JHR 41: 75-94 (2014) re JOURNAL OF | *reerrtieved opencoss ural
Sirsa ae (4) Hymenoptera

http://jhr.pensoft.net The insertional Society of ymenopterists. RESEARCH

Nesting biology of an Oriental carpenter bee,
Xylocopa (Biluna) nasalis Westwood, | 838,
in Thailand (Hymenoptera, Apidae, Xylocopinae)

Watcharapong Hongjamrassilp', Natapot Warrit'

| Center of Excellence in Entomology & Department of Biology, Faculty of Sciences, Chulalongkorn University,
Bangkok, Thailand 10330

Corresponding author: Natapot Warrit (Natapot.w@chula.ac.th)

Academic editor: Jack Neff| Received 10 May 2014 | Accepted 14 November 2014 | Published 22 December 2014
http://zoobank.org/CDF10DA4-7F4C-48EC-891 6-E029D77E1766

Citation: Hongjamrassilp W, Warrit N (2014) Nesting biology of an Oriental carpenter bee, Xylocopa (Biluna) nasalis
Westwood, 1838, in Thailand (Hymenoptera, Apidae, Xylocopinae). Journal of Hymenoptera Research 41: 75-94. doi:
10.3897/JHR.41.7869

Abstract

The biological study of wild non-Apis bees can provide useful information that may help with the pollina-
tion of food crops and native plants in areas where the keeping of honey bee colonies is restricted or affected
by CCD. Here, we describe the nesting biology of the Oriental large carpenter bee, Xylocopa (Biluna)
nasalis Westwood, 1838. An aggregation of more than 80+ bamboo nests of X. nasalis was discovered in
Suan Pheung district, Ratch Buri province, Thailand on the 25° of May 2012. We collected 27 nests from
the site to dissect, measure the external and internal nest architecture, and analyze the pollen composition
of the pollen masses. X. nasalis constructs linear unbranched nests with nest entrance mostly located at the
open-end of the bamboo culms. The nest length and the branch diameter of the nest entrance (excluding
nesting edge) are 25.40 + 6.95 cm and 17.94 + 6.00 mm, and the maximum number of provisioned cells
is 8. A biased sex ratio of 89: 14 is reported, with up to 7 adults inhabiting in a single nest. 29 pollen
types were identified from 14 pollen masses using an acetolysis method and visualization under both light
microscope and scanning electron microscope. 13 pollen types were considered as major pollen sources
(contribute > 1% in total pollen volume); however, only 10 can be identified to family and generic levels.
The dominant pollen sources are of the families Elaeagnaceae (Elaeagnus cf. latifolia), Euphorbiaceae (C7o-
ton), Fabaceae (Senna siamea and Cassia), Fagaceae (Lithocarpus and Castanopsis), and Lythraceae (Trapa)
which are mostly native to the region of Southeast Asia. The nesting architectural details should prove to
be beneficial to beekeepers and researchers who are interested in trapping and studying X. nasalis, and the
polylectic behavior of X. nasalis can be highly valuable for future crop pollination strategies, particularly
for plants that require sonication of their poricidal anthers.

Copyright W. Hongjamrassilp, N.Warrit. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

76 W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

Keywords
Carpenter bee, nesting biology, Thailand, bamboo, pollen

Introduction

Because of the declining honey bee population worldwide resulting from the condition
known as Colony Collapse Disorder (CCD; Oldroyd 2007, van Engeldorp et al. 2008,
Ratnieks and Carreck 2010), the use of widespread pesticides (Hopwood et al. 2012),
climate changes (Bartomeus et al. 2011), and the increase in monotonous agricultural
landscapes that reduce the biodiversity and the availability of foods for bees, the study
of wild and/or domesticated non-Apis bees can provide useful information for com-
plementary bee species that may help with the pollination of food crops in areas where
keeping of honey bees colonies are being affected or restricted (Chagnon et al. 1993,
Wilmer et al. 1994, Javorek et al. 2002, Hoehn et al. 2008, Brittain et al. 2013). Until
now, only a handful of non-Apis bee species have been used extensively in agriculture,
e.g., Bombus terrestris (Linnaeus, 1758), Megachile rotundata (Fabricius, 1787), No-
mia melanderi Cockerell, 1906, Osmia rufa (Linnaeus, 1758), and some stingless bee
species (Westerkamp and Gottsberger 2000, Hogendoorn et al. 2006, Greenleaf and
Kremen 2006, Slaa et al. 2006, Hoehn et al. 2008). These bees have been shown to be
effective pollinators, as good as, if not better than, honey bees on certain crop plants
(Greenleaf and Kremen 2006).

