutritional resources which would otherwise be directed towards the flight muscles are instead directed to the reproductive system. o i-3 o s IO O Cn o P H* P H* 33 3 cr 3 O 3 W 3 h-1 P 3 to P O P P O 1-* H- 3 H H ct- O 3 P W P H* M P cn ci- , O 03 P id H* t—»• 3 O H* O CO 3* M 3 o 3 H* t-D c O O CO 0 ►3 O 3 O 03 3 < 3 c H* P 3 O' 01 3 P 3 H* M P P P o O’ c t 3 H H* M P O P 01 3 3 P P ci- H* + * 3 < * Length body/ cq P * Length longest embryo

3 + * * * P < * Length body/ 33 P 3 Length forewing O P- c o + * rt- < Length body/ No. embryos H* P O with pigmented eyes 3 H- 3 1 3 < ♦ • Length body/ Week ct* P > > 0T • « P P t—1 1 P > CD • • H*0 P 01 1 3 Embryo no./ Week o < • • M> P > > t3 « • P o 3* Ovariole no./ Embryo no. 3 H* 3 P P

VjJ No. of samples

66

Ovariole number/ Embryo Ovariole no, Embryo no./ Week no./ Embryo Length longest embryo/ No. embryo/ No. Length longest embryos eyes pigmented with Co O M Length forewing/ Embryo Length no, forewing/ W M Crt *TJ Length body/ Week body/ Length with pigmented eyes with pigmented Length body/ No. No. body/ embryos Length Length Length forewing Length body/ Length Length body/ Length embryo Length longest 1 * * Week no./ Ovariole

3 d 0 01 3 H- t— O O ct- CD O t3 v>i v>i -p- samples No. of □ CD d d M 3 O ^ Id 1

—1 O d cl* CD P3 H* d o o t ct- H* O O *■*} O a H* *-i CD

Table 4.20 Correlation of variables relating to reproduction in alatae of two species of Pterocommatinae 001 APHIDINAE The material studied comprised 35 different alate morphs of 19 species, eight monoecious and 11 heteroecious. Of these 19> 16 were collected in sufficient numbers for the correlation coefficients between the various variables recorded to be calculated (Table 4-.21) Wing length increased with body length in all but two species. In 10 species the length of the largest embryo increased with body length. With very few exceptions, the maximum length to which the embryos grew was less in alatae than it was in the equivalent wingless morphs, as in the Lachninae and Pterocommatinae. The correlation between wing length and embryo number was calculated to investigate whether those individuals with a greater reproductive investment, i.e. greater embryo numbers, had a lower investment in locomotion and dispersal, the "oogenesis-flight" syndrome of Johnson (1969). Dixon (1975) for example, found that brachypterous individuals of Drepanosiphum platanoidis had greater powers of reproduction than those with normally developed wings. In the present investigation however, eight of the ten species in which there was a relationship between wing length and embryo number showed an increase in embryo number with wing length. Therefore, either wing length is not a reliable parameter for the assessment of flight potential, or the "oogenesis-flight" syndrome does not apply to these species. No obvious differences were found between monoecious and heteroecious aphids in the patterns of correlations, and the largest embryos-of both were around the same size, measuring 31% of adult body length in monoecious species compared with 32% for heteroecious species. Numbers of ovarioles were less variable in alatae than in apterae, both within samples and across the whole subfamily, with a range of from 8 to 11 (Table 1.22), which was the same as that found in the Lachninae.

s p e c i e s No. of alate morphs studied. Heteroecious or monoecious No. of samples Ovariole no./ Embryo no. Embryo no./ Week Ovariole no./ Week embryos with pigmented eyes Length longedt embryo/ No. Length forewing/ Embryo no. pigmented eyes Length body/ Week Length forewing Length body/ No. embryos with Length body/ Length body/Length longest embryo i ft * •# » ■» * 2 __ > s : > • rjn > > 3* ' i id i » •» * « * ♦ S3 __ o \ i 0 4 3 : r o P 3 01 H- ►3 ft ft S3 0 4 o n P P O H- PO 3 > > 01 I-1 3 • 1 • ft ft * ft ft ft ft ft * ft ft «* ft * * * «> * « • 'ft ft « w __ l OH OH n ? P 3 H- 3* p M P 3 01 3 O O H- P 3 i * * * ft * ft ft ft ft ft ft ft •• ft ft __ i n 0 4 o P- H- O P PP- f-1 p p V-f 3 3* p P H- o 03 71 s r * * * • * V* VJ1 H- ct- 3 O 3 < p 3 3" H- P a 73 n n i * « * ft * •» # «* * * * * * * * * * * * * * * * S3 S3 r o r o __ i PP H- 3 P P P rt- P 3 a M • 71 ►d l * *« * * * * * * S3 __ CT* l 0 4 r ? n r P i— > o P rf- 3 o CD H- t- 1 N OP p 5- n << 1 i * ft * ** * ** ** * ** * * ♦ * * * ** * * «- S3 r o __ l __ r ? i i— \ 3 01 3 S' o p- H* rt- 01 01 3 3 P- P 3 3 O O H* 3 f 73 : ** 3 ro r o ct 3 01 CD P 3 P t—' CD H 3 P 3 H- i— i 3 “ P O O TJ 3 * * * •* ** * 2 2 r o H* H- 3 3 CD H- M 01 2 3 * **** ** * * **** * *** s POO o o ' H* O M H- s «« 73 l ** * ** * * ♦ ft ** * ft __ ro t r ? 0 4 r o p 01 CD o 3 S' 3 H* 2 P O o 3 01 r) 3 • * * * * * ft * * s -p - 0 4 «J 1—1 P *1 CD H- O ►1 u CD e C3 01 •d l 1 « * ft * ** * ft * ft * * • ft K r o V>4 __ i—i m --- 3 1 3 “ 3 p 0 01 H- 1 o t-1 hs fa

Direction of correlation +ve +ve +ve -ve ~va +ve -ve -ve +ve (square brackets indicate that the direction of the correlation is contrary to that for the remaining samples) 2 01 2 Table 1.22. Mean numbers of ovarioles, embryos and embryos per ovariole in aphidine alatae. Data for the equivalent wingless morph are shown in brackets.

SPECIES MEAN OV. MEAN EMB. NO. MB. NO. NO. / OV. Aphis euonymi 12 (12) 31 (40) 2.8 (3.3) A. farinosa 10 (12) 36 (69) 3.6 (5.8) A. epilobiara 10 (10) 28 (34) 2.8 (3.2) A. epilobii 10 (10) 27 (33) 2.7 (3.3) A. grossulariae 9 (10) 25 (35) 2.8 (3.5) A. salicariae 10 (10) 38 (41) 3.8 (4.1) A. fabae (D* 12 (14) 15 (80) 3.8 (5.7) !! (2) 12 (13) 51 (78) 4.3 (6.5) Dysaphis crataegi (2) 10 (10) 31 (11) 3.4 (4.1) D. plantaginea (D 13 (16) 35 (120) 2.7 (7.5) Brachycaudus helichrysi (D 11 (12) 29 (82) 2.6 (6.8) ii (2) 8 (8) 18 (15) 2.3 (5.6) B. klugkisti 10 (11) 23 (62) 2.3 (5.6) Longicaudus trirhodus .(D 11 (12) 39 (58) 3.5 (4.8) Hyperomyzus lactucae (D 10 (10) 29 (59) 2.9 (5.9) ii (2) 10 (10) 19 (65) 4-9 (6.5) Cavariella aegopodii (D 10 (10) 12 (52) 4.2 (5.2) ii (2) 10 (10) 39 (60) 3.9 (6.0) Myzus persicae (D 11 (10) 16 (59) 4.2 (5.9) it (2) 10 (10) 11 (75) 4-4 (7.5) Macrosiphum rosae (D 13 (14) 72 (92) 5.5 (6.6) i i (2) 13 (13) 57 (53) 4.4 (4-D Macrosiphoniella artemisiae 14 (14) 69 (84) 4.9 (6.0) M. minefolii 13 (12) 56 (58) 4.3 (4.8) M. oblonga 12 (12) 11 (75) 3.7 (6.3) *(1 = developing on primary host, 2 = developing on secondary host)

For each species the winged morph was less fecund than its wingless counterpart, except for the summer generations of M. rosae, where the difference in embryo number was very small. In most cases, alatae had the same or slightly smaller ovariole numbers than the equivalent apterae, and the greater fecundity of the aptera was due to a greater number of embryos per ovariole. A comparison of the mean numbers of eggs per ovariole in alate virginoparae of Aphidinae and those of Macrosiphinae, using a Student's T test, revealed no significant difference.

CHAITOPHORINAE Correlations between the variables studied for the Chaitophorinae, Drepanosiphinae, Anoeciinae and Pemphiginae are shown in Table 1.23 Of the four species of Chaitophorinae studied in this survey, Periphyllus hirticomis was found to produce alatae infrequently, and so was not included in the analysis, and only three Chaitophorus capreae alatae were xJ • CO CO o • CD CD d O •H d S -> 43 o d d CO d d B to d d d o <0 <0 w d CD d X> CD PH E e a d • <0 <0 CD ho CD <1) •H o d hO o CD a o 4> d S5 XJ d 43 P h •H CO \ 10 •H <0 • CP CO 0) \ 43 \ CD d 43 d hO ^ 4-> 4-3 4-3 4-3 43 -> o o CD Ph

* * ♦ 2 1 M Chaitophorus leucomelas * * * * * * * * * *** * * * 6 2 M Periphyllus testudinacea *

* * * * * * * * * 6 M Drepanosiphum platanoidis * * * ♦ * * * t [**] 3 1 *► 1 --- __ *** * * * * * ♦ * * * M 1 Eucallipterus tiliae 1 7 3 * * * ♦ # * * * * * * 4 * * * ♦ * * 4 M Euceraphis punctipennis * * * * * + 3 * * * 2 1 M Phyllaphis fagi

|-* * +-j * * * ♦ * ♦ * * * H Anoecia corni * * * * * + * 5 3

* * * * 2 1 H Erisoma ulmi * 1 1 H Pemphigus bursarius N.A. N.A. N.A. * * * * * ** 1 M Pemphigus spjrrothecae ♦ 3 4ve (square brackets Direction of correlation 4ve 4ve 4ve -ve -ve 4ve -ve -ve that the direction of the correlation is contrary to that for the remaining samples)

Table4-23 Correlation of variables relating to reproduction in alatae of 10 species of Aphididae O dissected. Body length and wing length were correlated in both P. testudinacea and C. leucomalas alatae, and the largest embryos were shorter in alatae than in the apterae of all three species, as in most of the other subfamilies. Ovariole numbers in chaitophorine alatae showed little variation either within or between samples. In all three species, alatae had a lower embryo number than apterae, and since ovariole numbers were the same in both morphs, the lower embryo numbers of alatae were due, as in the Aphidinae, to their having fewer embryos per ovariole.

DREPANOSIPHINAE With the exception of Phyllaphis fagi, all viviparae (including fundatrices) of the species of Drepanosiphinae studied were alatae. D. platanoidis, E. tiliae and E. punctipennis all showed a seasonal reduction in both ovariole number and embryo number (Table 1.23). P. fagi was one of the few examples of a species in which the alatae were as fecund as their wingless counterparts. In P. fagi, fundatrices are wingless, and wingless morphs are produced during the season, whereas in the remaining three species all morphs except oviparae are winged. Ovariole numbers showed very little variation in these species (Table 1.21). Apart from E. punctipennis. which showed no variation in ovariole number within each morph, variation in fundatrices was greater than in the succeeding generations.

Table 1.21 Distribution of ovariole numbers in drepanosiphine alatae. SAMPLE OVARIOLE NO. SPECIES MORPH SIZE 8 9 10 11 12 13

D. platanoidis Fund. 27 - - 1 3 22 1 n Virg. 16 - 1 15 -- - E. punctipennis Fund. 12 -- 12 -- - tt Virg. 17 17 ---- - E. tiliae Fund. 13 — 3 8 1 1 — it Virg. 23 23 -- ---

Compared with the alatae of Aphidinae, embryo numbers in alate Drepanosiphinae were high, due to their having greater numbers of embryos per ovariole rather than greater numbers of ovarioles (Table 1.25). Table 4..25. Mean ovariole numbers, embryo numbers and numbers of embryos per ovariole in alatae of three species of Drepanosiphinae.

SPECIES MORPH MEAN MEAN NO. EMB. 0V. NO. MB. NO. /OV. Drepanosiphum platanoidis Fund. 12 79 6.6 It IT Virg. 10 40 4.0 Euceraphis punctipennis Fund. 10 57 5.7 ii it Virg. 8 30 3.8 Eucallipterus tiliae Fund. 10 94 9.4 i i it Virg. 8 59 7.4

However, the reproductive advantage that fundatrices had over later generations was achieved by having both greater ovariole numbers and a greater embryo:ovariole ratio. Undeveloped ovarioles and regressing embryos were also found in this subfamily, and the occurrence of these is discussed in section 4.1.4.

THELAXLNAE, MINDARINAE & ANOECIINAE In Glyphina betulae and Thelaxes dryophila all of the offspring of the winged morphs are sexuals. Similarly, Heie (1980) considers all alate Mindarinae also to be sexuparae, and therefore these are all dealt with in section 4*1 .3.3, which deals with the reproductive anatomy of the pre-sexual generation. In Anoecia comi, summer alatae were larger, and had longer forewings than spring alatae, and their embryos grew to a greater length, but in neither sample of alatae did the maximum embryo size approach that of the apterae. As in the apterae of this species, embryos grew to a large size, averaging 44% of parental length in spring alatae and 4-0% in summer alatae. These were among the largest embryos, relative to parental length, found in the Aphididae as a whole. Very little variation in ovariole number was found, 16 out of 20 individuals having ten ovarioles and the remainder having nine. Potential fecundity was slightly greater in the spring alatae than in the summer alatae, but in both cases, alatae had lower numbers of embryos than apterae.

PEMPHIGINAE No significant correlations were obtained between any of the variables studied in Eriosoma lanuginosum alatae, and few correlations were found in the three remaining species (Table 4*23). The embryos contained within an adult pemphigine alata are all at the same stage of development, with pigmented eyes and fully formed appendages, irrespective of their position in the ovariole. This phenomenon, which is characteristic of alate Pemphiginae, was investigated more closely in E. ulttri. For each of the four larval instars, a sample of ten individuals was dissected to examine the reproductive system. A similar series of dissections were made of the larval instars of alatae A. pisum (Aphidinae) for comparison. Whereas each successive A. pisum instar had a higher number of embryos than the previous one (Fig. 4.7), those of E. ulmi showed virtually no variation in embryo number at all (Fig. 4.8). At birth, the first instars of E. ulmi. already have their full complement of embryos, and there is no increase in embryo number from this developmental stage onwards. Because the germaria have already disappeared at birth, it would be necessary to study the alatae while they are still in the embryonic stage in order to find out how this synchronous development of embryos is achieved. This curious phenomenon has so far only been encountered in pemphigine alatae. It presents a strategy which enables the alata, after completing migration, to deposit all of her progeny in rapid succession without the necessity of feeding from the secondary host in order to provide nutrition for still developing embryos.

ALATAE: SUMMARY Only two out of 49 alate morphs, representing 38 species, were found to have a higher potential fecundity than the equivalent wingless morph. In most cases ovariole numbers were the same or very similar in both apterae and alatae, and the greater potential, fecundity of apterae was the result of their having greater numbers of embryos per ovariole. It would appear that there are two basic strategies available to the developing virginoparous aphid; to develop into either a wingless, fecund adult, or to develop into a winged, less fecund adult. The rates of assimilation and conversion of the nutritional resources available to aphids limit the number of embryos which a presumptive alata is capable of developing because so much of the energy is channelled into the development of the wings, their associated muscles, and the fat body which is needed to sustain prolonged flight. In most species the largest embryos of alatae were slightly smaller than in the equivalent wingless morph, particularly in the Lachninae, and it may be that by producing slightly smaller offspring, alatae are able to produce more offspring than their lower fecundity would otherwise allow. Alatae show less interspecific variation in reproductive potential than do either apterae or fundatrices. In the whole of the Aphidoidea, ovariole 108 109

A First instar B Second instar Third instar 4.8 OVARIESOF SUCCESSIVE C D Fourth instar INSTARS OF Eriosoma ulmi ALATAE E Adult numbers of alatae varied from eight to fourteen, except in Pterocommatinae, where alatae of the three species studied had mean ovariole numbers of 15, 19 and 20. Alatae were more fecund in Pterocommatinae and Drepanosiphinae than they were in other subfamilies, possibly because in these two subfamilies the alatae do not need to complete a period of flight activity before they begin to reproduce. Those heteroecious species having two winged virginoparous morphs did not all show the same seasonal trends in potential fecundity; in some the summer alata was more fecund than the spring migrant, while in others the opposite was true. In the heteroecious Aphididae, as well as in the Adelgidae and Phylloxeridae, the generation .which gives rise to either one or both of the sexual morphs is winged, but because of the special nature of these morphs they are discussed separately from the alate virginoparae, in the following section which deals with the pre-sexual generation. 4.1.3.3 SEXUPARAE Sexuparae were studied in 24- of the species involved in this survey. Ten species had wingless sexuparae, and were monoecious. Of the remaining 14, seven were the gynoparae of heteroecious Aphidinae and the remainder were winged sexuparae of either monoecious or heteroecious species. WINGLESS SEXUPARAE Table 4.26. Mean embryo and ovariole numbers and embryo lengths in the wingless sexuparae of the species studied, compared with the same data for the equivalent virginoparae (shown in brackets).

SPECIES MEAN MEAN MEAN EMBRYO EMBRYO NO. OV. NO. LENGTH (urn) Cinara pini 17 (31) 9 (12) 1360 (1103) Lachnus roboris 46 (55) 14 (14) 1703 (1316) Maculolachnus submacula 8 (26) 11 (12) 1470 (1339) Pterocomma populeum 36 (82) 14 (15) 1113 (998) P. salicis 58 (71) 18 (19) 1120 (816) Aphis farinosa 46 (69) 11 (12) 632 (674) Macrosiphoniella artemisiae 39 (84) 14 (14) 686 (822) M. millefolii 49 (58) 12 (12) 936 (878) Chaitophorus capreae 25 (47) 8 (8) 547 (594) C. leucomelas 30 (36) 8 (8) 625 (660) Periphyllus hirticornis 53 (53) 11 (13) 969 (984)

In all species but one, Periphyllus hirticornis, wingless sexuparae had fewer embryos than wingless virginoparae of the same species (Table 4*26). In many cases ovariole numbers were also lower in sexuparae than in virginoparae, and even where ovariole numbers were the same in both morphs, sexuparae always had fewer embryos. In Lachninae and Pterocommatinae, the largest embryos of sexuparae had both a mean and a maximum length greater than that of the largest embryos of wingless virginoparae. The largest embryos dissected out of these samples would have been mostly oviparae (see section 1.3), so it appears that in these two subfamilies the new-born oviparae are larger than new-born virginoparae. The wingless sexuparae of Lachninae, Pterocommatinae and monoecious Aphidinae are morphologically indistinct from the wingless virginoparae, and although the sexuparae of the species studied had larger embryos and lower potential fecundities than the virginoparae, their reproductive systems did not appear to be specialised in any particular way for the production of sexuals. A detailed study of the changes in the reproductive system associated with the transition from parthenogenetic to sexual reproduction was carried out in the monoecious aphidine, Acyrthosiphon pisum, and is described below in section 4.2.2 » WINGED SEXUPARAE

Table 4.27. Mean embryo and ovariole numbers for winged sexuparae (including gynoparae) of the species studied. Where available, data for virginoparous alatae of each species, occurring on the same host, are also Included in brackets. Figures in square brackets refer to the source of the virginoparous alata, i.e. primary or secondary host.

SPECIES MEAN EMBRXO NO. MEAN OV. NO.

Aphis fabae 14 (51) 11 (12) [2] Dysaphis plantaginea 16 (35) 8 (13) [1] D. crataegi 25 (34) 10 (10) [2] Brachycaudus helichrysi 11 (18) 8 ( 8) [2] Longicaudus trirhodus 15 (39) 10 (n) [1] Myzus persicae 37 (44) 10 (10) [2] Hyperomyzus lactucae 35 (49) 10 (10) [2] Cavariella aegopodii 31 (4D 10 (10) [2] Periphyllus testudinacea 17 (69) 8 (14) Eucal]ipterus tiliae 61 (59) 8 ( 8) Thelaxes dryophila 18 9 Glyphina betulae 36 10 Mindarus abietinus 12 11 Anoecia comi 21 (41) 9 (10) [2] Pemphigus spirothecae 8 8

Like the wingless sexuparae, embryo numbers were lower in winged sexuparae, including gynoparae, than in the equivalent winged virginoparous morphs (Table 4*27). The only exception was Eucallipterus tiliae. Unlike the wingless sexuparae, the largest embryos were not consistently larger than the embryos of the equivalent virginoparae, being larger in some species and smaller in others, but without any pattern consistent with either the taxon or the type of life-cycle. As with the wingless sexuparae, ovariole numbers in winged sexuparae and gynoparae were usually similar to those found in the previous generations, and the often striking differences in fecundity were due to a reduction in the numbers of embryos per ovariole. Absorption of the oldest embryos by summer morphs, presumably aestivating sexuparae, was found in both D. platanoidis and E. punctipennis. A number of Drepanosiphinae are known to enter reproductive diapause in summer, when host quality is poor, and resume reproduction in late autumn to give birth to the sexuals. It seems likely therefore that those embryos which were being absorbed were presumptive virginoparae, whereas the intact embryos were the presumptive sexuals. The reproductive system of such an individual is shown in Fig. 4*9. 113

Fig. 4.9 Reproductive system of Euceraphis punctipennis (?)sexupara. Absorption of most advanced embryos (indicated with arrows) appears to be occurring.

Fig. 4.10 Ovarioles of Drepanosiphum platanoidis sexupara. Unusual structures on youngest embryos indicated. In addition to the phenomenon described above, sexuparae of Drepanosiphum platanoidis, collected 9.XI.82, had small structures attached to the least developed embryo in each ovariole (Fig. 4-«10). These structures are apparently undescribed, and their function is unknown, although it may be nutritive. Dissections of the sexuparae of a number of species revealed evidence of absorption or arrested development of oocytes which had been ovulated either prior to or after the ovulation of the successfully developing oocytes. This was shown particularly in a number of aphidine gynoparae, including Dysaphis crataegi, Hyperomyzus lactucae and Macrosiphum rosae. Adult gynoparae of the above aphidine species had one (occasionally two) fully developed embryo(s) in each ovariole, a string of either undeveloped oocytes or regressed embryos occupying the remainder of the ovariole (fig. 4-.11). The same type of ovariole was previously found in Myzus persicae gynoparae (Searle & Mittler,1982 p.216). This type of embryo absorption was rather different from that found in early summer morphs of Drepanosiphinae about to enter reproductive diapause, in which the most advanced embryos were absorbed. Both these kinds of embryo absorption were, however, observed during an experimental study of the changes in the reproductive system of Myzus persicae during the transition from parthenogenetic to sexual reproduction (section U.2.2). V

FIG. 4.11 OVARY OF H. I actucae gynopara showing undeveloped embryos or oocytes in each ovariole 4.1.3.4 OVIPARAE

Table 4*28. Summary of modal ovariole numbers in oviparae of Aphidoidea compared with those of wingless virginoparae and fundatrices. (In heteroecious species, marked with an asterisk, the ovariole number of the wingless virginopara on the secondary host is used for comparison.)

