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Diversity and trap-nesting studies of Singaporean Megachile bees to inform monitoring and management of tropical pollinators Soh Jia Yu, Eunice A thesis submitted to the Department of Biological Sciences National University of Singapore in partial fulfilment for the Degree of Bachelor of Science with Honours in Life Sciences

Cohort AY2013/2014 S2

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Acknowledgements

I express gratitude to the following people, without whom this project would not have been possible: 

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Asst. Prof. John S. Ascher for being there from the start to the end: inception of project idea, fieldwork, advice on bee taxonomy, specimen curation to report writing, Ms. Samantha Lai for approval of permit [NParks’ Permit number: NP/RP14-025] and park managers of Dairy Farm Nature Park, HortPark, Pasir Ris Park, Sungei Buloh Wetland Reserve, Pulau Ubin, Lower Peirce Reservoir Park, Upper Seletar Reservoir Park, Bishan Park for allowing me to sample in their parks, Ms. Lua H. K., curator in LKCNHM, for allowing us to visit the museum to examine specimens, Mr. Zestin W. W. Soh for sharing his reference collection and Grammatophyllum speciosum Megachile, and advice on finding Megachile, Mr. John X. Q. Lee for sharing the discovery of Megachile atrata, and his specimens, and company during fieldwork, Mr. C. Barthléméy for advice on trap-nesting, Prof. Meier (Evolutionary Biology Laboratory) for the usage of laboratory facilities to barcode the bees and Mr. Wong Wing Hing for advising and guiding the molecular work, Mr. Foo Maosheng for allowing me to ride on the lab’s permit for trap-nesting, and Ms. Jayanthi Puniamoorthy for help on logistics and teaching me how to use the VDI, Assoc. Prof. Hugh L. W. Tan, Dr. Chong Kwek Yan and Mr. Alex Yee from the Botany Laboratory for plant identifications, advice and help for site selection, Mr. Justin Tan for generously sharing specimens, Dr. Daniel J. J. Ng and Dr. Hwang Wei Song for advice on science, Dr. Cheong Loong Fah for allowing me join his collecting field trips, Asst. Prof. Darren C. J. Yeo for encouraging me to pursue ant and insect work, Friends from NUS EVB Life Science – Xin Rui, David Tan, Wei Wei, Kang Rui, Zi Yun, Huiqing, Mingkai, Dickson, Joan, Kar Mun, Ming Li, Boon Hong, Kenny and Darren Yeo for encouragement, help and advice, Fellow human bee-ings from the same lab – Jonathan, Shao Xiong, Kia Yi, Si Hao and Cheryl for running alongside a similar journey, Family and friends (Siân, Fion, Eugene and Auntie Zenda) for their kind understanding.

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Table of Contents

Acknowledgements ......................................................................................................................... i Table of Contents........................................................................................................................... ii List of Appendices...................................................................................................................iii Word Count .............................................................................................................................iii Abstract ......................................................................................................................................... iv 1. Introduction ................................................................................................................................1 2. Materials and Methods ..............................................................................................................6 2.1 Documenting diversity, spatiotemporal distribution and floral resources of Megachile ....... 6 2.2 Imaging Megachile species .................................................................................................... 9 2.3 DNA barcoding of Megachile species.................................................................................... 9 2.4 Recording leaf resources for nest lining ............................................................................... 11 2.5 Trap-nesting using bamboo .................................................................................................. 11 2.6 Documenting nests of Megachile ......................................................................................... 16 3. Results and observations..........................................................................................................17 3.1 Megachile of Singapore........................................................................................................ 17 3.1.1 Species diversity ............................................................................................................ 17 3.1.2 DNA barcoding ............................................................................................................. 20 3.1.3 Spatiotemporal distribution ........................................................................................... 22 3.1.4 Floral and leaf resources................................................................................................ 25 3.2 Trap-nesting in Singapore .................................................................................................... 29 3.2.1 Species diversity of trap-nested arthropods................................................................... 29 3.2.2 Ants occupying trap-nests ............................................................................................. 33 3.2.3 Tanglefoot did not deter ants ......................................................................................... 35 3.2.4 Preferred microhabitats and hole diameters .................................................................. 36 4. Discussion ..................................................................................................................................38 4.1 Singaporean Megachile – a synoptic representation of divergent lineages .......................... 38 4.2 Utility of diagnostic and identification tools ........................................................................ 39 4.3 Plant resources of Megachile ............................................................................................... 42 4.4 Potential drivers of seasonal Megachile flight activity ........................................................ 45 4.5 Trap-nesting in Singapore .................................................................................................... 47 4.6 Implications for monitoring and management ..................................................................... 55 References .....................................................................................................................................59

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List of Appendices     



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Appendix I – a review of published trap-nesting studies for bees and wasps Appendix II – pan trap trial Appendix III – sampling sites and habitat association of Megachile species Appendix IV – detailed procedures for DNA extraction and sequencing for cox1, sequence lengths and supplementary distance analyses Appendix V – behavioural observations of Megachile foraging from nest  In-situ observations of Megachile (Callomegachile) disjuncta  In-situ observations of Megachile (Creightonella) atrata  Comparison of foraging behaviour Appendix VI – descriptive nest bionomics of Megachilidae  Nest bionomics of Megachile (Aethomegachile) laticeps  Nest bionomics of Megachile (Creightonella) atrata  Nest bionomics of Anthidiellum (Pycnanthidium) smithii  Nest bionomics of Heriades (Michenerella) sp. 1  Comparison of nest bionomics Appendix VII – short notes on nesting sites of other Megachile  Nest site of Megachile disjuncta and Megachile fulvipennis  Nest site of Megachile umbripennis Appendix VIII – plates of megachilid habitus and genitalia Appendix IX – plates of representative trap-nests and a summary of nest contents

Word Count    

Introduction: 1340 Methods: 2459 Results and observations: 2750 Discussion: 5447 Total: 11 996

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Abstract

Megachilids are managed as pollinators in temperate agro-ecosystems but lack of knowledge of their diversity, identification and life history impeded similar applications in the tropics. Twentyone Megachile species belonging to seven subgenera were recorded in Singapore. Four subgenera are new records to Singapore. To facilitate identification of Megachile, diagnostic resources were improved through DNA barcoding and high resolution imaging. Megachile were seasonally abundant in August 2014. Megachile were recorded to collect pollen from five plant families attributed to three pollination syndromes. Leaf-cutter Megachile use leaves from plants of 17 families in nest construction. Bamboo trap-nests (1261 internodes) were deployed at nine sites with 4.83% occupancy by 12 solitary bees and wasps, including three megachilids (Megachile laticeps, Anthidiellum smithii, Heriades (Michenerella) sp. 1). They were more likely to occupy trap nests placed in open areas at low heights. Ants affected success of trap-nesting bees and wasps, occupying 30.13% of the internodes, largely in urban sites. Cavity diameter is correlated to size of megachilid occupant. A new host-kleptoparasitic association was made on rearing Coelioxys confusa from trap-nests of M. laticeps. Megachile atrata nests were discovered at Thalassina mounds in back mangroves, a new association with Crustacea. Anthidiellum is first recorded for Singapore. Keywords: megachilidae, flowers, barcoding, taxonomy, ants, Anthidiellum, Megachile laticeps, new records

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1. Introduction Bees depend on flowers for pollen and nectar resources, and are the most important pollinators of the angiosperms [1], suggesting that bees have coevolved with their floral hosts [2], sometimes exhibiting one-to-one specialisations [3]. They are key vectors in ensuring genetic diversity of flowering plants in many ecosystems [4], including tropical rainforests [5] and agricultural systems [6]. Most bee research focuses on eusocial apines (i.e., Apis and Meliponini) [7] as they are generalist flower visitors and have economic value as providers of honey and pollinators of crops; far fewer studies are conducted on solitary bees. However, awareness of the need to maintain and promote the wild solitary bee diversity is growing, corollary to recent scientific reports that show the importance of solitary bees: specialist solitary bees can pollinate plants more efficiently [8]. In turn, this decreases complete dependence on the honey bee Apis for pollination [9]. This study focused on the solitary leaf-cutter and resin bees Megachile sensu lato. This hyperdiverse and cosmopolitan genus of bees includes 1550 described species [10]. The genus was traditionally split into three genera based on mandibular morphologies correlated with nesting lining material [1,11]: typical leaf-cutters Megachile, atypical leaf-cutters Creightonella, and resin-collectors Chalicodoma sensu Michener. These were eventually combined into a single genus because species possessing morphological characters of both Megachile and Chalicodoma were discovered [1]. Furthermore, the three lineages are not independently monophylectic but more recently, these have been proposed to be split once again into four genera based on morphological phylogenetic analyses: Chalicodoma, Megachile, Thaumatosoma and Matangapis sensu Gonzalez [11]. Despite varied nesting

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lining materials used by Megachile, many species make their nest in pre-existing cavities for nesting and can do so gregariously, increasing their potential to be managed pollinators [13]. The pollination efficacy of Megachile as floral specialists, with their readiness to utilize man-made substrates for nesting confers high utility in management [14]. This is carried out in North America, where the alfalfa leaf-cutter bee Megachile rotundata, was accidentally introduced from Europe and is now managed at large commercial scales for the pollination of alfafa Medicago sativa (Fabaceae), widely-cultivated due to its importance as livestock feed. These bees pollinate M. sativa much more efficiently than honey bees do as they are able to thrust the lateral petals and ventral keel of the papilionaceous flowers downwards so as to expose the stamens [15-17]. Similar flowers exist in the tropics but as yet Megachile bees are not widely managed there. In Singapore, Megachile bees could be monitored and potentially managed, to augment genetic diversity of fragmented forest habitats by ensuring pollination transfer and connectivity between them, and the pollination of urban edible or endangered plants. Three main overlapping themes were central to understanding the monitoring and management of Megachile in this study: 1) investigating their species diversity and spatiotemporal distribution in Singapore, 2) documenting their nest resource requirements, 3) deploying trap-nests and improving protocols for these as a first step towards the management of this genus. Managing Megachile as pollinators remains a black box until a rigorous inventory is made and sound taxonomy established. Conversely, the Indo-Malayan bee fauna has been described as “at present one of the least understood” [18]. Several taxonomists worked on bees in the region between 1775 to 1927 including J. C. Fabricius in the late 18th century, C.

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T. Bingham, P. Cameron, W. H. Ashmead, H. Friese and T. D. A. Cockerell in the late 19th and early 20th century. The latter two workers were the most prolific describers of bees of all time and each described vast numbers of Megachile from many regions [19,20]. Even so, species are not readily identified due to brief species descriptions (e.g., for M. alticola in [18]), ambiguities in species delimitation (e.g., unresolved synonymy in M. umbripennis and M. lerma in [21]) as well as the practical issues of scattered type specimens. In Singapore, the first inventory of Hymenoptera and other insects was made by A. R. Wallace, and his bees were studied by Frederick Smith of the British Museum [22]. Cockerell later described Megachile species from Singapore (i.e., M. ramera [23]) and reported at least one previously known species (M. subrixator) [24]. After a long gap in research, three modern studies [25-27] focused on the ecology of bees in Singapore, of which an inventory of floral relationships of bees in the parks of Singapore was the first to report a substantial number of megachilid bees [27] but a comprehensive species inventory had yet to be consolidated. Thus, considerable species diversity reported herein for Megachile [28] which demonstrates the potential for diverse Megachile species to be managed. In lieu of a species inventory, diagnostic tools are improved; these include cox1 barcodes allow for the matching or confirmation of sex associations between the male and females of the same species as they are dimorphic [29] and high resolution imagery which allow for dissemination on the world wide web. With advances in diagnostic tools and the restudy of type specimens, such impediments can slowly be overcome through the enhanced dissemination of knowledge [30] and integrative taxonomy [31]. Kremen [8] suggests that maintaining wild pollination populations of wild pollinators first requires knowledge of their resource requirements, which then allows management of landscapes for their specific food, nesting and breeding resources. Thus, this study also

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investigates resources utilized in nest construction and provisioning which can help illuminate management strategies for resources in proximity to nesting sites [32]. These include specific type of pollen, nest lining materials and nesting substrates used [33]. Such information is derived from life history studies of, e.g., plant resources and nest bionomics. Although many Megachile nest in pre-formed cavities, some nest the ground [34], and it is essential to have basic comparative information about their behaviour. Trap-nesting [35] is effective for monitoring cavity-nesting bees and wasps [36] and obtaining information on ecological associates and life history [37]. Trap-nests that are implemented long-term and in high densities provide nesting sites for a variety of bees which has the potential to increase pollinator diversity and abundance [38]. Trap-nests are also portable, allowing for translocation of bees. However, trap-nests are employed mostly in temperate regions [36] (mainly in N. America and Europe), and less so in the subtropics (except Brazil and Hong Kong) and tropics [39-41]. Thus, I test the effectiveness of bamboo internodes at different microhabitat and trap placements, and attempt to rear the occupants for identification. In addition, I hypothesize that since temperatures do not fluctuate so much in the tropics, the need for an east-facing trap-nest opening is not essential as opposed to results in a temperate study [42]. Further, by following protocols for bamboo trap-nests implemented successfully in subtropical Hong Kong [37], direct comparisons can be made between Asian cities. The overall goals of this study are to summarize up-to-date biodiversity information of Megachile leaf-cutter and resin bees and test the efficacy of trap-nesting, to inform their management and monitoring. To achieve this aim, I have done the following:

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1) Compile, validate, and summarize records of Megachile species for Singapore based on specimens available in local museum collections and fieldwork (see Appendix III). The resulting database, hosted on Google Fusion Tables, is used to produce distributional maps, determine flight activity and estimate species richness. 2) Improve diagnostic tools by a) creating a high-resolution image library including habitus photographs of Megachile species with selected diagnostic characters (e.g., female mandibles and male genitalia) to facilitate identification and enable taxonomic efforts such as the construction of online species pages and dynamic identification keys (Appendix IX), and b) generating a diagnostic mitochondrial cytochrome oxidase 1 (cox1) DNA barcode library for Megachile species to confirm sex associations based on morphology and to facilitate future ecological studies (e.g., of immature stages). 3) Investigate and summarize what is known about the plant resources for Megachile, including species specificities in floral resources and leaf resources to understand their required nesting resources. 4) Test efficacy of a bamboo trap-nest set-up and explore microhabitat variables which may encourage the occupancy of bees and wasps.

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2. Materials and Methods 2.1 Documenting diversity, spatiotemporal distribution and floral resources of Megachile Reference collections. Singaporean Megachile bees were examined from several collections (Table 1). Species identifications were made or verified by John S. Ascher (National University of Singapore (NUS)), who searched for and studied megachilids in the Smithsonian Institution and the American Museum of Natural History to facilitate bee identifications. Stephan Risch of Germany was consulted regarding recognition of difficult species and their subgeneric positions. Zestin W. W. Soh, a student at Imperial College London, examined and imaged type specimens of megachilids in the Natural History Museum, London. Label information such as date of collection, collector(s), locality and notes (e.g., floral associates) were recorded. Table 1. Collections examined during this study and their abbreviated names. Abbr. MIP ZRC ZWWS

Full collection name Mangrove Insect Project (Evolutionary Biology Laboratory, NUS) Lee Kong Chian Natural History Museum Zoological Reference Collection Zestin W. W. Soh 2012 flower visitor survey reference collection

Choice of sampling methods. Several methods have been used by melittologists to sample bees in various habitats, namely malaise traps that intercept bees in flight, pan traps and sugar baiting which attract bees, trap-nesting and netting at flowers [36]. Prior studies using sugar baiting as a method to sample bees did not attract megachilids [25]. Malaise traps yield few megachilids [43]. A preliminary trial with pan traps yielded no bees (see Appendix IV). I therefore chose netting at flowers as the optimal method to sample megachilids in addition to trap-nesting.

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Netting at flowers. A significant event prior to the start of the study was the drought of 61 days from 13 January – 16 March 2014, reportedly the longest since 1869 in Singapore [44]. Few bees were observed to be foraging during that period, except Ceratina and Nomia (pers. obs.); many plants were observed to be wilting and were not flowering. Thus, sampling efforts with netting for the present study focused on May–August 2014 after conditions improved. Sampling was done during the day, both in mornings and afternoons. Megachile are known to prefer hot and are repelled by cold and rainy days (J. S. Ascher, pers. comm.). Sampling localities. Sampling was conducted across the island of Singapore, which has a tropical climate. Numbered 1-km were overlaid on the map of Singapore and were assigned a habitat type as defined by and based on Yee et al. [45] with QGIS 2.2.0-Valmiera [46]. To ensure that all habitat types were represented, at least one grid of a habitat type was sampled (see Appendix III for sampled grids). Sampling events. A non-random area with flower patches within the 1-km grid was sampled as bees aggregate at patches with high floral density [47]. For each sampling event, the grid number, time spent in grid (rounded off to the nearest half hour), survey start time and GPS coordinates were recorded. Floral associates, i.e. the flowering plants attracted Megachile (for pollen which is carried on its ventral scopa and/or nectar, or no reward), were also noted. Only plants up to two metres tall, such as shrubs, creepers, climbers, low epiphytes and treelets or trees with low hanging flowers were surveyed. Z. W. W. Soh contributed survey data for Megachile at Grammatophyllum speciosum in August 2014. The resulting compilation of floral associates is qualitative for each species. Specimen vouchers. Specimens collected were pinned and placed in the Insect Diversity Laboratory (IDL) reference collection (National University of Singapore) for study.

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Vouchers will be permanently deposited in the ZRC. Mites living on the bees, found mainly on the declivity of the propodeum (M. laticeps, M. disjuncta, M. umbripennis, M. atrata, M. tricincta, M. stulta) were preserved in molecular-grade absolute ethanol (Merck Chemicals, Singapore) for taxonomic and phylogenetic study by Pavel Klimov [48]. Species richness estimation. Individual-based rarefaction and estimator curves (Chao1) were computed with by EstimateS v. 9 [49] by pooling specimens collected from field work along with MIP, ZRC and ZWWS reference collections. Only the bias-corrected non-parametric Chao1 estimator (not ACE) was computed since all rare species were singletons (i.e., there were no doubletons) [49]. The estimated species richness is unlikely to be biased by inclusion of museum specimens as can occur [50] because the ZRC specimens were a relatively small fraction of specimens used in the estimation (48 specimens of 481 specimens), and included only one singleton [50]. Flight activity. The wing wear of specimens collected during fieldwork in May to August 2014 was examined and scored based on the extent of tatter on the left forewing, using a standardised scale of one to six; a wing wear of 0–2 is a proxy for emergence of less than six days [51]. Abundance of Megachile were normalised by hour for each sampling event (i.e., catch effort per hour) for all bees and a subset for bees with wing wear from 0–2. These were compared alongside the flower-visitor survey of parks in 2012 (Z. W. W. Soh, unpublished). Measuring specimens. The body length and intertegular distance were measured with a pair of vernier calipers for the sex of each species. Data management and mapping. Species records from reference collections and field surveys were assigned a latitude and longitude. To consolidate all data, these were keyed into

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an Excel spreadsheet with the following columns (see Table 1). These were ported onto Google Fusion Tables geodatabase for visualization of distribution of Megachile species. Table 2. The various attributes and their corresponding purpose in the database. Domains of the attributes are in brackets and additional description of the attributes are in square brackets both contiguous with the attribute name. Purpose Systematic information

Columns names with domains based on Structured Query Language (SQL) terminology species(varchar), family(varchar), genus(varchar), “bee”(varchar) [= whether it is a bee, wasp, or other taxa for quick sorting purposes]

Identification information

IDlastCheckedOn(date) [= date of accurate determination], IDlastCheckedBy(varchar) [= subsequent determination or verification – may or may not have a label], detBy(varchar) [= first accurate determination]

Data entry

dataKeyedInBy(varchar), dataKeyedInOn(date)

Spatial information

x(number), y(number), scale [= accuracy of scale] (numerical)

Collection information

day(number), month(char), year(number), time(varchar), country(varchar), gender(char), coll(varchar) [= repository name], RefNo(varchar) [= unique identifier numbers]

*in decimal degrees (based on WGS84)

Supplementary notes(varchar) [= notes on the labels, including floral associates and information comments in square brackets], wingwear1-6(number) [= wing wear based on classification by Mueller and Mueller [51]], photograph(varchar) [= whether the specimen is fit for photography], text(varchar) [= concatenated text formatted to summarize collection information] 2.2 Imaging Megachile species Pinned specimens (with mandibles opened, and genitalia and mouthparts exposed where possible) were imaged for each Megachile species (Visionary Digital Imaging Systems) in at least three views (head, plan and profile). 2.3 DNA barcoding of Megachile species For DNA barcoding, the standard mitochondrial cytochrome c oxidase 1 (cox1) gene was to 1) generate a diagnostic database of Megachile species for future work (e.g., larvae-