The large carpenter bees of the genus Xylocopa Latreille, 1802 (Hymenoptera;
Apoidea) have recently received attention due to their pollination capabilities. The use
of large carpenter bees to assist with pollination of greenhouse tomatoes and honeydew
melons in Australia and Israel has been reported (Hogendoorn et al. 2000, Sadeh et al.
2007, Keasar 2010). In Brazil, where passion fruits are one of the main exported fruit
crops of the nation, studies of using X. (Veoxylocopa) grisescens Lepeletier, 1841 and X.
(N.) frontalis (Olivier, 1789) to pollinate the flowers, instead of using manual labor,
have shown promising results in increasing the fruit sets and quality and reducing the
production costs (Junqueira et al. 2012, Yamamoto et al. 2012).

Carpenter bees can be found throughout the tropical and subtropical parts of the
world (Hurd and Moure 1963, Gerling et al. 1989). These are large and robust bees
that many novices regularly confuse with bumble bees (Bombus) due to their similar
sizes and shapes. There are currently ca. 470 species described with 32 subgenera rec-
ognized in a single genus (Michener 2007, Ascher and Pickering 2013). Most Xylocopa
species are known to excavate their nests in dead or decaying woods, with the excep-
tion of the subgenus Proxylocopa Hedicke, 1938 which excavates nests in the soil (Got-
tlieb et al. 2005). There are two main types of nests among the wood-nesting Xylocopa
species: (1) unbranched or linear nests in which the tunnel runs in the same direction
as the nest entrance or at most with a single right angle corner from the nest entrance

Nesting biology of X. nasalis Tal

and (2) branched nests which consist of at least two tunnels or more although with
only one nest entrance (Gerling et al. 1989).

One subgenus of Oriental Xylocopa, Biluna Maa, 1938, comprises five to nine
species (Michener 2007, Ascher and Pickering 2013). Its distribution ranges from
India and Sri Lanka to Southeast Asia and Japan. Species of Bi/una are only known to
construct unbranched nest in bamboo culms (Maa 1946, Maeta et al. 1985; Hurd and
Moure 1963). Xylocopa (Biluna) nasalis Westwood, 1838, is a species commonly found
throughout Southeast Asia. It superficially resembles the sympatric species X. (Mesotri-
chia) latipes Drury, 1773 and X. (M.) tenuiscapa Westwood, 1840 because of the pres-
ence of black pubescence on the mesosoma and their large size (21-35 cm in length).
Males of Biluna lacks both a basitibial plate and a spine on the outer apex of the hind
tibia, while the females have a dense mat of short setae on the middle tibia and lack
an apical middle tibial spine. The behavior, biology and natural history of X. nasalis,
is poorly known even though it is commonly found throughout rural and agricultural
areas in Southeast Asia. Boontop et al. (2008) briefly described the nesting biology of
X. nasalis studied in Kasetsart University, Kamphaengsaen Campus, Nakhonpathom
province, Thailand, though their account lacks many nest architectural details and,
importantly, the palynological data on plant food sources. Here, we extend Boontop
et al. (2008)’s work via reporting the finding of a nest aggregation of X. nasalis in Suan
Pheung district, Ratch Buri province (~100 km southeast of Nakhonpathom provy-
ince), Thailand, along with details of other nest architectural components, its floral
preferences, and some behavioral observations at the nest entrance. We anticipate that
by providing such detailed nesting biology and pollen food sources of a local large
carpenter bee from an area with poorly known mellitological data (such as Southeast
Asia), it will stimulate interest and provide practical information for local bee keepers
and bee researchers to consider Xylocopa to be an important native pollinator for cer-
tain crops and endemic plants in the near future.