SPECIES OV. NO. 0V. NO. 0V. NO. OVIPARA APTERA FUNDATRIX Cinara pini 14 12 22 C. stroyani 8 13 22 Lachnus roboris 14 14 14 Maculolachnus submacula 12 12 14 Pterocomma salicis 19 19 21 P. populeum 14 15 34 Aphis farinosa 8 12 — A. salicariae 10 10 * 10 A. fabae 12 12 * 20 Dysaphis plantaginea 9 10 * 22 D. crataegi 9 10 * 14 Brachycaudus klugkisti 9 11 * — Longicaudus trirhodus 10 10 * 20 Hyperomyzus lactucae 10 10 * 14 Cavariella aegopodii 10 10 * 14 %-zus persicae 10 10 * 15 Macrosiphum rosae 12 13 * — M. millefolii 12 12 — Macrosiphoniella oblonga 12 12 — M. artemisiae 14 14 — Chaitophorus capreae 8 8 8 C. leucomelas 8 8 14 Periphyllus testudinacea 10 14 14 P. hirticomis 11 12 26 Drepanosiphum platanoidis 10 10 12 Euceraphis betulae 8 8 10 Eucalli.pterus tiliae 8 8 10 Phyllaphis fagi 10 10 12 Thelaxes dryophila 4 10 10 Glyphina betulae 7 10 — Mindarus abietinus 5 12 — Anoecia comi 4 10* 8 Pemphigus spirothecae 1 10 8

In Lachninae, Pterocommatinae, Aphidinae, Chaitophorinae and Drepanosiphinae ovariole numbers in the oviparae were the same or slightly lower than in the equivalent virginoparae. In Thelaxinae, Mindarinae, Anoeciinae and Pemphiginae, however, the number of ovarioles was much lower in the ovipara (Table 4*28), because in these four subfamilies, the oviparae are dwarfish and larviform. Each of the individuals of Pemphigus oviparae had only one ovariole containing one egg, but in the other species with dwarfish oviparae, the number of ovarioles often exceeded the total number of eggs, one or two of the ovarioles being undeveloped (see Fig. 4.1). By dissecting the young embryos within the ovarioles of autumn migrants of Briosoma lanigerum, Baker (1915) showed that when the oviparae are in an early embryonic stage they contain eight germaria, and these must correspond to the eight ovarioles and germaria of the virginoparae. In later embryos, most ovarioles are in the process of degeneration, and only two develop. Of these, one eventually degenerates, leaving the one in which the egg is developed. In most Thelaxes oviparae dissected, four ovarioles were found, but usually only two of these contained eggs, the other two being vestigial or undeveloped. Anoecia and Mindarus oviparae nearly always contained undeveloped ovarioles, and it may be that oviparae of the Thelaxinae, Mindarinae and Anoeciinae are similar to those of Pemphigus in this respect, but that the vestigial ovarioles persist, and the final number of ovarioles which develop eggs successfully is less rigidly fixed than in the Pemphiginae. Considering the Aphidoidea as a whole, differences in ovariole numbers between apterae and oviparae reflected differences in gross morphology; for example, where oviparae were morphologically very similar to apterae, they had the same number of ovarioles. This corresponds to the relationship between fundatrices and apterae, where fundatrices which closely resembled their descendants often had the same or very similar numbers of ovarioles. Thus there is a scale of degrees of specialisation of both morphology and reproductive function, with those species with the same ovariole number in all morphs at one end, e.g. Lachnus roboris, and those which have a 50- or 60-fold decrease in ovariole number between the fundatrix and the ovipara at the other; e.g. Thecabius (data from Tashev & Markova, 1982) and Adelges. With very few exceptions the number of eggs developing in oviparae was smaller than the number of embryos developing in viviparae of the same species. However, comparisons between their potential fecundities are difficult because of the very different roles played by the two morphs. The oocytes of an ovipara grow to a considerable size, due to the assimilation of yolk via the nurse cells, before they can be fertilized and begin to develop as embryos, whereas the oocytes of a vivipara mature and start embryonic development soon after they are extruded from the germarium. Presumably because the eggs of oviparae need to be connected to the germarium by trophic cords throughout their growth phase prior to fertilization and development, the rate of ovulation is slower in oviparae than in viviparae, the number of ovulations is lower, and ovulation continues for longer into adult life. Samples of adult oviparae of the same species collected at different times often had strikingly different numbers of eggs, older adults having greater numbers of eggs which had completed vitellogenesis. Thus estimates of potential fecundity of oviparae made from dissections are likely to underestimate their true potential fecundities, and comparisons between the potential fecundities of oviparae are impracticable unless the differences are very obvious. Dissections of the oviparous fundatrices of phylloxerids and adelgids revealed that the greater fecundities of these morphs compared with the later oviparous generations are achieved by increases in the numbers of ovarioles rather than by increasing the numbers of eggs per ovariole. Possibly, therefore, in oviparae of Aphididae the number of ovarioles is probably a better measure of the relative fecundity, than the number of eggs which are present at any one time, apart from in those species with dwarfish oviparae. 4.. 1.4 GENERAL DISCUSSION

Having considered all of the morphs which occur in the various taxa within the Aphidoidea it is clear that every species studied shows a seasonal decrease in potential fecundity, either as a result of decreasing ovariole numbers, decreasing embryo numbers or both. These changes are more marked in some taxa than in others, and this is associated with the degree to which each morph is specialised for its particular role in the life-cycle of the species. These seasonal changes in ovariole and embryo numbers of successive morphs, and the extent to which they are "programmed", in the sense of Wellings et al (1980), to anticipate changes in host-plant quality, or are direct responses to environmental stimuli are discussed below.

Ovariole number i. Seasonal reduction in ovariole number. Differences in ovariole number between different aphid morphs were first recorded for three successive generations of Hormaphis hamamelidis (Lewis and Walton, 1958). It was already known, however, that pemphigine oviparae contained a single ovariole, whereas virginoparae contained eight (Baker, 1915). Evidence resulting from detailed work on seasonal changes in ovariole numbers and adult weight of aphids (Dixon and Dharma, 1980; Wellings et al, 1980; Leather and Wellings, 1981; Leather et al, 1983) indicated that the number of ovarioles is independent of adult weight, and is genetically programmed to anticipate predictable changes in host plant quality (Dixon and Wellings, 1982). However, it was suggested from recent work on Rhopalosiphum padi that ovariole numbers in the early generations of emigrants are dependent on adult weight, which decreases as a result of declining host plant quality (Wiktelius and Chiverton, 1985).

In each of 43 species studied in this survey, there occurred at least one seasonal reduction in ovariole number. In only five species was it shown that the ovariole number is constant throughout the season. The seasonal patterns of changing ovariole number were of three main types

A: Where the major reduction in ovariole number occurred at the beginning of the season, between the fundatrix and the fundatrigenia, as in all four Drepanosiphinae studied as well as in a number of heteroecious Aphidinae; B: Where there were major reductions in ovariole number both at the beginning and at the end of the season, as in heteroecious Pemphiginae, Adelgidae and Phylloxeridae; C: Where a reduction in ovariole number occurred only at the end of the season, between the sexupara and the ovipara, as in Thelaxes dryophila and Anoecia comi.

Many species did not conform strictly to one of these three types, but fitted in somewhere in between, nevertheless 'showing a seasonal reduction in ovariole number. The five species which did not show any seasonal reduction in ovariole number were Lachnus roboris, Chaitophorus capreae, Aphis epilobii, A. salicariae and Macrosiphoniella artemisiae, species from diverse taxa, and with different life-cycles (four monoecious and one heteroecious species) and host plant associations. The fundatrices of these species are relatively unspecialised and resemble the wingless virginoparae morphologically. It might therefore be reasonable to suggest that these species represent the primitive condition for their taxa, where the fundatrix has not yet become specialised in either her external morphology or her reproductive potential. However, this would mean that specialisation of the fundatrix has evolved in at least five separate lineages, and this seems unlikely when one considers that the primitive Adelgidae and Phylloxeridae are highly polymorphic in terms of both external morphology and reproductive potential, and that this phenomenon also occurs in all the aphid subfamilies treated in this study. One may accept, therefore, that seasonal changes in ovariole number are a normal condition of the Aphidoidea, and that stability of ovariole number in these five species is probably a secondarily derived condition. Further support for this argument comes from the results of studies on ovariole number in Pemphigus spyrothecae. Host alternation is found in almost all of the pemphigines. The fundatrices are much larger than the later generations, have shorter legs, and are usually much more fecund. In P. spyrothecae the fundatrix is not typical of the Pemphiginae, and has both an ovariole number and a reproductive potential characteristic of a pemphigine virginopara. Host-alternation is absent in this species, and it has an abbreviated life-cycle. The capacity for producing a distinct fundatrix morph has thus been lost in this species. It is possible that this loss has also occurred in the other species which have constant ovariole number. It is nevertheless puzzling that these five species seem to have no particular property in common, and even include a heteroecious species, Aphis salicariae. It is of interest that Leather (unpublished observations) recorded the fundatrices of this species in Finland as having 14 ovarioles and the remaining morphs 10, whereas all morphs of the British species have only 10 ovarioles. It may be that the loss of the capacity for fundatrix production is variable; i.e. it is complete in P. spyrothecae, but only occurs in certain populations of A. sali cariae, or that under certain environmental conditions the developmental sequence which leads to a fundatrix is ’’re-routed", and a wingless virginopara is produced, although the genotype retains the capacity for the production of a true fundatrix. In such species, the degree to which the fundatrix resembles a fundatrix typical for its taxon may depend upon environmental conditions or the effect of stimuli received during embryonic or nymphal development. In order to determine whether seasonal variation in ovariole number is simply another aspect of seasonal morphological polymorphism, or whether it is under separate control, it is necessary to consider the occurrence of undeveloped or vestigial ovarioles and variation in ovariole numbers within morphs. ii. Vestigial or undeveloped ovarioles. The adult oviparae of Pemphiginae are known to contain only one ovariole, and this has been recorded in the genera Briosoma, Forda, Prociphilus and Pemphigus (Baler, 1915; Pergande, 1912; Toth, 1937; and the present study). According to Toth (1937) oviparae of Pemphigus spyrothecae develop two ovarioles, of which one degenerates. Baker (1915) records a similar phenomenon in Eriosoma lanigerum where seven of the original eight ovarioles degenerate during embryonic development. Similarly, in the present study, immature Mindarus oviparae had between seven and 12 ovarioles, whereas the adults had between one and eight. Van Rensburg (1981) records the presence of undeveloped ovarioles in Cinara cronartii apterae. In the present study, degenerate or undeveloped ovarioles were frequently found in a number of different taxa, and in different morphs. It often requires very careful scrutiny to see these undeveloped ovarioles, as they are so small, and it may be that in older adults they have disappeared completely. It also seems likely that in addition to undeveloped ovarioles persisting into adult life other ovarioles may have been present either in earlier instars or during embryonic development which have completely disappeared, as in Baker’s description of Pemphigus oviparae. iii. Variation in ovariole number within morphs. The amount of variation in ovariole number for a particular sample of a particular morph was not constant throughout the Aphidoidea studied. Ovariole numbers were very stable in all morphs of Drepanosiphinae and in the post-fundatrix generations of Pemphiginae (except P. spyrothecae) and Chaitophorinae. A particular ovariole number seems to be associated with most morphs of most species studied. Where the ovariole number was stable, it was invariably an even number, often 8 (the most common), 10, 12 or 14» However, where the ovariole number of a morph of a particular species was found to vary, this variation was usually continuous, with odd numbers of ovarioles occurring frequently. In Pterocomma pilosum and P, salicis almost half the individuals dissected contained odd numbers of ovarioles. Odd numbers of ovarioles were frequently found in Brachycaudus klugkisti and in Hyzus persicae. Some distributions of ovariole numbers are shown in table 4-.29 (overleaf). Table 4*29 Distributions of ovariole numbers in combined samples of post-fundatrix winged and wingless virginoparae in those species having a modal ovariole number of 10.

SPECIES N OVARIOLE NUMBER 7 8 9 10 11 12 13 14

Cinara pilicomis 23 — —— 11 2 6 2 2 Cinara stro.yani 10 —— '— 10 —— — — Aphis epilobiaria 31 —— 3 26 2 — — — Aphis epilobii 46 — — 2 42 2 — — — Aphis grossulariae 30 1 1 2 25 1 —— — Aphis salicariae 34 —— — 34 ———— Aphis farinosa 6 — —— 4 2 ——— Rhopalosiphum insertum 12 — 1 — 11 —— — — Brachvcaudus klugkisti 10 —— 1 7 2 —— — Dysaphis crataegi 50 1 1 4 44 — — — — Dysaphis plantaginea 10 — — — 10 ——— — Longicaudus trirhodus 20 —— 1 19 — — — — Hyzus persicae 32 — — 1 31 ——— — Hyperomyzus lactucae 63 —— 1 59 3 —— — Cavariella aegopodii 78 —— 3 74 1 —— — Drepanosiphum platanoidis 11 — —— 11 —— — — Phyllaphis fagi 34 — 1 3 30 —— — — Thelaxes dryophila 19 — — — 19 —— —— Anoecia comi 78 — 4 15 59 — — —— Pemphigus spyrothecae 20 — 1 2 16 1 — ——

TOTALS 617 2 9 38 542 16 6 2 2

Table 4-29 shows that of all the individuals belonging to morphs which have a modal ovariole number of 10 only 12% have ovariole numbers other than 10. In most species there are more individuals with fewer than 10 ovarioles than there are with more than 10. The same would have been true if individuals belonging to morphs which had a modal ovariole number of 12 or 14 had been selected. It seems probable therefore that virginoparae have a characteristic ovariole number, and that individuals belonging to the same morph and of the same generation which have, however, fewer than the characteristic number of ovarioles have undergone the loss of one or more ovarioles during their larval or nymphal development. In general there appear to be characteristic ovariole numbers for particular morphs, but their expression may be subject to variation. Ovarioles may degenerate during embryonic or nymphal development as a result of environmental influences acting directly on the larva or via her mother. Thus the ovariole number is essentially another aspect of seasonal polymorphism, but may be altered under certain circumstances. Lack of development, or regression, of potential ovarioles may explain low ovariole numbers in the oviparae of Thelaxinae, Anoeciinae and Pemphiginae, and late-season virginoparae in other taxa, but how do fundatrices of various taxa come to have very high ovariole numbers? If all variation in ovariole number between the individuals of a species was the result of ovariole degeneration, it would be necessary to hypothesize that at some stage in the embryological or nymphal development of every morph there exists the capacity to develop the maximum number of ovarioles found in that species. This number is usually that found in the fundatrix. This study has shown that there is a close relationship between the morphological specialization of the fundatrix and her ovariole number, throughout the Aphidoidea. This relationship suggests that exceptionally high ovariole numbers are a specialization peculiar to fundatrices, and that their occurrence is controlled in a different way from the variability in ovariole number which is found in subsequent morphs. How the high numbers of ovarioles in fundatrices is achieved is not understood, although an embryological study of, for example, pemphigine fundatrices might make this clear. Although there do not appear to be any fixed rules for all Aphidoidea, ovariole numbers generally are stable and genetically programmed for each morph. In those species which show considerable variation in ovariole number within morphs, i.e. some of the Iachninae, Pterocommatinae and Aphidinae, this variability is probably achieved by the lack of development or regression of one or more potential ovarioles. In cases where many individuals occur which have more than the modal ovariole number, it is possible that those having the modal number have all undergone the loss of some of their ovarioles (this may have occurred in C. pilicornist table 4.29). Dixon & Dharma (1980) showed that the ovariole number of alate exules of A. fabae varies from 6 to 12, and it was suggested that this variation was an aspect of the "phenotypic plasticity" of the species, enabling it to cope with a varying environment by adopting a variety of reproductive strategies. Alatae of Rhopalosiphum padi were also found to show considerable variation in ovariole number, 10-16 for emigrants and 6-10 for alate exules (Walters & Dixon, 1983). Within each of the two morphs, alatae with fewer ovarioles had different patterns of flight behaviour than those with greater numbers. These results again suggested that mixed reproductive strategies were being employed by the particular morph, enabling the species to exploit a variable environment. Leather et al (1983) showed that on poor quality hosts those R. padi with the highest numbers of ovarioles suffered the highest mortalities. Ward and Dixon (1984.) have applied the phrase uspreading the risk" to succinctly explain variation in ovariole number both within and between the morphs of a' particular species. Kidd and Cleaver (1986) have questioned whether variation in ovariole number within morphs is actual1y an evolved reproductive strategy which anticipates variations in environmental quality. They cite a number of authors who have found that alate exules of Aphis fabae consistently contain 12 ovarioles, in contrast to Dixon and Dharma's figure of 6-12. They suggest that the ability to vary the number of ovarioles within a morph is not a widespread phenomenon for this species, and that interclonal variation is responsible for these conflicting results. My own results for A. fabae, showing ovariole numbers for this morph with a range of 11-13 and a mean of 11.9> also suggest that perhaps the clone which Dixon & Dharma used was rather unusual. In the present study, variation in ovariole number within morphs was found to occur most frequently in Cinarini (Lachninae), Pterocommatinae and Aphidinae. The morphologically primitive Drepanosiphinae showed the least amount of within-clone variation, as did the Iachnini, which are generally regarded by aphid systematists to be more primitive than the Cinarini (see also section 6); however, Pterocommatinae is also generally regarded as a primitive branch of the Aphidinae. It may be, however, that within-clone variation in ovariole number has evolved separately as a reproductive strategy in those lineages which have undergone rapid speciation and colonization of new habitats comparatively recently in the evolutionary history of the Aphidoidea. Further studies are necessary to show whether or not intra-clonal variation in ovariole number is an evolved reproductive strategy. The lack of any consistent patterns of intra-clonal variation in ovariole number, and the existence of differences in the extent of variation between closely related species, suggest that if the ability to vary the ovariole number is indeed an evolved reproductive strategy, it is used only under certain conditions and not as a matter of course. In many species, the potential to vary the ovariole number may not therefore be realised unless the aphids are subjected to a variety of environmental conditions, and if variation 1 2 6

then results, it must be shown not to have arisen as a direct result of stress, starvation or other adverse influences. Studies on ovariole numbers in embryos of morphs known to exhibit considerable variation in ovariole number would help to answer some of these questions.

Embryo number

In recent years, a number of authors have attempted to use embryo counts as measures of the reproductive potential of aphids. This method has some serious drawbacks, and Adams and van Emden (1972) have pointed out, with reference to Aphis fabae, that the relationship between embryo number and fecundity is still uncertain. However, recent studies (Wratten, 1977; Kempton et al., 1980; Brown & Llewellyn, 1985; Llewellyn & Brown, 1985a, 1985b) suggest that there is a close relationship between embryo number and fecundity. In those species where few or no ovulations occur during adult life, embryo counts are a good measure of reproductive potential, but only if all of the embryos, and not just those with pigmented eyes, are taken into account. In many individuals, it is possible to determine whether ovulations are complete, from the size of the smallest embryo and the condition of the germarium. The reproductive potential of adults in a state of reproductive diapause, aestivation or embryo absorption cannot be assessed by this method. Embryo numbers decreased as the season progressed in all but five of the 39 species with sample sizes sufficiently large and representative of all or most morphs. None of the five exceptions (Pterocomma pilosum, Myzus persicae, Hyperomyzus lactucae, Macrosiphonie 11a oblonga and Periphyllus testudinacea) was among those mentioned above in which ovariole number was constant throughout the season, and (with the possible exception of M. persicae and H. lactucae) they show no taxonomic and biological affinities. Clearly, in at least 10 (about 25%) of the species surveyed, there does not appear to be a direct relationship between ovariole and embryo number. In fact 13 species, all well represented by large samples of all or most morphs, failed to show any correlation between these two variables. Therefore, an increase in ovariole number does not necessarily result in an increase in embryo number, and increases in ovariole number perhaps result only in increases in reproductive rate, rather than in potential fecundity. This is, however, mere speculation, and further studies on reproductive rate and fecundity in relation to both embryo number and ovariole number would be of great value. 1 2 7

4.2 INFLUENCE OF THE ENVIRONMENT ON REPRODUCTION Various environmental factors can have an effect on the way aphids reproduce, and this has been demonstrated in the past by various workers investigating the effects of different environmental agencies. By rearing Aphis fabae on different varieties of field beans (Vicia faba), Banks (1965) concluded that fecundity was affected by nutrition. More recently, Llewellyn & Brown (1985a) showed that numbers of embryos were affected by host-plant differences in 63% of alatae and in 75% of the apterae which they examined. Nutrition and effects of crowding can influence the production of either apterae or alatae (Mittler & Klein jan, 1970; Bonnemaison, 1968). Since alatae of nearly all species covered in the survey (section 4 »1 ) had fewer embryos than the equivalent wingless morph, the reproductive system can thus also be affected in this way, albeit as a secondary effect. Temperature and night-length are responsible for morph determination, particularly the induction of the sexual phase of the life-cycle, in many species. As the category of the progeny is thus determined, the ovariole and embryo number of the parent may also be affected. Gynoparae of some species of Aphidinae are known to contain fewer embryos, on average, than equivalent winged virginoparae (Blackman, 1975; Searle & Mittler, 1982). Parasitization can affect the reproductive system in different ways, depending on the stage of development at which the aphid is parasitized. Campbell & Mackauer (1975) found that when adult aphids were parasitized by Aphidius smithi (Braconidae), the aphids were still able to reproduce for some days. However, aphids parasitized at first instar were unable to complete their development or reproduce (Campbell & Mackauer, 1975; Soldan & Stary, 1981). The following section deals very briefly with the effects of host-plant differences on aphid reproduction, using some of the data from the systematic survey in section 4 .1.2. The effects of photoperiod on reproduction in a monoecious and a heteroecious aphidine are investigated, and the effects of parasitization on the reproductive system of different instars of the pea aphid, Acyrthosiphon pisum are also examined.

4.2.1 HOST-PLANTS AND APHID REPRODUCTION In order to assess the influence of host-plant differences on aphid reproductive performance it would be necessary to compare numbers of progeny or embryo numbers of samples of the same morph of the same species on different hosts. Ideally, the samples to be compared should be collected at the same time of year to exclude any effects of seasonality in plant growth, and the plants should be growing under similar conditions in the same geographical area. In the systematic survey of ovariole and embryo numbers (section 4*1.2) there are no instances in which all of the above conditions are fulfilled, but there are a few cases of the same morph of the same species occurring at the same time of year on different host plants, and numbers of ovarioles and embryos can in such cases be compared.

Pterocomma salicis Fundatrices of P. salicis were collected within a period of three weeks from Salix cinerea and S. viminalis from Kew Gardens and Putney Common respectively, both sites within a few miles of each other, in S.W. London. Data concerning body lengths, ovariole and embryo numbers and sizes of largest embryos are summarized in table 4*30.

Table 4 • 30 Data concerning reproduction in P. salicis fundatrices from two different species of Salix.

HOST N MEAN BODY OVARIOLE NUMBER EMB. NO LGSTEMB LENGTH mm MEAN RANGE MODE MEAN MEAN^un +/- +/- +/- +/- Cl Cl Cl Cl S. cinerea 6 3.4-9 0.15 21.9 3.0 16-24. 22/24 88 21 1243 81 S. viminalis 10 3.37 0.11 21.5 1.2 19-24 21 108 11 1117 79

No significant differences between means were found for any of the variables studied. Thus, the data from these two small samples suggest that neither species of willow is a more suitable host for P. salicis fundatrices.

Macrosiphum rosae Wingless virginoparae of M. rosae were collected from Chelsea Physic Garden, London SW3 on the same date (19th July) in two successive years. Samples were collected from Dipsacus and Scabiosa. Data for these two samples are summarized in table 4-*31 • Table 4*31 Mean body lengths, ovariole and embryo numbers and sizes of largest embryos in M. rosae virginoparae on different secondary hosts.

HOST N MEAN BODY OVARIOLE NUMBER EMB. NO LGST EMB. LENGTH mm MEAN RANGE MODE MEAN MEAN jam +/- +/- +/- +/- Cl Cl Cl Cl Dipsacus 12 2.01 0.17 12.4 0.59 10-13 12 57 3 693 23 Scabiosa 17 2.29 0.11 12.6 0.51 11-15 12 50 4 786 59

Despite being significantly smaller (P<0.01), virginoparae on Dipsacus had a greater embryo number (P<0.05). Actually, mean embryo numbers were similar in both samples, $7 for the Dipsacus sample, and 50 for the Scabiosa sample, but variation in embryo number was very slight in both samples, hence the significant differences between the means. M.rosae may indeed be able to produce more embryos on Dipsacus, but there is always the problem with field-collected material that one cannot know how many progeny have already been deposited. A slight difference in the average age of the adults from the two samples could have caused a difference in mean embryo number (as discussed on p.83 ).

Periphyllus testudinacea Alatae were collected on Acer campestre and A. pseudoplatanus from Chelsea Physic Garden and Barnes Common (SW London) respectively, within a month of each other. Data for the two samples are summarized in table 4-.32.

Table 4*32 Mean body lengths, ovariole and embryo numbers and sizes of largest embryos of P. testudinacea- alatae from two species of Acer.

HOST N MEAN BODY OVARIOLE NUMBER MB.NO LGST EMB. LENGTH mm MEAN RANGE MODE MEAN MEAN pm +/- +/- +/- +/- Cl Cl Cl Cl A. campestre 10 2.59 0.20 14.0 14 85 4 628 27 A. pseudoplatanus 10 2.26 0.07 14.1 0.2 14-15 14 75 3 684 51

Alatae from A. pseudoplatanus were smaller (P<0.01) and had fewer embryos (P<0.001) than alatae from A. campestre and their largest embryos were larger (P<0.05). A. campestre is the usual host of P. testudinacea in England (Stroyan, 1977), colonies being found less frequently on A. pseudoplatanus, and the data in table 4«32 suggest that A. campestre may be a more suitable host as regards the reproductive capabilities of P. testudinacea. The presence of larger embryos in the A. pseudoplatanus population suggests that the differences in body length and embryo number are not due to a difference in the average age of the adults from the two colonies. However, the production of fewer progeny, which are larger at birth, by the A. pseudoplatanus population may be a particular reproductive strategy employed in response to some other environmental influence, and not simply a reflection of poorer host-plant quality. Colonies of P. testudinacea are also occasionally found on Aesculus hippocastanum, a tree from another plant genus. It would be interesting to compare the reproductive performance of P. testudinacea on these three hosts, using much larger samples.

Anoecia corai Alatae of A. corni were collected from two different Coraus species from Chelsea Physic Garden and Spencer Road during May 1984.. Data for the two samples are summarized in table 4*33.

Table 4..33 Mean body lengths, numbers of ovarioles and embryos and sizes of largest embryos in A. comi alatae from different Comus species.