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adult matching), 2) and to make or confirm sex associations for these sexually dimorphic bees. Two questions were asked: 1) can the species be grouped perfectly within a) a local samples and b) based on a global database based on uncorrected distance thresholds and 2) what distance cluster threshold is optimal for species delimitation? The right mid-leg of a bee was removed and stored in molecular-grade absolute ethanol (Merck Chemicals, Singapore) at -20ºC for subsequent DNA extraction [52]. Legs were from specimens frozen for one day or when the specimen was alive but inactivated by cooling. They were mechanically crushed with forceps to expose muscle tissue. Cox1 barcodes were obtained via phenol-chloroform extraction of genomic DNA, polymerase chain reaction (PCR) amplification of cox1, chain termination and sequencing (details in Appendix V). Overall, protocol used from PCR to sequencing were similar to Lim [53]. For PCR, the primers pairs used were LCOHym forward and Nancy reverse [54], or Lep F1 and Lep R1 reverse [55] when the former did not work (Table V-1). Cycling temperatures were 95 ºC to activate Taq polymerase, and 34 cycles of 95 ºC for 45 seconds (denaturation), 50 ºC for 30 seconds (annealing) and 72 ºC for one minute (extension). Forward and reverse sequences were edited by visually comparing chromatogram signals of nucleotides and made into contigs in Sequencher® [56]. Cox1 sequences for Singaporean species previously sequenced and available through on BOLD, i.e., for M. laticeps and M. umbripennis, were downloaded [57], as were sequences for selected other Megachile cox1 sequences [38,58-62] searched on GenBank with the keywords ‘COI’ and ‘Megachile’. These were downloaded with the package ‘ape’ [63] via GenBank accession numbers in R v.3.1.0 [64]. These were included in the analyses to test the generality of

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intraspecific and interspecific distances. Alignment of sequences was done in MAFFT v.7 (default settings) [65]. The data was well aligned with no indels. MEGA v.6.06 [66] was used to check for indels and to summarize the number of polymorphic sites at the first, second and third codons, and the corresponding amino acids. TaxonDNA v.1.6.2 [67] was used to compare pairwise uncorrected distances between barcodes of individuals [68] and cluster species at distance thresholds of a range from 0.5 to 12% to identify a ‘best close match’ threshold. 2.4 Recording leaf resources for nest lining During field surveys (both trap-nesting and netting at floral associates), plants were also surveyed for cut leaves. Such cuts were characterized by an oblong shape (for cell lining) or a smaller, circular shape (for cell cap or nest closure), characteristic of leaf-cutter bee damages [69]. Only plants with more than three cuts on a single branch of leaves were considered to be a resource. To prevent double-counting, each site was only assessed for leaf damage once. Diagnostic photographs of damage by Megachile leaf-cutter bees (in Megachile (Group 1 or Group 3) were taken. Plants were identified to the lowest taxonomic level possible by a botanist (Assoc. Prof. Hugh Tan, NUS) and with a local plant guidebook [70]. These were compiled by frequency of occurrences by plant species. 2.5 Trap-nesting using bamboo Acquisition of dried bamboo. Non-pithy bamboo was cut from bamboo groves in Singapore, either dried or fresh, though the former was preferred. Fresh bamboo was air-dried for at least three weeks in an open but sheltered area. Dried bamboo was cut with secateurs or tree saws at internodes to lengths of 12–15 cm, such that one end was sealed by a node and the other was open. Bamboo internodes have internal entrance diameter ranged from 1 to 17

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mm with a mean of 6.16 mm, and were sorted into two size classes based on internal entrance diameter: small (1–10 mm) and large (11–20 mm). Trap-nest set-up. Bamboo internodes were tied together with raffia string; approximately 15 internodes were tied together for the small size class, and eight for the large size class. This ensured that the total diameter of the trap-nests were similar and equally visible, and was not a potential confounding variable. A bundle of bamboo pieces is henceforth referred to as ‘trap-nest’, whereas an individual bamboo piece is referred to as an ‘internode’. Sampling duration and localities. Nine study sites were chosen (Fig. 1, Table 3) on the island of Singapore. Five sites were located in gardens: behind University Hall near National University of Singapore (UH), Butterfly Garden and Kampong Daze at HortPark (HP), Tampines EcoGreen (TE), Kitchen Garden at Pasir Ris Park (PR) and Butterfly Hill at Pulau Ubin (PU), and these were considered urban as they were managed by humans. The mangrove site was at Sungei Buloh Wetland Reserve (SB). Three forest sites include a mature secondary forest edge near Wallace Education Centre at Dairy Farm Nature Reserve (DF), Nee Soon Swamp Forest near pipeline (NS) and primary lowland dipterocarp forest at Bukit Timah Nature Reserve, Fern Valley (BT). These sites were chosen on the basis that Megachile were previously recorded or seen (Z. W. W. Soh and J. X. Q. Lee, pers. comm), that known floral associates of Megachile were present (Chong, K. Y., pers. comm.) or cut leaves were observed (pers. obs.). The trap-nesting study was carried out in two phases, from 23 March to 10 May 2014 (seven sites) and 2 July to 29 August 2014 (two sites) (Table 3).

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Fig. 1. Sites where trap-nesting was conducted. Black circles (●) are sites sampled from March to May 2014. Grey squares ( ) the sites sampled from July to August 2014.

Table 3. Abbreviated names and coordinates of the nine trap-nesting sites. Site Dairy Farm Nature Reserve near Wallace Education Learning Lab Butterfly Garden and Kampong Daze, HortPark* Sungei Buloh Wetland Reserve near the mangrove arboretum* south of Tampines Eco Green* Kitchen Garden, Pasir Ris Park* Butterfly Hill, Pulau Ubin* behind University Hall, National University of Singapore* Fern Valley, Bukit Timah Nature Reserve^ Nee Soon Swamp Forest near the pipeline^ *surveyed from end March to May 2014 ^ surveyed from July to August 2014

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Abbr.

Latitude (dd)

Longitude (dd)

DF HP SB TE PR PU UH BT NS

1.406194444 1.279425 1.447294444 1.364525 1.379830556 1.402355556 1.297175 1.351129 1.37863

103.7744861 103.7983083 103.7298194 103.9475833 103.9517361 103.9663917 103.7767528 103.773848 103.805541

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Sampling design for the sites. Within each site, the 17–18 bamboo trap-nests were randomly tied to trees, except at BT and NS where 13 and 12 trap-nests respectively. Each site had an approximately equal number of small and large size class trap-nests. A trap-nest is tied with raffia string 10–178 cm above ground in an oblique manner such

Fig. 2. Trap-nest unit tied onto a tree.

that rain water would not accumulate (Fig. 2). A subset of trap-nests were chosen at random for the application of Tree Tanglefoot (USA), a mechanical barrier of ants, on the area preceding the trap-nest (in three urban sites TE, UH, PU). A few layers of cling wrap was placed on the structure before the application of a thick layer of Tanglefoot. Trap-nests were monitored three times, at intervals of three to four weeks. On the first monitoring session (the session after the trap-nest was put up), the angle – assessed with a compass, canopy cover – assessed with a densiometer (Forestry Suppliers Inc., Jackson, Mississippi) [71] and height of the trap-nest were measured. Nests utilized by bees or wasps which are recognized by plugs at the opening of the internode were harvested for rearing. Those occupied by ants were enumerated by ant morphospecies or species, and internode diameter. Few other insects were seen inside the internodes (only three Perisphaerus (Blaberidae) at BT) and were not considered in this study. Testing for microhabitat and trap placement. The first aim of trap-nesting was to determine if direction, canopy cover and height affected the number of internodes occupied by solitary bees or wasps (pooled from all the monitoring sessions), as a proxy for the tendency to nest. Generalised Linear Mixed-effects Models (GLMM) with the Laplace

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estimation method and poisson errors [72] was used as the response variable (number of internodes occupied by solitary bees or wasps) is count data and not normally distributed. Site was used as a random effect. Direction (east- or west- facing), canopy cover (classified as open or closed based on a threshold of 50% canopy cover) and height were fixed effects. The model was fitted up to a two-way interaction and subsequently simplified using a chi-squared test first for the random effects before simplifying the fixed effects via stepwise backward selection. Akaike Information Criterion (AIC) were also compared for all preceding models when the best model was obtained. The package ‘lme4’ [73] was used to run the GLMM in R. All variables are not collinear and data are not overdispersed. The marginal and conditional (R2GLMM(M or C); variance explained by fixed effects only and variance explained by both fixed and random effects, respectively) for mixed-models were calculated to explore the fit of the model [74]. Traps where ants occupied all the holes or were entirely damaged were removed during the monitoring sessions, and excluded from the GLMM analyses. Testing for the effectiveness of Tree Tanglefoot. The second aim of the study was to determine if the use of Tree Tanglefoot deterred ants. A GLMM with the Laplace estimation method and binomial errors [72] was used to model the response variable (presence or absence of ants nesting). Site was a random effect and the usage of Tanglefoot was a fixed effect. The data are not overdispersed. All R code used for data exploration and statistical analyses are on GitHub. Rearing solitary aculeates and recording nest bionomics. The occupied bamboo internodes were split longitudinally (John X. Q. Lee, pers. comm.), and nest contents and architecture within were examined and photographed periodically (see Appendix VI, Appendix VIII). Nests were reared at room temperature in Ziploc bags for identification purposes. After eclosion, individuals were pinned and identified with the help of J. X. Q. Lee,

Diversity and trap-nesting studies of Singaporean Megachile

16

R. Kendrick, A. Teo, J. S. Ascher and Z. W. W. Soh. Selected larvae were fixed in Kahle’s solution (70% ethanol: acetic acid: formaldehyde, 18:1:1) and preserved in 70% ethanol for study by J. G. Rozen, Jr. from the American Natural History Museum. 2.6 Documenting nests of Megachile Nest cavities were obtained by trap-nesting or discovered through opportunistic searching. All nest cavity diameters were measured and nesting activity were observed where possible (see Appendix V, VI, VII). Cavity diameters were correlated with body size and intertegular length of female Megachile.

Diversity and trap-nesting studies of Singaporean Megachile

17

3. Results and observations 3.1 Megachile of Singapore 3.1.1 Species diversity In total, 21 species of Megachile occur in Singapore and is the most species rich megachilid genus in Singapore as it is globally. Other genera in the family recorded from Singapore include Anthidiellum (Anthidiini, 1 sp., genus newly recorded for Singapore), Euaspis (Anthidiini, 1 sp., a cleptoparasite of Megachile), Heriades (Osminii, 1 sp.), Lithurgus (Lithurgini, 2 spp.) and Coelioxys (Megachilini, 2 spp., kleptoparasites of Megachile) (see Appendix VI, Fig. VI-1)). Of the 21 species, 15 are positively identified to species and the other six morphospecies, all with subgeneric placements and two of which are placed in species-groups [28] (Table 5). These six morphospecies likely pertain to described but poorly known species. Based on local records, four Megachile species (including morphospecies) are only known from males, while three are only known from the females. Seven species are singletons collected only once. Only ten species were present among accessioned material in the ZRC; the other 11 species were represented only in recent collections. Selected high-resolution images of Megachile known from Singapore are available in Appendix VIII and additional images available on Dropbox; all Megachile known from Singapore are represented except for M. (Eutricharaea) sp. 2, known only from a unique male specimen in the ZRC. Singaporean Megachile species belong to seven phylogenetically divergent subgenera [11], including Aethomegachile (6 spp., 3 new species records for Singapore, and first report of this recently expanded subgenus for Singapore), Eutricharaea (3 spp., 1 new species record for Singapore), Paracella (1 sp., new subgeneric record for Singapore), Creightonella (1 sp.),

Diversity and trap-nesting studies of Singaporean Megachile

18

Chelostomoda (1 sp., new subgeneric record for Singapore), Alocanthedon (1 sp., new subgeneric record for Singapore), Callomegachile (8 spp., 5 new species records for Singapore), after historical literature and recent published and unpublished surveys were considered [25-27]. The interspecific body size variation is marked as well, ranging from 7.7 mm (female M. subrixator) to 20.9 mm (female M. indonesica). With reference to a modern classification based on the only Megachile-specific phylogeny tested with morphological characters (sensu Gonzalez [11]), Chalicodoma, Megachile sensu Gonzalez, Thaumatosoma are present in Singapore, whereas Matangapis is not. These are only discussed here as they reflect phylogenetic relationships but are not applied to species names in the rest of the report; these generic updates have not been used widely in the literature [12] perhaps due to the widespread use of traditional names (sensu Michener [1]) or the convenience of a single large genus Megachile sensu lato. Species which use resin in nest construction belong to a group comprising of the closely-related subgenera Callomegachile and Alocanthedon (Fig. VII-4 A–C) [75] and are in Chalicodoma. Subgenera Aethomegachile, Eutricharaea and Paracella are characterised by a complete cutting edge in the interspaces of the mandibles (Fig. VII-4 A–C) used to cut leaf fragments with smooth edges (Fig. VII-1) and are classified under Megachile sensu Gonzalez. Other Megachile bees belonging to the dissimilar subgenera Creightonella and Chelostomoda, cut irregular leaf fragments to line their nest along with masticated leaves. Creightonella is characterised by incomplete cutting edges in the second, third and fourth interspace of the mandibles (Fig. VII3D). Gonzalez [11] placed Creightonella within Megachile sensu Gonzalez as it is sister to all the other leaf-cutting Megachile [76]. Chelostomoda has an incomplete cutting edge in the second interspace [11] (Fig. VII-5 D) and is placed in Thaumatosoma sister to all Megachile sensu lato except the unusual Megachile (Matangapis) of Borneo [18]. The more narrowly-

Diversity and trap-nesting studies of Singaporean Megachile

19

delimited genera proposed (e.g., Alocanthedon [77]), while monophyletic, require further testing with increased taxon sampling and molecular data as they may be nested within other large subgenera (e.g., Callomegachile). Species identifications were challenging for Megachile with similar habitus or striking sexual dimorphism. The plan habitus can be strikingly similar across phylogenetically divergent Megachile and could be an example of Müllerian mimicry. Three groups are present in Singapore including species with 1) black-and-orange metasoma (Fig. VI-6, E, G-J) (3 spp.), 2) black-and-white metasoma (Fig. VI-6 A-D, F) (3 spp.), and 3) black habitus with orange wings (Fig. VI-7) (5 spp.). These can be attributed to divergent subgenera by mandibular morphology (dentition and presence of cutting edges in females; Fig. VI-4, Fig. VI-5) and can be diagnosed by their mesosomal sculpturing and genitalia morphology (Fig. VI-10) (e.g., M. conjuncta and M. laticeps males; Fig. VI-10 A, B). In contrast, the sexes of Megachile can be remarkably dimorphic in colour pattern (e.g., see Fig. VI-6 E-F for a highly dimorphic species, Megachile conjuncta) and associating the sexes with certainty required expert taxonomic opinion (S. Risch, pers. comm.). Based on the 481 specimens examined, the Chao1 estimator (Fig. 3), does not plateau but increases with a sharp slope; an estimated species richness cannot yet be inferred for Singapore.

Diversity and trap-nesting studies of Singaporean Megachile

Fig. 3. Individual-based rarefaction curve based on 481 specimens.

20

3.1.2 DNA barcoding In total, 58 sequences of 27 species were compared. Twenty sequences from Singapore belonging to 12 species were generated, with replicates for four species (M. atrata, M. disjuncta, M. tricincta, M. subrixator) (Table V-1). Thirteen additional non-Singaporean sequences for M. umbripennis and M. laticeps, species found in Singapore, were from BOLD. From GenBank, 33 additional identified Megachile sequences from the Holarctic belonged to 14 species. The aligned sequence was 639 bp but the total number of sites excluding external gaps of 136 bp and missing data (3 bp) was 500 bp (due to a shorter cox1 sequence of M. tuberculata; see sequence lengths in Table V-1). There were no indels present in the sequences. Sequences are available on Github. Of the 639 bp, 193 sites were variable across the sequences, with 70 of 213 variable amino acids. The third codon position had the highest number of variable sites (n=182), followed by the first (n=73) and the second (n=21) (Table 4). The number of variable sites corresponding to an amino acid change was high at the first (n=54) and third (n=54) codon position but was considerably greater in proportion at the first position (0.74) than at the third position (0.30). Notably, the change in amino acid could have been contributed at the first, second or third positions. For example, at positions 79–81 (AAA which codes for Lysine), the polymorphic sites were at the second (A-U transversion for M. fulvipennis; resulting in a synonymous change to Isoleucine) and third (A-U transversion for M. laticeps; resulting in a synonymous change to Asparagine) codon positions. Polymorphisms at a site can also be nonsynonymous at a site with variable amino acid. For example, at positions 40–42 (AUU which codes for Asparagine), the polymorphic site in the third position (U-C transition for M.

Diversity and trap-nesting studies of Singaporean Megachile

21

latimanus) is a non-synonymous change. However, these were not accounted for as only coarse patterns of synonymous and non-synonymous mutation would suffice in this context.

Table 4. Number of variable sites at the first, second and third positions, and their correspondence to a variable amino acid (a. a.) site.

Total number of base pairs External gaps or missing data Variable sites Variable sites corresponding to an a.a. variable site Proportion of sites Minimum variable sites that are non-synonymous

Codon position 1st 2nd 3rd 213 213 213 45 46 48 73 21 182 54 20 54 0.74 0.95 0.30 19 1 128

When different species cluster thresholds were applied to the local species and a combined local, BOLD and GenBank, the optimal thresholds with the highest number of ‘perfect’ clusters and lowest number of ‘split’ and ‘lumped’ clusters ranged from 3–5 % (Fig. 4). A few observations were made. Firstly, two locally occurring species, M. tricincta and M. atrata, appear to have high intraspecific variation (2.16% and 2.80% respectively) as compared to other Megachile species with multiple sequences available (Table IV-3). Specimens for the two species were collected in a similar area. Secondly, Palearctic M. (Xanthosarus) willughbiella and Nearctic M. (X.) frigida was the only pair of species which were lumped at the 3% threshold but were split at the 2% threshold. Third, when comparing sequences for geographically distant species of M. umbripennis, the distance was 1.54% between Singaporean and Fijian specimens (the latter population is thought to be a recent invasion from Southeast Asia [57]).

Diversity and trap-nesting studies of Singaporean Megachile

22

Fig. 4. Number of Megachile species (n=27) that are perfect, split or lumped at different species cluster thresholds for A, local sequences only (12 species, with 4 species that have more than one sequence), B, local, BOLD and GenBank sequences (with 27 species, with 16 species that have more than one sequence).

3.1.3 Spatiotemporal distribution Megachile can be found in managed greenery, mangroves, old secondary and primary forests of Singapore. Three of the most common Megachile (i.e., M. disjuncta, M. laticeps, M. umbripennis) were found island-wide (Fig. 5). Megachile laticeps in particular occupy a wide range of habitats, except scrubland (Table III-2). Megachile atrata was found at coastal areas including back mangroves, where it was also found excavating its nest burrows in mud lobster Thalassina (Crustacea: Decapoda) mounds (see Appendix VI and VII).

Diversity and trap-nesting studies of Singaporean Megachile

23

Fig. 5. Island-wide distribution of the three most commonly occurring species, A, Megachile laticeps, B, M. disjuncta, C, M. umbripennis. Each yellow dot ( ) represents a species occurrence based on a specimen record. Source: Megachile of Singapore Google Fusion Tables mapped in the course of this study.

Seasonality was observed in the flight activity of Megachile in 2014; flight activity and newly-emerged bees both showed in increasing trend from the months May to August (Fig. 6). The highest number of species was also obtained in August (16 species). The flight activity in the months of May and June 2014 were of similar and comparable values to the study conducted in 2012 (Z. W. W. Soh, unpublished data).

Diversity and trap-nesting studies of Singaporean Megachile

24

No. of sampling events Total no. of sampling hours

2012 ■ 2014

3

4

4







2012 ■ 2014

7

8

8







9 2

11 20

– 7

(0,1,1)

(9,10,1)

(6,1,0)

(0,4,4)

(18.5,16.5,2.5)

(7.5,1.5,0)

18 8

22 37.5

– 9

– 15

(8,7,0)

– 28.25

(17,0,11.25)

Fig. 6. Flight activity and newly-emerged bees for Megachile at the generic level, across two surveys spanning February to June 2012 (■) and May to August 2014 ( ). Flight activity based on mean number of Megachile individuals per hour (± S. E.) for 2012 and 2014 surveys; newly-emerged bees based on bees with wing wear 0–2 ( ) for the 2014 survey. Table summarizes sampling effort (number of sampling events and total number of hours); in brackets are the respective values stratified by habitat types: urban, forest and mangroves.

Diversity and trap-nesting studies of Singaporean Megachile

25

3.1.4 Floral and leaf resources Floral resources of Megachile Megachile visit a variety of plants for nectar, as Megachile bees were not carrying pollen when visiting these plants and only collect pollen from plants which conform to pollination syndromes (5 families, 7 spp.; Table 5). The smaller-sized Eutricharaea and Paracella collected pollen from flowers of Asteraceae and Muntinga calabura (Muntingiaceae). The larger-sized Aethomegachile, Alocanthedon [77] and Callomegachile collected pollen from flowers of Fabaceae, Vitex (Lamiaceae) and Memecylon (Melastomaceae) (Fig. 7). The flowers, or parts of the flower, are brilliantly coloured in shades of purple, blue, yellow or orange. The pollen hosts, which generally have clustered, upward projecting stamens and have anthers of similar heights, can be grouped into three pollination syndromes based on flower morphology: 1) papilionaceous Fabaceae type which require a tripping mechanism to expose the stamens [17], 2) Lamiaceae-Memecylon type with exposed stamens and do not require tripping (Fig. 7), and 3) Asteraceae-Muntinga type with many exposed florets or stamens.

Fig. 7. Memecylon flowers.

