Sociality of Spiders of the Genus Anelosimus (Araneae: Theridiidae), with Focus on Species in Southeast and East Asia

SOCIALITY OF SPIDERS OF THE GENUS ANELOSIMUS (ARANEAE: THERIDIIDAE), WITH FOCUS ON SPECIES IN SOUTHEAST AND EAST ASIA

GOH SEOK PING

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

NATIONAL UNIVERSITY OF SINGAPORE

2014

SOCIALITY OF SPIDERS OF THE GENUS ANELOSIMUS (ARANEAE: THERIDIIDAE), WITH FOCUS ON SPECIES IN SOUTHEAST AND EAST ASIA

GOH SEOK PING B.Sc. (Hons.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2014

DECLARATION

I hereby declare that this thesis is my original work and it has been written by me in its entirety except the phylogenetic tree in Chapter 1 which was analyzed by Dr. Ingi Agnarsson from the University of Vermont, USA. I have duly acknowledged all the sources of information which have been used in the thesis.

This thesis has also not been submitted for any degree in any university previously.

Goh Seok Ping 31 May 2014

i ACKNOWLEDGEMENTS

To God, the Creator of all things.

To my parents, for giving me the freedom and support I needed to follow my interest.

To Matthew, thank you for your patience, support, encouragement and ‘naggings’, which I needed.

To my family and friends, Thank you for standing by me and believing in me. Your prayers and encouragement have carried me this far.

I would like to thank many who have helped me in this dissertation:

Special thanks to my supervisor Assoc. Prof. Li Daiqin, for allowing me to pursue my topic of interest and supporting me in the completion of this dissertation.

Thanks to fellow lab mates (past and present): Shiyu, Junhao, Diego, Zhanqi, Shichang, Jeremy, Laura, Stanley, Mindy, Joelyn, Jiafen, Ganison and Yuan Ting for making lab a fun filled place and for the company through lunches, tea breaks and late nights. Special thanks to Junhao, Laura, Shiyu, Wendy, Xu Xin and Yingying for enduring the hot sun, downpours, mosquitoes and leeches with me during field trips.

Thanks to Mr. Chong and Mdm. Loy, for their help in usage of the S.E.M.

I would also like to thank the National Parks Board and Agri-Food & Veterinary Authority of Singapore for the issuing of research and import permits.

For those who taught and inspired me, Dr. Ingi Agnarsson and lab members, for an opportunity to work and learn in the lab and for providing me with invaluable advice and specimens from Borneo and Indonesia.

David Court, for his help and enthusiasm and in providing specimens from Singapore.

Dr. Matjaž Kuntner and members of EZ lab, for the opportunity to participate in 27th European Congress of Arachnology and for inspiring me with his passion for research and his encouragement.

Prof. Rudolf Meier and Dr. Kwong Shiyang, for guiding me and getting me started on the daunting task of molecular genetics.

ii TABLE OF CONTENTS

SUMMARY vi LIST OF TABLES x LIST OF FIGURES xii

INTRODUCTION Sociality in the animal kingdom 1 Evolution of sociality in spiders 3 The genus Anelosimus 8 Research objectives 10 References 13

CHAPTER 1: SYSTEMATICS AND PHYLOGENY OF SOUTHEAST AND EAST ASIAN SPIDERS OF THE GENUS ANELOSIMUS (ARANEAE: THERIDIIDAE), WITH THE DESCRIPTIONS OF TEN NEW SPECIES Abstract 28 Introduction 29 Materials and Methods 31 Spiders collection 31 Taxonomy 31 Phylogenetics 32 Results 36 Morphology 36 Phylogeny 36 Natural History 37 Discussion 40 Taxonomy 43 References 123

CHAPTER 2: NATURAL HISTORY AND BEHAVIOURAL OBSERVATIONS OF ANELOSIMUS SPECIES WITH FOCUS ON SOCIAL AND REPRODUCTIVE TRAITS Abstract 129 Introduction 131 Materials and Methods 137 Colony survey in the field 137 Spider collection and maintenance in laboratory 137 Behavioural observations in laboratory 142 Reproductive traits 145 Data analyses 145 Results 148 Colony survey in the field 148 Colony structure 152 Life cycle 153 Maternal care 155 Cooperative prey capture and food sharing 156 Natal dispersal 160 Reproductive traits 161

iii Discussion 171 References 183

CHAPTER 3: SENSORY RECEPTORS AND SOCIALITY IN SPIDERS OF THE GENUS ANELOSIMUS (ARANEAE: THERIDIIDAE) Abstract 192 Introduction 193 Materials and Methods 198 Study subjects 198 Sample preparation 198 Data analyses 199 Results 201 Tactile hair 201 Chemoreceptor 204 Slit sensillum 205 Trichobothrium 207 Discussion 216 References 224

CHAPTER 4: ENVIRONMENTAL FACTORS AFFECTING THE GEOGRAPHICAL DISTRIBUTION OF ANELOSIMUS SPECIES Abstract 233 Introduction 234 Materials and Methods 238 Species and spatial data 238 Environmental data 240 Data processing and analyses 241 Results 244 Species distribution 244 Variation partitioning 245 Environmental variables 246 Discussion 252 References 257

CHAPTER 5: PARASITES AND SOCIALITY OF ANELOSIMUS SPIDERS Abstract 266 Introduction 267 Materials and Methods 272 Nest collection and examination 272 DNA extraction and detection of Wolbachia 276 Data analyses 277 Results 278 Nest contents 278 Wolbachia detection 279 Discussion 284 References 290

iv OVERALL DISCUSSION Overall Discussion 301 Future directions 305 References 307

APPENDIX Table 1 310 Table 2 312

v SUMMARY

The formation of groups is pervasive in the animal kingdom. Groups formed may be

temporal or permanent and are sometimes made up of related individuals or unrelated

ones. The purpose of group formation can also vary from predator defense and brood

fostering to increased foraging efficiency. While group formation is generally rare in

spiders, the occurrence of closely related social spider groups is exceptionally unusual.

Out of 44,000 spider species, less than 25 species have been documented as social.

Social behaviour is believed to have evolved 12 times in spiders, of which three

derivations have occurred in the family Theridiidae. To date, the genus Anelosimus

(Araneae: Theridiidae) consists of 64 nominal species, of which six are social, 14

subsocial and five solitary living, while the sociality of the remaining species are

unknown. Whilst intensive work has been conducted on spiders of this genus in North

and South America, little is known about Anelosimus in Asia. A comprehensive study of spiders of this genus is important due to its cosmopolitan distribution as well as its composition of spiders with a spectrum of social behaviour. Therefore, the goal of this thesis is to supplement the knowledge of spiders of the genus Anelosimus, with

concentration on species found in Southeast and East Asia. While 13 species of

Anelosimus has been described in Southeast and East Asia to date, the natural history

of many remains undetermined. Moreover, almost half of these species were described before the year 2000. With the advancement of technology, I thus redescribed 11 of these species, supplementing past taxonomic descriptions with photographs, updates of missing male/female descriptions and information on natural history. Furthermore, I also included description of nine new species collected from

Malaysia and Indonesia.

vi In order to gain a better understanding of the evolution of sociality within this genus of spiders, I further examined the natural history of ten species of Anelosimus collected from China, Malaysia and Singapore, with special focus on traits such as habitat and colony structure, maternal care, cooperative behaviour, dispersal and a

number of reproductive traits. I determined that seven of these species are solitary

living, while the remaining three species are subsocial. Solitary species dispersed

from the natal nest by 3rd instar but exhibited prey sharing amongst siblings before

dispersal. In contrast, subsocial species disperse from the natal nest before reaching

maturity, between 4th to 6th instars. Females of all species studied displayed maternal

care through guarding of egg sacs and provision of prey for 2nd instar spiderlings.

Interestingly, I found that even solitary Anelosimus possessed traits such as reduced

dispersal distance and maternal care that suggest that this group of spiders may be predisposed to the development of social behaviour.

In Chapter 3, I looked at the sensory receptors of eight Anelosimus species (six solitary and two subsocial) to determine if a reduced sensitivity to external signals corresponds with the evolution of social behaviour. I found that subsocial species possessed lower mechanoreceptor densities as compared to solitary species although there is no significant difference in total mechanoreceptor densities between solitary and subsocial spiders.

Besides morphological and behavioural disposition to the evolution of sociality, abiotic factors in the habitat may also affect the evolution of social behaviour in Anelosimus. Social spiders tend to occur in lowland tropical rainforests, while subsocial species occur at higher latitudes and altitudes. A number of environmental factors similarly vary according to changes in latitudes and altitudes.

Therefore, in Chapter 4, I analyzed the worldwide distribution of 35 Anelosimus

vii species and determined that solitary species tend to occur in habitats with high rainfall

and temperature, subsocial species occur in areas with low rainfall and temperatures

while social species are commonly found in areas with high rainfall and intermediate

temperatures. Possible explanations for this observation are associated with ability of

web structure to withstand rain damage, prey abundance and effects of temperatures

on metabolism and growth of spiders.

Finally, parasitism has been closely associated with social groups across

different animal taxa. The rate of parasitism is therefore examined in Chapter 5. In

this chapter, I investigated the prevalence of parasites in nests of eight Anelosimus

species, including six solitary and two subsocial, and tested for the presence of the

endosymbiotic bacteria Wolbachia in 37 species of Anelosimus. Results obtained

suggest that cooperative web maintenance from juveniles in subsocial species may aid

in reducing the number of parasites in the bigger nests of subsocial spiders.

Meanwhile due to solitary spiders’ preference in constructing webs on small

wildflowers, there are higher numbers of parasitic organisms such as mites,

lepidopteran larva and hymenopterans in their nests. Wolbachia infection was also not

found to be widespread amongst Anelosimus species tested. However, infection appears to be more prevalent in social species. As Wolbachia has been shown to induce female biased sex ratio and other endosymbionts such as Cardinium has been shown to affect the dispersal distance of spiders, it is pertinent to further investigate the role of endosymbionts in the development of social behaviour in this group of spiders.

Overall, my research on Anelosimus species in Southeast and East Asia has revealed a high concentration of solitary species and an absence of social species in this region. However, field surveys conducted were not comprehensive and it is

viii expected that more species of Anelosimus will be discovered with more widespread surveys. In spite of this, by examining solitary and subsocial species of Anelosimus in this region, I have determined that subsocial species have a lower density of mechanoreceptors, which may result in lowered sensitivity and aggression towards conspecifics. Furthermore, rainfall and temperature have also been found to affect the distribution of solitary, subsocial and social spiders. Lastly, the role of endosymbionts such as Wolbachia in the development of sociality should be further investigated.

ix LIST OF TABLES TABLE PAGE TITLE NO. NO.

1.1 Primer sequences and sources, and optimal annealing temperatures. 35

2.1 List of Anelosimus species collected with GPS coordinates and 139 elevation of collection sites.

3.1 Results from ANOVAs testing the differences in the mean tactile 203 hair density on Legs among eight species of Anelosimus.

3.2 Results from ANOVAs testing the differences in the mean total 203 number of tactile hairs per spider on Legs among eight species of Anelosimus.

3.3 Results from ANOVAs testing the differences in the mean 204 chemoreceptor density on Legs among eight species of Anelosimus

3.4 Results from ANOVAs testing the differences in the mean total 205 number of chemoreceptor on Legs among eight species of Anelosimus

3.5 Results from ANOVAs testing the differences in the mean slit 206 sensillum density on Legs among eight species of Anelosimus

3.6 Results from ANOVAs testing the differences in the mean total 207 number of slit sensilla on Legs among eight species of Anelosimus

3.7 Results from ANOVAs testing the differences in the mean 208 trichobothrium density on Legs among eight species of Anelosimus

3.8 Results from ANOVAs testing the differences in the mean total 209 number of trichobothria on Legs among eight species of Anelosimus

4.1 List of 35 Anelosimus species partitioned into three groups 239 according to level of sociality.

TABLE TITLE PAGE NO. NO.

4.2 Principal component scores and loadings of PC1–3, based on four 243 selected environmental variables.

4.3 Variables selected from forward selection and included in variation 246 partitioning

5.1 List of Anelosimus species nests examined. Localities, GPS 274 coordinates, elevation, habitat type and number of nests collected for each species are as listed.

5.2 Total count of non-Anelosimus inhabitants in the nests of 8 species 280 of Anelosimus according to type and average number of individuals per nest. Nest intruders are in bold

5.3 List of Anelosimus species tested for presence of Wolbachia using 282 amplification of wsp gene. Absence of band in electrophoresis of PCR product indicates absence of Wolbachia. Presence of strong or faint bands indicates presence of Wolbachia and these are in bold.

xi LIST OF FIGURES FIGURE PAGE TITLE NO. NO.

1.1 Phylogeny of Anelosimus based on Bayesian analyses of the 39 concatenated molecular matrix. Branches are coloured according to geographical distribution. Branches are proportional to length as estimated in MrBayes, outgroups are omitted for clarity, see Figure 1–20 for full phylogeny. Social species are indicated with a red circle, subsocial with a blue circle, solitary with an open circle and species with unknown social behaviour lack a symbol. Numbers on nodes are posterior probability support values. Scale shows expected substitutions per site.

1.2 Anelosimus cameronis, sp. nov. (A) Female. Female epigynum: 47 (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Ventral; (E) Ectal. (F) Male. (G) Female lateral; (H) Male lateral. Scale bars as indicated below each photograph.

1.3 Anelosimus cameronis sp. nov. Male palp: (A) Ectal; (B) 48 Subventral; (C) Ventral and (D) Details of sclerites, cymbial hood (smaller arrow) and embolus (larger arrow). Scale bar as indicated in each photograph.

1.4 Anelosimus chonganicus. (A) Female. Female epigynum: (B) 52 Ventral; (C) Dorsal, cleared. Male palp: (D) Ventral; (E) Lateral. (F) Male. (G) Female lateral. (H) Male lateral. Scale bars as indicated below each photograph.

1.5 Anelosimus chonganicus. Male palp: (A) Ventral and (B) details 53 of sclerites, cymbial embolic groove (smaller arrow), and embolus (larger arrow). Scale bars as indicated in each photograph.

1.6 Anelosimus subcrassipes. (A) Female. Female epigynum: (B) 56 Ventral; (C) Dorsal, cleared, (D) Female lateral. Scale bars as indicated below each photograph.

1.7 Anelosimus exiguus. (A) Female. Female epigynum: (B) Ventral; 60 (C) Dorsal, cleared. (D) Male palp ventral. (E) Male. (F) Female lateral. (G) Male lateral. Scale bars as indicated below each photograph.

1.8 Anelosimus exiguus. Male palp: (A) Ventral; (B) Ectal and (C) 61 Details of sclerites. Arrow indicates the embolus. Scale bars as indicated in each photograph.

xii

FIGURE PAGE TITLE NO. NO.

1.9 Anelosimus kohi. (A) Female. Female epigynum: (B) Ventral; 65 (C) Dorsal, cleared. Male palp: (D) ventral; (E) lateral. (F) Male. (G) Female lateral. (H) Male lateral. Scale bars as indicated below each photograph.

1.10 Anelosimus kohi. Male palp: (A) Ventral; (B) Details of sclerites. 66 Arrows indicate embolus. Scale bar as indicated in each photograph.

1.11 Anelosimus parvulus sp. nov. (A). Female. Female epigynum: 69 (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

1.12 Anelosimus parvulus sp. nov. Male palp: (A) Subventral; (B) 70 Ventral and (C) Details of sclerites. Arrows indicate the emobolus. Scale bar as indicated in each photograph.

1.13 Anelosimus membranaceus. (A). Female. Female epigynum: (B) 74 Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

1.14 Anelosimus membranaceus. Male palp: (A) Ventral; (B) 75 Subventral and (C) Details of sclerites. Arrows indicate emobolus. Scale bars as indicated in each photograph.

1.15 Anelosimus taiwanicus. (A) Female. Female epigynum: (B) 79 Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Lateral. (F) Male. (G) Female lateral. (H) Male lateral. Scale bars as indicated below each photograph.

1.16 Anelosimus taiwanicus. Male palp: (A) Ventral; (B) Ectal and 80 (C) Details of sclerites. Arrows indicate the emobolus. Scale bars as indicated in each photograph.

1.17 Anelosimus kinabaluensis, sp. nov. (A) Female. (B) Male palp, 84 Ventral; Female epigynum: (C) Ventral; (D) Dorsal, cleared. (E) Female lateral. Scale bars as indicated below each photograph.

xiii FIGURE PAGE TITLE NO. NO.

1.18 Anelosimus kinabaluensis sp. nov. Male palp: (A) Ventral and 85 (B) Details of sclerites. Arrow indicate the embolus. Scale bars as indicated on each photograph.

1.19 Anelosimus floreni sp. nov. (A) Female. Female epigynum: (B) 88 Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

1.20 Anelosimus floreni sp. nov. Male palp: (A) Ventral and (B) 89 Details of sclerites. Arrows indicate embolus. Scale bars as indicated in each photograph.

1.21 Anelosimus cochlianis, sp. nov. (A) Female. Female epigynum: 93 (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

1.22 Anelosimus cochlianis sp. nov. Male palp: (A) Ventral and (B) 94 Details of sclerites. Arrow indicate embolus. Scale bar as indicated in each photograph.

1.23 Anelosimus hiatus, sp. nov. (A) Female. Female epigynum (B) 97 Ventral; (C) dorsal, cleared. (D) Female lateral. Scale bar as indicated below each photograph.

1.24 Anelosimus seximaculatum. (A) Female. Female epigynum: (B) 101 Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

1.25 Anelosimus seximaculatum. Male palp: (A) Ventral and (B) 102 Subventral. Arrows indicate embolus. Scale bars as indicated in each photograph.

1.26 Anelosimus helix. (A). Female. Female epigynum: (B) Ventral; 106 (C) Dorsal, cleared. (D) Male. Male palp: (E) Mesal; (F) Ventral. (G) Lateral. (H) Female lateral. (I). Male lateral. Scale bars as indicated below each photograph.

xiv FIGURE PAGE TITLE NO. NO.

1.27 Anelosimus helix sp. nov. Male palp: (A) Ventral and (B) Details 107 of sclerites. Arrow indicate embolus. Scale bar as indicated in each photograph.

1.28 Anelosimus albus, sp. nov. Female only. (A) Female. Epigynum: 110 (B) Ventral; (C) Dorsal, cleared. (D) Female lateral. Scale bars as indicated below each photograph.

1.29 Anelosimus circumdantus, sp. nov. (A) Male. (B) Male lateral. 113 Male palp: (C) Mesal; (D) Ventral; (E) Lateral. Scale bars as indicated below each photograph.

1.30 Anelosimus circumdantus sp. nov. Male palp: (A) Ventral and 114 (B) Subventral. Arrow indicate embolus. Scale bar as indicated in each photograph.

1.31 Anelosimus bali. (A) Female. Female epigynum: (B) Ventral; (C) 117 Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

1.32 Anelosimus bali. Male palp: (A) Ventral and (B) Details of 118 sclerites, cymbial embolic groove (smaller arrow), and embolus (larger arrow). Scale bars as indicated in each photograph.

1.33 Anelosimus deelemanae, sp. nov. Female only. (A) Female. 121 Female epigynum: (B) Ventral; (C) Dorsal, cleared. (D) Female lateral. Scale bars as indicated below each photograph.

1.34 Full phylogeny including all outgroups and posterior probability 122 support for each clade.

2.1 (A) Glass cage (110 x 55 x 155 mm) used for dispersal 147 experiments. (B) Glass cage was placed within a translucent plastic container (190 x 115 x 155 mm) in a ‘cage-within-cage’ setup used to determine the natal dispersal.

2.2 (A) Web of Anelosimus chonganicus. (B) Web of A. kohi. (C) 150 Web of Anelosimus sp. B. (D) Web of Anelosimus sp. A. (E) Flowers on which Anelosimus sp. C were found. (F) Ixora plant on which A. taiwanicus were found.

xv FIGURE PAGE TITLE NO. NO.

2.3 Plants species on which Anelosimus species were found. (A) 151 Scleria bancana. (B) Cyperus rotundus. (C) Cyanthillium cinereum. (D) Stachytarpheta jamaicensis. (E) Chamaesyce thymifolia. (F) Eragrostis tenella. All photos provided by (Kwan, 2013).

2.4 Colony structure of Southeast and East Asian Anelosimus species 154 encountered during field surveys. Percentage of spiders in different life stages and groups represented by different colour codes. Sample sizes as indicated below species name.

2.5 Anelosimus kohi female and spiderlings sharing a housefly. 158

2.6 Typical sequence of behaviour and movement during prey 159 capture in Southeast and East Asian Anelosimus. Observations conducted under laboratory conditions.

2.7 Box plot of number of days taken for spiderlings to disperse from 165 natal nest under laboratory conditions. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Different lower case letters above column indicates statistically significant difference (p < 0.05). Circle indicates outliers. Asterisks above each column indicate omission from statistical analyses due to small sample sizes. Sample sizes as indicated below species name

2.8 Mating position of four Anelosimus species. (A) A. chonganicus, 166 (B) A. parvulus, (C) A. taiwanicus and (D) A. kohi.

2.9 Boxplot of copulation duration (min) of seven species of 167 Anelosimus examined. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Sample sizes as indicated below species name

2.10 Mean (± s. e.) number of spiderlings emerged from each egg sac. 168 Different lower case letters above column indicates statistically significant difference (p < 0.05). Asterisks above each column indicate omission from statistical analyses due to small sample sizes. Sample sizes as indicated below species name.

xvi FIGURE PAGE TITLE NO. NO.

2.11 (A) Boxplot of clutch (number of egg sacs) produced per female. 169 (B) Boxplot of time (d) for incubation of eggs. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Different lower case letters above each column indicates statistically significant difference (p < 0.05). Circle indicates outlier. Asterisks above column indicate omission from statistical analyses. Sample sizes as indicated below species name

2.12 (A) Mean (± s. e.) interval (d) between hatching of spiderlings 170 and production of subsequent egg sac. (B) Mean (± s. e.) interval (d) between clutches. Different lower case letters above each column indicates statistically significant difference (p < 0.05). Asterisks above column indicate omission from statistical analyses. Sample sizes as indicated below species name

3.1 Scanning electron microscopy photographs showing sensory 200 receptors of spiders indicated by white arrows. (A) Tactile hairs, (B) Chemoreceptors, (C) Slit sensilla, (D) Trichobothria. Scale bars as indicated on each photograph.

3.2 Mean density of sensory receptors in eight species of 210 Anelosimus. (A) Mean ( s.e.) density of tactile hair; (B) Mean ( s.e.) density of chemoreceptor; (C) Mean ( s.e.) density of trichobothrium; and (D) Mean ( s.e.) density of slit sensillum. Different lower cases indicate significant difference (p < 0.05).

3.3 Mean total number of receptors per spider of eight species of 211 Anelosimus. (A) Mean ( s.e.) number of tactile hairs; (B) Mean ( s.e.) number of chemoreceptors; (C) Box plot of number of slit sensilla; (D) Mean ( s.e.) number of trichobothria. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. No significant differences were found in mean receptor number in all species

xvii FIGURE PAGE TITLE NO. NO.

3.4 Mean (± s.e.) tactile hair density of eight species of Anelosimus. 212 (A) Mean (± s.e.) density of tactile hair on Legs I; (B) Box plot of the density of tactile hair on Legs II; (C) Mean (± s.e.) density of tactile hair on Legs III; and (D) Box plot of the density of tactile hair on Legs IV. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Different lower cases indicate significant difference (p < 0.05).

3.5 Mean chemoreceptor density of eight species of Anelosimus. (A) 213 Mean (± s.e.) density of chemoreceptor on Legs I; (B) Mean (± s.e.) density of chemoreceptor on Legs II; (C) Mean (± s.e.) density of chemoreceptor on Legs III; and (D) Mean (± s.e.) density of chemoreceptor on Legs IV. No significant differences were found.

3.6 Mean slit sensillum density of eight species of Anelosimus.(A) 214 Mean (± s.e.) density of slit sensillum on Legs I; (B) Mean (± s.e.) density of slit sensillum on Legs II; (C) Mean (± s.e.) density of slit sensillum on Legs III; and D. Mean (± s.e.) density of slit sensillum on Legs IV. Different lower case letters indicate significant differences (p < 0.05).

3.7 Mean trichobothrium density of eight species of Anelosimus. (A) 215 Mean (± s.e.) density of trichobothrium on Legs I; (B) Mean (± s.e.) density of trichobothrium on Legs II; (C) Mean (± s.e.) density of trichobothrium on Legs III; and (D) Mean (± s.e.) density of trichobothrium on Legs IV. Different lower case letters indicate significant differences (p < 0.05).

4.1 Global distribution of Anelosimus species examined in this study. 244 Each circle represents one data point. Legend as shown. Image by Reto Stockli NASA’s Earth Observatory, using data courtesy of USGS.

xviii FIGURE PAGE TITLE NO. NO.

4.2 Results of variance partitioning to separate purely environmental 246 (grey), spatially structure environmental (white) and purely spatial (patterned) components of variance explaining the distribution of Anelosimus species. The environmental variables are composed of synthetic variables PC1 and PC3, while spatial variables consist of geographical latitude and selected PCNM variables. Unexplained variance is represented as “Other factors” (black).

4.3 Gradient of environmental variables. (A) Annual mean 248 temperature and (B) Annual precipitation, with distribution of Anelosimus mapped. Legend as shown.

4.4 Gradient of environmental variables. (A) Maximum temperature 249 of warmest month and (B) Minimum temperature of coldest month, with distribution of Anelosimus mapped. Legend as shown.

4.5 Boxplot of (A) Annual mean temperature, (B) Maximum 250 temperature of warmest month, (C) Minimum temperature of coldest month, and (D) Annual precipitation of localities of social, subsocial and solitary Anelosimus species. . Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Circles outside boxplot represent outliers.

4.6 Plot of mean of synthetic variables, PC1 to PC3 extracted from 251 PCA of four environmental variables (mean annual temperature, maximum and minimum temperatures and annual precipitation). Higher PC1 and PC2 values represent gradients of increasing temperatures, while higher PC3 represents gradient of increasing precipitation.

5.1 Nest of the (A) subsocial Anelosimus kohi (Image by Kuntner, 273 2011) and the (B) solitary living A. chonganicus.

xix FIGURE PAGE TITLE NO. NO.

5.2 Length of non-Anelosimus individuals found in the nests of 8 281 species of Anelosimus. Shaded bars represent the maximum length of insects found (* denotes values have been scaled down by factor of 10), black bars represent minimum length and white bars represent average length of individuals. Subsocial species indicated with an asterisk beside species name.

5.3 Average nest intruders density per nest of 8 species of 281 Anelosimus. Subsocial species indicated with an asterisk beside species name.

GENERAL INTRODUCTION

General Introduction

GENERAL INTRODUCTION

Wilson (1971) stated the basis of sociality as “reciprocal communication of a cooperative nature”, highlighting the importance of communication and cooperation in the evolution of sociality. The origin and mechanisms involved in the evolution of sociality in the animal kingdom have intrigued researchers for decades. Considering the myriad of animal taxa exhibiting social behaviour and the variety of social groupings displayed in these organisms, it is no wonder that researchers are still far from understanding the evolution of sociality. Scott (1956) defined a social group as consisting of members of a population or subgroup of the same species. In light of

Wilson’s (1971) statement, ‘social groups’ in this thesis is further refined to refer to groups consisting members of the same species, which exhibit cooperative behaviours benefiting one another. ‘Cooperative behaviours’ follow the definition by Whitehouse

& Lubin (2005), as adapted from Packer & Ruttan (1988), and refer to behaviours exhibited in the presence of a conspecific. Social groups have been observed in birds

(Grzybowski, 1983; Møller, 2006), fishes (Jacoby et al., 2011; Reddon et al., 2014), insects (Hermann, 1979; Tabadkani et al., 2012), mammals (Ebensperger &

Blumstein, 2006; Silk, 2007; Kutsukake, 2009) and spiders (Avilés, 1997;

Whitehouse & Lubin, 2005; Miller, 2006; Lubin & Bilde, 2007). Although social groups in these animal taxa share some similarities, many differences exist among them as well. Variations amongst social groups include proximity between members, period of interaction and function of aggregation (Pitcher et al., 1983; Warburton &

Lazarus, 1991; Bejder et al., 1998; Krause & Ruxton, 2002).

Functions of social groups vary and result in the formation of different social systems. Groups may be formed for purposes such as resource guarding (Lindström,

General Introduction

1986; Mosser & Packer, 2009), defense against predators (Strassmann et al., 1988;

Hass & Valenzuela, 2002; Machado, 2009; Beauchamp, 2010), brood fostering

(Riedman, 1982; Avilés, 1997; Armitage, 1998), increased foraging efficiency

(Packer et al., 1990; Blundell et al., 2001; Dechmann et al., 2010) or for higher

reproductive success (Kappeler & van Schaik, 2001; Silk, 2002; Lehmann et al.,

2008; Betram, 2009 ). Social mammals such as lions and baboons form social groups

consisting of single male with multiple closely related females, extracting benefits

from group living through increased foraging efficiency (Caraco & Wolf, 1975;

Packer et al., 1990; Kappeler & van Schaik, 2001) and dominance in mating rights

(Kappeler & van Schaik, 2001; Lehmann et al., 2008; Betram, 2009). On the other hand, social ungulate groups are made up of a diverse mix of sexes and age classes

(Leuthold & Leuthold, 1975; Lingle, 2003). Individuals in such groups benefit from reduced predation risk through dilution effects and group vigilance (Geist, 1974; Kie,

1999; Lingle, 2003). The highest level of sociality is demonstrated in eusocial organisms such as ants, termites and wasps. In these social systems, a social group usually comprises a single reproductive female, the queen, and sterile daughters functioning as workers that cooperate in foraging, defense and nest maintenance

(Wilson & Hölldobler, 2005; Nowak et al., 2010).

Despite the prevalence of social groups in various animal taxa, it remains rare in spiders (Avilés, 1997; Whitehouse & Lubin, 2005; Lubin & Bilde, 2007). Out of

44 000 species of spiders described to date (Platnick, 2014), less than 0.5% have been found living in social groups (Whitehouse & Lubin, 2005; Yip & Rayor, 2013).

Group living in spiders is present in three main types, ‘colonial’ (Whitehouse &

Lubin, 2005; Lubin & Bilde, 2007), ‘subsocial’ and ‘social’ spiders (Avilés, 1997;

Whitehouse & Lubin, 2005; Lubin & Bilde, 2007; Agnarsson et al., 2007). ‘Colonial’

General Introduction spiders construct individual webs within a communal silk framework, exhibits territorial behaviour and aggression towards conspecifics. Cooperation among colonial spiders is limited to communal web construction and occasionally, in prey capture or sharing only (Uetz & Hieber, 1997; Whitehouse & Lubin, 2005). Groups of

‘subsocial’ spiders are made up of a female and her offspring that disperse from the natal nest just before reaching sexual maturity. Cooperative behaviour such as prey sharing (i.e. individuals that did not participate in prey capture are allowed to feed on the prey) and cooperative prey capture (i.e. two or more individuals participate in prey capture through either biting or contributing silk for prey wrapping) is observed in these spiders (Whitehouse & Lubin, 2005; Yip & Rayor, 2013). Groups of ‘social’ spiders consist of multiple adult females that cooperate in construction and maintenance of a silken nest as well as in prey capture (Avilés, 1997; Whitehouse &

Lubin, 2005; Lubin & Bilde, 2007). Brood fostering is also exhibited in some species

(Marques et al., 1998; Ainsworth et al., 2002; Avilés & Purcell, 2012). Social spiders are characterized by inbreeding and female-biased sex ratios (Avilés, 1997;

Whitehouse & Lubin, 2005; Agnarsson, Avilés, et al., 2006; Lubin & Bilde, 2007;

Avilés & Purcell, 2012). The subjects of interest of this thesis are the subsocial and social spiders, therefore, further discussion on spider sociality will focus mainly on these groups.

Despite their rarity, social spiders are exceptionally valuable model organisms in the study of the evolution of social behaviour, as sociality has arisen multiple times in independent families (Avilés, 1997; Whitehouse & Lubin, 2005; Agnarsson, Avilés, et al., 2006; Lubin & Bilde, 2007; Johannesen et al., 2007). Twenty five species of social spiders are found in six families across nine genera (Avilés, 1997; Whitehouse

& Lubin, 2005; Lubin & Bilde, 2007), representing 14 independent origins of

General Introduction

sociality (Agnarsson, Avilés, et al., 2006; Agnarsson et al., 2007; Avilés & Purcell,

2012). The evolution of sociality in spiders is closely associated with maternal care

and inbreeding (Lubin & Bilde, 2007; Avilés & Purcell, 2012). One hypothesis for the

evolution of sociality in spiders is that sociality in evolved as a result of subsocial

behaviour where extended maternal care resulted in delayed dispersal and promotion

of cooperative behaviour among juveniles (Avilés, 1997; Whitehouse & Lubin, 2005;

Lubin & Bilde, 2007). Finally, as dispersal from natal nest is delayed and eventually

eliminated in some colonies, inbreeding develops (Avilés & Purcell, 2012).

Even though social behaviour in spiders is believed to have evolved through subsocial behaviour, a number of biotic and abiotic factors have also been found to contribute to the formation of groups (Pruitt & Riechert; Powers & Avilés, 2007;

Purcell & Avilés, 2007, 2008; Guevara & Avilés, 2007; Pruitt, 2010; Purcell, 2011;

Majer et al., 2013a, 2013b). Factors such as behavioural dispositions (Pruitt et al.,

2008; Pruitt, 2010; Pruitt & Riechert, 2010), elevation (Purcell & Avilés, 2007;

Avilés et al., 2007), plant structures (Purcell & Avilés, 2007; Guevara & Avilés,

2007), prey and predator type and abundance (Powers & Avilés, 2007; Guevara &

Avilés, 2007; Purcell & Avilés, 2008), rainfall and temperatures (Purcell & Avilés,

2008; Majer et al., 2013a; 2013b) are known to affect the evolution of sociality through promoting a decrease in outbreeding and dispersal events (Lubin & Bilde,

2007). This is reflected in the distribution of social species, with a high concentration of social spiders occurring in lowland tropical forest (Powers & Avilés, 2007; Avilés et al., 2007) and a higher number of subsocial species occurring at higher latitudes and altitudes (Purcell & Avilés, 2007, 2008).

General Introduction

Prey Abundance

Both social and colonial spiders have been observed to occur in habitats with high

insect abundance and large size (Rypstra, 1986; Powers & Avilés, 2007; Mestre &

Lubin, 2011). High nutritional intake is necessary to sustain colonies of social spiders

which can comprise of between twenty to tens of thousands of individuals (Nentwig

& Christenson, 1986; Avilés & Tufiño, 1998; Avilés & Salazar, 1999; Avilés, 2011).

Guevara & Avilés (2007) confirmed that insects in lowland forests are larger than

those occurring at higher altitudes. The success of social spiders in lowland forests

has also been attributed to their ability to capture larger insects because of cooperation

in prey capture and larger web structures (Nentwig, 1985; Powers & Avilés, 2007;

Yip et al., 2008). Prey abundance has been associated with reduced aggression

amongst conspecifics and delayed dispersal in spiders, thus prolonged exposure to

constant supply of food is hypothesized to contribute to the evolution of sociality in spiders (Krafft et al., 1986; Rypstra, 1986; Ruttan, 1990; Gundermann et al., 1993).

Defense against Predators

Besides benefiting from increased efficiency in prey capture, social spiders are hypothesized to have evolved as a means of defense against predators (Henschel,

1998; Purcell & Avilés, 2008; Purcell, 2011). Ants are voracious raiders of nests and

are commonly found in lowland tropical forest which social spiders inhabit

(Hölldobler & Wilson, 1990; Purcell & Avilés, 2008). Predation by ants has been

found to lead to the formation of larger colonies of Stegodyphus dumicola (Eresidae)

(Henschel, 1998). Similarly, Purcell & Avilés (2008) also found that ants in lowland forests may hinder the persistence of subsocial spiders which form smaller colonies as opposed to the larger colonies of social spiders.

General Introduction

Parasitism

Other than being attacked by ants and other predators, spiders are also affected by

parasites or parasitoids such as nematodes, wasps and endosymbiotic bacteria (Allard

& Robertson, 2003; Rowley et al., 2004; Goodacre et al., 2006; Eberhard, 2010;

Foelix, 2011). Parasitism is closely associated with group living, although the

relationship is not always linear (Côté & Poulin, 1995; Smith & Schwarz, 2006;

Rifkin et al., 2012; Lutermann et al., 2013). Social spiders sharing a communal web

may be more conspicuous and vulnerable to attacks by parasites (Hieber & Uetz,

1990; Purcell, 2011). In addition, frequent contact among spiders through cooperative

behaviour also increases transmission of contact parasites (Ezenwa, 2004); the similar

genetic material among these heavily inbred societies further exacerbates this (Liersch

& Schmid-Hempel, 1998). Infection of spiders by endosymbiotic bacteria such as

Cardinum and Wolbachia has been found to bring about behavioural and

physiological changes. Of particular interest are the female biased sex ratio induced in

infected individuals and reduced dispersal distances, both of which are characteristic

traits observed in social spiders (Stouthamer et al., 1999; Goodacre et al., 2009;

Vanthournout et al., 2011).