HOST N MEAN BODY 0VARI0LE NUMBER EMB. NO LGST EMB LENGTH mm MEAN RANGE MODE MEAN MEAN pm +/- +/- +/- " +/- Cl Cl Cl Cl C. alba 10 1.39 0.05 9.8 0.3 9-10 10 45 5 603 36 C. hemsleyi 10 1.39 0.05 9.8 0.3 9-10 10 44 4 628 36

No differences were found between any of the variables studied for the two samples, but what is more remarkable is the great similarity between the two samples. 131

DISCUSSION The few examples above serve to illustrate that in some instances, differences in host-plant can have an effect on reproductive parameters of the aphid, whether the differences are at the genus- or species-level of the host. Few conclusions can be drawn from these examples, and further, detailed comparisons of the same morph of a species living on different hosts would yield useful information on the relationship between reproduction and host-plant specificity in the Aphidoidea. Brown and Llewellyn (1985) and Llewellyn and Brown (1985s) have attempted to relate variation in aphid reproductive potential to differences in host-plant species and growth form. Reproductive potential was measured by dissecting aphids which had been previously preserved in alcohol, and counting the total number of embryos. To evaluate differences in reproductive potential due to differences in host-plant species, data were recorded for seventeen aphid species, each of which was recorded from more than one host-plant. A total of 30 comparisons were made, and^ in 21 of these, total embryo content was found to be affected by differences in host plant species. Seven of the comparisons concerned Aphis fabae and five concerned A. pomi. Of the comparisons of A. fabae on different hosts, a number very probably concerned different morphologically indistinguishable subspecies or species all belonging to the A. fabae group. Slight differences in the life-cycles of these species or subspecies could also be responsible for differences in reproductive potential. Some of the comparisons were of different morphs of the same species on the primary and on the secondary hosts, for instance Cavariella pastinaceae on Salix and Heracleum, and Macrosiphum rosae on Rosa and Scabiosa. The problem with this last type of comparison is that the morph in question is preadapted to use different kinds of hosts during particular phases of its life-cycle, and so differences between the reproductive potential of the two samples may not be due to host-plant differences alone. Whatever the difficulties associated with this kind of study, it has shown that in many of the comparisons made, differences in host plant species are connected with differences in embryo number, and this is perhaps hardly surprising. In order to investigate the relationship between aphid reproductive potential and plant growth form Brown and Llewellyn (1985) divided the host plants in their extensive data base into nine categories, based on phenology and growth form. They used the following categories: annuals, biennials/perennials, grasses, ferns, evergreens, deciduous shrubs and deciduous trees. They further combined the first four categories under herbaceous plants, and the last three under woody plants. Embryo numbers of alatae and apterae feeding on plants in the nine categories were sorted into months of the year. Significant differences between samples from different host categories were evaluated within each month, and differences between samples from the same host category in successive months were also evaluated. In summary, the following conclusions were reached concerning the relationship between aphid reproduction and plant growth form: 1) Species on grasses and ferns have a low reproductive potential. 2) Seasonal fluctuations in embryo number are greatest in aphids on annual plants 3) The absence of observations for aphids on annuals early in the season indicates a slower colonization of this growth form than that of longer-lived herbs. 4) An increase in embryo number of aphids on biennials and perennials during September suggests -that the aphids may benefit from the mobilization of plant food reserves prior to winter, as is the case in tree-dwelling aphids. 5) Apterae on shrubs have a significantly higher potential fecundity than those on trees. 6) Potential fecundity is higher in aphids on herbaceous plants.

Using data from the survey (section 2.1), I have classified host plants in the same way and the resulting data for embryo numbers of apterae for the months May to September are compared with the equivalent data from Brown & Llewellyn (1985) in table 4*34* Since I did not examine any aphids on ferns, this category is ommitted. The data in Table 4«34 from my own results do not support any of the above statements. The only exception is that aphids on herbaceous plants are potentially more fecund (or at least have greater numbers of embryos) than those on woody plants, at least from June onwards. Actually, neither set of data tells us anything about either seasonal trends in aphid reproduction, or about differences in reproductive potential due to differences in host plant category. In both data sets the samples are of unequal sizes, are not replicates of each other with regard to the species included in each sample and furthermore contain a mixture of morphs and species with very different life-cycles. It is inevitable that the pooling of data from such a diversity of sources yields different results depending on the relative abundance of species having certain kinds of life-cycles. Table *4.5*4 Mean numbers of embryos (+/- standard errors) in apterae associated with different host plant categories. Data from survey, with data from Brown 8c Llewellyn, 1985/ in brackets.

PUNT CATEGORY MAY JUNE JULY AUGUST SEPTEMBER

Annuals 6 8 . 5 2 +/-2 . 0 6 70. *40 +/-**. 1 0 55.90 +/-2 . 9 0 (.— -- ,) (55.5*f + A 1 . 9 D (60.95 +/-1 .9 1) (*49.00 +/-2.57) (8 1 . 1 6 +/-*f.26)

Biennials/ 55.70 +/-2 . 8 0 61.10 +/-2 . 3 0 *48.20 +/-1.70 5 8 . 0 0 +/-5.60 35.90 +/-2 . 7 0 Perennials (70.8** +/A.W (5 5 . 5 7 4 /-I.9 0 ) (5 7 .3 5 +/-1.61) 0*48.89 +/-1.53) (60.60 +/-2 .0 9 )

____ Grasses 56.70 +/-**. 1 0 5 2 . 0 0 +/-8.50 *49.80 +/-5.20 (— — ) (58. 9 6 +/-2.3D (27.50 +/-8.*40) (55.11 +A3.56) (— --- )

2 .0 8 57.28 *40 88 Total herbaceous 5 8 . 3 7 +/-2.29 61.82 +/_ 50.67 +/- 1-'>3 +/-3-26 • +/ _ 2.63 (70.8** + / - k M ) (**8.19 +/-1.58) (58.*49 +/-1 .2 8 ) (*4 8 .9 2 +/-1.32) (6 *4 .6 5 +/-1.96) K\ (A O + I Evergreens *4 8 .0 0 +/-2.00 5**.*K) +/-3 . 2 0 2 9 . 1 0 +/-1.3 8 *4 7 .0 0 • 16.50 +/-2 .78 (6 6 . 8 0 +/-5.02) (5 0 . 6 0 (------) (------) • +/-1 .6 5 ) (62.9*4 +/-5.86)

Deciduous 97.70 +/-5.*K) *48.22 +/-2 . 7 1 4 5 . 5 0 +/-5.80 1 9 . 1 1 +/-1.86 20.00 +/-**. 00 shrubs (6 0 . *+9 +/-3-W (58.57 +/-2.57) (**7.66 +/-1.*42) (5 0 . 8 1 +/-2 .1 6 ) (*42.50 +/-**. **9 )

Deciduous 1 1 5 . 0 0 +/A.60 8 2 . 1 0 + /-5.80 6 5 . 6 0 +/-*4.20 *4 8 .6 0 +/-3 .0 0 *47.10 +/-2.90 trees 6 1 . 6 5 +/-4. 75) (*40.29 +/-1 .82) (*49.19 +/-1.71) (*4*4 .6 2 +/-5.W (36.06 +/-2 .0 5 )

Total woody plants 9 2 . 8 7 +/-3.19 76*78 +/-'*. 87 56-*49 +/"5.07. , *43 • 57 +/-2.35v 37-83 + A 5 . 1 1 (6 1 . 6 1 +/-2.**9) (50.72 +/-1.69) (55.06 +/-2.07) (*47.31 +/-7 .0 7 ) (3 6 .74 +/-1 .9 2 ) 133 134

4-2.2 THE EFFECTS OF PHOTOPERIOD ON THE REPRODUCTIVE SYSTEM IN A MONOECIOUS AND A HETEROECIOUS APHID

INTRODUCTION In many aphid species the seasonal production of sexual morphs is partly under photoperiodic control. Theoretically, under constant conditions of long day-length, and given adequate food, holocyclic species may reproduce indefinitely by parthenogenesis. Similarly, if aphids collected in the field early in the season are placed in artificial short-day conditions, the production of sexuals may be induced many months before they would occur in the field. Lees (1960) has discussed the existence of an "interval timer" which operates to prevent the production of sexuals under natural short-day conditions in the spring. The sequence of different morphs produced by virginoparae transferred from long- to short-day conditions has been studied for species of Aphidinae under various regimes of day-length and temperature (Blackman, 1975; de Fluiter, 1950; Kenten, 1955; Lamb & Pointing, 1975; Lees, 1960). However, changes in the gross morphology of the reproductive system, including records of any differences in ovariole number or embryo number between virginoparae and sexuparae are referred to infrequently in the literature. Blackman (1975) noted that gynoparae of M. persicae contained few embryos, and Hardie (1980b) compared the reproductive systems of winged virginoparae and gynoparae of A. fabae. The reproductive systems of these two morphs were also compared and illustrated by Searle & Mittler (1982) for M, persicae. These authors all noted that ovarioles of gynoparae differed in form from those of winged virginoparae in the heteroecious species studied. The present study was carried out in order to investigate and compare changes in the gross morphology of the reproductive system during the transition from parthenogenetic to sexual reproduction in a monoecious and a heteroecious aphid.

MATERIALS & METHODS To investigate reproductive changes in a monoecious aphid, pea aphids (Acyrthosiphon pisum) from a clone established at the British Museum (Natural History) in 1981, from an original culture from Ashurst Lodge, Imperial College, Silwood Park, Ascot, Berkshire, were used. Peach-potato aphids (Myzus persicae), from a clone established at the BM(NH) in 1969, collected originally from Gosfield, Essex, were used as the heteroecious species. Fifty adult pea aphids were allowed to reproduce on broad bean leaves (Vicia faba, Sutton Dwarf variety) in excised leaf cages (see p.32 ) for 24 hours at 20°C under an L16:D8 (long-day) regime. Larviposition occurred every 4.-6 hours, so this resulted in a culture of approximately 200 first instar aphids, with dates of birth synchronized to within 24 hours of each other. These were kept in 20 cages at a density of 10 aphids a leaf under long-day conditions until they had reached fourth instar. Five aphids were then removed from each cage and transferred to another set of cages in an L12:D12 (short-day) regime at 16°C. Fresh leaves were provided every two days. At the third instar the morph of each aphid became distinguishable as either an aptera or an alata. At this stage, therefore, both the long- and the short-day cultures were subdivided according to morph, in order to Investigate the possible effects of the morph of the mother on the reproductive system of her progeny. Thus four cultures of adult individuals belonging to different categories were obtained: LD-winged, LD-wingless, SD-winged, SD-wingless. On reaching adulthood, each of these four cultures was allowed to reproduce for 24 hours and their progeny were maintained under the same conditions as the parents. From each of the four cultures a sample of ten individuals was removed at each instar stadium, approximately every 24-48 hours. These samples were dissected as described in section 3-3-1 and the following variables were recorded: i) Length of body, ii) Number of ovarioles. iii) Number of embryos, iv) Number of embryos with discernible eyes, v) Length of the largest embryo.

In addition, the appearance of the ovarioles and embryos was noted, as well as any other observations of interest. Each reproductive system dissected was preserved in Dubosq-Brasil Bouin for further study if necessary, and some were cleared in Methyl-Benzoate Celloidin and mounted on slides. For M. persicae the experimental procedure followed that described above for A. pi sum, but all individuals were reared on potato leaves (Solanum tuberosum). RESULTS: Acyrthosiphon pisum Under short-day conditions, all the progeny of both winged and wingless mothers of A. pisum grew into wingless adults (sexuparae), which in turn gave birth exclusively to oviparae during the first few days after reaching adulthood. Under long day conditions progeny of both winged and wingless mothers developed into both winged and wingless adults. This sequence of morphs is summarised below.

LONG-DAY SHORT-DAY

GENERATION 1ST INSTAR U V + W

4TH INSTAR uv+wv----

ADULT uv+wv UV+WV 2ND / \ GENERATION UV+WV UV+WV UV UV (Sexuparae) (Sexuparae) 3RD •L i* GENERATION 0 0

Studies described earlier in this thesis, on many aphid species, have shown that there are often major differences between the reproductive systems of apterae and alatae, alatae generally containing fewer embryos. Therefore, since all the sexuparae were wingless, the data recorded concerning their reproductive systems was only compared with that obtained for the wingless virginoparae from long day conditions. Presumptive alatae cannot be distinguished from presumptive apterae until the third instar; however, differences between their respective reproductive systems are also not apparent until after this stage, so inclusion of data for first and second instar alatae among that for apterae cannot have affected the results substantially. A total of four different categories of wingless morph were therefore studied from each instar and compared:

1. Long-day: Wingless virginoparae from wingless mothers. 2. Short-day: Wingless sexuparae from wingless mothers.

3. Long-day: Wingless virginoparae from winged mothers. 4. Short-day: Wingless sexuparae from winged mothers. 137

The data obtained for each instar of each of these four categories are tabulated below (Tables 4-.35 - 4-38) and shown graphically in Figs. 4-12- 4.13. All individuals dissected but one contained 14 ovarioles, and so ovariole number data are not included below.

Table 4*35 Long-day: Wingless virginoparae from wingless mothers

INSTAR N MEAN BODY EMBRYO NO. EMBRYOS SIZE LGST EMBRYO LENGTH mm NO. MEAN WITH EYES MEAN pm MAX p m +/- Cl +/■- CI MEAN +/- CI +/-■ CI I 10 1.10 0.05 36.1 2.1 0 122 7 141 II 10 1.70 0.08 51.0 3.7 0 236 21 281 III 5 2.21 0.53 63.0 ■11.5 0 306 75 375 IV 4 2.67 0.41 78.8 '14.5 0 504 61 547 A 10 3.62 0.15 106.5 2.3 20.0 2.2 963 73 1100

Table 4-36 Short-day: Wingless sexuparae from wingless mothers

INSTAR N MEAN BODY EMBRYO NO. EMBRYOS SIZE LGST EMBRYO LENGTH mm NO. MEAN WITH EYES MEAN um MAX pm +/- ci +/■- CI MEAN +/- CI +/-- CI I 10 1.13 0.03 35.4 1.5 0 132 8 141 II 10 1.53 0.07 47.3 1.4 0 209 6 219 III 10 1.97 0.07 57.3 2.4 0 286 23 313 IV 10 2.41 0.14 66.3 3.4 0 397 22 453 A 10 3.38 0.12 83.8 2.1 16 .9 1 .0 852 30 968 1 0 CD ^ Long-day: Wingless virginoparae from winged mothers

INSTAR N MEAN BODY EMBRYO NO. EMBRYOS SIZE LGST' EMBRYO LENGTH mm NO. MEAN WITH EYES MEAN jjm MAX pm +/- ci +/- CI MEAN +/- CI +/-- CI I 10 0.89 0.03 28.6 1.0 0 108 4 109 II 10 1.37 0.06 37.1 1.4 0 197 9 219 III 10 1.90 0.10 50.9 2.2 0 322 24 391 IV 6 2.46 0.17 65.0 5.6 0 482 35 516 A 10 3.19 0.24 79.0 3.7 14.9 2.3 939 66 1094

Table 4-38 Short-day: Wingless sexuparae from winged mothers

INSTAR N MEAN BODY EMBRYO NO. EMBRYOS SIZE LGST EMBRYO LENGTH mm NO. MEAN WITH EYES MEAN pm MAX pm +/- CI +/- CI MEAN +/- ci +/-- CI I 10 0.94 0.07 26.2 2.6 0 114 13 156 II 10 1.49 0.14 35.9 2.4 0 230 14 250 III 10 1.78 0.11 46.1 2.6 0 322 22 359 IV 10 2.94 0.15 67.0 4.7 7 .3 1.6 666 55 750 A 10 3.53 0.12 72.0 3.4 1 7 .5 1.7 1023 23 1078 LONG day : wingless mothers

SHORT DAY:

LONG DAY: WINGED m o t h e r s

* SHORT d a y :

IV A INSTAR

FIG.4.12 Mean numbers of embryos in successive insfars of A. pisum ...... LONG day; WINGLESS MOTHERS

------SHORT day:

------LONG DAY; WINGED MOTHERS

• --- • SHORT DAY;

INSTAR

FIG. 4.13 Mean sizes of largest embryos in successive instars of A. pisu m 139 Mean body lengths, embryo numbers (fig. -4-12) and sizes of largest embryos (fig. 4» 13) differed between categories for each instar. A two-way analysis of variance was therefore carried out to discover which of the differences between the means of the three variables were significant. Further analysis of variance on the data for body length, embryo number and size of largest embryo was used to assess the influence of day-length and the effect of having winged or wingless mothers. The results of the two-way ANOVA are summarized in tables 4-39—4«4i•

Table 4*39 Significant differences between mean body lengths of A. pisum under different day-lengths and from either winged or wingless mothers.

SOURCE OF VARIATION INSTAR I II III IV A DAILENGTH F 1,37 ———— =4.39* ——— — MORPH OF MOTHER F 1,37 F 1,37 F 1,32 F 1,28 — =85.47*** =15.42*** =14.65*** =8.03** —

Table 4*40 Significant differences between mean numbers of embryos of A^ pisum under different day-lengths and from either winged or wingless mothers.

SOURCE OF VARIATION INSTAR I II III IV A DAILENGTH — F 1,37 F 1,32 — F 1,37 — =6 .60* =4.39* — =66.02*** MORPH OF MOTHER F 1,37 F 1,37 F 1,32 — F 1,37 =98.20*** ==142.26*** =54.54*** — ==115.60***

Table 4.41 Significant differences between mean sizes of largest embryos of A. pisum under different day-lengths and from either winged or wingless mothers.

SOURCE OF VARIATION INSTAR I II III IV DAILENGTH F 1,37 =$.25* MORPH OF MOTHER F 1,37 F 1,32 F 1,28 F 1,37 = 19. 14 *** =4.76* =29.24*** =7.00*

Differences between the mean body lengths (table 4*39)» mean numbers of embryos (fig. 4*12, table 4.40) and mean sizes of largest embryos (fig. 4.13, table 4»41) of individuals belonging to each instar from the four different categories did not occur in a way that was consistent with the progression of successive instars. For example, differences between mean bodylengths of samples from each of the four categories occurred at every larval instar, but adults from each treatment were approximately the same length. Similarly embryo numbers showed significant differences between treatments in adults and in all instar stadia except the fourth. Where differences between samples from the four categories were found, the effect of whether the mothers had been winged or wingless was more important than the effect of day-length (tables 4»39, 4-.40, 4*41 )• At birth, individuals from winged mothers were smaller, had fewer embryos and had smaller embryos than those from wingless mothers. Their development appears to have lagged behind that of the individuals from wingless mothers, at least for the first two or three instars. Later in their development, however, their mean body lengths and embryo sizes approached, and in some cases overtook, those of the individuals from wingless mothers. Embryo numbers of individuals from winged mothers remained, however, lower than those of individuals from wingless mothers, except at fourth instar. Most of the variation between samples from the four treatments can, therefore, be attributed to a slight lagging behind in the development of those individuals which came from winged mothers. Therefore, although short day-length (or, more correctly, long night-length) is the factor responsible for the production of sexuparae, and consequently, sexuals, it does not appear to have a consistent effect on bodylength, embryo number or size of largest embryos, in this species. The apparent effects of day-length on embryo number of adults (table 4»39) may have been due to the development of individuals under short-day conditions lagging behind that of those from long-day conditions, as the development of the former would probably have been slowed down by the lower temperatures which they experienced. Inconsistencies in the data may have been caused by a lack of uniformity in the nutritional quality of the leaves on which the aphids were feeding, perhaps contributing further to the development of individuals from particular categories lagging behind that of those from other categories. Myzus persicae Under short-day conditions, all the progeny of wingless mothers of M. persicae grew into winged adults (gynoparae), which in turn gave birth exclusively to oviparae during the first few days after reaching adulthood. Data concerning the successive instars of the Essex clone are summarized in table 4*42.

Table 4*42 Mean body lengths, embryo numbers and sizes of largest embryos for successive instars of gynoparae of M. persicae.

INSTAR N MEAN BODY EMBRYO NO. EMBRYOS SIZE LGST EMBRYO LENGTH mm NO. MEAN WITH EYES MEAN um MAX um i + o i-i +/- Cl MEAN +/- Cl +/- Cl I 10 0.73 0.03 20.1 1.1 00.0 — 119 4 125

II 10 1.35 0.06 26.5 2.6 00.0 — 189 18 219

III 10 1.17 0.08 29.0 2.1 00.0 — 242 24 312 IV 10 1.70 0.13 40.9 1 .0 1.8 1.0 403 21 422 A 10 1.98 0.06 37.1 2.0 9.0 0.8 655 29 734

However, under long-day conditions, only wingless progeny were produced. Various methods of inducing the production of winged virginoparae under long-day conditions were attempted, including crowding of the adults, which Awram (1968) found was the most important cause of alata production in this species, and manipulation of the temperature regime, but none met with success. This problem was also encountered by Searle & Mittler (1982). Therefore, although complete sets of data were obtained concerning body length, ovariole and embryo number and sizes of largest embryos for successive instars of gynoparae, data for winged virginoparae from long-day conditions, for comparative purposes, were not obtained. During the course of the systematic survey (section 4*1)» a colony of M. persicae with plenty of winged virginoparae was found in Wisley Gardens, Surrey. Data concerning the reproduction of adult alatae from this colony were therefore compared with those for the gynoparae from the Essex clone,using Student’s T-tests. Significant differences between the two samples are summarized in table 4*43 (overleaf). Table 4*43 Mean body lengths, embryo numbers and sizes of largest embryos of field-collected winged adults compared with those of gynoparae induced under short-day conditions

VARIABLE MEAN T (18 D.F.) LEVEL OF LONG DAY SHORT DAY SIGNIFICANCE BODY LENGTH 2.05 mm 1 .98 mm 2.22 P<0.0$ EMBRYO NUMBER 43.7 37.1 3.40 P<0.01 NO. EMBRYOS WITH EYES 15.1 9.0 6.12 P<0.001 SIZE OF LGST EMBRYO 684 pm 655 pm 0.95

Field-collected alatae were, on average, slightly longer and had a greater total number of embryos and more embryos with pigmented eyes than gynoparae. The numbers of embryos in the successive instars of gynoparae continued to increase until fourth instar, but decreased significantly (P<0.01) between fourth instar and adult. This decrease was due to the gradual absorption of the oldest embryos, i.e. those which had been ovulated first. Absorption of the oldest embryos began to be discernible in dissected aphids at the second instar, and continued into adulthood. Between fourth instar and adulthood, some of these embryos were completely absorbed, resulting in the drop in mean embryo number. The ovarioles of a typical third instar presumptive gynopara are shown in fig. 4 *1 4 * In addition to absorption of the first-ovulated embryos, younger embryos ceased developing at a very early stage. Thus the sequence of embryonic development in most ovarioles was for the first-ovulated embryo to be absorbed before the gynopara reached adulthood, the second-ovulated embryo to develop normally, becoming an embryonic ovipara with pigmented eyes, and those embryos subseqently ovulated to be absorbed. One ovariole of an adult gynopara of M. persicae is shown in fig. 4*15.

DISCUSSION: Acyrthosiphon pisum In the present work, all sexuparae of A. pisum were wingless. White (1946) also found that a higher proportion of apterae were produced under short-day conditions than under long-day conditions in A. pisum. Young (1972) described the suppression of alata production in monoecious aphids under short-day conditions as "ecologically sensible", as in such species Fig. 4.14 Ovarioles of third instar presumptive gynopara showing absorption'of first-ovulated embryos (indicated by arrows).

Fig. 4.15 One ovariole of adult gynopara, showing absorption or non-development of later ovulated embryos. 145 there is no need for a migration back to the primary host. Alate sexuparae do occur however in some monoecious species, e.g Thelaxes dryophila and Phylloxera glabra, although it does appear that in general, the sexuparae of monoecious species are wingless. Mackay, Reeleder & Lamb (1983) were able to induce the production of both winged and wingless sexuparae in A. pi sum. They monitored the offspring of both morphs and found that the winged forms gave birth almost exclusively to oviparae, rather like gynoparae in heteroecious species. They concluded that the production of these winged "gynoparae" conferred no obvious advantages on a monoecious species, and was in some ways disadvantageous. However, in my opinion, a period of dispersal before the birth of the sexual generation, particularly in species where the males are wingless, would increase the chances of cross-fertilization with other clones. It is also possible that the apparent production of gynoparae in a monoecious aphid may be a relic of a previously heteroecious life-cycle. The exclusively wingless sexuparae which were induced by short-day conditions in the present work, were apparently very similar to ordinary wingless virginoparae, and the few differences found between sexuparae and wingless virginoparae appear to have been due to the development of the former lagging slightly behind that of the latter. Sharma et al (1973) found, however, that maximum fecundities of A. pisum occurred at 16-hour photoperiods, and declined with diminishing photoperiod. Lamb & Pointing (1975) found that the rate of reproduction of A. pisum was not affected by different photoperiods. Reproductive rate in aphids is thought to be correlated with ovariole number (Wellings et al, 1980) and in the present study, ovariole numbers of A. pisum were found not to be affected by changing photoperiod, which could explain Lamb & Pointing’s findings. Orlando (1972a) described differences between the ovarioles of parthenogenetic first instar Megoura viciae from long-day conditions and those from short-day conditions. He found that germaria of those under short-days were larger, and the ovarioles contained fewer embryos than those under long days. In the present study, germaria were not measured, but no differences in embryo numbers were found between first instar parthenogenetic females from long- and short-day conditions. 146

Myzus persicae In the present study, most adult M. persicae gynoparae contained only one or two developed embryos per ovariole, those embryos which had been subsequently ovulated being in the process of absorption (fig. 4-.15). This was previously observed in the gynoparae of other Aphidinae (p.l15). Searle & Mittler (1982) also observed this phenomenon in M, persicae gynoparae, and found that whereas the ovarioles of alate virginoparae contained an average of four embryos of normal appearance, in successive stages of development, those of gynoparae contained a single developed embryo with (usually two) abnormal follicles occupying the remainder of the ovariole. Blackman (1975) stated that the occurrence of a small number of embryos in mature M. persicae gynoparae suggested that only one or two embryos were developing in each ovariole. This arrest of oogenesis he associated with the development of flight-related systems, consistent with the oogenesis-flight syndrome theory of Johnson (19&9). The fact that fewer embryos develop successfully in gynoparae than in winged virginoparae could be due to gynoparae having to fly further to find the primary host than winged virginoparae which are looking for another secondary host. This would also be consistent which the oogenesis-flight syndrome theory. Indeed, a comparison of winged virginoparae from the secondary host with gynoparae, using data from the systematic survey (section 4*1-2), shows that gynoparae had consistently fewer embryos than virginoparae (table 4»44)» These data are for total numbers of embryos, some of which may, in gynoparae, be destined for regression or non-development.