Several species, discovered by Z. W. W. Soh, were attracted to flowers of the Tiger Orchid Grammatophyllum speciosum (Orchidaceae) which emits a scent to attract bee pollinators without a reward (Z. W. W. Soh and Yam T. W., pers. comm.); these medium- and large-sized Megachile are likely to be the principle pollinators of G. speciosum (Z. W. W. Soh and colleagues, in prep.).

Diversity and trap-nesting studies of Singaporean Megachile

26

Table 5. Floral resources of Singaporean Megachile. Numerous pollen hosts for certain species suggests their polylecty. A question mark (?) indicates that the pollen host currently not known. Species

Floral resources

Megachile (Aethomegachile) borneana M. (Aet.) nr. borneana M. (Aet.) conjuncta M. (Aet.) laticeps

Asystasia gangetica+

M. (Aet.) ramera M. (Aet.) fusciventris sp.-group M. (Alocanthedon) cf. indonesica M. (Callomegachile) fulvipennis M. (Cal.) disjuncta M. (Cal.) biroi sp.-group M. (Cal.) stulta M. (Cal.) nr. stulta M. (Cal.) ornata M. (Cal.) tuberculata M. (Cal.) umbripennis M. (Chelostomoda) moera M. (Creightonella) atrata M. (Eutricharaea) sp. 1 (nr. subrixator, white scopa) M. (Eut.) sp. 2 (crenulated T6) M. (Eut.) subrixator M. (Paracella) tricincta

Asystasia gangetica+, Cratoxylum cochinchinense Crotolaria retusa*+, Grammatophyllum speciosum# Vitex trifolia*+, Memecylon caeruleum*, Asystasia gangetica, Bidens pilosa, Cratoxylum cochinchinense, Grammatophyllum speciosum# Asystasia gangetica+, Grammatophyllum speciosum# Cratoxylum cochinchinense, Asystasia gangetica Grammatophyllum speciosum#, likely Memecylon caeruleum^ Cratoxylum cochinchinense, Asystasia gangetica, Grammatophyllum speciosum# Crotolaria pallida+*, Memecylon caerulea*, Vitex trifolia*, Ageratum conyzoides, Antigonon leptopus, Syzygium zeylanicum, Premna foetida, Grammatophyllum speciosum Grammatophyllum speciosum# Muntingia calabura*, Bidens pilosa*, Cratoxylum cochinchinensis, Grammatophyllum speciosum# Cratoxylum cochinchinensis Grammatophyllum speciosum# Grammatophyllum speciosum# Vitex trifolia*+, Premna foetida, Antigonon leptopus, Asystasia gangetica, Grammatophyllum speciosum# unknown Peltophorum pterocarpum*, Luffa aegyptica+, Asystasia gangetica+, Grammatophyllum speciosum# Bidens pilosa*, Synedrella nodiflora* ?** Muntinga calabura*, Bidens pilosa*, Dendrolobium umbellatum@, Cuphea hyssopiflora, Turnera subulata, Acacia confusa Muntinga calabura,* Bidens pilosa *

*Pollen host !Pollen hosts from more than two families **A singleton museum specimen @A likely pollen host though only males were observed on Tioman Island in July 2014 +Based on data from Soh and Ngiam (2013) [27] ^Based on Engel and Gonzalez (2011) [75] # Unpublished data collected by Z. W. W. Soh and colleagues (in prep.)

Diversity and trap-nesting studies of Singaporean Megachile

No. of pollen hosts ?

2!

? ?

? ? ? ? 5!

2

!

1 1

? ? ? ? ?

2 2! 2!

?

27

Leaf resources of leaf-cutting Megachile Plants cut by Megachile came from a large variety of species (at least 48 spp. from 17 families) based on field observations of cut leaves but the highest proportion are from Fabaceae (43.7% of the occurrences observed; Fig. 10). Of the observed plants, species most often cut include Dendrolobium umbellatum (six occurrences), Bauhinia (climber) sp. (10 occurrences in total), Caesalpinia crista (four occurrences), Archidendron clypearia (three occurrences) (Fabaceae), and Cratoxylum (2 spp., C. cochinchinense and C. formosum) (seven occurrences) (Hypericaceae). Megachile species are observed to cut two types of leaves (see Table VI-1). The first type, cut by M. (Aethomegachile) laticeps, are usually softer leaves that have matte upper surface across all three nests from different sites. Leaf cuts are made with smooth edges with circular or oblong shapes (Fig. VI-3). These are likely to be generalizable to the rest of the bees in Aethomegachile. The second type, cut by M. (Creightonella) atrata, tend to be leaves which are thicker and has a waxier upper surface; this was consistent across all three nest clusters examined. Cut leaves of M. atrata also have jagged edges and are rounded (Fig. VI7), is distinctive of Creightonella [1]. Megachile atrata was also observed in the act of cutting a Syzygium (Myrtaceae) leaf (Fig. 8) at St. John’s Island.

Fig. 8. Syzygium leaves cut by Megachile (Creightonella) atrata. Cuts are rounded with jagged edges. Diversity and trap-nesting studies of Singaporean Megachile

28

Fig. 9. Leaves of Dendrolobium umbellatum (Fabaceae) that are cut by Megachile. Inset is a close-up of the leaves which are cut in different ways (oblong or circular) different purposes in lining the nest (i.e., for the sides or for the cap respectively). Photograph taken at Lazarus’ Island on 8 Sept 2014.

Fig. 10. Percentage of plants, sorted by family (in pie chart) and order (by colour, see legend) which are cut by Megachile species (n=93 occurrences). Number of species in each family are stated if multiple species of plants are cut. Diversity and trap-nesting studies of Singaporean Megachile

29

3.2 Trap-nesting in Singapore 3.2.1 Species diversity of trap-nested arthropods Of 1261 bamboo internodes, 4.83% (n = 61) were occupied by solitary bees and wasps. Thirteen wasps were unidentifiable due to mortality at the larval stage. Forty-eight of these internodes were reared to adults and identified. These comprised 13 species belonging to six bee and wasp families (Megachilidae, Colletidae, Crabronidae, Sphecidae, Pompilidae and Vespidae) (Table 7; Fig. 11). The five species of bees included two colletid species (Hylaeus aff. penangensis and Hylaeus sp. 2), and four megachilid species (Heriades (Michenerella) sp. 1, Anthidiellum smithii, Megachile laticeps, Coelioxys confusa), with C. confusa a newly confirmed kleptoparasite of M. laticeps. The seven species of wasps included three vespids (Allorhynchium argentatum, Eumenes sp. 1, Rhynchium haemorrhoidale), two crabronids (Trypoxylon sp. 1 and Trypoxylon sp. 2), one sphecid (Isodontia severini) and one pompilid (Auplopus sp. 1). Other arthropods also occupied the bamboo trap-nest internodes. These included two species of erebid moths – both of which pupated in the cavity (a caterpillar moult was shed in the internode), a spider (Siler semiglacus) and its nest, and a termite species (Microcerotermes sp.) built its nest with chewed wood at the entrance. Notably few Megachile occupied bamboo internodes despite the many locallyoccurring species recorded from floral netting surveys and represented in local reference collections (Table 7). In particular, DF and BT are known to have the highest number of species of 12 and 10 respectively.

Diversity and trap-nesting studies of Singaporean Megachile

30

Fig. 11. Trap-nested bee and wasp larvae. A, Hylaeus aff. penangensis, B, Megachile laticeps (post-defecating larvae), C, Heriades (Michenerella) sp. 1, D, Trypoxylon sp. 1. The site with the greatest number of internodes occupied by solitary bees and wasps was NS (n =19 internodes) (Table 6). The site with the lowest was BT (n = 0 internodes) due to rain seeping into the bamboo internodes when inspected on the second and third monitoring session. Ants (Formicidae) nested in 30.13% (n = 380) of the internodes. Ants were also found mainly in urban (PU, UH, HP, TE, PR) and mangrove (SB) sites (81% of 380; mean = 57 ± 9 internodes per site) as compared to the forest sites (NS, BT, with the exception of DF as it was at the edge of the forest) (mean = 0.5 internodes for NS and BT; DF = 33 internodes) (Table 6). 7.59% of the trap-nests (n = 11) were also damaged by an animal (suspected to be the long-tailed macaque Macaca fascicularis in NS and the plaintain squirrel Callosciurus notatus in SB and UH).

Diversity and trap-nesting studies of Singaporean Megachile

31

Table 6. Number of occupied internodes and trap-nests, and species richness of locally-known Megachile in the various sites. Internodes with cavity renters (all arthropods but not including ants) Internodes with solitary bees and wasps Internodes with solitary bees

SB

PU

UH

HP

TE

PR

DF

NS

8

9

6

6

1

9

8

19

66

8

8

6

6

9

6

18

61

5 1 1 35

1 1 69

3 3 1 33

1

15 7 4 38

145 128 120 165 177 1261

3

Internodes with ants

4

98

3 2 2 64

Total number of internodes Trap-nest with solitary bees and wasps Trap-nests with ants Trap-nests that have at least one internode scratched open by an animal Trap-nest units

104

122

158

142

3

5

4

6

10

15

13

7

17

17

18

17

17

Species richness cavity renters Species richness of solitary bees and wasps Species richness of solitary bees

4

4

4

3

1

4

3

4

Internodes with Megachilidae Internodes with Megachile

1

Species richness of cavity-renting Megachile Species richness of Megachile, based on netting at floral resources Presence (×) of Megachile atrata*

Total

4

2

7

31

11

8

1

81

5

4

11

17

17

12

5

4

2

16

3

5

3

1

12

2 2

2 1

1 1

2 2

5 4

1

1

1

1

16

2

Species richness of Megachilidae

Species richness of ants

39

BT

2

3

5

3

5

3

2

3

1

5

4

5

8

6

12

1

×

×

×

*a ground-nesting Megachile

Diversity and trap-nesting studies of Singaporean Megachile

×

13

145

13 10

18 4

32

Table 7. Trap-nesting species in the various sites. Of 68 bamboo internodes which were occupied with individuals, 62 were solitary aculeates. Species Bees Anthidiellum smithii (Megachilidae) Hylaeus aff. penangensis (Colletidae) Hylaeus sp. 2 (Colletidae) Heriades (Michenerella) sp. 1 (Megachilidae) Megachile laticeps (Megachilidae) Wasps Allorhynchium argentatum (Vespidae) Auplopus sp. 1 Eumenes sp. 1 (Vespidae) Isodontia severini (Sphecidae) Rhynchium haemorrhoidale (Vespidae) Trypoxylon sp. 1 (Crabronidae) Trypoxylon sp. 2 (Crabronidae) Solitary wasp unknown due to larval mortality* Moths (not nesting but pupated in the bamboo internode) Erebidae sp. 1 Erebidae sp. 2 Others (nest/egg sac was present in the bamboo internode) Microcerotermes sp. 1 (Isoptera: Termitidae) Siler semiglaucus (Arachnida: Salticidae) Species richness of trap-nesting individuals Species richness of solitary bees and wasps Species richness of bees (including kleptoparasites) Species richness of Megachilidae

SB

PU

3

1 1 2 1

UH

HP

1

4

2^

1

3 3

1

1 1

2

TE

PR

DF 2

1

1

1 1

1

5

1

1

9 2 2

4 4

1 4 2

NS

9 1

1 4 5 2 1

3 3 2 1

1

5 5 1 1

4 3 3 2

2 1

BT

Total 15 2 5 3 1 4 46 1 1 6 9 1 10 5 13 3 2 1 2 1 1 16 13 5 3

* nests of wasps and were obtained as young instars – their mortality could be due to the sensitivity of instars at the stage to movement, touch or temperature, and there was no evidence of any parasitoids or cleptoparasites; ** nests were not counted in the total number of occupied internodes as they were not retrieved for rearing; ^ one nest was parasitized by Colioxys confusa

Diversity and trap-nesting studies of Singaporean Megachile

33

3.2.2 Ants occupying trap-nests Occupation by ants prevented bees from occupying the trap-nest cavities. Ants occupied a wide range of cavity diameter, overlapping with trap-nested Megachile laticeps, with no apparent preference (Fig. 12). At each site, median of three ant species were present (Table 6). The most commonly occurring species is Dolichoderus thoracicus (DT) (n = 140 internodes; Fig. 13B), followed by Crematogaster sp. 1 (n = 47 internodes), Tapinoma melanocelphalum (T1) (n = 46 internodes), a tramp species [78] (Fig. 12). The morphospecies from Crematogaster (Fig. 13B), Camponotus, Leptogenys, Tetramorium, Tetraponera, Technomyrmex and Monomorium. All ants belong to genera with arboreal species [79]; the arboreal ants could have been foraging on or inhabiting the trees but can be inconspicuous and not observed when the trap-nests were tied onto the tree.

Fig. 12. Boxplot for diameter of holes occupied by Megachile laticeps (Meg), and various ant morphospecies (arranged in decreasing abundance). The number of occupied holes are reflected below the abbreviated species names. ABBREVIATION OF ANT SPECIES/MORPHOSPECIES: DT – DOLICHODERUS THORACICUS, C1 – CREMATOGASTER SP. 1, T1 – TAPINOMA MELANOCEPHALUM, C3 – CREMATOGASTER SP. 3, TET1 – TETRAMORIUM SP. 1, TEC1 – TECHNOMYRMEX SP. 1, C4 – CREMATOGASTER SP. 4, C2 – CREMATOGASTER SP. 2, MF – MONOMORIUM FLORICOLA, TEP1 – TETRAPONERA SP. 1, CA1 – CAMPONOTUS SP. 1, L1 – LEPTOGENYS SP. 1.

Diversity and trap-nesting studies of Singaporean Megachile

34

Fig. 13. A, Nest made in bamboo by Crematogaster sp. 4 and B, nest in bamboo by Dolichoderus thoracicus.

Some solitary aculeates nested in trap-nests where ants were present. Overall, ants occupied 58.27% (81 of 139) of the trap-nest (units) (Table 6). Twelve of 94 trap-nests with ants had solitary aculeates nesting, totalling to 30% of the nesting solitary aculeates (n=21 internodes); seven out of 12 known trap-nested bee and wasp species nested in trap-nests where ants were present (Table 7). These included Hylaeus (with Crematogaster sp. 1), Eumenes (with Tapinoma melanocephalum), Isodontia (with Dolichoderus thoracicus, Tapinoma melanocephalum), Trypoxylon (with Dolichoderus thoracicus, Tapinoma melanocephalum, Camponotus sp. 1), Auplopus (with Tetramorium sp. 1). Those that did not nest with ants include the large vespids (Allorhynchium argentatum and Rhynchium haemorrhoidale), and the megachilids (Heriades (Michenerella) sp. 1, Megachile laticeps and Anthidiellum smithii).

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Table 8. Number of bamboo internodes occupied by solitary aculeates where ants were present and not present in the same trap-nest unit.

Species not nesting with ants in any trap-nest unit – > 5 spp. Allorhynchium argentatum (Eumeninae) Anthidiellum smithii (Megachilidae) Heriades (Michenerella) sp. 1 (Megachilidae) Megachile laticeps (Megachilidae) Rhynchium haemorrhoidale (Vespidae) Unknown wasps due to mortality Species with individuals nesting with ants – 7 spp. Auplopus sp. 1 (Pompilidae) Eumenes sp. 1 (Vespidae) Hylaeus aff. penangensis (Colletidae) Hylaeus sp. 2 (Colletidae) Isodontia severini (Sphecidae) Trypoxylon sp. 1 (Crabronidae) Trypoxylon sp. 2 (Crabronidae) Total

With ants

21 1 4 4 2 3 4 3 21

Without ants 22 1 2 1 4 1 13 18 2 1 1 6 6 2 40

Total 22 1 2 1 4 1 13 39 1 6 5 3 9 10 5 61

3.2.3 Tanglefoot did not deter ants The application of Tanglefoot did not significantly deter presence of ants (Table 9). Of the 20 trap-nests with Tanglefoot applied in PU, TE and UH, 16 had ants nesting in them. Of the 114 trap-nests without Tanglefoot applied, 64 had ants nesting in them. Table 9. The presence or absence of nesting ants based on the presence or absence of Tanglefoot in GLMM with binomial errors. Parameter Intercept Presence of Tanglefoot σ2 intercept Nsite = 9

Coefficient (β) 0.2306 0.2775 2.7198

SE 0.7884 1.1056

p > 0.05 > 0.05

Ntrap-nests = 109

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z 0.7884 1.1056

36

3.2.4 Preferred microhabitats and hole diameters Based on the model with the lowest AIC, the main effects of height, canopy cover and the two-way interaction of height and canopy cover were significant predictors of the number of internodes occupied by a solitary aculeates (Table 10). Direction was not a significant and was not included as a variable in the final model. The R2GLMM(M) (the variance explained by fixed effects) is 19.47% and R2GLMM(C) (the variance explained by fixed and random effects) is 49.19%. The observed values and predicted values based on the model are visualised in Fig. 14. Holding all other variables constant, for every increase in 1 cm, the mean number of internodes occupied by a solitary aculeate decreases by 1.09 (Table 10). On the average, trapnests in an area with a closed canopy have 2.51 fewer internodes occupied by solitary aculeates as compared to areas with an open canopy. The positive interaction between height and canopy cover suggests that height has less of an effect when in the shade. This could be an artefact of the distribution of predicted data (low number of nesting individuals in open areas at greater heights) rather than an interpretable phenomenon (Fig. 14). Table 10. Number of bamboo internodes occupied by solitary aculeates based on by direction, height and canopy cover in a GLMM with poisson errors. Parameter Intercept Height (cm) Canopy cover (closed) Height × canopy cover (closed) σ2intercept Nsite = 9

Coefficient (β) 2.85541 -0.03641 -3.25656 0.03204 3.3109

* parameter is statistically significant

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SE 1.05200 0.01029 1.11202 0.01078

p < 0.01* < 0.01* < 0.01* < 0.01*

Ntrap-nests = 109

z 2.714 -3.540 -2.929 2.973

37

Fig. 14. Interaction plot between canopy cover and height featured with A, confidence intervals based on fixed-effects only and B, confidence intervals with the inclusion of uncertainty from random effect of site. Triangles ( ) represent observed data and circles ( ) represent predicted data. There is a positive correlation between body length and intertegular distance of the female bee with median cavity size occupied (R2body.length = 0.7524, R2intertegula.length = 0.5677; Fig. 15 A, B).

Fig. 15. Scatter plot of A, body length of female, B, intertegular distance of female against median cavity diameter for six megachilid species (see Appendix IV and Appendix VII, Table VII-1). Diversity and trap-nesting studies of Singaporean Megachile

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4. Discussion 4.1 Singaporean Megachile – a synoptic representation of divergent lineages The total number of Singaporean Megachile species stands at 21 (Table 5) and are a synoptic representation of divergent Megachile groups – this suggest high phylogenetic diversity. Nest studies of M. (Aethomegachile) laticeps and M. (Creightonella) atrata also suggest that they are divergent as they line with leaves distinctly from each other (Table VI1). Four species were previously recorded in the 19th and early 20th centuries by earlier workers, including M. conjuncta, M. atrata, M. ramera and M. subrixator [28]. Contrary to extensive faunal extinctions in other taxa (e.g., birds [80]), all four species are still extant in Singapore as they were found in recent surveys [27] and fieldwork during the course of this study. No large-scale, modern diversity study has been published for Megachile in this region; for that reason, diversity-based comparisons with other regional fauna cannot be made. Nevertheless, the sharp increase in slope for the species estimator curve (Chao1) (Fig. 3) suggests that the inventory of Megachile is not complete for Singapore. The number of new species records is very likely to increase if a wider range of sampling methods are employed (e.g., using a tropics net to reach flowers high in trees) with multi-year of sampling. Diversity studies of bees are still in their infancy in Singapore as it is with the Old World tropics. While Michener [1] notes the low diversity of bees in the tropics relative to other biomes, this phenomenon could also be due to the lack of efficient sampling methods in the canopy, especially since it is the “center of most plant activities” [81], and the short flowering duration of tropical rainforest plants [81].