Abiotic Factors

Biotic factors are often influenced by abiotic factors. For example, prey abundance is

associated with the productivity of a habitat (rate of biomass generated within the

ecosystem) (Allaby, 2010; Majer et al., 2013a; 2013b), which is heavily influenced by

temperature and rainfall (Purcell & Avilés, 2008; Purcell, 2011). Consequently, social

spiders are often found in areas with high temperature and rainfall (Purcell, 2011).

Purcell & Avilés (2008) found that rainfall intensity is higher at lowland forest as

General Introduction

compared to higher elevation forests. They hypothesized that high rainfall intensity

may damage smaller web structures of subsocial spiders, whereas large webs of social

spiders are able to withstand damage induced by heavy rainfall. Besides affecting

productivity of the habitat (O’Donnell & Ignizio, 2012), temperature also influences

the metabolic and growth rates of organisms (Gillooly et al., 2002; Jones et al., 2007;

Riechert & Jones, 2008; Pruitt et al., 2011a). Jones et al. (2007) suggested that lower

temperatures at higher latitudes promote brood fostering in populations of the

subsocial species Anelosimus studiosus as chances of female surviving beyond

offspring emergence are lower, thus juveniles benefit from alloparental care in social

groups. Interestingly, recent work has uncovered that tolerance and aggression in A.

studiosus is modified by changes in temperature effected through changes in

metabolic rates (Pruitt et al., 2011a).

Besides biotic and abiotic factors that may contribute to the development of

sociality in spiders, recent work on behavioural traits of spiders have uncovered

differences in aggression levels among individuals that may be closely associated

with the evolution of sociality (Pruitt et al., 2008, 2012; Pruitt, 2010; Pruitt &

Riechert, 2010a; 2010b; Grinsted et al., 2013; Keiser et al., 2014). Differences in

levels of aggression have been closely related to task differentiation in some social

spiders. Aggressive individuals attack prey more frequently than docile ones

(Settepani et al., 2012; Grinsted et al., 2013; Keiser et al., 2014). Consequently,

aggressive individuals also respond more quickly to vibrations caused by prey as

compared to docile ones (Pruitt et al., 2008). Pruitt et al. (2012) suggested that

repeated evolution of reduced aggression, together with increased within-species

variation in aggression may contribute to the evolution of sociality in spiders.

General Introduction

Comparisons between social and subsocial spiders have also confirmed that social

species are less aggressive to prey (Pruitt et al., 2011b).

While there is general consensus on the ‘subsocial’ pathway of evolution of

sociality in spiders, various abiotic and biotic factors contribute as push and pull

factors in the natal dispersal and development of maternal care, both of which are

crucial in the evolution of sociality in spiders.

Study System

Spiders of the genus Anelosimus (Araneae: Theridiidae) are distributed globally and

display a spectrum of social behaviour, from solitary to social (Nentwig &

Christenson, 1986; Avilés & Gelsey, 1998; Marques et al., 1998; Agnarsson & Zhang,

2006; Agnarsson et al., 2006a; 2007; 2010; 2012a; Avilés et al., 2007). Out of 64

nominal species, six are social, 14 subsocial and five solitary living while the sociality

of the remaining species are unknown (Avilés, 1997; Whitehouse & Lubin, 2005;

Agnarsson et al., 2006b; Lubin & Bilde, 2007; Agnarsson, 2012b). Phylogeny of

Anelosimus has revealed subsociality as an ancestral state in this group of spiders

while solitary behaviour may have evolved secondarily (Agnarsson, 2006, 2012b;

Agnarsson et al., 2006b; 2007).

All six social Anelosimus species are found in South America, with four

located in lowland tropical rainforests and two (A. guacamayos and A. oritoyacu)

found at higher elevation (Avilés, 1997; Marques et al., 1998; Agnarsson, 2006;

Avilés et al., 2006; 2007). Colonies of social Anelosimus range from 20 to 50 000

individuals and comprise of juveniles, adult females and adult males (Avilés, 1997).

Colonies also usually display a various degrees of female-biased sex ratio (Vollrath,

1986; Avilés & Salazar, 1999; Avilés et al., 2007; Avilés, 2011). Subsocial

General Introduction

Anelosimus are found in North and South America (Avilés & Gelsey, 1998; Marques

et al., 1998; Powers & Avilés, 2003; Avilés & Bukowski, 2006; Rao & Aceves-

aparicio, 2012). In South America, subsocial species have been found to inhabit

habitats at higher elevation as compared to social species (Guevara & Avilés, 2007;

Avilés et al., 2007; Purcell & Avilés, 2008). Intriguingly, the subsocial A. studiosus

has been found to form multi-female colonies at higher latitudes (Furey, 1998). This

phenomenon has been attributed to brood fostering amongst females, an adaptation to

increase offspring survival under a colder climate where females may not survive beyond egg eclosion (Jones et al., 2007). Finally, only three species of solitary

Anelosimus (A. ethicus, A. nigrescens and A. pacificus) are found in Central and

South America (Agnarsson, 2004; Agnarsson et al., 2006a; Guevara et al., 2011).

Maternal care in these species is rudimentary and spiderlings are able to emerge from egg sacs without aid from females (Agnarsson et al., 2006a). Due to the small number of solitary species as compared to social and subsocial species, little is known about the life history of solitary Anelosimus species.

Even though intensive research has been conducted on spiders in this genus, as highlighted, the bulk of which has been done on New World species (North and South

America). Little is, however, known of Anelosimus species in the Old World (Africa and Asia). Attempts to rectify this can be seen in recent work where 22 new species of

Anelosimus were described from Africa, Madagascar, Asia, Australia and Papua New

Guinea (Agnarsson & Kuntner, 2005; Agnarsson & Zhang, 2006; Agnarsson et al.,

2010; Zhang et al., 2011; Agnarsson, 2012b). Although many of these new species have been phylogenetically placed and speculatives of the level of their sociality have been made, the natural history of most of these species remains largely unknown and is an area that deserves to be explored further. Being a model organism, a

General Introduction

comprehensive understanding of Anelosimus would go a long way in deciphering the

evolution of sociality in spiders.

Objectives

The primary aim of this study is to gain an understanding of the distribution and

sociality of Anelosimus species in Asia, with focus on species from Southeast and

East Asia. This was firstly achieved through field surveys in China, Peninsula

Malaysia, East Malaysia, Singapore and Taiwan. In addition, preserved specimens

from Indonesia and Sabah, Malaysia from the personal collection of Deeleman-

Reinhold were also examined. All species collected and examined were photographed

and described taxonomically. Furthermore, DNA extract were acquired whenever

possible and sequenced to obtain an understanding of the phylogenetic relationships

amongst species in Asia and globally (Chapter 1). In order to determine the natural history and sociality of Anelosimus species in Asia, ten species were collected and observed under laboratory conditions. To assess the level of sociality displayed in these species, it is pertinent to examine and compare the social traits displayed in these spiders to determine their level of sociality. This was accomplished by assessing the following traits (including social traits): (1) habitat structure, (2) colony structure,

(3) presence of maternal care, (4) cooperation (e.g. in web maintenance and prey capture), (5) timing of dispersal (i.e. time elapsed between egg-sac emergence and dispersal from the natal nest), (6) mating duration, (7) clutch size (i.e. number of hatchings per egg sac), (8) number of clutches (i.e. number of egg sacs produced by a female in her lifetime), (9) interval between clutches, and (10) interval between hatching of first clutch and production of a subsequent clutch. As maternal care is

General Introduction

significant in the development of sociality, many of the traits examined are related to

it.

Upon determination of the sociality of Asian Anelosimus, I was interested in

examining if morphological differences exist in the transition from subsocial to

solitary living (and vice-versa) in this group of spiders. As studies have shown that

social species are less aggressive and less responsive towards vibrations of prey and foreign spiders than subsocial species (Pruitt et al., 2011b), a reduction in sensitivity

towards external stimuli may contribute to increased tolerance towards conspecifics sharing a common web in more social species. To achieve this, I examined the sensory receptor count and densities (chemoreceptor, slit sensillum, tactile hair and trichobothrium) of eight species (two subsocial and six solitary) of Asian Anelosimus spiders (Chapter 3).

Although morphological transition may affect the sensitivity and behaviour of spiders, external abiotic factors in the habitat may similarly exert pressure on group living and the evolution of sociality (Purcell, 2011). To determine if sociality is affected by abiotic factors, I examined the worldwide distribution of social, subsocial and solitary Anelosimus and took into consideration any variation in temperature, rainfall and seasonality among the localities (Chapter 4).

Finally, besides morphological adaptations and environmental factors, external biotic factors may also affect the success of group living. One important factor is parasitism. Sociality is frequently associated with parasitism and the success or failure of social groups sometimes hinges on the parasite load of a colony (Rifkin et al.,

2012; Lutermann et al., 2013). The relationship between social groups and parasitism has never been closely examined in Anelosimus. Therefore, in Chapter 5, I examined the ectoparasite load and prevalence of the endosymbiotic bacteria Wolbachia in

General Introduction

Anelosimus of different levels of sociality. This was accomplished by examining the nest contents of eight species of Anelosimus and using Polymerase Chain Reaction

(PCR) amplification of the wsp gene (Goodacre et al., 2006) to determine the prevalence of Wolbachia in 37 species of social, subsocial and solitary species of

Anelosimus.

General Introduction

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CHAPTER 1

SYSTEMATICS AND PHYLOGENY OF SOUTHEAST AND EAST ASIAN SPIDERS OF THE GENUS ANELOSIMUS (ARANEAE: THERIDIIDAE), WITH THE DESCRIPTIONS OF TEN NEW SPECIES

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

ABSTRACT

Spiders of the genus Anelosimus are distributed globally and display a spectrum of social, subsocial and solitary behaviour. Therefore, this group of spiders has been regarded as a model system and extensively studied to elucidate the evolution of sociality in spiders.

However, despite numerous studies on Anelosimus species in the Neotropics, little is known of the spiders of this genus in Asia. To date, only 16 species of Anelosimus have been described in Asia, with sparing information on the social behaviour of these spiders. In this chapter, I described ten previously undescribed species of Anelosimus (A. albus sp. nov., A. cameronis sp. nov., A. circumdantus sp. nov., A. cochlianis sp. nov., A.deeleemanae sp. nov.,

A. floreni sp. nov., A. habitus sp. nov., A. helix sp. nov., A. kinabaluensis sp. nov. and A.

parvulus sp. nov.) from Southeast and East Asia. In addition, male-female pairings of two

species (A. membranaceus Zhang, Liu & Zhang and A. seximaculatum Zhang, Liu & Zhang)

are corrected and the males of A. bali Agnarsson and A. subcrassipes Zhang, Liu & Zhang

are described for the first time. Phylogenetic analysis also confirms the inclusion of A. albus

sp. nov., A. cameronis sp. nov., A. habitus sp. nov., A. helix sp. nov., A. kinabaluensis sp.

nov., A. parvulus sp. nov., A. subcrassipes Zhang, Liu & Zhang and A. taiwanicus Yoshida in

the genus Anelosimus and suggest the occurrence of a reversal from subsocial to solitary

living in Asian Anelosimus species. Contrary to solitary species in the New World that occur

at higher latitudes, solitary species in Asia are instead found at forest edges in low latitude

tropical rainforests. Meanwhile, subsocial species in Asia are found near beachfront habitats

in contrast to occurrence at higher altitudes in other parts of the world. Therefore, the

evolution of social behaviour in the genus Anelosimus in the Old World differs from that in

the New World.

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

INTRODUCTION

The spider genus Anelosimus Simon, 1891 occurs worldwide, having been described from

Africa, Asia, Australia, Europe, North and South America (Platnick, 2014). This genus of

spiders also contains a mix of social, subsocial and solitary species (Rypstra & Tirey, 1989;

Ito & Shinkai, 1993; Furey, 1998; Marques et al., 1998; Avilés & Salazar, 1999; Powers &

Avilés, 2003; Agnarsson & Kuntner, 2005; Agnarsson & Zhang, 2006; Coddington &

Agnarsson, 2006; Avilés & Bukowski, 2006; Avilés, 2011; Agnarsson, 2012; Yip & Rayor,

2013) and has been frequently used as a model organism in the study of the evolution of

sociality in spiders (Avilés, 1997; Avilés & Bukowski, 2006; Powers & Avilés, 2007; Avilés

et al., 2007; Yip et al., 2008; Pruitt & Riechert, 2011; Guevara & Avilés, 2011; Corcobado et

al., 2012; Samuk & Avilés, 2013; Majer et al., 2013). As a model, it is therefore pertinent to

obtain a comprehensive understanding of all species within this genus.

Despite its global distribution, taxonomic description and behavioural studies of

Anelosimus species have largely been focused in the Neotropics (Levi, 1963; Brach, 1977;

Avilés, 1997, 2011; Avilés & Salazar, 1999; Agnarsson, 2005, 2006; Avilés et al., 2007; Rao

& Aceves-aparicio, 2012), with only a handful of Old World taxonomic description and

behavioural study (Yoshida, 1986; Ito & Shinkai, 1993; Zhu, 1998; Agnarsson & Kuntner,

2005; Agnarsson & Zhang, 2006; Agnarsson et al., 2010; Zhang et al., 2011; Agnarsson,

2012). Interestingly, the majority of Neotropical species have been described as subsocial or

social whereas recent studies suggest that a number of Old World species are solitary living

(Agnarsson & Kuntner, 2005; Agnarsson & Zhang, 2006; Agnarsson et al., 2010; Agnarsson,

2012). The imbalance of knowledge between Neotropical and Old World species thus

contributes to an asymmetry of understanding between social and solitary species. To fully

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

grasp the intricacies of the evolution of sociality in spiders, it is thus crucial to fill the gap of

knowledge in Old World Anelosimus species.

In Asia, a total of 16 species of Anelosimus have been described from China,

Indonesia, Japan, Malaysia, and Singapore (Yoshida, 1986; Zhu, 1998; Agnarsson &

Kuntner, 2005; Agnarsson & Zhang, 2006; Agnarsson et al., 2010; Zhang et al., 2011;

Agnarsson, 2012). Many of these species were described before 2000, supported by taxonomic drawings. With the advancement of technology, the use of high resolution

photography would greatly enhance the taxonomic identification of these species. Here, I

describe 18 species of Anelosimus found in Asia, particularly Southeast and East Asia. Of the

18 species described, males of two species (A. bali and A. subcrassipes) are newly described, male-female pairing of two species are corrected (A. membranaceus and A. seximaculatum),

ten species are novel species. In addition, I report the biology of three subsocial species (A.

kohi, A. subcrassipes and A. taiwanicus) and six solitary species (A. cameronis, A. exiguus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum).

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

MATERIALS AND METHODS

Spider Collection and Maintenance in the Laboratory

Spiders were collected in the field at various localities around East and Southeast Asia

(indicated under each species description) between 2009 and 2011. All collections were made either between 0800 h and 1800 h or between 2000 h to 2200 h. Collection of spiders was achieved either by hand or with the use of sweep nets.

Taxonomy

Specimen preparation and images

Examination of specimens was accomplished using an Olympus SZX16 dissecting microscope (Olympus, Centre Valley, PA). Measurements were taken in mm, using K2

Infinity long distance microscope with a micrometer eyepiece. Length of prosoma and abdomen were obtained from lateral view of specimens. Width of both segments were measured in dorsal view. Length of legs were measured after detachment from prosoma. All measurements were taken at the widest points. Palps of males were immersed in concentrated

KOH (~1g mL-1) for 1 min, followed by immersion in distilled water for expansion of palps

to examine palp morphology (Coddington, 1990). Female genitalia were excised using sharp

needles and immersed in concentrated KOH (~1g mL-1) for 5 min, followed by immersion in

distilled water for digestion of excess tissue to facilitate viewing of the morphology of the

epigynum. Excised epigyna were then immersed in 75% ethanol for examining both the

ventral and dorsal views.

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

To prepare samples for photography, anatomical preparations were mounted on clear hand

sanitizers, 65 – 70% ethanol gels, and covered with 70% ethanol (Agnarsson, 2012).

Photographs were taken using a visionary digital imaging system with core component of a

Canon 5D digital camera body (Canon, Tokyo) and a K2 Infinity microscope (Boulder, CO)

equipped with Olympus metallurgical objectives. Multiple sequential images were taken and combined using Helicon Focus 4.0 (Kharkov, Ukraine) and processed with Photoshop CS3

(Adobe, San Jose, CA) for alteration to contrast, brightness and removal of background blemishes. Adobe Illustrator CS6 was used to combine plates.

Scanning electron microscopy

Palps were excised and air dried for 15 min. Upon drying, individual palps were mounted on

an aluminium stub using a conductive adhesive strip and sputter coated with platinum using

JEOL JFC 1600 Pt Fine coater for 4 min. After sample preparation, palps were examined and photographed under the scanning electron microscope JEOL JSM 6510 SEM.

Phylogenetics Specimens collected in the field were fixed in 95% ethanol. Other specimens were obtained from the personal collection of Deeleman-Reinhold and were kept in 75% ethanol. Voucher specimens will be deposited in the National Museum of Natural History (NMNH),

Smithsonian Institution or Lee Kong Chian Natural History Museum (LKCNHM) (formerly

Raffles Museum), National University of Singapore.

One whole leg was excised from each specimen and macerated with a sterile plastic pestle, after 5 s drying in liquid nitrogen. Qiagen DNeasy Blood and Tissue Kit (Hilden,

Germany) was used for the extraction of genomic DNA, with an incubation period of 48 h at

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

55oC in lysis buffer and Proteinase K. Standard Polymerase Chain Reaction (PCR) amplifications were carried out using PuReTaq Ready-To-Go PCR Beads (GE Healthcare) with 1 µL of genomic DNA extract, 22 µL of nuclease-free dH2O, 1µL each of 10µM forward and reverse primers. A total of four gene regions were amplified, one mitochondrial gene (CO1) and three nuclear genes (ITS, 16S and 28S). Primer pairs and annealing temperatures are presented in Table 1.1. Genes were selected for their universality, variability and ease of amplification (Mardulyn & Whitfield, 1999; Agnarsson, 2010).

Eppendorf Mastercycler EP S Thermal Cycler was utilized in PCR amplification.

Cycling conditions began with initial denaturation step at 94oC for 2 min, followed by 34

cycles of 20 s at 94oC, 35 s at 48 – 50oC (depending on primers used; Table 1.1) and 30 s at

65oC and a final extension step of 3 min at 72oC. PCR products were verified on 1%

agarose/TBE electrophoresis gel. PCR product purification and Sanger sequencing were

outsourced to Beckman Coulter Genomics and Macrogen, USA. The same primers used for

PCR amplification were used for sequencing. Contigs were assembled from forward and

reverse ABI chromatograms using Phred (Green & Ewing, 2002) and Phrap (Green, 1999)

via Mesquite (Maddison & Maddison, 2011) using the Chromaseq module (Maddison &

Maddison, 2011). and trimmed of low-quality ends using Chromaseq Version 1.01

(Maddison & Maddison, 2011).

Mitochondrial (16S and CO1) and nuclear (ITS2 and 28S) sequences were obtained

for Anelosimus albus, A. cameronis, A. chonganicus, A. exiguus, A. helix, A. kinabaluensis, A.

kohi, A. membranaceus, A. parvulus, A. seximaculatum, A. subcrassipes and A. taiwanicus.

These data were incorporated into an existing Anelosimus phylogeny (Agnarsson et al., 2007;

2010; Agnarsson, 2012). DNA extraction was unsuccessful in A. floreni, A. circumdantus and

A. deelemanae as these specimens are old and have not been well preserved.

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

The total dataset consist of 79 terminal taxa representing 42 species of Anelosimus.

Alignment and analysis of molecular data is modelled after previous studies (Agnarsson et al.

2007, 2010a; Agnarsson 2012) and summarized as follow:

Sequences were aligned in ClustalW (Thomspon et al., 1994) with gap opening and

extension cost of 24/6 (Agnarsson et al., 2007). The protein coding CO1 sequences aligned

with no gaps and no stop codons. The remaining non-protein coding genes were adjusted

manually after Clustal alignment (Agnarsson et al., 2007). All genes were subsequently

concatenated into a four gene matrix in Mesquite (Maddison & Maddison, 2011) and

exported to jModeltest 0.1.1 (Posada, 2008) for analysis and model selection.

The matrix was partitioned by non-protein coding (28S, 16S, ITS2) and protein

coding (CO1) gene. The CO1 gene was further partitioned by codon position, resulting in a

total of six partitions. jModeltest 0.1.1 (Posada 2008) was used to select a suitable model

among the 24 models implemented in MrBayes for each partition. Model selected for each

partition was: 28S, COI1st, COI2nd, 16S = GTR+I+G; COI3rd, ITS2 = GTR+G. The

concatenated matrix was then analyzed in MrBayes (Huelsenbeck & Ronquist 2001;

Ronquist & Huelsenbeck 2003). The Bayesian analysis was run for 10 000 000 generations,

with all base frequencies estimated from the data and parameter estimates unlinked (‘unlink

statefreq= (all) revmat= (all) shape= (all) pinvar= (all)’). The first 5 000 000 were then

discarded as ‘burn-in’, after which stationarity was achieved.

Chapter 1 – Systematics of Southeast and East Asian Anelosimus species

Table 1.1. Primer sequences and sources, and optimal annealing temperatures.

Annealing Gene Forward Sequence Reverse Sequence Reference temperature

16S 16SF CTAAGGTAGCATAATCA 16SR ATGATCATCCAATTGA Agnarsson et al., 2007 48oC T

28S 28SC GGTTCGATTAGTCTTTCGC 28SO GAAACTGCTCAAAGG Whiting et al., 1997 50oC C TAAACGG

ITS ITS4 TCCTCCGCTTATTGATATG ITS5.8 GGGACGATGAAGAAC Agnarsson, 2010 47oC C GCAGC

CO1 LCOI1490 GGTCAACAAATCATAAAG C1-N-2776 GGATAATCAGAATAT Folmer et al., 1994 48oC ATATTGG CGTCGAGG Hedin & Maddison, 2001

Systematics of Southeast and East Asian Anelosimus species

RESULTS

Morphology All species examined here share similar morphological features that are distinctive of

Anelosimus. With the exception of Anelosimus albus (Figure 1.16), the characteristic abdominal patterns (Figure 1.2A) and thickened, curved femur I (Figure 1.1D) are present in all species. The 18 species examined also display simple palp and epigynum structures.

Phylogeny The phylogeny (Figure 1.1) here is similar to previous phylogenies of Anelosimus

(Agnarsson, 2012). Besides A. subcrassipes Zhang, Liu & Zhang and A. taiwanicus Yoshida

(Yoshida, 1986), all new species (A. albus sp. nov., A. cameronis sp. nov., A. hiatus sp. nov.,

A. helix sp. nov., A. kinabaluensis sp. nov., A. membranaceus Zhang, Liu & Zhang, A. parvulus sp. nov. and A. seximaculatum Zhang, Liu & Zhang) were recovered in the ‘Solitary

II’ clade which also contains the solitary Papua New Guinea species (A. potmosbi Agnarsson and A. pomio Agnarsson) and the subsocial A. eidur Agnarsson. In this phylogeny, A. rupununi Levi, A. luckyi Agnarsson, A. subcrassipes, A. taiwanicus and A. pratchetti

Agnarsson are sister to the rest of the Anelosimus species. The solitary A. pacificus Levi, A. ethicus Keyserling, A. nigrescens Agnarsson and A. decaryi Agnarsson form the ‘Solitary I’ clade and is sister to the subsocial A. kohi Yoshida from Southeast Asia. Together, this group is sister to the New World ‘eximius lineage’ (Agnarsson, 2006) and two species from

Madagascar (A. may Agnarsson and an undescribed Anelosimus species). The ‘Solitary II’ clade is made up of nine solitary species (A. cameronis, A. chonganicus Zhu, A. exiguus

Yoshida, A. kinabaluensis, A. membranaceus, A. parvulus, A. pomio, A. potmosbi and A. seximaculatum,) and one subsocial species (A. eidur), and the behaviourally unknown A. agnar Agnarsson, A. albus, A. bali Agnarsson, A. hiatus, A. helix and A. linda Agnarsson.

Systematics of Southeast and East Asian Anelosimus species

The ‘Solitary II’ clade thus represent a second reversal from subsociality to solitary living

that has been demonstrated in the A. pacificus (Agnarsson, Barrantes, et al., 2006) in the

‘Solitary I’ clade.

Natural History

Webs of Anelosimus cameronis, A. parvulus, A. chonganicus, A. exiguus, A. membranaceus ,

A. seximaculatum and A. kinabaluensis are similar to webs of the solitary A. pacificus Levi,

with loose threads of web enveloping the tip of a branch or flowers (Agnarsson, Barrantes, et

al., 2006). When webs were located at the tip of branches, a retreat is commonly formed by

spiders using silk to roll up a single leave partially or to adhere two leaves together. Webs of

these spiders usually consist of single adult male, single adult female, female with egg sac or

female with spiderlings of instar II to IV. Instar stages of spiderlings are estimated based on

comparison with laboratory reared juveniles. Individual spiderlings above instar III were also found dispersed from the natal nest. Under laboratory conditions, females were observed capturing prey for spiderlings and spiderlings of instar II and III were observed sharing prey

(Chapter 2). In addition, spiderlings of instar II and III were also observed cooperating in prey capture. Laboratory observations also revealed that dispersal in these species occur at instar III and cooperative behaviour ceased upon dispersal (Chapter 2).

Webs of A. kohi encountered in the field varied, some were ‘basket webs’ while others resembled webs of A. pacificus (Agnarsson, Barrantes, et al., 2006; Agnarsson et al.,

2010). ‘Basket webs’ of A. kohi usually consist of a female with late instar juveniles (instar V or VI) or multiple late instar (instar V or VI) females. Webs containing single male, single female or female with egg sac, single female with spiderlings (instar II to IV), pair of adult male and female are similar to webs of solitary Anelosimus. These are usually found on tips

Systematics of Southeast and East Asian Anelosimus species

of branches with threads of silk enveloping surrounding leaves and branches. Similar to the

‘basket webs’, these webs also sometimes contain detritus and silk is used to adhere dried

leaves together, forming a retreat for spiders. Under laboratory conditions, females were

observed capturing prey which were shared amongst spiderlings of instars II to IV. In

addition, cooperative prey capture has also been observed in juveniles beyond instar IV. Prey

sharing among adults was also observed (Chapter 2).

Webs of A. subcrassipes and A. taiwanicus typically consist of a single adult male, single adult female, female with egg sac or individual juveniles of instar IV to VI. Webs constructed by these species are similar to those of the solitary species and A. pacificus

(Agnarsson, Barrantes, et al., 2006; Agnarsson et al., 2010), except that these webs were

about twice to thrice bigger. In the field, juveniles of instar II to IV were never encountered

and laboratory observations indicates that dispersal occurs after instar IV. Similar to A. kohi,

females were observed capturing prey for juveniles of instar II to IV, with sharing of prey

amongst spiderlings. Cooperative prey capture was also seen in spiderlings beyond instar IV

and amongst adults.

Anelosimus cochlianis and A. floreni were collected through canopy fogging in secondary tropical rainforests. Natural history of the remaining species is unknown.

Systematics of Southeast and East Asian Anelosimus species

Kochiura rupununi luckyi subcrassipes taiwanicus pratchetti Solitary I pacificus ethicus nigrescens decaryi

kohi

Anelosimus Madagascar 040A may

eximius domingo jabaquara ‘eximius’ lineage dubiosus studiosus vierae arizona analyticus jucundus baeza elegans

guacamayos oritoyacu tosum Solitary II cameronis linda membranaceus agnar parvulus seximaculatum eidur hiatus albus Asia, Indonesia and Papua helix New Guinea chonganicus Americas potmosbi Africa and Madagascar bali Australia pomio exiguus kinabaluensis 0.1

Figure 1.1. Phylogeny of Anelosimus based on Bayesian analyses of the concatenated molecular matrix. Branches are coloured according to geographical distribution. Branches are proportional to length as estimated in MrBayes, outgroups are omitted for clarity, see Figure 1.20 for full phylogeny. Social species are indicated with a red circle, subsocial with a blue circle, solitary with an open circle and species with unknown social behaviour lack a symbol. Numbers on nodes are posterior probability support values. Scale shows expected substitutions per site.

Systematics of Southeast and East Asian Anelosimus species

DISCUSSION

Field observation and laboratory observations suggest that A. cameronis, A. chonganicus, A. exiguus, A. kinabaluensis, A. parvulus, A. membranaceus and A. seximaculatum are solitary while A. kohi, A. subcrassipes and A. taiwanicus are subsocial.

Phylogeny and biogeography

As with previous phylogenies (Agnarsson & Kuntner, 2005; Agnarsson & Zhang, 2006;

Agnarsson, Avilés, et al., 2006; Agnarsson et al., 2010; Agnarsson, 2012), this phylogeny supports the monophyly of Anelosimus and the placement of ten new species (A. subcrassipes, A. taiwanicus, A. parvulus, A. cameronis, A. membranaceus, A. seximaculatum,

A. hiatus, A. albus, A. helix and A. kinabaluensis) in the genus Anelosimus. The majority of the new species are placed within the ‘Solitary II’ clade, these species were collected from

China, Malaysia, Indonesia, Singapore and Papua New Guinea and nine out of 16 species in this clade display solitary behaviour.

Similar to prior work by Agnarsson (Agnarsson & Kuntner, 2005; Agnarsson &

Zhang, 2006; Agnarsson, Avilés, et al., 2006; Agnarsson et al., 2010; Agnarsson, 2012), the phylogeny supports the ‘eximius lineage’ clade that consists of all New World social and subsocial species. The New World solitary A. pacificus, A. ethicus and A. nigrescens is also grouped together with the Madagascan solitary A. decaryi. This phylogeny further supports the independent colonization of Madagascar by two lineages of Anelosimus (Agnarsson et al.,

2010), with the placement of A. decaryi in the ‘Solitary I’ clade and A. may sister to the

‘eximius lineage’.

Systematics of Southeast and East Asian Anelosimus species

However, as with the phylogeny by Agnarsson (2012), this phylogeny remains perplexing in terms of biogeographical distribution of a few Anelosimus species. Firstly, the

Old World A. luckyi, A. subcrassipes and A. taiwanicus and Australian A. pratchetti are placed sister to the remaining Anelosimus species with undetermined phylogeographical implications. The placement of A. pratchetti sister to A. subcrassipes and A. taiwanicus suggest long distance dispersal. Secondly, the Asian A. kohi is nested among New World and

Madagascan species, again, suggesting long distance dispersal. However, these discussions on biogeography of Anelosimus are merely speculative and the inclusion of more Anelosimus species from Australia and Papua New Guinea would aid in clarifying any biogeographic hypotheses.

Phylogeny, habitat type and the evolution of sociality

The findings of this study indicate that seven Asian species (A. cameronis, A. chonganicus, A. exiguus, A. kinabaluensis, A. parvulus, A. membranaceus and A. seximaculatum) are solitary living and are placed phylogenetically within the ‘Solitary II’ clade which contains all species from Papua New Guinea and the majority of species from Asia. Within this clade, only the

Papuan A. eidur is subsocial while the behaviour of the remaining species remain unknown.

Based on the phylogenetic placement of these species, they are likely to be either solitary or subsocial. Since the ancestral state of Anelosimus is likely to be subsocial (Agnarsson et al.,

2007; Agnarsson, 2012), both ‘Solitary I’ clade and the Asian ‘Solitary II’ clade thus likely represent two independent secondary reversal to solitary living in Anelosimus.

Incidentally, five solitary species (A. chonganicus, A. exiguus, A. parvulus, A. membranaceus and A. seximaculatum) share similar habitats in low latitude tropical habitat at forest edges with little to zero canopy cover. Meanwhile the remaining two solitary species

(A. pomio and A. potmosbi) are found at coastal habitats. In contrast, with the exception of A.

Systematics of Southeast and East Asian Anelosimus species

decaryi, the ‘Solitary I’ clade group species (A. ethicus, A. nigrescens and A. pacificus) occur

at higher latitudes in Europe and southern South America. The geographical location of

solitary species in Asia thus appears to run contrary to the pattern observed in the Americas

where solitary species are typically found at higher latitudes.

Previous work has shown that the distribution of social and subsocial Anelosimus is

influenced by prey size and abundance (Powers & Avilés, 2007; Guevara & Avilés, 2007).

Agnarsson (2012) therefore, hypothesized that exposure to sun and sea spray may result in

prey of smaller sizes at beachfront habitats, preventing the development of social behaviour

in the solitary A. amelie and A. decaryi in Madagascar and A. pomio and A. potmosbi in

Papua New Guinea. Similarly, the five solitary species here were found along edges of the

forests or in open field with little canopy cover, where environment could be just as harsh as

beachfront habitats.

Despite the potentially harsh environment of the beachfront forests, the subsocial A.

kohi, A. subcrassipes and A. taiwanicus are found in these habitats. Anelosimus subcrassipes was found on vegetation along beachfront at subtropical latitude, A. taiwanicus was among

Ixora sp. in a landscaped garden near the coast, while A. kohi was found at the edge of low

latitudinal mangrove forest. These habitats are similarly exposed to sunlight and sea spray but

such harsh conditions did not hinder the development of subsocial behaviours. Taken

together, it appears that the development of sociality in Asian Anelosimus may be very

different from the New World species and more complex than previously hypothesized. More

information on the social behaviour and habitat of the remaining species within the ‘Solitary

II’ clade would help shed light on the influence of habitat type on the development of social

behaviour in this group of spiders.

Systematics of Southeast and East Asian Anelosimus species

TAXONOMY

All holotypes are deposited in National Museum of Natural History (NMNH), Smithsonian

Institution or Lee Kong Chian Natural History Museum (LKCNHM), National University of

Singapore. Previously described species have been included to complement lack of

photographs or offer initial description of sexes or female/male pairing.

Abbreviations: CD, copulatory ducts; FD, fertilization ducts; E, embolus; MA, median

apophysis; t, tegulum; ST, subtegulum; CH, cymbial hood; C, cymbium.

Family Theridiidae Sundevall, 1833

Refer to Agnarsson (2004) for diagnosis

Genus Anelosimus Simon, 1891

All species described here possess classic Anelosimus features: (a) longitudinal abdominal pattern (e.g. Figures 1.2A, F), but unusual in A. floreni (Figure 1.19A) and A. albus (Figure

1.28A) (b) thick and curved femur I (e.g. Figure 1.2G, H) and (c) absence of visible colulus but with a pair of colulus setae. Palps of all species treated here are secondarily simplified with the absence of a conductor and tegular apophysis.

Systematics of Southeast and East Asian Anelosimus species

Species descriptions

Anelosimus cameronis sp. nov.

(Figures 1.2, 1.3)

Material examined

One male holotype and five male and six female paratypes from MALAYSIA, Cameron

Highlands, 4°28'48.90"N 101°23'3.40"E, 1466 m elevation, 30.x.2009, coll. S.P. Goh, D. Li,

J.H. Tang and D.P. de Araujo.

Diagnosis

Anelosimus cameronis (Figure 1.2A, D) can be distinguished from other species by the dark colouration of its prosoma and external and internal epigynum structures (Figure 1.2B, C), with simple fertilization ducts and convoluted copulatory ducts.

Description Female (holotype)

Total length 2.60. Prosoma 1.00 long, 0.90 wide, dark brown throughout. Abdomen

1.90 long, 1.40 wide, 1.50 high, pattern as in male and (Figure 1.2A). Eyes subequal, diameter ~0.07. Leg I femur 1.27, patella 0.20, tibia 1.05, metatarsus 1.00, tarsus 0.56. Femur

I thickened, about five times longer than wide, curved. Leg formula 1423. Leg colour similar to male except lighter at base of legs. Epigynum as in (Figure 1.2B, C) with lightly sclerotized copulatory plate and convoluted copulatory ducts.

Systematics of Southeast and East Asian Anelosimus species

Male (paratype)

Total length 2.03. Prosoma 1.02 long, 0.85 wide, orange with dark band in the centre and near the eyes. Abdomen 1.17 long, 0.98 wide, 0.95 high, pattern as in (Figure 1.2F), with red colouration on the longitudinal band at the centre. Eyes subequal, diameter ~0.09. Leg I femur 1.37, patella 0.40, tibia 1.17, metatarsus 0.91, tarsus 0.52. Femur I thickened, about four times longer than wide, curved. Leg formula 1243. Leg base colour orange, femur I dark orange brown in colour. Palps as diagnosed (Figures 1.2D, E; 1.3), with spiralling embolus forming 1.5 spirals of unequal diameters around the tegulum before extending upwards slightly beyond the cymbium.

Variation

Female: Total length 1.70 – 2.60. Prosoma 0.90 – 1.00 long, 0.70 – 0.90 wide.

Distribution

Only known from type locality.