Methods

Nesting site and nest dissections

We discovered a nesting aggregation of Xylocopa nasalis in a makeshift roof structure
(Figure 1; 80+ nests) made from bamboo culms (tribe Bambuseae) at a local restau-
rant in Suan Pheung district, Ratch Buri province, Thailand (13°33'32.4138"N and
99°21'32.3202"E) on the 25" of May 2012. The collecting of the nests was done in
the early month of the Monsoon season in Thailand, when flowers were abundantly
blooming. Almost 95% of the bamboo culms were occupied by X. nasalis. We also
observed some unidentified megachilids using some of the bamboo culms for nesting
as well. The bamboo culms ranged in size of outer diameter ~ from15.80 to 29.39 mm
and 3.00 to 3.25 m in length (n = 27) and were situated approximately 2.50 m above
the ground. We collected 27 nests (with all of the bee inhabitants) by plugging the nest

78 W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

Vang eere sv
doe ie —
a eae TT ae | we

SU UOT TEN TEL ET A (b)
ewe

Oe ”

, SS]
mers oS ea =o Lala Sly © ‘ Lo Lhe oi Osa . r
\ i= | j Pl ;
4 I } 4 I

| j
| | /
MWitj@ek

Figure |. Nesting habitat of X. nasalis; A nesting habitat of X. nasalis on a makeshift roof of a restaurant
in Suan Pheung district, Ratch Buri province, Thailand. The red arrows indicate locations where the
bamboo culms were arranged ca. 2.50 m above the ground (la and Ib). At the nest entrance, the female
of X. nasalis was dehydrating the nectar previously foraged (Ic).

—
ied}
~—

Vestibulum length

Ja}WWeIp
youeig

Inner most cell length

Figure 2. Nesting architecture of X. nasalis; Dissected nests of X. nasalis revealing the nest structure
inside the bamboo culm and its residents. Measurements of the nest parameters are shown in Table 1.
The diameters of the nests (excluding the nest thickness) were measured at the nest entrance, followed by
the vestibulum (antechamber) length, cell length, and the inner most cell length, respectively (2a). Cells
containing larvae with pollen masses and their feces were collected and weighted (2b).

entrances with cotton balls and sealing them with duct tape. Nests were brought back
to the Department of Biology, Chulalongkorn University, Thailand, and preserved in
a -20 °C freezer for later dissection. We dissected each nest and recorded the following

Nesting biology of X. nasalis 72

data: measurements of the external and internal nest structures using a vernier caliper
and tape measurement (Figure 2), numbers of individuals at different life-stages, num-
bers of provisioned cells, fresh weights of the pollen balls in each cell, feces’ weights,
and sex ratio of adults. Since the data on X. nasalis cell lengths cannot be assumed to
be drawn from any given probability distribution, the non-parametric Kruskal-Wallis
test was employed to test whether there are differences among the average cell lengths
of 54 cells measured; the Mann-Whitney U test was used to test the difference between
the average inner most cell length of all 27 nests and the average cell lengths. The two
statistical tests were performed using the program SPSS ver. 20.0 (IBM Corp. 2001).
Fourteen pollen balls from six nests were collected for pollen analysis. All remaining
nests were sketched and photographed. All specimens (including eggs, larvae, and pu-
pae) were preserved in 95% Ethanol and deposited at the Natural History Museum of
Chulalongkorn University, Bangkok, ‘Thailand for future genetic analyses.

Pollen analyses

For pollen analysis, we employed the acetolysis method (Erdtman 1960) with a
modification at the end of the process where we re-suspended the decanted pol-
len samples in a benzene solution and topped up with silicone oil in a vial. The
pollen-benzene solution was air-dried for one week or until the benzene solution
was completely evaporated leaving only the pollen samples preserved in silicone oil
for examination under light microscope (Olympus CH-BI45-2). For preparing the
pollen pictures to be captured and identified under a scanning electron microscope
(Hitachi Tabletop Microscope TM-100), an additional step was performed before
pollen samples were re-suspended in benzene solution: 70% ethanol was added and
the pollens were mixed in the solution, the pollen was pelleted by centrifugation, and
the ethanol was then discarded (we sometimes repeated additional steps with 95%
and absolute ethanol).

Before counting the pollen grains, we mixed the vial containing pollen grains sub-
merged in silicone oil to obtain a homogenous pollen suspension. Ten drops of the
pollen suspension were removed and placed on microscopic slides and each aliquot was
spread to an area of ca. 30 x 30 mm. Three slides per pollen mass were used for the
examination. We counted 300 pollen grains for each slide, which provide a total pollen
count of 900 grains from a single pollen mass. Since there is no published exhaustive
key for the pollens endemic to western Thailand, we were limited in resources to ac-
curately identify most pollens to the specific level. We followed the pollen identifica-
tion guides from various authors whose works were on the melittopalynology of the
Asian Tropics, i.e., Huang (1972), Tissot et al. (1994), Nagamitsu et al. (1999), and
Jongjitvimol and Wattanachaiyingcharoen (2006), which allowed us to identify most
pollen types to family and genus. We also identified the plant food sources in the area
of western Thailand using published botanical keys provided by Hanum and van der
Maesen (1977), Gardner et al. (2000), Smitinand (2001), and Phengklai (2006), to

80 W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

corroborate with our pollen data. The plant classification system of the Angiosperm
Phylogeny Group (2009) was followed.