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4.2 Utility of diagnostic and identification tools Notably, the cox1 barcodes were very variable (30.20% of the sites are polymorphic) for a dataset of 27 species from 58 sequences. Acknowledging the species included in this analysis belong to a hyperdiverse genus of bees and are represented by divergent lineages from several biogeographic regions (N. America, Europe and Oriental; Table IV-2), it would be anticipated. Additionally, Sheffield et al. [62] also noted an elevated divergence in cox1 for bees and attributed it to higher rates of molecular evolution of the mitochondrial genome for bees (Apis) relative to flies (Drosophilia) [82]. The polymorphisms are biased towards the third codon position, and largely are non-synonymous mutations (at least 128 of 182 polymorphic sites; Table 4). This explains the higher number and proportion of mutations accumulated in the third position [83]. Cox1 barcodes diagnose between species at an often-used 3% K2P distance threshold [55]. In this study, the optimal uncorrected distance threshold was 3% for all Megachile species (Fig. 4; except one pair, see discussion below). The accuracy and error of barcoding is also dependent on how the barcodes are utilized. Assuming the database was created for regional identification (e.g., within a region for a region of a localised area), haplotypes of widespread species from geographically distant localities need not be considered and overlaps between intraspecific and interspecific thresholds would likely decrease [84]. A local sample of the bee barcodes in Nova Scotia, Canada [38] showed that the mean K2P intraspecific distance was 0.48% (n = 122 species); in this study, the mean uncorrected intraspecific distance for Singaporean species was 1.93% (n= 4 species; see Table VI-3) but within 3%. On the other hand, if the barcodes were to be used as a global database for species identification, haplotypes of a species from geographically-distant localities may have diverged beyond the Diversity and trap-nesting studies of Singaporean Megachile

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stipulated 3% threshold [85], though this was not the case in Fijian and Singaporean M. umbripennis (Table VI-3). At present, only 27 spp. of 1516 species in the genus [10] have a cox1 barcode and are annotated with a positive species identification. Greater taxon sampling (increasing number of species) and species coverage (number of specimens per species for a localised area and of those that are geographically distant) are necessary to judiciously assess if the 3% can be used as a diagnostic tool for both regional and global samples. High error rates in species barcodes is resultant from overlapping maximum intraspecific and minimum interspecific thresholds, attributed to sequences of geographically-distant specimens of conspecifics [67] and such rates were lower for the regional studies [54,62]. The 3% threshold did not apply to two species, M. (Xanthosarus) willughbiella and M. (X.) frigida (i.e., 7% of the barcoded Megachile species). These were lumped at the 3% threshold but formed distinct species clusters at the 2% threshold (Fig. 4). The species tagged to the GenBank accessioned numbers are unlikely to be misidentified as they were verified by taxonomists. These species are commonly occurring [10], morphologically distinct [29,86] and allopatric in distribution [54,60,62]. This suggests that the thresholds may not always apply especially against a global database of barcodes [67]. In this case, species distances are closer than expected. Firstly, this phenomena usually pertains to cryptic [62] or very recentlydiverged [54] species. Indeed, the two species are from the same subgenus and could be closely related (J. S. Ascher, pers. comm.). Secondly, selective sweeps could prevent fixation of novel alleles when it decreases fitness of the carrier (e.g., [87]), particularly in cox1 as it codes for a key protein involved in respiration [88]. To affirm such hypotheses in species delimitation, more genes should be analysed in combination with phylogeographic evidence. Alternatively, if they were the same species exhibiting phenotypic plasticity, mating

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hybridisation experiments could be conducted to test for this. For diagnostic purposes based on a regional sample, a 3% threshold would still be effective since M. wulliughbiella and M. frigida are found in different biogeographic regions, of the Palarctic and Nearctic respectively (see Table IV-2). For diagnostic purposes of local Megachile, the dimorphic sexes of Singaporean Megachile (M. atrata, M. tricincta, M. subrixator, M. disjuncta, M. subrixator) were confirmed. With barcodes for M. ornata and M. sp. 1 (nr. subrixator), matching of the other sex will be possible in the future. Cox1 barcodes can put a positive species identification to larval stages (e.g., [89]) since larval mortality (13 of 61 occupied internodes) can be substantial when obtained in trap-nests. Moreover, barcoding also may allow preservation of the larvae at various instars to study an additional suite of morphological characters. Due to the lack of sample sizes from trap-nesting study, barcoding of Megachile larvae was not conducted. The usage of high resolution imagery and cox1 gene are complementary if disseminated on online platforms to facilitate not only the management and monitoring of bees but to further taxonomic [33] and ecological studies [62] as well. High-resolution imagery has provided a complementary way to identify these bees based on morphology. The creation of online and interactive keys such as on platforms such as ScratchPad [90] and DiscoverLife [91] may trump traditional dichotomous taxonomic keys as they allow for usage of multiple characters at an instance.

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4.3 Plant resources of Megachile Megachile are known to trip the papilionaceous Fabaceae effectively [17], visit Polygalaceae [92], Asteraceae and Lamiaceae [11] for pollen. Similarly, Singaporean Megachile visit on plants belonging to three pollination syndromes [93] (Table 5). These include flowers whose stamens are clustered and upward projecting, and the anthers of similar heights (e.g., Fig. 7), allowing the non-parasitic megachilids to collect pollen efficiently on the ventrally-positioned scopal hairs of (Fig. VIII-1A, D, F, H, I; i.e., not Euaspis or Coelioxys). Singaporean Megachile with known pollen hosts are polylectic (i.e., do not specialize on a group of plants), as they use various species from different families (subsets of combinations of Fabaceae, Asteraceae, Lamiaceae, Melastomataceae, Muntingiaceae). Polylectic species include M. laticeps, M. disjuncta, M. umbripennis, M. stulta, M. tricincta and M. subrixator (Table 5). Bee larvae of oligolectic megachilid species have been shown experimentally to survive poorly on pollen of plants which are not their pollen host [94]. Many of these Asteraceae (e.g., Bidens pilosa, Ageratum conyzoides, Synedrella nodiflora) and M. calabura are exotic to Singapore and come from the Americas [95], further validating that these bee species, presumably native, can collect pollen from exotic plants to provision cells for their progeny. Species whose host plants are currently unknown are likely to conform to the abovementioned pollination syndromes but they could also be strongly oligolectic. If and when their nests are obtained, pollen studies and further morphological study of female Megachile can be conducted. Some Megachile have very specialized morphology, allowing it

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to collect pollen from a specific group of pollen host (e.g., modified hairs above the clypeus of M. mitchelli [11]; hindleg brushes for M. pilicrus [96]). If specialists do exist, the flight activity of the bee and flowering phenology of the plant are likely to be very closely tied with each other. No such specializations are recorded in Singapore yet. Overall, pollen seems to be an important resource and could also be limiting. The size, and consequently fitness [97], of the offspring corresponds to amount of provisioning it gets [98] and female Megachile also invest a large proportion of its life collecting pollen [16]. Moreover, wet and/or colder weather causes nectar and pollen production of flowers decrease [7], making it a resource less consistently available. Megachile atrata and M. disjuncta collect pollen on all observed mornings, suggesting that it is most available in the morning (see Appendix V) and could be due to anthesis of flowers. Consequently, pollen may be a limiting resource in nest construction; further studies are required to demonstrate that there exists competition for pollen which has been shown between non-native honey bees and bumble bees [99]. Megachile are known to cut leaves of both Fabaceae [100] and Rosaceae [101] leaves but few Rosaceae occur in tropical environments naturally [102]. In this study, Megachile used leaves of 17 families to line their nests (Fig. 10) but there are a high number of occurrences for cuts leaves of Fabaceae and Hypericaceae. As a large number of plant species from different families are used, Megachile are not likely to be constrained for the type of leaves used in contrast to pollen resources. However, at a given site only a subset of plants were usually observed to be cut. Bees are central place foragers [7] and return to the same area for resources. Since Megachile are flower visitors of Fabaceae and Hypericaceae (Cratoxylum cochinchinense), it is possible that they return to the same plant and use leaves Diversity and trap-nesting studies of Singaporean Megachile

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of the same plant to line their nests. In contrast, Megachile avoid leaves which are high in serotonin [103] and leaves (or leaflet) have area greater than 1 cm2 [104]. Studies conducted in the Neotropics and Switzerland found similar results of a wide variety of plant species used in leaf cutting but with a few species cut with high occurrences [100,105]. Since a wide array of species from different families can be used and leaf availability is not seasonal, leaf resources are not likely to be limiting. In general, Megachile were rarely observed in the act of cutting leaves – it was only observed on one fleeting event at St. John’s Island (Fig. 8) throughout the entire field season. Evidence based on cut leaves require further validation in the future by identifying the leaves used in the nest construction from the trap-nest of the bees. Other Megachile and megachilids use different lining materials. During this study, resin resources were not documented, and could be a limiting factor in the construction of nest for the resin bees. It is evidently a key resource for megachilids such as Heriades (Michenerella) sp. 1, Anthidiellum smithii and other resin Megachile (Appendix VII), and Megachile, like M. (Callomegachile) pluto which collects resin from dipterocarp trees [106]. It is currently uncertain how Megachile bees select their materials as nest linings but Messer showed that dipterocarp resin used to line nests of M. pluto had anti-microbial properties [107]. A similar proposition can be made with the leaves used to line nests. Plants which are cut with high numbers of occurrences (Dendrolobium umbellatum (Fig. 9), Archidendron clypearea, Caesalpinia crista, Bauhinia spp. (climbers and B. kockiana) and Cratoxylum spp.) can be investigated further with regards to similar properties. Fungi were observed growing on the exterior of the leaf cell but not at the pollen resources (see M.

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laticeps, Fig. VI-4) whereas if fungal growth within the cell can contribute to larval mortality (see M. atrata, Fig. VI-5). 4.4 Potential drivers of seasonal Megachile flight activity Seasonal abundance of Hymenoptera known in the tropics; Tyalianakis et al. conducted their study at Ecuador over a land-use gradient and found temporal seasonality to be significant [39] whereas Momose et al. observed the seasonal increase in nest density of giant honey bees Apis dorsata in dipterocarp forests of Lambir Hills, Sarawak [92]. In Singapore, highest Megachile activity was observed in August in a sampling season spanning from May–August 2014; relatively lower activity was also observed from January–June 2012 (Fig. 6). Thus, there is seasonality in Megachile activity, though a multi-year time series is necessary to ascertain if 1) this trend is replicable, 2) if the high activity in August 2014 is a truly a peak, 3) if so, are there more than one peaks in a year, or 4) do peaks occur supraannually, 5) whether these phenomenon occurs are parallel in both urban and natural areas. Such patterns in seasonality could occur due to various reasons. Firstly, it could be due to annual patterns in weather, which directly or indirectly affects abundance and/or flight season of bees. Bivoltinism was observed in February and August in Chandigrah, India [108] for two M. (Callomegachile) spp. where the maximal temperatures reaches 44°C in the summer and is likely the direct cause for bee mortality or diapause. In Singapore, where temperature does not fluctuate to a large extent, it is unlikely that temperature affects the flight activity or population dynamics of the Megachile. However, rainfall or drought could be an indirect driver of bee flight activity, as elaborated in the subsequent point.

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Secondly, as many tropical rainforest plants flower only for a short period of time [7], pollen and nectar resources are only available to the bees when these flowers appear. This causes seasonal availability of food resources for the bees and provisioning resources for bee progeny. Tropical rainforests are known for their synchronous but pulsed flowering phenology of diverse groups of plants [109,110]. In monsoonal rainforests, this is in response to annual weather patterns, such as a marked transition from a drier to wetter season [111] (e.g., in India [112] and Laos [113]). Non-monsoonal tropical forests also show seasonality in flowering, in a phenomenon known as general flowering (GF), occurring at irregular intervals of a few years. What triggers GF is still uncertain but is usually preceded by drought [114]. In Singapore, GF events were also observed this year, in 1987 [115], 1990, 1996 and in 2005 (S. K. Y. Lum, pers. comm.) suggesting that it occurs every few years. Some understorey plants flower supra-annually but not in synchrony with GF [81]. Based on the study conducted at the non-monsoonal tropical rainforest at Lambir Hills, Sarawak, Megachile bees appeared both in the canopy and understorey during GF and outside GF, though rarely in the former, as its host plants were generally in the understorey and had shorter flowering cycles [116]. This suggests that there exists a correlation with appearance of Megachile flight activity and the flowering of its host plants but is unlikely to be correlated with GF. Could Megachile seasonality be driven by flowering phenology in Singapore? Earlier in the year (March – May 2014), mass flowering was observed in both wayside and rainforest canopy trees of Singapore after a drought (January – March 2014) [117]. Correspondingly, newly-emerged Megachile (i.e., bees with wing wear 0–2) were observed to be highest in August 2014 (Fig. 6). It is likely that females are likely to be provisioning the nests for these bees a total of five to seven weeks before (i.e., late May to June 2014); it would take one to

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two weeks to construct the nest and four to five weeks for the development from egg to adult (observed in M. laticeps – see Appendix VI and M. fulvipennis – see Appendix VII). Flowering is a plausible driver of seasonality in Megachile, and whether increased Megachile flight activity is driven by the increased availability of pollen resources, is a question that cannot yet be answered as it was not rigorously quantified. To test the drivers of bee flight activity, the first approach would be to detect if there is a correlation with abiotic factors (rainfall and temperature) and processes (e.g., El Niño) and flowering phenology of managed and natural vegetation (noting which are pollen or nectar hosts, or do not offer a reward) [118]. Monitoring bee flight activity is ideally corroborated with passive methods such as trap-nesting, as netting flowering plants could potentially bias the sampling with false absences. Phenology of insects and flowers are poorly recorded in the tropics [119], and could be locality-specific [81]. Species-specific flowering phenology is scantly recorded in Singapore [115,120] but is likely to be differ for the wayside trees, ornamental shrubs, rainforest canopy trees (S. K. Y. Lum, pers. comm.) and understorey plants [121]. Understanding such drivers would help illuminate potential allocation of management efforts. 4.5 Trap-nesting in Singapore Universally, trap-nesting studies have low percentage of cavity occupation by bees and wasps – not exceeding 19% (except in Jayasingh and Freeman [40]), and can be as low as 0.01% (see Appendix I). This could be due to a greater availability of nesting sites relative to the abundance of solitary bees and wasps. It may secondarily be due to the trap-nest design. Sheffield et al. suggested that cavities in very close proximity discourage bees from

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occupying in the same trap-nest [38] even though they can nest gregariously. Most other bamboo or reed trap-nest studies bundle approximately 200 internodes together without leaving gaps in between, and is used as a standardised monitoring method across many European studies, which may explain the low nesting rates [122]. Consequently, they used a modified set-up, leaving gaps in between the cavities (Fig. 16) and achieved higher proportion of occupied cavities. Remarkably, Jayasingh and Freeman also achieved a very high (and the highest) percentage of occupied cavities (44%); they placed individual internodes and wood pieces (i.e., not bundled) in walls and on wooden structures in 112 sites [40]. In this study, I obtained a 4.83% bee and wasp occupancy and had bundled 8 or 15 internodes together.

Fig. 16. Sheffield et al.’s trap-nest design with a polystyrene cover in the front of a milk carton tube, where paper tubes were placed inside the box. Source: Sheffield et al. (2008), used without permission. Trap-nesting communities are generally not speciose, where host species richness (i.e., not including parasitoids) does not exceed 40 (see Appendix I). There appears no significant trend with species richness and latitude in highly replicated studies sampling across different habitat types: Albrecht et al. obtained 39 species of solitary bees and wasps in temperate

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Switzerland (44 sites) [123], whereas Tylianakis et al. obtained 31 species in tropical Ecuador (48 sites) [39]. The sampling for this study conducted over two months for each of the nine sites. To comprehensively sample the trap-nesting community, a larger spatial and temporal coverage would be necessary. Studies conducted over a longer period of time and with more sites had higher species richness (e.g., Albrecht et al. obtained 39 species over two years [123]; Frankie et al. obtained more than 30 species in a subtropical over two years [124]). Studies which were conducted through one sampling season usually obtained fewer species (10–14 species; see Appendix I). Species richness obtained in this study was 12 from six solitary bee and wasp families (Table 7), and is comparable for the sampling effort. Temporal replication is necessary as Tylianakis et al. showed that solitary bee and wasp abundance is seasonal through trap-nesting [39]. In Singapore, Megachile appears to be seasonally abundant as well. Spatial replication is necessary so as to encompass various habitat types which may harbour different species assemblages. Large-scale factors such as the landscape, e.g., habitat type and distance to the forest, could determine species assemblage present. Klein et al. showed that solitary bee abundance and wasp richness was negatively correlated with distance to forest, due to nest site and food availability respectively [41]. Barthléméy also conducted his study only in an intact forest (Sha Lo Tong and Pak Sha O; Fig. 17) which may explain the higher species richness and abundance of occupied trap-nests obtained [37], whereas I conducted my study in sites of urban greenery and mangroves, in addition to forest sites.

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Fig. 17. Barthléméy’s sampling sites represented by an orange star ( ) in Sha Lo Tong and Pak Sha O, Hong Kong. Source: Google Maps Ants were found to be the biggest obstacle in trap-nesting. The ants occupied 30.13% of the bamboo internodes and were present in 58.27% of the trap-nests in this study (Table 6), and did not show a preference for a cavity diameter (Fig. 12). They were especially prevalent in managed gardens, mangrove and forest-edge sites. A small proportion of the trap-nests were also damaged other animals (7.59%) which presumably were looking for food (e.g., larvae present in the trap-nests). Only one study in the subtropics by Miyano and Yamaguchi quantified the presence of the ants at trap-nests [125]. They replicated their study across three sites and found that the ants were present in the forest-edge sites, whereas the urban site (at a gazebo) had no ants. Most other studies, notably from the tropics and subtropics [37,41,124,126], made cursory statements on the treatment of ant-occupied trap-nests (e.g., removing them) and attempted to prevent them with a sticky substance (e.g., TangleFoot or glue). It is very likely that ants do not occupy many trap-nests in temperate sites, and do so in sites which are disturbed by humans in subtropics and tropics (such as in managed greenery and forest edges), as was Diversity and trap-nesting studies of Singaporean Megachile

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found in this study. Further, ant communities in such human-modified habitats may exhibit unique ant assemblages, typically that of tramp species [127]; Tapinoma melanocephalum, was the third most commonly occurring species in all of the sites (Fig. 12), is one of such species [78]. Miyano and Yamaguchi suggested that bees and wasps may actively avoid nesting near ants [125]. Evidence from this study suggests that this is so, as the majority (40 of 61 trap-nested bees and wasps) did not nest in a trap-nest with ants (Table 8). Thus, ants not only compete with solitary bees and wasps for nest sites by occupying the nesting site but also deter nesting bees and wasps from nesting in their vicinity. It is also very probable that they do so via the chemical pheromones [128]. Twenty-one solitary bees and wasps successfully completed their nests in ant-occupied trap-nests, suggesting that the ants found in the same trap-nests as these nesting bees and wasps did not actively attack the provisioning individual, or rob the contents of these nests. The ants may have done so for other incomplete nests but was not quantified in this study. However, preventing or avoiding ants in trap-nests tied to trees were a challenge in urban sites. The application of Tanglefoot did not significantly decrease the presence of ants in three sites of this study over a period of two months (Table 9) and a large proportion of the trap-nests (16 of 20) where Tanglefoot was used still had ants in them. Miyano and Yamaguchi used a paired experimental set-up of ten replicates in three sites, with and without Tanglefoot on a wooden stake [125]. While they showed that Tanglefoot significantly prevented ants from nesting in trap-nests over a period of five months, ants still occupied 15% of the trap-nests with Tanglefoot, in sites where ants were present. Reapplication of Tanglefoot, or Vaseline on a regular basis (e.g., fortnightly) may prevent nesting ants as their Diversity and trap-nesting studies of Singaporean Megachile

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efficacy is be compromised with rain (washed away) and insolation (drying up) in the tropics (pers. obs.) where rainfall is higher and photoperiod is longer. Using wire sheaths to hang the nest from a tree may also decrease ant colonization of the trap-nests [37]. Avoidance of the ants by attaching trap-nests to wooden stakes or walls rather than to trees where arboreal ants may already be inhabiting may decrease the proportion of trap-nests with ants. Rain seeped into bamboo internodes at BT; none of the internodes were occupied by bees, wasps or ants. Thus, preventing rain is important in tropical trap-nesting studies. Barthléméy [37] suggested that thick bamboo is necessary to prevent seepage of water but this does not appear to effectively deter rain from accumulating in the trap-nesting unit (pers. obs.). Roofing over the trap-nest unit is not used in many studies (see Appendix I) and may be effective for shading purposes rather than rain prevention. Many studies place trap-nests in plastic tubes, tins or milk cartons, and these may provide some shelter against rain. To discourage fungi from growing due to accumulated moisture, a fungicide may also be sprayed [129]. Solitary aculeates reportedly do not have a preference for the type of nesting substrates [103]. Nesting substrates used in trap-nesting studies are broadly categorized into 1) wooden blocks drilled with holes, 2) bamboo or reed internodes, or 3) man-made paper or cardboard tubes. Gaston et al. showed that wooden blocks with drilled holes worked best in temperate gardens, followed closely by bamboo internodes [42]; they showed that paper tubes, on the other hand, performed exceptionally poor and was corroborated by Westphal et al. [36], presumably because they are much more pliable. Wooden blocks are not easily constructed as they require specialized drilling equipment with a drill bits of special sizes (diameters ranging from 4–12 mm). They also cannot be easily split to explore the nest contents unless split Diversity and trap-nesting studies of Singaporean Megachile

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beforehand or a paper tube is placed in the cavity but they can be reused. On the other hand, bamboo or reed internodes are the best compromise if nest are to be split opened for examination and are relatively easier to construct. Small-scale factors such as microhabitat have been shown to play a part in encouraging solitary bees and wasps to nest [103]. The variables shadiness and height were significant contributors to the number of internodes occupied by solitary aculeates (Table 10); whereas direction of the trap-nest was not significant, concurring with the hypothesis proposed for the tropics. The model suggests that the number of nesting solitary aculeate is significantly more when in the open than in the closed areas. This could be due to a greater visibility of the trap-nest in open areas as compared to in the closed areas. The model also suggests that aculeate Hymenoptera prefer trap-nests at lower heights in the open, where height has a greater effect in the open than in the shade. This could mimic their natural nest sites, as a M. laticeps nest (with one incomplete cell) was found on a broken twig on the projecting out of the ground at MacRitchie Reservoir Park (pers. comm., S. X. Chui). However, generalizations on the favourable nesting sites of solitary aculeates should be interpreted with caution. The fixed effects of model only explained 19.43% of the variation, does not appear to fit the observed data very well by visual inspection, especially for predictions in the shade (Fig. 14 A, B). It could be that solitary aculeates do not have a preference at the scale of the microhabitat, or that not all solitary aculeates may have a similar microhabitat preference. However, this was not statistically tested here due to low sample sizes obtained. Furthermore, the random effects of site contributed a substantial amount of variation as well (29.8%). The unexplained variation and variation from random effects is likely to be contributed by more important but complex variables, which cannot be modelled Diversity and trap-nesting studies of Singaporean Megachile

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(e.g., the ants). Thus, the best model obtained with the lowest AIC, should be used prudently for management practices. Cavity diameter corresponds positively to the body size of the bee. Evidently, there is a positive linear correlation with body size and the usage of hole diameter (Fig. 15A), suggesting that cavity size is needs to be optimised to suit the target species. However, the bamboo internodes of the trap-nests ranged from 1–17 mm; this may also partially account for low rates of occupation by Megachile, particularly so for the medium- and large-sized species. Lastly, not all Megachile may nest in pre-existing cavities, and is exemplified in Singapore by M. atrata which excavates nests in the ground. Ground-nesting Megachile are not exceptional (e.g., 14 of 38 Canadian Megachile are known to nest exclusively in soil) [29,34,130]. Such species require different monitoring and management strategies, e.g., augmenting nest sites with terracotta saucers [131].