Table 4-.44- Embryo numbers, ovariole numbers and numbers of embryos per ovariole in winged virginoparae compared with those of gynoparae in heteroecious Aphidinae

SPECIES EMBRYO NO OVARIOLE NO NO EMBS/0V. WV(2) G WV(2) G W(2) G

Aphis salicariae 38 36 10 10 3.8 3.6 Brachycaudus helichrysi 18 11 8 8 2.3 1.4 Dysaphis crataegi 34 25 10 10 3.4 2.5 Hyzus persicae 44 37 10 10 4*4 3.7 Hyperomyzus lactucae 49 35 10 10 4.9 3.5 Cavariella aegopodii 39 28 10 10 3.9 2.8

Furthermore, the production of embryos determined as oviparae may tax the nutritional resources of the mother more than production of embryonic virginoparae, thus constraining the mother to produce fewer progeny. In addition to the non-development of those embryos ovulated after the normally developed embryos in each ovariole, absorption of those embryos ovulated before the one or two developed embryos was also found (fig. 4-.14-) • It may be that these embryos would have been determined as viviparae, and that following exposure of the mother to a critical number of short days, these embryos began to be absorbed, with the diversion of nutrients to the next ovulated embryo in each ovariole, determined as an ovipara. Absorption of their embryos by aphids was noted by Ward & Dixon (1982) in Megoura viciae which had been starved for five days. In this instance the smallest embryos in each ovariole were selectively absorbed. In A. pisum parasitized at fourth instar by Bphedrus plagiator all embryos degenerated except those few which had almost completed their development (Polaszek, 1986a). This degeneration of embryos may also have been absorption due to nutrient stress. It appears that gynoparae are able selectively to absorb embryos under conditions other than those of nutrient stress. Some older embryos are absorbed, and eggs ovulated later fail to develop. The appearance of the undeveloped follicles is rather similar to that of the single abortive eggs found by Crema (1979) in Megoura viciae. However, the positions of the abortive eggs observed by Crema coincided with those of eggs normally determined as males, and they are thought to occur as a consequence of the cytogenetic changes that occur during male determination. Further research is necessary to discover what is • the purpose of absorption or non-development of particular oocytes or embryos in gynoparae. The study of serial sections with light microscopy, and ultrastructural studies with transmission electron microscopy would perhaps help in answering this question. 4.2.3 THE EFFECTS OF PARASITIZATION ON THE FEMALE APHID REPRODUCTIVE SYSTEM

INTRODUCTION During the course of the systematic survey a number of samples collected were found to contain individuals 'which had been parasitized by Hymenoptera. Many of these individuals appeared externally to be healthy, but their reproductive systems had been substantially affected by parasitization. The reproductive systems of parasitized aphids had undergone considerable degeneration in many cases, although the rest of the internal anatomy appeared normal. A controlled investigation into the effects of parasitization by two species of aphidiine Braconidae was therefore carried out. The development of ichneumonid and braconid larvae within their hosts has been described for a number of host/parasitoid associations. These descriptions vary concerning the feeding methods of the larvae and the effects of their feeding on the organs of the host. Many studies of ichneumonoid parasitoids contain references to large, spherical, or less regularly shaped cells, found inside the haemocoel of their parasitized hosts. These cells have been given a number of names: teratocytes, trophocytes, macroblastomeres, enlarged trophamnion cells and giant cells; and various functions have been ascribed to them, e.g. as a source of nutrition for the developing larva (Tremblay, 1966; Vinson, 1970) or as a means of protecting the larva from the host’s immune response (Salt, 1971). It has been shown that in both species of parasitoid used in this study the teratocytes develop from the trophamnion which surrounds the parasitic larva in the early stages of its development (Ivanova-Kasas, 1956).

MATERIALS AND METHODS Cultures of Ephedrus plagiator (Nees) and Aphidius ervi Haliday were supplied by Dr. W. Powell of Rothamsted Experimental Station, Harpenden, Hertfordshire. The parasitoids were reared on pea aphids, Acyrthosiphon pisum, from the same clone described in section 4*2.2. Cultures of 100-200 first instar aphids, bom within 24 hours of each other were obtained as described in section 4*2.2. Because the ovarioles of alatae develop more slowly than those of apterae, inclusion of presumptive alatae among the host aphids would have increased the range of the measurements. To avoid this problem, alatae were selected as parents, because few of their progeny are alate. These were randomly divided into a control culture and an experimental culture, and the aphids put into excised leaf cages in groups of 20, and maintained at 20 1°C under an L16:D8 (long-day) regime. Three experiments were carried out. Two cultures of first instar pea aphids were subjected to parasitization by Bphedrus plagiator and Aphidius ervi respectively, and a third culture was first allowed to develop to fourth instar before being subjected to parasitization by E. plagiator. Four adult parasitoids were introduced into each of the experimental cages for six hours, while the unparasitized (control) aphids were left to develop normally. Samples of ten aphids were removed from control and parasitized cultures after 21+ hours, and thereafter at 21+ or 4-8 hour intervals, and immediately dissected in saline. The first two or three samples may have contained a few presumptive alatae as it is difficult to distinguish them from presumptive apterae until the third instar. However, this is unlikely to have affected the results, because differences between the ovarioles of the two morphs are not apparent until the third instar. When the two morphs became distinguishable from each other, only apterae were selected. Records were made of the length of the aphid’s body, the numbers of ovarioles and embryos, the number of embryos with pigmented eyes, and the lengths of the smallest and largest embryo. In some cases the dimensions of the parasitoid larvae and serosal cells were recorded. Differences between the data from control aphids and parasitized aphids were evaluated for each age-interval using Student1 s T - tests. Ovarioles, giant cells and parasitoid larvae were drawn at each stage of development and then preserved in Dubosq-Brasil Bouin fixative. Ovarioles were then washed in absolute alcohol, treated with methyl-benzoate celloidin for 21+ hours and mounted on slides in Canada balsam for examination and photography using phase-contrast illumination. From each sample to be dissected two or three individuals were preserved, undissected in Dubosq-Brasil Bouin fixative. After at least 21+ hours, these were then transferred into a series of acetone solutions of increasing concentrations and dried using critical point drying apparatus. Aphids were then partially dissected to reveal parasitoid larvae and coated with gold-palladium for examination under the scanning electron microscope. Photomicrographs were taken of parasitoid larvae and teratocytes at magnifications from 40X to 50,000X. 150

RESULTS A. First instar aphids i) The effects of parasitization on the reproductive system. Aphids which had been subjected to parasitization at first instar by either of the parasitoid species showed significant reductions in both embryo number (Figs. 4..l6a, 4-»l6d) and the size of the largest embryo (Figs. 4.16b, 4-«l6e) 24. hours after exposure to parasitoids. At this stage A. ervi hosts had on average 13% fewer embryos than the control aphids (P<0.001) and the largest embryos of parasitized aphids were 29% smaller than those of control aphids (P<0.0001). For E. plagiator these figures were 26% (P<0.0001) and 30% (P<0.0001). Differences in both embryo number and maximum embryo length between parasitized and unparasitized aphids increased as the experiment progressed. At no time did the maximum embryo length of aphids exposed to parasitoids at the first instar exceed 280 pm, whereas the maximum embryo length reached 1250 pm in the control aphids. Differences in the length of the body between parasitized and control aphids became significant after three days (P<0.0001) with E. plagiator (Fig. 4-»l6f) and after nine days (P<0.0001) with A.ervi (Fig. 4--16c). With both parasitoids the host’s reproductive system persisted intact, though in a diminished form, until the end of the experiment, 24.-4-8 hours before larval pupation (Fig. 4-»17). No disruption in the ovariole sheath was observed, and all embryos were still connected to each other nine days after the start of the experiment. All germaria persisted throughout this time and individuals dissected the day before pupation of the parasitoid contained the normal ovariole number of 14-. ii) Development of parasitoid larvae. In the first samples dissected, 24. hours after oviposition, parasitoid eggs were still very small and escaped detection in most cases. After three days the eggs had increased sufficiently in size so as to be easily located, and by this time the surface of the egg consisted of a serosa composed of closely packed spherical cells (Fig. 4-»18a). After four or five days the serosal cells had increased greatly in size (Fig. 4-«18b). At eclosion, five to six days after parasitization, these cells separated from ech other, at first in clumps and then individually (Fig. 4-*18c), and became distributed throughout the host’s haemocoel, increasing to five times their original diameter over the next five to six days (Fig 4-»18d). During their development larval parasitoids were not observed attacking the tissues of their hosts, although two individuals were observed eating the i f .f jg rkn ie cnrl aphids. control line:broken in A. E mb' y A A. pisum, A. 16 Changesinmeanembryo (Anumber &D), maximumembryo size (B &length(C body E) & and F), with following parasitization at the first instar byfirstinstar the following at parasitization oi lc p oltcr Doyi q r a si 1 1 2 o 1 1 on . ervi A. (A, B & C and and B (A,& C D E.plagiator as fe p oil ion oiio iilu fo po after Days D, E D,aphids& Solid line:F). parasitized 95^0 confidence limtky confidence 151 152

being parasitized at first instar by A. ervi. Embryos and germaria are still connected by the intact ovariole sheath, but embryos have degenerated considerably.

Fig. 4.18 A: Egg of E. plagiator three days after oviposition, clearly showing serosal cells. B: Five days old E . plagiator larva within the serosa C: Five days old E. plagiator larva hatching from the serosa the cells of which are beginning to disperse. D: Large serosal cell from A . pisum nine days after parasitization at first instar by A. ervi. dispersed and enlarged serosal cells. These cells decreased in number towards the end of the experiment. In many cases up to five larvae developed' to the eclosion stage in a single host, though only one of these was able to complete its development and pupate. Supernumerary larvae did not develop beyond the first instar, and dead supernumerary larvae were frequently found in the haemocoel. These may have died as a result of direct attack by the successfully developing larva, or by passive, chemical poisoning. The completion of larval, parasitoid development was signified by sudden'changes in the appearance of the host, aphids containing A. ervi pupae turning bronze in colour, and those parasitized by E. plagiator turning black. No aphids parasitized at the first instar reached adulthood or reproduced.

B. Fourth instar aphids i) The effects of parasitization on the reproductive system. Differences in embryo number between parasitized and unparasitized aphids became significant after three days (P<0.001), but ceased to be significant after nine days (Fig. 1.19a), since by this time the number of larvipositions by unparasitized aphids had resulted in a reduction in embryo number to the level of that of the parasitized aphids. After 21 hours there was no size difference between the largest embryos of parasitized aphids and those of the control aphids (Fig. 1.19b), and the ovaries of some fourth instar aphids contained apparently healthy embryos in positions corresponding to those of degenerating embryos in other ovarioles (Fig. 1.20). After three days all the embryos in parasitized aphids were degenerating and any healthy embryos, corresponding to those observed in the previous sample, had presumably been bom. First instar offspring were now found in all cages, which by now contained a few adult aphids. However, because most cages contained some unparasitized aphids, it was impossible to tell whether these first instars were the offspring of recently parasitized aphids or of the few unparasitized individuals among them. In the ovaries of parasitized aphids, those embryos in which the appendages would normally be visible had become rounded, their limbs had become indistinguishable, and their eye pigment had dispersed. Adult aphids parasitized at fourth instar were not significantly smaller than control aphids (Fig. 1.19c), and all underwent a final moult. As with first instar aphids, the ovaries remained intact, with no disruption in the ovariole sheath. The germaria were present until just before pupation of the parasitoid. Body length (mm) Length of lorgeil embryo _(ym| _ Embryo number ih9% ofdne iis i . pisum, inA. limits, confidence 95% with aiu mro size embryo maximum i. .9Cags nma eby ubr (A), number embryo mean in Changes 4.19 Fig. broken line: control aphids. control line: aphids, broken parasitized line: Solid plagiator. E. following parasitization at fourth instar by instar fourth at parasitization following 2 I - 123456789 as fe poroiilnafion offer Days (B) (B) n oy egh (C), length body and A. pisum parasitized at fourth instar fourth at parasitized pisum A. Fig. 4.20 One ovary of an adult an of ovary One 4.20 Fig. positions in other ovarioles are ovarioles other in positions embryos, while those in corresponding in those while embryos, instar, showing two apparently healthy apparently two showing instar, degenerating.

154

ii) Development of parasitoid larvae. Larval development closely followed that described above for parasitized first instars. The serosal cells became dispersed through the host’s haemocoel and increased five times in diameter over the nine days of the experiment. One aphid, dissected nine days after being subjected to parasitization, turned black in the 30 minutes between removal of the sample and dissection, signifying the completion of the development of the larva within. The pupating larva had consumed the entire contents of the body cavity apart from a small white mass, probably the meconium. Supernumerary larvae, killed at the eclosion stage, were presumably also consumed by the successful larva before its pupation. All other aphids from this sample still contained two intact, though reduced, ovaries with seven ovarioles each.

C. Scanning electron micrography of parasitized aphids Low-power photomicrographs showed parasitoid larvae present within the aphid's haemocoel, surrounded by teratocytes (fig. 4-.21). At increasing magnifications the surfaces of the teratocytes were seen to consist of ramifying microvilli (figs 4-.22-4.2k) •

DISCUSSION

i) The effects of parasitization on the reproductive systems of first and fourth instar aphids. With first instar aphids, there were significant reductions in the host's embryo number and maximum embryo length with both parasitoid species only 24 hours after parasitization, three to four days before hatching of the parasitoid larva. This suggests either a) that a substance which arrests embryonic development is injected with the egg by the mother parasitoid during oviposition, or b) that secretions from the egg itself cause these effects. Substances injected with the egg by the female parasitoid, which interfere with the reproductive activity of the host, are known to occur in other parasitic hymenoptera. The scelionid Telenomus heliothidis injects such a substance into the egg of its host Heliothis virescens (Strand et al., 1983), and the ichneumonid Campoletis sonorensis injects a virus into its host at oviposition (Edson et al., 1981). 156

Fig. 4.21 Ventral view of aphid abdomen, partially dissected, revealing a parasitoid larva lying within the haemocoel, surrounded by teratocytes. Fig. 4.22 Detail of 4.21, showing cuticular sculpturing of pare.sit.cid larva. Fig. 4.23 Teratocytes, xl,000 (lower picture= detail) Fig. 4.24 Detail of the surface of a single teratocyte (xl5,000) showing ramifying microvilli. The largest embryos of fourth instar aphids were apparently unaffected for the first two days following exposure to parasitoids, contrary to the findings for first instar aphids. At the moment of parasitization, the largest embryos were already at an advanced stage of development, with their appendages formed, and with their cuticle presumably thick enough to resist any parasitogenic effects. It is likely that these embryos completed their development and were bom, but that any advanced embryos which had not yet formed a thick enough cuticle had already begun to be absorbed. If the resistant embryos were all bom by the third day after parasitization, then one would expect a drastic reduction in maximum embryo length after this time, ■ since only the embryos susceptible from the moment of parasitization remained, and this is indeed what happened (Fig. 4-19b). The appearance of the degenerate embryos of parasitized fourth instar aphids corresponded closely to the descriptions by Soldan & Stary (1981), but, as was also the case with first instar aphids, their descriptions of degenerating ovarioles cannot be reconciled with my own observations. The persistence of the reproductive organs until at least 24- hours before larval pupation suggests they are not directly attacked until this time, contrary to the findings of Couchman & King (1977), working with Diaeretiella rapae and Brevicoryne brassicae t who put forward the hypothesis that "....[first instar] larvae migrate towards host embryos and apply their raouthparts to them. It seems likely that movements of the mandibles would disrupt the ovariole wall and membranes of the host embryos enabling the larvae to suck out the contents by pumping movements..." The present study confirms both the presence of giant cells, as described by a number of workers on this subject (Sluss & Leutenegger, 1968; Smith, 1952; Spencer, 1926; Tremblay, 1966; Tremblay & Iaccarino, 1971), and the fact that they originate from the serosa. Vinson (1970) and Tremblay & Iaccarino (1971) have suggested that these cells have a secretory phase followed by a trophic phase. The rapid increase in the size of the egg immediately after oviposition, coupled with the enlargement of the serosal cells, suggests that the trophic function may start immediately after oviposition. This would account for the significant effects of parasitization long before eclosion of the larval parasitoid. Both A. ervi and E. plagiator induced similar degenerative changes in their host’s embryos. I have observed similar effects in, for instance, the gynoparae of some Aphidinae which were absorbing some of their embryos. Thus comparable effects on the reproductive system can be generated in ways other than by parasitization. The degenerative changes in the reproductive system were confined to the embryos themselves, the gross structure of the ovariole 158 sheaths, germaria and terminal filaments being unaffected until just before pupation of the larval parasitoid. These discoveries, a) that there is an effect on the reproductive system of the host -within 24 hours of oviposition by the parasitoid and b) that only the embryos are affected, conflict with the findings of Soldan & Stary (1981) concerning Aphidius smithi and Acyrthosiphon pisum. They did not observe any parasitogenic effects until 4-5 days after infestation, after which time they observed disintegration of the "interfollicular zones", and the complete disappearance of the germarium. In the present study, embryonic degeneration was clearly an indirect effect of parasitization. Soldan & Stary (1981) consider that this degeneration could be due to a diversion of nutrients resulting in starvation of the embryos. It may however have resulted from interference with the aphid’s hormone levels, which Johnson (1958, 1959) showed to be the factor responsible for morphological changes in parasitized aphids, or it may have resulted from a combination of these two related factors.

ii) Development of parasitoid larvae. Both A. ervi and E. plagiator belong to the subfamily Aphidiinae within the ichneumonoid family Braconidae (van Achterberg, 1984) Vinson (1970), in a study of the development of the braconid Cardiochiles nigriceps inside its host Heliothis virescens, describes the occurrence of trophic cells within the parasitized host which are derived from the embryonic membrane of the parasitoid egg, and which increase in diameter from 14 /mi to 350 p m in ten days. After the egg hatches these cells disperse and enlarge, becoming the main food source for the developing larva. Smith (1952) and Sluss & Leutenegger (1968) found that this also occurred in two species of beetle parasitized by euphorine braconids, which are considered by some authors to be closely related to the Aphidiinae (Mackauer & Stary, 1967; Mackauer, 1968). According to Tremblay & Iaccarino (1971) the larvae of Aphidiinae and Euphorinae feed in the way described above by Vinson, and Schlinger & Hall (1960) described the larva of Praon palitans, an aphidiine parasitoid of the spotted alfalfa aphid, as feeding almost exclusively on enlarged "trophamnion" cells. The most favourable situations for parasites are those where they are able to complete their development inside the host with the minimum amount of interference with the host's biology. In this way food will continue to be provided and defence responses are less likely to be elicited. By obtaining nutrients from the host via the parasite’s serosal cells a direct onslaught on the host’s tissues is avoided, and the present study suggests that this is the feeding method for all larval instars except the last in both Bphedrus plagiator and Aphidius ervi. iii) Scanning electron micrography of parasitized aphids The appearance of the teratocyte surface covered with what appear to be microvilli (figs 4-.21-4..24) accords with the description of teratocyte ultrastructure in Aphidius matricariae (Tremblay & Iaccarino, 1971). The microvilli observed by these authors, however, appear to be smaller, perhaps a quarter of the diameter of the ones which I observed. These differences may be due to differences between the ages, and hence developmental stages, of the teratocytes, or to the different Aphidius species. The presence of microvilli, confirmed during this experiment, is in agreement with the absorptive function of teratocytes which follows a supposed secretory phase, proposed by Sluss & Leutnegger (1968) and Vinson (1970). 5. EXTERNAL GENITALIA IN FEMALE APHIDS

INTRODUCTION The external genitalia of female Aphididae are extremely reduced, consisting simply of a transverse ventral cleft, the genital opening, situated between the anal and genital plates (the latter also known as the subgenital plate) (Heie, 1980). Among the Aphidoidea, only Adelgidae are equipped with a true ovipositor, and this is similar in structure to that found in Aleyrodidae. Situated between the genital opening and the anal plate are the so-called rudimentary gonapophyses. These are small tubercles covered with short setae, and they vary in number from 0-4. according to species. In the past, the number of rudimentary gonapophyses has been used to characterize higher taxa of aphids, originally by van der Goot (1913). He divided aphids into 12 tribes, which approximate to the families of Bbmer (1952) or the subfamilies of Eastop (1977), and recorded the number of rudimentary gonapophyses for each tribe. Ilharco (1966) referred to the importance of the numbers of rudimentary gonapophyses as a character which can be used to aid the seperation of Callaphidinae (=Drepanosiphinae) into a number of tribes. Hille Ris Lambers (1968) also discussed this character when referring to the systematic position of the unusual genus Neuquenaphis. In his reclassification of the higher taxonomic categories of the Aphidoidea, Heie (1980) drew attention to the number of rudimentary gonapophyses as a character which, if taken into consideration, leads to the removal of the genus Drepanosiphum from Callaphidinae sensu lato.

METHODS During the dissection of Lachnus roboris virginoparae, as part of the survey, all individuals were found to be equipped with four processes projecting below the genital opening, two median processes and two larger lateral processes. These appeared to be associated with the rudimentary gonapophyses. The presence or absence of these genital processes was investigated in other Lachninae, and then in a number of species representative of the other aphid subfamilies. Using a Cambridge S180 stereoscan a total of 18 species of Aphidoidea were examined. The presence of genital processes, their number (where present), and the number of rudimentary gonapophyses were recorded. Wherever possible, the alate virginoparous morph was studied, as this perhaps represents the most morphologically primitive morph. The genital processes were found not to be visible in slide preparations, apart from in some Lachninae. Numbers of rudimentary gonapophyses were, however, recorded from slide preparations in a further 30 species. Where possible four virginoparae of each species were examined and in some cases oviparae were also examined. •: Oviparae of 4-5 species representative of all the subfamilies of Aphidoidea were examined for the presence of an ovipositor.

RESULTS Numbers of genital, processes or rudimentary gonapophyses in the Aphidoidea.

LACHNINAE Species belonging to this subfamily had the most well-developed genital processes. Two large lateral processes and two smaller median processes were found in Tuberolachnus salignus (fig..5.1), Lachnus roboris (fig. 5.2) and Maculolachnus submacula (fig. 5»3)» Four similar genital processes were also visible in slide preparations of Stomaphis quercus and Longistigma caryae. All individuals with four genital processes also had four well-defined rudimentary gonapophyses lying slightly posterior to the processes. No Cinara or Schizolachnus species were examined for the presence of genital processes, but slide preparations revealed three rudimentary gonapophyses in both genera, as documented by previous authors. Four genital processes, with the middle pair partialy fused, appeared to be present in a reduced form in Bulachnus rileyi (fig. 5.-4) • Four rudimentary gonapophyses were seen in slide preparations, although in some individuals the central pair appeared as a single gonapophysis. Essigiella knowltoni had four rudimentary gonapophyses. According to van der Goot (1913) all lachninae have three rudimentary gonapophyses.

PTEROCOMMATINAE A single species Pterocomma rufipes was studied. Three genital processes were present (figs 5»5, 5•6), a single median process and two lateral processes. As in the Lachninae, these lay anterior to the three rudimentary gonapophyses, but were not as prominent.

APHIDINAE A single species, Aphis fabae was studied. Three genital processes and rudimentary gonapophyses were found (fig. 5.7), arranged very similarly to those found in Pterocomma rufipes. Van der Goot (1913) recorded Aphidinae as having three rudimentary gonapophyses. 30HM Figs 5.1-5.7 Scanning electron micrographs of the genital area in winged female aphids.

5.1 Tuberolachnus salignus 5.2 Lachnus roboris 5.3 Maculolachnus submacula 5.4 Eulachnus rileyi 5.5 Pterocomma rufipes 5.6 Pterocomma rufipes 5.7 Aphis fabae 1 6 3

GREENIDEINAE Two species, Greenidea ficicola and Greenideoida elongata were studied from slide preparations. Both had four rudimentary gonapophyses.

ANOMALAPHIDINAE Two species, Anomalaphis comperei and Schoutedenia lutea, were studied from slide material. No rudimentary gonapophyses were found in Anomalaphis. Five Schoutedenia specimens had three rudimentary gonapophyses,and one appeared to have only two.

CHAITOPHORINAE Stereoscans of Periphyllus testudinacea showed that genital processes were absent. All individuals of this species, as well as specimens of Chaitophorus and Sipha had four rudimentary gonapophyses, although in some individuals the central pair were partially fused.