Natural history

Web structure resembles A. pacificus from Costa Rica (Agnarsson et al., 2006), occupied by single adult male or female, sometimes female with egg sac. Webs of A. cameronis were found in open fields or on vines growing on fences with little to no canopy cover. Absence of observations of cohabiting juveniles indicates that this species is solitary.

Systematics of Southeast and East Asian Anelosimus species

Etymology

The specific epithet is named after Cameron Highlands, where it was collected.

Systematics of Southeast and East Asian Anelosimus species

Figure 1.2. Anelosimus cameronis, sp. nov. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Ventral; (E) Ectal. (F) Male. (G) Female lateral; (H) Male lateral. Scale bars as indicated below each photograph. A B C 47

Systematics of Southeast and East Asian Anelosimus species

Figure 1.3. Anelosimus cameronis sp. nov. Male palp: (A) Ectal; (B) Subventral; (C) Ventral and (D) Details of sclerites, cymbial hood (smaller arrow) and embolus (larger arrow). Scale bar as indicated in each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus chonganicus Zhu, 1998

(Figures 1.4; 1.5)

Anelosimus chonganicus Zhu, 1998: 291, figs 197A–B; Zhang, Liu & Zhang, 2011:

55, figs 15–18

Material examined

Five male and 19 female paratypes from CHINA, Xiamen Botanical Gardens, 24°26'22.50"N

118° 6'0.30"E, 50 m elevation, 15.x.2009, coll. S.P. Goh and L-M Y.L. Yap. One male and

one female paratypes from CHINA, Hainan, Jian Feng Ling, 18°42'36.84"N 108°47'14.04"E,

179 m elevation, 25.vi.2011, coll. S.P. Goh and D. Li. One male and one female paratype from TAIWAN, Yang Ming Shan, 25°8'59.40"N 121°30'46.98"E, 284 m elevation,

10.vi.2010., coll. S.P. Goh, R.C. Chung and C.P. Liao.

Diagnosis

Anelosimus chonganicus (Figure 1.4A, F, G, H) is similar to a number of male Anelosimus

species with spiral shaped embolus. It differs from A. cameronis (Figure 1.2), A. pomio

Agnarsson (Agnarsson, 2012) and A. seximaculatum (Figure 1.24) in having more embolus

spirals, about 2.5, spiralling more evenly and away from the tegulum (Figure 1.4D, E). The

embolus of A. chonganicus is also about ½ as thick as that of male A. seximaculatum. Palps

are also lighter in colour than A. cameronis. Females of A. chonganicus can be differentiated

from other Anelosimus by the copulatory plate of the epigynum. Internal epigynum is similar to A. potmosbi Agnarsson (Agnarsson, 2012), except in A. chonganicus, the gap between the

spermathecae and copulatory ducts is shorter (Figure 1.4B, C).

Systematics of Southeast and East Asian Anelosimus species

Description

Female (paratype)

Total length 2.38. Prosoma 1.15 long, 0.93 wide, brown with dark band in centre and

around eyes area. Abdomen 1.72 long, 1.18 wide, 1.20 high, pattern as in (Figure 1.4A). Eyes

subequal, diameter ~0.07. Leg I femur 1.38, patella 0.47, tibia 1.15, metatarsus 0.75, tarsus

0.50. Femur I thickened, about four times longer than wide, curved. Leg formula 1423. Leg

colour similar to male except lighter. Epigynum consist of simple fertilization and copulatory ducts as diagnosed in Zhu, 1998 and in (Figure 1.4B, C).

Male (paratype)

Total length 1.72. Prosoma 0.98 long, 0.77 wide, orange with dark band in the centre and around rim. Abdomen 1.02 long, 0.74 wide, 0.63 high, pattern as in (Figure 1.4F). Eyes

subequal, diameter ~0.08. Leg I femur 1.08, patella 0.37, tibia 1.01, metatarsus 0.72, tarsus

0.44. Femur I thickened, about four times longer than wide, curved. Leg formula 1243. Leg

base colour light yellow, femur I dark orange brown in colour except at base and distal end of

leg segments darkened. Palps as diagnosed in Zhang et al., 2013 and (Figures 1.4D, E; 1.5),

with spiralling embolus completing 2.5 spirals of the same diameter, extending upwards from

the base of the embolus and ending at the tip of the cymbium.

Variation

Female: Total length 1.60 – 3.00. Prosoma 0.80 – 1.20 long, 0.80 – 0.93 wide. Male: Total

length 1.40 – 1.90. Prosoma 0.80 – 1.00 long, 0.70 – 0.90 wide.

Systematics of Southeast and East Asian Anelosimus species

Distribution

China (Hainan, Fujian), Taiwan (Yang Ming Shan, Ken Ding), Thailand (Khao Sam Roi Yat

National Park).

Natural history

Almost all webs were found constructed on small wildflowers (0.5 – 1.5 m) above ground, along forest edges or on roadsides. Individual webs were often found clustered in a small area. Webs frequently consist of a single adult male, single adult female, female with an egg sac or female with juveniles between instar II – III. The instar stages were approximated by size comparisons with laboratory reared specimens.

Systematics of Southeast and East Asian Anelosimus species

A B

FD CD

F D E

E CH

G H

Figure 1.4. Anelosimus chonganicus. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Ventral; (E) Lateral. (F) Male. (G) Female lateral. (H) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

MA T

Figure 1.5. Anelosimus chonganicus. Male palp: (A) Ventral and (B) details of sclerites, cymbial embolic groove (smaller arrow), and embolus (larger arrow). Scale bars as indicated in each photograph.

Anelosimus subcrassipes Zhang, Liu & Zhang (2011)

(Figures 1.6; 1.7)

Anelosimus subcrassipes, Zhang, Liu & Zhang, 2011: 52–53, fig 7–9.

Material examined

One female holotype from CHINA, Pu Tuo Island, 29°59'19.90"N 122°23'7.20"E, 13 m

elevation, 20.x.2009, coll. S.P. Goh.

Systematics of Southeast and East Asian Anelosimus species

Diagnosis

Anelosimus subcrassipes (Figure 1.6A, D) can be distinguished from other Anelosimus species by its larger size and configuration of epigynum, with two large spermathecae above the copulatory ducts (Figure 1.6B, C). A. subcrassipes is similar to A. crassipes Bösenberg &

Strand and A. taiwanicus Yoshida (Yoshida, 1986) but differs in the distance between copulatory ducts. Male is distinct from A. taiwanicus and A. crassipes in the anti-clockwise turn of the embolus (Figure 1.6E).

Description

Female (paratype)

Total length 4.18. Prosoma 1.60 long, 1.20 wide, light brown with dark band in centre. Abdomen 2.48 long, 1.85 wide, 1.80 high, pattern as in (Figure 1.6A). Eyes subequal, diameter ~0.11. Leg I femur 1.76, patella 0.43, tibia 1.55, metatarsus 1.06, tarsus 0.54. Femur

I thickened, about four times longer than wide, curved. Leg formula 1423. Legs light brown in colour, darkening at distal end of femur and distinct dark band on distal end of metatarsus I and IV. Epigynum as diagnosed in Zhang et al. (2011) and in (Figure 1.6B, C), lightly sclerotized, with coiling pair of copulatory ducts positioned just next to each other.

Distribution

Only known from type locality

Systematics of Southeast and East Asian Anelosimus species

Natural history

Web structure resembles A. kohi Yoshida (Agnarsson & Zhang, 2006) from Singapore. A. subcrassipes was collected on vegetation along the coast on tips of branches of trees at height of between 1.5 to 2.0 m. Webs consisted of a single adult male, single adult female, female with egg sac or female with juveniles of instars II – IV. Instar stages of juvenile estimated by comparison with laboratory reared specimens.

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.6. Anelosimus subcrassipes. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared, (D) Female lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus exiguus Yoshida, 1986 (Figures 1.7; 1.8)

Anelosimus exiguus Yoshida, 1986: 37–38, figs 13–16. Zhu, 1998: 289, figs 195A–D.

Material examined

One male and one female paratypes from CHINA, Hai Nan Island, Jian Feng Ling,

18°42'36.84"N 108°47'14.04"E, 179 m elevation, 30.iv.2009, coll. S.P. Goh, F.X. Liu, Y.Y.

Ren and S.P. Yin.

Diagnosis

Anelosimus exiguus is distinct from other Anelosimus species due to the shape of its embolus

(Figure 1.7D, E) which curves around its base. Males are similar to A. dude Agnarsson

(Agnarsson & Zhang, 2006) and A. monskeyensis Agnarsson (Agnarsson & Zhang, 2006) but can be distinguished by the short length of its embolus that unlike these two species, does not reach the cymbium. Females of A. exiguus differs from other Anelosimus in its external copulatory plate and in its internal genitalia (Figure 1.7B, C).

Description

Female (paratype)

Total length 2.39. Prosoma 0.94 long, 0.86 wide, light brown with dark band in centre and near eyes area. Abdomen 1.78 long, 1.43 wide, 1.36 high, pattern as in (Figure 1.7A).

Eyes subequal, diameter ~0.08. Leg I femur 1.28, patella 0.29, tibia 1.08, metatarsus 0.78,

Systematics of Southeast and East Asian Anelosimus species tarsus 0.53. Femur I thickened, about five times longer than wide, curved. Leg formula 1243.

Legs light brown in colour, darkening at distal end of each segment. Epigynum consist of simple fertilization and copulatory ducts as diagnosed in Yoshida, 1986 and in (Figure 1.7B),

C, with a pair of lightly sclerotized opening and pair of spermathcae visible through the cuticle.

Male (paratype)

Total length 1.77. Prosoma 0.81 long, 0.69 wide, light orange brown with dark band in centre. Abdomen 1.11 long, 0.81 wide, 0.86 high, pattern as in (Figure 1.7F). Eyes subequal, diameter ~0.07. Leg I femur 1.20, patella 0.20, tibia 0.88, metatarsus 0.69, tarsus

0.48. Femur I thickened, about five times longer than wide, curved. Leg formula 1243. Legs light brown in colour, darkening at distal end of each segment, most distinct in femur I. Palp as diagnosed in Yoshida, 1986 and in (Figure 1.7D, E; 1.8), with a short curved embolus around its base.

Distribution

China (Hainan), Japan.

Natural history

Webs of A. exiguus were often found in wild flowers along walking paths of Jian Feng Ling

National Park. Unlike the sympatric A. chonganicus, the abundance of A. exiguus was lower and webs were further apart. Webs were consisted of a single adult male, single adult female, female with egg sac or female with juvenile at instar II stage. The lack of observations of late

Systematics of Southeast and East Asian Anelosimus species instar spiderlings with female indicates that this species is solitary living. Instar stages of juvenile estimated by comparison with laboratory reared specimens.

Systematics of Southeast and East Asian Anelosimus species

Figure 1.7. Anelosimus exiguus. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. (D) Male palp ventral. (E) Male. (F) Female lateral. (G) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.8. Anelosimus exiguus. Male palp: (A) Ventral; (B) Ectal and (C) Details of sclerites. Arrow indicates the embolus. Scale bars as indicated in each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus kohi Yoshida, 1993

(Figures 1.9; 1.10)

Anelosimus kohi Yoshida, 1993: 8, figs 1–3. Agnarsson & Zhang, 2006: 21, figs 2A–

E, 3A–I.

Material examined

Three male and five female paratypes from SINGAPORE, Mandai Mangroves, 1°26'26.68"N

103°45'55.52"E, 0 m elevation, 30.i.2009, coll. S.P. Goh.

Diagnosis

Anelosimus kohi (Figure 1.9A, F, G, H) is distinct from other Anelosimus species due to its

non-twisted, curved embolus (Figure 1.9D, E). Male is similar to A. nelsoni Agnarsson

(Agnarsson & Zhang, 2006) but can be distinguished by the position of the embolus base on

the apex of the tegulum. Females of A. kohi is unique in possessing the two pairs of spermathecae or accessories (Figure 1.9B, C).

Description

Female (paratype)

Total length 4.76. Prosoma 2.06 long, 1.44 wide, light brown with dark band in centre. Abdomen 3.01 long, 2.32 wide, 2.14 high, pattern as in (Figure 1.9A). Eyes subequal, diameter ~0.10. Leg I femur 2.61, patella 0.72, tibia 2.27, metatarsus 2.19, tarsus 0.91. Femur

I thickened, about six times longer than wide, curved. Leg formula 1423. Legs light brown in

Systematics of Southeast and East Asian Anelosimus species colour, darkening at distal end of femur and tibia. Distinct band at distal end of tibia I to IV.

Epigynum is lightly sclerotized with two pairs of spermathcae (or accessories) as diagnosed in Yoshida, 1993; Agnarsson & Zhang, 2006 and in (Figure 1.9B, C).

Male (paratype)

Total length 3.05. Prosoma 1.43 long, 1.01 wide, light orange brown with slightly darkened band in centre. Abdomen 1.75 long, 1.12 wide, 1.23 high, pattern as in (Figure

1.9F). Eyes subequal, diameter ~0.10. Leg I femur 1.95, patella 0.45, tibia 1.75, metatarsus

1.72, tarsus 0.75. Femur I thickened, about six times longer than wide, curved. Leg formula

1423. Legs light orange brown in colour, with slight darkening at distal end of each segment in Legs I and II. Palp as diagnosed in Yoshida, 1993; Agnarsson & Zhang, 2006 and in

(Figures 1.7D, E; 1.8), with a simple, slightly curved embolus, extending towards the cymbium but does not reach the tip.

Variation

Female: Total length 3.50 – 4.76. Prosoma 1.60 – 2.09 long, 1.20 – 1.60 wide. Male: Total length 2.50 – 3.12. Prosoma 1.20 – 1.60 long, 1.00 – 1.10 wide. Colouration on abdomen ranges from yellowish with distinct red longitudinal band to dark brown with dark longitudinal band and white spots on the sides.

Distribution

Singapore (Mandai, Pulau Ubin), Malaysia (Johor), Thailand (Khao Sam Roi Yat National

Park).

Systematics of Southeast and East Asian Anelosimus species

Natural history

Anelosimus kohi are found in mangrove habitats on Avicennia officinalis and Bruguiera cylindrica. Webs were found at the tip of branches enveloping several leaves and often littered with detritus. Similar to A. subcrassipes, webs of A. kohi consisted of a single adult male, single adult female, female with egg sac, females with juveniles between instar II to V.

In addition, webs consisting of a single adult male and adult female were also sometimes encountered. Anelosimus kohi is believed to be subsocial due to the late natal dispersal (instar

IV – VI), cooperative prey capture and prey sharing observed in juveniles of instars II to IV.

Under laboratory conditions, adults were also observed engaging in cooperative prey capture and prey sharing. Instar stages of juvenile estimated by comparison with laboratory reared specimens.

Systematics of Southeast and East Asian Anelosimus species

Figure 1.9. Anelosimus kohi. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) ventral; (E) lateral. (F) Male. (G) Female lateral. (H) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

A B

T MA

Figure 1.10. Anelosimus kohi. Male palp: (A) Ventral; (B) Details of sclerites. Arrows indicate embolus. Scale bar as indicated in each photograph.

Anelosimus parvulus sp. nov. (Figures 1.11; 1.12)

Material examined

Three male and eight female paratypes from SINGAPORE, opposite Tampines EcoGreen Park,

1°21'48.94"N 103°56'39.15"E, 15 m elevation, 30.x.2009, coll. S.P. Goh.

Systematics of Southeast and East Asian Anelosimus species

Diagnosis

Anelosimus parvulus (Figure 1.11A, F, G, H) is distinguished from other Anelosimus by its extremely short emobolus (Figure 1.11D, E). Females of A. parvulus differs from other

Anelosimus in external and internal genitalia. Internal epigynum of A. parvulus is similar to

A. bali Agnarsson (Agnarsson, 2012), A. potmosbi Agnarsson (Agnarsson, 2012) and A. chonganicus Zhu (Zhu, 1998) but differ in the position of the fertilization ducts which are just beside the spermathecae (Figure 1.11B, C). Epigynum structures are highly similar to A. linda Agnarsson (Agnarsson & Zhang, 2006) but phylogenetic analysis places them as two distinct species (Agnarsson, per. comm.)

Description

Female (paratype)

Total length 1.92. Prosoma 1.01 long, 0.84 wide, light brown with dark band in centre and around eyes. Abdomen 1.23 long, 0.92 wide, 0.83 high, pattern as in (Figure 1.11A).

Eyes subequal, diameter ~0.08. Leg I femur 1.28, patella 0.39, tibia 1.04, metatarsus 0.83, tarsus 0.30. Femur I thickened, about five times longer than wide, curved. Leg formula 1243.

Legs pale yellow in colour, darkening at distal end in femur I. Epigynum consists of simple fertilization and copulatory ducts as diagnosed in (Figure 1.11B, C), with a pair of lightly sclerotized opening and faintly visible spermathcae through the cuticle.

Male (holotype)

Total length 1.91. Prosoma 1.08 long, 0.82 wide, light orange brown with faint dark spot at near the pedicel. Abdomen 1.06 long, 0.80 wide, 0.82 high, pattern as in (Figure

1.11F). Eyes subequal, diameter ~0.09. Leg I femur 1.64, patella 0.38, tibia 1.24, metatarsus

0.92, tarsus 0.53. Femur I thickened, about five times longer than wide, curved. Leg formula

1243. Legs pale yellow in colour, with slight darkening at distal end of each segment in Legs 67

Systematics of Southeast and East Asian Anelosimus species

I and II. Palp as diagnosed in (Figures 1.11D, E; 1.12), with a short, simple embolus and sclerotization of part of the tegulum margin.

Variation

Female: Total length 1.70 – 2.10. Prosoma 0.80 – 1.10 long, 0.60 – 0.90 wide. Male:

Total length 1.50 – 1.92. Prosoma 0.90 – 1.17 long, 0.70 – 0.83 wide.

Distribution

Singapore (Mandai and Tampines), Malaysia (Johor and Pahang).

Natural history

Webs of A. parvulus were found in abundance on wildflowers, Cyanthillium cinereum. These were located in open fields with zero canopy cover. Individual webs were in close proximity and webs were often clustered in patches of wildflowers. Anelosimus parvulus is a solitary species as webs encountered consisted of only a single adult male, single adult female, female with egg sac or female with juveniles of instar II or III. Laboratory observation also revealed that juveniles disperse from natal nest between instar II and III. Instar stages of juvenile estimated by comparison with laboratory reared specimens.

Etymology

The specific epithet is a reference to ‘parvulus’ which means tiny in Latin and refers to the extremely short embolus of male.

Systematics of Southeast and East Asian Anelosimus species

A B

FD CD

D E F G

H I

Figure 1.11. Anelosimus parvulus sp. nov. (A). Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.12. Anelosimus parvulus sp. nov. Male palp: (A) Subventral; (B) Ventral and (C) Details of sclerites. Arrows indicate the emobolus. Scale bar as indicated in each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus membranaceus Zhang, Liu & Zhang, 2011

(Figures 1.13; 1.14)

Anelosimus membranaceus Zhang, Liu & Zhang, 2011: 50–52, figs 5–6.

Anelosimus seximaculatum Zhang, Liu & Zhang, 2011: 53–55, figs 10–12.

Material examined

Seven male and 21 female paratypes from CHINA, Ba Wang Mountain, 18°42'36.84"N

108°47'14.04"E, 179 m elevation, 18.vi.2011, coll. S.P. Goh, D. Li, W.J. Gan and X. Xu.

Diagnosis

Anelosimus membranaceus (Figure 1.13A, G, H, I) is distinct from other Anelosimus by the

shape of the embolus which is short and hooked (Figure 1.13D, E, F). Male is similar to A. exiguus Yoshida (Yoshida, 1986) and A. parvulus (Figure 1.11) but can be distinguished by the curve (towards the cymbium) of the embolus, the convolution of the sperm ducts and the dark tegulum margin. Females differ from other Anelosimus in external and internal genitalia

(Figure 1.13B, C).

Description

Female (paratype)

Total length 2.13. Prosoma 1.02 long, 0.91 wide, brown with dark band in centre and around eyes. Abdomen 1.40 long, 1.08 wide, 0.96 high, pattern as in (Figure 1.13A). Eyes subequal, diameter ~ 0.08. Leg I femur 1.50, patella 0.42, tibia 1.30, metatarsus 1.15, tarsus

Systematics of Southeast and East Asian Anelosimus species

0.55. Femur I thickened, about five times longer than wide, curved. Leg formula 1243. Legs

pale yellow in colour at base, darkening at distal end of each segment in Legs I and II. Femur

I orange brown in colour. Epigynum consists of simple fertilization and copulatory ducts as

diagnosed in Zhang et al. (2011) and in (Figure 1.13B, C), with distinctly sclerotized pair of openings and pair of spermathecae visible through the cuticle. There is little to no gap between the spermathecae.

Male (paratype)

Total length 1.77. Prosoma 0.89 long, 0.71 wide, light orange brown with faint dark spot near the pedicel. Abdomen 0.99 long, 0.78 wide, 0.78 high, pattern as in (Figure 1.13G).

Eyes subequal, diameter ~0.08. Leg I femur 1.26, patella 0.35, tibia 1.05, metatarsus 0.61,

tarsus 0.37. Femur I thickened, about five times longer than wide, curved. Leg formula 1243.

Legs pale yellow in colour at base, with slight darkening at distal end of each segment, most

distinct in femur I. Palp as diagnosed in Zhang et al. (2011) and (Figure 1.13D–F; 1.14), with

a short embolus that curves towards the tip of the cymbium.

Variation

Female: Total length 1.90 – 2.80. Prosoma 0.85 – 1.20 long, 0.70 – 1.00 wide. Male:

Total length 1.40 – 1.9. Prosoma 0.70 – 1.00 long, 0.60 – 0.80 wide.

Distribution

China (Hainan), East Malaysia (Sabah), Peninsula Malaysia (Selangor).

Systematics of Southeast and East Asian Anelosimus species

Natural history

Anelosimus membranaceus were collected from wildflowers between 0.5 – 1.0 m high, growing along forest edges and roads with little canopy cover. Individual webs were often found in close proximity and in clustered patches. Webs consisted of single adult male, single adult female, female with egg sac, female with juveniles between instar II to IV. Field and laboratory observations indicates that A. membranaceus is solitary due to early dispersal

(Instar II to III) and lack of cooperation amongst siblings beyond instar IV. Instar stages of juvenile estimated by comparison with laboratory reared specimens.

Comments

Egg sacs produced by female A. membranaceus were allowed to hatch under laboratory conditions. Juveniles were reared to adulthood to confirm the male conspecific of this species.

Systematics of Southeast and East Asian Anelosimus species

A B

FD CD

D E F

H I

Figure 1.13. Anelosimus membranaceus. (A). Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

A B

T T

Figure 1.14. Anelosimus membranaceus. Male palp: (A) Ventral; (B) Subventral and (C) Details of sclerites. Arrows indicate emobolus. Scale bars as indicated in each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus taiwanicus Yoshida, 1986

(Figures 1.15; 1.16)

Anelosimus taiwanicus Yoshida, 1986: 32 – 26, figs 4–8. Zhu, 1998: 290–291, figs

196A–D.

Material examined

Four male and five female paratypes from TAIWAN, Ken Ding National Park, 21°57'42.30"N

120°48'43.00"E, 225 m elevation, 14.vi.2010, coll. S.P. Goh, R. C. Cheng and C.P. Liao.

Diagnosis

Anelosimus taiwanicus (Figure 1.15F–H) can be distinguished from other Anelosimus species

by the shape of its embolus which spirals 3 times clockwise on the plane of the embolus base

around its base (Figure 1.15D, E). Male is similar to A. biglebowski Agnarsson (Agnarsson &

Zhang, 2006), A. dude Agnarsson (Agnarsson & Zhang, 2006) and A. eidur Agnarsson

(Agnarsson, 2012) but can be distinguished by the higher number of spirals and the smaller diameter of the spiral in relation to the tegulum. Females are different from other Anelosimus in internal and external female genitalia (Figure 1.15B, C), which consists of convoluted copulatory ducts below the spermathecae.

Description

Female (paratype)

Systematics of Southeast and East Asian Anelosimus species

Total length 3.51. Prosoma 1.40 long, 1.20 wide, brown with dark spot in centre and tip. Abdomen 2.76 long, 2.29 wide, 1.78 high, pattern as in (Figure 1.15A). Eyes subequal, diameter ~0.11 Leg I femur 1.72, patella 0.60, tibia 1.51, metatarsus 1.15, tarsus 0.63. Femur

I thickened, about five times longer than wide, curved. Leg formula 1243. Legs light brown in colour at base, darkening at distal end of femur and tibia. Distinct band at distal end of tibia. Epigynum as diagnosed in Yoshida, 1986 and in (Figure 1.15B, C), consisting of convoluted copulatory ducts just beneath the pair of spermathecae.

Male (paratype)

Total length 2.67. Prosoma 1.63 long, 1.24 wide, orange brown with dark spot near the pedicel. Abdomen 1.36 long, 1.11 wide, 0.71 high, pattern as in (Figure 1.15F). Eyes subequal, diameter ~0.12. Leg I femur 2.01, patella 0.72, tibia 1.97, metatarsus 1.79, tarsus

0.76. Femur I thickened, about five times longer than wide, curved. Leg formula 1243. Legs light orange brown in colour at base, darkening at distal end of each segment, most distinct in legs I and II. Palp as diagnosed in Yoshida, 1986 and (Figurse 1.15D, E; 1.16), with the embolus forming 2.5 to 3 tight spirals around the base before extending beyond the cymbium.

Variation

Female: Total length 2.50 – 3.57. Prosoma 1.40 – 2.70 long, 1.00 – 1.30 wide. Male:

Total length 3.10 – 4.20. Prosoma 1.00 – 1.80 long, 0.80 – 1.27 wide.

Distribution

Taiwan (Ken Ding National Park).

Systematics of Southeast and East Asian Anelosimus species

Natural history

Anelosimus taiwanicus was collected in bushes of Ixora sp. All specimens were collected in one patch of bushes. Webs of A. taiwanicus enveloped several leaves at the tip of branches with a small amount of detritus. Webs consisted of single adult female or female with juveniles between instar III to IV. Laboratory observations indicate that A. taiwanicus is subsocial due to delayed juvenile dispersal at instar IV and cooperation amongst siblings up to instar V.

Systematics of Southeast and East Asian Anelosimus species A B

C FD CD

D E F

G H

Figure 1.15. Anelosimus taiwanicus. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Lateral. (F) Male. (G) Female lateral. (H) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.16. Anelosimus taiwanicus. Male palp: (A) Ventral; (B) Ectal and (C) Details of sclerites. Arrows indicate the emobolus. Scale bars as indicated in each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus kinabaluensis sp. nov.

(Figures 1.17; 1.18)

Material examined

One male holotype, three male and four female paratypes from MALAYSIA, Sabah, Mount

Kinabalu National Park 6° 2'13.20"N 116°33'0.66"E, 2100 m elevation, 18.vi.2011, coll. S.P.

Goh.

Diagnosis

Anelosimus kinabaluensis (Figure 1.17A, G) can be distinguished from other Anelosimus

species by external and internal structure of the female epigynum (Figure 1.17B, C). The

external structure of the epigynum is similar to A. seximaculatum (Figure 1.24) but can be

distinguished from it by the internal structure of the epigynum and lighter body colouration in

A. kinabaluensis. Male is distinct in dark colouration of the palps, with a curved embolus

turning clockwise (Figures 1.17D–F; 1.18). Male is similar to A. circumdantus (Figure 1.29)

and A. exiguus Yoshida (Yoshida, 1986) but differs in the length of the embolus.

Description

Female (holotype)

Total length 2.35. Prosoma 1.04 long, 0.88 wide, dark brown with dark spot in centre

and tip. Abdomen 1.53 long, 1.19 wide, 1.23 high, pattern as in (Figure 1.17A). Eyes

subequal, diameter ~0.08. Leg I femur 1.33, patella 0.41, tibia 1.14, metatarsus 0.99, tarsus

Systematics of Southeast and East Asian Anelosimus species

0.55. Femur I thickened, about six times longer than wide, curved. Leg formula 1423. Legs light yellowish brown in colour at base, darkening at femur, patella and tibia. Epigynum consists of simple fertilization and copulatory duct as diagnosed in (Figure 1.17B, C, 1.), with a pair of distinctly sclerotized openings.

Male (paratype)

Total length 2.23. Prosoma 0.83 long, 0.79 wide, dark brown. Abdomen 0.95 long,

0.70 wide, 0.70 high, pattern similar to female holotype (Figure 1.17A). Eyes subequal, diameter ~0.07. Leg I femur 1.32, patella 0.46, tibia 1.86. Legs in poor condition. Palp is roundish and dark as diagnosed in (Figures 1.17D–F; 1.18), with the embolus turning downwards towards the tegulum before curving upwards towards the cymbium, stopping a short distance from the tip.

Variation

Male: Prosoma length 0.83 – 1.08, width 0.79 – 0.91. Abdomen length 0.95 – 1.20, width 0.70 – 0.95. Femur I 1.32 – 1.37. Female: Prosoma length 1.03 – 1.20, width 0.74 –

1.20. Abdomen length 1.45 – 1.78, width 1.19 – 1.57. Femur I 0.93 – 1.41.

Distribution

Only known from type locality.

Systematics of Southeast and East Asian Anelosimus species

Natural history

Anelosimus kinabaluensis were collected from wildflowers located along roadside with zero canopy cover and from canopy fogging. Webs consisted of single adult male, single female, female with egg sac and female with instar II juveniles. Instar stages of juvenile estimated by comparison with laboratory reared specimens.

Etymology

The specific epithet is named after Mount Kinabalu, where it was collected.

Systematics of Southeast and East Asian Anelosimus species

Figure 1.17. Anelosimus kinabaluensis sp. nov. (A) Female. (B) Male palp, Ventral; Female epigynum: (C) Ventral; (D) Dorsal, cleared. (E) Female lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.18. Anelosimus kinabaluensis sp. nov. Male palp: (A) Ventral and (B) Details of sclerites. Arrow indicate the embolus. Scale bars as indicated on each photograph.

Anelosimus floreni sp. nov.

(Figures 1.19; 1.20)

Material examined

One male holotype, four male and two female paratypes from MALAYSIA, Sabah, Mount

Kinabalu National Park 6° 6'N 116°50'E, 500 – 700 m elevation. 2.iii.1997, coll. A. Floren.

Systematics of Southeast and East Asian Anelosimus species

Diagnosis

Anelosimus floreni (Figure 1.19A, G, H, I) is distinct from other Anelosimus species in the

shape of its embolus in the male (Figure 1.19D–F), which is spiral shaped, completing 3 coils

along the plane of the embolus base. The male is similar to A. taiwanicus Yoshida (Yoshida,

1986), A. eidur Agnarsson (Agnarsson, 2012), A. biglebowski Agnarsson (Agnarsson &

Zhang, 2006), and A. dude Agnarsson (Agnarsson & Zhang, 2006), but can be distinguished

by the number of spirals and the diameter of the spirals in relation to the size of the palps.

Females also differ from other Anelosimus species in external and internal epigynum

structures (Figure 1.19B, C), with distinct convoluted copulatory ducts around the

spermathecae.

Description

Female (paratype)

Total length 2.10. Prosoma 0.94 long, 0.75 wide, dark brown. Abdomen 1.36 long,

1.02 wide, 1.11 high, pattern as in (Figure 1.19A). Eyes subequal, diameter ~0.07. Leg I

femur 1.16, patella 0.28, tibia 0.90, metatarsus 0.85, tarsus 0.46. Femur I thickened, about

five times longer than wide, curved. Leg formula 1243. Legs light yellowish brown in colour

at base, darkening at femur I. Epigynum as diagnosed in (Figure 1.19B, C), with lightly

sclerotized opening and a pair of spermathecae visible through the cuticle. Copulatory ducts are long and convoluted around the spermathcae.

Male (holotype)

Total length 1.73. Prosoma 0.86 long, 0.77 wide, dark brown. Abdomen 1.14 long,

0.87 wide, 0.78 high, pattern as in (Figure 1.19G). Eyes subequal, diameter ~0.09. Leg I

Systematics of Southeast and East Asian Anelosimus species

femur 1.27, patella 0.25, tibia 1.08, metatarsus 0.71, tarsus 0.51. Femur I thickened, about four times longer than wide, curved. Leg formula 1243. Legs orange brown in colour, darkening on distal ends of each segment, most distinct in legs I and II. Palp as diagnosed in

(Figures 1.19D–F; 1.20), with embolus forming 3 spirals of diameter similar to the width of the palps.

Variation

Male: Total length 1.57 – 1.80. Prosoma 0.70 – 0.92 long, 0.58 – 0.78 wide. Abdomen

0.83 – 1.15 long, 0.62 – 0.89 wide, 0.66 – 0.79 high. Leg I femur 0.83 – 1.35. Female: Total length 1.99 – 2.11. Prosoma 0.94 – 1.12 long, 0.74 – 0.78 wide. Abdomen 1.32 – 1.36 long,

1.03 wide, 0.99 – 1.11 high. Leg I femur 1.03 – 1.17.

Distribution

Only known from type locality.

Natural history

Anelosimus floreni were collected from canopy fogging in five, 15 and 40 year old secondary forest.

Etymology

The specific epithet, a noun in apposition, is a reference to the collector A. Floren.

Systematics of Southeast and East Asian Anelosimus species

A B

G D E F

H I

Figure 1.19. Anelosimus floreni sp. nov. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

Figure 1.20. Anelosimus floreni sp. nov. Male palp: (A) Ventral and (B) Details of sclerites. Arrows indicate embolus. Scale bars as indicated in each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus cochlianis sp. nov.

(Figures 1.21; 1.22)

Material examined

One male holotype, four male and two female paratypes from MALAYSIA, Sabah, Mount

Kinabalu National Park 6° 6'N 116°50'E, 500 – 700 m elevation. 16.ii.1997 – 2.iii.1997, coll.

A. Floren.

Diagnosis

Anelosimus cochlianis (Figure 1.21A, F, G, H) is distinct from other Anelosimus species in the shape of the embolus which is ‘corkscrew’ shaped (Figure 1.21D, E). Male is similar to

A. luckyi Agnarsson (Agnarsson, 2012) but A. cochlianis can be distinguished by its longer embolus and the presence of a cymbial hood. Females also differ from other Anelosimus species in external and internal epigynum structures (Figure 1.21B, C), with sclerotized epigynum margin and a pair of spermathcae visible through the cuticle.

Description

Female (paratype)

Total length 2.22. Prosoma 0.93 long, 0.83 wide, orange brown with dark band in centre and near eyes. Abdomen 1.46 long, 1.24 wide, 1.18 high, pattern as in (Figure 1.21A).

Eyes subequal, diameter ~0.08. Leg I femur 1.10, patella 0.21, tibia 0.81, metatarsus 0.66, tarsus 0.48. Femur I thickened, about six times longer than wide, curved. Leg formula 1423.

Systematics of Southeast and East Asian Anelosimus species

Legs light yellowish brown in colour at base, darkening in femur I. Epigynum consists of simple fertilization and copulatory ducts as diagnosed in (Figure 1.21B, C), with sclerotized epigynum margin and pair of spermathcea visible through the cuticle.

Male (holotype)

Total length 2.12. Prosoma 0.93 long, 0.72 wide, light orange brown. Abdomen 1.03 long, 0.73 wide, 0.75 high, pattern as in (Figure 1.21F). Eyes subequal, diameter ~0.07. Legs are in poor condition. Palp is roundish and as diagnosed in (Figures 1.21D, E; 1.22), with a short ‘corkscrew’ shaped embolus that extend to the tip of the cymbium.

Variation

Male: Total length 2.12 ˗ 2.18. Prosoma 0.79 – 0.93 long, 0.58 – 0.72 wide. Abdomen

0.79 – 1.08 long, 0.62 – 0.73 wide, 0.66 – 0.75 high. Female: Prosoma 0.79 – 1.01 long, 0.66

– 0.84 wide. Abdomen 1.12 – 1.46 long, 0.79 – 1.25 wide, 0.83 – 1.18 high.

Distribution

Malaysia, Sabah, (Mount Kinabalu National Park and Crocker Range Park).

Natural history

Anelosimus cochlianis were collected from canopy fogging in a five year old secondary forest.

Systematics of Southeast and East Asian Anelosimus species

Etymology

The specific epithet is a reference to ‘cochlia’ which means ‘twist’ in Latin and is a reference to the twisted shape of the embolus.

Systematics of Southeast and East Asian Anelosimus species

A B

FD CD

D E F G

CH E

H I

Figure 1.21. Anelosimus cochlianis sp. nov. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

A B

T CH

Figure 1.22. Anelosimus cochlianis sp. nov. Male palp: (A) Ventral and (B) Details of sclerites. Arrow indicate embolus. Scale bar as indicated in each photograph.

Anelosimus hiatus, sp. nov.

(Figure 1.23)

Material examined

One female holotype CHINA, Hainan, Ba Wang Mountain 19° 7'15.81"N109° 5'0.51"E, 299

m elevation. 29.vi.2011, coll. S.P. Goh and D. Li.