Fig. 18. Schematic diagram summarizing factors affecting the species richness and number of trap-nesting individuals. Diversity and trap-nesting studies of Singaporean Megachile

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4.6 Implications for monitoring and management Even as high occupancy by Megachile was not achieved with bamboo trap-nests, much insight was gained to trap-nesting, and this complemented fieldwork of netting bees at flowers and observations of bees at their nesting sites to inform the monitoring and management of Megachile. A few general statements can be made about the status and resource requirements of Megachile in Singapore, 1) more species can still be discovered (Fig. 3) Megachile have seasonal flight activity (Fig. 6), 3) Megachile collect pollen from flowers that conform to three pollination syndromes (Table 5), 4) leaves collected are not constrained taxonomically (Fig. 10). To address the low proportion of nesting by solitary bees and wasps in trap-nests, more optimization and a radically-improved trap-nest design is necessary. At present, many variables contribute to encouraging bees and wasps to occupy the trap-nest cavities. Key practices related to important variables include: 1) the prevention of ants in urban sites by a) tying trap-nests to a wall or wooden stake rather than tree (Fig. 19A), b) tying trap-nests via a wire sheath or string (Fig. 19B), c) reapplication of Tanglefoot on a frequent basis (e.g., fortnightly), 2) the prevention of rain seepage by placing internodes in a plastic tube, tin or milk carton, and 3) solitary bees and wasps may be encouraged to nest by a) placing internodes individually rather than bundling them together (as in Jayasingh and Freeman [40]), b) place bamboo internodes with the right diameters (values can be predicted based on the correlation; see Table 11) and c) possibly by placing the trap-nest in open areas at a low height, Should trap-nests be placed in areas with an open canopy the trap-nests should not be

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in direct sun but shaded with a roof or under a tree [124]. The seasonality of Megachile may affect the number of nesting individuals, thus, temporal replication is crucial. Since ants are less prevalent in forest sites, preventive measures may be less necessary.

Fig. 19. A trap-nest design with reed or bamboo internodes in plastic tubes, A, by tying trapnest to a wooden stake, B, by tying the trap-nest via a string and securing it from the bottom. Source: Christoph Scherber laboratory web page, used without permission. Table 11. Predictions for optimal trap-nest cavity diameters for each species based on correlation in Fig. 15A. Cavity diameter size 9 mm 15 mm 22 mm

Species M. moera, M. sp. 1, M. stulta, M. subrixator, M. tricincta M. fulvipennis, M. ornata, M. ramera, M. conjuncta, M. fusciventris sp.-group M. cf. indonesica, M. tuberculata.

Thus far, one Megachile species (M. laticeps) has been successfully trap-nested in Singapore, in both managed gardens (HP, UH) and mature secondary forest edge (DF). This is one of the most commonly occurring Megachile species in Singapore and found in the most number of habitat types, except scrublands (Fig. 5). Since it can persist in a wide range of environments, it is likely to be managed with ease and can be translocated with trap-nests. It could be used as a pollinator of plants with flowers of appropriate pollination syndromes, in community garden initiatives such as the Community in Bloom initiative by National Parks’ Diversity and trap-nesting studies of Singaporean Megachile

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Board [132], or as a pollinator of native plants that are ‘critically endangered’ like Memecylon [95] which it is known to visit routinely (see Google Fusion Tables records). Other smallsized Megachilinae, Heriades (Michenerella) sp. 1 and Anthidiellum (Pycnanthidium) smithii), and two species of Hylaeus (Colletidae) also occupied trap-nests, and these could also be potentially manageable species. Megachile laticeps is particularly desirable for management purposes; the multiple layers of cell leaf lining may help buffer against movement and changes in temperature, and individual cells can be detached from each other [16]. Through trap-nesting, Coelioxys (Allocoelioxys) confusa was confirmed as a kleptoparasite of M. laticeps. Kleptoparasitic bees like Coelioxys lack pollen-carrying structures, and are likely to be less effective pollinators than their hosts. Should host bees be managed on a larger scale, preventing these kleptoparasitoid bees will be a cause for concern [133]. Other nest associates in the M. laticeps nest include tachinid fly pupae and a microlepidoptera larvae. However, their presence was not directly linked to bee mortality, suggesting that they are only inquilines. Further study is necessary to ascertain the effect of their presence in the nest as they may be parasites as flies, such as conopids, are known parasitoids of Megachile [134]. Further hypotheses posited from this study include 1) flight activity and voltinism of Megachile is correlated with flowering of floral resources, 2) certain species are specialists (if successfully trap-nested, pollen contents should be analysed [135] preferably including analyses of DNA for identification and population analysis). Longer-term is necessary to further validate the trends observed in this study. Applications of trap-nesting are many, and have been used in other studies to examine sex ratios and nest aggregations [136], maternal Diversity and trap-nesting studies of Singaporean Megachile

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investment [137], oligolecty and polylecty [138], food webs in community ecology [139], and evolution of kleptoparasitic behaviour [140]. The potential for trap-nesting is great and is of interest in both science and management but require optimisation.

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References 1. Michener CD (2007) The Bees of the World 2nd ed.; Michener CD, editor. Baltimore, Maryland, USA: John Hopkins University Press. 992 p. 2. Proctor CD (1978) The pollination of flowers by insects. In: Richards AJ, editor. Linnean Society Symposium Series. Newcastle upon Tyne: Academic Press. pp. 104-114. 3. Shimizu A, Dohzono I, Nakaji M, Roff DA, Miller DG, et al. (2014) Fine-tuned bee-flower coevolutionary state hidden within multiple pollination interactions. Scientific Reports 4. 4. James RR, Pitts-Singer TL (2008) Bees in nature and on the farm. In: James RR, Pitts-Singer TL, editors. Bee Pollination in Agricultural Ecosystems. New York, USA: Oxford University Press. pp. 145-165. 5. Corlett RT (2004) Flower visitors and pollination in the Oriental (Indomalayan) Region. Biological Reviews 79: 497-532. 6. Klein A-M, Vaissiére BE, Cane JH, Steffan-Dewenter I, Cunningham SA, et al. (2006) Importance of pollinators in changing landscapes for world crops. Proceedings of the the Royal Society B 274: 303-313. 7. Roubik DW (1989) Ecology and natural history of tropical bees. Cambridge: Cambridge University Press. 8. Kremen C (2008) Crop pollination services from wild bees. In: James RR, Pitts-Singer TL, editors. Bee Pollination in Agricultural Ecosystems. New York, USA: Oxford University Press. pp. 145-165. 9. Garibaldi LA, Carvalheiro LG, Leonhardt SD, Aizen MA, Blaauw BR, et al. (2014) From research to action: enhancing crop yield through wild pollinators. Frontiers in Ecology and the Environment 12: 439-447. 10. Ascher JS, Pickering J (2014) Discover Life Bee Species Guide and World Checklist (Hymenoptera: Apoidea: Anthophila). Draft 40 26 July 2014. 11. Gonzalez VH (2008) Phylogeny and classification of the bee tribe Megachilini (Hymenoptera: Apoidea, Megachilidae), with emphasis on the genus Megachile. Kansas: University of Kansas. 274 p. 12. Niu Z, Wu Y-R, Zhu C-d (2012) A review of Megachile (Chelostomoda) Michener (Megachilidae: Megachilini) known from China with the description of a new species. Zootaxa 3267: 55-64. 13. Stephan WP (1981) The design and function of field domiciles and incubators for leafcutting bee management (Megachile rotundata (Fabricius)). In: Agricultural Experiment Station OSU, editor. Corvallis, Oregon. 14. Bohart GE (1972) Management of wild bees for the pollination of crops. Annual Review of Entomology 17: 287-312. 15. Pitt-Singer TL, Cane JH (2011) The alfalfa leafcutting bee, Megachile rotundata: the world's most intensively managed solitary bee. Annual Reviews of Entomology 56: 221-237. Diversity and trap-nesting studies of Singaporean Megachile

60

16. Pitts-Singer TL (2008) Past and present management of alfafa bees. In: James RR, Pitts-Singer TL, editors. Bee Pollination in Agricultural Ecosystems. New York, USA: Oxford University Press. pp. 145-165. 17. Córdoba SA, Cocucci AA (2011) Flower power: its association with bee power and floral functional morphology in papilionate legumes. Annals of Botany 108: 919-931. 18. Baker DB, Engel MS (2006) New subgenus of Megachile from Borneo with arolia (Hymenoptera: Megachilidae). American Museum Novitates 3505: 1-11. 19. Rasmussen C, Ascher JS (2008) Heinrich Friese (1860-1948): Names proposed and notes on a pioneer melittologist (Hymenoptera, Anthophila). Zootaxa 1833: 1-118. 20. Zuparko RL (2006) The Published Names of T. D. A. Cockerell. Part I. Hymenoptera. 21. Gupta RK (1993) Taxonomic studies on the Megachilidae of North-Western India. Jodhpur, India. 291 p. 22. Smith F (1857) Catalogue of the hymenopterous insects collected at Sarawak, Borneo; Mount Ophir, Malacca; and at Singapore, by A.R. Wallace. Journal of the Proceedings of the Linnean Society of London Zoology 6: 42-88. 23. Cockerell TDA (1918) Descriptions and records of bees - LXXX. Annals and Magazine of Natural History 9: 390-384. 24. Cockerell TDA (1918) The megachilid bees of the Philippine Islands. The Philippine Journal of Science 13. 25. Liow LH (2001) Bee diversity along a gradient of disturbance in tropical lowland forests of Southeast Asia. CMB:s Skriftserie 3: 101-130. 26. Chong ESM (2010) Bee visitors to Melastoma malabathricum in Singapore. Singapore: National University of Singapore. 41 p. 27. Soh ZWW, Ngiam RWJ (2013) Flower visiting bees and wasps in Singapore Parks. Nature in Singapore 6: 153-172. 28. Ascher J, Risch S, Soh ZWW, Lee JXQ, Soh EJY (submitted) Megachile leaf-cutter and resin bees of Singapore (Hymenoptera: Apoidea: Megachilidae). Raffles Bulletin of Zoology. 29. Sheffield CS, Ratti C, Packer L, Griswold T (2011) Leafcutter and mason bees of the genus Megachile Latreille (Hymenoptera: Megachilidae) in Canada and Alaska. Canadian Journal of Arthropod Identification 8. 30. Carvalho MRd, Bockmann FA, Amorim DS, Brandão CRF, de Vivo M, et al. (2007) Taxonomic impediment or impediment to taxonomy? A commentary on systematics and the cybertaxonomic-automation paradigm. Evolutionary Biology 34: 140-143. 31. Schlick-Steiner BC, Steiner FM, Seifert B, Stauffer C, Christian E, et al. (2010) Integrative taxonomy: a multisource approach to exploring biodiversity. Annual Review of Entomology 55: 421-438. 32. Gathmann A, Tscharntke T (2002) Foraging ranges of solitary bees. Journal of Animal Ecology 71: 757-764.

Diversity and trap-nesting studies of Singaporean Megachile

61

33. Ivanochko M (1979) Taxonomy, biology and alfafa pollinating potential of Canadian leafcutter bees - genus Megachile Latrielle (Hymenoptera: Megachilidae). Montreal, Canada: McGill University. 410 p. 34. Eickwort GC, Matthews RW, Carpenter J (1981) Observations on the nesting behavior of Megachile rubi and M. texana with a discussion of the significance of soil nesting in the evolution of megachilid bees (Hymenoptera: Megachilidae). Journal of the Kansas Entomological Society 54: 557-570. 35. Krombein KV (1967) Trap-nesting wasps and bees: life histories, nests, and associates. Washington: Smithsonian Press. 36. Westphal C, Bommarco R, Carré G, Lamborn E, Morison N, et al. (2008) Measuring bee diversity in different European habitats and biogeographical regions. Ecological Monographs 78: 653-671. 37. Barthléméy C (2012) Nest Trapping, a simple method for gathering information on life histories of solitary bees and wasps. Bionomics of 21 species of solitary aculeate in Hong Kong. Hong Kong Entomological Bulletin 4: 3-37. 38. Sheffield CS, Kevan PG, Westby SM, Smith RF (2008) Diversity of cavity-nesting bees (Hymenoptera: Apoidea) within apple orchards and wild habitats in the Annapolis Valley, Nova Scotia, Canada. Canadian Entomologist 140: 235-249. 39. Tylianakis JM, Klein AM, Tscharntke T (2005) Spatiotemporal variation in the diversity of hymenoptera across a tropical habitat gradient. Ecology 86: 3296-3302. 40. Jayasingh DB, Freeman BE (1980) The comparative population dynamics of eight solitary bees and wasps (Aculeata; Apocrita; Hymenoptera) trap-nested in Jamaica. Biotropica 12: 214-219. 41. Klein A-M, Steffan-Dewenter I, Buchori D, Tscharntke T (2002) Effects of land-use intensity in tropical agroforestry systems on coffee-visiting and trap-nesting bees and wasps. Conservation Biology 16: 1003-1014. 42. Gaston KJ, Smith RM, Thompson K, Warren PH (2005) Urban domestic gardens (II): experimental tests of methods for increasing biodiversity. Biodiversity and Conservation 14: 395-413. 43. Puniamoorthy J, Grootaert P, Foo M, Ascher J, Meier R. Mangrove Insect Project (MIP): species discovery, inventory and habitat assessment; 2014; Signapore. 44. BBC (2014) February was Singapore's driest month since 1869. BBC News Business. 45. Yee ATK, Corlett RT, Liew SC, Tan HTW (2011) The vegetation of Singapore―An updated map. The Gardens’ Bulletin, Singapore 63: 205-212. 46. QGIS Development Team (2014) QGIS Geographic Information System. Open Source Geospatial Foundation Project. 47. Seifan M, Hoch E-M, Hanoteaux S, Tielbörger K (2014) The outcome of shared pollination services is affected by the density and spatial pattern of an attractive neighbour. Journal of Ecology 102: 953-963.

Diversity and trap-nesting studies of Singaporean Megachile

62

48. Klimov PB, OConnor BM (2008) Morphology, Evolution, and Host Associations of BeeAssociated Mites of the family Chaetodactylidae (Acari: Astigmata); Burch JB, editor. Ann Arbor, USA: Museum of Zoology, University of Michigan. 736 p. 49. Colwell RK (2013) EstimateS: Statistical estimation of species richness and shared species from samples. Version 9 and earlier. User’s Guide and application. 50. Grytnes J-A, Romdal TS (2008) Using museum collections to estimate diversity patterns along geographical gradients. Folia Geobotanica 43: 357-369. 51. Mueller UG, Wolf-Mueller B (1993) A method for estimating the age of bees: age-dependent wing wear and colouration in the wool-carder bee Anthidium manicatum (Hymenoptera: Megachilidae). Journal of lnsect Behavior 6: 259-536. 52. Quicke DLJ, Belshaw R, Lopez-Vaamonde C (1998) Preservation of hymenopteran specimens for subsequent molecular and morphological study. Zoologica Scripta 28: 261-267. 53. Lim GSM (2009) Can DNA sequences help with sorting biodiversity samples? Singapore: National University of Singapore. 149 p. 54. Magnacca KN, Brown MJF (2012) DNA barcoding a regional fauna: Irish solitary bees. Molecular Ecology Resources 12: 990-998. 55. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proceedings of the Entomological Society of London Series B 270. 56. Gene Codes Corporation Sequencher®. Ann Arbor, Michigan, USA. 57. Davies OK, Groom SVC, Ngo HT, Stevens MI, Schwarz MP (2013) Diversity and origins of Fijian leaf-cutter bees (Megachilidae). Pacific Science 67: 561-570. 58. Sedivy C, Praz CJ, Müller A, Widmer A, Dorn S (2008) Patterns of host-plant choice in bee of genus Chelostoma: the constraint hypothesis of host-range evolution in bees. Evolution 62: 2487-2507. 59. Sedivy C, Dorn S, Widmer A, Müller A (2012) Host range evolution in a selected group of osmiine bees (Hymenoptera: Megachilidae): the Boraginaceae-Fabaceae paradox. Biological Journal of the Linnean Society 108: 35-54. 60. Stahlhut JK, Fernández-Triana J, Adamowicz SJ, Buck M, Goulet H, et al. (2013) DNA barcoding reveals diversity of Hymenoptera and the dominance of parasitoids in a subarctic environment. BMC Ecology 13. 61. Françoso E, Arias MC (2013) Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini-barcode. Molecular Ecology Resources 13: 844-850. 62. Sheffield CS, Hebert PN, Kevan PG, Packer L (2009) DNA barcoding a regional bee (Hymenoptera: Apoidea) fauna and its potential for ecological studies. Molecular Ecology Resources 9: 196-207. 63. Paradis E, Claude J, Strimmer K (2004) APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20: 289-290. 64. R Core Team (2014) R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. Diversity and trap-nesting studies of Singaporean Megachile

63

65. Katoh S (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772-780. 66. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30: 27252729. 67. Meier R, Shiyang K, Vaidya G, Ng PKL (2006) DNA barcoding and taxonomy in Diptera: A tale of high intraspecific variability and low identification success. Systematic Biology 55: 715-728. 68. Srivathsan A, Meier R (2011) On the inappropriate use of Kimura-2-parameter (K2P) divergences in the DNA-barcoding literature. Cladistics 28: 190-194. 69. Maeta Y, Yamaguchi T, Goubara M (1997) The unusual nest of a leaf-cutting bee, Megachile igniscopata Cockerell, form the Iriomote Island, southernmost Japan (Hymenoptera: Megachilidae). Japanese Journal of Entomology 65: 1-6. 70. Boo CM, Omar-Hor K, Ou-Yang CL (2003) 1001 Garden Plants In Singapore. Singapore: National Parks Board. 71. Korhonen L, Korhonen KT, Rautiainen M, Stenberg P (2006) Estimation of forest canopy cover: a comparison of field measurement techniques. Silva Fennica 40: 577-588. 72. Bolker BM, Brooks ME, Clark CJ, Geange SW, Poulsen JR, et al. (2009) Generalized linear mixed models: a practical guide for ecology and evolution. Trends in Ecology and Evolution 24: 127-135. 73. Bates D, Maechler M, Bolker B, Walker S (2014) Linear mixed-effects models using Eigen and S4. R package version 1.1-7. 74. Nakagawa S, Schielzeth H (2012) A general and simple method for obtaining R2 from generalized linear mixed-effects models. Methods in Ecology and Evolution 4: 133-142. 75. Engel MS, Schwarz M (2011) Two species of Alocanthedon from Indonesia and Malaysia (Hymenoptera: Megachilidae). Entomofauna 32: 429-436. 76. Michener CD (1965) A classification of the bees of the Australian and South Pacific regions. Bulletin of the American Museum of Natural History 130: 1-362. 77. Engel MS, Gonzalez VH (2011) Alocanthedon, a new subgenus of Chalicodoma from Southeast Asia (Hymenoptera, Megachilidae). ZooKeys 101: 51-80. 78. Sarnat EM (2008) PIA Key: Identification guide to invasive ants of the Pacific Islands, Edition 2.0, Lucid v. 3.4. Center for Plant Health Science and Technology, University of California Davis. 79. Antwiki Contributors (2014) Antwiki. 80. Brook BW, Sodhi NS, Ng PKL (2003) Catastrophic extinctions follow deforestation in Singapore. Nature 424: 420-426. 81. Sakai S, Momose K, Yumoto T, Nagamitsu T, Nagamasu H, et al. (2005) Plant reproductive phenology and General Flowering in a mixed dipterocarp forest. In: Roubik DW, Sakai S,

Diversity and trap-nesting studies of Singaporean Megachile

64

Karim AAH, editors. Pollination Ecology and the Rainforest Sarawak Studies Ecological Studies. New York, USA: Springer. pp. 49-64. 82. H. CR, C. CY, G. MA (1989) The CO-I and CO-II region of the honeybee mitochondrial DNA: evidence of variation in insect mitochondrial evolutionary rates. Molecular Biology and Evolution 6: 399-411. 83. Pisani D, Carton R, Campbell LI, Akanni WA, Mulville E, et al. (2013) An overview of arthropod genomics, mitogenomics, the evolutionary origins of the arthropod proteome. In: Minelli A, Boxshall G, Fusco G, editors. Arthropod Biology and Evolution: Molecules, Development, Morphology. Heidelberg, Germany: SpringerLink. 84. Ratnasingham S, Hebert PDN (2013) A DNA-based registry for all animal species: The Barcode Index Number (BIN) System. PlosOne. 85. Tan DSH, Ang Y, Lim GS, Ismail MRB, Meier R (2009) From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences, morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera). Zoologica Scripta 39: 51-61. 86. Bees, Wasps and Ants Recording Society (2013) Megachile willughbiella. 87. Hurst GDD, Jiggins FM (2005) Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts. PNAS 272: 1525-1534. 88. National Center for Biotechnology Information (2014) MT-CO1 mitochondrially encoded cytochrome c oxidase I. Washington, USA. 89. Curiel J, Morrone JJ (2012) Association of larvae and adults of Mexican species of Macrelmis (Coleoptera: Elmidae): a preliminary analysis using DNA sequences. Zootaxa 3361: 5662. 90. Smith VS, Rycroft S, Scott B, Baker E, Livermore L, et al. (2012) Scratchpads 2.0: a virtual research environment infrastructure for biodiversity data. 91. Schuh RT, Hewson-Smith S, Ascher JS (2010) Specimen databases: A case study in entomology using web-based software. American Entomologist 56: 206-216. 92. Momose K, Karim AAH (2005) The plant-pollinator community in a lowland dipterocarp forest. In: Roubik DW, Sakai S, Karim AAH, editors. Pollination Ecology and the Rainforest Sarawak Studies Ecological Studies. pp. 65-72. 93. Fenster CB, Armbruster WS, Wilson P, Dudash MR, Thomson JD (2004) Pollination syndromes and floral specialisation. Annual Review of Ecology, Evolution, and Systematics 35: 375-403. 94. Praz CJ, Müller A, Dorn S (2008) Specialised bees failed to develop on non-host pollen: do plants chemically protect their pollen. Ecology 89: 795-804. 95. Chong KY, Tan HTW, Corlett RT (2009) A checklist of the total vascular plant flora of Singapore: Native, Naturalised and Cultivated Species. In: Tan HTW, editor. Raffles Museum of Biodiversity Research: Singapore. pp. 186.