DREPANOSIPHINAE Three species were studied with the stereoscan, Drepanosiphum platanoidist Myzocallis castanicola (fig. 5«8) and Therioaphis ononidis (fig. 5.9)* Genital processes were not found in any of these species. Numbers of rudimentary gonapophyses were recorded from these and from a further 10 species. Crypturaphis gras s ii t Antalus albatus, Neophyllaphis michelbacheri, Neuquenaphis papillata, Oestlundiella flava, Tamalia coweni, Paoliella papillata and Sensoriaphis nothofagi all had four rudimentary gonapophyses. Drepanosiphum platanoidis had three rudimentary gonapophyses. Euceraphis betulae, Myzocallis castanicola and Therioaphis ononidis all had two rudimentary gonapophyses. However, the number of rudimentary gonapophyses was found to vary in some of the species. For example, oviparae of Crypturaphis grassii had only three rudimentary gonapophyses, the median ones presumably having become fused. One Oestlundiella flava alata had four rudimentary gonapophyses, whereas in another these had all apparently become fused to form a single gonapophysis. Oviparae of this species had either one or three gonapophyses. Similarly, in Therioaphis some individuals had a single gonapophysis (e.g. fig. 5»8) whereas the remainder had two. Paoliella carpinganae oviparae had a single gonapophysis. According to van der Goot (1913) > Callipterina (=Drepanosiphinae as used here, minus the genus Drepanosiphum) have two rudimentary gonapophyses. In

Heie's reclassification of the Drepanosiphinae and Chaitophorinae (1980, 1982), a third subfamily, Phyllaphidinae, is recognised, and the three subfamilies are seperated partly on the number of rudimentary gonapophyses. Heie characterized the Drepanosiphinae as having three gonapophyses. However, of the six tribes which he included in this subfamily only one, Drepanosiphini, contains species with three gonapophyses. He pointed out, however, that the middle rudimentary gonapophysis may be subdivided in some non-Scandinavian species of Drepanosiphinae.

PHLEOMIZINAE, MINDARINAE, THELAXINAE Phleomyzus passerinii, Mindarus abietinus Glyphina betulae and Thelaxes dryophila are all without genital processes and have two rudimentary gonapophyses (figs 5.10, 5.11 > 5.12). The same number was found in Kurisakia onigurumii, which may be closely related to Thelaxes. In certain Mindarus individuals there were one or two groups of hair bearing tubercles between the gonapophyses. Where there was only one group, as in a single individual of this species from India, it appeared as a third, median rudimentary gonapophysis, very similar to the two lateral ones. Van der Goot (1913) recorded Mindarinae as having (?)'’ rudimentary gonapophyses.

ANOECIINAE No genital processes were found in Anoecia corai (fig. 5.13). The number of rudimentary gonapophyses was difficult to determine, certain individuals appearing to posess two (e.g. fig. 5.13)» and others having simply an irregular row of hairs which in some cases could be interpreted as consisting of four gonapophyses. Aiceona .japonica alatae had three well-defined rudimentary gonapophyses.

HORMAPHIDINAE Three species were studied from slide preparations, Euthoracaphis umbellulariae, Hammamelistes spinosus and Hormaphis betulinus. All had two rudimentary gonapophyses.

PEMPHIGINAE Genital processes were not found in Eriosoma ulmi alatae (fig. 5.1-4). Numbers of rudimentary gonapophyses were difficult to determine in most pemphigine specimens. They were absent in Eriosoma ulnri. Some specimens of Pemphigus bursarius appeared to have two, whereas in others the number was impossible to determine, although the area between the subgenital and anal plates was clearly visible. Pachypappa populi and Thecabius affinis alatae had three rudimentary gonapophyses, though in certain specimens the middle one clearly consisted of two fused gonapophyses, and so such specimens could be interpreted as having four. Prociphilus fraxini had three well defined rudimentary gonapophyses, with the median one well developed and heavily sclerotized.

Summary: Table 5.1 Numbers of genital processes and rudimentary gonapophyses in virginoparous females of 48 genera of Aphididae.

SUBFAMILY GENUS NO. OF GENITAL NO. OF RUDIMENTARY PROCESSES GONAPOPHYSES

Lachninae Lachnus 4 4 Longistigma 4 4 Maculolachnus 4 4 Stomaphis 4 4 Tuberolachnus 4 4 Essigiella 4 Eulachnus 3/4 3/4 Cinara 3 Schizolachnus 3 Pterocommatinae Pterocomma 3 3 Aphidinae Aphis 3 3 Greenideinae Greenidea 4 Greenideoida 4 Anomalaphidinae Anomalaphis 0(?) Schoutedenia 3/2 Chaitophorinae Periphyllus 0 4 Chaitophorus 4 Sipha 4/3 Drepanosiphinae An talus 4 Crypturaphis 4/3 Drepanosiphum 0 3 Eucallipterus 2 (also with 3) Euceraphis 2 Israelaphis 3/4 (Ilharco, 1966) Myzoca.ll i s 0 2 Neuquanaphis 4 Neophy 13 aphis 4 Qestlundiella 4 (also 1 or 3) Paoliella 4 (also 1 or 3) Phyllaphis 2 Sensoriaphis 4 Therioaphis 0 2 (also 1 or 3) 1 6 7

Table 5.1 (Continued) SUBFAMILY GENUS NO. OF GENITAL NO. OF RUDIMENTARY PROCESSES GONAPOPHYSES Phleomyzinae Phleomyzus 0 2 Mindarinae Mindarus 0 2 (also 3 or 4-) Thelaxinae Glyphina 0 2 Thelaxes 0 2 Kurisakia 2 Anoeciinae Anoecia 0 ?0,2,4 Aiceona 3 Hormaphidinae Euthoracaphi s 2/1 Hammamelistes 2 Hormaphis 2 Pemphiginae Eriosoma 0 0 Forda ?2 Pachypappa 3 (?4) Pemphigus ?2 Prociphilus 3 Thecabius 3 (?4)

The presence of an ovipositor or ovipositor--like structure in Aphidoidea

The oviparae of the following 4-5species of Aphidoidea were examined for the presence of an ovipositor or similar structure: Adelges cooleyi, Anoecia comi, A. zimitzi, Aphis fabae, Boemerina occidentalis, Cervaphis quercus t Cinara pini Crypturaphis grassii, Drepanosiphoniella aceris, Drepanosiphum platanoidis, Buca.1 ~1 i pterus tiliae, Eulachnus agilis, Glyphina betulae, Greenideoida elongata, Lachnus roboris, Lizerius brasiliensis, Monaphis antenna ta, Mindarus abietinus, M._obliquus, Moritziel 1 a corticalis t Neophyllaphis brimblecombei , Neuquenaphis papil1ata, N. schlingeri, Oestlundiella flava, Paoliella browni, P._harteni, P. papilla ta, Paratrichosiphum tattakanum, Pemphigus bursarius^P. spyro thecae, Phleomyzus passerinii, Phyllaphis fagi, Phylloxera glabra, Pineus orientalis, Pterocalli.s alni, Pterocomma salicis, Sensoriaphis furcifera S. nothofagi, Stegophylla essigi, Stomaphis yanonis, Subsaltusaphis paniceae, S. rossneri, Thelaxes dryophila, Therioaphis ononidis t Thripsaphis brevicomis # An ovipositor, or ovipositor-like structure was found in only five of the species examined, the two Adelgidae and three Paoliella species. Oviparae of Adelges cooleyi and Pineus orientalis had a true ovipositor resembling the ovipositor of parthenogenetic, oviparous adelgid morphs. In Mindarus abietinus and M. obliquus a highly sclerotized structure was found which appeared to be mainly internal, but with two pointed lateral extensions apparently projecting externally as an ovipositor-like structure. Illustrations of oviparae of M. abietinus by Niisslin (1900) and of M. japonicus by Sorin (1962) both depict this structure, but in neither description is is clear to what extent it is an internal or an external structure. Nusslin described it as the "chitinspange" or "chitin-clasp". Stereoscan photographs were therefore taken of this structure (fig. 5.15) and it appears from these that the sclerotized structure is not in fact an ovipositor-like organ. Slide preparations of Paoliella browni, P. harteni and P. papillata oviparae revealed the presence of a small, highly sclerotized and pointed structure, situated anterior to the rudimentary gonapophyses (fig. 5.16). This structure was not found in males or virginoparae. The appearance of the structure suggests that it may be used for pricking the substrate of the host-plant before oviposition. However, it would be necessary to examine whole specimens, possibly with SEM, in order to get any real indication of what its true purpose might be. A single ovipara of Lizerius brasiliensis also appeared to be equipped with such a structure, but in other oviparae of the same species its presence or absence could not be stated with any certainty. 1 6 9

Fig. 5.15 Scanning electron micrograph of the genital region in a Mindarus abietinus ovipara. (See text)

Fig. 5.16 The last few abdominal segments of a Paoliella harteni ovipara (from a slide preparation). 6 . COMPARATIVE ANATOMY OF THE MALE APHID REPRODUCTIVE SYSTEM

6.1 INTRODUCTION

Comparative studies of male genitalia have formed the bases of taxonomic and phylogenetic studies of many groups of insects. The Aphidoidea, however, are polymorphic, and in almost all species males occur only during a limited period of the year. In addition, within the sexual generation, most species appear to have a sex-ratio strongly biased towards oviparae. The comparative rarity of the male morph, compared with the abundant viviparous morphs, has meant that both the taxonomy and the construction of higher classifications of Aphidoidea have generally been based on the morphology of the females. The currently accepted systems of higher classification of the Aphidoidea, i.e. those of Eastop (1977), Heie (1980) and Remaudiere & Stroyan (1981), while possessing some stability and predictive value, contain a number of subfamilies of uncertain position, e.g. Thelaxidae, Mindaridae, Anoeciidae and Phleomyzidae, none of which contains more than two genera. In addition, the large subfamily Drepanosiphinae (=Drepanosiphidae of Heie) is impossible to characterize to the exclusion of species from other subfamilies, and is almost certainly polyphyletic. Ideally, a higher classification reflects the course of evolution of the group concerned. In order to attempt to reconstruct the phylogeny of the group it is necessary to assess characters as either primitive or derived, based on the putative morphology of the hypothetical common ancestor of the group and that of the its sister group. Various attempts have been made to reconstruct the phylogeny of the Aphidoidea, using characters including presence of a 3-lensed eye in the larva (Hill e Ris Lambers, 1961), parasitoid data (Mackauer, 1965) and combinations of morphological and life-cycle data (Baker, 1920; Takahashi, 1931; Heie, 1967). There is little agreement between any of these reconstructions, although most authors placed Pemphiginae close to Hormaphidinae, far away from Lachninae. At present there is very little evidence for singling out a particular genus or subfamily as representing the most primitive state. The Steraorrhyncha comprises four superfamilies, and the problems involved in deducing their relationships have been pointed out by Hennig (1981). However, Schlee (1969a, 1969b) in his careful study of both the external and internal morphology of all the groups of Steraorrhyncha, concluded that coccids are the sister group of aphids, and that these two groups together, "Aphidiformes”, are the sister group of the "Psylliformes" (=psyllids + whiteflies). He also pointed out (1969a) similarities between the wing venation of the margarodid Sphaeraspis priaskaenis (Coccoidea) and that of Aphididae. In the present study, the male internal genitalia of 4-5 aphid species in 33 genera were studied, representing both the Adelgidae and Phylloxeridae and all but one of the thirteen subfamilies of Aphididae. The exception was Greenideinae for which only slide mounted males were available.The external genitalia of 4-0 species were also examined and figured, including representatives of males of all the subfamilies. Internal genitalia were mostly studied by dissection, occasionally by serial sectioning, and external genitalia by observation with both light microscopy and scanning electron microscopy, (see section 2). Because of the rarity of the male morph, sample sizes consisted of six individuals or fewer in most cases (in the following section, sample sizes are included under each species). Some species were studied from a single specimen only. The external genitalia of eight species were studied from slide mounted specimens. The purpose of these surveys was to see if any characters could be found which might be of use in deducing the phylogenetic relationships between the taxa, and, subsequently, in the construction of a more stable higher classification of the Aphidoidea. The external genitalia of various species representing the other three superfamilies of Sternorrhyncha were studied from preserved material and from a few records in the literature. The internal genitalia of three species of Coccoidea and one species of Aleyrodoidea were also examined for any similarities with those of Aphidoidea. Descriptions of the internal genitalia of other hemipteran groups, obtained from the literature, included: Heteroptera: Rhodnius (Bonhag & Wick, 1953); Peloriidae (Pendergrast, 1962); Auchenorrhyncha: Euscelis (Kunze, 1959)» Alnetoidea (Bednarczyk, 1983); Psylloidea: Psylla (Glowacka & Klimaszewski, 1969). In the following sections, the general morphologies of the male genitalia of Aphidoidea are first described (section 6.2), and individual species are then studied systematically (6.3)• The morphologies of the male genitalia of other Hemiptera are then compared with those of Aphidoidea (6.4-). This is followed by a discussion of which genitalia characters should be interpreted as primitive and which are derived, and how this affects any attempts to deduce the relationships between the taxa involved (6.5). The bearing which the present work has on the higher classification of the Aphidoidea is discussed in section 7. 6.2 MALE GENITALIA

6.2.1 External genitalia The external genitalia (figs 6.1, 6.2) in the form most commonly occuring in alate male aphids consist of a pair of sclerotized, setose claspers (also referred to in the literature as parameres or operculae) ■which are probably used in gripping the female during copulation, a pair of valves and an unsclerotized aedeagus. The claspers vary in form between subfamilies, or they may be absent. They normally remain visible in slide-mounted aphids. In a few genera the claspers are reduced or little developed, but the sclerotized region in front of each clasper is produced forwards, forming a pair of finger-like process (e.g. figs 6.7, 6.31). I have termed these structures the anterior genital processes. The valves lie posterior to the claspers (terminology of Balbiani, 1869). These are absent in some genera, but where they are present, they vary little in form between both genera and subfamilies. They are usually weakly sclerotized for the most part, and take part in copulation and in everting the aedeagus and maintaining it in position. While the aedeagus is withdrawn the valves cover the genital opening. The valves are most strongly sclerotized at their articulations with the rest of the genital apparatus, and the remaining weakly sclerotized part is often not visible in slide-mounted specimens. In many species the distal ends of the valves have a small number of circular pits, which may have a sensory function. In most species the valves remain in contact with each other along their posterior margins at all times. The aedeagus is entirely unsclerotized, but in many species the basal part, or conjunctiva, can be differentiated from the distal part, or vesica (terminology from Bonhag and Wick, 1953). The aedeagus is normally withdrawn within the body, and eversed only during copulation. According to Balbiani (1869), when the aedeagus is everted, the ejaculatory duct and the ducts of the accessory glands and vasa deferentia are pulled into it as a result. The shape of the aedeagus varies between species, but is normally not visible in slide preparations. 173

1 0 0 y r )

Figs 6.1, 6.2. Megoura viciae, scanning electron micrographs of male external genitalia. A: Claspers B: Valves C: Subgenital plate D: Anal plate E: Cauda F: Aedeagus

Fig. 6.3 M. viciae- internal genitalia. 6.2.2 Internal genitalia The internal reproductive systems of male aphids (Fig. 6.3) consist typically of a pair of testes each consisting of follicles the number of which varies between species. The two testes may, however, be fused and so appear as a single organ. Vasa efferentia lead out of the testes, becoming vasa deferentia which lead into the ejaculatory duct. These are usually considerably broader at their junctions with the testes, and may function in such cases as seminal vesicles. A pair of accessory glands also opens into the ejaculatory duct, although these are absent in some taxa. The size and shape of the testis follicles and accessory glands varies according to the age of the adult male. In general as the adult male gets older, the testis follicles become smaller, and the accessory glands become slightly larger. Too much importance, therefore, should not be attached to their dimensions when making comparisons. However, in a few cases, e.g. among the Chaitophorinae, the accessory glands are clearly reduced, irrespective of the age of the adult aphid.

6.3 SYSTEMATIC SURVEY

APHIDIDAE: LACHNINAE The external genitalia of five species from four genera and the internal genitalia of six species from the same four genera were studied.

Lachnus roboris (males winged) External genitalia (4 specimens; fig. 6.4a): Claspers present but rather small, separate. Valves present. Aedeagus curved backwards, conjunctiva and vesica clearly distinct. Internal genitalia (4 specimens; fig. 6.4b): Testes fused together, total of 14 testis follicles, seven per testis. Accessory glands absent. Glowacka et al (1974b) found both this species and L. longirostris to have seven follicles per testis, and accessory glands absent. However, according to Glowacka et al, the testes are separate in L. roboris but fused in L. longirostris. Serial sections through L. roboris were therefore carried out, and these showed that the ends of the vasa efferentia are fused, sharing a common lumen where they join the testes (fig. 6.5).

Maculolachnus submacula (males wingless) External genitalia (3 specimens; fig. 6.6a): Claspers present but reduced. Sclerotized region, anterior to and between the claspers, strongly 175

Fused, testes

0.1 mm

3K

Lachnus roboris internal genitalia Fig. 6.4-b 1 76

Fig. 6.5 Serial sections through male reproductive system of Lachnus roboris showing fusion of vasa efferentia. A: Vasa efferentia clearly separate. 3: Beginning of the junction between the two vasa efferentia. Spermatozoa can be seen passing from one canal to the other. C: Vasa efferentia fused, joining up with the ducts from the testis follicles. (Stained with haemotoxylin & eosin and photographed with interference contrast microscopy) 177

0.1 mm

Fig. 0.6a Maculolacnnus submacula external genitalia

Fig. 6.6b Maculolachnus submacula internal genitalia produced forwards, forming a single pointed structure. Valves present, most strongly sclerotized along their posterior margins. Aedeagus extruding perpendicular to abdomen, curving backwards for the distal quarter of its length . Internal genitalia (4 specimens; fig. 6.6b): Testes fused, total of 12 testis follicles, six per testis. Many of the testis follicles are rather irregularly shaped. In three out of four specimens, two follicles (one from each testis) were conspicuously smaller than the remaining ten. Accessory glands absent. Wojciechowski (1977) studied the internal genitalia of this species by serial sectioning, and the present study confirms his findings.

Stomaphis quercus (males wingless, examined from slide preparations only) External genitalia (12 specimens; figs 6.7a, 6.7b): The sclerotized external male genitalia of this genus are extremely unusual. Claspers undeveloped. Large, paired anterior genital processes present, these with short hairs along their entire length (c.f. S. yanonis, Cinara, Schoutedenia, Greenidea). Valves present, separate (i.e. not in contact along their posterior margins), finger-like in form, unlike the flattened valves of other genera; covered with longish hairs along their entire length. Aedeagus (visible in one specimen) short and straight. Internal genitalia: The male internal genitalia of Stomaphis querceus consist of fused testes of four follicles each (Wojciechowski, 1977).

Stomaphis yanonis (males wingless, examined from slide preparations only) External genitalia (4- specimens; fig. 6.7c): Very similar to S. quercus, but anterior genital processes much longer and without hairs distally, these covered instead with fine denticles. Valves fused, free only at their tips, differing from the valves of other aphid genera to an even greater extent than those of S. quercus. In this species the form and sculpturing of the anterior genital processes are reminiscent of those of the "claspers" of male Pemphigus (fig. 6.54b) and the ends of the "penis sheath" of Margarodes (fig. 6.60f).

Cinara pini (males wingless) External genitalia (5 specimens; fig. 6.8a): Claspers present but reduced. Paired short anterior genital processes present, resembling those of C. boemeri (fig. 6.8b). Valves present, sclerotized along their posterior margins. Aedeagus hook-shaped, curving sharply upwards for the final quarter of its length. 179

Anterior genital processes Anterior genital processes Fig. 6.7bs. quercus external genitalia Fig. 6.7c S. yanonis external genitalia 180

Fig. 6.8a Cinara nini external genitalia

Fig. 6.8c C. pini internal genitalia 181 final quarter of its length. Internal genitalia (5 specimens; fig. 6.8c): Testes fused, total of 10 testis lobes, five per testis, constant for- five specimens. Accessory glands absent. Buckton (1882) described and figured an aphid testis, of a species he called Lachnus pinicola, which also consisted of five follicles. The species may have been Cinara pinicola, or is perhaps more likely to have been C. pini. The species C. nuda, possibly also C. pini (Eastop, personal communication), was found to have separate testes of five follicles each (Glowacka et al, 1974-b).

Cinara stroyani (males wingless) External genitalia (2 specimens; figs 6.9a, 6.9b, 6.9c): Structure of claspers and valves as for C. pini. Anterior genital processes present, longer than those of C. pini. Aedeagus extending forwards from the abdomen at an approximately 4-5° angle. Internal genitalia (3 specimens; fig. 6.9d): Specimens with 8, 9 and 10 follicles respectively. Testis fused. Testis follicles varying greatly in size. Accessory glands absent. A species called first C. stroyani by Wojciechowski (1977) and then C. viridescens (a synonym of C. pilicomis) by Szelegiewicz & Wo jciechowski (198$) also had fused testes of four follicles each.

The male genitalia of a further three Cinara species have been studied by other authors. C. bogdanowi (=€. pruinosa) had fused testes of three follicles each, C. juniperi and C. mordwilkoi both had fused testes of four follicles each (Wojciechowski, 1977).

Eulachnus rileyi (males winged) External genitalia (1 specimen; fig. 6.10a): Claspers extremely reduced, virtually undetectable, but well-developed anterior genital processes present. Aedeagus straight, very short, projecting vertically downwards from the abdomen. Internal genitalia (2 specimens; fig. 6.10b): Testes separate, with two follicles each. Accessory glands absent.

Eulachnus agilis (Males winged) Internal genitalia (1 specimen; fig. 6.10c): As for E. rileyi. In both species of Eulachnus, the testes were clearly separated, with no indication even of accretion, although Glowacka et al (1974b) found the 1 8 2

0.1 nnn

6 '9 ^ PojVerior Vie-Vil Fused testes

0.1 mm

^5 t ' 9 <*‘ Vasa deferentia

Ejaculatory duct Anterior genital processes Fig. 6.9a, 6.9b, 6.9c Cinara stroyani external genitalia Fig. 6.9d C. stroyani internal genitalia 183

Fig. 6.10a Eulachnus agilis external genitalia

0.1 mm testes

Fig. 6.10b E. agilis internal genitalia Fig. 6.10c E. rileyi internal genitalia 184 testes to be fused in E. agilis, and Wo j ciechowski (1977) found them to be fused in E. rileyi. It seems unlikely, though possible, that both of these authors have wrongly interpreted their serial sections, so it may be that the fusion of the testes is a rather inconstant and unreliable character in this genus (cf. L. roboris, above).

Summary External genitalia The claspers in the Lachninae are small in most species examined, and are positioned laterally. In Eulachnus and Stomaphis they are either completely undeveloped, or have been lost, secondarily. This is in contrast to the situation in Aphidinae and Drepanosiphinae, where in most species examined the claspers are large, and positioned at the anterior end of the genital area. It may therefore be that the claspers are non-functional in Lachninae. Anterior genital processes are present in Cinara and Eulachnus, and are particularly well-developed in Stomaphis. It is possible that these extended regions of the genital apparatus perform the same function, presumably that of gripping the female, as the claspers do in other aphids. The single anterior genital process in Maculolachnus may represent the fusion of paired processes. Similar anterior genital processes are also present in other aphid subfamilies, and the nclaspersn of certain genera (e.g. Pemphigus) may be derived from homologous structures, and not from such true claspers as occur in Aphidinae and Drepanosiphinae. The various modifications of the sclerotized external genitalia are discussed in detail in section 6.5. Valves were present in all species studied. These are rather more setose than in other subfamilies, particularly in Stomaphis, where they are also unusually shaped. The shape of the aedeagus is very variable between the different genera examined, and also between two different species of Cinara. However, within each species the shape of the aedeagus varies very little.

Internal genitalia A striking characteristic of the internal genitalia of Lachninae is the complete absence of such accessory glands as are found in the other aphid subfamilies, first reported by Glowacka et al (1974b), and recorded for 14- species to date. The role of accessory glands in insect reproduction has been reviewed by Leopold (1976). In most insects they are large, paired structures, opening either into the vasa deferentia or directly into the ejaculatory duct. Their function is to produce various secretions which 185 protect and facilitate transfer of the gametes. In certain cases, the secretory products of the accessory glands have also been shown to be capable of inducing behavioural changes, and to stimulate development of the gonads. Like the Lachninae, some muscoid Diptera lack distinct accessory glands and their function is taken over by glandular areas incorporated into the wall of the ejaculatory duct (Leopold, 1976). According to Wojciechowski (1977) the function of the the accessory glands in Lachninae has been adopted by the cells of the internal layer of the vasa deferentia walls. This may also be case among certain species of Pemphiginae (q.v.).

PTEROCOMMATINAE Both the external and internal genitalia of two species of Pterocomma were examined.

Pterocomma populeum (males winged) External genitalia (2 specimens; fig. 6.11a): Claspers present, large, separate. Valves present. Aedeagus shaped like an inverted question mark. Internal genitalia (3 specimens; fig. 6.11b): Testes fused, 10 follicles, five per testis. Testis follicles long and tapering to a point. Accessory glands present, small.

Pterocomma salicis (males wingless) External genitalia (2 specimens; figs 6.12a, 6.12b): Claspers present, large, more widely separated than in P. populeum. Valves present. Aedeagus similar in shape to that of P. populeum though not as strongly recurved at the distal end, and tapering gradually to a point. Internal genitalia (5 specimens; fig. 6.12c): Testes fused, total number of follicles in the five specimens examined: 8, 8, 7, 7, 6. Testis follicles long and tapering to a point, as in P. populeum. Accessory glands present, small. Tannreuther (1907) recorded this species as having 10 testis follicles, five per testis. However, as stated in section 1.6.2, according to cytological studies by Blackman (1985) this was probably a misidentification, and this could explain the discrepancy between the range of follicle numbers found in the present study, and Tannreuther’s result. However, the follicle number does seem to be very variable in this species. If larger samples of some of the other species had been available, perhaps similar variation may also have been found. 1 8 6

Fig. 6.11a Pterocomma po-puleum external genitalia

Fig. 6.11b P. poDuleum internal genitalia 187

6.12c P« sali.cis uit/CrnsJ. ggni’fcsJjLcL 188

Summary The external genitalia of the Pterocommatinae studied differ from those of the Lachninae in having much larger claspers, which are placed further forward than those of Lachninae. Anterior genital processes, found in some lachninae, are absent. However, in P. salicis the claspers are wider apart than in P. populeum, with a sclerotized region between them, though this is not extended forwards. The internal genitalia differ most markedly from those of Lachninae in having discernible accessory glands. The testis follicles are long and tapering, like those of some Drepanosiphinae, and are also reminiscent of those of Psylla fusca (fig. 6.61a).

