EXAMENS ARBETE
Naturvård och Artmångfald 180 hp
Bumblebees in a region of northwestern Scania:
Is species number correlated to the number of flowering angiosperms and does gene flow occur between four locations?
Linnea Dahlgren
Biologi 15 hp
Halmstad 06-02-2014
1 Abstract
Pollination, one of our ecosystem services, is considered to be in critical condition due to a worldwide reduction in pollinators and their biodiversity. As the agricultural landscape becomes more and more intense, the pollinators lose important food and living resources.
In temperate ecosystems, bumblebees (Bombus spp) are an important group of wild pollinators, and as with pollinators in general, they are declining in both abundance and richness, in Sweden as well as other countries.
The purpose of this study was to see if bumblebee species number of a location ZDV linked to the location’s number of flowering angiosperm species in northwestern Scania when
examining eight locations, and to see if gene flow existed between four chosen locations.
The result of this study suggests that it is not possible to tell from the flowering
angiosperm species how many bumblebee species that will be abundant, but that it might be possible to tell the number of bumblebee individuals. With the number of bumblebee species, the abundant Fabaceae species was more important than the total number of flowering angiosperms of the location. The number of abundant Fabaceae species was strongly correlated to the bumblebee diversity index of the locations, indicating that it is a group of flowers closely linked to bumblebees.
To see if gene flow occurred between the chosen locations, mtDNA sequences were
compared in neighbor joining trees. The result showed that though some tendencies of
isolation existed, gene flow seemed to occur in general between the locations in that
fragmented and human dominated landscape of northwestern Scania.
2 1. Introduction
Many ecosystem services, i.e. processes in ecosystems that clearly benefit mankind, may be about to disappear (Daily 1997; Palmer et al. 2004). One of the ecosystem services that are considered to be in critical condition is the pollination of our crops (Corbet et al. 1991;
Williams 1994; Ingram et al. 1996; Matheson et al. 1996; Allen-Wardell et al. 1998; Kearns et al. 1998; Kremen & Ricketts 2000; Kevan & Phillips 2001; Kremen et al. 2002; Steffan- Dewenter et al. 2005; Ricketts et al. 2008; Steffan-Dewenter & Westphal 2008; Potts et al.
2010).
Most plants depend on animals as pollinators (Burd 1994; Kearns et al. 1998; Klein et al.
2007; Larson & Barrett 2000; Ashman et al. 2004), therefore it is believed that a reduction in pollinator species can lead to a corresponding reduction in plant species (Biesmeijer et al.
2006). It is also believed that an unsuccessful pollination in a fragmented landscape can be a major threat to the reproduction of wild plants (Persson 2011).
To humans, pollination is an important ecosystem service since animal pollination enables sexual reproduction in many of our crops (McGregor 1976; Crane & Walker 1984; Free 1993;
Williams 1994; Nabhan & Buchmann 1997; Westerkamp & Gottsberger 2000). About 35% of the world production of crops, fruits and vegetables depend on pollinators to form fruit and set seed, and the production of 39 of the globally leading 57 single crops increase in the presence of pollinating animals (Klein et al. 2007).
Worldwide, honeybees (Apis spp) are the most valuable pollinator economically when it comes to monocultures (McGregor 1976; Watanabe 1994). It is nowadays quite common in some places that our crop pollination is handled by domesticated honeybees (Klein et al.
2007). However, honeybee colonies are declining in many parts of the world (Williams et al.
1991; Matheson et al. 1996; Delaplane & Mayer 2000), partly due to the dispersal of pests such as parasitic mites (Downey & Winston 2001; Chen et al. 2004). In North America, much of the honeybee colonies were eliminated in the Colony Collapse Disorder (Stokstad 2007), and it was then realized how big of a risk it is of relying on one single pollinator.