Diversity and trap-nesting studies of Singaporean Megachile

65

96. Müller A, Bansac N (2003) A specialized pollen-harvesting device in western palaearctic bees of the genus Megachile (Hymenoptera, Apoidea, Megachilidae). Apidologie 35: 327-337. 97. Jong-Yoon K (2003) Female size and fitness in the leaf-cutter bee Megachile apicalis. Ecological Entomology 22: 275-282. 98. O'Neill KM, Pearce AM, O'Neill RP, Miller RS (2010) Offspring size and sex ratio variation in a feral population of alfalfa leafcutting bees (Hymenoptera: Megachilidae). Annals of the Entomological Society of America 103: 775-784. 99. Vergara CH (2008) Environmental impacts of bees introduced for crops pollination. In: James RR, Pitts-Singer TL, editors. Bee Pollination in Agricultural Ecosystems. New York, USA: Oxford University Press. pp. 145-165. 100. Genaro JA (1996) Plantas usadas por abejas del genero Megachile para construir las celdillas de sus nidos (Hymenoptera: Megachilidae). Carribean Journal of Sciences 32: 365-368. 101. Chang YZ, Chen HM, Qi RS (1989) Ornamental pest - studies on leafcutting bees Megachile subtranquilla Yasumatsu. Acta Agriculturae Universitatis Pekinensis 15: 208-213. 102. Stevens PF (2001 onwards) Angiosperm Phylogeny Website. Version 12. 103. Morato EF, Martins RP (2006) An overview of proximate factors affecting the nesting behavior of solitary wasps and bees (Hymenoptera: Aculeata) in preexisting cavities in wood. Neotropical Entomology 35: 285-298. 104. Horne M (1995) Leaf area and toughness: effects on nesting material preferences of Megachile rotundata (Hymenoptera: Megachilidae). Annals of the Entomological Society of America 88: 868-875. 105. Bilinski M, Gosek J, Kuna K, Kaczmarska K, Jablonski B (1980) Rosliny wykorzystywane przez miesiarki (Megachile Latr.). Pszczelnicze zeszyty naukowe 24: 97-112. 106. Messer AC (1984) The world's largest bee rediscovered living communally in termite nests (Hymenoptera: Megachilidae). Journal of the Kansas Entomological Society 57: 165-168. 107. Messer AC (1985) Fresh dipterocarp resins gathered by megachild bees inhibit growth of pollen-associated fungi. Biotropica 17: 175-176. 108. Kumari P, Kumar NR (2014) Studies on Megachile Latreille subgenus Callomegachile Michener (Hymenoptera: Megachilidae) from Chandigarh and Haryana plains, India. Zootaxa 2814: 591-599. 109. Sakai S (2001) Phenological diversity in tropical forests. Population Ecology 43: 77-86. 110. Hamann A (2004) Flowering and fruiting phenology of a Philippine submontane rain forest: climatic factors as proximate and ultimate causes. Journal of Ecology 92: 24-31. 111. van Schiak CP, Terborgh JW, Wright SJ (1993) The phenology of tropical forests: adaptive significance and consequences for primary consumers. Annual Review of Entomology 24: 353-377. 112. Aravind NA, Ganeshaiah KN, Shaanker RU (2013) Indian monsoons shape dispersal phenology of plants. Biology Letters 9: 20130675.

Diversity and trap-nesting studies of Singaporean Megachile

66

113. Kato M, Kosaka Y, Kawakita A, Okuyama Y, Kobayashi C, et al. (2008) Plant–pollinator interactions in tropical monsoon forests in Southeast Asia. American Journal of Botany 95: 1375-1394. 114. Harrison RD (2005) A severe drought in Lambir Hills National Park. In: Roubik DW, Sakai S, Karim AAH, editors. Pollination Ecology and the Rainforest Sarawak Studies Ecological Studies. New York, USA: Springer. pp. 65-72. 115. Corlett RT (1990) Flora and reproductive phenology of rain forest at Bukit Timah, Singapore. Journal of Tropical Ecology 6: 55-63. 116. Momose K, Yumoto T, Nagamitsu T, Kato M, Nagamasu H, et al. (1998) Pollination biology in a lowland dipterocarp forest in Sarawak, Malaysia. I. Characteristics of the plantpollinator community in a lowland dipterocarp forest. American Journal of Botany 85: 1477-1501. 117. Online community of Singapore Flowering (2014) Singapore Flowering. 118. Shivanna KR, Tandon R (2014) Reproductive ecology of flowering plants: a manual; Shivanna KR, Tandon R, editors. New Dehli, India: Springer. 119. Wolda H (1988) Insect seasonality: why? Annual Review of Ecology and Systematics 19: 118. 120. Rao AN, Wee YC (1989) Singapore trees. Singapore: Singapore Institute of Biology. 121. Yumoto T, Nakashizuka T (2005) The canopy biology program in Sarawak: scope, methods, and merit. In: Roubik DW, Sakai S, Karim AAH, editors. Pollination Ecology and the Rainforest Sarawak Studies Ecological Studies. New York, USA: Springer. pp. 13-21. 122. Tscharntke T, Gathmann A, Steffan-Dewenter I (1998) Bioindication using trap-nesting bees and wasps and their natural enemies community structure and interactions. Journal of Applied Ecology 35: 708-719. 123. Albrecht M, Duelli P, Schmid B, Müller CB (2007) Interaction diversity within quantified insect food webs in restored and adjacent intensively managed meadows. Journal of Animal Ecology 76: 1015-1025. 124. Frankie GW, Thorp RW, Newstrom-Llyod LE, Rizzardi MA, Barthell JF, et al. (1998) Monitoring solitary bees in modified wildland habitats: implications for bee ecology and conservation. Environmental Entomology 27: 1137-1148. 125. Miyano S, Yamaguchi T (2001) Ants reduce nest building activities of tube-nesting wasps and bees (Hymenoptera). Entomological Science 4: 243-246. 126. Tylianakis JM, Tscharntke T, Lewis OT (2007) Habitat modification alters the structure of tropical host–parasitoid food webs. Nature 445. 127. Krushelnycky P, Holway D, LeBrun E (2010) Invasion processes and causes of success. In: Lach L, Parr C, Abbott K, editors. Ant Ecology. Oxford, UK: Oxford University Press. 128. Holldobler B, Wilson EO (1990) The Ants. Berlin, Germany: Springer. 732 p. 129. Staab M, Ohl M, Zhu C-D, Klein A-M (2014) A unique nest-protection strategy in a new species of Spider Wasp. PlosOne 9: e101592. Diversity and trap-nesting studies of Singaporean Megachile

67

130. Alqarni AS, Hannan MA, Gonzalez VH, Engel MS (2014) Nesting biology of the leafcutting bee Megachile minutissima (Hymenoptera: Megachilidae) in Central Saudi Arabia. Annals of Entomological Society America 107: 635-640. 131. Sheffield CS, Wilkes MA, Cutler GC, Hermanutz L (2014) An artificial nesting substrate for Osmia species that nest under stones, with focus on Osmia inermis (Hymenoptera: Megachilidae). Insect Conservation and Diversity. 132. National Parks Board (Singapore) (2013) Community in Bloom. 133. Cane J, Schiffhauer D, Kervin LJ (1996) Pollination, foraging, and nesting ecology of the leaf-cutting bee Megachile (Delomegachile) addenda (Hymenoptera: Megachilidae) on Cranberry Beds. Ecology and Population Biology 89: 361-367. 134. Cardoso CF, Silveria FA (2012) Nesting biology of two species of Megachile (Moureapis) (Hymenoptera: Megachilidae) in a semideciduous forest reserve in southeastern Brazil. Apidologie 43: 71-81. 135. Muller A, Diener S, Schnyder S, Stutz K, Sedivy C, et al. (2006) Quantitative pollen requirements of solitary bees: Implications for bee conservation and the evolution of bee– flower relationships. Biological Conservation 130: 604-615. 136. Paini DR (2004) Nesting biology of an Australian resin bee (Megachile sp.; Hymenoptera: Megachilidae): a study using trap nests. Australian Journal of Entomology 43: 10-15. 137. Seidelmann K (2014) Optimal progeny body size in a solitary bee, Osmia bicornis (Apoidea: Megachilidae). Ecological Entomology Early view. 138. Praz C (2008) Floral specialization in solitary bees: a case study of the osmiine bees. Zurich, Switzerland: ETH Zurich. 131 p. 139. Laliberté E, Tylianakis JM (2010) Deforestation homogenizes tropical parasitoid-host networks. Ecology 91: 1740-1747. 140. Rozen JG, Kamel SM (2008) Hospicidal behavior of the cleptoparasitic bee Coelioxys (Allocoelioxys) coturnix, including descriptions of its larval instars (Hymenoptera, Megachilidae). American Museum Novitates 3636: 1-15. In Appendices 141. Cane JH, Minckley RL, Kervin L (2001) Sampling bees (Hymenoptera: Apiformes) for pollinator community studies: pitfalls of pan-trapping. Journal of Kansas Entomological Society 73: 208-214. 142. Grismer JL, Grismer LL, Das I, Yaakob NS, Lim BL, et al. (2004) Species diversity and checklist of the herpetofauna of Pulau Tioman, Peninsular Malaysia, with a preliminary overview of habitat utilization. Asiatic Herpetological Research 10: 247-279. 143. Michener CD (1979) Biogeography of bees. Annals of the Missouri Botanical Gardens 66: 298-347.

Diversity and trap-nesting studies of Singaporean Megachile

68

144. Krombein KV (1950) The aculeate Hymenoptera of Micronesia II. Colletidae, Halictidae, Megachilidae, and Apidae. Proceedings of the Hawaiian Entomological Society 15: 101142. 145. Michener CD, Szent-Ivany JJH (1960) Observations on the biology of a leaf-cutter bee Megachile frontalis in New Guinea. Papua New Guinea Agricultural Journal 13: 22-35. 146. Willmer PG, Stone GN (2011) Incidence of entomophilous pollination of lowland coffee (Coffea canephora); the role of leaf cutter bees in Papua New Guinea. Entomologia Experimentalis et Applicata 50: 113-124. 147. Griswold T (2001) Two new species of trap-nesting Anthidiini (Hymenoptera: Megachilidae) from Sri Lanka. Proceedings of Entomological Society of America 103: 269-273. 148. Krombein KV, Norden BB (2001) Notes on trap-nesting Sri Lankan wasps and bees (Hymenoptera: Vespidae, Pompilidae, Sphecidae, Colletidae, Megachilidae). Proceedings of the Entomological Society of Washington 103: 274-281. 149. Müller A (2013) Palaearctic Osmiine Bees. ETH Zürich. 150. Lieftinck MA (1954) Bij het nest van een Javaans harsbijtje. Idea 10: 20-25.

Diversity and trap-nesting studies of Singaporean Megachile

Appendices Appendix I – a review of published trap-nesting studies for bees and wasps Table I-1. Table summarizing trap-nesting studies reviewed, sorted by year. Information not stated in the paper is represented with an em-dash (–). Information not possible to estimate or calculate is represented with a question mark (?). Monitoring intensity 2–4 wks



Trap units per site –

Cavities per unit 1

40

10

6

180

6083; 3075 lost 43200

0.007%

19

Locality

Climate

Jayasingh and Freeman (1980)

Population biology

Jamaica

Tropics

2 yrs

Gathman et al. (1994)

Germany

Temperate

5 mths

Once at the end

Frankie et al. (1998); Kim and Thorp (2001)

Diversity over agricultural landuse gradient Diversity baseline, invasion ecology

California

Subtropics

2 yrs during summer

3 wks

Wood

6

2

9

12

648

?

At least 30 spp.

Miyano and Yamaguchi (2001)

Ants prevent trap-nesting bees/wasps

Japan

Subtropics

5 mths

Reed

3

3

10

74

740

5–50%/site

Klein et al. (2002)

Diversity over agricultural landuse gradient

Indonesia

Tropical monsoon

5 mths

1 mth once and at the end 2 wks

Reed

12



6

200

14400

SteffanDewenter (2002); SteffanDewenter (2003) Paini (2004)

Ecology

Germany

Temperate

7 mths

Once at the end

Reed

15



8

150

Nesting biology of Megachile Diversity

Australia

Mediterra nean Subtropics semi-arid

1 yr

3–8 weeks 1 mth

6

1

80

4

2

Urban ecology, diversity

Britain

Temperate

3 yrs during summer

Pine wood Bamboo; cardboar d tubes Paper straws; wood; bamboo

20

1

Gaston et al. (2005)

Brazil

4–6 mths

Once at the end

Total no of cavities

No of host spp. 11

Question

Bamboo, pine wood Reed

No of habit -ats

Successful solitary aculeate nesting 44%

Study authors

Aguiar et al. (2005)

Trapnest material

No of site -s 112

Study duration

Structure placed on Walls, wooden structures Wooden post

Cavit -y diameter –

Rain cover/ others No

mixed

Tin

Trees



11

Wooden post

mixed

No/ moved blocks when ants nested Plastic containe -r

13617 brood cells

10

Trees



18000

1640 brood cells

24

Wooden post



Plastic tubes/ sticky glue –

4

1920

45%



Shrubs

mixed

No

1

370

1322

20%

14

?

mixed

2

70

2800

1–30%

12

?



Plastic tarpaulin No

Tylianakis et al. (2005); Tylianakis et al. (2006)

Ecology

Ecuador

Tropics

Buschini (2006)

Diversity

s. Brazil

Albrecht et al. (2007)

Diversity, food webs

Switzerla nd

Temperate coastal Temperate

Loyola and Martins (2008) Sheffield et al. (2008)

Diversity

s. Brazil

Subtropics

Diversity over agricultural landuse gradient

Canada

Temperate

Gazola and Garófalo (2009)

Diversity

c. Brazil

Holzschuh et al. (2010)

Diversity over agricultural landuse gradient

Sabino and Antonini (2011)

Everaars et al. (2011) Barthléméy (2012) Cardoso and Silveria (2012)

1 yr

1 mth

Reed

48

4

9

200

86400

15047 bee/wasp individuals

31

Trees, wooden post



2 wks

Wood

6

3

48

12

1152

11

Once at the end

Reed

44

2

2

200

10400

? 1.5m high Wooden post

mixed

5 mths for 2 yrs 11 mths 3 yrs, Apr – Sep

10%; bees only 19%

mixed

Wooden roof

3–4 days Once at the end

Wood

9

1

5

30

1350

0.10

11

Pole

mixed

No

Paper straws

24

4

6

80

11520

0.03%

13 bees spp.

Trees, fence post

mixed

Subtropics

2 mths

1 mth

3

2

variable

variable

4259

0.09%

12

Trees

simila -r

Germany

Temperate

2 yrs

Once at the end

Bamboo, black cardboar d tubes Reed

Milk carton/ Tanglefo -ot Hard plastic

92

2

2

180

33120

11193 bee/wasp individuals

18

Wooden post



Nesting biology of Megachile

Brazil

Subtropics

4 yrs

2 weeks

1

1

50

?



?

?

Ground

mixed

Landscape and microhabitat preference of Osmia bicornis Nest bionomics

Germany

Temperate

4 mths

Once at the end

Cardboa rd tubes and wooden blocks Bamboo

Chipboa -rd roof for shading No

1

1

350

33

11550

N. A.

No

Subtropics

6 yrs

?

Bamboo

2



?

7

957

simila -r

No

Nesting biology & seasonality of 2 spp. of Megachile

s. Brazil

Subtropics

1 yr

1 mth

Bamboo

1

1

276

6

1656

0.01% for 2 Megachile spp.

4 Meg achi le

Tree, balcony, roof, others Trees and others (but with wire sheath) Trees

mixed

Hong Kong

46% of trap-nest units occupied 34%

mixed

No

39

21

No/ fungicide, Tanglefoot applied No

Appendix II – pan trap trial Methods Soufflé cups were spray-painted with Pylox ultraviolet-yellow spray paint and left to dry for at least half-an-hour. Water is filled half-way mark of 3-oz soufflé cups. A drop of soap was placed decrease tension on the surface of the water. Each soufflé cup was placed five metres apart from each other, outside the grass patch at University Hall. These were following protocols on Bee Inventory Plot Bee Bowls (see http://online.sfsu.edu/beeplot/, pan traps are also known as ‘bee bowls’). Twelve bowls were placed 5 m apart along a linear transect. Bowls were placed in a shaded area to prevent evaporation of liquids. Bowls were checked at 12-hour intervals.

Fig. II-I. A photograph of the pan trap and the trapped insects. Descriptive results Small-bodied parasitic wasps, ants, planthoppers, calliphorid and dolichopodid flies, and butterflies were caught from the traps within 12 hours. After 24 hours, more insects were caught in the traps. After 36 hours, water dried up. This suggests that 24 hours would be an optimal time for the bee bowls collection. No bees were obtained in the traps throughout the observation periods. Discussion Bee bowls seem to be more effective with small-bodied insects, as well as large insects which drown easily in water (e.g., butterflies, blow flies). It has been suggested by Cane [141] that bee bowls are less attractive to bees where floral resources are available, and may be placed at an elevated height to improve number of bee samples. DNA of specimens should not degrade within 24 hours and specimens should be placed in at least 95% ethanol (Wilson, J. J., pers. comm.).

Appendix III – sampling sites and habitat association of Megachile species Background According to Yee et al. (2011), 38.85% of Singapore’s land is not vegetated while 55.48% is vegetation, comprising of 27.45% managed greenery, 19.64% young secondary forest, 5.92 % scrubland, 1.37% old secondary forest, 0.16% primary hill dipterocarp forest, 0.91% mangrove forest and 0.39% freshwater swamp. Methods Sampling was done to in various habitat types and in places in Singapore which there were no local records of bees (see Table IV-1). The longtitude and latitude of species occurrences were plotted on QGIS 2.2.0-Valmiera where species occurrences were matched to habitat types by visually comparing an overlain layers of the 1-km habitat-stratified grid vectors and Open Street Map park vectors (Fig. III-1) and knowledge of the landuse of the collecting locality. A matrix of species and the seven habitat types was generated to summarize absence-presence species-habitat associations (Table III-2), to be interpreted only as a cursory and preliminary assessment of habitats occupied by each species. A similar approach was applied in Grismer et al. [142].