Since pollens are diverse in their shapes and sizes, to accurately identify which pol-
len type contributes the most to the bee diets, one should not depend only on the most
number of grain counts alone. Buchmann and O’Rourke (1991) suggested weighing
the volume of pollens with the percentage of the pollen counts to achieve a more reli-
able estimation of the type of pollens that contribute to the pollen masses. To obtain
the volumes of each pollen type, we measured the longitudinal axis (p) and equatorial
axis (e) lengths from 30 grains of each pollen type then calculated the mean values.
Pollen dimensions are categorized into two types: spherical and elliptical forms. The
following formulas were used for the calculation of the pollen volumes: spherical form
= 1/6np° and elliptical form = 1/6ze’*p. Contributing pollen types were subjectively
categorized into two groups — the “major” and “minor” pollen sources based on their
percentage of total volume. The major pollen sources are defined as contributing in X.
nasalis diet = 1% of the total pollen volume, whereas minor pollen sources are those
that are accounted < 1% of the total pollen volume.

We also observed some behaviors exhibited by the bees on the day before we col-
lected the bamboo nests. These behaviors were related to their nesting habits and are
briefly discussed in the next section.

Results

Nest architecture and contents

Nests of Xylocopa nasalis nest are strictly unbranched. The provisioned cells are sepa-
rated by partitions made from bamboo particles excavated by the founding female. All
of the nest entrances are located at the end of the bamboo culms, except for a couple
of nests that the bees excavated from the undersides. A summary of nest architectural
details is provided in Table 1. The average total nest length (including the vestibulum
(antechamber) length) is 38.35 cm. The average nest length (measured from the nest
entrance to the end of the innermost cell) is ca. 25.40 + 6.95 cm. The mean branch di-
ameter of the nests (excluding nest thickness) is 17.94 + 6.00 mm. The number of cells
per nest ranged from 0-8 cells with an average cell number around 3 per nest. There is
a difference in terms of the average individual cell lengths among cells from 27 nests (x7
= 28.11, p = 0.021), though the significance value is fairly weak. On the contrary, the
innermost cell lengths were tested to be strongly different from other cells (U < 0.0001,
p<0.0001). The average number of individual adult bees found per nest ranged between
1 and 7 individuals (mean + s. d.: 3.24 + 1.90) with a sex-ratio bias of 7.989: 14. We
found the average number of pupae and post-defecating larvae: larvae: eggs as follow
1.15: 0.69: 0.04; however, we did not find any nest that had all life stages of the bees
present at once. Three of the 27 nests contained eggs; the mean fresh weight of their

Nesting biology of X. nasalis 81

Table |. Nesting structure measurements of X. nasalis; Summary of the measurements of nesting
architecture of X. nasalis (n = 27) from Suan Pheung district, Ratch Buri province, Thailand (13°33'
32.4138"N and 99°21'32.3202"E).

Nest Characters nanges Max. Min
(Mean + S.D.)

Vestibulum length (cm) 12.95 + 67.80 31.00 5.04
Nest length (cm) 25.40 + 6.95 36.25 10.00
Number of cells / nest 2.83 + 2.55 8 0
Inner most cell length* (mm) 32.75 + 11.06 55:30 15.00
Individual cell length (except *) (mm) 23.25 + 3.88 41.00 17.00
Branch diameter at nest entrance (mm) 17.94 + 6.00 30.70 11.00
Nest thickness (mm; measured at the entrance) 4.66 + 0.79 6.80 3.30
Partition thickness (mm) 0.88 + 0.27 1.60 0.50
Pollen weight / cell (g) (n = 3) 137+ 0.13 152 £20
Feces’ weight / cells (g) (n = 6) 0.24 + 0.23 0.86 0.01
Number of adult individuals 3.24 + 1.90 7 1
Number of female adults 3.19 + 2.04 7 1
Number of male adults 0.40 + 0.70 2 0
Number of pupa and post-defecating larva 1.15 + 2.41 - 0
Number of larva 0.69 + 1.38 5 0
Number of eggs 0.04 + 0.19 3 0

unconsumed pollen masses was 1.37+0.13 g (n = 3). The average weight of the feces in
the cells of post-defecating larvae averaged 0.24 + 0.23 g (n = 6).