Systematics of Southeast and East Asian Anelosimus species

Diagnosis

Anelosimus hiatus (Figure 1.23A, C) is distinct from other Anelosimus species in the internal structure of the epigynum (Figure 1.23B). Female is similar to A. helix (Figure 1.26) and A. seximaculatum (Figure 1.24) but A. hiatus can be distinguished by the gap between the coils of the pair of copulatory ducts (Figure 1.23B).

Description

Female (holotype)

Total length 2.85. Prosoma 1.23 long, 1.00 wide, orange brown with dark band in centre and near eyes. Abdomen 2.08 long, 1.64 wide, 1.59 high, pattern as in (Figure 1.23A).

Eyes subequal, diameter ~0.09. Leg I femur 1.25, patella 0.50. Femur I thickened, about five times longer than wide, curved. Leg formula 1423. Legs light brown in colour at base, darkening in femur I. Epigynum consists of simple fertilization and convoluted copulatory ducts as diagnosed in (Figure 1.23B), with a gap between the pair of copulatory ducts.

Distribution

Only known from type locality.

Natural history

Anelosimus hiatus was collected on wildflowers along the forest edge with zero canopy cover.

Systematics of Southeast and East Asian Anelosimus species

Etymology

The specific epithet is a reference to ‘hiatus’ which means ‘gap’ in Latin and is a reference to the gap between the copulatory ducts in the female epigynum.

Systematics of Southeast and East Asian Anelosimus species

Figure 1.23. Anelosimus hiatus sp. nov. (A) Female. Female epigynum (B) Ventral; (C) dorsal, cleared. (D) Female lateral. Scale bar as indicated below each photograph.

Systematics of Southeast and East Asian Anelosimus species

Anelosimus seximaculatum Zhu, 1998

(Figures 1.24; 1.25)

Theridion seximaculatum Zhu, 1998: 141, 84A–C.

Anelosimus seximaculatum Zhang, Liu & Zhang, 2011: 53, 10–14.

Anelosimus membranaceus Zhang, Liu & Zhang, 2011: 50, 1–4.

Material examined

One male paratype from MALAYSIA, Sabah, Observation Hill behind Kota Kinabalu city,

5°58'47.94"N 116° 4'43.20"E, 45 m elevation, 2.vi.2011, coll. S.P. Goh. Three male and four

female paratypes from CHINA, Hainan, Ba Wang Mountain 19° 7'15.81"N109° 5'0.51"E, 299

m elevation. 29.vi.2011, coll. S.P. Goh and D. Li. Two males and four females paratype from

CHINA, Hainan, Jian Feng Ling, 18°42'36.84"N 108°47'14.04"E, 179 m elevation. 27.vi.2011,

coll. S.P. Goh and D. Li. 13 males and ten females paratype from TAIWAN, Wu Lai,

24°50'17.30"N 121°31'41.40"E, 219 m elevation. 11.vi.2010, coll. S.P. Goh.

Diagnosis

A. seximaculatum (Figure 1.24A, G) is distinct from other Anelosimus species in the shape of

the embolus, which is spiral shaped with the spirals rising towards the cymbium,

perpendicular to the embolus base (Figure 1.24D–F). Male is similar to A. chonganicus Zhu

(Zhu, 1998) and A. helix (Figure 1.26) but can be distinguished by the lower number of spirals, about 1.5 times, the looser coils of the spirals and the clockwise turn of the embolus.

Females of A. seximaculatum can be differentiated from other Anelosimus by the copulatory

Systematics of Southeast and East Asian Anelosimus species plate of the epigynum, with a pair of distinctly sclerotized openings, with dark colour spermathecae visible through the openings and long convoluted copulatory ducts (Figure

1.24B, C).

Description

Female (paratype)

Total length 2.80. Prosoma 1.21 long, 1.03 wide, dark brown with black streaks in centre and near the rim. Abdomen 1.71 long, 1.59 wide, 1.44 high, pattern as in (Figure

1.24A). Eyes subequal, diameter ~0.09. Leg I femur 1.50, patella 0.50, tibia 1.16, metatarsus

0.82, tarsus 0.57. Leg formula 1243. Legs pale yellow in colour with dark bands on distal ends of femur and tibia. Epigynum as diagnosed in Zhang et al., 2011 and in (Figure 1.24B,

C) with a pair of sclerotized opening and convoluted copulatory ducts.

Male (paratype)

Total length 2.45. Prosoma 1.21 long, 0.98 wide, orange brown with streaks of black colouration in centre and near eyes. Abdomen 1.50 long, 1.31 wide, 1.18 high, pattern as in

(Figure 1.24F). Eyes subequal, diameter ~0.09. Leg I femur 1.55, patella 0.49, tibia 1.16, metatarsus 0.90, tarsus 0.57. Leg formula 1243. Femur I four times as long as wide. Legs orange brown in colour, darker in femur I. Palps as diagnosed in Zhang et al. (2011) and in

(Figures 1.24D–F; 1.25), with a spiral shaped embolus that coils clockwise from the base towards the tip of the cymbium.

Systematics of Southeast and East Asian Anelosimus species

Variation

Male: Total length 2.00 – 2.90. Prosoma 1.27 – 1.51 long, 0.87 – 1.11 wide. Femur I

1.67 – 2.14. Female: Total length 2.46 – 3.33. Prosoma 1.11 – 1.59 long, 1.03 – 1.19 wide.

Femur I 1.67 – 2.06.

Distribution

China (Hainan). Taiwan (Wu Lai). Malaysia (Sabah).

Natural history

Webs of A. seximaculatum were located on wildflowers (0.2 – 1.5 m) growing along edge of trails or roads with little to no canopy cover. Webs consisted of single adult male, single adult female, female with egg sac, female with juveniles of instar II to III or juveniles between instar III to V. Laboratory observation of A. seximaculatum reveals it as solitary living with juveniles dispersing from natal nest between instar II and III and little cooperation amongst juveniles beyond instar III.

Comments

Males were collected at same locality as females. Egg sacs produced by female A. seximaculatum were also allowed to hatch under laboratory conditions. Juveniles were reared to adulthood to confirm the male conspecific of this species.

100

Systematics of Southeast and East Asian Anelosimus species

Figure 1.24. Anelosimus seximaculatum. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

101

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.25. Anelosimus seximaculatum. Male palp: (A) Ventral and (B) Subventral. Arrows indicate embolus. Scale bars as indicated in each photograph.

Anelosimus helix sp. nov.

(Figures 1.26; 1.27)

Material examined

One male holotype and one female paratype from MALAYSIA, Sabah, Mount Kinabalu

National Park, Poring Hot Springs 6° 2'N 116°50'E, 500 – 700 m elevation. 27.ii.1996, coll.

A. Floren.

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Systematics of Southeast and East Asian Anelosimus species

Diagnosis

Anelosimus helix (Figure 1.26A, D) is distinct from other Anelosimus species in the shape of

the embolus, which is spiral shaped with the spirals rising perpendicular to the embolus base

(Figure 1.26E–G). Male is similar to A. chonganicus Zhu (Zhu, 1998) and A. seximaculatum

(Figure 1.24), but can be distinguished by downward turn (towards the embolus base) of the

2nd coil and the clockwise spiral of the embolus. The cymbium is also longer relative to its

width in A. helix as compared to A. seximaculatum. Females of A. helix can be differentiated

from other Anelosimus by the copulatory plate of the epigynum, with a pair of sclerotized

openings and long convoluted copulatory ducts (Figure 1.26B, C). External epigynum

structure of A. helix is similar to A. kinabaluensis but can be distinguished by its internal epigynum structure.

Description

Female (paratype)

Total length 2.29. Prosoma 1.19 long, 0.90 wide, dark brown with black streaks in centre and near the rim. Abdomen 1.31 long, 1.11 wide, 0.98 high, pattern as in (Figure

1.26A). Eyes subequal, diameter ~0.08. Leg I femur 1.03, patella 0.37, tibia 0.91, metatarsus

0.69, tarsus 0.49. Leg formula 1243. Legs pale yellow in colour with dark bands on distal ends of femur and tibia. Epigynum as diagnosed in (Figure 1.26B, C) with a pair of lightly sclerotized opening, pair of spermathcae barely visible through the cuticle and convoluted copulatory ducts.

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Systematics of Southeast and East Asian Anelosimus species

Male (holotype)

Total length 1.23. Prosoma 0.66 long, 0.50 wide, orange brown with streaks of black

colouration in centre and near eyes. Abdomen 1.04 long, 0.50 wide, 0.79 high, pattern as in

(Figure 1.26D). Eyes subequal, diameter ~0.05. Leg I femur 0.98, patella 0.26, tibia 0.83,

metatarsus 0.69, tarsus 0.42. Leg formula 1243. Femur I four times as long as wide. Legs

orange brown in colour, darker in femur I. Palps as diagnosed in (Figures 1.26E–G; 1.27),

spiral shaped with a clockwise turn, completing about 1.5 turns.

Distribution

Malaysia, Sabah (Crocker Range Park and Mount Kinabalu National Park).

Natural history

Webs of A. seximaculatum were located on wildflowers (0.2 – 1.5 m) growing along edge of

trails or roads with little to no canopy cover. Webs consisted of single adult male, single adult female, female with egg sac, female with juveniles of instar II to III or juveniles between

instar III to V. Laboratory observation of A. seximaculatum reveals it as solitary living with

juveniles dispersing from natal nest between instar II and III and little cooperation amongst

juveniles beyond instar III.

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Systematics of Southeast and East Asian Anelosimus species

Comments

105

Systematics of Southeast and East Asian Anelosimus species

E CH

Figure 1.26. Anelosimus helix sp. nov. (A). Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. (D) Male. Male palp: (E) Mesal; (F) Ventral. (G) Lateral. (H) Female lateral. (I). Male lateral. Scale bars as indicated below each photograph.

106

Systematics of Southeast and East Asian Anelosimus species

A B

MA T

Figure 1.27. Anelosimus helix sp. nov. Male palp: (A) Ventral and (B) Details of sclerites. Arrow indicate embolus. Scale bar as indicated in each photograph.

Anelosimus albus sp. nov. (Figure 1.28)

Material examined

One female holotype MALAYSIA, Sabah, Tawau Hills Park 6° 2'N 116°50'E. 06.ix.2009, coll.

A. Floren.

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Systematics of Southeast and East Asian Anelosimus species

Diagnosis

Anelosimus albus (Figure 1.28A, D) is distinct from other Anelosimus in the external and internal structure of the epigynum (Figure 1.28B, C) which consist of a lightly sclerotized copulatory plate and long convoluted copulatory ducts that are visible through the cuticle.

Internal epigynum structure is similar to A. nelsoni Agnarsson (Agnarsson & Zhang, 2006) but differs in the position of coils around the spermathecae.

Description

Female (holotype)

Total length 1.85. Prosoma 0.91 long, 0.70 wide, orange brown with dark band in centre and near eyes. Abdomen 1.31 long, 1.11 wide, 0.98 high, pattern as in (Figure 1.28A).

Eyes subequal, diameter ~0.09. Leg I femur 0.90, patella 0.20, tibia 0.86, metatarsus 0.91, tarsus 0.50. Femur I about five times as long as wide. Legs orange brown in colour.

Epigynum as diagnosed in (Figure 1.28B, C), consisting of lightly sclerotized copulatory plate with visible copulatory ducts and spermathcea through the cuticle and long convoluted copulatory ducts which coils around the spermathecae.

Distribution

Only known from type locality.

Natural history

Unknown.

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Systematics of Southeast and East Asian Anelosimus species

Etymology

The specific epithet references “albus” which is white in Latin and refers to the unusual white colouration of the abdomen of the type specimen.

109

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.28. Anelosimus albus sp. nov. Female only. (A) Female. Epigynum: (B) Ventral; (C) Dorsal, cleared. (D) Female lateral. Scale bars as indicated below each photograph.

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Systematics of Southeast and East Asian Anelosimus species

Anelosimus circumdantus sp. nov.

(Figures 1.29; 1.30)

Material examined

One male holotype and one male paratype from INDONESIA, East Bali, Kintamani. 2.viii.1992, coll. C.L. Deeleman.

Diagnosis

Anelosimus circumdantus (Figure 1.29A, D) can be differentiated from other Anelosimus

species by the spiral coil of the embolus which turns forms 2 complete turns at its base, with

the wider second coil extending below the base. Male is similar to A. floreni (Figure 1.19)

and A. eidur Agnarsson (Agnarsson, 2012) but can be distinguished by the number of spirals,

more elongated cymbium and the shape of the sperm duct.

Description

Male (holotype)

Total length 1.95. Prosoma 0.98 long, 0.79 wide, light orange brown. Abdomen 1.09

long, 0.86 wide, 0.87 high, pattern as in (Figure 1.29A). Eyes subequal, diameter ~0.07. Leg I

femur 1.37, patella 0.37, tibia 1.08. Legs in poor condition. Palps as diagnosed in Figures

1.29B, C, 1.30, with a spiral shaped embolus completing 2 spirals around its base.

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Systematics of Southeast and East Asian Anelosimus species

Distribution

Only known from type locality.

Natural history

Unknown.

Etymology

The specific epithet references “circumdant”, which means encircled in Latin and refers to the coiling of the embolus around its base.

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Systematics of Southeast and East Asian Anelosimus species

Figure 1.29. Anelosimus circumdantus, sp. nov. (A) Male. (B) Male lateral. Male palp: (C) Mesal; (D) Ventral; (E) Lateral. Scale bars as indicated below each photograph.

113

Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.30. Anelosimus circumdantus sp. nov. Male palp: (A) Ventral and (B) Subventral. Arrow indicate embolus. Scale bar as indicated in each photograph.

Anelosimus bali Agnarsson, 2012

(Figures 1.31; 1.32)

Anelosimus bali Agnarsson, 2012: 10, 14: figs 5D–F.

Material examined

Two male and three female paratypes from Indonesia, Lombok, Kuta. 10.xi.1990, coll. S.

Djojosudharmo.

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Systematics of Southeast and East Asian Anelosimus species

Diagnosis

Anelosimus bali (Figure 1.31A, G, H, I) can be distinguished from all other Anelosimus

species by the shape of the embolus which is spiral shaped, forming 2 complete coils

beginning from the apex of the embolic base and reaching just beyond the tip of the cymbium

(Figure 1.31D–F). Male is similar to A. chonganicus Zhu (Zhu, 1998) but larger and can be

distinguished by the slightly uneven diameters of the coil. Females differ in external

epigynum structure from other Anelosimus species, with lightly sclerotized epigynum margin

forming an incomplete heart shape and a pair of spermathcae clearly visible through the

cuticle. Internal epigynum structure resembles A. chonganicus Zhu (Zhang et al., 2011) and

A. pomio Agnarsson (Agnarsson, 2012) but can be distinguished by the wider distance

between the spermathcae and the shorter distance between the copulatory ducts and the

spermathcae.

Description

Female (paratype)

Total length 2.07. Prosoma 0.98 long, 0.77 wide, orange brown, darkening at centre

and near eyes. Abdomen 1.25 long, 0.92 wide, 0.94 high, pattern as in (Figure 1.31A). Eyes

subequal, diameter ~0.07. Epigynum consists of simple fertilization and copulatory ducts as diagnosed in Agnarsson (2012) and in (Figure 1.31B, C), with a lightly sclerotized heart

shaped epigynum.

Male (paratype)

Total length 2.60. Prosoma 1.17 long, 0.93 wide, orange brown, darkening at centre

and near eyes. Abdomen 1.53 long, 1.19 wide, 1.27 high, pattern as in (Figure 1.31G). Eyes

115

Systematics of Southeast and East Asian Anelosimus species subequal, diameter ~0.08. Legs in poor condition. Palps as diagnosed in (Figures 1.31D–F;

1.32), with a spiral shaped embolus completing 2 coils and a cymbium hood.

Variation

Female: Total length 2.02 – 2.35. Prosoma 1.03 – 1.06 long, 0.66 – 0.76 wide.

Abdomen 0.99 – 1.29 long, 0.83 – 0.92 wide, 0.91 – 0.99 high.

Distribution

Indonesia, Bali and Lombok.

Natural history

Unknown

116

Systematics of Southeast and East Asian Anelosimus species

A B

C CD FD

D E F G

E CH

H I

Figure 1.31. Anelosimus bali. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. Male palp: (D) Mesal; (E) Ventral; (F) Lateral. (G) Male. (H) Female lateral. (I) Male lateral. Scale bars as indicated below each photograph.

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Systematics of Southeast and East Asian Anelosimus species

A B

Figure 1.32. Anelosimus bali. Male palp: (A) Ventral and (B) Details of sclerites, cymbial embolic groove (smaller arrow), and embolus (larger arrow). Scale bars as indicated in each photograph.

118

Systematics of Southeast and East Asian Anelosimus species

Anelosimus deelemanae sp. nov.

(Figure 1.33)

Material examined

One female holotype from Indonesia, Bali, Cocos Grove. 2.viii1992, coll. C.L. en P.R.

Deeleman.

Diagnosis

Anelosimus deelemanae (Figure 1.33A, D) is distinct from other Anelosimus species by the external and internal epigynum structure (Figure 1.33B, C), which is lightly sclerotized, with faintly visible spermathcea through the cuticle and long convoluted copulatory ducts. Female is similar to A. albus (Figure 1.28) and A. floreni (Figure 1.19) but can be distinguished by a smaller gap between spermathcae and the convolution of the copulatory ducts on the ventral side of the spermathecae.

Description

Female (holotype)

Total length 3.08. Prosoma 1.41 long, 1.01 wide, light orange brown. Abdomen 1.67 long, 1.32 wide, 1.32 high, pattern as in (Figure 1.33A). Eyes subequal, diameter ~0.09. Leg I femur 1.61, patella 0.50, tibia 1.41, metatarsus 1.20, tarsus 1.08 Leg formula 1423. Legs yellowish in colour, darkening orange brown band at distal end of tibia, metatarsus and

119

Systematics of Southeast and East Asian Anelosimus species tarsus. Epigynum as diagnosed in (Figure 1.33B, C), consisting of lightly sclerotized copulatory plate and long convoluted copulatory ducts.

Distribution

Only known from type locality.

Natural history

Unknown

Etymology

The specific epithet is a reference to “C.L. Deeleman”, the collector of the holotype specimen.

120

Systematics of Southeast and East Asian Anelosimus species

A B

C CD

Figure 1.33. Anelosimus deelemanae sp. nov. Female only. (A) Female. Female epigynum: (B) Ventral; (C) Dorsal, cleared. (D) Female lateral. Scale bars as indicated below each photograph.

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Systematics of Southeast and East Asian Anelosimus species

Figure 1.34. Full phylogeny including all outgroups and posterior probability support for each clade.

122

Systematics of Southeast and East Asian Anelosimus species

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CHAPTER 2

NATURAL HISTORY AND BEHAVIOURAL OBSERVATIONS OF ANELOSIMUS SPECIES WITH FOCUS ON SOCIAL AND REPRODUCTIVE TRAITS

Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders

ABSTRACT

Sociality in spiders is a rare occurrence and much attention has been paid to

deciphering the steps of its evolution in spiders. Social behaviour and its evolution in

spiders of the genus Anelosimus (Araneae: Theridiidae) from Americas and Africa

have been the subject of numerous studies, but little is known about the natural

history and sociality of Asian Anelosimus species. In this study, I investigated natural

habitat, colony structure, life history and behavioural traits, focusing on maternal care,

natal dispersal, cooperative prey-capture and food sharing of ten species of

Anelosimus in Southeast and East Asia. I also examined reproductive traits of these

species, including copulation duration, clutch size (number of hatchlings per egg sac

per female), number of clutches produced in life time, incubation time, interval

between clutches and interval between hatching of spiderlings and production of

subsequent clutch. Mating behaviours of seven species were also described. All

species of Asian Anelosimus studied were found in areas with open canopy, exhibited

egg-sac guarding and maternal provision of food beginning at 2nd instars. Anelosimus

cameronis, A. chonganicus Zhu, A. exiguus Yoshida, A. kinabaluensis, A.

membranaceus Zhang, Liu & Zhang, A. parvulus and A. seximaculatum Zhang, Liu &

Zhang and are categorized as ‘solitary’ species due to their early dispersal at 2nd and

3rd instars and lack of cooperative behaviour beyond 3rd instars. Meanwhile A. kohi

Yoshida, A. subcrassipes Zhang, Liu & Zhang and A. taiwanicus Yoshida are

classified as ‘subsocial’ as a result of late dispersal between 4th and 6th instars and

continued cooperative behaviour beyond 3rd instars, even among spiderlings of different developmental stages. I also found that solitary species exhibited longer copulation duration, produced more clutches but with smaller clutch size, had shorter

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intervals between clutches and between hatching of spiderlings and production of the

subsequent clutch as compared to subsocial species. I discuss possible reasons for the

observations of these differences in behavioural and reproductive traits among the species examined and compare them with what has been documented in other species of Anelosimus known in the Americas.

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INTRODUCTION

Benefits of group living in spiders include communal brooding, cooperative prey-

capture, defence against predators and sharing of resources (Buskirk, 1981; Avilés,

1997; Whitehouse & Lubin, 2005; Lubin & Bilde, 2007). This is advantageous to

spiders due to access to larger prey (Nentwig, 1985; Powers & Avilés, 2007; Yip et

al., 2008), cooperation in guarding of egg sacs and feeding of young spiderlings, resulting in greater offspring survival (Christenson, 1984; Salomon & Lubin, 2007).

Yet, despite the benefits of sociality, it remains a rare occurrence in spiders. Less than

0.5% of spiders amongst 44 000 species (Platnick, 2014) adopts this form of lifestyle

(Burgess & Utez, 1982; Avilés, 1997; Whitehouse & Lubin 2005; Lubin & Bilde,

2007; Bilde & Lubin, 2011; Yip & Rayor, 2013).

Spiders are considered group living when multiple individuals of the same species share a web (Buskirk, 1981). Group living in spiders occurs in a few forms and these can be classified into three broad categories. ‘Non-territorial permanent-

social’ (Avilés, 1997), also known as ‘quasi-social’ or simply ‘social’ (Wilson, 1971)

spiders refer to group-living spiders that share a common web and exhibit cooperation

in prey-capture, brood care, web construction and web maintenance. These spiders

display a lack of dispersal and spiderlings usually do not disperse from the natal nest

(Avilés, 1997; Whitehouse & Lubin, 2005; Lubin & Bilde, 2007; Bilde & Lubin,

2011). ‘Non-territorial periodic-social’ (Avilés, 1997) or ‘subsocial’ (Wilson, 1971) spiders refers to groups that are formed by an individual mother and her progeny, which disperse before independent reproduction (Avilés, 1997; Whitehouse & Lubin,

2005; Lubin & Bilde, 2007; Bilde & Lubin, 2011; Avilés & Hardwood, 2012; Yip &

Rayor, 2013). Finally, ‘territorial’ (Avilés, 1997) or ‘communal’ (Wilson, 1971)

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders

spiders are those that aggregate but maintain individual web, exhibiting minimal

direct cooperation (Avilés, 1997; Uetz & Hieber, 1997; Whitehouse & Lubin, 2005;

Lubin & Bilde, 2007; Bilde & Lubin, 2011).

Social species are found mainly at low altitudes in the tropics, especially in the

Amazonian region of South America (Avilés, 1997; Powers & Avilés, 2007;

Whitehouse & Lubin, 2007). With fewer than 25 species, social spiders are atypical

even among group living spiders (Whitehouse & Lubin, 2007). However, these

spiders are diverse and are found in 11 genera of eight different families, with the

highest number in the family Theridiidae (Agnarsson et al., 2006a). While communal

spiders are believed to have evolved parasocially (Whitehouse & Lubin, 2005; Lubin

& Bilde 2007), phylogenetic analyses have revealed that social species are closely

related to subsocial species and may have evolved from subsocial ancestors (Avilés,

1997; Agnarsson et al., 2006a; Whitehouse & Lubin, 2007).

Subsocial spiders have been defined as ‘spiders in which spiderlings persist together with parent even after gaining independence in prey capture but disperse before production of their own progeny’ (Yip & Rayor, 2013). Under this definition, there are about 70 species of subsocial spiders in 16 families with high concentration in families Theridiidae and Eresidae (Yip & Rayor, 2013). These species are found in a wide range of habitats, overlapping with social species in the tropics but also persisting in the subtropics. Subsocial spiders are also more commonly found at

higher elevation than social species (Avilés et al., 2007; Guervara & Avilés, 2007;

Purcell & Avilés, 2008).

Differences in habitat preferences of social and subsocial species have led to speculation that ecological factors may contribute to or hinder the development of sociality. Recent studies have also unveiled effects of temperature and prey sizes on

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the level of sociality and group sizes in some species (Avilés & Tufino, 1998; Furey,

1998; Jones & Reichert, 2008). These indicate that both genetics and ecology play a

role in shaping and maintaining social behaviour of spiders (Yip & Rayor, 2013).

With a myriad of factors influencing sociality and variation in social behaviour of

spiders, much remains to be unveiled about this unique group of spiders.

The spider genus Anelosimus Simon, 1891, from the family Theridiidae has been considered an excellent model system for the study of social evolution in spiders

(Avilés et al., 2007; Guevera & Avilés, 2007; Purcell & Avilés, 2008; Avilés &

Harwood, 2012; Samuk & Avilés, 2013). Recent studies have revealed that maternal

care and a lack of offspring discrimination are ancestral states in this genus of spiders

which predisposes these spiders to the development of social behaviour (Agnarsson et

al., 2006a; Yip & Rayor, 2013; Samuk & Avilés, 2013). This genus is of great interest

also because it contains a mixture of solitary, subsocial, and social species, presenting

a spectrum of social behaviour among closely related species. Although this gradient

is also displayed in some species in the families Agelenidae, Dictynidae, Eresidae,

Scytodidae, Sparassidae, and Uloboridae, Anelosimus has the largest number of

subsocial and social species known (Avilés, 1997; Agnarsson, 2006; 2012a; 2012b;

Lubin & Bilde, 2007; Bilde & Lubin, 2011; Yip & Rayor, 2013).

Anelosimus spiders typically build three-dimensional webs and can be found

in a wide range of habitats, from lowland tropical forests to montane forests (Avilés,

1997; Agnarsson & Kuntner, 2005; Agnarsson, 2006; 2012a, 2012b; Agnarsson &

Zhang, 2006). To date, the genus Anelosimus contains six social species. With the

exception of A. guacamayos Agnarsson and A. oritoyacu Agnarsson, all social

Anelosimus species occur in lowland tropical rainforest in South America (Avilés et

al., 2007; Purcell, 2011). Colony structure of social species in this genus varies and

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders can consist of a single female with her spiderlings or up to tens of thousands of spiders (Vollrath, 1986; Avilés, 1997; Avilés et al., 2007). In all six social species, cooperation in foraging, web maintenance and alloparental brood care are also present

(Buskirk, 1981; Christenson, 1984; Avilés, 1997; Samuk & Avilés, 2013). These species also exhibit female biased sex ratio as a result of inbreeding (Avilés, 1997;

Whitehouse & Lubin, 2007). Dispersal in these spiders is accomplished through colony fission as a result of mechanical breakage of web of a single colony or by mass scattering of groups of mated females (Avilés & Tufino, 1998; Avilés et al.,

2006; Avilés & Harwood, 2012). However, differences exist even amongst these social species: A. eximius Keyserling is the only species observed to exhibit coordinated web movement in prey capture (Vollrath, 1984; Whitehouse & Lubin,

2007); A. guacamayos females with egg sacs maintain greater distances between conspecifics on the web as compared to A. eximius and A. domingo Levi (Avilés et al.,

2007; Avilés & Harwood, 2012) and form colonies with smaller number of individuals (Avilés et al., 2007). On top of these, the extent of aggregation in these species is also affected by environmental factors: A. eximius is observed to adopt a more subsocial living with increase in elevation (Agnarsson, 2006; Avilés et al.,

2007).

Subsocial spiders form the majority of species in this genus, with 16 species found in the Americas, Madagascar, Australia and Asia (Agnarsson & Kuntner, 2005;

Agnarsson & Zhang, 2006; Agnarsson, 2012a; Yip & Rayor, 2013). These 16 species are found in various habitats ranging from tropical forests to montane forests (Avilés et al., 2007; Yip & Rayor, 2013). Typical colonies of subsocial species consist of a single female and spiderlings that disperse just before reaching maturity. However, dispersal is not always consistent, such as in A. studiosus Hentz, in which nests of

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders multiple females are common at higher latitudes, indicating a lack of dispersal after reaching maturity (Riechert & Jones, 2008). Dispersal distances of these spiders have also been found to be short (< 50 cm) and it is thus suggested that resource competition instead of inbreeding avoidance is a cause of dispersal (Powers & Avilés,

2003; Corcobado et al., 2012). Unlike social species, cooperation in subsocial spiders, including Anelosimus occurs only amongst siblings and maternal female (Avilés,

1997; Whitehouse & Lubin, 2007; Yip & Rayor, 2013).

The disparities in behaviour amongst social and subsocial species of

Anelosimus have been attributed to various ecological factors such as prey size, growth rate of spiderlings, presence of predators, maternal longevity, temperature, precipitation and habitat structure (Avilés, 1997; Avilés et al., 2007; Powers & Avilés,

2007; Purcell & Avilés, 2007; 2008; Purcell, 2011; Avilés & Hardwood, 2012).

Indeed, as social behaviour is well developed in spiders of the genus Anelosimus, comparative studies of social behaviour of these spiders displaying different levels of sociality have sought to unveil the steps in the evolution of social behaviour within this genus and also in spiders.

Thus far, the majority of Anelosimus species described are found in North and

South America, with a few species recently described from Africa and Asia

(Agnarsson & Kuntner, 2005; Agnarsson, 2006; 2012a; 2012b; Agnarsson & Zhang,

2006; Agnarsson et al., 2010). Most Anelosimus species found in North and South

America are social or subsocial (Christenson, 1984; Rypstra & Tirey, 1989; Marques et al., 1998; Agnarsson, 2006; Avilés et al., 2007), with only three species, A. ethicus

Keyserling, A. nigrescens Keyserling and A. pacificus Levi described as solitary living (Agnarsson, 2006b; 2012a; 2012b). Meanwhile, phylogenetic analysis has revealed that Asian Anelosimus form an Old World Clade consisting of a mixture of

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subsocial and solitary species (Agnarsson, 2012b). Among the nine species included

in the analysis, little is known about the sociality and life histories of five species

(Agnarsson, 2012b). An additional seven species of Asian Anelosimus species have

also been described taxonomically but little is known about their life histories and

sociality (Yoshida, 1986; Agnarsson & Zhang, 2006; Zhang et al., 2011).

In this study, I conducted laboratory and field observations and experiments

on ten poorly known Anelosimus species from Southeast and East Asia. I aimed to document the life history of these ten species and determine whether they are solitary,

subsocial or social, with particular attention paid to habitat structure, colony structure, maternal care, cooperation, dispersal, and a number of reproductive traits, including mating, clutch size (number of hatchlings/egg sac), number of clutches (i.e., egg sacs), interval between clutches and interval between hatching and subsequent clutch. I aimed to obtain some comparative data to determine whether the species possess suites of traits that define them as solitary, subsocial or social species and also to

unveil the steps in the evolution of social behaviour in this genus of spiders. Of the

ten species studied, three species (A. cameronis, A. kinabaluensis and A. parvulus) are

newly described (Chapter 1) while the natural history of the remaining seven species

(A. chonganicus, A. exiguus, A. kohi, A. membranaceus, A. seximaculatum, A.

subcrassipes and A. taiwanicus) is unknown.

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MATERIALS AND METHODS

Colony Surveys in the Field

For simplicity, I referred to all webs occupied by Anelosimus spiders as ‘colonies’ (Li

et al., 1999; Yap & Li, 2009). I conducted 3–7 day surveys at each of the respective

locations (Table 2.1) to study the colony structure of each of ten Anelosimus species.

How long a survey lasted depended on the species, habitat, and the number of

colonies available. During field surveys, information on the occupants of every

Anelosimus colony encountered was recorded. The majority of spiders were found

along forest edges with little canopy cover. Spiders were commonly found on shrubs

and wildflowers, between 1.0 m to 1.8 m above ground level. The following

information were recorded for every colony of each species: (a) life stage of spider(s),

(b) number of individuals in the same colony, and (c) colony structure, where a

colony consists of a spider(s) living in an individual web, void of connections with

other webs (Li et al., 1999; Marques et al., 1998). The life stages of spiders were

determined by comparison with laboratory-bred spiderlings of identified instars.

Spider Collection and Maintenance in the Laboratory

For the purpose of observation under laboratory conditions, spiders were collected in

the field at various localities around East and Southeast Asia (Table 2.1) between

2009 and 2011. All collections were made either between 0800 h and 1800 h or

between 2000 h to 2200 h. Collection of spiders was achieved either by hand or with the use of sweep nets. Spiders collected were transported back to the laboratory where they were kept individually in transparent plastic cylindrical containers (65 ×

85 mm) with holes drilled for the provision of food and water. They were fed a diet of

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houseflies (Musca domestica), fruit flies (Drosophila melanogaster) or crickets

(Gryllus bimaculatus) provided ad libitum once a week to ensure optimal growth (Li

et al, 1999). Water was constantly available through a cotton dental roll dipped in a

water reservoir located at the bottom of the cage. They were housed in a controlled

laboratory environment (temperature: 25 ± 1°C; relative humidity: 60–80%;

photoperiod: 12L: 12D, with lights turned on at 0800 h and off at 2000 h daily.

All laboratory observations of spiders were conducted between 0800 h and

1800 h. Observations were only conducted on spiders that had been housed in the

laboratory for more than a week to ensure acclimatization. Spiders observed were

randomly chosen and no individuals were used for observation more than once per

day or for multiple observations for the same set of experiments.

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Table 2.1. List of Anelosimus species collected with GPS coordinates and elevation of collection sites.

Species Location GPS coordinates Elevation /m

Anelosimus cameronis Cameron Highlands, Peninsular Malaysia 4°28'48.90"N 101°23'3.40"E 1466

Xiamen, China 24°26'22.50"N 118° 6'0.30"E 50 Hainan, China 18°42'36.84"N 108°47'14.04"E 179

Anelosimus chonganicus Yang Ming Shan, Taiwan 25° 8'59.40"N 121°30'46.98"E 284 Ken Ding, Taiwan 21°57'42.00"N 120°48'43.00"E 225 Khao Sam Roi Yat National Park, 12°14'45.56"N 99°57'27.06"E 0 Thailand

Zhejiang, China 29°59'19.90"N 122°23'7.20"E 13 Anelosimus subcrassipes Zhejiang, China 29°50'36.00"N 122°16'59.90"E 7.9

Anelosimus taiwanicus Ken Ding, Taiwan 21°57'42.30"N 120°48'43.00"E 225

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Table 2.1 (Continued)

Species Location GPS coordinates Elevation /m

Jason Bay, Peninsular Malaysia 1°50'32.22"N 104° 8'47.70"E 4

Mandai Mangrove, Singapore 1°26'26.68"N 103°45'55.52"E 0 Anelosimus kohi Pulau Ubin, Singapore 1°24'25.20"N 103°59'27.60"E 0

Khao Sam Roi Yat National Park, Thailand 12°14'45.56"N 99°57'27.06"E 0

Genting Highland, Peninsular Malaysia 3°21'7.32"N 101°47'13.62"E 655 Anelosimus parvulus Tampines, Singapore 1°21'48.94"N 103°56'39.15"E 15

Crocker Range, Sabah, East Malaysia 5°25'0.00"N 116° 8'0.00"E Anelosimus Hainan, China 18°42'36.84"N 108°47'14.04"E 179 membranaceus Ulu Gombak Biodiversity Research Centre, 3°18'31.32"N 101°44'37.58"E 762 Peninsular Malaysia

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Table 2.1 (Continued)

Species Location GPS coordinates Elevation /m

Hainan, China 18°42'36.84"N 108°47'14.04"E 179 Anelosimus Kota Kinabalu, Sabah, East Malaysia 5°58'47.94"N 116° 4'43.20"E 44.8 seximaculatum Wulai, Taiwan 24°50'17.30"N 121°31'41.40"E 219

Anelosimus Mount Kinabalu National Park, Sabah, East 6° 2'13.20"N 116°33'0.66"E 2100 kinabaluensis Malaysia

Anelosimus exiguus Hainan, China 18°42'36.84"N 108°47'14.04"E 179

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Behavioural Observations in the Laboratory

Observations of colony composition during field survey provided information on the possible social levels displayed by the spiders examined. Colonies of social species contain multiple adult females and spiderlings (Lubin & Bilde, 2007); subsocial colonies consist of single adult female and late instar spiderlings. Late instar spiderlings refer to spiderlings beyond 4th instar stages (Yip & Rayor, 2013). Lastly, a nest of solitary species contains single adult female and 2nd or 3rd instar spiderlings.

While field surveys offered in-situ observation of the colony structure of Anelosimus species studied, laboratory observations of the following behavioural and reproductive traits were also examined to obtain a comprehensive understanding of the social structure of the study species.