Table III-1. Grids of habitat types (not including young secondary forest and managed greenery) in Singapore and sampling areas in this study. Numbered 1-km grids are based on different habitat types matched by eye to Yee et al. (2011). Grid numbers in bold have been surveyed prior this study. Underlined are the grids which are sampled in the course of this study. Military areas and other inaccessible areas are in square brackets. Habitat Old secondary forest

1 km-grids ID numbers North of Upper Seletar Reservoir: 197, 198, 241, 242, 243, 244; South of Upper Seletar Reservoir: 334, 378, 379, 381, 423, 424, 425, 426; Dairy Farm Nature Park: 427

Primary dipterocarp forest Mangrove/ coastal

Bukit Timah Nature Reserve: 512, 557, 558 Central Catchment Nature Reserve: 333, 526, 562, 563

Freshwater swamp forest Scrubland

South of Upper Seletar Reservoir: 335, 336, 380

Other areas sampled previously

Lorong Danau: 458; HortPark: 687; Toh Tuck: 645; West Coast: 691, 736; Pasir Panjang & Bukit Merah: 828, 1151, 784, 785, 786; Singapore Botanic Gardens: 651, 696; Toa Payoh Town Park: 609; Bedok: 618, 619; Pasir Ris: 439, 440, 441, 485; Pulau Ubin: 305

Other areas sampled in this study but previously with no bee records

Young secondary forest - Mandai secondary forest: 287, 288; Rifle Range and Jelutong Tower: 515; MacRitchie Reservoir Park: 562; Chestnut forest: 422; Kent Ridge: 782; Green corridor: 557; Managed greenery - Clean Tech Park: 504; Chinese Garden: 507; Zhenghua Park: 421; Clementi Woods: 736; West Coast Park: 781; Ang Mo Kio Town Park: 431

Sungei Buloh Wetland Reserve: 101, 102, 103, 146; Sungei Khatib Bongsu: 159, 160; Pulau Ubin: 260, 261, 262; Labrador Nature Park: 920

Tuas: 814; Kranji: 147, 148; Sembawang: [150, 200, 201]; Punggol: 255; Lorong Halus: 391, 392; Tampines Eco Green: 484; Changi Airport: [490, 535, 537, 579, 580, 582, 626, 642, 669, 670, 671]

Fig. III-1. Map of Singapore showing various vector layers, including square grids of 1 km and 100 m created with QGIS. 26 grids out of 563 grids were previously sampled. Each 1 km grid is further split into 100 m square grids, which is labelled horizontally by alphabets and vertically by numbers (see inset). Jurong Island, Pulau Semakau and Pulau Tekong were left out of ‘grids’ in the map either because it is off-limits or very industrialised. Basemap by OpenStreetMap. GRIDS BY COLOUR: BLUE GRIDS – FRESHWATER SWAMP FOREST; LIGHT GREEN GRIDS – MANGROVES; DARK GREEN GRIDS – SCRUBLAND; RED GRIDS – OLD SECONDARY FOREST. YOUNG SECONDARY FORESTS AND MANAGED GREENERY ARE NOT SHADED BY COLOUR. OTHER SYMBOLS: PINK STAR – TRAP-NESTING SITES; YELLOW DOTS – BEE SPECIMENS FROM ‘BEEDAT’, GREEN LINES – PARK CONNECTORS (GOOGLE MAPS PUBLIC MAP), BLUE LINE – GREEN CORRIDOR; GREEN SHAPE VECTORS – MANAGED PARKS (OPEN STREET MAP VECTOR DATA).

Table III-2. A preliminary assessment of habitat occupied by Megachile species. THE FOLLOWING ABBREVIATIONS APPLY: MG – MANAGED GREENERY; SC – SCRUBLAND (ABANDONED), YSF – YOUNG SECONDARY FOREST; OSF – OLD SECONDARY FOREST; PD – PRIMARY DIPTEROCARP FOREST; SF – FRESHWATER SWAMP FOREST; MN – MANGROVES OR COASTAL AREAS.

MG Megachile (Aethomegachile) borneana* M. (Aet.) nr. borneana* M. (Aet.) conjuncta M. (Aet.) laticeps M. (Aet.) ramera+ M. (Aet.) fusciventris sp.-group* M. (Alocanthedon) cf. indonesica M. (Callomegachile) fulvipennis M. (Cal.) disjuncta M. (Cal.) biroi sp.-group M. (Cal.) stulta M. (Cal.) nr. stulta* M. (Cal.) ornata M. (Cal.) tuberculata M. (Cal.) umbripennis M. (Chelostomoda) moera+ M. (Creightonella) atrata M. (Eutricharaea) sp. 1 (nr. subrixator, white scopa) M. (Eut.) sp. 2 (crenulated T6)* M. (Eut.) subrixator M. (Paracella) tricincta 21 species in total

SC

YSF

OSF

PD

SF

MN

× × ×

× ×

× × × × × × × 11

× ×

×

×

× × × × ×**

×

× × ×

×

× ×

×

×

5

11

× × × × × × × ×

× × × × × × × × ×

× ×

× ×

× ×

× ×

× 8

×

Singaporean voucher in LKCNHM

× × × × ×

×

× 12

Singleton collection in Singapore ×

× 5

3

×

× × × ×

×

×

6

10

* only known from the male in Singaporean records; + only known from the female in Singaporean records; **new record in Nov 2014

1

No. of habitat types occupied 1

3 4 74 1 7 13 36 100 8 25 1 1 6 100 1 26 13

2 1 6 1 3 3 3 5 3 4 1 1 3 4 1 4 3

1 22 39 482

1 4 3

Specimen s from Singapore

Appendix IV – detailed procedures for DNA extraction and sequencing for cox1, sequence lengths and supplementary distance analyses Tissue digestion was performed in 900 µl of CTAB digestion buffer and 20 µl of Proteinase K solution for 15 h with regular vortexing. 500 µl of phenol-chloroform was used for the elution of the DNA was done in two repeated steps where centrifugation was done in between (13400 RPM, 10 min). After spinning, the top chloroform layer, containing DNA, was removed in each step before addition of the subsequent phenolchloroform layer. To precipitate the DNA, 80% ethanol was added to the aqueous phase and left to settle for 10 min. After which, it was centrifuged at 13400 RPM for 30 min. Ethanol was removed and dried, leaving a DNA pellet at the base of the Eppendorf tube. After adding 20 µl Rnase-free water, the concentration of DNA was quantified with a NanoDropTM Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA). Each PCR reaction comprised of 0.15 µl ExTaq TM (TaKaRa, Kyoto, Japan) or 0.25 µl BioReady rTaq (Bulldog Bio, Portsmouth, USA), 2.5 µl 10x buffer, and 2 µl 2mM dNTP mixture, provided by TaKaRa or BioReady, 1 µl 100 µM primer for both forward and backward direction, and 90 µl Dnase-free sterile RO water (10 × dilution), totalling a volume of 25 µl per Eppendorf tube. The primers used were either LCOHym forward and Nancy reverse or Lep F1 and Lep R1 reverse. Cycling temperatures were: 95ºC to activate Taq polymerase, and 34 cycles of 95ºC for 45 seconds (denaturation), 50ºC for 30 seconds (annealing) and 72ºC for one minute (extension). Amplification products were kept on hold at 20ºC until they were retrieved for gel electrophoresis to confirm successful amplification. Purification was performed by Sure Clean (Bioline, Randolph, MA). Cycle sequencing was then performed in 10-μL volumes with BigDye ver. 3.1 (Applied Biosystems, Foster, CA) for both forward and reverse directions used according to specifications by manufacturer. A final purification was done with CleanSEQ® kit (Agencourt® Bioscience Corporation, Beverly, MA). Sequencing was carried out in an ABI PRISM ® 3130 xL Genetic Analyzer (Applied Biosystems, Foster, CA).

JModelTest2 was used to choose the best nucleotide substitution model based on Aikaike Information Criterion (AIC) for ML and Bayesian analyses. Bayesian analysis was performed with BEAST (nucleotide substitution model GTR I+G; clock model relaxed uncorrelated lognormal; tree prior coalescent: constant population size; MCMC chain length of 10 million with 1000 burn-in) for comparison purposes. DnaSNP Unweighted Pair Group Method with Arithmetic Mean (UPGMA) (Kimura 2-parameter, 1000 bootstraps) (explore distance under a strict molecular clock), Maximum Parsimony (1000 bootstraps, tree bisection and reconnection heuristic algorithm, with the MaxTrees set at 1000) (MP) (explore steps), and Maximum likelihood (GTR I+G substitution model, 500 bootstraps, 1+2, 3 position codon) (ML) trees (explore phylogenetic relationships)

Specimens sequenced or to be sequenced in Table V-1. Species without fresh specimens for barcoding include: M. (Aethomegachile) borneana, M. (Aet.) nr. borneana, M. (Aet.) conjuncta, M. (Aet.) ramera, M. (Callomegachile) nr. stulta, M. (Chelostomoda) moera, M. (Eutricharaea) sp. 2 (crenulated T6). Table IV-1. Barcoded specimens (12 species) with the cox1 gene. Only M. umbripennis and M. laticeps has been sequenced in Davies et al. [57] and is publicly available on GenBank. Collection information can be retrieved on Google Fusion Tables. DNA concentration was measured with NanoDropTM Spectrophotometer. Maximum number of base pairs (bp) is 652 and the minimum (M. tuberculata) is 500. Specimen code

Species

Sex

Batch03-016 PRPB005 Batch03-011 Batch04-030 NUS-9Jun2014b Batch03-002 Batch03-003 PGA-003 Batch03-015 CGA-011 DFB-013 Batch04-009 DFB-027 Batch03-005 CGA-006 CGA-012 CTA-007 PGA-004 MDA-010 PRP-A023

Megachile atrata Megachile atrata Megachile biroi sp.-group Megachile disjuncta Megachile disjuncta Megachile fulvipennis Megachile fusciventris sp.-group Megachile (Eutricharaea) sp. 1 (nr. subrixator) Megachile ornata Megachile subrixator Megachile stulta Megachile tricincta Megachile tricincta Megachile tuberculata Megachile umbripennis Megachile (Eutricharaea)^ Megachile (Eutricharaea)^ Megachile (Eutricharaea)^ Megachile (Eutricharaea)^ Megachile (Eutricharaea)^

M F F M F M M F F F F F M M F M M M M M

^matched to M. (Eutricharaea) subrixator

DNA conc. after extraction (ng/ʮl) 49 60.8 8 4.1 15.2 22.5 34.5 3.7 81.9 3.9 4.3 8.5 1.6 26.9 8.9 2.4 2.1 4.4 6.4 0.9

Forward primer

Backward Primer

Length of sequence (bp)

LCO Hym LCO Hym LCO Hym LCO Hym LCO Hym LCO Hym Lep1 LCO Hym LCO Hym LCO Hym Lep1 LCO Hym LCO Hym LCO Hym LCO Hym Lep1 Lep1 Lep1 Lep1 Lep1

Nancy Nancy Nancy Nancy Nancy Nancy R1 Nancy Nancy Nancy R1 Nancy Nancy Nancy Nancy R1 R1 R1 R1 R1

642 652 610 631 649 650 647 652 619 599 628 652 649 503 649 652 652 652 620 643

Table IV-2. Comparing interspecific uncorrected distance. Species and the nearest neighbor by the least percentage dissimilarity. The dominant geographical region was obtained from species distribution maps on Discover Life [10] and following biogeographic regions by Michener’s Biogeography of bees [143]. % dissimilarity was underlined when less than 5% and bolded when less than 10%. Dominant Dominant Same Next closest species (by least Same Species geographical geographical % distance vice% distance) subgenera region region versa Megachile atrata@ Oriental Megachile anthracina Oriental 13.1* Megachile biroi sp.-group@ Oriental Megachile disjuncta Oriental 14.0* × Megachile centuncularis Holoarctic Megachile versicolor Palearctic × 9.13 Megachile disjuncta@ Oriental Megachile umbripennis@ Oriental 11.2* × × Megachile frigida Nearctic Megachile willughbiella Palearctic × × 2.36 @ Megachile fulvipennis Oriental Megachile tuberculata Oriental 13.1* × Megachile fusciventris sp.Oriental Megachile melanophaea Palearctic 11.7 group@ Megachile gemula Nearctic Megachile maritima Palearctic × × 5.67 Megachile inermis Palearctic Megachile maritima Palearctic × 9.92* Megachile laticeps@ Oriental Megachile (Eutricharaea) sp. Oriental ^ 12.0* 1 (nr. subrixator) Megachile latimanus Nearctic Megachile gemula Nearctic 6.46* × Megachile melanophaea Palearctic Megachile gemula Nearctic × 5.66 Megachile montivaga Nearctic Megachile frigida Nearctic × 8.63* Megachile ornata@ Oriental Megachile anthracina Oriental 14.7* Megachile pugnata Nearctic Megachile gemula Nearctic 9.13* Megachile stulta@ Oriental Megachile frigida Nearctic 11.15 Megachile (Eutricharaea) sp. Oriental 11.15* 1 (nr. subrixator) Megachile subrixator@ Oriental Megachile rotundata Holoarctic × × 8.01 Megachile tricincta@ Oriental Megachile gemula Nearctic 11.7 Megachile tuberculata@ Oriental Megachile anthracina Oriental 10.9* Megachile versicolor Palearctic Megachile centuncularis Holoarctic × 9.13 Megachile (Eutricharaea) sp. 1 Oriental ^ Megachile maritima Palearctic 8.69 (nr. subrixator) @ * species pairs from the same or overlapping (e.g., in the case of Nearctic or Palarctic with Holoarctic) geographical region ^ this was nominally assumed to be from the Oriental region since it was collected in Singapore; @ Singaporean Megachile

Table IV-3. Comparing intraspecific uncorreted distance for 16 species with multiple sequences. Comparisons with 0% dissimilarity are left as blank in the ‘maximum intraspecific distance’ column. Maximum intraspecific No. of No. of No. of Species distance (by % distance collecting Source of sequence sequences countries dissimilarity) localities Megachile atrata 2.80 2 1 1 This study Megachile centuncularis 2 1 ? GenBank Megachile disjuncta 2 1 2 This study Megachile frigida 0.32 5 1 ? GenBank Megachile gemula 2 1 ? GenBank Megachile inermis 3 1 ? GenBank Megachile laticeps 9 1 1 BOLD Megachile latimanus 2 1 ? GenBank Megachile subrixator 0.83 6 1 5 This study Megachile melanophaea 2 1 ? GenBank Megachile pugnata 2 1 ? GenBank Megachile rotundata 3 1 ? GenBank Megachile tricincta 2.16 2 1 1 This study Megachile umbripennis 0.15 5 2 2 This study (1); BOLD (4) Megachile versicolor 3 1 ? GenBank Megachile willughbiella 2 1 ? GenBank The following GenBank sequences (with accension numbers) were downloaded: Megachile anthracina KF861940.1, Megachile willughbiella JQ909794.1/ JQ909793.1, Megachile versicolor JQ909792.1/ JQ909791.1/ JQ909790.1, Megachile maritima JQ909789.1/ JQ909788.1, Megachile centuncularis JQ909787.1, Megachile frigida JX829541.1/ FJ582311.1/ FJ582310.1/ FJ582309.1/ FJ582308.1, Megachile pilidens JQ677597.1, Megachile rotundata FJ582328.1/ FJ582327.1/ FJ582326.1, Megachile melanophaea FJ582321.1/ FJ582320.1, Megachile gemula FJ582314.1/ FJ582313.1/ FJ582312.1, Megachile montivaga FJ582322.1, Megachile pilidens EU863056.1, Megachile pugnata FJ582325.1/ FJ582324.1/ FJ582323.1, Megachile latimanus FJ582319.1/ FJ582318.1, Megachile inermis FJ582317.1/ FJ582316.1/ FJ582315.1, Megachile centuncularis FJ582307.1

Appendix V – behavioural observations of Megachile foraging from nest Methods For species that could be observed at nests, in-situ observations were made at least from dawn to dusk at least 0650 to 1900 h for one day. Behavioural ethograms were made. Details recorded included behaviour (foraging or in the nest), type of foraging resource, weather, time of the day and duration were noted during observations. Oviposition events were inferred when the bee stayed in the nest for a long period of time and switched from foraging for pollen to lining material [130]. In-situ observations of Megachile (Callomegachile) disjuncta The following observations were made of an M. disjuncta female individual on five days (15 to16, 18 to 20 April 2014), where 15 April 2014 was the first day of nest construction. It stopped constructing the nest between 22 to 25 April 2014, suggesting that nest construction took place between seven to ten days. Four of four days the female was collecting pollen in the morning but was also seen collecting pollen on at 1531 h on 19 April 2014. The individual’s earliest observed foraging time was at 0751 h and latest at 1641 h throughout the observation period (sunrise at 0656 h, sunset 1907 h). On 18 April 2014, where an observation was made from dawn to dusk (0700 h to 1820 h), 17 foraging trips out of the nest was observed within the active foraging period of the individual from 0751 h to 1530 h (459 min). The individual collected either pollen, mud or a white substance at one time. The latter two are presumably nest-lining material. The individual collected nest-lining material on five foraging trips then to pollen on six foraging trips, before reverting back to nest-lining material for seven foraging trips. Foraging was halted at 1530 h when dark clouds started forming and rain ensued later. After the last pollen collection, it stayed for 15 min in the nest, likely for oviposition. Overall, pollen collection took 18.25 ± 1.61 min (n = 19) and stayed in the nest for 4.05 ± 0.23 min (n = 16). Nest-lining collection of mud or a white substance (8.21 ± 1.23 min, n = 25) took a shorter time and the individual stayed in the nest for more variable periods of time (6.14 ± 2.32 min, n = 25). The individual foraged on sunny and cloudy days. Multimedia: http://tinyurl.com/mdisjuncta In-situ observations of Megachile (Creightonella) atrata Observations of M. atrata individual 1 were made for a full day (0743 to 1858 h) and of M. atrata individual 2 from 0753 to 1355 h (Fig. V-1). Individuals 1 and 2 were in the process of nest construction whereas individual 3 had just started excavating its nest. M. atrata individual 3 was observed to start excavating a fresh hole at 1648 h, starting bringing leaves in at 1804 h at intervals of one to two minutes till 1855 h, and stopped activity at 1855 h on 2 June 2014 (sunrise 0657 h, sunset 1908 h). The individuals either collected pollen or leaves (masticated or a whole piece) at one time. Individual 1 made 61 foraging trips. At first activity, it made two trips to collect leaves, presumably for the innermost leaf lining before making ten trips to collect pollen and then switching to leaves for the rest of the day. For individual 1, pollen collection (mean = 12.58 ± 1.69 min, n = 10), and stayed in the nest (mean = 3.37 ± 0.67 min, n = 8) except after the last collection where it stayed for 23 min for oviposition. Masticated leaves were collected 6.88

± 1.51 min (n=4) and stayed in the nest 6.17 ± 1.52 min (n=4). Leaf pieces were collected in 4.29 ± 4.71 min (n=14) and stayed in the nest for variable periods (i.e., 4.82 ± 3.36 (n=15)). Other behaviour included carrying mud out of the nest. Individual 2 made eight trips to collect pollen in the morning, before switching to leaves for the rest of the observation period (n = 32). For individual 2, pollen collection (mean = 13.13 ± 4.09 min, n = 8), and stayed in the nest (mean = 3.05 ± 0.52 min, n = 7) except after the last collection where it stayed for 26 min. Individual 3 had only started excavating the nest. It took mud from the mound and dumped the mud approximately 10 cm away from the mound. As such, a tumulus was not formed outside the nest as sand was not kicked out.

Fig. V-1. Megachile atrata individual 2 activity, A, carrying mud out, B, carrying masticated leaf in, C, scopa full of orange pollen, D, coming out of the nest. Photographs by S. X. Chui. Comparison of foraging behaviour The foraging behaviour of M. (Callomegachile) disjuncta and M. (Creightonella) atrata are summarized in Table V-1Table. Descriptions of foraging activity is described to a greater extent for individual bee species in Appendix VI. The total time required for provisioning a cell (i.e., total time to collect pollen) is approximately the same for M. disjuncta and M. atrata (~2-3h) since a larger bee (i.e., M. atrata) can carry correspondingly more pollen. M. disjuncta foraged mostly in the morning for pollen, whereas M. atrata forages solely in the morning for pollen based on the observed individuals. M. disjuncta was observed to take a shorter total amount of time to construct a cell as compared to M. atrata. This could be due to the variability in amount of time used to collect lining material, as lining is less intricate in resin bees (cf. Chalicodoma sp. [a Megachile (Callomegachile)] in [37]) than in than in leaf-cutter Creightonella bees, which lines its nest with a layer of leaves, then masticated leaves and another layer of leaves. Foraging activity is weather dependent and is halted during rain.

Table V-1. Comparison of foraging behaviour from the nest for Megachile disjuncta and M. atrata.

Amount of time required for construction of a cell (min) Number of trips required for the provisioning of one cell Earliest observed time for foraging (h) Latest observed time for foraging (h) Period of the day where it was seen collecting pollen Mean time required for a pollen collection trip (min) Mean time spent in nest after a pollen collection (min) Mean time required for collection of mud/other lining material (min) Mean time spent in nest after collection of mud/ other lining material (min) Mean time required for leaf collection (min) Mean time spent in nest after leaf collection (min)

Megachile disjuncta (n=1 individual, 5 days) ~½ a day 6 0751 1641 Morning, afternoon 18.25 4.05 8.21

M. atrata (n=2 individuals, 1 day) ~¾ a day 8–10 0743 1858 Morning only 12.58–13.13 3.05–3.37 N. A.

6.14

N. A.

N. A. N. A.

1.34–4.29 1.67–4.83

Appendix VI – descriptive nest bionomics of Megachilidae Methods Standardised nest nomenclature (Fig. 20) were used to describe nest bionomics in bamboo internodes following Krombein Where possible, the gender of emerging bee was noted. For other solitary aculeates, only hole diameter (D), number of provisioning cells (PC), provisioning type, and partitioning (P) and closing plug (CP) material were noted (Appendix X).