Pollen analyses

A total of 29 pollen types were identified from the 14 pollen masses. We were able to
identify pollen grains from 13 families, including 12 identifiable genera (Table 2). For
three of the 13 plant families — Anacardiaceae, Araceae, and Cyperaceae — generic level
identification could not be confirmed. Brief descriptions of the 14 unidentified pollen
types are also given in Table 2. We consider 13 pollen types as “major” pollen sources,
whereas the other 16 pollen types are considered as “minor” pollen sources based on
their percentage total volumes of the diets (Table 2, Figure 3).

For the 13 pollen types classified as major pollen sources, we were able to identify
10 pollen types to their generic level and 2 of these to species (Elaeagnus cf. latifolia
Linnaeus and Senna siamea (Lam.) Irwin et Bradley). These include the family Acan-
thaceae (Thunbergia; 2.35%), Anacardiaceae (genus unknown; 4.68%), Elaeagnaceae
(E. cf. latifolia; 12.88%), Euphorbiaceae (Croton; 14.95%), Fabaceae (Cassia; 12.17%
and S. siamea; 12.91%), Fagaceae (Lithocarpus; 7.65% and Castanopsis; 3.22%), Ly-
thraceae (Trapa; 13.36%), and Theaceae (Schima; 6.42%). Three pollen types (all are

< 3% of total pollen volume) remain unidentified at any level (under “Unknowns” in

W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

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Nesting biology of X. nasalis

84 W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

Figure 3. Pollen grains collected by X. nasalis; Some representations of pollens collected from pollen
masses of X. nasalis. The “major” pollen sources: Fagaceae, Castanopsis sp. (3a); Elaeagnaceae, Elaeagnus
cf. latifolia (3b); Fabaceae, Cassia sp. (3¢ and 3d), Senna siamea (3e and 3f); Acanthaceae, Thunbergia
(3g and 3h).

Nesting biology of X. nasalis 85

Table 3. Dominant pollens foraged by X. nasalis; Dominant pollen types from 14 pollen masses deter-
mined by the highest percentage pollen grain count and percentage of pollen volume (sequential order

starting from the pollen mass number in the inner most cell (#1) proceeding to the nest entrance).

Nest Number/ Family: Genus/Speci Percentage of Percentage of
Pollen Mass number sie eae oa pollen grains pollen volume

1/1 FAGACEAE: Lithocarpus 83.3 52.6
1é2 FAGACEAE: Lithocarpus 70.1 54.5
1/3 FAGACEAE: Castanopsis DD.2 LNe7:
FABACEAE: S. siamea 22x 41.5
V4 56.4 87.7
1/5 ELAEAGNACEAE: E. cf. latifolia 76.5 65.1
2/1 ANACARDIACEAE 33.6 49.9
3/1 ELAEAGNACEAE: E. cf. latifolia 31.1 50
3/2 FAGACEAE: Lithocarpus 63.8 54.9
4/1 47.0 73.8
4/2 ARACEAE 1M I 23.3)
4/3 EUPHORBIACEAE: Crvoton 18.2 63.6
5/1 LYTHRACEAE: Trapa 49.6 74.8
5/2 45.5 37.3

THEACEAE: Schima 14.1 57
6/1 FAGACEAE: Lithocarpus 45.2 22
EUPHORBIACEAE: Croton 8.4 42.1

Table 2). Minor pollen sources that can be identified are of the families Araceae (genus
unknown; 0.81%), Caprifoliaceae (Sambucus; 0.17%), Cyperaceae (genus unknown;
0.03%), Juglandaceae (Engelhardtia; 0.22%), and Rhamnaceae (Ziziphus; 0.12%);
whereas nine minor pollen types could not be identified.

The dominant pollen types based on both the highest percentage pollen type
amount and total percentage of pollen volumes for each of the 14 pollen masses is
displayed in Table 3. Eight different families of plants were found to be the dominant
contributor to the 14 pollen masses based on the highest total percentage of pollen
volumes — Anacardiaceae (genus unknown), Araceae (genus unknown), Elaeagnaceae
(Elaeagnus cf. latifolia), Euphorbiaceae (Croton), Fabaceae (Senna siamea), Fagaceae
(Lithocarpus), Lythraceae (Trapa), and Theaceae (Schima).