Maternal care

Glass cages (110  55  155 mm) (Figure 2.1A) were used for the observation of maternal care of eggs and hatched spiderlings in seven species of Anelosimus (A. chonganicus, A. kohi, A. membranaceus, A. parvulus, A. seximaculatum, A. subcrassipes and A. taiwanicus). Upon production of an egg sac, females were transferred to the glass cage, housed individually and monitored daily. Behaviours exhibited by females such as egg sac guarding and care for the egg sac were recorded.

The number of hatched spiderlings, date of emergence and behaviour of spiderlings upon eclosion were recorded.

To determine if females were able to distinguish between egg sacs of their own and that produced by conspecifics, I conducted an egg-sac recognition experiment using A. kohi females (N = 18) and A. chonganicus females (N = 10) with egg sacs. Females were paired to match egg sac production dates to ensure egg sacs

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders exchanged were of the similar developmental stage. Each female was allowed to hold on to her own egg sac for at least three days prior to the experiment. To begin each test, each pair of females were exposed to carbon dioxide for 1 min to anaesthetize the female before the egg sacs were removed from the chelicerae. After removal, the egg sacs were switched between the two females of two different species (i.e. female A was given egg sac of female B and vice versa) and placed in the cage beside the anaesthetized female. Females were then monitored for 30 min or until she picked up the egg sac, whichever occurred first. Females were checked again after 24 h to determine if the egg sac was still held between chelicerae or had been rejected.

Females were considered to have failed to recognize their own egg sac when the foreign egg sac was held in chelicerae when 24 h had elapsed.

Maternal care for spiderlings include food provision through sharing of prey caught, capturing prey for spiderlings, regurgitation or matriphagy (Salomon et al.,

2005; Yip & Rayor, 2013). To observe maternal care for spiderlings, the same females used for observation of maternal care of egg sacs were used. Upon hatching of spiderlings, individual colonies were maintained in the same glass cages and placed individually in plastic containers (Figure 2.1B). Daily observations of the interactions between the female and its spiderlings were conducted until dispersal of spiderlings from the glass cage. During this period, fruit flies or houseflies (dependent on availability) were dropped into the glass cage followed by the observations of the behaviour and interactions of the female and spiderlings.

Natal dispersal

To determine whether and when the spiderlings dispersed from their natal nests, the same group of females used for the observation of maternal care were used. Using the

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders cage within a cage setup (Ruttan, 1990) (Figure 2.1B), once a female had produced an egg sac, she was placed in a glass cage (110  55  155 mm), which is subsequently placed in a plastic container (190  115  155 mm). Upon emergence of spiderlings, the corks on the sides and top of the inner glass cage were removed to allow free movement of spiderlings in and out of the inner cage. Spiderlings were considered as having dispersed once they moved from the inner glass cage to the outer plastic cage and remained outside for more than 24 h. The date of dispersal for the particular brood of spiderlings was taken as the date when half the brood had moved out of the inner cage. In addition, any parental or siblings interaction in the natal nest was recorded.

Cooperative prey-capture

Under laboratory conditions, I define a ‘colony’ as either a female with her offspring or a group of spiderlings or spiders produced by the same female (i.e. siblings).

Cylindrical cages (65  85 mm or 80  100mm) (larger cages were used for the larger species: A. kohi, A. subcrassipes and A. taiwanicus) were used in the setup for the observation of cooperative prey capture in a colony. One week before the observations, a colony was transferred to the cage and allowed to construct webs within the container. To observe the feeding and prey-capture behaviour, five houseflies or ten fruit flies were dropped into the cage at the same time and the activities of all the spiders were observed for at least 30 min or until they had captured and fed on prey for at least 15 min. Responses of spiders toward prey, including retreat, biting or wrapping with silk, together with the number of individuals and instar stages of spiderlings, were noted during the observations. Upon successful prey capture, spiders were observed to determine if prey sharing occurred; that is if spiders

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders shared prey with individuals that participated in prey capture and if spiders allowed individuals that did not participate in prey capture to feed on prey.

Reproductive Traits

Virgin females were placed in a petri dish (100  15 mm) for 1 h to allow for acclimatization and silk laying to allow deposition of pheromones before the introduction of a male into the same petri dish. Observations commenced upon the introduction of the male until the cessation of a successful mating or after 1 h. All observations were filmed using SONY HD Camcorders HDR-CX7 and recordings were reviewed to determine the duration of mating for each species. Mating were taken as having commenced when the female and the male were in contact and the female was stationary, exposing her epigynum to male’s palps and ceased when the male and the female separated.

Females that mated successfully under laboratory conditions were monitored daily for information on the following variables for each species: (a) clutch size, (b) number of clutches produced in life time, (c) the interval between egg-laying and hatching of spiderlings), (d) the interval between clutches, and (e) the interval between hatching of spiderlings and the subsequent clutch.

Data Analysis

All data collected were tested for normality using Shapiro-Wilk tests. When the data was normally distributed, one-way ANOVA was conducted to determine if any significant difference existed for behavioural and reproductive traits amongst species of Anelosimus examined. Post-hoc multiple comparison tests were then further performed using Games-Howell tests. If the data was not normally distributed or

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders sample size was small, Kruskal-Wallis tests were conducted instead. Post-hoc multiple comparison tests were also performed with adjusted significance levels. All statistical analyses were performed on IBM SPSS Statistics for Macintosh, Version 22.

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Figure 2.1. (A) Glass cage (110 x 55 x 155 mm) used for dispersal experiments. (B) Glass cage was placed within a translucent plastic container (190 x 115 x 155 mm) in a ‘cage- within-cage’ setup used to determine the natal dispersal.

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RESULTS

Colony survey in the field

Microhabitat

Collection sites of all species of Asian Anelosimus studied are presented in Table 2.1.

Anelosimus cameronis, A. chonganicus, A. exiguus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum and were found in similar microhabitats. All species were frequently found on the underside of small wildflowers or on the tip of branches with small leaves (Figure 2.2). Anelosimus chonganicus, A. membranaceus, A. parvulus and A. seximaculatum were frequently collected from Chamaesyce thymifolia, Cyanthillium cinereum, Cyperus rotundus,

Eragrostis tenella, Scleria bancana and Stachytarpheta jamaicensis (Figure 2.3).

These species were often located on parts of plants between 0.3 m and 1.5 m above ground. In contrast, A. kohi and A. subcrassipes were collected along coastal habitats.

Anelosimus subcrassipes were found in beach forests near the coast and were collected from tips of branches 1.5 m above ground. In Singapore, A. kohi were collected from branches of Avicennia officinalis and Bruguiera cylindrica. Lastly, A. taiwanicus were collected from Ixora plant species located in a landscaped garden, about 1.3 m above ground level.

Web Structure

The web structures of A. cameronis, A. chonganicus, A. exiguus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum are similar and are usually spun as a sheet, covering the tips of branches and leaves or the underside of flowers (Figure

2.2A–E). The webs of A. subcrassipes consisted of silk covering the branches and

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders leaves, but these webs were often bigger and included more layers of leaves as compared to webs of A. cameronis, A. chonganicus, A. exiguus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum. Lastly, the webs of A. kohi were more varied, with webs of individuals constructed with silk spun around the leaves at tip of branches and webs of colonies were bigger and frequently littered with more layers of detritus than the remaining species examined.

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A D

Figure 2.2. (A) Web of Anelosimus chonganicus. (B) Web of A. kohi. (C) Web of A. seximaculatum. (D) Web of A. membranaceus. (E) Flowers on which A. kinabaluensis were found. (F) Ixora plant on which A. taiwanicus were found.

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C Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders

A B

C D

E F

Figure 2.3. Plants species on which Anelosimus species were found. (A) Scleria bancana. (B) Cyperus rotundus. (C) Cyanthillium cinereum. (D) Stachytarpheta jamaicensis. (E) Chamaesyce thymifolia. (F) Eragrostis tenella. All photos provided by (Kwan, 2013).

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Colony Structure

A total of 343 colonies from ten species of Anelosimus were surveyed over a period of two years. However, colony composition recorded in this study may not reflect the actual composition of a natural population due to a number of factors: (a) some species (A. cameronis, A. kinabaluensis and A. taiwanicus) were not as abundant as the remaining seven species, (b) larger webs of later instar spiders are more visible and easily spotted, (c) not all microhabitats (e.g. canopy) that might contain

Anelosimus species were not surveyed. Nevertheless, the information gathered in this study provided some data on the types of aggregations that occur naturally in Asian

Anelosimus species and depicted the abundance of each aggregation type during the period of survey.

The colony composition and life stages of spiders within the colonies encountered during field observations are presented in Figure 2.4. Several patterns of colony composition were observed: (a) In all the species examined, 2nd instar spiderlings were never encountered alone or in groups in the absence of an adult female; (b) Colonies of A. cameronis, A. chonganicus, A. exiguus, A. membranaceus,

A. parvulus and A. seximaculatum were mostly composed of a single adult female and its 2nd instar spiderlings; (c) In A. kinabaluensis, A. kohi, subcrassipes and A. taiwanicus, colonies were also composed of a single female with its 3rd instar spiderlings; (d) Colonies of A. kohi and A. subcrassipes were also found to consist of spiderlings of 3rd instars or above, and (e) In A. kohi colonies were found comprising a mixture of multiple female adults and spiderlings of various instars (Figure 2.4).

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Life Cycle

All species of Anelosimus studied were multivoltine, producing between two to four egg sacs in a year and underwent between six to seven moults before reaching maturity, with males reaching maturity one instar before females.

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100.00

90.00

80.00

70.00

60.00

50.00

40.00

Percentage of colonies Percentage 30.00

20.00

10.00

0.00

Single female Male + Female Single male Single female with 2nd instar spiderlings Single female with spiderlings 3rd instar or above 2nd instar spiderlings 3rd instar above Groups with spiderlings 3rd instar above

Figure 2.4. Colony structure of Southeast and East Asian Anelosimus species encountered during field surveys. Percentage of colonies with spiders of different life stages and groups represented by different colour codes. Sample sizes as indicated below species name.

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Maternal Care

Females of all ten species of Anelosimus were observed carrying egg sacs in their chelicerae. Females held on to the egg sacs and released them only during prey capture and feeding. When females sensed vibrations on the web, they usually moved towards the egg sacs, picking them up immediately. In the field, there were no occurrences of encounters with egg sacs left unattended.

The results from egg-sac recognition experiment showed that (a) females of A. kohi and A. chonganicus attempted to steal egg sacs of other females when their egg sacs were removed experimentally; (b) females of A. kohi did not appear to recognize or grant exclusive care to egg sacs produced by themselves as a significant number of females accepted foreign egg sacs (χ2 = 4.418, df = 1, p < 0.0001, N = 18). In contrast, females of A. chonganicus did not show a significant tendency in acceptance or rejection of foreign egg sacs (χ2 = 1.05, df = 1, p = 0.527, N = 10). The rejected egg sacs were either left unattended or torn by A. chonganicus females within 24 h. It was also noted that egg sacs that were left unattended due to maternal death usually did not hatch and were often found covered in mould.

In all ten species, spiderlings underwent one moult in the egg sacs before emergence. Spiderlings that emerged from egg sacs were observed staying near females. Females were also observed capturing prey for spiderlings, either leaving the prey for spiderlings to share or sharing the prey with spiderlings (Figure 2.5). Often, during prey capture, the 2nd instar spiderlings did not approach prey on the web while females attacked the prey. After subduing prey, females were observed plucking on the web, thereafter spiderlings approached prey and began feeding. Females were observed capturing prey for spiderlings to feed on up to the 4th instars in A. kohi, A. subcrassipes and A. taiwanicus.

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Cooperative Prey-Capture and Sharing

All the ten species of Anelosimus exhibited prey-sharing behaviour in 2nd and 3rd instars where prey were mostly captured by maternal females. In all the ten species, spiderlings at 2nd instars from the same brood usually congregated and fed on one or two prey items captured by the female. Prey-sharing continued in 3rd instars in all the ten species, with group sizes in the range of 2 – 15 spiderlings per fruitfly. In A. chonganicus, A. kohi, A. seximaculatum, A. subcrassipes and A. taiwanicus, prey- sharing was observed beyond 4th instars. In these five species, prey-sharing occurred even amongst 5th and 6th instar spiderlings, with group sizes ranging from two to nine spiderlings. Prey-sharing also occurred in related males and females of A. kohi, with group sizes ranging from two to ten. Occasionally, prey items shared by adults or later instar spiderlings were broken up and fed on individually or in smaller groups of two to three individuals. Prey-sharing amongst spiderlings of different instars (e.g., 3rd and 4th instar spiderlings) or between non-maternal adult and spiderlings was sometimes observed in A. kohi, A. subcrassipes and A. taiwanicus. Spiderlings of A. parvulus were observed capturing prey without aid of maternal female at 2nd instars while the remaining nine species displayed this behaviour only at 3rd instars.

The 3rd instar spiderlings of all ten species were also observed participating in cooperative prey-capture. Multiple individuals took turns biting the prey item and throwing silk on the prey. Feeding occurred once the prey was immobilized.

Individuals that did not participate in prey-capture were allowed to feed on the same prey item. In A. kohi, A. subcrassipes and A. taiwanicus, cooperative prey-capture occurred beyond 3rd instars. In these species, cooperative prey-capture also occurred in adults and amongst a mixture of spiderlings, adult males and females under laboratory conditions.

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In general, all the ten species of Anelosimus examined exhibited similar prey- capture sequence (Figure 2.6). When prey items were dropped into cages, they either advanced and attacked the prey immediately or retreated. Retreats were frequently observed when prey were struggling and created high intensity vibrations on webs.

Spiders attacked through a cycle of biting, throwing silk onto prey and retreating from the prey items until preys were completely immobilized. Once preys were immobilized, spiders proceeded to feed on them immediately. Occasionally, spiders ignored prey items that were too large (e.g., crickets). In such situations, they retreated from the prey and these prey items were usually able to break free from web as they struggled.

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Figure 2.5. Anelosimus kohi female and spiderlings sharing a housefly.

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Prey dropped into cage

Retreat from prey Attack prey

Biting and throwing silk onto prey Ignore prey

Wrap prey in silk for

complete immobilization

Feed

Figure 2.6. Typical sequence of behaviour and movement during prey capture in Southeast and East Asian Anelosimus. Observations conducted under laboratory conditions.

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Natal Dispersal

Spiderlings of A. cameronis, A. exiguus and A. kinabaluensis did not survive well under laboratory conditions, hence these species yielded few to no subjects for testing to determine natal dispersal behaviour. Data, were, however collected for the remaining seven species. Spiderlings of A. chonganicus, A. membranaceus, A. parvulus and A. seximaculatum dispersed between four and seven days of emergence from the egg sacs, after reaching their 3rd instars. In contrast, spiderlings of A. kohi, A. subcrassipes and A. taiwanicus were observed leaving their natal nest at later instars, between 4th and 6th instars, about 16 to 20 days after emergence from the egg sacs

(Figure 2.7). In all the species observed, there were usually two to three spiderlings that dispersed one to two days earlier than the majority of spiderlings. In addition, there were also one to two spiderlings that remained in the natal nest for prolonged period of time after all other spiderlings had dispersed.

There was a significant difference in the ranks of mean time taken for spiderlings to disperse from their natal nests amongst seven species examined

(Kruskal-Wallis test: H = 43.8, df = 5, p < 0.001; Figure 2.7). Spiderlings of A. parvulus were the earliest to disperse from the natal nest (mean  s.e. = 4.3  0.3 d) while spiderlings of A. subcrassipes remained in the natal nest for the longest (20.7 

2.7 d). Post-hoc pairwise analyses revealed no significant difference in dispersal time of spiderlings of A. kohi and A. taiwanicus. Spiderlings of both species dispersed significantly later than those of A. chonganicus, A. parvulus and A. seximaculatum.

Spiderlings of A. parvulus dispersed significantly earlier than those of A. chonganicus,

A. kohi, A. membranaceus, A. seximaculatum, A. subcrassipes and A. taiwanicus was left out the analysis due to small sample size (N = 3).

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In the field, in all species, spiderlings were often found in close proximity to one another. They were often collected on adjacent flowers or leaves. Species such as

A. chonganicus, A. membranaceus and A. seximaculatum were even observed maintaining individual webs on the same stalk of flower.

Reproductive Traits

In these observations, information or statistical analyses on certain species were absent due to small sample sizes which was due to their low survivorship under laboratory conditions. Species examined for each reproductive trait are clearly listed in the corresponding charts for each trait.

Mating

Mating behaviour of seven species of Anelosimus, A. cameronis, A. chonganicus, A. kohi, A. membranaceus, A. parvulus, A. seximaculatum and A. taiwanicus was observed and these species were found to adopt the same position during mating

(Figure 2.8). Females were seldom aggressive towards males when males approached.

Males were observed plucking on the webs of females using the first two legs when in close proximity to females. If a female was receptive, she would raise her prosoma, allowing the male to insert its palps into her epigynum. Other than plucking, no obvious courtship displays were observed in all species examined. In addition, it appeared that males were more successful in locating and copulating with females when females were allowed to lay silk in the petri dishes prior to the introduction of males, indicating that chemoreception is important in mate recognition in these species.

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When females rejected males, males usually plucked more intensely on the webs before approaching females again. Due to small sample size (N=1), A. cameronis was excluded from analysis. Copulation duration differed significantly among the six species (Kruskal-Wallis test: H = 28.2, df = 5, p < 0.001; Figure 2.9).

Post-hoc multiple pairwise comparisons showed that the copulation duration of A. kohi was significantly shorter than that of A. chonganicus, A. membranaceus, A. parvulus, A. seximaculatum and A. taiwanicus, Duration of copulation of A. membranaceus and A. seximaculatum were similar and significantly longer than that of A. kohi, A. parvulus and A. taiwanicus. Copulation duration of A. chonganicus was also not significantly different from that of A. membranaceus and A. seximaculatum.

No significant difference in copulation duration was found among A. chonganicus, A. parvulus, and A. taiwanicus. In all seven species, males were used in multiple experiments and hence demonstrated ability to mate with multiple females.

Clutch Size

There was a significant difference in the clutch size (number of hatchlings per egg sac per female) among eight Anelosimus species examined (ANOVA: F6, 228 = 9.1, p <

0.001). A. chonganicus had the smallest clutch size (mean  s.e. = 14.8  0.7) while A. taiwanicus had the largest clutch size (27.7  3.3). The majority of species examined had mean clutch size of 15 to 20 (Figure 2.10). A. cameronis and A. subcrassipes were excluded from statistical analyses due to small sample size. Games-Howell post- hoc multiple paired comparisons revealed no significant differences in clutch size among A. chonganicus, A. exiguus, A. kohi, A. membranaceus, A. parvulus, and A. seximaculatum. Mean clutch size of A. taiwanicus was, however, significantly larger

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Number of Clutches

There was a significant difference in the mean number of clutches (i.e., number of egg sacs) produced by females in their life time among seven species examined (Kruskal-

Wallis test: H = 19.8, df = 6, p = 0.003; Fig. 11A). On average, females of A. kohi produced significantly fewer clutches as compared to A. chonganicus, A. exiguus, A. membranaceus, A. parvulus, and A. seximaculatum (Figure 2.11A). No significant differences in the number of clutches were found among A. chonganicus, A. exiguus,

A. membranaceus, A. parvulus, A. seximaculatum and A. taiwanicus.

Time for Spiderlings to Hatch

There was a significant difference in mean time taken for egg incubation amongst seven species examined (Kruskal-Wallis test: H = 17.2, df = 7, p = 0.016; Figure

2.11B). A. exiguus had the shortest egg incubation time while A. cameronis had the longest egg incubation period. Post-hoc pairwise comparison revealed that egg incubation time of A. exiguus and A. membranaceus were significantly shorter than A. cameronis and A. kohi. No significant differences were found in egg incubation time among A. chonganicus, A. exiguus, A. membranaceus, A. parvulus, A. seximaculatum and A. taiwanicus.

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The Interval between Egg-Hatching and Subsequent Clutch

There was a significant difference in the mean interval between egg-hatching and subsequent clutch among five Anelosimus species examined (ANOVA: F4, 82 = 7.49, p

< 0.001). Anelosimus exiguus had the shortest interval between egg-hatching and subsequent clutch production, while A. kohi had the longest interval (Figure 2.12A).

Games-Howell post-hoc multiple paired comparisons revealed that A. kohi had significantly longer interval than A. chonganicus, A. membranaceus and A. parvulus.

A. seximaculatum also had significantly longer interval than A. chonganicus and A. parvulus. No significant differences were found in the interval of A. chonganicus, A. membranaceus and A. parvulus.

Interval between Clutches

There was a significant difference in the mean interval between clutches among five

Anelosimus species examined (ANOVA: F4, 74 = 7.71, p < 0.001). A. exiguus had the shortest interval between clutches, while A. taiwanicus had the longest interval between clutches (Figure 2.12B). Games-Howell post-hoc multiple pairwise comparisons revealed that A. kohi had a significantly longer interval between clutches as compared to A. chonganicus and A. parvulus. There was no significant difference in interval between A. kohi, A. membranaceus and A. seximaculatum. There were also no significant differences in the interval between clutches among A. parvulus, A. membranaceus and A. seximaculatum.

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Figure 2.7. Box plot of number of days taken for spiderlings to disperse from natal nest under laboratory conditions. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Different lower case letters above column indicates statistically significant difference (p < 0.05). Circle indicates outliers. Asterisks above each column indicate omission from statistical analyses due to small sample sizes.

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A B

C D

Figure 2.8. Mating position of four Anelosimus species. (A) A. chonganicus, (B) A. parvulus, (C) A. taiwanicus and (D) A. kohi.

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Figure 2.9. Boxplot of copulation duration (min) of seven species of Anelosimus examined. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Sample sizes as indicated below species name.

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Figure 2.10. Mean (± s. e.) number of spiderlings emerged from each egg sac. Different lower case letters above column indicates statistically significant difference (p < 0.05). Asterisks above each column indicate omission from statistical analyses due to small sample sizes. Sample sizes as indicated below species name.

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Figure 2.11. (A) Boxplot of clutch (number of egg sacs) produced per female. (B) Boxplot of time (d) for incubation of eggs. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Different lower case letters above each column indicates statistically significant difference (p < 0.05). Circle indicates outlier. Asterisks above column indicate omission from statistical analyses. Sample sizes as indicated below species name.

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Figure 2.12. (A) Mean (± s. e.) interval (d) between hatching of spiderlings and production of subsequent egg sac. (B) Mean (± s. e.) interval (d) between clutches. Different lower case letters above each column indicates statistically significant difference (p < 0.05). Asterisks above column indicate omission from statistical analyses. Sample sizes as indicated below species name.

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DISCUSSION

In this study, I examined the life history of ten species of Anelosimus from Southeast and East Asia and determined whether they were solitary, subsocial or social by examining their habitat, colony structure, maternal care, cooperation, dispersal, and a number of reproductive traits. Results revealed similarities and differences in behavioural and reproductive traits among the ten species of Anelosimus that allow the categorization of these ten species into “solitary” and “subsocial” species. All species examined exhibited similar web architecture, multivoltine life cycles and basic maternal care of egg-sac guarding and prey provision for 2nd instar spiderlings.

Extended maternal care, later natal dispersal and cooperative behaviour beyond 4th instars were only observed in A. kohi, A. subcrassipes, and A. taiwanicus, suggesting that they are subsocial. In contrast, the remaining seven species (A. cameronis, A. chongancius, A. exiguus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum) are solitary as they dispersed early and showed little cooperation beyond 3rd instars.

Although subsocial Anelosimus in the New World are usually found at higher altitudes and latitudes (Guevera & Avilés, 2007), both subsocial and solitary species in this study have been found at edges of lowland tropical rainforests. Of the ten species studied, only A. subcrassipes was collected from higher subtropical latitude while the remaining nine species were collected in the tropical region. Among species collected within the tropical latitudes, A. cameronis and A. kinabaluensis were collected at higher elevation (above 1,000 m). Among the three subsocial species, two species (A. kohi and A. taiwanicus) are found at lowland tropical habitat. Furthermore, the remaining seven solitary species of Anelosimus are also found along forest edges

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders of lowland tropical forests. Therefore, subsocial Anelosimus in Southeast and East

Asia are not confined to higher latitudes and altitudes. Moreover, similar to its social congeners, both solitary and subsocial Anelosimus have been found to occupy forest edges of lowland tropical forests. Thus, the ecological effect on the sociality of

Anelosimus species may be more complicated than previously thought.

Habitat Preferences and Web Structure

The ten species of Anelosimus studied here exhibited similar patterns in habitat preferences that may possess an abundance of small preys. All species were found in areas with open canopy, along forest edges Anelosimus cameronis, A. chongancius, A. exiguus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum were found on wildflowers or tips of branches located along roads or trails where webs are exposed to sunlight for long period, while A. kohi, A. subcrassipes and A. taiwanicus were also frequently located along the edges of beach vegetation. This preference for forest edge vegetation is similar to A. guacamayos, A. jucundus O.P.-Cambridge, A. pacificus and A. rupununi (Nentwig & Christenson, 1986; Avilés & Salazar, 1999;

Agnarsson et al., 2006b; Avilés et al., 2007) and may be due to higher abundance of small insect prey at forest edges (Gibbs & Stanton, 2001; Molnar et al., 2001). A lack of large sized prey may hinder the development of sociality in the habitats of the ten species of Anelosimus studied here. Powers and Avilés (2007) found that insect sizes were larger in tropical lowland habitats where social species were commonly found and postulated that prevalence of social species in tropical lowland habitats is due to lack of large insects at higher altitudes. Guevera and Avilés (2007) further confirmed this hypothesis through findings that social species of Anelosimus captured larger preys at lowland tropical forest. Therefore, the absence of larger insects at forest

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders edges favoured by the ten species of Anelosimus encountered here may contribute to a lack of further development of social behaviour. Furthermore, the webs of these species do not conform to the typical basket web of social and subsocial Anelosimus species (Brach, 1977; Rypstra & Tirey, 1989; Agnarsson & Zhang, 2006). Instead, webs were small, three-dimensional, enveloping parts of surrounding vegetation, similar to that of the solitary A. pacificus (Agnarsson et al., 2006b). The small web size and lack of overhanging threads that are effective in the capture of large flying prey (Nentwig, 1985) may further reduce the efficacy of these species to capture larger prey item, hampering the formation of social groups as sociality has been found to be enhanced by high productivity and a high prey abundance (Powers & Avilés,

2007; Majer et al., 2013).

Natal Dispersal

Results from the field surveys of colony structure revealed variations in dispersal patterns and tolerance levels among ten Asian Anelosimus species. One of the determining factors of the sociality of spiders is how early spiderlings leave the natal nest (Avilés, 1997; Whitehouse & Lubin, 2005; Lubin & Bilde, 2007, 2011;

Corcobado et al., 2012; Yip & Rayor, 2013). A study of seven social and subsocial

Anelosimus species revealed that there is a gradual reduction in dispersal ability in subsocial to social species (Corcobado et al., 2012), resulting in the lack of dispersal even after maturation in social species. In the present study, field observations show that the solitary species, A. cameronis, A. chonganicus, A. exiguus, A. membranaceus,

A. parvulus and A. seximaculatum dispersed from the natal nests some time between

2nd and 3rd instars as there were no encounters of 4th instar or above spiderlings with adult females during field surveys. These observations also corroborates with

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders laboratory experiments in which A. chonganicus, A. membranaceus, A. parvulus and

A. seximaculatum were found to disperse between four to seven days after eclosion, at

2nd or 3rd instars. In contrast, encounters of A. subcrassipes, A. kohi and A. taiwanicus colonies with females and 3rd instar spiderlings or above were recorded. Laboratory experiments also revealed that these three species dispersed later, between 16 to 20 days after eclosion, at 4th instars or later. Dispersal at 4th instars or beyond is similar to that observed in the subsocial A. vierae Agnarsson (Avilés & Gelsey, 1998). These evidences support A. kohi, A. subcrassipes and A. taiwanicus as species that display a higher level of sociality as compared to the solitary species. Anelosimus kinabaluensis is postulated to be a solitary species as well due to the observation of singleton 2nd instar spiderlings during field survey. Although studies have found that abundance or lack of prey has an effect on the gregarious phase of spiderlings (Gundermann et al.,

1993; Kim, 2000), prey abundance is not a factor of consideration here as all colonies were fed with the same frequency and prey.

Dispersal distance in species of Anelosimus species examined here were observed to be short. In field surveys, A. chonganicus, A. membranaceus, A. parvulus and A. seximaculatum were observed in close proximity to conspecifics, sometimes even sharing the same floral inflorescence in the wild. Similarly, in A. kohi, A. subcrassipes and A. taiwanicus, multiple individuals were often found on the same plant. Why would these species leave their natal nests but not disperse far away from the natal nest? The subsocial A. studiosus is known to exhibit an innate tendency to aggregate, apart from any environmental cues (Furey, 1998). The close proximity at which A. chonganicus, A. kohi, A. membranaceus, A. parvulus, A. seximaculatum, A. subcrassipes and A. taiwanicus remain to the natal nest even after dispersal may indicate that these species possess a similar innate tendency to aggregate. Short

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(Nentwig & Christenson, 1986; Avilés & Gelsey, 1998; Powers & Avilés, 2003). In view of Corcobado et al.’s (2012) observation that a reduction in dispersal ability may contribute to the development of sociality, the short dispersal distance observed here suggests that solitary spiders of the genus Anelosimus may have retained this ancestral social trait (Agnarsson et al., 2006; Yip & Rayor, 2013; Samuk & Avilés, 2013).

Maternal Care

The subsocial route of evolution of sociality in spiders involves prolonging the gregarious phase of the offspring as a result of maternal care, eventually leading to the elimination of dispersal in social species (Avilés, 1997, Whitehouse & Lubin, 2005;

Lubin & Bilde, 2007), therefore, the duration and type of maternal care given plays a crucial role in the determination of sociality in spiders. Guarding of egg sac is a basic form of maternal care in spiders and this was observed in all ten species studied; egg sacs were carried in the chelicerae of maternal females. However, even solitary species of spiders guard their egg sacs and newly hatched spiderlings (Foelix, 2011).

Nevertheless, it has been postulated that the absence of offspring or egg sac recognition is an important factor contributing to the evolution of social behaviour

(Samuk & Avilés, 2013). The failure to recognize egg sacs would promote cooperative brood care as observed in A. eximius and A. rupununi (Christenson, 1984;

Avilés & Salazar, 1999). Samuk & Avilés (2013) tested for discrimination of egg sacs in three subsocial and three social species and found no indication of egg sac discrimination in these species. Anelosimus kohi’s acceptance of foreign egg sacs resembled the behaviour of the social A. domingo Levi, A. eximius, A. guacamayos

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Rao & Aceves–Aparicio, 2012; Samuk & Avilés, 2013). Meanwhile, unlike A. kohi, A. chonganicus did not accept all foreign egg sacs presented but results did not indicate significant difference in either acceptance or rejection of foreign egg sacs. The solitary living A. chonganicus may be demonstrating egg sac discrimination but testing on a larger sample size would be needed to confirm this hypothesis. This trait should also be further tested in the remaining species to determine if discrimination of offspring is an obstacle preventing the development of subsocial behaviour in the solitary types of Anelosimus species. It should however be noted that solitary theridiids such as Faiditus ululans O.P.-Cambridge (formerly Argyrodes ululans) and

Theridion grallator Simon similarly demonstrate a lack of egg sac recognition in experimental removal of egg sacs (Gillespie, 1990; Cangialosi, 1990).

Furthermore, while most species of Anelosimus require assistance from adults during eclosion, spiderlings of A. parvulus were observed on one occasion emerging from egg sac without aid. Viera et al. (2007) suggest that more social spiders would exhibit an absence of endogenous mechanism for egg sac opening to allow non- maternal females to care for and open egg sacs (Gonzaga & Vasconcellos-Neto; 2001).

Therefore, the possible egg sac recognition in A. chonganicus and emergence of A. parvulus spiderlings without aid indicates a lower level of sociality in these species.

Maternal care also consists of provision of prey and the availability of food resource in the natal nest contributes to the extension of gregarious phase of spiderlings. Similar to the solitary A. pacificus, females observed in this study captured prey and allowed spiderlings up to the 3rd instar to feed without sharing prey item with the spiderlings (Agnarsson et al., 2006b). Unlike spiderlings of A. eximius

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1986), the 3rd instar spiderlings of all species examined here were observed successfully attacking and feeding on small prey items such as fruitflies and fruitfly larva. Furthermore, 2nd instar spiderlings of A. parvulus were also observed capturing prey without aid from maternal female. The cessation of dependence on maternal provision could have contributed to the early dispersal observed in A. parvulus and the dispersal of spiderlings at 3rd instars in A. chonganicus, A. membranaceus and A. seximaculatum. This also indicates that spiderlings of these species are better developed and lack a dependence on food provision from mother or other adults.

However, it is noted that cessation of maternal food provision at 4th instar did not lead to immediate dispersal of spiderlings of A. kohi, A. subcrassipes and A. taiwanicus.

This may be due to the cooperative behaviour observed in these species.

Cooperative Prey-Capture and Communal Feeding

The prey-capture strategies adopted by ten Anelosimus species examined were similar and all species demonstrated cooperative prey-capture and communal feeding at early instar stages of 2nd and 3rd instars. While maternal care for young spiderlings is common in theridiids and is observed even in solitary spiders such as Faiditus ululans and Theridon grallator, cooperative prey-capture amongst young spiderlings was not recorded in these species. Instead, spiderlings of T. grallator were unable to capture prey and subsisted on prey provided by females (Gilliespie, 1990; Ciangialosi, 1990).

Similarly, spiderlings of social A. eximius are only able to participate in prey capture after 3rd instar and newly emerged spiderlings have to be fed through regurgitation by adults (Christenson, 1984). The ability to capture prey is a prerequisite for the cessation of parental care and dispersal. All species examined in this study

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However, why do spiderlings of A. kohi, A. subcrassipes and A. taiwanicus delay dispersal even after gaining independence from maternal female in prey capture? In these three species, although spiderlings were able to capture prey items independently a few days after eclosion, females continued to capture prey for their spiderlings up to 3rd instars. Therefore, it may be beneficial for spiderlings to remain in the natal nest as parental care persists. Beyond 3rd instars, cooperative prey-capture was observed in 4th and 5th instar spiderlings in these three species and there were many instances of spiderlings sharing in feeding without participating in prey capture.

Hence, delayed dispersal in A. kohi, A. subcrassipes and A. taiwanicus before 3rd instar could be due to the benefits of parental food provision and beyond which, cooperation amongst siblings in prey capture.

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Reproductive Traits

Subsocial A. kohi, A. subcrassipes and A. taiwanicus have much shorter copulation duration as compared to the remaining solitary species. Duration of copulation plays a role in determining the success of copulation in a number of species (Siva-Jothy &

Tsubaki, 1989; Castagnoli & Liguori, 1991; Andres & Rivera, 1999) and there are a number of factors affecting copulation duration. In the wolf spider Pardosa milvina

Hentz, copulation duration is influenced by the mating status of females and the presence of cues from females (Rystra et al., 2003). In phytoseiid mites, when copulation is disrupted, there is a reduction in the amount of sperm transferred and fecundity is reduced (Castagnoli & Liguori, 1991). The same study also found that fecundity of species with the longest mating duration is most affected when copulation is disrupted. In addition, in the fly Cyrtodiopsis whitei, which forms groups while roosting, copulation duration is shorter in the presence of other males

(Lorch et al., 1993). Since the present study is the first intra-specific comparison of the copulation duration in the genus Anelosimus, it is premature to determine what factors could have influenced the duration. One can only speculate that competition for parentage in solitary species has resulted in prolonged mating time. More information on the mating duration of other Anelosimus species and experimental manipulations are needed to ascertain why subsocial species have shorter copulation duration compared to solitary species.

The reproductive traits of clutch size, number of clutches produced in life time, time for spiderlings to hatch, the interval between clutches, and the interval between egg hatching and the subsequent clutch are often closely related and influenced by the same set of factors. Discussions on the sociality of spiders have frequently included the costs and benefits of group living and the reproductive benefit is one area that has

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders often been highlighted (Kullmann, 1972; Smith, 1982; Siebt & Wickler, 1988; Lubin

& Bilde, 2007; Jones & Riechert, 2008; Yip & Rayor, 2013). In general, it is believed that higher investment in parental care may result in fewer offspring (Shine, 1978;

Schneider 1996). This is observed in the social spider Stegodyphus mimosarum Pavesi in which fewer eggs are produced in larger colonies (Siebt & Wickler, 1988).

Accordingly, it is expected that solitary spiders would produce more offspring as compared to subsocial and social ones. In the present study, offspring production was measured through two avenues, the mean number of clutches (i.e., egg sacs) produced by a female in her lifetime and mean clutch size (i.e., number of hatchlings). The clutch size of subsocial A. taiwanicus was significantly larger than those of solitary species examined and clutch size of A. kohi was also larger than the solitary species.

This seems to be contrary to the hypothesis of reduction in progeny numbers with increased in parental care. However, there is also no distinct pattern of decreasing clutch size with increasing sociality observed between social and subsocial

Anelosimus species. The clutch sizes of social species A. dubiosus, A. eximius, A. guacamayos and A. rupununi vary from eight to 53 (Vollrath, 1985; Marques et al.,

1998; Avilés & Salazar, 1999; Avilés et al., 2007), while subsocial A. jabaquara Levi and A. vierae produce 27– 31 spiderlings per clutch (Marques et al., 1999; Viera et al.,

2007).