Fig. 20. Schematic diagram of the nest architecture with nest bionomics terminology, abbreviated. Abbreviations used VC IC PC PP CP P

Vestibulary cell Intercalary cell Provisioning cell Preliminary plug Closing plug Cell partitioning

Nest bionomics of Megachile (Aethomegachile) laticeps M. (Aethomegachile) nest bionomics have not been recorded in the literature except for a brief note by Krombein [144] that it nests in dead reeds and lines its nest with leaves of Pithecellobium dulce (Fabaceae) in Micronesia. In this study, three nests were recovered from trap-nesting. Nest 1 (ref: UHAunk-2) had two cells (diameter: 8 mm, length: 250 mm, height: 170 cm, direction: 164º, on tree) and was retrieved at an edge to Adinandra belukar secondary rainforest (Kent Ridge, lat., lon.: 1.297126, 103.776906) on 29 Apr 2014. It was incomplete and was partially destroyed by another animal. Nest 2 (ref: HPA002-1) had four cells (diameter: 9 mm, length: 282 mm, height: 172 cm, direction: 196º, on tree) but the first cell was not occupied by any larvae, and was retrieved from a shady area at a managed garden (HortPark, lat., lon.: 1.279034, 103.7996) on 10 May 2014. Nest 3 (ref: DFA014-1) had five cells (diameter: 7 mm, length: 368 mm, height: 45 cm, direction: 106º, on tree) and was retrieved at a sunny area at the edge of a secondary forest near a hill dipterocarp primary rainforest (Dairy Farm Nature Park, lat., lon.: 1.359152, 103.776844) on 10 May 2014. The cells were in a linear series, with individual cells wrapped in leaves and an outer layer of interlocking leaves (three large oval leaves interlocking two cells) (Fig. VI-1). The entirety of the nest can either be flush to the end of the bamboo internode (DFA014-1) or found in the middle (HPA002-1, UHAunk-2).

Fig. VI-1. A, Photograph of the nest in its entirety. B, The leaves used to cap the nest. SCALE BAR: 10 MM. Cell length ranged from 16 to 19 mm (n=4) with Individual cells slightly concave on one end (Fig. VI-2 A–C) and convex on the other side such that the preceding cell fits into the concavity of the following. Nest are sealed with two circular leaf pieces. A dissected cell showed that it contained orange-coloured pollen mass on the upper layer and a lower mud layer. Cells had 14 large oval leaf pieces (n=1) as inner leaf linings and eight to nine pieces (n=2) of circular leaf pieces as a cap. The large oval leaf pieces of the inner lining decreased in length and width towards the inner layers. There was no leaf cap for the concave end of the leaf cell. Some fungus (Fig. VI2D) were observed growing at the cell cap and at the edge of the outer interlocking leaves and some of the inner interlocking leaves but not the inner-most ones.

Fig. VI-2. Megachile laticeps nest. A, Schematic diagram of the outermost cell comprising (a) leaf cap, (b) leaf lining, (d) young instar and (d) pollen and mud layers. B, an entire cell, C, cross section of a cell, D, Layers of leaf lining for one cell (nest 3, cell 1). Fungi growing on the cap. A post-defecating larvae spun a cocoon (Fig. VII-3A,B). The emerging adult chewed a hole at the top of the leaf cap (Fig. VI-3C) when emerging. Males were always observed to be nearest to the rear and the last cell, a female (nest 1: ♂ Coelioxys confusa, ♀; nest 2: ×, ♂, ♂, ♀; nest 3: ♂, ♂, ♂, ♀, ×; × indicates that the larvae did not emerge). Defecation of a post-defecating larvae did so outside the cocoon. Nest 1 cell 1 (outermost) was parasitized by a male Coelioxys confusa. A microlepidoptera larvae (Fig. VII-4A) and tachinid fly (Fig. VI-4B) pupal cases (Fig. VI-4C) were present in nest 3.

Fig. VII-3. A, Cocoon spun by a post-defecating larva. B, Post-defecating larva, C, Circular leaf cap chewed open by emerging male adult. SCALE BAR: 10 MM.

Fig. VI-4. Nest associates of M. laticeps. A, A microlepidoptera larvae present in the inner oval leaf layers of nest 1, cell 1. B, Tachinid fly which emerged from nest 3, profile and dorsal views. C, Pupal case of emerged tachinid fly and fungi growing on outer interlocking leaves. SCALE BAR: 10 MM. Hatching is expected to take place relatively quickly. Larval growth from early to late instars took place quickly, over a period of a few days. The pupal stage is approximately a month though this was not carefully recorded. Nest bionomics of Megachile (Creightonella) atrata Megachile atrata nesting was last recorded from Medan, Indonesis [as M. tuberculata, a misidentification] [145]and more recently nesting of M. frontalis, its sister species, was recorded [145,146]. Megachile atrata were found nesting in mud lobster (Thalassina sp., Crustacea) mounds (Fig. VII-5) in back mangrove at Pasir Ris Park (lat., lon.: 1.3792, 103.9523) on 9 June 2014. Little sea water reaches the channels at the highest tide. Mud skippers (Gobiidae), mangrove crabs (Episesarma spp.), Telescopium telesopium and Terebralia palustris suggestive of a wet mangrove habitat, and mangrove fauna (Sonneratia and Rhizophora) and mangroveassociated plants (Hibiscus tiliaceus) were present. Three nests were excavated. Nest 1 had three cells (cell 1 – d: 10.6 mm, l: 25mm; cell 2 – d: 10.5 mm, l: 22 mm; cell 3 – d: 11 mm, l: 25 mm) and nest 2 had one cell (cell – 12.6 mm; 37 mm) (Fig. VII-6 inset). Both nest 1 and 2 were incomplete and did not have a leaf cap at the end of the nest. Nest 3 was complete and was capped by a circular leaf piece (entrance hole diameter: 17.1 × 18.4 mm) and had six cells and two cells from an older nest (characterised by black fungus within the cell) (Fig. VI-5A). Mortality of bees could be attributed to fungal growth. Two cells from an old nest were noticed to have a black fungus growing (Fig. VI-3E), whereas a fresh cell had fungus growing only on the cap (Fig VII-5B). When five cells were left in an enclosed, plastic container, three cells grew mould after a day. This species was unsuccessfully reared to adulthood (a late-instar individual dissected from its cell was kept alive for at least 6 days from 9 to 15 June 2014 but was found dead by 18 June 2014) and thus the duration for pupation is not known.

Fig. VI-5. A, Cell with pollen provisions all ingested by larva. B, Cell provision with black fungus. F, Fungi growing on the inner leaf-lining cap. SCALE BAR: 10MM.

Fig. VI-6. Nesting site of Megachile atrata in a mud lobster mound. The black arrow points to nest 1. (Inset) photograph of cells. SCALE BAR: 10 MM. The cells were in a linear series. Similar to M. laticeps, they were found interlocked with each other and had a convex rear and concave front (Fig. VI-7 A–C). However, the nest was found at an angle in the mud lobster mound. The mud lobster mound substrate was crumbly and damp, and excavation of the nest was done with ease.

Fig. VII-7. Megachile atrata nest cells. A, Schematic of an individual cell with M. atrata larva. B, Photograph of an intact cell, C, cross section of a cell of a post-defecating larva 1. SCALE BAR: 10MM.

Cells ranged from 25 to 37 mm (n = 4 cells). A cell had 14 pieces of large, circular leaf pieces (n=1) and two to four small, circular leaf pieces (n = 5 cells) for the outer leaf lining, followed by a dark-brown layer of masticated leaves and an inner leaf layer, comprising of a cap and a leaf lining. The layers differed from M. laticeps which only had one outer layer made out of leaves. As larvae were collected for further morphological study and not reared, gender arrangement within each nest was unknown. However, the youngest instar was found nearest to the end of the nest. When opened, the larvae, young and old instars were found feeding continuously on the pollen provisions. A thin, top layer of pollen seemed to be semi-liquid (Fig. VII-8A), suggesting a mixture of nectar and pollen, and the bottom layer a solid pollen layer. The colour of pollen were sometimes consistently of one colour, and other times variegated in layers. When cells of later instars were opened, the liquid mixture of nectar and pollen was consumed, leaving a hard layer of pollen (Fig. VII-8B). The post-defecating larvae had its head on the bottom of the cell (Fig. VI-8C).

Fig. VI-8. Megachile atrata larvae at different stages. A, A young instar (flat) floating atop the nectar-pollen mixture, B, An older instar feeding on pollen, C, A post-defecating larvae with its head on the bottom of the cell.

Nest bionomics of Anthidiellum (Pycnanthidium) smithii An Anthidiellum smithii nest, a first record of the genus for Singapore, was recovered and studied on 11 June 2014 (ref: DFA014-3). It was retrieved at a sunny area at the edge of a secondary forest near a hill dipterocarp primary rainforest (Dairy Farm Nature Park, lat., lon.: 1.359152, 103.776844) and had four cells (diameter: 3.3 mm, length: 23 mm, height: 45 cm, direction: 106º, on tree). The pollen provisioning cells were in total 19 mm long, each cell were 6 to 6.5 mm in horizontal length, similar to in A. butarsis and A. krombeini. There were four cells with partitions of the white substance, and coated internally with resin. In Sri Lanka, trap-nesting Anthidiellum (Pycnanthidium) used resin for cell partitions with a closing plug made of mud and resin (in A. butarsis) and a debris-resin complex or resin (in A. krombeini), and with description of the bionomics [147,148]. Provisioned cells were in a linear series, placed obliquely to each other and contained dry, solid pollen. The cells were not flush to the end of the bamboo internode. The nest was plugged with resin (Fig. VI-9). This was followed by a partition made up of a complex of resinous-masticated white substance 5.5 mm (IC) from the entrance, followed by another similar partition 17.5 mm from the entrance (IC). The white substance and resin were then interspersed as particles throughout the nest after the second partition, for 34 mm, before the pollen provisioning cells.

Fig. VI-9. Anthidiellum smithii nest. (A) The entire nest, (B) zoom in on the cells. ‘S’ and ‘E’ demarcate the start and end of each of the four cells respectively. Bamboo opening is on the right. SCALE BAR: 10 MM.

Nest bionomics of Heriades (Michenerella) sp. 1 Heriades (Michenerella) sp. 1 were nesting in bamboo internodes (diameter: 4 mm, length: 208 mm, height: 95 cm, direction: 99º, on wooden pole), retrieved from a managed garden within a coastal park (Pasir Ris Park, lat., lon.: 1.37901, 103.949748). There were seven cells in the nest and was not flushed to the end of the bamboo internode. The nest was plugged with a black resin. There was a partition made out of black-coloured resin 11 mm into the entrance (VC). After the seventh provisioning cell, there was an empty cell with pollen and white material (IC), which was 12 mm long and ended with a concave resin partition (Fig. VI-10A). The cells were found in a linear series and were partitioned by the same black-coloured resin. The cells was each 7.5 to 10 mm long, increasing in length towards the end. The pollen was wet on the end nearer to the entrance, and dry at the bottom and was orange (Fig. VI-10B). The nesting biology of Palearctic H. (Michenerella) is poorly known [149]. An Indonesian species (H. (M.) othonis) excavated nests in pithy stems rather than a trap nest [150].

Fig. VI-10. Heriades (Michenerella) sp. 1. A, The entire nest. B, Larva feeding on pollen provisioning. C, Pupa in cocoon with defecation surrounding the top. Bamboo opening is on the right. SCALE BAR: 10MM.

Comparison of nest bionomics A comparison of nest bionomics is presented in Table VI-1. M. atrata and M. laticeps use leaves to line their nest while Heriades sp. 1 and A. smithii both use resin. Further, M. laticeps, H. sp. 1, A. smithii are cavity renters of bamboo internodes whereas M. atrata nests in the soil. The development time of M. laticeps from egg to adult is approximately four to five weeks. Only nest associates were discovered for M. laticeps, notably including Coelioxys confusa, a kleptoparasite from the same tribe. Table VI-1. Comparison of the nest bionomics characteristics for megachilids. Nest terminologies abbreviated are described in the methods.

Nesting substrate Lining or partition type Shape of leaves for cell lining No. of leaves for side lining of cells Number of cells in each cell-cluster Length of each cell (mm) CP type Presence of PP Presence of VC Presence of IC(s) Flushed to the end of internode Pollen type

Megachile atrata (n=3) Side of mud lobster mound Lining with green, thick leaf pieces and black masticated leaves Round, jagged edges

M. laticeps (n=3) Bamboo internode Lining with green, thin (but browning) leaf pieces

14

Rectangular, smooth edge 14

6

Anthidiellum smithii (n=1) Bamboo internode

Heriades sp. 1 (n=1) Bamboo internode Partition with resin Partition with and whiteresin masticated complex N. A. N. A. N. A.

N. A.

4–5

4

8

25–37

16–19

6–6.5

7.5–10

Leaf No No No N. A.

Leaf No No No Yes

Resin No Yes No No

Resin Yes Yes Yes No

Semi-liquid and dry

Unknown

Dry

Insect associates

None

None

Pupa

In a cocoon

Coelioxys confusa, Tachinidae fly pupa, microlepidoptera larvae

Semi-liquid and dry None

Unknown

In a cocoon

In a cocoon

Appendix VII – short notes on nesting sites of other Megachile Methods The foraging behaviour or nest bionomics of these Megachile were not recorded in great detail due to the late discovery of nest and/or inaccessibility of nest contents.

Nest site of Megachile disjuncta and Megachile fulvipennis (see record on LKCNHM Singapore Biodiversity Records) Megachile disjuncta (lat., lon.: 1.2966, 103.7827) nested horizontally in a rectangular hole in a wooden bench (7 mm × 9 mm) (Fig. VII-1A, B) on the third storey of non-air conditioned area, sheltered part of a building in April 2014. The same hole was utilized by Megachile fulvipennis in 23 August 2014 (Fig VII-1 C; Fig. VII-2) and finished the nest on 21 September 2014. Megachile fulvipennis individual(s) had emerged by 31 October 2014 (may also have been 1–2 days earlier).

Fig. VIII-1. A, the cavity which M. disjuncta nested in. B, M. disjuncta entering the cavity (see video: https://www.youtube.com/watch?v=eBrNwdU4B5U). C, the cavity plugged by Megachile fulvipennis.

Fig. VII-2. Female M. fulvipennis individual which was caught for identification and later released. Nest site of Megachile umbripennis Three M. umbripennis females (lat., lon.: 1.2966, 103.7827) were observed building nests on 13 Apr, 14 Apr and 25 May 2014 respectively. The bees were nesting horizontally in a rectangular hole in a wooden bench (5 mm × 10 mm) on the third storey of non-air conditioned area, sheltered part of a building. It was observed to mud, lap up salts from the wooden bench (Fig. VII-3), collecting and depositing pollen in the cavity.

Fig. VII-3. M. umbripennis (ESJYBee006) lapping up salts from the bench (see video: http://www.youtube.com/watch?v=0_mkLRH9MgU).

Appendix VIII – plates of megachilid habitus and genitalia

Fig. VIII-1. Representative genera of the family Megachilidae in Singapore. Lateral habitus of A, Anthidiellum (Anthidiini), C, Euaspis (Anthidiini), D, Heriades (Osminii), F, Lithurgus (Lithurgini), H, Coelioxys (Megachilini), I, Megachile. B, Wing of Anthidiellum. Note the short prestigma and stigma, a diagnostic feature of the tribe Anthidiini (see Fig. VIII-2 for an annotated wing venation). E, Tarsus of Heriades. Note the arolia present on all legs, a diagnostic feature of the tribe Osminii (with the exception of Megachile (Matangapis) which also has arolia on all legs). G, Head of Lithurgus. Note the notch on the head, a diagnostic feature of females in the genus. The black arrows point to diagnostic features. Images of these bees in other views and the other sex are available on Dropbox. SCALE BAR = 1 MM.

Fig. VIII -2. Wings of Megachile with annotations based on Michener (2007).

Fig. VIII-3. Mandibles of Megachile with leaf-cutting edge. A, M. (Aethomegachile) laticeps, B, M. (Eutricharaea) subrixator C, M. (Paracella) tricincta, and Group 3 Megachile, D, M. (Creightonella) atrata.

Fig. VIII-4. Mandibles of Megachile without cutting edge or incomplete cutting-edge. A, M. (Alocanthedon) cf. indonesica B, M. (Callomegachile) disjuncta, C, M. (Callomegachile) stulta, D, M. (Chelostomoda) moera (the white arrow points to the incomplete cutting edge at the second mandibular interspace).

Fig. VIII-5. Megachile with black-and-white metasoma (first row, A-D; second row, F) and Megachile with black-and-orange metasoma (second row, E-J except F). Inset for the species with black-and-white metasoma shows sculpturing on the scutum and for selected black-and-orange metasoma shows gonoforceps of male genitalia. Species names appear below each diagram. SCALE BAR = 1MM.

Fig. VIII-6. Megachile with black metasoma and orange wings. Inset shows the sculpturing of the scutum. Scale bar = 1mm. Names of each species are appended below each diagram. SCALE BAR = 1 MM.

Fig. VIII-7. Megachile with either a singleton collection in surveys from 2012-2014 (A, B, C, E, G) and/or forest-associated species (B-G). The images of each species are of the plan, profile, head from top to bottom. Species names appear below each diagram. Black arrows point to diagnostic features. SCALE BAR = 1 MM.

Fig. VIII-8. Small leaf-cutting Megachile species from the subgenus Eutricharaea (A–D) and Paracella (E). The images of each species are of the plan, profile, head from top to bottom. Names of each species are appended below each diagram. The black arrows point to diagnostic features. SCALE BAR = 1MM.

Fig. VIII-9. Genitalia of selected Megachile males (A, C, D, E, F, G, H, I – dorsal view, B – ventral view). Names of each species are appended below each diagram. The white arrows point to diagnostic features.

Appendix IX – plates of representative trap-nests and a summary of nest contents Trap-nest present represent the trap-nesting arthropods, except the megachilids (see Appendix VII instead), and are not presented for Hylaeus sp. 2, Pompilidae sp. 1 (both not photographed), Siler semiglaucus and nests which had no emerging adults (100% mortality). ALL SCALE BAR = 10 MM.

Fig. IX-1. Hylaeus aff. penangensis HPA001-1, det. by J. S. Ascher. Pupae did not spin a cocoon.

Fig. IX-2. A, Trypoxylon sp. 2 (black, small) SBA005-2, B, Trypoxylon sp. 1 (orange T2-T4, large) NS04-1, det. by EJYS. Pupae of both species were enclosed in a cocoon.

Fig. IX-3. Isodontia severini (Kohl) PRA012-2, det. by J. X. Q. Lee. Pupae was enclosed in a cocoon.

Fig. IX-4. Rhynchium haemorrhoidale (Fabricius) SBA013-1, det. by EJYS. Pupae did not spin a cocoon.

Fig. IX-5. Allorhynchium argentatum (Fabricius) PRA015-3, det. by Z. W. W. Soh and EJYS. Pupae did not spin a cocoon.

Fig. IX-6. Eumenes sp. 1 HPA007-1, det. by EJYS. Pupae did not spin a cocoon.

Fig. IX-8. Erebidae sp. 1 DFA012-1, det. by R. Kendrick.

Fig. IX-9. Erebidae sp. 2 NS04-3, det. by R. Kendrick.

Fig. IX-9. Microcerotermes TEA011-1 individual (left), 5 mm-diameter hole plugged (right). Det. by Theodore Evans Laboratory. Table IX-1. Trap-nesting species, with diameter ranges, number of cells, closing plug, partitioning material and provisioning type. Diameter range (median) (mm)

Number of cells (median)

Erebidae sp. 1 (Lepidoptera) Erebidae sp. 2 (Lepidoptera) Eumenes sp. 1 (Vespidae) Heriades (Michenerella) sp. 1 (Megachilidae)

4.0 5.0 2.5–4.0 (2.8) 4.0

1 1 1–5 (3) 7

Resin and mud ?^ Resin-white material complex Mud Mud Mud Resin

Hylaeus aff. penangensis (Colletidae) Hylaeus sp. 2 (big) (Colletidae) Isodontia severini (Sphecidae)

1.0–3.0 (2.5) 2.0–4.0 (4) 3.0–8.0 (4.8)

6, 7 5–10 (8) 1–3 (1)

Cellophane Cellophane Plant wool

Cellophane Cellophane Plant wool

Megachile laticeps (Megachilidae) Microcerotermes sp. 1 (Isoptera: Termitidae) Rhynchium haemorrhoidale (Vespidae) Siler semiglaucus (Arachnida: Salticidae) Trypoxylon sp. 1 (with orange T2-4, big) (Crabronidae) Trypoxylon sp. 2 (black, small) (Crabronidae)

7.0–9.0 (8.5) 5.0 7.5 8.5 4.0–8.5 (7.7)

4, 5 N. A. 1 an egg clutch 4–8 (6)

Leaves Soil Mud N. A. Mud

Leaves N. A. Mud N. A. Mud

N. A. N. A. Caterpillars Pollen (semi-liquid and solid) Pollen (semi-liquid) Pollen (semi-liquid) Cockroaches (Blattellidae) Pollen (unknown) N. A. ?* N. A. Spiders (Salticidae)

3–7 (5)

Mud

Mud

Spiders (Salticidae)

Allorhynchium argentatum (Vespidae) Auplopus sp. 1 (Pompilidae) Anthidiellum smithii (Megachilidae)

4.6 5.0 3.3–4.4 (3.9)

2 1 1, 4 (N. A.)

2.0–4.0 (3.0)

Closing plug

*obtained at the pupal stage and provisioning resources were already consumed; was not documented ^

Partitioning material

Resin and mud ?^ Resin-white material complex N. A. N. A. Mud Resin

Provisioning type

Unknown Spiders (Ctenidae) Pollen (solid)