Not only does Xylocopa nasalis display polylecty as indicated by results of the pol-
len analyses in its foraging behavior, but each female also exhibited a broad host plant
range when foraging for pollen. Table 4 shows a foraging female that utilized 13 differ-
ent pollen types to construct 5 pollen masses in a single nest, with the dominant pollen
source for each pollen mass changing over time, e.g., Cells 1 and 2 are dominated by
Lithocarpus, whereas Cell 4 and 5 are dominated by Elaeagnus cf. latifolia. Castanopsis
pollens are found throughout all five pollen masses.

We also observed some notable nest-entrance behaviors by the bees. Competition
for nests seemed to be very high at the nest site despite the abundance of available bam-

86 W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

Table 4. Pollen composition from a single nest of X. nasalis; Pollen composition from a single nest (nest

#1; Table 3) of X. nasalis. P represents a percentage of the given pollen grains in a pollen mass, whereas

V represents a percentage of the pollen volume. Cell numbers are arranged from as in Table 3.

Family:
Genus/Species
FAGACEAE

Cell 2

Cell 3

Cell 4

Cell 5

7 ee RC ae al ae ee a

Castanopsis
Lithocarpus
FABACEAE
Cassia

S. slamea

| 83.3 | 52.6 |

70.1

15.7

ELAEAGNACEAE

E. cf. latifolia

CAPRIFOLIACEAE
Sambucus
THEACEAE

Schima

ACANTHACEAE

Thunbergia

TRAPACEAE

Trapa

UNKNOWNS
Triangular and tripolate

Triangular and inaperture

Three furrows triangular and
tricoplate

Irregular and inaperture

Figure 4. Nest defending postures of X. nasalis;
lis to repel other conspecifics in the aggregated nesting site. The bee blocking the entrance via protruding
her head out from the nest entrance (4a). Guarding the entrance by using the dorsal side of her metasoma

to block the invaders (4b).

eri
Three furrows and tricoplate td ||| Sea ieee |S Oe, ; ;

Defending posture tactics performed by females X. nasa-

Nesting biology of X. nasalis 87

boo culms. Two defending posture tactics were observed. The most common defense
posture is that of a female blocking the entrance with her head (Figure 4a), although
sometimes we observed a female bees using her metasoma to block the nest entrance

(Figure 4b).

Discussion

Boontop et al. (2008) provided a brief nest architectural description of 20 Xylocopa
nasalis communal nests collected from Nakhon Pathom province ~100 km southeast
of our collecting site, though they only reported the nest total lengths (described as
“Internode length”), size of the nest entrances, diameter of the bamboo nests, and the
sex ratio. Here, we also reported additional detailed nest characteristics that were unde-
scribed previously. The total nest length averaged at 38.35 cm, whereas Boontop et al.
(2008) found theirs to be 32.63 cm. The branch diameters of the nests are also similar
between our work and that of the previous authors, 17.94 and 15.60 mm, respectively;
however, the sex ratio between female and male bees from this observation is about
twice to what was earlier described (89: 14 vs. 49: 1¢). The difference in the number
of female to male bees can be explained by the collecting date, which may correspond
to a later period of colony development, where most of the sister bees have emerged
and stay together inside the nest, whereas male bees may have departed right after
emerging from their cocoons or there is a sex ratio bias in egg-laying by the mothers.
Observations on such activity are needed to test these hypotheses about the skew sex
ratio in the nest. The three unconsumed pollen masses have an average fresh weight of
1.37+0.13 g compared to 1.16 and 1.09 g in X. (Ctenoxylocopa) sulcatipes Maa, 1970
and X. (Koptortosoma) pubescens Spinola, 1838, species found in the desert area of the
Middle East (Gerling et al. 1989).