In terms of the number of clutches produced, the solitary species in this study were observed to produce higher number of clutches per female (between 2.4 and 2.9 egg sacs) than the subsocial A. kohi and A. taiwanicus, which produced between 1.7 to

1.8 egg sacs per female. This is consistent with the hypothesis of reduction in progeny number with increasing sociality, however, the subsocial A. vierae is found to produce an average of 2.7 egg sacs per female (Viera et al., 2007), which is comparable to the

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders numbers found in solitary species in this study. Parental care is not the only factor influencing clutch size, it has also been found that female body size (Simpson, 1995), adult longevity (Martin et al., 2001), hunting strategies (Simpson, 1995), parasites and predators (Smith, 1982) are also able to affect clutch sizes. The interplay between these factors and level of parental care given could thus result in the lack of clear patterns in number of clutches and clutch sizes observed in Anelosimus species.

Intervals between clutches and between egg hatching and the subsequent clutch are expected to decrease with the level of sociality. Consistent with this prediction, the time intervals between clutches and between hatching of first egg sac and the subsequent clutch was found to be shorter in solitary Anelosimus species than in subsocial Anelosimus species examined here. The subsocial A. kohi and A. taiwanicus in this study had 25.3 d and 30 d between the clutches, while the solitary species had averages of between 15 d and 24 d between clutches. The longer interval between clutches may be due to several factors such as the longer maternal care given to the subsocial species (up to 4th instars) and later juvenile dispersal (16 – 20 d after hatching). As females carry their egg sacs in their chelicerae during egg sac attendance, a longer interval between clutches may allow females to be free to capture prey for spiderlings in the subsocial species. In contrast, spiderlings of solitary species disperse from the natal nest early and maternal food provision ceases at 2nd or 3rd instars, this thus allows the females to invest in the production of another egg sac earlier than subsocial species.

With the exception of A. cameronis, the incubation time of eggs of the remaining eight Anelosimus species in this study were similar, between 11 d and 13 d.

This period of incubation is shorter than the subsocial A. vierae (17.2 d) (Viera et al.,

2007) and social A. eximius (20–30 d) (Vollrath, 1985). Viera et al. (2007) found that

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Chapter 2 – Natural History and Behavioural Observation of Asian Anelosimus spiders the difference in incubation time of egg sacs may be related to ambient temperature.

In this study, eggs of A. cameronis had the longest incubation time and this species is also found at higher elevation where lower temperature may contribute to slower development of embryos. Due to the lack of information on the incubation time of other Anelosimus species, it is difficult to determine if the length of incubation is associated with levels of sociality.

Conclusion

Upon examination of a number of behavioural and reproductive traits in the ten species of Anelosimus collected in Southeast and East Asia, three species (A. kohi, A. subcrassipes and A. taiwanicus) can be considered subsocial, while the remaining seven species (A. cameronis, A. chonganicus, A. exiguus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum) are solitary. The solitary

Anelosimus species described in this study had also exhibited maternal care, cooperative and sharing behaviour typical of social and subsocial species during early instar stages. Dispersal distances of these species were also found to be short. As such it seems that solitary species of Anelosimus likely retained certain ancestral traits

(cooperative prey capture and reduced dispersal distance) that are characteristic of subsocial spiders. The differences in the reproductive traits of clutch sizes, interval between clutches, incubation time and copulation duration may be due to differences in level of sociality or other ecology and phylogenetic factors that need to be further explored. The lack of social species and abundance of solitary species in Southeast and East Asia remains to be further investigated.

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SENSORY RECEPTORS AND SOCIALITY IN SPIDERS OF THE GENUS ANELOSIMUS (ARANEAE: THERIDIIDAE)

Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus

ABSTRACT

Sensory receptors play an important role in communications for purposes of

reproduction, food detection, predator avoidance and social interaction. Spiders that

have poor eye sight primarily rely on tactile, chemical, seismic or/and acoustic signals

for communications and possess legs that are arrayed with a mixture of tactile hairs,

chemoreceptors, slit sensilla and trichobothria for the detection of these signals.

Densities of these receptors are known to vary greatly with species, habitat types and

life history. The genus Anelosimus consists of solitary, subsocial and social species and these are found in epigean habitats ranging from montane forest to tropical

rainforests. Recent studies have revealed differences in responses and aggressiveness

to conspecifics and prey between subsocial and social species of Anelosimus.

Subsocial species are able to respond more quickly to the presence of prey on webs as

compared to social species. In this study, I examined and compared the overall

receptor densities of tactile hair, chemoreceptor, slit sensillum and trichobothrium in

eight species of Asian Anelosimus, of which two are subsocial while the remaining

are solitary species. I also compared these sensory receptor densities across Legs I to

IV amongst the eight species. I found that mechanoreceptor (tactile hair, slit sensillum

and trichobothrium) densities of subsocial species were significantly lower than those

of solitary species, but no significant difference was found in chemoreceptor densities

between solitary and subsocial species examined. I discuss possible explanations for

the findings of this study, including how lowered receptor densities may contribute to

the evolution of social behaviour in spiders.

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INTRODUCTION

Spiders communicate through a variety of signals: visual, acoustic, chemical and

vibration (Gaskett, 2007; Foelix, 2011). These signals are emitted and received in the process of communication for a myriad of purposes: mating, prey detection and capture, predator avoidance and cooperation (Barth, 2002; Foelix, 2011; Uhl, 2013).

The forms of communication used often depend on the physiology of spiders, the

environment and the function of the signal.

A typical spider leg is covered with a multitude of receptors, including tactile

hairs, chemoreceptors, trichobothria and slit sensilla. Tactile hairs are scattered all

over a spider’s body and are used in the detection of mechanical changes caused by

vibrations, touch or air flow (Barth, 2002, 2004; Barth & Dechant, 2003; Foelix,

2011). Chemoreceptors are curved hairs with rounded open ends and are concentrated

on the distal ends of spiders’ legs. These receptors are responsible for the detection of

both air borne and contact chemicals that are important in the distinction between

prey, predators and conspecifics (Trabalon, 2013). Trichobothria are highly sensitive

hairs similar to the filiform hairs found in insects (Barth, 2004). These hairs are

different from tactile hair in their attachment to the cuticle and their length;

trichobothria are attached perpendicularly to the cuticle of spiders and are longer,

increasing the sensitivity of these receptors, allowing them to detect even minute air

current flows (Barth, 2002; Foelix, 2011). Slit sensilla are made up of a thin sheet of

membrane that distorts when pressure is exerted on the exoskeleton of the spiders or

when vibrations are detected from the substrate. These receptors are highly sensitive,

being able to detect substrate vibrations of 10–6 to 10–7 cm (Barth, 2002, 2004, 2012;

Foelix, 2011).

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The densities of receptors vary between and within species, and also depend on the type of signals each utilizes and receives. The wandering spider Cupiennius salei (Ctenidae), for example, possesses the highest recorded number of trichobothria

(936) and 3,500 slit sensilla on its legs and pedipalps (Barth et al., 1993) as it relies heavily on vibrational signals to differentiate between vibrations caused by ambient air and those caused by a mate or a prey (Barth, 2002). In Liphistius spiders, males have been deduced to rely heavily on chemoreception in search of mates and are found to possess an exceptionally high density of chemoreceptors (2,000 hairs on one tarsus) (Foelix & Erb, 2010). On top of this, the arrangement of trichobothria in spiders also appears to be species specific (Lehtinen, 1980), indicating a possible adaption of density of this receptor to the habitat or life history of the species.

As predators, it is often necessary for spiders to be able to identify prey quickly to secure capture. In solitary spiders, this is achieved through a general aggression towards both prey and conspecifics, resulting in cannibalism of conspecifics that wander into their territories (Foelix, 2011). In contrast, subsocial and social species are defined by their aggregation and cooperation among conspecifics.

For aggregation to occur, individuals of a species must therefore tolerate close proximity of conspecifics (Choe & Crespi, 1997). Hence, aggressive response in social or subsocial species should only be directed towards prey, this thus requires distinction between conspecifics and prey.

Furthermore, it is also necessary for social/subsocial species to distinguish between kin and non–kin to reduce the cost of group living. The cost of social living is often mitigated by inclusive fitness, where benefits are given preferentially to related individuals, aiding the propagation of shared genes (Howard & Blomquist,

2005; d’Ettorre & Lenoir, 2010). Discrimination between kin and non–kin was

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observed in a number of social and subsocial species. For example, spiderlings of the

subsocial Stegodyphus tentoriicola (Eresidae) fed more from shared prey items in the

presence of non–siblings, resulting in unbalanced growth amongst spiderlings (Ruch

et al., 2009). Similarly, the subsocial Stegodyphus lineatus feeds more effectively,

gaining more weight, in sibling groups as compared to unrelated spider groups

(Schneider & Bilde, 2008). Social species such as Diaea ergandros (Thomisidae) and

Delena cancerides (Sparassidae) preferentially cannibalize unrelated spiderlings over

siblings when starved (Beavis et al., 2007; Evans, 2008).

Changes in level of aggression and tolerance are related to modification of cues. In the spider Tegenaria atrica (Agelenidae), females are never aggressive towards pre–dispersal spiderlings, but are unable to tolerate the presence of post- dispersal spiderlings (Pourie & Trabalon, 1999). This behavior was associated with a change in the pheromones of spiderlings (Pourie & Trabalon, 1999). Similarly, spiderlings of the solitary species, Hogna helluo (redescribed as Tigrosa helluo)

(Lycosidae) preferentially cannibalize non–related conspecifics during gregarious phase at 2nd instars. However, this preferential treatment is lost after dispersal at 3rd

instar (Roberts et al., 2003). In the subsocial crab spider Diaea socialis, despite a lack

of capture web, spiderlings were found to be attracted to siblings and remain close to

the natal nest through an attractant pheromone (Evans & Main, 1993). These studies

suggest that cues are given off by spiders which promote aggregation, and the

breakdown of tolerance corresponds to the loss or modification of these signals.

Similarly, spiderlings of the subsocial Stegodyphus lineatus has also been shown to

possess different pheromone profiles that allow discrimination between kin and non-

kin. In addition, these changes in pheromones profile are also found around the period

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of dispersal, further indicating a role of pheromones in regulation of tolerance and

aggregation in this species (Grinsted et al., 2011).

In the spider genus Anelosimus, various studies have shown that signaling

plays a crucial role in shaping tolerance and prey-capture behaviour. Anelosimus

eximius is less cohesive, maintaining greater distance between individual spiders in

the presence of unrelated spiders (Mailleux et al., 2008). The pheromones adhering to the silk of A. eximius during dispersal are suggested to be means by which spiders distinguish between kin and non-kin (Mailleux et al., 2008). Anelosimus eximius also coordinate prey-capture sequence through feedback of the intensity of vibrations elicited by prey and from spiders involved in capture and transport of prey (Vakanas

& Krafft, 2001, 2004).

Social and subsocial species of Anelosimus have also shown variation in their responses to prey presence on the web. For example, the subsocial A. baeza shows a

higher level of participation; with individuals responding quickly to the presence of

prey on webs while the social A. dubiosus is less aggressive in its response (Harwood

& Aviles, 2013). There is an increasing gradient of participation in prey capture

following decrease in the level of sociality observed. The latency in responding to

presence of prey on webs by social/subsocial species may be associated with a

decrease in sensitivity towards external stimuli produced by prey movement.

Social and subsocial spiders are known to use vibrational and/or chemical

signals in the coordination of cooperation and kin recognition (Vakanas & Krafft,

2001, 2004; Mailleux et al., 2008; Harwood & Aviles, 2013). Furthermore, specific

vibrational or chemical cues have also been shown to affect the level of cohesion in

social species (Mailleux et al., 2008; Grinsted et al., 2011). Variation in sensitivity

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While production of these signals has been confirmed in these species, the ability to detect these signals remains to be explored. The vision of spiders from the family Theridiidae are not known to be good due to the small size and simple structure of the eyes (Homann, 1971). Therefore, in this study, I investigated whether a variation in density of receptors exists in subsocial and solitary species of Asian

Anelosimus species. This was achieved by examining the densities of tactile hair, chemoreceptor, trichobothrium and slit sensillum on the legs of eight species of

Anelosimus exhibiting solitary or subsocial behaviour (Chapter 2) using scanning electron microscopy (SEM). I hypothesized that subsocial species of Anelosimus may have a higher density of receptors due to: (a) a need to distinguish related and unrelated conspecifics and (b) to distinguish between prey and conspecifics.

Meanwhile, solitary species exhibit early dispersal from the natal nest and do not gain any benefits from communal living and reduction in aggression. Therefore, distinguishing between conspecifics and prey or between related and unrelated conspecifics may not be important.

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MATERIALS AND METHODS

Study Subjects

Eight species of Anelosimus collected in Southeast and East Asia were used in this

study. Of the eight species studied, six species, Anelosimus cameronis, A.

chonganicus, A. kinabaluensis, A. membranaceus A. parvulus and A. seximaculatum

are solitary living while two species, A. kohi and A. taiwanicus, are subsocial (Chapter

2). For each species examined, two adult males and two adult females were randomly picked either from a population captured in the wild or laboratory grown population.

Sample Preparation

The specimens were killed by immersion in 75% ethanol to clean off any residue on the spiders. Four legs (Legs I to Legs IV) were excised from the respective coxa of each specimen, yielding the tarsus, metatarsus, tibia, patella and femur of Legs I to

Legs IV. These leg segments were then air dried for 15 min. Upon drying, individual

legs were mounted on an aluminum stub using a conductive adhesive strip and sputter

coated with platinum using JEOL JFC 1600 Pt Fine coater for 4 min.

After sample preparation, the spider legs were examined under the scanning

electron microscope JEOL JSM 6510 SEM. Multiple photographs of each section of

leg segments were captured to obtain a composite of the entire leg of each specimen.

The total numbers of each type of receptors (tactile hair, chemoreceptor,

trichobothrium and slit sensillum; Figure 3.1) from each photograph were counted

and the area of the leg was measured using Image J (Schneider, 2012). A total of

~ 9, 000 photographs were taken and examined.

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Data Analyses

To compare the abundance of each type of receptors found in each species of spiders,

contrasts were made by examining densities and total count of each receptor types on

the legs of spiders. The densities of receptors (tactile hair, chemoreceptor,

trichobothrium and slit sensillum) of Legs I to IV were obtained through dividing the

total number of receptors on the tarsus, metatarsus, patella, tibia and femur of each leg

by the total surface area of the leg. The total number of each receptors per spider were

obtained by collating the total number of receptors on Legs I to IV of each spider.

Data (i.e., densities and counts) collected were tested for normality using

Shapiro-Wilk and subsequently analyzed using either parametric or non-parametric methods whichever was applicable. One-way ANOVAs or Kruskal-Wallis tests were used to determine whether significant differences exist in the density and counts of receptors (tactile, chemoreceptor, trichobothria and slit sensilla) among the eight species and between individual legs (Legs I to IV) of each species. When a significant difference was detected, post–hoc tests (Tukey’s HSD or Dunnett’s) were performed.

All data are reported as mean ± s.e. and all analyses were performed on untransformed data. All statistical analyses were completed using IBM SPSS

Statistics Version 22 (IBM Corp., USA).

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A B

C D

Figure 3.1. Scanning electron microscopy photographs showing sensory receptors of spiders indicated by white arrows. (A) Tactile hairs, (B) Chemoreceptors, (C) Slit sensilla, (D) Trichobothria. Scale bars as indicated on each photograph.

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RESULTS

A total of eight species of Anelosimus species were examined, with four samples (two

males and two females) per species. Four legs (Legs I – Legs IV) were examined for

each sample.

Tactile Hair

There was a significant difference in the mean density of tactile hair among the eight

Anelosimus species (ANOVA: F7, 24 = 18.4, p < 0.001; Figure 3.2A). Anelosimus

cameronis had the highest density of tactile hair (mean ± s.e.: 0.00064 ±

0.000062/m2) while A. kohi had the lowest mean tactile hair density (0.00016 ±

0.00001/m2). The mean tactile hair density of A. kohi and A. taiwanicus were significantly lower than that of the other six species. Mean tactile hair densities of A. chonganicus and A. seximaculatum were also significantly lower than A. cameronis.

No significant differences were observed in the mean tactile hair density between A. chonganicus and A. seximaculatum, between A. kohi and A. taiwanicus, as well as among A. cameronis, A. kinabaluensis, A. membranaceus and A. parvulus. In contrast, no significant differences were found in the mean total number of tactile hair per spider among the eight species (ANOVA: F7, 24 = 1.74, p = 0.147; Figure 3.3A).

Comparisons of the mean tactile hair density on Legs I to IV across species revealed significant differences among species (Figure 3.4, Table 3.1). Both A. kohi

and A. taiwanicus possessed a lower mean tactile hair density as compared to the remaining six species on Legs I to Legs IV (Figure 3.4). On Legs I, both A. kohi and

A. taiwanicus had significantly lower tactile hair density than the six solitary species

(Figure 3.4A). The mean tactile hair density on Legs I of A. chonganicus was

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus significantly lower than that of A. cameronis. No significant differences in tactile hair densities were observed between A. kohi and A. taiwanicus and among A. chonganicus, A. membranaceus A. parvulus and A. seximaculatum. The mean tactile hair density on Legs II of A. kohi was significantly lower than the other seven species.

Similarly, A. taiwanicus had lower mean tactile hair density on Legs II as compared to the six solitary species. There were no significant differences in tactile hair densities in Legs II of the six solitary species (Figure 3.4B). Mean tactile hair densities on Legs III of both A. kohi and A. taiwanicus were significantly lower than those of A. cameronis and A. kinabaluensis. While the mean tactile hair density on

Legs III of A. cameronis and A. kinabaluensis was significantly higher than that of the other six species. There were no significant differences in tactile hair densities on

Legs III of A. chonganicus, A. kohi, A. parvulus, A. membranaceus, A. seximaculatum and A. taiwanicus (Figure 3.4C). Lastly, mean tactile hair density on Legs IV of A. kohi was significantly lower than the other six species. Similarly, A. taiwanicus had lower mean tactile hair density on Legs II as compared to the six solitary species. No significant differences were found in tactile hair densities among the six solitary species. The differences in mean tactile hair densities in Legs IV were similar to those observed in Legs II. No significant differences were observed in the total number of tactile hairs in the eight species across Legs I to IV (Table 3.2).

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Table 3.1. Results from ANOVAs testing the differences in the mean tactile hair density on Legs among eight species of Anelosimus. Leg Test Statistic df p

I One–way ANOVA 19.2 7, 24 < 0.001

II Kruskal–Wallis 22.4 7, 7 0.002

III One–way ANOVA 6.06 7, 24 < 0.001

IV Kruskal–Wallis 21.2 7, 7 0.004

Table 3.2. Results from ANOVAs testing the differences in the mean total number of tactile hairs per spider on Legs among eight species of Anelosimus.

Leg Test Statistic df p

I Kruskal–Wallis 10.182 7, 7 0.179

II One–way ANOVA 0.992 7, 24 0.460

III One–way ANOVA 1.795 7, 24 0.135

IV Kruskal–Wallis 8.801 7,7 0.267

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Chemoreceptor

Overall, there was no significant difference in the mean chemoreceptor density among

the eight species examined (ANOVA: F7, 24 = 0.856, p = 0.554; Figure 3.2B). A. taiwanicus has the lowest mean chemoreceptor density (0.00011 ± 0.00004/m2) and

A. cameronis has the highest mean chemoreceptor density (0.00029 ± 0.00014/m2).

No significant difference was observed in the mean total number of chemoreceptors per spider in the eight species examined (ANOVA: F7, 24 = 1.0398, p = 0.398; Figure

3.3B).

Similarly, when the mean chemoreceptor densities on Legs I to Legs IV were

compared among species, no significant differences were found (Figure 3.5; Table

3.3). No significant differences were observed in the mean total number of

chemoreceptors in the eight species across Legs I to IV (Table 3.4).

Table 3.3. Results from ANOVAs testing the differences in the mean chemoreceptor density on Legs among eight species of Anelosimus. Leg Test Statistic df p

I Kruskal–Wallis 5.24 7, 7 0.631

II One–way ANOVA 0.416 7, 24 0.883

III One–way ANOVA 1.99 7, 24 0.099

IV One–way ANOVA 0.458 7, 24 0.855

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Table 3.4. Results from ANOVAs testing the differences in the mean total number of chemoreceptor on Legs among eight species of Anelosimus.

Leg Test Statistic df p I One–way ANOVA 0.775 7, 24 0.614

II One–way ANOVA 1.208 7, 24 0.337

III One–way ANOVA 1.546 7, 24 0.200

IV Kruskal–Wallis 6.022 7, 7 0.537

Slit Sensillum

A significant difference in the mean slit sensillum density was found among the eight

species (ANOVA: F7, 24 = 9.67, p < 0.001; Figure 3.2C). A. kohi had the lowest mean

slit sensillum density (0.000011 ± 0.000002/m2), while A. cameronis had the highest mean density of slit sensillum (0.000046 ± 0.000006/m2). The mean slit sensillum

densities of A. kohi, A. parvulus and A. taiwanicus were significantly lower than those of A. cameronis, A. chonganicus, A. kinabaluensis and A. seximaculatum. There were no significant differences in slit sensillum densities among A. kohi, A. parvulus, A. seximaculatum and A. taiwanicus and among A. cameronis, A chonganicus, A.

kinabaluensis and A. membranaceus (Figure 3.2C). No significant difference was

found in the mean total number of slit sensilla per spider among the eight species

(Kruskal-Wallis test: H = 12.22, df = 7, p = 0.094; Figure 3.3C).

Comparisons of the mean slit sensillum densities on Legs I to IV across

species revealed significant differences among species (Figure 3.6, Table 3.5). On

Legs I, the mean slit sensillum densities of A. kohi and A. taiwanicus were

significantly lower than those of A. cameronis, A. kinabaluensis and A.

membranaceus. There were no significant differences found in Legs I slit sensillum

densities among the six solitary species and among A. kohi, A. parvulus, A.

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seximaculatum and A. taiwanicus (Figure 3.6A). Slit sensillum density on Legs II of

A. kohi was significantly lower than that of A. cameronis. There were no significant

differences in slit sensillum densities on Legs II among the remaining six species

(Figure 3.6B). Although slit sensillum densities on Legs III were found to be

significantly different across species, pairwise comparisons did not reveal any

significant difference between species (Figure 3.6C, Table 3.5). On Legs IV, slit

sensillum density of A. kohi was significantly lower than that of A. cameronis, A.

chonganicus, A. membranaceus and A. seximaculatum. There were no significant

differences in slit sensillum densities of Legs IV among A. cameronis, A.

chonganicus, A. kinabaluensis, A. membranaceus, A. parvulus, A. seximaculatum and

A. taiwanicus (Figure 3.6D). No significant differences were found in the mean total

number of slit sensilla in the eight species across Legs I to IV (Table 3.6).

Table 3.5. Results from ANOVAs testing the differences in the mean slit sensillum density on Legs among eight species of Anelosimus. Leg Test Statistic df p

I One–way ANOVA 6.24 7, 24 < 0.001

II One–way ANOVA 3.78 7, 24 0.007

III One–way ANOVA 3.25 7, 24 0.014

IV One–way ANOVA 4.79 7, 24 0.002

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Table 3.6. Results from ANOVAs testing the differences in the mean total number of slit sensilla on Legs among eight species of Anelosimus.

Leg Test Statistic df p I One–way ANOVA 2.437 7, 24 0.049

II Kruskal–Wallis 12.231 7, 7 0.093

III Kruskal–Wallis 8.177 7, 7 0.317

IV Kruskal–Wallis 15.294 7, 7 0.032

Trichobothrium

There was a significance difference in the mean trichobothrium density among the

eight species (ANOVA: F7, 24 = 5.74, p < 0.001; Figure 3.2D). A. kohi had the lowest

mean trichobothrium density (0.0000023 ± 0.0000002/m2), while A. cameronis had the highest density of trichobothrium (0.0000093 ± 0.0000021/m2). Overall, the

subsocial A. kohi and A. taiwanicus had lower mean density of trichobothrium than

the six solitary species. Trichobothrium density of A. kohi was significantly lower

than that of A. cameronis, A. kinabaluensis and A. membranaceus. Both A. kohi and

A. taiwanicus had significantly lower trichobothrium densities than A. cameronis.

There were no significant differences in trichobothrium densities among A.

chonganicus, A. kohi, A. parvulus, A. seximaculatum and A. taiwanicus and among A.

chonganicus, A. kinabaluensis, A. membranaceus, A. parvulus, A. seximaculatum and

A. taiwanicus (Figure 3.2D). No significant difference was found in the mean total

number of trichobothria per spider in the eight species (ANOVA: F7, 24 = 0.659, p =

0.704; Figure 3.3D).

Comparisons of the mean trichobothrium densities on Legs I to IV across species revealed significant differences among species in Legs III and IV and non– significant differences among species in Legs I and II (Figure 3.7, Table 3.7). In Legs

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III, trichobothrium density of A. kohi was significantly lower than that of A. cameronis and A. membranaceus. Trichobothrium densities of both A. kohi and A. taiwanicus were also significantly lower than those of A. cameronis. No significant differences were found in Legs III trichobothrium densities among A. chonganicus, A. kinabaluensis, A. kohi, A. parvulus and A. seximaculatum (Figure 3.7C). In Legs IV, trichobothrium densities of A. kohi and A. taiwanicus were significantly lower than those of A. cameronis, A. chonganicus and A. parvulus. No significant differences were found in Legs IV trichobothrium densities among A. kinabaluensis, A. kohi, A. membranaceus, A. seximaculatum and A. taiwanicus (Figure 3.7D). Comparison of mean total number of trichobothria across Legs I to IV also yielded no significant differences (Table 3.8).

Table 3.7. Results from ANOVAs testing the differences in the mean trichobothrium density on Legs among eight species of Anelosimus. Leg Test Statistic df p

I Kruskal–Wallis 12.3 7, 7 0.092 II One–way ANOVA 1.52 7, 24 0.21

III One–way ANOVA 4.30 7, 24 0.004

IV One–way ANOVA 6.37 7, 24 < 0.001

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Table 3.8. Results from ANOVAs testing the differences in the mean total number of trichobothria on Legs among eight species of Anelosimus. Leg Test Statistic df p I Kruskal–Wallis 6.036 7, 7 0.536

II Kruskal–Wallis 9.928 7, 7 0.193

III Kruskal–Wallis 3.193 7, 7 0.867

IV Kruskal–Wallis 2.405 7, 7 0.934

In the comparison of the overall sensory receptors densities among the eight species of Anelosimus, the two subsocial species (A. kohi and A. taiwanicus) were consistently found to possess lower densities of tactile, trichobothrium and slit sensilla compared to the six solitary species. Among the six solitary species, A. cameronis and A. kinabaluensis were also repeatedly found to have higher densities of sensory receptors compared to other four species. In contrast, no obvious pattern is present in the mean total number of receptors per spider in the eight species of

Anelosimus.

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus

A c b, c b, c b b b, c

a a

* *

B a

a a a a a a a

2 m

s/µ r * *

ecepto

f r C b o

o. b b a a, b b n

M a a

* * a, b, c c D b, c b, c a, b, c a, b, c a, b a

* *

Figure 3.2. Mean density of sensory receptors in eight species of Anelosimus. (A) Mean ( s.e.) density of tactile hair; (B) Mean ( s.e.) density of chemoreceptor; (C) Mean ( s.e.) density of trichobothrium; and (D) Mean ( s.e.) density of slit sensillum. Different lower cases indicate significant difference (p < 0.05). Subsocial species indicated with asterisk.

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus

* *

* C

Mean total receptor number per spider receptor Mean total

* * D

* *

Figure 3.3. Mean total number of receptors per spider of eight species of Anelosimus. (A) Mean ( s.e.) number of tactile hairs; (B) Mean ( s.e.) number of chemoreceptors; (C) Box plot of number of slit sensilla; (D) Mean ( s.e.) number of trichobothria. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. No significant differences were found in mean receptor number in all species. Subsocial species indicated with asterisk.

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus

c, d A b, c, d c, d b, c, d b b, c

a a

* * c c c B c c c

a b 2 m µ

* * c C c a, b a, b a, b a, b

Mean no. of tactile hair/ a a

D c c c c c c a b

* *

Figure 3.4. Mean (± s.e.) tactile hair density of eight species of Anelosimus. (A) Mean (± s.e.) density of tactile hair on Legs I; (B) Box plot of the density of tactile hair on Legs II; (C) Mean (± s.e.) density of tactile hair on Legs III; and (D) Box plot of the density of tactile hair on Legs IV. Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Different lower cases indicate significant difference (p < 0.05). Subsocial species indicated with asterisk.

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus

* * B

2 /µm *

chemoreceptors

Mean no. of

* * D

Figure 3.5. Mean chemoreceptor density of eight species of Anelosimus. (A) Mean (± s.e.) density of chemoreceptor on Legs I; (B) Mean (± s.e.) density of chemoreceptor on Legs II; (C) Mean (± s.e.) density of chemoreceptor on Legs III; and (D) Mean (± s.e.) density of chemoreceptor on Legs IV. No significant differences were found. Subsocial species indicated with asterisk.

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus

c c A b, c c

a, b, c a, b, c a, b a

* *

B b a, b a, b a, b a, b a, b a, b a 2 m

* *

C a a a a a

Mean no. of slit sensilla/ a a a

* *

D b b b a, b b

a, b a, b a

* * Figure 3.6. Mean slit sensillum density of eight species of Anelosimus.(A) Mean (± s.e.) density of slit sensillum on Legs I; (B) Mean (± s.e.) density of slit sensillum on Legs II; (C) Mean (± s.e.) density of slit sensillum on Legs III; and D. Mean (± s.e.) density of slit sensillum on Legs IV. Different lower case letters indicate significant differences (p < 0.05). Subsocial species indicated with asterisk. 214

Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus

a A a

a a a a a a

* a B a

a 2 a a m a a µ a

* *

C c b, c a, b, c a, b, c Mean no. of trichobothria/ a, b, c a, b, c

a a, b

* * D b a, b a, b b b a, b a a

* *

Figure 3.7. Mean trichobothrium density of eight species of Anelosimus. (A) Mean (± s.e.) density of trichobothrium on Legs I; (B) Mean (± s.e.) density of trichobothrium on Legs II; (C) Mean (± s.e.) density of trichobothrium on Legs III; and (D) Mean (± s.e.) density of trichobothrium on Legs IV. Different lower case letters indicate significant differences (p < 0.05). Subsocial species indicated with asterisk.

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DISCUSSION

The majority of social spiders construct webs for prey capture and as a form of retreat

(Aviles, 1997; Whitehouse & Lubin, 2005). The spider web is an effective medium

for the transmission of vibrational and chemical cues (Krafft & Cookson, 2012). The

findings of this study showed that subsocial species of Asian Anelosimus possess

lower densities of mechanoreceptors (tactile hair, trichobothrium and slit sensilla) as

compared to solitary species. Both A. kohi and A. taiwanicus, which are subsocial species had lower densities of tactile hair, trichobothrium and slit sensilla than the solitary species, A. cameronis, A. chonganicus, A. kinabaluensis, A. membranaceus,

A. parvulus and A. seximaculatum. Furthermore, amongst solitary species, A. cameronis and A. kinabaluensis had the highest densities of mechanoreceptors. No obvious patterns of chemoreceptors densities were observed. The results of this study does not seem to support the hypothesis that subsocial species possess higher densities of sense receptors due to a need to distinguish between related and unrelated conspecifics and between conspecifics and prey. Perhaps there are other more important factors influencing the densities of receptors in this group of spiders. In addition, no significant differences were detected in mean total number of receptors

(tactile hairs, chemoreceptors, slit sensilla and trichobothria) among the eight species.

I adopted the method of comparison by Willemart & Gnaspini (2003) where comparisons between species were made using densities of receptors and highlight the disparity between densities of receptors and total number of receptors in the comparisons made among the eight species.

The observation of lower densities of mechanoreceptors in subsocial species than in solitary species in this study suggests that subsocial species may be less

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus sensitive to mechanical signals (vibrations, touch or sound) as compared to solitary species. This corroborates the findings of Pruitt et al. (2008) where the comparison between social and asocial A. studiosus revealed that social individuals of A. studiosus show delayed response to vibrations caused by prey. The sensitivity and speed of response to stimuli may thus be closely related to the level of sociality in Anelosimus species. Moreover, as compared to the more social A. dubiosus and A. jabaquara, individuals in colonies of the subsocial A. baeza are also found to respond to prey vibrations more readily (Harwood & Aviles, 2013). Marques et al. (1998) also found that while the less social A. jabaquara attacked and killed female A. dubiosus that were dropped into their webs, the more social A. dubiosus did not exhibit the same level of aggression towards females of A. jabaquara dropped into their webs. Instead,

A. dubiosus females are even observed participating in prey capture with the foreign

A. jabaquara females. These observations suggest that the more social species of

Anelosimus are less aggressive towards conspecifics and foreign invaders and they may be less sensitive to mechanical or chemical signals produced by other individuals in the web. The occurrence of high number of kleptoparasites in webs of social and subsocial spiders further attests to this hypothesis. Webs of the social spider, A. eximius frequently contain the kleptoparasite Argyrodes ululans (Theridiidae)

(Cangiolosi, 1990), while webs of the subsocial A. studiosus also commonly contain

Anyphaenidae, Salticidae, Philodromidae, Araneidae, Tetragnathidae or Agelenidae spiders (Perkins et al., 2007). This indicates a possible link between the level of aggression and sensitivity to densities of receptors. Lower densities of receptors may therefore be an adaptation to lower level of aggression exhibited by more social species to promote tolerance among individuals as the number of spiders sharing a

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web increase. To further test this hypothesis, mechanoreceptor densities of social

species would need to be examined.

Although pheromones are known to affect cohesion and group living in

spiders (Roland, 1984; Evans & Main, 1993), there is a lack of significant difference

in the chemoreceptor densities between solitary and subsocial species in this study.

This may indicate a distinction in the function of mechanical signals and chemical

signals in this group of spiders. Chemoreceptors are used for the detection of both

contact and air borne chemicals. Unlike mechanical signals, chemical signals can be

long lived and can be transmitted through long distances (Wyatt, 2003; Gaskett, 2007;

Foelix, 2011; Ulh, 2013). Furthermore, pheromones produced are usually species

specific and effective even in small quantities (Pollard et al., 1987; Schulz & Toft,

1993; Wyatt, 2003; Gaskett, 2007). Pheromones are also often used during

reproduction where males search for and evaluate viable females for mating (Ross &

Smith, 1979; Gaskett, 2007; Uhl, 2013). The lack of clear patterns of chemoreceptor

densities variation in accordance to level of sociality in this study suggests that all the

eight species of Anelosimus examined here may also rely on pheromones in seeking

and assessing potential mate instead. Therefore, the lack of significant differences in chemoreceptor densities of the eight species of Anelosimus examined may reflect a

lack of reliance on chemical communications in mediating group living with

increasing sociality.

The type of habitat in which species are found also influences densities of

receptors and sensitivity towards stimulus. Spiders are known to adapt signal

transmission according to the environment (Taylor & Jackson, 1999). Both A.

cameronis and A. kinabaluensis possess high densities of mechanoreceptors and

chemoreceptors and are also found at higher elevations, where the environment is

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very different from that of lowland tropical forests where the remaining six species

were found (Chapter 2). Willemart & Gnaspini (2003) found that the density of

sensory receptors of cave dwelling species that reside near streams is lower than that

of epigean harvestmen and they hypothesized that this may be due to higher food

abundance or reduction in predation pressure. As compared to tropical lowland

forests, the temperate forests where A. cameronis and A. kinabaluensis were found

often contain less insects and insects that are smaller in size (Olson, 1994; Aviles et

al., 2007; Guevara & Aviles, 2007). The smaller sizes of prey in temperate

environment may make detection harder, leading to development of higher sensitivity,

and hence higher receptor densities in A. cameronis and A. kinabaluensis.

Nevertheless, it is important to note that sensory receptors are also influenced

by predation pressure (Holling, 1961; Willemart & Gnaspini, 2003). In a study by

Purcell & Aviles (2008), it was found that there is a higher abundance of ants in

lowland tropical forests as compared to high elevation cloud forests. Hence, with

lower predation pressure, the densities of sensory receptors may be lower in A.

cameronis and A. kinabaluensis, yet this is not observed in this study. Mate searching

is another factor (Foelix, 2011; Trabalon, 2013), however, the distance between nests of A. cameronis and A. kinabaluensis was similar to that observed in A.

membranaceus, A. parvulus and A. seximaculatum that possess lower densities of

receptors. Nonetheless, the abundance of these two species was the lowest amongst

the eight species examined here and this indicates that mate searching will be more

difficult in these two species. Thus although distance between mates in A. cameronis

and A. kinabaluensis is similar to that of A. chonganicus, A. membranaceus and A.

seximaculatum, the low abundance of conspecifics may mean a need to invest in the

development of more receptors to increase sensitivity to cues for the detection of

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Chapter 3 – Sensory Receptors and Sociality in Spiders of the Genus Anelosimus mates. Therefore, the need to adapt to the low prey abundance, small prey size and mate searching may be the possible driving force for the development of higher densities of sensory receptors in these two solitary species.