APHIDINAE Both the internal and external genitalia of 17 species in 11 genera were examined. Because of the great similarity between both the external and internal genitalia of most species examined, the results are summarised below, and any exceptional species are described in greater detail. External genitalia: The sclerotized external genitalia differed very little between species. The claspers were large, separate and positioned at the anterior end of the genital area. The only exception was Cavariella aegopodii in which the claspers were greatly reduced and positioned laterally (fig. 6.20). Valves were present in all species. The shape of the aedeagus was the main feature of the external genitalia which differed markedly between species, and this is therefore figured for all 17 species (figs 6.13 - 6.29). In all species, the conjunctiva of the aedeagus was distinguishable from the vesica. In all but two species the conjunctiva occupied a small part of the proximal part of the aedeagus, with the vesica occupying the remainder. In Dysaphis crataegi ssp. kunzei (fig. 6.27) and Amphorophora tuberculata (fig. 6.29), however, the conjunctiva was much more extensive. The size of the aedeagus relative to the rest of the body was similar in all species except for Longicaudus trirhodus (fig. 6.26), in which it was extremely small. Compared with the Lachninae, the external genitalia of Aphidinae are less specialised, having many features in common with those of Drepanosiphinae (q.v.), and differing little from the hypothetical plesiomorphic condition. The claspers are large in both Aphidinae and Pterocommatinae, whereas they tend to be reduced in the Lachninae. Anterior genital processes, found in some Lachninae, are absent 189

Fig. 6.15 A. farinosa external genitalia Fig. 6.16 A. nraeterita external genitalia 190

Fig. 6.19 M. oblonga external genitalia 191

Fig. 6.20 Cavariella aegopodii 1 9 2

Fig. 6.24 Pleotrichophorus sp. Fig. 6.25 Myzus nersicae external genitalia external genitalia

Fig. 6.26 Lonqicaudus trirhodus Fig. 6.27 Dysaphis crataegi external genitalia external genitalia

-RSrrtig. 6.28a 00 Megoura viciae . . external genitalia Fig. 6.29 ------Amnhorophora tuberculata external genitalia in Aphidinae. The sclerotized external genitalia of Cavariella aegopodii differ from those of the rest of the Aphidinae, forming an apparently more specialised condition. The aedeagus in the Aphidinae, as in the Lachninae, appears to come in a variety of shapes and sizes, though with little variation (albeit with small sample sizes) within each particular species. Of the species examined, the shape of the aedeagus was similar in Macrosiphoniella artemisiae and the closely related M. millefolii, differing from that of M, oblonga, in which the distal aedeagus was rather more curved. The aedeagus of Dysaphis crataegi resembled that of Amphorophora tuberculata, although these two species are not closely related. There was a large amount of variation in the shape of the aedeagus between different species of Aphis. In the three genera where more than one species was examined, i.e. Aphis, Macrosiphoniella and Macrosiphum, differences in the shape of the aedeagus between congeneric species were as great as differences between species from different genera. However, in order to properly evaluate differences within species and between closely related species, larger sample sizes would be necessary. Balbiani (1869) also described and figured the external genitalia of M. millef olii. There are some differences between his figures and my own, for example, the valves in his illustrations appear deeply pigmented and covered with setae. Of the 17 species of Aphidinae studied in the present survey none had valves which were pigmented throughout, and setae were only ever present at the proximal end of each valve. The shape of the extruded aedeagus in Balbiani' s drawing accords well with my own findings. Baker & Turner (1916) attempted to illustrate the external genitalia of Aphis pomi, but their drawing is of doubtful accuracy. Internal genitalia: In all 17 species examined the testes were fused and accessory glands were present. 13 of the 17 species had six testis follicles, three per testis. The remaining four species, with different numbers of testis follicles, did not share any unique taxonomic or life-cycle characteristics. The arrangement of the internal genitalia in all the aphidine species was approximately as for Megoura viciaes in fig. 6.3. Some characteristics of the male Aphidinae studied are summarised below (table 6.1). Table 6.1 Sample sizes, presence of wings and numbers of testis follicles in 17 species of Aphidinae.

SPECIES N Winged/Wingless No. testis follicles

Aphis epilobiaria 1 Winged 8, 4 per testis Aphis fabae 5 Winged 6, 3 per testis Aphis farinosa 1 Wingless 5 Aphis praeterita 2 Winged 6, 3 per testis Macrosiphoniella artemisiae 1 Winged 6, 3 per testis Macrosiphoniella millefolii 2 Winged 6, 3 per testis Macrosiphoniella oblonga 3 Wingless 6, 3 per testis Macrosiphum rosae 2 Winged 6, 3 per testis Macrosiphum albifrons 5 Winged 6, 3 per testis Cavariella aegopodiae 1 Winged 5 Hyperomyzus lactucae 1 Winged 7 Pleotrichophorus sp. 1 Alatiform 6, 3 per testis Ifyzus persicae 11 Winged 6, 3 per testis Longicaudus trirhodus 1 Winged 6, 3 per testis Dysaphis crataegi ssp kunzei 2 Winged 6, 3 per testis Megoura viciae 5 Winged 6, 3 per testis Amphorophora tuberculata 5 Alatiform 6, 3 per testis

The internal male reproductive systems of three of the above species have been studied previously by other workers. Macrosiphoniella millefolii was first studied by Balbiani (1869), then by Glowacka (1974a). Although all three studies were carried out without knowledge of the others, the results are all in agreement. Bochen et al (1975) also looked at M. artemisiae, and found the reproductive system to comprise two fused testes of three follicles each, as above. Balbiani (1869) examined and figured the internal reproductive systems of immature stages of both nAphis persicaeM and Uroleucon .jaceae (Balbiani does not give an author for the former species, but it seems likely to represent either Myzus persicae or a Brachycaudus species (Eastop & Hille Ris Lambers, 1976). He found that in embryonic "A. persicae11 males the testes comprise three follicles each, but are not yet fused at this stage. In first instar U. jaceae the testes are fused and comprise five follicles each, although an aberrant individual was figured by Balbiani, having six follicles per testis. Leuckart (in Balbiani, 1869) described the testes of Aphis (=Rhopalosiphum) padi as consisting of a single structure, divided into six 195 lobes. In the present study, the internal genitalia of M. persicae were studied in greater detail than those of the other species. Serial horizontal sections of the testes of first to fourth instar larvae, recently moulted adults, and adults after 5 days days of confinement with oviparae, were prepared. Sections were stained using Haemotoxylin and Eosin (see section 3.4-) and photographed at each developmental stage (figs 6.30a - 6.30f). In first instars, the testes were already formed, and consisted of three follicles each. However, the two testes were still rather widely separated, and accessory glands were not yet present, as found by Balbiani (1869). Each follicle was clearly composed of a number of approximately spherical cysts, each of which contained the developing spermatocytes. Within each cyst a clones of cells was seen to be developing in synchrony, but each cyst was at a slightly different stage of development (see particularly fig. 6.30c). Dividing spermatocyte nuclei were observed in testis cysts of first, second and third instar larvae. The testes of third instar larvae contained cysts in which the tails of the spermatids were forming (fig. 6.30c). The heads of these spermatids were in contact with the cyst wall, as described by Philipps (1970). In the testes of fourth instars (fig. 6.30d), the head-ends of the spermatids had become condensed, talcing up the Haematoxylin stain very strongly, and the tails were extended behind, all the tails in one cyst lying parallel to one another. The testis follicle cysts of adult M. persicae (fig. 6.30e) were rather more elongate than those of earlier instars, and spermatozoa were also visible within the proximal ends of the vasa efferentia, these spermatozoa appearing to be more elongate than those still within the testes. At this stage the cyst walls appeared to be starting to break down. In the testis follicles of older adults, the cyst walls had broken down almost completely (fig. 6.30f) and the individual spermatozoa had become lengthened, resembling the spermatozoa to be found within the vasa efferentia. Balbiani (1869) studied the develelopment of the sperm in Drepanosiphum platanoidis. He also found that the spermatids within each cyst develop in synchrony, and observed the formation of the tails coupled with the elongation of the cyst.

The following two subfamilies are not represented in Europe, but a sample of male Schoutedenia lutea was collected in Sydney, Australia, and very kindly sent to me by Dr. Dinah Hales. Male Greenideinae were studied from slide preparations. 196

Fig. 6.30 a-f. Horizontal serial sections through testis follicles of successive instars of Myzus persicae. a. 1st instar b. 2nd instar c . 3rd instar d. 4th instar e. Young adult f. Old adult ANOMALAPHDINAE

Schoutedenia lutea (males winged) External genitalia (3 specimens; figs 6.31a, 6.31b): Claspers present, but positioned laterally and reduced. Well-developed anterior genital processes present, as in some Lachninae. Valves present, large. Because specimens arrived already preserved in Bouin fixative, it was not possible to study the unsclerotized external genitalia. Internal genitalia (3 specimens; fig. 6.32): Because the material was already preserved, dissections were extremely difficult, and the morphology of the internal genitalia was not always clear. Testes separate and apparently each with three follicles. However, a number of smaller, less clearly defined, follicles may also have been present. Vasa efferentia/deferentia broadly expanded for most of their length, but particularly at their junctions with the testes. Accessory glands present, rather small, though this may simply have been because the sample contained only young adults.

GREENTDEINAE

Studied from slide preparations only. Greenidea anonae (males winged) External genitalia (six specimens; fig. 6.33): Claspers reduced and positioned laterally like those of S. lutea, but larger. Anterior genital processes present, as in S. lutea, but these narrower and more finger-like. Valves present, aedeagus not visible in slide preparations. The external genitalia of this species were illustrated by Raychauduri (1956). 1 9 8

Fig. 6.31. Schoutedenia lutea male external genitalia. A: Claspers B: Anterior genital processes C: Valves. (Lateral view / Anterior view). lo^

Testes

Fig. 6.33 Greenidea anonae. external genitalia (after Raychauduri, 1956)

Fig. 6.32 S. lutea male internal genitalia 199

CHAITOPHORINAE Four species from two genera were studied.

Chaitophorus capreae (males wingless) External genitalia (3 specimens; fig. 6.34a): Glaspers large, separate, positioned forward of the genital area. Valves present. Aedeagus very large in proportion to the body, very thick along its entire length. Internal genitalia (3 specimens; fig. 6.34b): Testes separate, with three follicles each. Accessory glands present, ducts of accessory glands rather short.

Chaitophorus leucomelas (mail.es winged) External genitalia (2 specimens; fig.6.35a): Claspers large, separate, positioned forward of the genital area. Valves present. Aedeagus rather shapeless. Membranous area in front of the subgenital plate clearly distended. Internal genitalia (2 specimens; fig. 6.35b): Testes separate, with three follicles each. Accessory glands greatly reduced.

Periphyllus hirticomis (males winged) External genitalia (3 specimens; fig. 6.36a): Very similar to those of C. leucomelas, valves extending slightly further forwards around the base of the aedeagus. Aedeagus more slender and much more curved than that of C. leucomelas, tapering gently. Membranous area in front of subgenital plate clearly distended. Internal genitalia (2 specimens; fig. 6.36b): Testes separate, three distinctly tapering follicles each. Accessory glands greatly reduced.

Periphyllus testudinacea (males winged) External genitalia (4 specimens; fig. 6.37a): Almost identical to those of P. hirticornis, but aedeagus more strongly reflexed. Membranous area in front of the subgenital plate very strongly distended. Internal genitalia (3 specimens; fig. 6.37b): Testes separate, three follicles each. Accessory glands greatly reduced.

Summary External genitalia: C. leucomelas shares with the two Periphyllus species the presence of a distended membranous area in front of the subgenital plate, but does not share this character with C. capreae. C. capreae is also the only species in which males were wingless. It is 200

Fig. 6.34a Chaitonhorus eanreae external genitalia

Fig. 6.34b C. canreae internal genitalia 201

0.1 nim

Fig. 6.35a Chaito-phorus leucomelas_ external genitalia 202 conceivable that the presence of an eversible membranous area could be an artifact due to the method of preparation of the specimens, but this seems very unlikely to have occurred only in these three species. Eversible adhesive vesicles have been described from viviparae of Neophyllaphis brimblecombei and other Neophyllaphis species (White & Carver, 1971). These are present between the abdominal sternites, and serve to allow the aphid to adhere to the substrate, or to right itself from a supine position. The largest vesicle is in front of the subgenital plate, as in the males in the present study. There is considerable doubt as to whether the structure in Chaitophorinae can be considered to be homologous with that in Neophyllaphis, although Dr. Dinah Hales (nee White) was shown the photograph in fig. 6.37a, and remarked that the distended area superficially resembled the eversible vesicles of Neophyllaphis species. Examination of alate viviparae of both Periphyllus and Chaitophorus species did not reveal any similar structures, and they appear, therefore, to be unique to males. Males of the three Chaitophorinae in which the vesicles are present were not observed in copula, but it may be that the position differs from that adopted by most aphids where the male approaches the ovipara from the rear and mounts her (illustrated in Heie, 1980 p.4-6). In these three species the membranous area may be involved in mounting and copulation. Internal genitalia: In all three species the testes were separate and each consisted of three follicles. Szelfgiewicz & Wojciechowski (1985) studied P. coracinus, P. villosus, C. nassonowi, C. populialbae and C. populeti and also found the testes to be separate in all five species. However, the two Periphyllus species had testes of four follicles each, and the Chaitophorus species had testes of three follicles each. In my own studies, accessory glands were reduced in all four species, markedly so in the three species which also shared the possession of the distended membranous area. According to Szelegiewicz and Wo jciechowski normal accessory glands were present in the two species of Periphyllus which they studied, but were reduced in all three Chaitophorus species.

DREPANOSIPHINAE The external genitalia of six species in five genera were studied in detail, a further five species being studied from slide preparations only. Species were selected original1y for their ease of collection. However, the unusual genus Neophyllaphis has, in the past, been the subject of speculation on aphid phylogeny (Hille Ris Lambers, 1984.; Heie, 1967) and it was thought worthwhile therefore to obtain specimens. Dr Dinah Hales kindly collected and sent N. brimblecombei from Sydney, Australia. N. podocarpi were collected in Chile by Mr. D. Hollis, BM(NH).

Drepanosiphum platanoidis (males winged) External genitalia (3 specimens; fig. 6.38a): Claspers large, separate, valves present. Aedeagus large, conjunctiva not clearly discernible from vesica. Internal genitalia (2 specimens; fig. 6.38b): Testes separate, three large, tapering follicles per testis. Accessory glands present. The internal reproductive system of D. platanoidis was also studied by Balbiani (1869) and by KLLmaszewski et aL (1973)- Both authors found it to consist of separate testes of three follicles each. Buckton (1882) mislabelled drawings of M. millefolii male genitalia, copied from Balbiani, as D. platanoidis.

Eucallipterus tiliae (males winged) External genitalia (3 specimens; fig. 6.39a): Clapers large, separate, valves present. Aedegus of medium size, smoothly curved, conjunctiva not clearly discernible from vesica. Internal genitalia (3 specimens; fig. 6.39b): Testes fused, each consisting of a single follicle only. Accessory glands present.

Euceraphis be tula e (males winged) External genitalia (2 specimens; fig. 6.40a): Claspers large, separate, valves present. Aedeagus rather sharply angled, conjunctiva clearly discernible from vesica. Internal genitalia (4 specimens; fig. 6.40b): Testes fused in three of the four specimens examined, apparently separate in one, but the separation may have been due to an error made during dissection. Three long, tapering follicles per testis. Accessory glands present. In the single individual in which the testes were apparently separate, no rupture of the either the vasa efferentia or testis follicles was evident, which would be expected if fused testes had been forced apart. Szel^giewicz & Wojciechowski (1985) use three distinct terms to describe separation of the testes: separate, fused and accreted. "Accreted" means that the walls of the vasa efferentia are fused but at no point do the vasa efferentia themselves actually share a common lumen, and this may have been the situation in E. betulae. Only one of the 29 species which were surveyed by Szelegiewicz & Wojciechowski was described as having testes accreted. 2 0 5

0.1 mm

0

Fig. 6.38a Drepanosiphum platanoidis

external genitalia

Accessory gland

Fig. 6.38b D. platanoidis internal genitalia 2 0 6

Fig. 6.39a Eucallinterus tiliae external genitalia

Fig. 6.39b E. tiliae internal genitalia 2 0 7

Fig. 6.10a Euceranhis betulae external genitalia

Fig. 6.10b E. betulae internal genitalia This was Symydobius oblongus, which is rather closely related to E. betulae, and shares the same host.

Phyllaphis fagi (males winged) External genitalia (2 specimens; fig. 6.4.1a): Claspers large, separate, valves present. Aedeagus rather sharply angled, conjunctiva clearly discernible from vesica. Internal genitalia (2 specimens; fig. 6.4.1b): Testes fused, three long, tapering follicles per testis. Accessory glands present.

Neophyllaphis brimblecombei (males winged) External genitalia (2 specimens; fig. 6.42a): Claspers large, separate. Valves present, form of aedeagus not clear since specimens were preserved in Bouin's fixative before the genitalia had been properly extruded. Internal genitalia (4- specimens; fig. 6.42b): Testes separate, three follicles per testis, one large follicle and two rather smaller ones. Accessory glands absent. Vasa efferentia/defferentia expanded along their entire lengths.

Neophyllaphis ?po do carpi (males winged) Internal genitalia (2 specimens; fig. 6.42c): Testes fused, three follicles of approximately equal size per testis. Accessory glands present. It is perhaps remarkable that the internal genitalia of the two species of Neophyllaphis differ from each other in almost every way possible. Only in having a total of six testis follicles are they similar, and this single character is also shared by very many other aphid species.

Tuberculoides ammlatus (males winged) Internal genitalia (3 specimens): Testes fused, three follicles per testis. Accessory glands present.

The external genitalia of a further five species were studied from slides. These five species were selected as representatives of unusual genera: Israelaphis, Tamalia, Neuquenaphist Lizerius, and Paoliella, the phylogenetic affinities of which have, in the past, been difficult to deduce. Remaudiere (in Remaudiere & Stroyan, 1984) considering all aphids to belong to one family, Aphididae, regarded Israelaphis and Tamalia as being sufficiently unusual as to warrant placement in subfamilies of their own. The remaining three genera he placed with Pterasthenia, An talus and 2 0 9

0.1 mm

Fig. 6.4.1a Phyllanhis fagi external genitalia 210

Fig. 6.42a Neoohyllaohis brimblecombei, SM of external genitalia

Fused testes

Fig. 6.42b N. brimblecombei internal genitalia Fig. 6.42c N. ?-Dodocaroi

internal genitalia 211

Sensoriaphis in a single subfamily, Neuquenaphidinae. The genera Tamali a and Neophyllaphis are unusual in having both sexuals winged (cf. Fhleomyzus). The external genitalia of Israelaphis tavaresi (fig. 6.43) were extremely unusual in that the claspers were very large, lobe-like, and directed backwards, apparently covering the genital opening. The anal plate was similarly specialised into two lobes which were directed forwards, again apparently covering the genital opening. The valves in this species were large, together forming an almost complete cylinder. The males are wingless. The males of the remaining four species were all winged except for those of Lizerius ocoteae. The external genitalia (figs 6.44-6.47) were rather similar in all of these species, with the claspers large and separate, and the valves present, resembling those of other Drepanosiphinae. In Neuquenaphis edwardsi the claspers were situated laterally, and in Tamalia coweni they were smaller than in the other species. The internal genitalia of a further four species of Drepanosiphinae have been studied by other authors. GZowacka et al (1974a) found the testes of Symydobius oblongus to be accreted and to consist of three follicles each. Fused testes, of two follicles each, were found in Iziphya bufo. In both species accessory glands were present. Karwanska (in Szelegiewicz & Wojciechowski, 1985) found the internal genitalia of both Betulaphis quadrituberculata and B. helvetica to consist of separate testes of two follicles each, with accessory glands present.

Summary External genitalia: The external genitalia of the Drepanosiphinae resemble those of the Pterocommatinae and Aphidinae, with the claspers large and separate, and the valves present. In general the claspers are larger than those of the Aphidinae, except in Tamalia, where they are rather small. The only really exceptional species, of those examined, is Israelaphis tavaresi, in which the genitalia have undergone a great deal of spe cialization. Internal genitalia: The Drepanosiphinae appear to have an extremely diverse range of types of internal genitalia. Both fused and separate testes occur, even in the same genus (Neophyllaphis) and possibly even in the same species (E. betulae). Six of the seven species studied had testes of three follicles each, this number of testis follicles also being very frequent among the Aphidinae. All species except Neophyllaphis ~ brimblecombei had accessory glands present. 212

Fig. 6 .LX Neucyiienanhls ed ward si external genitalia 2 1 3

Fig. 6 .4.6 Tama!ia coweni external genitalia

^g. 6.17 Paella harteni external genitalia

Fig. 6.18 Phleomyzus passerinii external genitalia PHLEOMXZINAE

Phleomyzus passerinii (males winged) External genitalia (studied from slides; fig. 6.43): Very large, separate, claspers present. Well-sclerotized valves also present. The external genitalia of this species, as they appear on slides, are very similar to those of the Drepanosiphinae examined, particularly Paoliella (cf. fig. 6.4-7).

MINDARINAE

Mindarus abietinus (males dwarfish, wingless) External genitalia (5 specimens; fig. 6.49a): Claspers present, strongly reduced and positioned laterally. Seperate valves present, but these very weakly sclerotized. Aedeagus tapering, extruded more or less vertically from the adbomen. Conjunctiva discernible from vesica. Internal genitalia (2 specimens; fig. 6.4-9b): Testes fused to form a single globular organ. Vasa efferentia very broad, accessory glands large and very broad at their proximal ends. The internal genitalia of male embryos and third instar males of this species were previously studied and illustrated by Niisslin (1910), whose findings coincide well with my own. The accessory glands of the third instar male in his illustration are, as expected, very much smaller than those which I found in adult males.

THELAXINAE

Glyphina be tula e (ma3.es small, wingless) External genitalia (4- specimens; figs 6.50a, 6.50b, 6.50c): Claspers fused into a single setose forward-pointing structure (figs 6.50a, 6.50b). Separate valves such as those found in Drepanosiphinae and Aphidinae either absent or modified and fused, apparently replaced by an extension of the anal plate (figs 6.50a, 6.50c). Aedeagus large, rather shapeless and pointing backwards. It is not clear whether the setose, forward-pointing structure at the anterior end of the genital area actually represents the claspers, which 2 1 5

Fig. 6.4.9a Mindarus abietinus external genitalia

Testis 0.1 mm

Fig. 6.4.9b M. abietinus internal genitalia 2 1 6

Ejaculatory duct 217 have become fused, or whether it is derived from the fusion of anterior genital processes such as those found in male Schoutedenia, Greenidea and Cinara, the claspers being either lost or undeveloped (cf. Pemphigus). Internal genitalia (2 specimens; fig. 6.50d): Testes completely fused, but apparently consisting of two, or possibly three, follicles. In the second individual the testes were less fusiform, consisting apparently of two follicles (dotted line in fig. 6.50d). Accessory glands present, rather small.

The Taxes dryophila (males dwarfish, wingless) External genitalia (4- specimens; fig. 6.51a): Claspers absent. Valves absent, their function perhaps performed by a sclerotized region at the rear base of the aedeagus, which appears as an extension of the anal plate. Aedeagus s-shaped, curved first forwards and then backwards on itself. - Internal genitalia (2 specimens; fig. 6.51b): Testes connected by a junction of the vasa efferentia. One follicle per testis, rather similar to E. tiliae (q.v.). Accessory glands present, well developed. The Thelaxinae currently contains only the genera Glyphina and Thelaxes (although I would consider that Kurisakia, which shares a number of morphological characters with Thelaxes, also belongs here, which conclusion was also reached independently by Remaudiere & Stroyan (1984-)). In this respect it is remarkable just how different the morphology of both the internal and external genitalia of Thelaxes and Glyphina are.

ANOECIINAE

Anoecia corn! (males dwarfish, wingless) External genitalia (4- specimens; figs 6.52a, 6.52b, 6.52c): Claspers absent, replaced by a single forward-pointing structure (figs 6.52a, 6.52b), slightly similar to the structure in Glyphina, but without setae and more sharply pointed. Valves appear from scanning electron micrographs to be absent, but the regions to either side of the downwardly-extended anal plate are weakly sclerotized (figs 6.52a, 6.52c). These are also visible in slide-mounted specimens. Aedeagus extruding downwards from the abdomen, tapering to a point. As with Glyphina, it is difficult to state whether the forward-pointing structure at the anterior end of the genital area actually represents the claspers, which have become fused, whether it is derived from the fusion of anterior genital processes or whether it has arisen in some other way. Fig. 6.51a Thelaxes dryophila external genitalia

Fig. 6.51b T.~ dryophila internal genitalia 2 1 9

Fig. 6.52a, 6.52b, 6.52c Anoecia corn! external genitalia

Fig. 6.52d A. comi internal genitalia Fig- 6.53 Hamamelistes sninosus internal genital! Internal genitalia (3 specimens; fig. 6.52d): Testes fused to form a single globular organ. Vasa efferentia broad, accessory glands present, small. Leuckart (reported in Balbiani, 1869) studied this species in 1858. He found the testes to be very strongly fused, almost completely spherical in form. Dr. R.L. Blackman (unpublished observations) also examined the internal genitalia of this species, with the same results. The external genitalia of Aiceona .japonica were described by Takahashi (1960). The description suggests removal of the genus from Anoeciinae, and its transfer to Greenideinae.