Maeta et al. (1985) reported finding of a nesting aggregation of another Biluna
species, Xylocopa tranquebarorum tranquebarorum, in Szechungchi near Henchun,
Taiwan. This Biluna species also nested in bamboo culms though the nest entrances
were excavated exclusively from the underside (the authors found only five nests in
successive internodes of a single culm. In contrast to the previous finding, we found
the nest entrance of X. nasalis to be mostly at the end of the bamboo culms, but we
also observed that a couple of the nest entrances were on the underside of the culms
excavated by the bees as well. This observation suggests that both X. nasalis and X.
tranquebarorum tranquebarorum can excavate nest entrance from the underside of the
bamboo culms, which might be a behavior shared by members of the subgenus Biluna.
This tedious nest entrance excavation of a smooth and hard surface such as the sides
of bamboo culm may be explained by the compensation that the bees will receive
after the initial perforation of the culm with the omission of the need for later heavy-
ily burrowing (Iwata 1938). However, if the ends of the bamboo culms are open and

88 W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

exposed, the bees may choose not to allocate their energy in the excavation of the un-
dersides of the culms as seen in this study.

Xylocopa nasalis is polylectic with a diverse group of pollens collected. This is con-
sistent with the described foraging behaviors of other carpenter bee species (Hurd and
Moure 1963, Gerling et al. 1989, Burgett et al. 2005). Interestingly, the family Fagace-
ae, particularly of the genera Lithocarpus and Castanopsis, constitutes abundant pollen
sources for X. nasalis. Both pollen types can be accounted for 26.43% and 13.77% ,
respectively, in terms of total grains count; however, their pollen sizes are twice or thrice
smaller than other pollen types found in this study (Table 2), thus the total percent-
age pollen volumes contribute to the bee diets are rendered to only 7.65% and 3.22%.
Lithocarpus and Castanopsis are evergreen genera of large shrubs and trees that can reach
more than 20 m in height. Records show that there are 56 species of Lithocarpus and
33 species of Castanopsis indigenous to Thailand (Phengklai 2006), though there is no
available information pertaining to a reliable identification of the pollen species of both
plant genera in the area of study. Many species of the lowland Lithocarpus and Castanop-
sis flowers bloom abundantly during the beginning of the dry season to the end of mon-
soon period (March—October), which corresponds to the time of our collecting. It is
known that species of both Lithocarpus and Castanopsis are pollinated via insects (Nixon
1989, Manos and Steele 1997, Manos et al. 2001), therefore it is evident that we should
consider X. nasalis as one of the important pollinators for genera of large endemic trees
that constitute the deciduous and evergreen landscape in the area of central Thailand. In
addition, Burgett et al. (2005) reported the importance of Lithocarpus and Castanopsis
pollens as two of the top food sources of the night-flying carpenter bee X. (Vyctomellita)
tranqueberica (Fabricius, 1804) as well. They suggested that these pollen types serve as
the primary pollen sources for X. tranqueberica found in northern Thailand, second
to the introduced plant species of Casuarina Linnaeus, 1753, which is heavily planted
throughout Thailand for reforestation (Burgett et al. 2005).

Another group of large trees that also benefit from Xylocopa nasalis visitation is
Senna siamea (Fabaceae), and other related but unidentified species in the genus Cas-
sia. Senna siamea is an indigenous evergreen tree found throughout Thailand and other
neighboring countries in South and Southeast Asia; locals use its leaves mainly for
consumption; it is seldom used as fodder for animals and intercropping. The flowering
period of this species is documented to be during March to September or otherwise
year round, if the hot and humid weather permitted (Hanum and van der Maesen
1997, Sosef et al. 1998). Both S. siamea and Cassia have poricidal anthers, which
require a sonication or “buzz-pollination” from floral visitors to extract pollen from
their anthers and thus eventually affect pollination (Buchmann and Hurley 1978, Bu-
chmann 1983). Visitations by carpenter bees, which are known for their abilities to
vibrate their thoraces at the pores of the flowers’ anthers to release the pollens (King et
al. 1996, King and Buchmann 2003), are crucial for the reproductive successes in these
plant genera. Xylocopa nasalis may as well be an important pollinator of this group of
large trees in this area.

Nesting biology of X. nasalis 89

From the analyses of the pollen volume, we found that Croton (Euphorbiaceae)
contributes the highest volume (14.95%). It is important to note that though this genus
was found for only 2.80% of the total pollen count (Table 2), the relatively large size
of Croton makes it become one of the most important food sources for Xylocopa nasalis.
In Thailand, there are about 30 species of Croton (Chayamarit and Welzen 2005). The
genus has a reputation of containing biomedical-active compounds such as alkaloids
and terpenoids (Rizk, 1987) that have potential values to the pharmaceutical industries.