The examination of tactile hair and slit sensilla on individual legs revealed that densities of these two types of mechanoreceptors are the highest on Legs III of all eight species of Anelosimus. In harvestmen, Legs I and II have frequently been accorded more importance in detection of signals from the environment, while Legs

III and IV are typically utilized in locomotion (Hoenen & Gnaspini, 1999; Elpino–

Campos et al., 2001). During locomotion, spiders are often seen waving Legs I and II in the air and subsequently ‘tasting’ these legs with the purpose of detecting pheromones (Suter et al., 1987; Gaskett, 2007; Foelix, 2011; Krafft & Cookson, 2012) or probing the environment (Schuch & Barth, 1990; Foelix, 2011). In contrast, Legs

III, being the shortest leg amongst the four legs examined in all eight species, were never observed being waved in the air by the eight species of Anelosimus. Under all circumstances, Legs III regularly remain firmly on the substrate which spiders are on.

Given the constant contact with the substrate, it is no surprise that Legs III have the highest number of tactile hairs and slit sensilla for detection of mechanical signals.

However, surprisingly, in the comparison of tactile hair and slit sensilla densities across legs in the eight species, there were no significant differences between subsocial and solitary species. Instead, subsocial species are shown to possess significantly lower densities of tactile hair than solitary species in Legs I, II and IV.

This may suggest a difference in functionality in different legs in Anelosimus species.

Multimodal communication is common in spiders: wolf spiders utilize both visual and seismic cues in courtship (Gordon & Uetz, 2011, 2012; Hebets et al., 2013;

Uetz et al., 2013); web-building spiders use both pheromones and visual cues to

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attract prey or as defense against predators (Chou et al., 2005; Nakata, 2009; Tan et

al., 2010). Therefore, a myopic focus on differences in a single type of receptor may

not yield a complete understanding of the adaptation to group living in Anelosimus

species. Social and subsocial spiders may utilize both mechanical and chemical

signals for communication. Hence, the lower density of mechanoreceptors observed

in subsocial species in this study does not necessarily indicate a lowered overall

sensitivity towards environmental or conspecific signaling in more social species.

Examination of the sensory receptors of these eight species of solitary and

subsocial Anelosimus also did not reveal any specialized receptors in any individuals.

While social insects such as ants and termites engage in task differentiation in social colonies (Theraulz et al., 1991; Karsai & Philips, 2012), task differentiation and castes have not been observed in social spiders (Ainsworth et al., 2002; Settepani et al., 2012). Moreover, while social insects communicate through seismic cues or sound to alert nest mates to sources of food and invasion by predators (Ayasse et al., 2001;

Cocroft & Rodriguez, 2005; Drosopoulos & Claridge, 2005; O’Donnell & Bulova,

2007; Matthews & Matthews, 2010), no studies have observed similar phenomenon in social spiders. In Anelosimus eximius, the apparent coordination in prey capture appears to be mediated solely through the vibrations caused by prey struggling on the web (Vakanas & Krafft, 2001, 2004). Hence, it is not surprising that unlike social insects, social spiders do not possess modification to their sensory receptors to allow for specialized task performance or specialized coordination within the web.

Therefore, unlike social insects, communication in social spiders may not be as extensive or crucial.

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While a clear pattern of lower receptor densities in subsocial species (A. kohi

and A. taiwanicus) than solitary species (A. cameronis, A. chonganicus, A. kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum) has been observed in tactile hair, slit sensilla and trichobothrium, significant differences between these two groups of spiders are only seen in tactile hair density. Meanwhile, it should be noted that variation between individuals within species appears to be larger in densities of chemoreceptor, slit sensilla and trichobothrium. This could likely have contributed to the failure to detect significant differences between subsocial and solitary species and may be rectified with increased sample sizes.

Finally, examination of the mean total number of receptors in each spider also revealed no significant differences among the eight species studied. The two subsocial species (A. kohi and A. taiwanicus) are larger than the remaining six solitary species

(see Chapter 1) and this may have contributed to lower density of mechanoreceptors in these two species. However, the same pattern is not observed in chemoreceptor densities. Neither is there any observed correspondence between size and receptor densities among the other six solitary species. Therefore, differences in densities of receptors in Anelosimus likely still reflect a reduction in sensitivity and an adaptation to sociality.

This is the first study into the receptor densities of spiders of the genus

Anelosimus. The findings from this study have shown a lower density of mechanoreceptors in subsocial spiders, A. kohi and A. taiwanicus. Lower number of mechanoreceptors may result in reduced sensitivity towards vibrational signals transmitted through the web. Reduced sensitivity to mechanical signals may be an adaptation to reduce aggression and promote tolerance as subsocial and social species spend longer period of time in aggregation. Examination and comparison of receptor

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Behavioural tests on the sensitivity of Anelosimus species towards simulated mechanical signals would also be highly informative.

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

ABSTRACT

Social spiders occupy a wide geographical range and the development of sociality has

occurred several times in different spider families. However, the majority of social spiders remain confined to lowland tropical rainforests. Lowland tropical rainforests are characterized by a number of common traits and these are speculated to contribute to the evolution of sociality in spiders. In the genus Anelosimus, social species are constrained to lowland tropical rainforests, subsocial congeners inhabit habitats at higher altitude and little is known of the habitat preference of solitary species. In this study, I examine the environmental variables associated with habitats of Anelosimus to determine if the development of sociality is connected with particular sets of environmental factors. Through examination of six social, 17 subsocial and 12 solitary species and analysis using the method of variation partitioning, I determined that temperature and precipitation are associated with distribution of social types in Anelosimus. Social spiders are more likely to occur in habitats of high rainfall and intermediate temperatures. Subsocial species are restricted to habitats with low rainfall and temperatures whereas solitary species tend to occur in environments with high rainfall and high temperatures. Prey abundance, rain damage, behavioural changes and differential development rates are possible factors driving these patterns of distribution.

Finally, I also discuss other possible factors that may account for the distribution pattern seen in Anelosimus.

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INTRODUCTION

Formation of groups occurs in the animal kingdom for various reasons ranging from predator

defense and risk dilution to increased foraging efficiency (Krause & Ruxton, 2002). Groups

formed may be either temporal such as flocks of foraging birds or permanent like in elephant

herds and social insects (Rubenstein & Kealey, 2012). The formation or breakdown of

groups are usually dependent on the balance of cost and benefits of group living (van Veelen

et al., 2010; Port et al., 2011; Tabadkani et al., 2012). Permanent groups are often

characterised by behaviours that increase benefits of aggregation, such as cooperative

foraging to increase nutritional intake or nest guarding and maintenance for enhanced defence

(Dugatkin, 1997; Rubenstein & Rubenstein, 2013). Often, animal groups are formed as a

response to biotic and abiotic conditions present in their habitats (Krause & Ruxton, 2002).

Unsurprisingly, environmental conditions of a species’ habitat often contributes to the

development of adaptive behaviour or physiological and morphological changes (Alcock,

2013). This also inevitably affects the distribution of species across different habitat ranges as

they evolve and adapt to local environmental conditions. The influence of environmental

factors on species distribution can be observed in many animal groups. Factors such as

temperature, precipitation, oxygen level, vegetation composition and canopy cover are known to play a major role in the distribution of various species (Cech et al., 1990; Entling et al.,

2007; Jiménez-Valverde & Lobo, 2007; Ziesche & Roth, 2008; Mattila et al., 2011; Blaser et al., 2013; Li et al., 2013).

Amongst spiders, aggregation is rare but occurs in a variety of forms: permanent social spiders, hereby ‘social’ spiders, form multi-female colonies with female biased sex ratio, they also display a lack of dispersal from the natal nest and cooperation amongst adults;

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‘subsocial’ or non-territorial periodic-social (Avilés, 1997) spiders form colonies consisting a single female and her offspring which disperse just before reaching sexual maturity (Avilés,

1997; Whitehouse & Lubin, 2005; Avilés & Harwood, 2012; Corcobado et al., 2012; Yip &

Rayor, 2013); and ‘colonial’ spiders form groups but maintain and defend individual webs and breed independently (Avilés, 1997; Whitehouse & Lubin, 2005). Although sociality in spiders is rare, it has evolved independently 18 times amongst at least seven different families

(Agnarsson et al., 2006b). Together with the assortment of social grouping in spiders and their diverse evolutionary origins, what are some factors that could influence the development of one form of social grouping over another? Most studies have focused on the social/subsocial groups, investigating phylogenetic and environmental conditions that predispose species to the development of social behaviour (Avilés, 1997; Avilés & Purcell,

2012; Pruitt et al., 2012; Majer et al., 2013a, b; Yip & Rayor, 2013). These studies have revealed that social spiders are mainly found in lowland forests in tropical and subtropical zones, whereas subsocial congeners commonly occur at higher altitudes and latitudes (Avilés et al., 2007; Powers & Avilés, 2007; Purcell, 2011). Incidentally, tropical and temperate habitats and elevational differences also present a gradient of environmental variables such as precipitation, seasonality and temperature (Blanckenhor & Demont, 2004; Purcell, 2011).

Both temperature and rainfall have been found to affect the distribution of other social arthropods. The southern harvester termite Microhodotermes viator is more abundant in areas with higher rainfall (Picker et al., 2006). The giant tropical ant, Parapnera clavata has also been found to favour habitats with high rainfall, high temperature and low seasonality

(Murphy & Breed, 2007). Sweat bees, Lasioglossum baleicum, also display social behaviour at habitats with higher temperatures in contrast to solitary behaviour in cooler climates

(Hirata & Higashi, 2008).

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

One of the aims of this study is to extend knowledge of how environmental conditions affect the geographical distribution patterns of sociality observed in the spider genus

Anelosimus (Araneae: Theridiidae). Anelosimus is a cosmopolitan group of spiders, distributed globally from Africa, Asia, Australia to North and South America (Agnarsson,

2006; 2012a; Agnarsson & Kuntner, 2006; Agnarsson & Zhang, 2006; Agnarsson et al.,

2006a). There are 64 nominal species within this genus (Platnick, 2014), of which six are social, 15 subsocial and eight solitary living (Agnarsson, 2012a; Chapter 2), while the sociality of the remaining species remains unknown. Anelosimus spiders thus represent a spectrum of social behaviour in spiders and their geographical distribution range presents an extensive variation of environmental factors. Therefore, using the genus Anelosimus, I seek to investigate whether geographical distribution patterns of social, subsocial and solitary

Anelosimus species may be related to temperature and rainfall.

Variation in rainfall and temperature results in the modification of ecological and environmental conditions. High rainfall and temperatures are often associated with increase productivity and high prey abundance (Janzen & Schoener, 1967; Wolda, 1978), often needed for the maintenance of group living (Dugatkin, 1997; Krause & Ruxton, 2002).

Consequently, prey availability is closely associated with the development of social behaviour in spiders (Avilés et al., 2007; Lubin & Bilde, 2007; Powers & Avilés, 2007). The prey availability hypothesis thus predicts that social species would occur in habitats with high prey abundance or large prey size (Powers & Avilés, 2007). High temperature has also been associated with high ant abundance (Kaspari et al., 2000) which has been attributed to affect the success of subsocial spiders in lowland rainforests (Purcell & Avilés, 2008). The ant predation hypothesis thus illustrates the absence of subsocial species in lowland rainforests.

Lastly, the rainfall hypothesis predicts that high intensity of rainfall may reduce the success

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Chapter 4 – Environmental factors affecting distribution of Anelosimus. of subsocial and solitary spiders that construct smaller and more fragile webs as compared to social species (Purcell & Avilés, 2008).

Examination of the distribution of Anelosimus and Stegodyphus (Eresidae) has disclosed a close association between high productivity, rainfall and sociality (Majer et al.,

2013a, b). However, Majer et al. (2013a) concentrated on species from Central and South

America, and included social and subsocial species and merely three species of solitary

Anelosimus. Unfortunately, other Anelosimus species from Africa, Asia and Australiasia have been neglected. Incidentally, these regions contain the majority of Anelosimus species that are solitary (Table 4–1). In this study, I examined the hypothesis that the geographical distribution patterns of sociality observed in Anelosimus species, at a wider geographical range, from Africa, Asia, Australasia and the Americas may be related to temperature and rainfall. This also included 17 solitary and subsocial species that were omitted from Majer et al. (2013a). I predict that social species of Anelosimus are more likely to occur in areas with high rainfall and temperatures, whereas non-social (subsocial and solitary) species may mainly occur in areas with lower rainfall and temperatures. This is the first study to include solitary and subsocial species of Anelosimus outside of Central and South America in the investigation of patterns of distribution. Since solitary species of Anelosimus build smaller webs than subsocial and social species, making their webs more vulnerable to high rain intensity and there is no high demand for prey availability as in social species, I further predict that solitary Anelosimus species should occur in areas with lowest rainfall. I tested these predictions by mapping the distribution of Anelosimus species onto a map of climatic variables to extract climatic conditions of the habitats.

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

MATERIALS AND METHODS

Species and spatial data

A total of 35 Anelosimus species were included in this analysis (Table 4.1). These 35 species

were selected because information on their point locality (latitude and longitude) and level of

sociality is available in literatures (Agnarsson, 2006; 2012a; 2012b; Agnarsson et al, 2006a;

2007; Chapter 2). Spiders were categorized into different social levels according to the level of cooperation exhibited and time of dispersal (Aviles, 1997). “Social” species do not disperse from the natal nest and adult females cooperate in web maintenance and prey capture; “subsocial” species disperse from the natal nest at late instars and cooperate in foraging during juvenile phases; “solitary” species disperse from the natal nest at early

instars. Spiders were assigned to each social group according to the study by Agnarsson

(2012a) and personal observations of A. cameronis, A. chonganicus, A. exiguus, A.

kinabaluensis, A. membranaceus, A. parvulus and A. seximaculatum (Chapter 2).

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Table 4.1. List of 35 Anelosimus species partitioned into three groups according to level of sociality.

Solitary Subsocial Social Anelosimus cameronis Anelosimus analyticus Anelosimus rupununi Anelosimus chonganicus Anelosimus arizona Anelosimus domingo Anelosimus ethicus Anelosimus baeza Anelosimus dubiosus Anelosimus exiguus Anelosimus eidur Anelosimus eximius Anelosimus kinabaluensis Anelosimus elegans Anelosimus guacamayos Anelosimus membranaceus Anelosimus jabaquara Anelosimus oritoyacu Anelosimus nigrescens Anelosimus jucundus Anelosimus pacificus Anelosimus kohi Anelosimus parvulus Anelosimus may Anelosimus pomio Anelosimus octavius Anelosimus potmosbi Anelosimus pratchetti Anelosimus seximaculatum Anelosimus sallee Anelosimus studiosus Anelosimus subcrassipes Anelosimus taiwanicus Anelosimus tosus Anelosimus vierae

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

Environmental data

Environmental variables were extracted for each point locality of Anelosimus from

WorldClim Global Climate Data Version 1.3 (Hijmans et al., 2005) environmental maps in

2.5 min resolution using DIVA-GIS Version 7.5.0 (Hijmans et al., 2004). A total of 19

climatic variables were examined (Appendix, Table 2), with four variables: (a) annual mean

temperature, (b) maximum temperature of the warmest month, (c) minimum temperature of

the coldest month, and (d) annual precipitation, selected after running through a principal

components analysis (PCA). These four variables were used to test the temperature and

rainfall hypotheses. All readings were calculated from weather stations with at least 10 years

records and are averages of monthly readings. Annual mean temperature was obtained from

the average of all monthly mean temperatures. Maximum and minimum temperatures were

calculated from the average of all maximum and minimum temperatures of all months.

Annual precipitation refers to the average sum of all total monthly precipitation in a year.

Data on temperatures gives an approximation of the total energy input to an ecosystem and

how species distribution can be affected by the amount of energy input and temperature

anomalies of the ecosystem (O’Donnell & Ignizio, 2012). Information on precipitation gives a prediction of how water availability can affect species distribution (O’Donnell & Ignizio,

2012).

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Data processing and statistical analyses

Species data

Geographical data of distribution of social type of Anelosimus species were transformed using Hellinger transformation. This transformation enables the preservation of Euclidean distances for redundancy analysis (RDA) and PCoA analyses (Legendre & Gallagher, 2001).

Environmental data

PCA revealed that four variables (annual mean temperature, mean maximum mean temperature of warmest month, mean minimum temperature of coldest month and annual precipitation) accounted for a significant amount of variance in climatic variables within the distribution ranges of Anelosimus (Appendix, Table 2). These four variables were thus selected for further analyses. To reduce correlated variation, another PCA was conducted on these four variables to generate three synthetic variables for further analyses. The three components generated accounted for 99.9% of the variation (Table 4.2). PC1 increases with rising annual mean temperature and minimum temperatures of coldest months. PC2 increases with rising annual mean temperature and maximum temperature of the warmest months. Thus both PC1 and PC2 represent a gradient of increasing temperature. PC3 increases with rising annual precipitation and represents a gradient of increasing precipitation.

Spatial data

Analysis of principal coordinates of neighbour matrices (PCNM) (Borcard & Lengendre,

2002) was used to explore spatial structures. Geographic coordinates of each species locality were used to generate a truncated matrix of Euclidean distances between localities. A

241

Chapter 4 – Environmental factors affecting distribution of Anelosimus. principal coordinates analysis (PCoA) was run on this matrix to generate PCNM variables.

Eigenvectors associated with positive eigenvalues were extracted and Moran’s I value was calculated for each eigenvector. Only eigenvectors with significant Moran’s I values were subjected to forward selection process.

Variation partitioning

The three synthetic variables (PC1–3) generated from the environmental variables through

PCA and the PCNM variables generated using PCoA on geographic coordinates were subsequently subjected to forward selection process (Dray et al, 2006) to reduce inflation of explained variation due to pure chance. Selected variables were subsequently used for variation partitioning (Borcard et al, 1992) to determine the influences of environmental (PC

1–3) and spatial (latitude/longitude and PCNM) variables on the distribution of social types of Anelosimus species.

All analyses were completed using IBM SPSS Statistics Version 22 (IBM Corp.,

USA) and R Version 3.0.2 (R Core Team, 2013), using library “ape” v. 3.0.11 (Paradis et al,

2004), “ade4” v. 1.6-2 (Dray & Dufour, 2007), “spdep” v. 0.5-71 (Bivand, 2014), “vegan” v.

2.0-1.0 (Oksanen et al. 2013), and packages “AEM” v. 0.5.1 (Blanchet et al., 2013);

“packfor” v. 0.0-8 (Dray et al. 2013); “PCNM” v. 2.1.2 (Legendre et al. 2013).

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Table 4.2. Principal component scores and loadings of PC1–3, based on four selected environmental variables.

PC1 PC2 PC3 (a) PCA result Eigenvalue 2.78 0.85 0.34 Proportion of variance explained 70.0 21.3 8.59 Cumulative variance explained 70.0 91.3 99.9

(b) Component loadings Annual mean temperature 0.73 0.65 0.22 Max Temperature of Warmest Month 0.27 0.96 - Min Temperature of Coldest Month 0.91 0.28 0.31 Annual precipitation 0.25 - 0.96

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RESULTS

Species distribution

Anelosimus species examined in this study are found mainly in East and South-east Asia, and

Central and South America. In addition, two species from Australia and Madagascar were also included in this analysis. There is however a lack of samples in mainland Africa, Central

Asia, Europe and North America. This lack is due to the absence of reliable natural history records or absence of records of Anelosimus species in these localities. Overall, the majority of Anelosimus species are found near the equator. Social species are only found in South

America, whereas the majority (58.3%) of solitary species are found in Asia. Subsocial species are found in all continents sampled (Figure 4.1).

Figure 4.1. Global distribution of Anelosimus species examined in this study. Each circle represents one data point. Legend as shown. Image by Reto Stockli NASA’s Earth Observatory, using data courtesy of USGS.

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

Variation partitioning

To establish the effects of environmental variables and spatial variables on the distribution of sociality in Anelosimus species, the method of variation partitioning (Peres-Neto et al., 2006;

Smith & Lundholm, 2010) was utilized. In forward selection of environmental variables, only

PC1 and PC3, representing gradients of increasing temperature and increasing precipitation, were significant (Table 4.3). In forward selection of spatial variables, only geographical latitude and PCNM variable 1 were found to be significant. (Table 4.3). Results of variation partitioning indicate that 10.1% of the distribution of Anelosimus species can be explained purely by the synthetic environmental variables (PC1 and PC3), while another small percentage (3.5%) by spatially structured environmental variables. Spatial influence on the distribution of these spiders is absent (Figure 4.2).

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

Table 4.3. Variables selected from forward selection and included in variation partitioning.

Variable R2 F p Environmental PC1 0.087 4.93 0.0115 PC3 0.082 5.05 0.0103

Spatial Latitude 0.155 9.55 0.001 PCNM variable 1 0.166 10.34 0.001

Explained variance (%) 10.1 0 3.5

Pure environment

Pure spatial

Spatially structured

environment

86.4 Other factors

Figure 4.2. Results of variance partitioning to separate purely environmental (grey), spatially structure environmental (white) and purely spatial (patterned) components of variance explaining the distribution of Anelosimus species. The environmental variables are composed of synthetic variables PC1 and PC3, while spatial variables consist of geographical latitude and selected PCNM variables. Unexplained variance is represented as “Other factors” (black).

Environmental variables

Examination of four environmental variables reveals that subsocial species tend to be found in areas with lower annual mean temperature, maximum temperature, minimum temperature and annual precipitation. In contrast, areas where solitary species are found tend to have higher annual mean temperature and a higher maximum and minimum temperature. Lastly,

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Chapter 4 – Environmental factors affecting distribution of Anelosimus. social species are often found in areas with intermediate temperatures and high annual precipitation (Figure 4.3, Figure 4.4 and Figure 4.5). This is also illustrated by the plot of

PC1–3 values in Figure 4.6, in which solitary species are represented by positive PC1–3 values, subsocial species are represented by negative PC1–3 values, and social species are represented by negative PC1 and PC2 values and high positive PC3 value.

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

Figure 4.3. Gradient of environmental variables. (A) Annual mean temperature and (B) Annual precipitation, with distribution of Anelosimus mapped. Legend as shown. 248

Chapter 4 – Environmental factors affecting distribution of Anelosimus.

Figure 4.4. Gradient of environmental variables. (A) Maximum temperature of warmest month and (B) Minimum temperature of coldest month, with distribution of Anelosimus mapped. Legend as shown.

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

A C o

erature/

Annual mean tem

C o

erature/ p

Max tem Max

C o

erature/ p

tem Min

/mm

itation p reci

Annual

Figure 4.5. Boxplot of (A) Annual mean temperature, (B) Maximum temperature of warmest month, (C) Minimum temperature of coldest month, and (D) Annual precipitation of localities of social, subsocial and solitary Anelosimus species. . Boxes show median (line within the box) and upper (25%) and lower (75%) quartiles, and whiskers indicate 5th and 95th percentiles. Circles outside boxplot represent outliers.

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

PC3

PC1

PC2

Figure 4.6. Plot of mean of synthetic variables, PC1 to PC3 extracted from PCA of four environmental variables (mean annual temperature, maximum and minimum temperatures and annual precipitation). Higher PC1 and PC2 values represent gradients of increasing temperatures, while higher PC3 represents gradient of increasing precipitation.

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DISCUSSION

The global distribution of Anelosimus species allowed the examination of the effects of spatial and environmental variables in structuring distribution of social, subsocial and solitary species. The results from this study indicate that spatial components have negligible effects on the distribution of social, subsocial and solitary Anelosimus. In contrast, the environmental factors, rainfall and temperature, have a significant impact. However, my results do not validate the hypothesis of a linear relationship of rainfall and temperature pattern with increasing level of sociality. Instead, I found that subsocial Anelosimus species are confined to regions of low rainfall and temperatures, whereas social and solitary species are commonly found in areas with high rainfall. The findings are congruent with previous studies where social species of Anelosimus and Stegodyphus are found to be confined to regions of high rainfall (Purcell & Avilés, 2008; Majer et al., 2013a, 2013b), whereas subsocial species are restricted to regions of low rainfall (Avilés et al., 2007; Purcell & Avilés, 2008).

High vs. low rainfall

Social Anelosimus are found in areas with high rainfall, whereas subsocial species are confined to areas of low rainfall. Colonies of social Anelosimus vary between a few to thousands of individuals (Avilés, 1997). With such a large number of individuals, a high prey availability is essential to sustain the colony. The importance of prey abundance in maintenance of group size is observed in the colonial web-building spider Cyrtophora citricola (Araneidae), where group sizes are larger at sites of greater prey abundance (Mestre

& Lubin, 2011). Studies have also confirmed that social species of Anelosimus reside in areas with larger insects and are successful in capturing these insects (Powers & Avilés, 2007). 252

Chapter 4 – Environmental factors affecting distribution of Anelosimus.

Insect size and abundance have been found to be affected by rainfall patterns, with high

insect abundance and large sizes occurring in regions of high rainfall (Janzen & Schoener,

1967; Guevara & Avilés, 2007; Powers & Avilés, 2007). Large web sizes are necessary to

capture large insects (Yip et al., 2008), thus indicating that a sizeable group size is essential

for thriving in regions of high rainfall where insects are larger (Guevara & Avilés, 2007). The

tendency for subsocial Anelosimus to occur in sites of low rainfall may thus reflect the

inability of their small web sizes to capture large prey that occur at sites of high rainfall. This

is confirmed by Powers & Avilés (2007) who found that subsocial spiders capture smaller prey as compared to their social congeners. The prevalence of social species and the absence

of subsocial species in regions of high rainfall thus likely reflects the influence of prey size

and abundance on the maintenance of large group sizes.

The prevalence of solitary species in areas of high rainfall thus presents a paradox to the prey size and abundance hypothesis. The occurrence of social and solitary species and the

absence of subsocial species in regions of high rainfall can also be examined through the rain

damage hypothesis (Riechert 1985; Riechert et al., 1986). High rain intensity causes damage

to webs of Anelosimus, ocassionally leading to break up of colonies (Avilés, 1997). The

damage induced by rainfall is found to be less on larger sized webs as compared to small

sized ones in the social spider Agelena consociata (Riecher 1985; Riechert et al., 1986). This

further supports the prevalence of social species with large web sizes in regions of high

rainfall. On the other hand, webs of solitary Anelosimus are smaller than subsocial and social

species, and their webs are also frequently found beneath small wildflowers or at the tip of

branches, incorporating rolled up leaves (Chapter 2). Purcell & Aviles (2008) found that the

performance of spiders with small webs can be improved when sheltered from rain. The

leaves and flowers that solitary Anelosimus incorporate in web building may similarly

provide structural shelter from rainfall, allowing these species to prevail in areas of high

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

rainfall. Furthermore, the small size of their webs may also reduce the mechanical pressure

exerted by the rain drops, thus reducing the damage inflicted on the web. In concert, these

attributes can contribute to the success of solitary species in areas of high rainfall.

High vs low temperatures

A negative relationship is shown between PC1 (gradient of increasing annual mean

temperature and max temperature) and increasing level of sociality. Solitary species of

Anelosimus is associated with regions of highest annual mean temperature while subsocial

and social species of Anelosimus are associated with regions of lower temperatures. High

temperature has been associated with increased aggressiveness in Anelosimus studiosus and

has been suggested to be the sole factor affecting behavioural tendency (social vs asocial) in

this species (Pruitt et al., 2011). At higher temperatures, A. studiosus females are less tolerant of conspecifics and distances between individuals are wider (Pruitt et al., 2011). The negative

relationship between temperature and social tendency has been attributed to differential

metabolism rates brought about by changes in temperatures (Pruitt et al., 2011). At higher

temperatures, metabolisms of ectotherms such as spiders will be higher, which results in a

higher demand for food resource (Gillooly et al., 2001). This higher demand for food

resource thus impedes the development of sociality since cannibalism is common among

spiders and conspecifics in close proximity will readily be consumed as prey (Wise, 2006).

Higher temperature also results in increased rates of growth and development, allowing organisms to reach maturity faster (Gillooly et al., 2002; Zuo et al., 2012). In A.

studiosus, a higher number of multi-female colonies occur at higher altitudes with cooler climates (Furey, 1998; Riechert & Jones, 2008). Occurrence of this phenomenon has been attributed to the benefits of brood-fostering (Jones et al., 2007; Jones & Riechert, 2008).

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Chapter 4 – Environmental factors affecting distribution of Anelosimus.

Under lower temperatures, brood maturity is delayed, thus increasing the probability of females dying before her progeny reaches adulthood, Jones & Riechert (2008) thus suggest that brood survivability is increased through brood-fostering. Conversely, under a high temperature environment, higher growth and development rates would allow females to produce multiple broods in her lifetime, without need for brood-fostering to improve survivability of progeny. Therefore, in a high temperature environment, the absence of benefit from brood-fostering, together with the cost of resource competition with multiple broods in the natal nest may lead to early natal dispersal and development of solitary behaviour in Anelosimus.

Other factors

Rainfall and temperature accounted for only 10.1% of the variance in distribution of

Anelosimus species. There are other factors that have been highlighted in previous studies that could affect the distribution of social and subsocial species. Ant predation is known to increase cooperative nesting in the allodapine bees Exoneura nigrescens (Zammit et al.,

2008). It is also linked to the failure of transplanted colonies of Anelosimus baeza in lowland rainforests (Purcell & Avilés, 2008). Social Anelosimus are also found to inhabit forest interior while non-social species show a preference for edge habitats (Purcell et al., 2012).

Vegetation structure in forest interior tends to be more stable and sturdier as compared to edge vegetation. Therefore, vegetation type seems to play an important role in distribution of social and non-social species (Purcell et al., 2012).

Current knowledge of the distribution of Anelosimus may be uneven. 57.1% (20 out of 35 species) of the species examined in this study are located in North and South America alone. In addition, all social species are described from South America while 58.3% of

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solitary species are found only in Asia. Meanwhile, subsocial species are found in all

continents surveyed. Moreover, the natural history and sociality of a large number of

described species of Anelosimus remain unknown. This may reflect a difference in

environmental conditions in the lowland rainforests of Asia and South America or an

incomplete sampling of Anelosimus. Nevertheless, the findings of this study gives a first glimpse of the effect of rainfall and temperature on the geographical distribution pattern of solitary Anelosimus.

Conclusions

The results of this study has illustrated the effects of rainfall and temperature on the global distribution of social, subsocial and solitary Anelosimus. Social species are associated with high rainfall and intermediate temperatures; subsocial species are associated with low rainfall and low temperatures; whereas solitary species are associated with high rainfall and high temperatures. The combination of rainfall and temperatures may play a role in the development of social behaviour in this group of spiders. These factors also serves to predict

the occurrence of social, subsocial and solitary spiders in various habitats.

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Chapter 5 – Parasites and sociality of Anelosimus spiders

ABSTRACT

The viability of social groups is frequently assessed by examining the cost and benefits of group living. One of the consequences of group living is parasitism, as a result of increased conspicuity of large groups and intimate interactions between individuals within groups. Spiders of the genus Anelosimus display a wide range of group sizes and sociality, with social species forming groups of up to tens of thousands of individuals, subsocial groups with 20–40 individuals and single individuals in solitary living species. These variations in group size and level of sociality displayed in these spiders have been attributed to factors such as prey availability, climatic variation and predator abundance. Although Anelosimus spiders are parasitized by wasps and moth larva, the impact of parasites on sociality has never been thoroughly investigated. In this study, I investigated the abundance of nest intruders of solitary and subsocial spiders in Southeast and East Asia and determined that nests of subsocial spiders harbour fewer intruders as compared to solitary species.

In addition, the prevalence of the endosymbiotic bacteria, Wolbachia was also assessed in wild populations of Anelosimus. Results indicate presence of Wolbachia in four social, two subsocial and one solitary species and highlight the need to examine the role of endosymbiotic bacteria on the development of sociality in this group of spiders.

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INTRODUCTION

Parasites are known to affect ecosystems in various ways and often play an integral

role in ecological relationships within the habitat (Hatcher et al., 2012). Parasitism affects the health and reproductive success of host organisms (Poulin, 2007), through the modification of host behaviour or physiology (Poulin, 2007; Adamo, 2012).

Rates of parasitism have frequently been associated with group sizes (Côté &

Poulin, 1995; Rifkin et al., 2012). The dynamics of group living allow easy spread of parasites due to higher incidences of contact with individuals and with faeces of conspecifics (Altizer et al., 2003). Therefore, parasitism is hypothesized to be higher in larger group sizes (Côté & Poulin, 1995; Rifkin et al., 2012). Moreover, the conspicuity of large groups may also attract more parasites (Krause & Ruxton, 2002).

In addition, certain social groups possess high rates of inbreeding, allowing infection rates to be higher as genetic similarity amongst group mates allows for easy propagation and adaptation of parasites (Liersch & Schmid-Hempel, 1998; Schmid-

Hempel, 2001). In contrast, the selfish herd and encounter-dilution effects hypothesis asserts that individuals benefit from group living due to lowered chances of infection

(Mooring & Hart, 1992). Furthermore, physiological and behavioural mechanisms such as allogrooming and immunity development in social groups may also be successful in curbing propagation of parasites within groups (Dugatkin, 1997;

Traniello et al., 2002).

Studies supporting both hypotheses abound. For example, ectoparasite loads are not found to be larger in marmots of larger group sizes or densities (Arnold &

Anja, 1993). The social parasitic allodapine bee Macrogalea maizina is also not more successful in larger host colonies and this is suggested to be due to better host defense in bigger colonies (Smith & Schwarz, 2006). In contrast, African bovids are known to

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harbor higher number of parasites at higher group sizes (Ezenwa, 2004).

Kleptoparasite load is also known to increase with growth in colony size in the orb-

weaving spider, Metepeira incrassata (Araneidae) (McCrate & Uetz, 2010). Similarly,

ectoparasite load is found to be greater in Galapagos’ hawk (Buteo galapagoensis)

from larger group sizes as compared to individuals from smaller groups (Whiteman &

Parker, 2004). These seemingly contradictory findings from these studies are attributed to different modes of transmission and specificity of parasites (Ezenwa,

2004).

Due to the close association between parasite load and group size, it is no

surprise that parasite infection has been shown to affect sociality in a number of

animal groups. Parasitism is often regarded as a cost of sociality, and is a contributing

factor in the assessment of the costs and benefits of social group formation (Côté &

Poulin, 1995; Liersch & Schmid-Hempel, 1998; Bilde et al., 2007; McCrate & Utez,

2009; Lutermann et al., 2013). Feeney et al. (2013) report that superb fairy-wrens

(Malurus cyaneus) may brood cooperatively as a form of defense against brood parasites. Other studies have shown that infected individuals that have been rendered

sterile by parasites salvage their reproductive output by exhibiting helping behaviour towards nest-mates (O’Donnell, 1997). Cohesive social activities such as

allogrooming in the social African ground squirrel are similarly adopted to control

ectoparasites infection (Hillegass et al., 2010).

As predators, spiders are at the top of food chains and only preyed on or parasitized by a number of organisms, consisting of flies, fungi, nematodes, wasps, to

birds and some mammals (Pizzi, 2009; Foelix, 2011; Evans, 2013). In addition, the presence of endosymbiotic bacteria such as Cardinium, Rickettsia, Spiroplasma and

Wolbachia has also been established in several spider families (Clark, 2007; Rowley

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et al., 2004; Duron et al., 2008; Martin & Goodacre, 2009; Perlman et al., 2010;

Wang et al., 2010). Spiders are affected by parasites in various ways. For example,

kleptoparasites pilfer food from spiders, reducing prey capture efficiency and

lowering nutritional input obtained (Davis, 2011; Whitehouse, 2011). Nematodes and

wasps consume eggs of spiders or lay eggs on spiders to allow their developing larva to feed directly on them (Foelix, 2011; Vetter et al., 2012). Certain wasps and symbiotic bacteria are also known to manipulate the behaviour of spider host

(Eberhard, 2000a, 2000b; Eberhard, 2001; Goodacre & Martin, 2013). In addition, symbiotic bacteria have also been shown to produce reduced dispersal distance, skewed sex ratio, castration, cytoplasmic incompatibility, parthenogenesis and feminization in spider hosts (Goodacre et al., 2009; Vanthournout et al., 2011;

Goodacre & Martin, 2012; 2013).

Likewise, group living spiders are also affected by the presence of parasites within the colony. Crouch & Lubin (2001) found that mass dispersal of the social spider Stegodyphus mimosarum (Eresidae) might be related to increased rates of parasitism in colonies. A number of observed traits of social spiders, such as inbreeding, skewed sex ratio and reduction in dispersal distance are similar to that observed in arthropods infected with endosymbiotic bacteria (Goodacre et al., 2009;

Vanthournout et al., 2011; Goodacre & Martin, 2012, 2013). The evolution of sociality is closely associated with the cost and benefits of group living (Whitehouse

& Lubin, 2005; Lubin & Bilde, 2007); therefore, it is pertinent to examine the effects of parasitism on sociality in the quest to understand the evolution of sociality in spiders.