HORMAPHIDINAE

Hamamelistes spinosus (males dwarfish, wingless) Internal genitalia (1 specimen; fig. 6.53): Testes separate, one large follicle per testis. Accessory glands present.

PEMPHIGINAE

Pemphigus spyrothecae (males dwarfish, wingless) External genitalia (5 specimens; figs 6.54a, 6.54b, 6.54c): Claspers separate and finger-like, without setae but with spinules (figs 6.54a, 6.54b). Valves absent, but the rear portion of the base of the aedeagus, extending from the anal plate, more strongly sclerotized than the remainder of the base of the aedeagus (figs 6.54a, 6.54c). Aedeagus bulbous, extruding vertically. Whether the claspers of P. spyrothecae are homologous with those found in the Drepanosiphinae and Aphidinae is not clear. Like the analogous structures in G. betulae and A. comi they may be derived from a different part of the genital apparatus. The spinulose "claspers" are reminiscent of the anterior genital processes of Stomaphis yanonis (fig. 6.7c) and of the lateral elements of the "penis sheath" in Margarodidae (fig. 6.60f). Baker (1915) illustrated the external genitalia of a male Eriosoma lanigerum (figs 6.55&» 6.55b), in which not only were separate clasp>ers present, but also a pair of large valves, which he referred to as "a large fan-shaped structure". The aedeagus in Baker’s drawing appears to be disproportionately small, much smaller than that of P. spyrothecae, and curved rather than bulbous. Internal genitalia (3 specimens; fig. 6 .56a): Testes fused to form a Fig. 6.54a, 6.54b, 6.54c Pemphigus sieyrothecae external genitalia

Fig. 6.56a P. suyrothecae internal genitalia 222 single globular structure, apparently of one follicle. Vasa efferentia first constricted and then expanded, vasa deferentia expanded at their junctions with the ejaculatory duct. Accessory glands absent. The internal genitalia of a male Prociphilus nidificus (=fraxini) embryo were studied by Niisslin (1910). He found that the testes were separate, in contrast to the situation found above for P. spyrothecae, and from his drawing, each testis appears to be made of up to five follicles. However, he found that accessory glands were absent, as in adult P. spyrothecae, and considered that the walls of the vasa efferentia/deferentia performed the function of the accessory glands. In order to make valid comparisons it would be necessary to study adult males, as the testes may become fused during development, and the number of testis follicles may become reduced. It is also possible that accessory glands could develop. Baker (1915) described and illustrated the internal genitalia of adult male Eriosoma lanigerum (fig. 6.56b). In this species the vasa efferentia/deferentia are separate along their entire length, but the testes are fused, although still distinguishable as two distinct organs. If the testes were separate, the internal genitalia of this species would greatly resemble those of Pineus orientalis. In contrast to the situation in Pemphigus two large, well-developed accessory glands are present.

ADELGIDAE: PINEINAE & ADELGINAE The external and internal genitalia of males of two species were studied and compared.

Pineus orientalis (males dwarfish, wingless) External genitalia (3 specimens; fig. 6.57a): Claspers absent. Valves absent, anal plate extended downwards. Aedeagus large in proportion to the body, pointing forwards. The internal genitalia of this species are similar to those of Trialeurodes vaporariorum (fig. 6.61b). Internal gentalia (2 specimens; fig. 6.57b): Testes separate, one follicle per testis. Vasa efferentia widely expanded. Accessory glands present.

Adelges cooleyi (males dwarfish, wingless) External genitalia (4 specimens; fig. 6.58a): Claspers absent, subgenital plate greatly enlarged and apparently fused with anal plate, forming a heavily sclerotized box-like structure (ah analogous structure in 2 2 3

Fig, 6.57a Pineus orientalis external genitalia Fig. 6.57b P. orientals internal genitalia.-.

0.1 mm

Fig. 6.58a Adelges cooleyi external genitalia pig. 6.58b A. cooleyi internal genitalia Oncopeltus has been termed the "genital capsule" try Bonhag & Wick, 1953)- Valves present, greatly extended, unsclerotized aedeagus absent. There are a great many important differences between the external genitalia of this species and those of P. orientalis, and these are discussed in section 6.5. Internal genitalia (2 specimens; fig. 6.58b): Testes separate, two follicles, a large one and a smaller one, per testis. Vasa efferentia/deferentia rather broad along their entire length. Accessory glands present. The internal genitalia of Chermes strobilobius (=Adelges laricis) were studied previously (ChoXodowsky, 1900). The original illustration (p. 279) depicts a single testis, with a single vas effererens/deferens, flanked by two accessory glands. A corrected illustration appears later in the same journal (p. 619), showing two testes, with the accessory glands in between them. This later drawing accords very well with my own findings for A. cooleyi, although the follicles of each testis are closer to each other in size. Unfortunately, the uncorrected version of ChoXodowsky1 s drawing was used by Szelegiewicz & Wojciechowski (1985) to illustrate internal genitalia in Adelgidae. Frolowa (1924.) found that spermatophores were present in older larvae of Adelges laricis and A. pectinatae. I found no such structures in either P. orientalis or A. cooleyi, and spermatozoa were observed passing down the vasa deferentia in the same way as in all other Aphidoideas studied. From Frolowa's illustrations, it appears that some other structures may have been wrongly interpreted as spermatophores.

PHYLLOXERIDAE

Phylloxera glabra (males dwarfish, wingless) External genitalia (5 specimens; fig. 6.59a, 6.59b): Claspers possibly fused, forming a single median process which is not setose, but has a serrated outline. Valves such as those found in Aphidinae or Drepanosiphinae absent, but an apparent extension of the anal plate fulfilling their function. Aedeagus bulbous, curved backwards at the end. Internal genitalia (2 specimens; fig. 6.59c): Testes separate, one follicle per testis. Vasa efferentia rather narrow compared with Adelges, accessory glands originating from vasa deferentia, rather than from the ejaculatory duct as in Adelgidae (q.v.). 2 2 5

Fig. 6.59a, 6.59b Phylloxera glabra external genitalia

0.1 mm

Fig. 6.59c P. glabra internal genitalia 226

6.4 MALE GENITALIA IN OTHER HEMIPTERA

6.4.1 External genitalia The external genitalia of Psylloidea (fig . 6.60a) and Aleyrodoidea (6.60b) are very sim ilar, consisting of a genital capsule (=hypandrium, subgenital plate), a pair of claspers (=forceps, parameres) and an a ed e ag u s.

In Coccoidea (fig . 6.60c) the male genitalia have been described as consiting of a conical penis sheath containing a tubular penis (Ossiannilsson et a l, 1956). However, the arrangement of the external genitalia in Coccoidea is clearly subject to a good deal of variation between fam ilies. For example, the "penis sheath" in Planococcus c itri

(figs 6.60d, 6 . 60e) is not an elongate structure, and includes a pair of lateral claspers and a pair of valves. The male genitalia of this species were also studied by Berlese (1893) (as Dactylopius c itri), and his illustra tio ns accord, w ell with my own. I have also studied slide-mounted Icerya nigroareolata (Margarodidae) male genitalia (fig . 6.60f) and in this species the "penis sheath" is clearly composed of two partially fused elements. These structures could conceivably be homologous with the anterior genital processes of some Aphididae.

6.4.2 Internal genitalia In their general structure, the internal reproductive organs of all male insects are similar. The principal elements are a pair of separate testes, consisting of a variable number of follicles or testicular tubules; vasa efferentia, later becoming vasa deferentia, leading from the testes to the ejaculatory duct; seminal vesicles, formed from part of the vasa deferentia; and paired, separate, accessory glands (Davey, 1 9 8 5 ). For this reason, it is not difficult to find apparent homologies between the internal genitalia of aphids and those of many other Hemiptera. The reproductive system of the milkweed bug, Oncopeltus fasciatus (Heteroptera), has been studied in great detail (Bonhag & Wick, 1953 ) > and differs little from the generalised system described herein for aphids. The testes are separate, and rather differently shaped from those of aphids, having the follicles in a fan-like arrangement. Each testis contains seven follicles, the same number as that found in Lachnus roboris, which was the highest number encountered in the Aphidoidea. The accessory glands in Oncopeltus are remarkably similar to those found in some Aphididae. The internal genitalia of Hemiodoecus veitchi (Coleorrhyncha: Peloridiidae) also consists of separate testes, each of one follicle, and a pair of 227

Fig. 6.60 External genitalia in other Sternorrhyncha 228

accessory glands (Pendergrast, 1962). Among the Auchenorrhyncha, Buscelis plebejus (Deltacephalinae) has separate testes, each of seven fo llicle s

(Kunze, 1959) as in Oncopeltus, and Populicerus populi (Idiocerinae) and Alnetoidea alneti (Typhlocybinae) each have separate testes of six and three fo llic le s respectively (Bednarczyk, 1983). In the Sternorrhyncha, the male reproductive system of the psyllid

Psylla fusca was studied by Glowacka and Klimaszewski (1969) (fig 6.61a). In this species the testes are separate and consist of six fo llic le s each. Two large accessory glands are also present, opening into the ejaculatory duct. In P. fusca, two large seminal vesicles occupy the position in which one would expect to find the accessory glands in Adelgidae, Phylloxeridae and Aleyrodoidea (see below), and it is possible that these organs are

homologous w ith the accessory glands in other Stemorrhyncha. The reproductive system of Trialeurodes vaporariorum (Aleyrodoidea) was

studied from ten specimens (fig . 6.61b). In this species the testes are separate, of one fo llic le each, and the accessory glands open onto the vasa deferentia, which are here expanded, possibly forming receptacula

semi rial i s. The accessory glands are separate fo r almost a ll th e ir length, but are fused for a very small portion of their distal ends only. Weber

(1935) described the internal genitalia of this species as having seperate accessory glands. It may be that the tips of the accessory glands become

accreted in older adults. Without this accretion, the internal genitalia of this species would bear a remarkable resemblance to those of both Pineus

orientalis and Phylloxera glabra (cf. figs 6.57b, 6.59c). Within Coccoidea, the internal genitalia of Coelostomidia sp. (fig . 6.61 c) and Icerya sp., (Margarodiae), and those of Planococcus c itri

(Pseuococcidae) (fig . 6.6ld), were studied. In both margarodids, the genitalia consisted of a sclerotized capsule, composed of both longitudinal and circular elements, packed w ith mature spermatozoa, apparently randomly

arranged. A coiled tubular structure, blunt at its distal end, was also present within this capsule, and this structure remained visible in many slide-mounted specimens. The genitalia of P. c itri (fig . 6.6ld) were easier to relate to those of other Sternorrhyncha. The testes in this species are extremely elongate and united by term inal filam ents. At the base of each

testis is an e llip tica l structure, resembling that identified as the accessory gland by Nur (1962) in his study of another member of the

Pseudococcidae, Pseudococcus obscurus. In the diaspid M ytilaspis fulva

(=Lepidosaphes beckii), the testes are separate and are of sim ilar proportions aphid testes, but, as in Planococcus, the accessory glands do

not form elongate outgrowths of the ejaculatory duct (Berlese, 1895). Fibrous capsule containing spermatozoa

6.61a Psylloidea, Psylla fusca 6.61c Coccoidea, Margarodidae Coelostomidia sp.

(after Glowacka & Klimaszewski, 1969)

6.61b Aleyrodoidea Trialeurodes vaporariorum 6.6ld Coccoidea, Pseudococcidae Planococcus citri 229

Fig. 6.61 Internal genitalia in other Sternorrhncha 230

6.5 DISCUSSION: The phylogenetic implications of this study

The preceding systematic survey of male genitalia in Aphidoidea has revealed a number of characters which are of potential use in deducing the

phylogenetic (genealogical) relationships between the included taxa. Such a reconstruction might then form the basis of a higher classification of the

Aphidoidea. The value of each character clearly varies according to the position in the phylogeny which is being considered. For example, w ithin the Aphidoidea, the whole subfamily Lachninae can be characterized by the absence of accessory glands, but this character is of no use in characterizing the tribe C inarini w ithin Lachninae. Here the useful terms "apomorphy" "synapomorphy" "plesiomorphy" and "symplesiomorphy" can be applied. Thus the character "absence of accessory glands" is a synapomorphy fo r Lachninae, and a symplesiomorphy fo r C inarini. These terms are defined

by Hennig (1979) and W iley (1981). Phylogeny is suggested by the presence of derived (apomorphic) characters shared by particular taxa, i.e . synapomorphies. In order to distinguish derived characters from prim itive (plesiomorphic) characters it is necessary to examine the homologous

character in the assumed sister-group to the taxon being studied. The assumed sister-group to Aphididae is either Phylloxeridae or Adelgidae, and

in both fam ilies the males are highly specialized. One fares little better going farther afield, to the Coccoidea, where once again the males have a highly specialized morphology.

In the following sections, however, the various characters relating to

aphid male genitalia are discussed, and attempts are made to categorize them as apomorphic or plesiom orphic, with reference to several out-groups. 6.5-1 External genitalia i. Claspers The various forms of the claspers in the families and subfamilies of Aphidoidea are summarized in Table 6.2.

T a b le 6 .2 FAMILY, SUBFAMILY FORM OF CLASPERS

Adelgidae Absent. Phylloxeridae Fused, probably not homologous with large claspers of Aphididae. Lachninae Separate. Often reduced. Pterocommatinae Large, separate. Aphidinae Large, separate. Greenideinae (incl. Anomalaphidinae) Small, separate. Chaitophorinae Large, separate. Drepano s iphinae Large, separate. Phleomyzinae Large, separate. Mindarinae Very small, separate. Probably homologous with large claspers of other subfamilies. Thelaxinae Absent, or, if present, then fused and possibly derived from anterior genital processes. Anoeciinae Absent or fused, derived from anterior genital processes? Pemphiginae Separate, derivation possibly as in Thelaxinae.

Large setose claspers occur in most genera of Pterocommatinae, Aphidinae, Chaitophorinae, Drepanosiphinae and Phleomyzinae, and the males in these subfamilies are never dwarfish. In Paoliella and Phleomyzus the claspers are perhaps the largest encountered among the Aphidoidea. The claspers appear to be greatly modified in Israelaphis, and reduced in Cavariella. In some Lachninae, Anomalaphidinae and Greenideinae, in which the males are also generally of normal size, the claspers are reduced, or possibly have been developed to a lesser extent. In Mindarus, having dwarfish males, the claspers are very small. Claspers are present in Pemphigus, but these are very different from the claspers found in the 232

subfamilies w ith normal-sized males. The remaining subfam ilies, as w ell as

Adelgidae and Phylloxeridae, which a ll have dwarfish males, have the

claspers either absent or apparently modified in some way. In all genera in which large, setose claspers occur the claspers are very similar in structure and general appearance, and they appear to be entirely homologous structures. Of the genera with dwarfish males, the claspers of Mindarus appear to be derived from large setose claspers. In the remaining genera they could equally be derived from anterior genital processes such as those found in Lachninae and Greenideinae. Particularly in Pemphigus, they appear more likely to have been derived from anterior genital processes. It is extremely d iffic u lt to classify the various forms of the claspers as either plesiomorphic or apomorphic since neither separate claspers, nor anterior genital processes are present in either Adelgidae or Phylloxeridae. In Coccoidea, a pair of lateral claspers, which resemble those of Aphididae, are present in Planococcus c itri (fig s. 6.60d, 6.60e).

In Margarodidae (fig . 6.60f) and some other Coccoidea (fig . 6.60c), no such claspers are present. Large, setose claspers are present in Aleyrodoidea

and Psylloidea, and are very sim ilar in structure in these two groups. However, these claspers (forceps, parameres) may not be homologous w ith the

large claspers present in Aphididae. The closest one gets to p syllid -like claspers among the Aphididae is , curiously enough, the valves of Stomaphis quercus (figs 6.7a, 6.7b; and see discussion on valves, below). However,

the presence in Coccoidea of claspers which resemble those found in many subfamilies of Aphididae suggests that the possession of claspers is plesiomorphic fo r Aphidoidea. The various modified nclaspers" found in Thelaxinae, Anoeciinae and Pemphiginae do not show any strong similarites with each other, and have developed in different ways. Their modification may be a result of the loss in overall body size. The different modifications of the claspers in these three subfamilies suggests that reduction in the size of males (and oviparae) has occurred independently In a number of distinct lineages within the Aphididae.

ii. Anterior genital processes These structures have never been desribed previously, although their

existence in Greenidea anonae was clearly known to Raychauduri (1956) (fig .

6.33). They are present in Stomaphis, Cinara, Eulachnus, Schoutedenia and Greenidea. They are not present in Pterocommatinae, Aphidinae,

Chaitophorinae, Drepanosiphinae and Phleomyzinae (i.e . those subfam ilies 233

having normal-sized males with large, setose claspers), or in Mindarinae.

Anterior genital structures are either specialized or absent in the dwarfish males of Thelaxinae, Anoeciinae and Pemphiginae. It is not clear whether the specialized structures in some of these subfam ilies are derived from the reduction of large setose claspers or from the fusion or modification of anterior genital processes. In Pemphigus the "claspers" appear to be derived from anterior genital processes. The absence of anterior genital processes from Adelgidae, Phylloxeridae and Coccoidea

suggests that either they are a synapomorphic feature of Aphididae, or that they have evolved more than once in the fam ily. The la te ra l elements of the so-called "penis sheath" in Margarodidae (fig . 6.60d) bear some resemblance both to the anterior genital processes of Stomaphis (fig 6.7) and to the

"claspers" of Pemphigus (fig s 6.54a» 6.54b), but this may be homoplasy. The absence of genital processes from both Lachnus and Longistigma, and their presence as apparently homologous structures in Stomaphis and

C inarini suggests monophyly of the la tte r two taxa. Although the genital processes appear sim ilar in Lachninae and Anomalaphidinae (cf. figs 6.7b, 6.31b), the two subfamilies d iffe r greatly

otherwise, and it seems like ly that the genital processes have evolved independently in these two lineages. The Anomalaphidinae and Greenideinae,

however, show several a ffin itie s and have been united in one subfamily, Greenideinae, by some authors, including, most recently, Remaudiere and

Stroyan (1984)• The occurrence of anterior genital processes in both Schoutedenia and Greenidea further supports this interpretation. The

unusual genus Aiceona is currently classified in Anoeciinae (Takahashi, 1960). Takahashi described the male genitalia as follow s: "Claspers with

numerous setae except on median part; produced parts rather slender, distinctly apart, distinctly longer than basal part, parallel, without setae." The apparent occurrence of both claspers and anterior genital processes in this genus, as well as the occurrence of alate oviparae suggests that Aiceona belongs in Greenideinae.

iii. Valves D istinct valves are present in a ll the genera with normal-sized males, and in Mindarus. These are the genera which also have large, setose claspers present. The valves are either setose or dotted with small

circular p its. In general, the valves are more setose in Lachninae than in the other subfam ilies. In Thelaxinae and Pemphiginae the valves appear to be replaced by, or modified into, a downward extension of the anal plate,

although in most instances this is not really clear. The valves are absent in Pineus but the structures present in Adelges could be homologous w ith the valves of Aphididae. They do not appear to be present in Phylloxeridae. In Planococcus (figs 6.60d, 6.60e) the valves seem sim ilar to those of Aphididae (cf. figs 6.60d & 6.47). Where they occur, the valves appear in almost a ll cases as flattened structures closely united with the base of the aedeagus, and attached to it. In slide-mounts of Stomaphis, however, the valves appear to be finger-like structures (fig . 6.7a), entirely covered with setae. In this species the valves may be less specialized, appearing not to be involved in eversion of the aedeagus, although this opinion is based only on a study of slide-mounted m aterial. The valves of Stomaphis resemble the parameres of Psylla species, and it may be that the valves in Aphididae are homologous w ith the parameres of Psylloidea and Aleyrodoidea. Thus the valves present in Aphididae could be derived either from such large, setose structures as are found in Stomaphis, or all valves, including those of Stomaphis could be derived from the type found in Planococcus. In either case their presence appears to be symplesiomorphic for Aphididae. The question now arises whether the absence or modification of valves in Glyphina, Thelaxes, Anoecia and Pemphigus represents synapomorphy in these four genera. In the latter three genera, males are dwarfish, and in Glyphina they are also rather small, so the reduction of valves could be due to the overall decrease in size. However, Mindarus, also has dwarf males, and valves are present. There is little evidence for interpreting the modification of the valves as either synapomorphy or homoplasy in these four genera. However, Baker’s illustrations of Eriosoma indicate the presence of valve-like structures, and if these are correct then loss of valves must have occurred separately in at least the Pemphigus lineage.

iv. Aedeagus Among Stemorrhyncha, the aedeagus is eversible only in Aphididae, Adelgidae and Phylloxeridae, a synapomorphy characterizing the Aphidoidea. The aedeagus in Aphidoidea shows few similarities to those of the other superfamilies of Sternorrhyncha. Within Lachninae, the various forms of the aedeagus are extremely diverse, whereas within Aphidinae, the shape is comparatively constant. The shape and structure of the aedeagus in Aphidinae seem to be more specialized than in Lachninae, Chaitophorinae and Drepanosiphinae. In these subfamilies, the aedeagus is, in general, more shapeless, although there are exceptions. In Mindarus and Glyphina the 235

aedeagus is also rather shapeless, whereas in Thelaxes and in the rem ining subfamilies of Aphididae, as w ell as in Phylloxera the aedeagus is small, but distinctively shaped. The presence of a large aedeagus in the adelgid Pineus orientalis and its apparent absence in Adelges cooleyi is rather strange, and suggests the more recent origin of Adelges. In Adelges the valves, or the structures which correspond to them, have been greatly elongated. The resulting organ might perhaps function in the same way as the sclerotized penis-sheath found in some Coccoidea.

6.5«2 Internal genitalia i. Accessory glands Accessory glands were present in all subfamilies and genera studied except the following:. Neophyllaphis brimblecombei, Pemphigus spyrothecae, and all Iachninae. The glands are reduced in Chaitophorinae. The presence of such glands in Psylla, Trialeurodes, Planococcus, Phylloxera, Pineus, Adelges and most Aphididae? suggests very strongly that the possession of accessory glands is plesiomorphic for both Aphidoidea and Aphididae. The loss of the accessory glands has probably occurred in at least three

separate lineages within the Aphididae. In Lachninae, absence of the accessory glands is presumably a derived character. W ithin Neophyllaphis,

N« ?podocarpi from Chile has accessory glands, but N. brimblecombei does not. Accessory glands have also been lost in the Pemphigus line w ithin

Pemphiginae, if Baker’s (1915) observation that Briosoma males have accessory glands is correct.

The accessory glands of Adelgidae, Phylloxera and Trialeurodes are very sim ilar in shape (cf. figs 6.57b, 6.58b, 6.59c & 6.61b), small and short. In a ll the subfamilies of Aphididae, however, the accessory glands are globular at their d istal ends and have long ducts at their proximal ends. The exception is Eriosoma (fig . 6.56b), in which the glands appear from Baker’s drawing to be very sim ilar in shape to those of Adelgidae.