One genus of an annual floating-leaved aquatic plant is also frequently visited by
Xylocopa nasalis. The pollens of water chestnut of the genus Trapa (Lythraceae) con-
tribute 13.36% of the total pollen volume in the bee diets. Smitinand (2001) listed
only three 77apa species in Thailand: 7. bicornis Osbeck, 1771, T: incisa Siebold &
Zuccarini, 1845, and 7. natans Linnaeus, 1753. However, the taxonomy of Trapa is
still in flux (Kadano 1987; Cook 1996; Takano and Kadano 2005), and a thorough
survey of this common aquatic plant in Thailand is needed, since the fruits of T° d7-
cornis are one of the important food crops in Thailand. Identification of Trapa pollens
to specific level can provide important information regarding which species X. nasalis
visit and pollinate.

Lastly, the main shrub species that X. nasalis visits for pollen is Elaeagnus cf. lati-
folia (Elaeagnaceae), a prominent shrub that has a native range in northern Thailand,
although it can be found throughout the country due to its high adaptability to various
soil conditions and habitats (Smitinand 2001). Other minor pollen-providing plants
that can be identified in this work such as Sambucus and Ziziphus, which are possibly
introduced into the area as ornamental plants, and which contribute less than 1% of
the pollen volume in a given pollen mass.

Our observations of nest-defending by resident females are consistent with the nest
defending postures reported in Xylocopa sulcatipes and X. pubescens in Israel (Gerling
et al. 1989) and in X. (Ctenoxylocopa) fenestrata (Fabricius, 1798) in India (Kapil and
Dhaliwal 1968a), though whether the guarding females are the progeny of the found-

ing female still needs to be investigated in X. nasalis.

Conclusion

In summary, our observations and dissections of Xylocopa nasalis nests agree with
known reports of other Xylocopa species (Hurd 1958, Hurd and Moure 1960, 1961,
1963, Kapil and Dhaliwal 1968a, 1968b, Michener 1974, Mordechai et al. 1978, Ger-
ling et al. 1989, Boontop et al. 2008). The X. nasalis nest is strictly unbranched. The
provisioned cells are separated via partitions made from bamboo particles excavated
by the founding female. The nesting architectural details provided within this work
should prove to be of beneficial to beekeepers and researchers who are interested in
trapping and studying X. nasalis. For further genetic and social behavioral studies, we
found that in a given nest, sister bees can tolerate and live inside the same nest with
up to 7 individuals along with their mother. Kinship analyses using molecular mark-

90 W. Hongjamrassilp & N. Warrit / Journal of Hymenoptera Research 41: 75—94 (2014)

ers such as microsatellite DNA will reveal interesting details pertaining to the social
structure in a single nest and the population structure of the bees living communally
in the same vicinity in Ratch Buri province, Thailand (W. Hongjamrassilp and N.
Warrit (unpublished data)). As for the pollens foraged by X. nasalis, the broad host
plants range can be highly beneficial for many crop pollinations, particularly for plants
that require the “buzz” pollination method by their pollinators (Keasar 2010). Flower
constancy and other related pollination studies are required for further justification
of using X. nasalis as future potential pollinator for agricultural and forest plants in
Southeast Asia.

Acknowledgements

We would like to thank two anonymous reviewers and the editor: Dr. Jack Neff,
Drs Deborah Smith, and Charles Michener for helping us to improve the manu-
script. This research cannot be completed without the assistances of undergraduate
and graduate students of the Department of Biology, Chulalongkorn University: Pat-
tarawit Engkananuwat, Suttimon Narongchaisarid, Thanawan Duangmanee, Narin
Chomphuphung, Nantikarn Thongcharon, Wassamon Suwannarat and Chatphagorn
Rangsri. Pollen identification was guided by Dr Chumpol Khunwasi, Department of
Botany, Chulalongkorn University, and Ms. Nungruthai Wichaikul. Dr Ajcharaporn
Piumsomboon graciously contacted the SEM facility at the Center of Nano-imaging,
Mahidol University, for us to produce the pictures of the pollen grains. We are grate-
ful to the assistance of Dr Orawan Duangphakdee and Mr. Preecha Rod-im of King
Mongkut’s University of Technology Thonburi for their hospitality and for providing
an opportunity to the authors to study the carpenter bees in Ratch Buri province. This
research is partially funded by the Thailand Research Fund (TRF#4MRG5380139)
and Grants for the Development of New Faculty Staff, Chulalongkorn University,
Thailand, to NW.

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