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The genus Anelosimus (Araneae: Theridiidae) consists of spiders exhibiting a

spectrum of social behaviour, comprising social, subsocial to solitary living (Avilés,

1997; Agnarsson & Kuntner, 2005; Agnarsson & Zhang, 2006; Agnarsson et al.,

2006; Agnarsson, 2012a, b; Yip & Rayor, 2013). Social Anelosimus species form

colonies ranging up to tens of thousands of individuals, exhibit female biased sex

ratio and an absence of dispersal from the natal nest (Avilés, 1997; Whitehouse &

Lubin, 2005; Lubin & Bilde, 2007). Subsocial species display delayed dispersal from

the natal nest, dispersing just before acquiring sexual maturity (Lubin & Bilde, 2007;

Yip & Rayor, 2013). Lastly, solitary species disperse from natal nests shortly after

egg-sac emergence (Agnarsson et al., 2006a). Social Anelosimus are believed to have

evolved from subsocial ancestors whereas solitary behaviour is gained secondarily

(Agnarsson et al., 2006a; 2006b). The evolution of social behaviour in this genus of

spiders has been associated with prey size and prey abundance (Guevara & Avilés,

2007; Powers & Avilés, 2007; Yip et al., 2008), habitat productivity (Majer et al.,

2013), maternal care (Yip & Rayor, 2013), and alloparental brood care (Marques et al.,

1998; Avilés & Salazar, 1999).

Parasites of Anelosimus include arachnid kleptoparasites (Cangialosi, 1990;

Gonzaga & Vasconcellos-Neto, 2001; Perkins et al., 2007; Viera et al., 2007), moth

larva (Robinson, 1977), and parasitic wasps (Avilés & Tufiño, 1998). Although

parasites have been shown to affect the behaviour and physiology of spiders, their

impact on Anelosimus sociality remains unknown. With the spectrum of social type

presented in the genus Anelosimus, they are thus prime candidates for the examination

of the effects of parasites on the evolution of sociality in spiders. Therefore, in this

study, I aimed to examine the parasite load of Anelosimus species to determine if differences exist amongst the social types by looking at the nest contents of eight

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Chapter 5 – Parasites and sociality of Anelosimus spiders species of Anelosimus. In addition, I also investigated the prevalence of the endosymbiotic bacteria Wolbachia in 37 species of Anelosimus with varying levels of sociality. I predicted a positive relationship between parasite load and sociality.

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MATERIALS AND METHODS

Nest collection and examination

A total of 81 nests of eight species of Anelosimus from 11 sites in East and Southeast

Asia were collected and examined (Table 5.1). All collections were conducted between 0800 h – 1600 h and 2000 h – 2300 h, in the years 2009 – 2011. The majority of spiders were collected from China, Malaysia and Singapore. All nests examined were built by wild populations of spiders. Only solitary and subsocial species were encountered in the geographical range surveyed. A typical nest consists of loosely spun silk enveloping vegetation such as wildflowers and leaves (Agnarsson et al.,

2006a; Figure 5.1). A nest is therefore defined as a combination of silk and encompassing plant material, devoid of other silk connections to other webs.

At each site, the nests of Anelosimus spiders were surveyed and randomly selected for collection. Only the nests of adult spiders were collected. To collect a nest, the resident spider was first removed. Care was taken to acquire the complete nest by cutting off an entire branch segment or flowers. The nest was subsequently placed in 70% ethanol. Nest contents were examined under a Leica MZ125

stereomicroscope, and sorted to the taxonomic rank of order. Body length between the

posterior tip of the head and the end of the abdomen of each specimen was also

measured using a stage micrometer. All measurements are taken to the nearest 0.1

mm. A total of 449 organisms were examined.

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A B

Figure 5.1. Nest of the (A) subsocial Anelosimus kohi (Image by Kuntner, 2011) and the (B) solitary living A. chonganicus.

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Table 5.1. List of Anelosimus species nests examined. Localities, GPS coordinates, elevation, habitat type and number of nests collected for each species are as listed. No. of nests Species Social type Location GPS Coordinates Elevation Habitat type collected Cameron Highlands, 4°28'48.90"N Tropical Moist Anelosimus cameronis Solitary 1466 Peninsular Malaysia 101°23'3.40"E Montane Forest 5 18°42'36.84"N Tropical Moist Hainan, China 50 108°47'14.04"E Lowland Forest 10 Anelosimus chonganicus Solitary 21°57'42.00"N Tropical Moist Ken Ding, Taiwan 225 120°48'43.00"E Lowland Forest 5 Mount Kinabalu National Anelosimus 6° 2'13.20"N Solitary Park, Sabah, East 2100 Tropical Moist kinabaluensis 116°33'0.66"E Malaysia Montane Forest 5 3°21'7.32"N Tropical Moist Mandai, Singapore 0 101°47'13.62"E Lowland Forest 10 Anelosimus parvulus Solitary 1°21'48.94"N Tropical Moist Tampines, Singapore 15 103°56'39.15"E Lowland Forest 10 Ulu Gombak Biodiversity Anelosimus 3°18'31.32"N Solitary Research Centre, 762 Tropical Moist membranaceus 101°44'37.58"E Peninsular Malaysia Lowland Forest 7 24°50'17.30"N Tropical Moist Wu Lai, Taiwan 121°31'41.40"E 219 Lowland Forest 3

Anelosimus Kota Kinabalu, Sabah, 5°58'47.94"N Tropical Moist Solitary 44.8 seximaculatum East Malaysia 116° 4'43.20"E Lowland Forest 6

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Table 5.1 (continued) No. of nests Species Social type Location GPS Coordinates Elevation Habitat type collected

1°26'26.68"N Tropical Mangrove Anelosimus kohi Subsocial Mandai Mangrove, Singapore 0 103°45'55.52"E Forest 15

21°57'42.30"N Tropical Moist Anelosimus taiwanicus Subsocial Ken Ding, Taiwan 225 120°48'43.00"E Lowland Forest 5

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DNA extraction and detection of Wolbachia

Specimens collected in the field were fixed in 95% ethanol. Other DNA extracts were

acquired from the personal collection of Agnarsson I, who utilized similar procedure

outlined as follows to obtain DNA extracts: one whole leg was excised from each

specimen and macerated with a sterile plastic pestle, after 5 s drying in liquid nitrogen.

Qiagen DNeasy Blood and Tissue Kit (Hilden, Germany) was used for the extraction

of genomic DNA, with an incubation period of 48 h at 55ºC in lysis buffer and

Proteinase K.

DNA extracted from 104 individuals (37 species) (Table 5.3) was tested for

Wolbachia using the method of polymerase chain reaction (PCR) amplification.

Primers used for the amplification of the cell surface protein gene wsp are as follows:

WSP-F (5’-TGGTCCAATAAGTGATGAAGAAACTAGCTA-3’) and WSP-R (5’-

AAAAATTAAACGCTACTCCAGCTTCTGCAC-3’) (Jeyaprakash & Hoy, 2000;

Goodacre et al., 2006). Standard PCR amplifications were carried out using PuReTaq

Ready-To-Go PCR Beads (GE Healthcare) with 1 µL of genomic DNA extract, 22 µL of nuclease-free dH2O, 1µL each of 10µM forward and reverse primers. Eppendorf

Mastercycler EP S Thermal Cycler was utilized in PCR amplification. Cycling

conditions began with initial denaturation step at 94oC for 1 min, followed by 35

cycles of 30 s at 94oC, 20 s at 55oC and 30 s at 72oC and a final extension step of 3

min at 72oC. PCR products were verified on 1% agarose/TBE electrophoresis gel.

PCR product purification and Sanger sequencing were outsourced to Beckman

Coulter Genomics and Macrogen, USA. The same primers used for PCR

amplification were used for sequencing. Contigs were assembled from forward and

reverse ABI chromatograms and trimmed of low-quality ends using Chromaseq

Version 1.01 (Maddison & Maddison, 2011).

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Data Analyses

For each location, the nest contents of identical species were combined for data analyses. The total number of each order of organisms and the average number of organisms per nest were calculated. Only whole, intact organisms were counted as this indicated that the spiders did not prey on these organisms. In addition, the longest, shortest and average lengths of non-Anelosimus inhabitants were calculated for each species. As no ectoparasites were directly observed on the bodies of spiders, organisms that are likely detrimental to the spiders were singled out. The class

Arachnida (mites and spiders) and the orders Hymenoptera and Lepidoptera were thus designated as “nest intruders” according to observations in previous studies on

Anelosimus species (Robinson, 1977; Cangialosi, 1990; Avilés & Tufiño, 1998;

Gonzaga & Vasconcellos-Neto, 2001; Perkins et al., 2007; Viera et al., 2007) and average counts of these categories were calculated for each species.

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RESULTS

Nest contents

Mites of the order Acari (Class Arachnida) and seven orders of insects: Collembola,

Diptera, Hemiptera, Hymenoptera, Lepidoptera, Pscoptera and Thysanoptera were

commonly encountered in the nests of both solitary and subsocial Anelosimus in East

and Southeast Asia (Table 5.2). Mites and aphids were the most commonly

encountered in nests. Most hymenopterans encountered were ants while only

lepidopteran larva or pupa were found in nests. Dipteran were only encountered four

times among all the nests surveyed and did not appear to be parasitic on spiders. Non-

Anelosimus spiders (Class Arachnida) were only encountered in the nest contents of A. chonganicus and A. kohi. The average number of non-Anelosimus inhabitants per nest ranges from 0.78 to 7.9. In contrast, most nests of subsocial Anelosimus contained about 20 spiders while nests of solitary Anelosimus contained a single Anelosimus individual. Nests of subsocial species (A. kohi and A. taiwanicus) did not contain more non-Anelosimus individuals as compared to solitary species.

The average length of non-Anelosimus nest inhabitants ranged from 0.28 to

1.47 mm and were smaller than resident spiders, which had average lengths between

1.6 to 3.5 mm (Chapter 1). Nests of A. cameronis contained inhabitants of the highest average length and nests of A. kinabaluensis contained inhabitants of lowest average length. The maximum length of nest inhabitants was found in the nest of A. kohi while that with the minimum length was found in the nest of A. kinabaluensis. The highest difference between maximum and minimum length of inhabitants was found in the nests of A. kohi, while the minimum difference was found in A. kinabaluensis (Figure

5.2).

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The highest density of nest intruders occurred in the nests of A. chonganicus, while the lowest density was found in A. taiwanicus (Figure 5.3).

Wolbachia detection

DNA extracted from all spider specimens used was successful in amplification of

CO1 gene (using LCO1490 and C1N227), indicating the success of DNA extraction.

Verification of the PCR product (~600 bp) from the amplification of the wsp gene indicates the possible presence of Wolbachia in seven species of Anelosimus (Table

5.3). Amongst these seven species, four are social, two are subsocial and the remaining species is solitary living. Infection also appeared to be more prevalent in A. studiosus, with individuals from three localities; Costa Rica, Ecuador and US testing positive. However, due to the small sample size of spiders tested for each species, the lack of wsp gene product in PCR amplification does not necessarily indicate absence of Wolbachia infection in the spider population.

Attempts to acquire the sequences of wsp PCR products failed and sequences obtained were of low quality, hence comparison of the strains of Wolbachia infection in Anelosimus could not be achieved.

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Table 5.2. Total count of non-Anelosimus inhabitants in the nests of 8 species of Anelosimus according to type and average number of individuals per nest. Nest intruders are in bold. No. of Types of non-Anelosimus inquilines Average Species nests no. per surveyed Lepidoptera Diptera Hemiptera Thysanoptera Pscoptera Collembola Arachnida Hymenoptera Others nest

Anelosimus 5 7 1 15 0 4 0 1 2 1 6.2 cameronis

Anelosimus 15 0 2 29 1 2 1 75 3 5 7.9 chonganicus

Anelosimus 5 0 0 0 0 0 0 15 0 0 3.0 kinabaluensis

Anelosimus 15 0 0 29 12 3 0 6 5 5 4.0 kohi

Anelosimus 10 0 0 26 6 3 1 9 16 1 6.2 parvulus

Anelosimus 7 1 1 0 2 0 7 8 0 1 2.9 membranaceus

Anelosimus 9 2 0 0 0 0 0 4 0 1 0.78 seximaculatum

Anelosimus 5 0 0 0 0 4 1 0 0 0 1.0 taiwanicus

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Chapter 5 – Parasites and sociality of Anelosimus spiders

1.60 Maximum length * Minimum length 1.40 Average length 1.20

1.00

0.80

Length/mm 0.60

0.40

0.20

0.00

Figure 5.2. Length of non-Anelosimus individuals found in the nests of 8 species of Anelosimus. Shaded bars represent the maximum length of insects found (* denotes values have been scaled down by factor of 10), black bars represent minimum length and white bars represent average length of individuals. Subsocial species indicated with an asterisk beside species name.

No. of nest intruders per 1

Figure 5.3. Average nest intruders density per nest of 8 species of Anelosimus. Subsocial species indicated with an asterisk beside species name.

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Table 5.3. List of Anelosimus species tested for presence of Wolbachia using amplification of wsp gene. Absence of band in electrophoresis of PCR product indicates absence of Wolbachia. Presence of strong or faint bands indicates presence of Wolbachia and these are in bold. Species Location Sociality PCR product Anelosimus amelie Madagascar Solitary - Anelosimus analyticus USA Subsocial - Anelosimus andasibe Madagascar Subsocial - Anelosimus arizona USA Subsocial - Anelosimus baeza Brazil Subsocial - Anelosimus baeza Costa Rica Subsocial - Anelosimus baeza Ecuador Subsocial - Anelosimus cameronis Malaysia Solitary - Anelosimus chonganicus China Solitary - Anelosimus domingo Ecuador Social - Anelosimus dubiosus Brazil Social Faint band Anelosimus elegans Ecuador Subsocial - Anelosimus ethicus Brazil Solitary - Anelosimus ethicus Uruguay Solitary Faint band Anelosimus exiguus China Solitary - Anelosimus eximius Brazil Social - Anelosimus eximius Ecudaor Social Faint band Anelosimus guacamayos Ecuador Social - Anelosimus jabaquara Brazil Subsocial Faint band Anelosimus jucundus Costa Rica Subsocial - Anelosimus jucundus Ecuador Subsocial - Anelosimus kinabaluensis Malaysia Solitary - Anelosimus kohi Singapore Subsocial - Anelosimus lorenzo Brazil Social - Anelosimus may Madagascar Subsocial - Anelosimus membranaceus Malaysia Solitary - Anelosimus misiones Argentina Unknown - Anelosimus nazariani Madagascar Subsocial - Anelosimus nigrescens Madagascar Solitary - Anelosimus oritoyacu Brazil Social - Anelosimus oritoyacu Ecuador Social Faint band Anelosimus pacificus Costa Rica Solitary - Anelosimus pacificus Ecuador Solitary - Anelosimus parvulus Singapore Solitary - Anelosimus rupununi Costa Rica Social - Anelosimus rupununi Ecuador Social Strong band Anelosimus salut Madagascar Unknown - Anelosimus seximaculatum Malaysia Solitary - Anelosimus subcrassipes China Subsocial -

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Table 5.3 (continued)

Species Location Sociality PCR product Anelosimus studiosus Costa Rica Subsocial Strong band Anelosimus studiosus Ecuador Subsocial Strong band Anelosimus studiosus USA Subsocial Strong band Anelosimus taiwanicus Taiwan Subsocial - Anelosimus tosus Ecuador Subsocial - Anelosimus vierae Uruguay Subsocial - Anelosimus vittatus Slovenia Solitary - Anelosimus vondrona Madagascar Subsocial -

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DISCUSSION

Through the examination of the nest contents of solitary and subsocial Anelosimus

species, I investigated if higher density of nest intruders is associated with increase in sociality. In addition, I also assessed the prevalence of the endosymbiont Wolbachia in wild populations of social, subsocial and solitary Anelosimus species. This was

conducted to determine if larger and more social colonies are more prone to infection

as compared to less social species. Results from this study indicate a negative

association between sociality and density of nest intruders (Figure 5.3). In contrast,

Wolbachia infection is found to be more prevalent in social Anelosimus species as compared to the less social species (Table 5.3).

Sociality and nest intruders

Taking into consideration the identity of non-Anelosimus inhabitants in nests of

Anelosimus, it is evident that not all are parasites of spiders. The most common orders of non-Anelosimus inhabitants in nests examined are the Acari and Hemiptera. The majority of hemipterans encountered were aphids, which are frequently preyed on by spiders (Sunderland et al., 1986; Toft, 1995; Roincé et al., 2012). Conversely, several species of mites are known to be parasites of spiders (Welbourn & Young, 1988;

Mąkol & Felska, 2011; Masan et al., 2012). Other orders such as Collembola, Diptera,

Psocoptera and Thysanopthera are not known to be parasitic and may be preyed on by spiders (Smithers, 1972; Lawrence & Wise, 2000; Agusti et al., 2003). Therefore,

only organisms from the Arachnid class, Acari and Araneae and insect orders

Hymenoptera and Lepidoptera are considered as possible parasites of Anelosimus in nests surveyed and designated as nest intruders.

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Contrary to my hypothesis, the subsocial species (A. kohi and A. taiwanicus) did not have a higher density of nest intruders as compared to the remaining six solitary species. Furthermore, although the web areas of the subsocial species are generally larger than solitary species (Chapter 2), the total count of non-Anelosimus inhabitants were also not higher in subsocial species (Table 5.2). Such observations may be due to two different speculations: (a) association with different plant parts between subsocial and solitary species and (b) web maintenance in subsocial spiders.

Dissimilar preferences in nest location may lead to a difference in nest contents of spiders. Nests of the six solitary species examined here were frequently found constructed on small wildflowers (Chapter 2). In contrast, nests of subsocial species were more frequently found on tree branches and leaves (Chapter 2).

Predatory mites from the genus Iphisesius have been found to favour depositing eggs on leaves near flower pollen patches (Faraji et al., 2001; 2002). In addition, mites have also been shown to supplement their diet through feeding on pollen (Van Rijn &

Tanigoshi, 1999). Similarly, ants, which make up the majority of hymenopterans encountered, feed on nectar from flowers (Blüthgen et al., 2003; 2004). The association of nests of solitary species with wildflowers may thus result in a higher count of mites and ants in these nests, as compared to the nests of subsocial species.

The presence of higher number of ants and mites at the preferred nest locations of solitary species could be a factor driving the early dispersal from natal nests as the small size of the spiderlings makes them more susceptible to attacks from nests intruders (e.g. parasites and predators) (Rayor & Uetz, 1993).

Although social groups may be associated with high parasite load, this observation is not consistent (Hieber & Uetz, 1990; Rifkin et al., 2012) and the findings of this study suggest the contrary. Similarly, negative relationships have also

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been shown in social groups of primates and ungulates (Altizer et al., 2003). This

reduction of parasite infection has been attributed to allogrooming (Altizer et al.,

2003). Web maintenance in subsocial spiders may be akin to allogrooming observed

in primates and ungulates, and could possibly lead to a reduction in nest intruders (e.g.

parasites) in nests. Spiderlings of the subsocial A. studiosus have been shown to participate in web maintenance from 3rd instar onwards (Jones & Parker, 2001).

Furthermore, Cangialosi (1990) also found that kleptoparasites are less effective in

larger colonies of A. eximius. Therefore, lower density of nest intruders in the nests of

subsocial species as compared to solitary species could likely be due to the

contribution of multiple spiderlings in web maintenance.

Prevalence of Wolbachia

Wolbachia infection is widespread amongst arthropods (Jeyaprakash & Hoy, 2000;

Clark, 2007) and transmission takes place through horizontal and vertical transfer

(Kremer & Huigens, 2011; Zug et al., 2012). Vertical transmission occurs through the

egg cytoplasm of an infected mother (Clark, 2007) while horizontal transmission

occurs through contact with infected individuals (Zug et al., 2012). As predators of

small arthropods, Anelosimus spiders are susceptible to horizontal transmission of

Wolbachia when they ingest prey or through infected parasites (Vavre et al., 1999;

Rowley et al., 2004; Goodacre et al., 2006). Unsurprisingly, Goodacre et al. (2006)

and Wang et al. (2010) found 30.6% and 22.6% Wolbachia infection in general

surveys of spiders. Amongst the spiders surveyed, only two theridiid species, Chrysso

octomaculatum Bösenberg & Strand 1906, previously Coleosoma octomaculatum

(Wang et al., 2010) and another unidentified theridiid spider (Goodacre et al., 2006)

tested positive for Wolbachia infection.

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The results of this study represent the first survey of Wolbachia in the theridiid genus Anelosimus and shows that 18.9% of species tested are infected. Intriguingly, amongst the infected, six species are social or subsocial. The social species A. dubiosus, A. eximius, A. oritoyacu and A. rupununi that were tested positive for

Wolbachia showed female biased sex ratio (Avilés, 1986; Marques et al., 1998;

Avilés & Salazar, 1999; Avilés & Purcell, 2011). Although skewed sex ratio in social spiders has been connected with high occurrence of inbreeding (Avilés, 1997; Avilés

& Harwood, 2012), the means through which this bias is achieved remains unknown

(Avilés et al., 2000). Meanwhile, sex ratio bias in two linyphiid species,

Pityohyphantes phrygianus C.L. Koch 1836 and Oedothorax gibbosus Blackwall

1941 have been attributed to Wolbachia infection and successfully reversed through

treatment (Goodacre & Martin, 2013). The prevalence of Wolbachia in social species

and the corresponding sex ratio bias thus calls for a re-evaluation of certain key traits

of social spiders, such as skewed sex ratio and reduced dispersal (Avilés 1997; Lubin

& Bilde, 2007) as these are typical traits observed in organisms infected by

endosymbionts such as Wolbachia (Clark, 2007; Vanthournout et al., 2011).

While female biased sex ratio is similarly observed in three other Anelosimus

species (A. domingo, A. guacamayos and A. lorenzo) (Avilés et al., 2000; 2007; Lubin

& Bilde, 2007) that tested negative for the presence of wsp gene, this does not

conclusively represent an absence of infection in them. As only a small number of

individuals were tested for each species, Wolbachia may turn up in these species if

more individuals are tested.

Besides its presence in social species, Wolbachia is also found in three populations of the subsocial A. studiosus. Anelosimus studiosus demonstrates differences in social behaviour, with the population at higher latitude in Tennessee,

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USA displaying social traits such as multi female colonies and female biased sex ratio

population (Riechert & Jones, 2008). Riechert & Jones (2008) attributed this to the

higher death rate of females at colder climate and the need for brood fostering.

Interestingly, the effects of Wolbachia infection have been found to be temperature dependent (Stouthamer et al., 1999). Stouthamer et al. (1990) found that exposure to

higher temperatures results in the elimination of Wolbachia. Furthermore, negation of

the impact of sex ratio bias of Wolbachia on its host was observed when female wasps

(Trichogramma) were exposed to higher temperatures (Louis et al., 1993). A lower

temperature allowing Wolbachia to mediate its effect on populations of A. studiosus at

higher latitude as compared to higher temperatures neutralizing the effects of

Wolbachia at lower latitude may thus explain the behavioural differences observed in

these two populations. Experimental transplantation of multi-female colonies to warm

or cold sites always resulted in multi-female colonies (Riechert & Jones, 2008), thus

disproving the hypothesis that higher temperatures at lower latitude sites resulted in

the removal of Wolbachia. However, there is no indication of the temperature spiders

are exposed to prior to transplant. Experiments conducted by Louis et al. (1993)

revealed that negation of the effects of Wolbachia only occurred after multiple

offspring generations. Therefore, without information on the temperatures at which

spiders are exposed to prior to transplantation and with observation conducted merely

on a single generation of juveniles, the role of Wolbachia in the social traits displayed

by A. studiosus at higher latitude should still be investigated.

Collectively, the findings of this study suggest that both ectoparasites and

endosymbionts (Wolbachia) exert contrasting forces on the development of sociality

in the genus Anelosimus. Specifically, higher abundance of ectoparasites in solitary

species promotes early dispersal, whereas infection with Wolbachia endosymbionts in

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social species may play a role in the development of female biased sex ratio. As a preliminary study of the prevalence of Wolbachia in Anelosimus, the findings here also confirms a need to take a closer look at the prevalence and effects of Wolbachia and other endosymbionts in spiders of the genus Anelosimus. This is especially so as numerous arthropod species have been found to suffer from multiple infections of endobacteria (Duron et al., 2008). Without a more comprehensive survey, with the inclusion of more individuals from different populations, any attempts to explain the possible role of Wolbachia in the evolution of sociality in Anelosimus remains purely speculative.

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General Discussion

OVERALL DISCUSSION

The new species discovered and information on the natural history of ten species

unveiled from this research highlights the lack of research on Anelosimus within this

region. Moreover, the distinct morphological differences and phylogenetic distance

between the Asian species and Neotropical and Madagascan species highlight the

importance to obtain an in-depth understanding of Asian Anelosimus species.

It has been hypothesized that spiders of the genus Anelosimus have evolved

from a subsocial ancestor (Agnarsson et al., 2010; Agnarsson, 2012a; 2012b).

Agnarsson (2012a) suggested that Anelosimus species in China and Bali might have

evolved from subsocial ancestral species originating from Papua New Guinea.

However, with the addition of more Asian Anelosimus species, the new phylogeny

reveals that colonization of China and parts of Southeast Asia (Malaysia and

Singapore) did not occur solely through the Papuan New Guinea species (Chapter 1).

Solitary species of Anelosimus are retrieved from the “Solitary II” clade, likely

representing a second reversal to solitary living from subsociality within the genus.

Behavioural and reproductive traits observed in solitary species of Anelosimus

in Southeast and East Asia corroborate with the reversal from subsocial to solitary

state hypothesis proposed by Agnarsson (2012a). Although solitary living, these

species (A. cameronis, A. chonganicus, A. exiguus, A. kinabaluensis, A.

membranaceus, A. parvulus and A. seximaculatum) display short dispersal distance.

Spiderlings of these species disperse from the natal nest by 3rd instar under laboratory

conditions. However, in field surveys, webs of these species are often clustered in

patches, suggesting short dispersal distance. With the exception of A. kinabaluensis, where in laboratory observations were not done, all solitary species exhibit maternal

301

General Discussion

care through guarding of egg sacs and provision of prey for spiderlings (3rd instar and

below). Additionally, cooperative prey-capture and food sharing were observed in

spiderlings at 2nd and 3rd instar. Similarly, subsocial species (A. kohi, A. subcrassipes

and A. taiwanicus) also displayed maternal care and cooperative prey-capture and

prey sharing among conspecifics but these persist beyond 3rd instar of spiderlings.

Comparisons between solitary and subsocial species reveal subtle differences between

these two groups of spiders, further suggesting that the evolution of sociality in these spiders involves gradual continuous behavioural change.

Since the ancestral state of Anelosimus is likely subsociality, what could be some factors promoting the reversal to solitary living in species within this region?

Two possible contributing factors are high temperature and high rainfall in Asian lowland tropical rainforests. High ambient temperature has been found to accelerate growth and increase metabolism (Gillooly et al., 2001, 2002; Pruitt et al., 2011). Jones

et al. (2007) suggested that slower growth rate of spiderlings of Anelosimus studiosus

at colder climate results in females rarely living beyond egg sac eclosion and could

promote brood fostering and social behaviour in these spiders. In contrast, at higher

ambient temperature, egg-incubation time is shorter and growth rates of spiderlings

are also accelerated, thus allowing females to guard egg sacs and provide for

spiderlings without reliance on conspecifics. This is further confirmed by the multiple

egg sacs produced by solitary Anelosimus observed in this study (Chapter 2).

Furthermore, accelerated growth also allows spiderlings to gain early independence in

nutrition acquisition, removing dependence on maternal provision and the need to

remain in the natal nest. Early independence of spiderlings of solitary species was

demonstrated in cooperative prey capture by 2nd instar spiderlings and independent

prey capture in 3rd instar spiderlings.

302

General Discussion

Finally, higher ambient temperature also leads to an increase in metabolism

(Gillooly et al., 2001, 2002; Brown et al., 2004) and thus a higher requirement for food resources. Since the average number of prey captured has been found to decrease with rising colony size (Jones & Parker, 2002), early dispersal from the natal nest in solitary species may be induced because of a higher demand for food resources.

Dispersal induced by resource competition is characterized by short dispersal distance

(Powers & Avilés, 2003; Johannesen et al., 2012) and is similarly observed in solitary species of Anelosimus in this study.

Distribution patterns of Anelosimus spiders have been found closely associated with rainfall patterns. The rain damage hypothesis states that webs of subsocial spiders are unable to withstand the mechanical damage brought about by heavy rainfall (Marques et al., 1998; Avilés & Tufiño, 1998; Crouch & Lubin, 2001; Purcell

& Avilés, 2008). In the Neotropical lowland forests, bigger webs of social species are able to counter the effects of mechanical damage by heavy rainfall (Purcell & Avilés,

2008). I propose that webs of solitary species of Anelosimus in this region are similarly able to resist rain damage through shielding using plant structures and the small size of the webs. If solitary species of Anelosimus in Asia evolved from subsocial ancestors from New Guinea where rainfall and temperature are lower

(Malhi & Wright, 2004), construction of smaller webs and early dispersal may be a likely response to the change in abiotic factors in Asia, eventually leading to the evolution of solitary behaviour.

The effect of parasitism on the transition from subsociality to solitary living is unknown. Nests of subsocial species in Asia have been found to harbour fewer nest intruders than solitary species. In addition, unlike in the Neotropics, none of the Asian

Anelosimus species tested are infected with the endosymbiont Wolbachia.

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A question remains pertaining to the absence of social Anelosimus in lowland tropical rainforests in Asia. The prevalence of solitary Anelosimus species in this region is peculiar especially when social congeners are aggregated in similar biome in the Neotropics. In response, I suggest two possible explanations: (a) incomplete field survey and studies; and (b) inherent differences of tropical rainforests in the

Neotropics and in Asia. The discovery of social spiders in the Neotropics is credited to extensive work in the past 15 years by Leticia Avilés (Avilés & Salazar, 1999;

Avilés et al., 2006, 2007; Avilés, 2011). In comparison, surveys for Anelosimus species have only been conducted sporadically in recent years, with emphasis only on taxonomic description (Agnarsson & Zhang, 2006; Zhang et al., 2011; Agnarsson,

2012a). Together with my own field surveys conducted from 2009 to 2011, coverage of these surveys is not extensive and certain habitat niches such as tree canopy were not examined.

On the other hand, the difference in sociality of Anelosimus species between

Asia and the Neotropics may be a reflection of the inherent differences existing between these habitats. While researchers have traditionally favoured highlighting similarities between Amazonian and Asian tropical rainforests, differences do exist between these forests (Malhi & Wright, 2004; Lafrankie, 2005). Corlett & Primack

(2006) pointed out differences in plant and animal communities among tropical rainforests in South America, Africa, Madagascar, Southeast Asia and New Guinea.

Even abiotic factors such as temperature and rainfall patterns vary among tropical rainforests in different continents (Malhi & Wright, 2004). For example, tropical rainforests in Africa and New Guinea are drier than those in Amazon and Southeast

Asia (Malhi & Wright, 2004). Similarly, seasonality in rainfall differs between

Amazonian rainforest and Southeast Asian rainforest (Lafrankie, 2005). The

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General Discussion

dissimilar distribution of Anelosimus species in both regions is definitely not the first reflection of inherent difference among these rainforests. Examination of plants, termites, small mammals and birds communities have also revealed divergence between rainforests in different continents (see review Primack & Corlett, 2005;

Lafrankie, 2005 ).

Future Directions

Although my work has revealed a number of solitary Anelosimus in Southeast and

East Asia and elucidated the natural history and behaviour of solitary species, field survey coverage was narrow and more work remains to be done. Large areas of forests in Asia such as in Cambodia, Japan, Myanmar, Thailand, Vietnam, and the

Philippines have yet to be surveyed. Moreover, tree canopies have not been included in past surveys and are areas that merit closer inspection.

Due to the distribution of known species of Anelosimus in Asia, behavioural observations have been lopsided, with more data collected on solitary species than subsocial species. With more extensive surveys, it is my hope that data collection on more subsocial species can be achieved. This would aid in a more in-depth study of the association between morphological changes in receptors densities and parasitism on the development of sociality in this genus of spiders.

Furthermore, given the extensive spread of suites of endosymbiotic bacteria in spiders (Goodacre et al., 2006; Duron et al., 2008; Martin & Goodacre, 2009; Wang et al., 2010), my work testing for the prevalence of Wolbachia in Anelosimus can be further expanded to include other endosymbionts such as Cardinum, Rickettsia and

Spiroplasma. In addition, although I have determined the presence of Wolbachia in four social and two subsocial species of Anelosimus, the effects of infection on spider

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General Discussion

behaviour and reproductive strategies remain unknown. Deliberate infection or

eradication of infection in Anelosimus and examination of its effect on the

reproduction and social behaviour of spiders should shed light on its influence on

evolution of sociality.

Despite having determined that temperature and rainfall have an effect on the

distribution of Anelosimus and evolution of sociality, some of the mechanisms

through which this is mediated remain unconfirmed. Future work should examine the

effects of long-term high/low temperature exposure on the metabolism and behaviour

of subsocial spiders. In addition, the mechanical damage induced by rainfall on

solitary nest of Anelosimus should also be investigated to confirm its ability to

withstand rain damage. Together, these would shed light on the evolution of solitary

and social species of Anelosimus from its subsocial ancestor.

Finally, comparative work can be done in another genus of spiders,

Stegodyphus (Eresidae) that similarly contain a mixture of social and solitary spiders distributed in Africa and Asia (Crouch & Lubin, 2001; Johannesen et al., 2007).

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General Discussion

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APPENDIX

Appendix

APPENDIX

Table 1. Colony structures of Anelosimus species encountered during field collection.

Nest Anelosimus Anelosimnus Anelosimus Anelosimus Anelosimus Anelosimus inhabitants cameroneanis chonganicus exiguus parvulus membranaceus seximaculatum Single individual 3 50 2 30 10 10 With mature male 0 5 0 3 0 0 With eggsac 2 20 1 14 3 3 With 2nd instar spiderlings 0 5 1 3 3 2 Mature female With 3rd instar or above spiderlings 0 0 0 0 0 0 With mixture of spiderlings of different instar 0 0 0 0 0 0 With mixture of spiderlings and adults 0 0 0 0 0 0

Single individual 2 20 2 10 5 4 Mature male With mature female 0 5 0 3 0 0 With spiderlings 0 0 0 0 0 0

2nd instar 0 3 0 5 3 0 Spiderlings 3rd instar and above 4 40 0 15 10 3

Groups with 2nd instar spiderlings 0 0 0 0 0 0 absence of adults 3rd instar and above spiderlings 0 0 0 0 0 0

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Table 1 (continued)

Anelosimus Anelosimus Anelosimus Anelosimus Nest inhabitants kinabaluensis subcrassipes kohi taiwanicus Single individual 5 4 15 3 With mature male 0 0 3 0 With eggsac 2 0 5 1 Mature female With 2nd instar spiderlings 0 0 2 0 With 3rd instar or above spiderlings 1 1 3 1

With mixture of spiderlings of different instar 0 0 4 0 With mixture of spiderlings and adults 0 0 2 0

Single individual 2 0 2 1 Mature male With mature female 0 0 3 0 With spiderlings 0 0 1 0

2nd instar 2 0 0 0 Spiderlings 3rd instar and above 3 5 19 2

Groups with absence 2nd instar spiderlings 0 0 0 0 of adults 3rd instar and above spiderlings 0 1 7 0

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Table 2. Principal component scores and loadings on environmental variation in distribution range of Anelosimus. Environmental variable PCA1 PCA2 PCA3 % Total variance explained 44.79% 21.76% 14.17% Annual mean temperature 0.840 0.482 -0.211 Mean Diurnal temperature Range -0.355 -0.141 -0.424 Isothermality (*100) 0.372 -0.655 -0.625 Temperature seasonality (S.D. *100) -0.452 0.618 0.628 Max Temperature of Warmest Month 0.521 0.810 - Min Temperature of Coldest Month 0.908 0.108 -0.352 Annual Temperature Range -0.607 0.555 0.423 Mean Temperature of Wettest Quarter 0.697 0.657 - Mean Temperature of Driest Quarter 0.826 0.288 -0.375 Mean Temperature of Warmest Quarter 0.583 0.785 0.11 Mean Temperature of Coldest Quarter 0.879 0.124 -0.447 Annual Precipitation 0.826 -0.293 0.373 Precipitation of Wettest Month 0.779 - 0.303 Precipitation of Driest Month 0.647 -0.453 0.411 Precipitation seasonality (Coefficient of Variation) -0.165 0.564 -0.278 Precipitation of Wettest Quarter 0.781 -0.117 0.308 Precipitation of Driest Quarter 0.656 -0.472 0.416 Precipitation of Warmest Quarter 0.450 -0.151 0.451 Precipitation of Coldest Quarter 0.784 -0.270 0.275

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