Additionally, the testes in these species, if one imagines them as being separate, greatly resemble those of both Pineus orientalis (fig . 6.57b) and Trialeurodes (fig . 6.61b). These characters relating to Briosoma, together

with the absence of accessory glands in Pemphigus, suggest that w ithin Pemphiginae, Pemphigus evolved more recently than Eriosoma. However, such speculation is hazardous when only two species of pemphigine have been studied, and in the knowledge that aphids w ithin the same genus may d iffe r

fundamentally in this character (viz. Neophyllaphis). 236

The swollen structures between the vasa deferentia and the ejaculatory duct in Pseudococcidae (fig . 6.6ld) have been described as accessory glands (Nur, 1962). However, they also resemble the proximal ends of the vasa deferentia in Trialeurodes (cf. fig . 6.61b) which function, perhaps, as seminal vesicles, and Berlese (1893) described homologous structures in Diaspididae as such. There do not appear to be any accessory glands in Margarodidae, and it may be that these have been lo st in the Coccoidea as a whole. A study of the internal genitalia of the remaining 16 or so fam ilies of Coccoidea might reveal males w ith accessory glands present.

ii. Fusion of the testes Two separate testes are present in Psylla (Weber, 1930; Glowacka &

Klimaszewski, 1969), Trialeurodes , Lepidosaphes (Coccoidea) (Berlese, 1895), Pineus, Adelges, Phylloxera and many genera of Aphididae. In Margarodidae, the internal genitalia appear to be highly specialized, and in Planococcus the testes are connected by their distal ends only. It is probable that the possession of separate testes is a plesiomorphy of Aphididae, and that the testes have become fused in a number of separate lineages (Table 6.3). This is borne out by the fact that fusion of the testes has occurred in a number of different ways:

i. Fusion of the proximal ends of the vasa efferentia. This is the most widespread form of fusion of the testes in Aphididae. Of the species studied, it occurs in four out of five genera of Iachninae, a ll Pterocommatinae and Aphidinae, seven out of nine genera of Drepanosiphinae, and in Thelaxes. This form of fusion of the testes has probably occurred in seperate lineages.

ii. Partial fusion of the testes. This is the situation in Briosoma, according to Baker’s (1915) drawings (fig. 6 . 56b). Here the vasa efferentia/deferentia remain seperate for their entire lengths. The testes are fused but are s till clearly distinguishable as two structures. The testes of Glyphina appear also to belong to this category. iii. Complete fusion of the testes, resulting in a single, globular te stis. This is the situation in Mindarus, Anoecia and Pemphigus. Data concerning fusion of the testes are summarized below (Table 6.3) 237

Table 6.3. Fusion and separation of the testes in Aphidoidea

TESTES VASA EFFERENTIA TESTES PARTIALLY TESTES COMPLETELY

SEPARATE FUSED ( i ) FUSED ( i i ) FUSED ( i i i )

P h y llo x e ra T h e la x e s E rio so m a Pem phigus

A d e lg e s Neophyllaphis G ly p h in a A n o e c ia

P in e u s P h y lla p h is M in d a ru s H orm aphis Tuberculoides Neophyllaphis E u c e ra p h is

B e tu la p h is Eucalli.pterus Drepanosiphum Aphis, Macrosiphum Chaitophorus (+ a ll other Periphyllus Aphidinae studied)

Schoutedenia Pterocom m a ? C in a ra C in a ra E u la ch n u s ?E u la ch nu s ?Lachnus S tom a p his Maculolachnus Lachnus

The testes are separate in Eulachnus (but see p.181 ), Schoutedenia, Chaitophorinae, Drepanosiphum, Betulaphis (Szelegievd.cz & Wojciechowski,

1985)> Neophyllaphis brimblecombei and Hamamelistes. Some species belonging to the firs t category above contain either some

individuals or populations with separate testes; e.g. Lachnus roboris, Eulachnus a g ilis , E. rile y i and possibly Euceraphis punctipennis. Also, of two species of Neophyllaphis one had fused testes while in the other they

were separate. It would appear that this form of fusion of the testes is not a stable character, and is therefore of doubtful use in the assessment of aphid phylogeny. P artial fusion of the testes (category ii. , above) has probably occurred independently in Eriosoma and Glyphina, although this would not necessarily be the case if the genus Thelaxes (category i) were older than Glyphina. Complete fusion of the testes must have occurred independently in Pempihiginae and Mindarinae+Anoeciinae, and comparison of other characters relating to genitalia in Mindarus and Anoecia, as w ell as other aspects of morphology, and biological inform ation, strongly suggests that these genera have also evolved fused testes independently from each

other. Complete fusion of the testes has probably, therefore, occurred 238

independently in a ll three genera in which it has been observed.

iii. Number of testis follicles The number of testis fo llic le s in Aphidoidea shows almost continuous variation, from one to 14, between genera. Numbers of fo llicle s in the subfamilies studied, as well as the few records in the literature, are

summarized below (Table 6.4-).

T a b le 6 . 4 . Numbers of species in each subfamily having particular numbers

of testis fo llicle s.

TOTAL NUMBER OF TESTIS FOLLICLES & FREQUENCY

SUBFAMILY 1 2 3 4 6 7 8 9 10 11 12 13 14

L a ch n in a e 2 1 4 1 2 1 1 Pterocommatinae 1 1 1 1

A p h id in a e 13 1 1 Anomalaphidinae ?1 Chaitophorinae 2 7 Drepanosiphinae 1 3 7 M in d a rin a e 1 T h e la x in a e 2 A n o e c iin a e 1 Hormaphidinae 1

Pemphiginae 1 1

The range of fo llic le numbers was greatest in Lachninae, followed by Pterocommatinae. Almost a ll Aphidinae had six fo llic le s . Chaitophorinae and

Drepanosiphinae mostly had four or six. Subfamilies with dwarf, or dwarfish, sexuales had only one or two fo lli cles. Odd numbers of fo llic le s were encountered rather infrequently.

The data in Table 6 .4 suggest that there is a correlation between fo llic le number and overall body size. This is due mainly to the large males of Lachnus having 14 fo llicle s, and dwarfish males from other subfamilies having one or two. However, there are many exceptions. For

example, w ithin Lachninae M. submacula has 12 fo llic le s whereas S. quercus has eight, and males are very sim ilar in size in these two genera. C.

stroyanit which has the smallest males of any Lachnine in the present survey, has up to 10 follicles, whereas the similarly sized E. agilis has only four. The drawing of comparisons is, however, complicated by the fact that certain of these species have winged males, whereas in others, males are wingless. Results of an extensive survey of testis follicle numbers in Miridae (Akingbohungbe, 1 983) suggested that follicle number was dependent on body size in this family, but the evidence in Aphididae is inconclusive. What do the data in Table 6 suggest about aphid phylogeny? The occurrence of one or two follicles is likely to be a consequence of the reduction in body size, and this has probably occurred in a number of separate lineages (see pp.244-245 ). Testes with a total of six follicles occur with remarkable frequency, particularly in Aphidinae, Chaitophorinae and Drepanosiphinae, although this may be largely due to the greater proportion of aphidine species among those surveyed. Pterocommatinae is often regarded as a sub-taxon of Aphidinae (Stroyan, 1984.; Heie, 1 9 8 5 ), containing species which are morphologically primitive and similar, perhaps, to the ancestors of Aphidinae. Follicle numbers are higher in some Pterocomma than in most Aphidinae, and it may be that a reduction in follicle number has taken place. According to Wojciechowski (1 9 7 7 ) similar reductions in testis follicle number, referred to as "processes of oligomerization", have occurred in Lachninae. Available evidence, including data concerning the male reproductive system, suggests that Lachnus is the most primitive of the lachnine genera studied in the present survey. As Lachnus also had the highest follicle number encountered among the Aphidoidea it seems probable that a testis follicle number of 14- (or more) is plesiomorphic for Aphididae, and that reductions have taken place in one or more lineages. The common occurrence of six follicles in Aphidinae, Chaitophorinae, Drepanosiphinae and possibly Anomalaphidinae must be, therefore, either synapomorphy or homoplasy. Many genera of Drepanosiphinae exhibit primitive characters not shared with either Aphidinae or Pterocommatinae, e.g. four rudimentary gonapophyses, sub-siphuncular wax-gland plates in oviparae and the presence of a three-lensed eye in the larvae. If the hypothesis is correct that the six follicles of Aphidinae are a result of a reduction in follicle number from a Pterocomma-like ancestor, then either the loss of the above plesiomorphies has occurred independently in Pterocommatinae and Aphidinae, or the occurrence of six testis follicles in Aphidinae and Drepanosiphinae (etc.) is homoplasy. It should be noted, however, that the primitive characters mentioned above occur in by no means all Drepanosiphinae. It should be added here that an equally valid argument for six follicles as the plesiomorphic state in Aphididae could also be put 240 forward. In conclusion, it appears that there may be a general relationship between follicle number and body size across Aphidoidea, but there are numerous exceptions. It seems probable that high numbers of testis follicles are plesiomorphic, with reductions having occurred in many separate lineages, i.e. within Lachninae, Pterocommatinae, Drepanosiphinae, Chaitophorinae and Pemphiginae.

6.5«3 Summary Analysis of characters relating to male aphid genitalia leads to the following conclusions regarding aphid phylogeny: i. Presence of claspers is plesiomorphic for Aphidoidea. Reduction or modification of these has probably occurred independently in the following lines: Adelgidae Phylloxeridae Lachninae Cavariella within Aphidinae Greenideinae + Anomalaphidinae Mindarinae Thelaxinae ?+Anoeciinae Pemphiginae ii. Anterior genital processes have evolved independently in a Cinarini/Stomaphis lineage within Lachninae, and' in Greenideinae/Anomalaphidinae. However, these may also have been present in ancestors of Thelaxinae, Anoeciinae and Pemphiginae, and might even be plesiomorphic for Aphidoidea. iii. Presence of valves is plesiomorphic for Aphidoidea. Their structure appears to be most primitive in Lachninae. Loss of the valves has occurred independently in Thelaxinae and Pemphiginae, possibly also in Anoeciinae. iv. Presence of accessory glands is plesiomorphic for Aphidoidea. These have been lost in three separate lineages, namely Lachninae, part of Neophyllaphis and the Pemphigus lineage within Pemphiginae. v. Fusion of the testes has occurred in different ways and in many distinct lines within Aphididae. This character is of little value in deducing aphid phylogeny. vi. The primitive number of testis follicles in Aphididae is probably high, possibly fourteen, as found in Lachnus roboris. However, reductions in testis follicle number have occurred in many separate lineages. 241

7. PHYLOGENY & HIGHER CLASSIFICATION OF APHIDOIDEA: GENERAL CONSIDERATIONS

The comparative morphology of male genitalia, when studied in isolation, is of little value in the reconstruction of phytogenies. Many other aspects of aphid morphology and biology may be of equal or greater importance in deducing phylogenetic relationships and constructing a higher classification. Some of these were given particular attention during the present study and are discussed below. Certain life-cycle characteristics are associated either d irectly or ind ire ctly with reproductive anatomy. For example, host-alternation in Pemphiginae, Hormaphidinae and Anoeciinae always involves sexuparae, and these give birth to dwarf or dwarfish sexuals. Reduction in the size of sexual morphs may then have a direct effect on characters associated w ith genitalia.

7.1 Host-alternation Among Stemorrhyncha, heteroecy occurs only in Aphidoidea. It has long been known that Adelgidae alternate between Picea and other conifers as hosts. Stoetzel (1985) discovered host alternation between different plant fam ilies in Phylloxeridae. In Aphididae the following subfamilies contain heteroecious species: Aphidinae, Anoeciinae, Hormaphidinae and Pemphiginae.

In most aphid subfam ilies which contain heteroecious genera the sexual part of the life-cycle, including the sequence of morphs, is essentially sim ilar, although both annual and biennial life-cycles occur. Winged sexuparae migrate from the secondary host to the primary host where they deposit the dwarf or dwarfish sexuales. However, the life -cycle is rather different in Aphidinae, which is the only subfamily containing species that are heteroecious and have alate males.

In the phylogenetic context, host-alternation appears to be prim itive in Aphididae. Loss of heteroecy has, therefore, occurred in a number of separate lineages; e.g. Phylloxeridae, Aphidinae, Anoeciinae and Pemphiginae, and in at least one further lineage which gave rise to the extant subfamilies which contain no heteroecious species, i.e . Lachninae, Drepanosiphinae etc. Acceptance of the hypothesis that the common ancestor of Aphididae was heteroecious raises the following questions: F irstly, is heteroecy involving both sexuparae and dwarfish sexuals the prim itive condition? Secondly, has heteroecy evolved more than once w ithin Aphididae? In answer to the first question, the previous study of male genitalia showed that it was the large males of certain monoecious Lachninae or Drepanosiphinae which had probably the most plesiomorphic genitalia characters. In addition, reduction in body size of the sexuales has clearly occurred in many lineages. Consideration of the different life-cycles of Anoeciinae (see 7 .5 ) suggests that extinct Anoecia species may have been both heteroecious and had normal-sized sexual morphs. Thus although conclusions must be, of necessity, based on rather flimsy evidence, it seems likely that the ancestor of Aphididae was heteroecious and had alate sexuparae which gave birth to normal-sized sexuals. Certain species of Hormaphidinae approach this condition, where the sexuals, though dwarfish, are quite large. The entirely monoecious Pterocommatinae are generally thought to represent primitive Aphidinae. Morphologically these two subfamilies are closer to the monoecious Lachninae, Drepanosiphinae and Greenideinae than to the heteroecious Hormaphidinae and Pemphiginae, suggesting that heteroecy could have evolved separately, and more recently, in Aphidinae.

7.2 Types of sexuparae In heteroecious aphids, either the sexuparae or the sexuals must be alate. Alate sexuparae, giving birth to dwarf males and females, are therefore normally associated with host alternation, and are found in heteroecious genera of Adelgidae, Phylloxeridae, Pemphiginae, Hormaphidinae and Anoeciinae, where they are the only morphs which give rise to the sexuals. However, alate sexuparae belonging to this category are also known to occur in the monoecious Phylloxera glabra and Thelaxes dryophila, where again they appear to be the only source of the sexuals. It is possible that the retention of an alate sexupara which produces dwarf sexuals may have resulted from the evolution of such monoecious species from heteroecious ancestors. Both winged and wingless sexuparae occur in Glyphina and Mindarus, but again, all of the winged morphs are sexuparae. Alate sexuparae giving rise to normal-sized sexuals occur in many genera of Drepanosiphinae, which are all, monoecious. In monoecious Aphidinae sexuparae are normally wingless, but winged sexuparae were found occasionally to occur in Acyrthosiphon pisum (Mackay et al, 1 9 8 3 )* These sexuparae, however, contained only the embryos of oviparae, thus resembling gynoparae of heteroecious Aphidinae in their biology.

7-3 Host-plant The subject of aphid evolution in relation to host-plant specificity has been dealt with in some detail by Eastop (1973, 1986). He suggested that aphids evolved on Hamamelidae or on an extinct group of Gymnospermae and transferred to Hamamelidae and Coniferae, and then from Hamamelidae to Coniferae, Dilleniidae and Rosidae, and further from Rosidae to Caryophyllidae and Asteridae. Unfortunately, there appears to be no system of plant family classification, let alone phylogeny, which is currently accepted by all the major authorities. For example, the system of Thorne (1980) places Urticales and Maivales in proximity within the superorder Malviflorae, whereas the systems of both Cronquist (1981) and Takhtajan (1980) place them in separate subclasses (corresponding to Thome's superorders). Each subfamily of Aphidoidea is broadly associated with a particular subclass of plants, at least as primary hosts, and these are summarized below (Table 7.1). Cronquist's (1981) classification is followed for angiosperms.

Table 7.1 Host-plant associations at the higher level in Aphidoidea APHID FAMILY, SUBFAMILY SUBCUSS OF HOSTS

Adelgidae Pinidae Phylloxeridae Hamamelidae, Dilleniidae Lachninae Pinidae, Hamamelidae, Rosidae Pterocommatinae Dilleniidae Aphidinae Rosidae, Asteridae Greenideinae (incl. Anomalaphidinae) Hamamelidae, Dilleniidae, Rosidae Chaitophorinae Dilleniidae, Rosidae, Commelinidae Drepanosiphinae Hamamelidae, Rosidae, Commelinidae Phleomyzinae Dilleniidae Mindarinae Pinidae Thelaxinae Hamamelidae Anoeciinae Rosidae, Commelinidae Hormaphidinae Hamamelidae, Asteridae Pemphiginae Dilleniidae, Hamamelidae.

In Table 7.1 a ll but one of the subclasses of dicotyledons are represented, and the remaining subclass, Magnoliidae, does contain a few host-plants of aphids. However, the main groups of aphid hosts fa ll into the Pinidae, prim arily Coniferales; Hamamelidae, prim arily Fagales; and

D illeniidae, prim arily Salicales. Within Pemphiginae Eriosomatini are associated w ith U rticales (Hamamelidae) whereas Pemphigini are on Salicales

(D illeniidae). If, as Eastop (1973) states, and as seems like ly, aphids

were on Hamamelidae before D illeniidae^ then the capture of hosts belonging 244 to the latter subclass must have occurred on a number of occasions, and this is, perhaps, not very suprising. However, according to Humphries (1982) Dilleniidae evolved before Hamamelidae, although this says nothing about the relative ages of Salicales and Fagales. At present, what little is known of host-plant phylogeny gives little information about aphid phylogeny, and were more information available, relationships between host-plants could only, at best, support morphological data and fossil evidence.

7-4 Alate sexual morphs According to Mordwilko (1928) nAt the beginning there existed in plant-lice but two forms of individuals, winged females and winged males, and several generations a year". Winged males occur in many species of Lachninae, Pterocommatinae, Aphidinae, Greenideinae, Chaitophorinae, Drepanosiphinae and Phleomyzinae. Winged oviparae occur, however, only in the following taxa: Greenideinae, Drepanosiphinae (Tamalia, Neophyllaphis and Drepanosiphum califomicum) and Phleomyzinae. There is little evidence to suggest that the occurrence of alate oviparae is a primitive characteristic of Aphididae. Even if this were the case, the possession of a single primitive character would not justify regarding these taxa as "relics of the past". Alate oviparae also occur in the genus Aiceona (Takahashi, 1960), currently classified among Anoeciinae. The occurrence of alate oviparae in this genus, coupled with the appearance of the male external genitalia (see section 6.5) supports the reclassification of this genus among Greenideinae.

7.5 Dwarf sexual morphs. In many species of Coccoidea, the males are dwarfish. Both males and sexual females are dwarfish in all Phylloxeridae, Adelgidae, Pemphiginae, heteroecious Anoeciinae and Thelaxes. In Hormaphidinae, monoecious Anoeciinae, Glyphina, some Chaitophorinae and Aphidinae, and Stomaphis, males are dwarfish or rather small but oviparae are either normal-sized or almost normal-sized. There is certain evidence, therefore, to suggest that the occurrence of dwarfish sexuals, or at least dwarfish males, might be a primitive aspect of aphid life-cycles. The similarities between the life-cycles of, for example, Phylloxera glabra and Thelaxes dryophila are striking. In addition, biennial life-cycles, with gall-formation, host-alternation and dwarfish sexuals with three-lensed eyes, occur in Adelgidae as well as Hormaphidinae and some Pemphiginae. It would be unwise, therefore, to dismiss completely the hypothesis that the ancestor of Aphididae also had dwarfish sexuals, or ju st dwarfish males, even though the previous survey of male genitalia (section 6) suggested that this is not the case. It does seem more lik e ly , however, that reduction in the size of the sexual morphs has occurred in many lineages. In the genus Anoecia both dwarf and normal-sized oviparae occur. The former in the heteroecious species and the la tte r in the monoecious species on Graminae and Car ex. Paul (1977) postulated that monoecious, holocyclic

Anoecia species are derived from previously heteroecious, holocyclic species which have firs t become anholocyclic on the secondary host and then evolved oviparae secondarily, from virginoparae. It seems more lik e ly that the ancestral Anoecia species was heteroecious and holocyclic, but that sexuparae gave b irth to dwarfish males and normal-sized oviparae. In the resulting lineages, dwarfish oviparae evolved in the heteroecious species in which this would be an advantage, but oviparae remained normal-sized in the monoecious lineage. The advantage to a species of having dwarf sexuals is presumably the reduction of th e ir developmental time, but why, as appears to be the case, should th is advantage be peculiar to heteroecious species? In a monoecious species the generations preceding the sexuals are obtaining a great deal of information about the nutritional state of the host on which the sexuals w ill later be living, and may therefore be better able to time the production of sexuals. Sexuparae of heteroecious species have no such inform ation, and may, therefore, often encounter primary hosts which are already well into the last stages of their seasonal development, and perhaps barely able to sustain the development of the sexuals. In such

cases, sexuals having a shorter developmental time would clearly be at an advantage. This would also be applicable for those monoecious species in which the sexuparae are always alate, e.g. Phylloxera glabra and Thelaxes dryophila. Heteroecious Aphidinae have been shown, in many cases, to respond to photoperiod, and this could explain why dwarf oviparae do not occur in these species, i.e . they are better able to time the production of

the sexuals. However, should a photoperiodic response ever be demonstrated in heteroecious Phylloxeridae, Adelgidae, Pemphiginae, Hormaphidinae or Anoeciinae, then some other explanation would become necessary. In some monoecious species, males are dwarfish whereas oviparae are more or less normal-sized, e.g. Stomaphis quercust Anoecia pskovica. The reduction of developmental time would bring such males more quickly to sexual m aturity and thus give them an advantage in competition fo r mating. 246

7.6 Reduction or loss of mouthparts and digestive tract in sexuals The reduction in overall body size of the sexuals has, in some species, been associated with the reduction or loss of mouthparts and digestive tra ct. This has occurred in males of some coccid genera, in the sexuals of Pemphiginae and Phylloxeridae and in the genus Stomaphis of Lachninae, i.e . in at least four separate lineages. Furthermore, N iisslin’s (1910) study of Mindarus obliquus revealed that, compared with the ovipara, the digestive system is greatly reduced in the male of this species. This suggests that Mindarus could represent a prim itive pemphigine, but there is little further supporting evidence for this idea.

7.7 The three-lensed eye The occurrence of the three-lensed eye as a larval character also present in some apterae has been the subject of debate on aphid phylogeny by Hi l i e Ris Lambers (1961), Mackauer (1965) and Heie (1967), of which the la st author has commented at great length on its phylogenetic significance.

Most alate adult aphids have a large multilensed eye with a three-lensed ocular tubercle next to it. It is thought that the three-lensed larval eye

is homologous w ith the adult’s ocular tubercle. In some species, immatures (and sometimes apterae) are equipped only with the three-lensed eye, whereas in others, larvae also have a m ultilensed eye. The former condition occurs in the larvae of some or a ll species of Aphidoidea in every subfamily except Chaitophorinae and Aphidinae. In Lachninae it occurs only in Trama. It is almost certainly a plesiomorphic character fo r Aphidoidea, and the acquisition, by the larva, of the m ulti-facetted eye has. probably occurred in many lineages.

Within the Drepanosiphinae, a few genera have larvae with just the three-lensed eye, and these include C eriferella, Lizerius, Neophyllaphist P aoliella, Parachaitophorus, and Tamalia. Interestingly, none of the hosts of these genera belongs to either Hamamelidae or Commelinidae, to which the

hosts of most other Drepanosiphinae belong. Other aspects of the morphology of this subfamily (see 7.8, 7.9) also suggest that it is polyphyletic.

7.8 Sub-siphuncular wax-gland plates in oviparae Drepanosiphinae have in the past, been defined partly by the oviparae having wax-plates on the ventral abdomen. The presence or absence of these

wax-plates was recorded in over sixty genera (Eastop & Polaszek, in press) and it was found that oviparae of well over half the genera of Drepanosiphinae studied lacked wax-plates. Wax-plates were also present in

the follow ing subfam ilies: Phleomyzinae, Mindarinae, Thelaxinae, Anoeciinae (monoecious spp.), Hormaphidinae and Pemphiginae. They were absent from

Fhylloxeridae and Adelgidae. A number of different shapes and types of fenestration of the wax-plates were found to occur, suggesting that wax-plates may have evolved more than once w ithin Aphididae.

7.9 Bilobed anal plate Like the presence of wax-plates in the ovipara, the possession of a bilobed anal plate has been used in the past as a characteristic of

Drepanosiphinae. Over fifty genera of Drepanosiphinae were therefore examined for the presence of this character, and it was absent in only nine. However, these nine included the genus Drepanosiphum, which gives its name to the subfamily and is also without wax-plates in the ovipara. A bilobed anal plate is also present in certain Hormaphidinae.

7.10 Number of rudimentary gonapophyses The survey data in section 5 suggest that the possession of four rudimentary gonapophyses is the plesiomorphic condition fo r Aphididae. This has been retained in either some or a ll species of Lachninae, Greenideinae, Chaitophorinae and Drepanosiphinae. Reductions have therefore taken place

in a number of lineages, and furthermore, the number can vary w ithin a

species • Thus although the number of rudimentary gonapophyses is a character worthy of consideration in the construction of a higher classification, particularly as an indication of which members of a particular subfamily are the more recently evolved, it has been used incorrectly in the past to categorize certain subfamilies.

7.11 Presence of an ovipositor-like structure The apparently ovipositor-like structure in Paoliella species (section 5) could be a plesiomorphy which has been lo st in a ll other aphid genera. Further studies are necessary to confirm the presence of this structure.

SUMMARY: Phylogeny While the survey of male genitalia (section 6) and this brief discussion of other morphological and life-cycle characteristics have drawn attention to some of the affinities which exist between certain genera and subfamilies, a number of equally plausible scenarios could be put forward for the evolution of the existing aphid subfamilies. Furthermore, characters can be found in almost any subfamily of Aphididae which suggest that it may be the most primitive. However, the polyphyletic nature of some 248

long-standing taxa has also been shown by the present survey. The taxon which is the most obviously polyphyletic is Drepanosiphinae, corresponding approximately to Callaphidinae or C allipterinae of other authors. Thirteen of the twenty subfamilies recognised in the most recent classification (Remaudi&re & Stroyan, 1984.) have been placed in Drepanosiphinae at one time or other, and eight of these have always previously been in Drepanosiphinae. This new classification may go a long way towards sorting out the confusion. However, the characters used fo r defining each subfamily

(except Tamaliinae) are not mentioned, and certain of the genera included together in some of the new subfamilies differ in one or more of the characters discussed above. The classification is based on morphological characters, and does not take any account of characters which could be extracted from a comparative study of genitalia, nor of anatomical characters (Remaudi&re, personal communication). The present work has served to confirm many of the existing divisions of aphid genera into subfamilies, but the plesiomorphic condition of male genitalia in a ll the drepanosiphine genera studied has meant that the survey has not helped in resolving the group in which most of the problems appear to lie .

There appears to have been a great deal of convergence in aphid evolution, particularly associated with reduction in the size of the sexual morphs and the consequent effect of this on the morphology. The present study, though aimed at resolving the problems of aphid higher classification, has served mainly to underline what Heie (1980) has so appositely called the "mosaic distribution of apomorphic characters". In order to get closer to a stable higher classifation, there is a necessity for analysis of a large number of characters, including life-cycle characters and anatomical features. Recently developed computerized methods of phylogenetic analysis would greatly fa c ilita te such studies. 249

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