Pollination is an important process in angiosperm flowers, being necessary for fertilization
(Campbell et al. 2008). It is the transfer of pollen from the anther of one plant to the stigma on
another plant. In angiosperm species pollination is most often received through biotic ways, as
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much as 80% of the species. The animals are either tricked to the flowers or rewarded with nectar. The nectar is produced by nectaries at the base of the flowers.
The reason that pollination is considered to be in danger is because a reduction of important pollinators and their biodiversity has been observed worldwide (Memmott et al.
2004; Biesmeijer et al. 2006; Fortuna & Bascompte 2006). The main reasons for the declines are considered to be habitat destruction and fragmentation as consequences of human activity (Buchmann & Nabhan 1996; Kearns et al. 1998; Biesmeijer et al. 2006).
Agriculture has become more intensive, which affects not only pollinators but also causes reductions and loss of biodiversity in other groups of organisms which live in the agricultural landscape (Stoate et al. 2001; Kleijn et al. 2009). The intensification of agriculture causes natural and semi-natural habitats in the landscape either to disappear, become fragmented or reduced in quality (Vandermeer & Perfecto 2007). The reason of why fragmentation and degradation are harmful to pollinators is most often because it comes with the loss or
dismemberment of important feeding and living resources (Hines & Hendrix 2005; Potts et al.
2005). When Klein et al. (2007) reviewed the studies done on the impact of agricultural intensification on pollination, all 16 studies found showed that the intensification have a negative impact on pollination.
Bumblebees (Bombus spp) are an important group of wild pollinators in temperate ecosystems (Corbet et al. 1991; Kremen et al. 2002; Memmott et al. 2004; Fontaine et al. 2006; Hegland
& Totland 2008) of both crops and wild flowers (Bommarco et al. 2012), and along with wild bees they have a considerable ecological function (Corbet et al. 1991; Allen-Wardell et al.
1998; Kremen et al. 2002; Klein et al. 2007).
In Sweden 29 native species of social bumblebees exist (Persson 2011). Bumblebees have two main resources they need: nectar and pollen from flowering plants since they feed exclusively on that, and secondly they need suitable nest sites within their foraging range (Ahrné 2009).
Social bumblebees live by an annual cycle around a queen (Goulson 2003; Benton 2006).
The queen wakes from hibernation sometime during the spring and begins the search for a
good location to place her nest. She lays her eggs in the nest and raises the first generation of
workers by herself. But when the first generation can begin foraging, the queen stays in the
nest and lays eggs while the workers ensure that enough food, to feed themselves and the new
generations of workers, arrive to the nest. Sometime during summer the intake of food to the
colony is high enough for sexual offspring to be produced. The sexual offspring consists of
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males and new queens, and once they have been raised the colony dies out. The males and new queens mate before autumn, where upon the males die. Queens on the contrary start foraging to create a reservoir of energy, and begin the search for suitable places to where they can hibernate while awaiting spring. During the lifetime of a queen, she therefore needs surmountable distance both to a partner, someplace for winter rest and someplace close to a large quantity of nectar and pollen to place her nest.
Social bumblebees have haplo-diploid sex determination, i.e. males develop from unfertilized eggs and thus are haploid while daughters, workers and new queens that is, are diploid as they develop from fertilized eggs (Goulson 2003; Benton 2006). So come it is possible for workers to lay haploid eggs that develop into males.
The sturdy size of bumblebees, their long tongues and high-frequenting humming to make pollen release from flowers, significantly increases the efficiency of pollen transfer with economically important crops such as tomatoes and berries (Cameron et al. 2011). Seed setting in red clover are entirely dependent on pollination by insects and happens mainly through the activity of bumblebees (Bommarco et al. 2012).
In recent decades a general decline and disappearance of bumblebees has been observed (Goulson et al. 2008; Williams & Osborne 2009; Cameron et al. 2011), and as with the other pollinators it is considered to be caused primarily by changes in land use and intensification, for bumblebees leading to loss of nest and floral resources (Carvell et al. 2006; Goulson et al.
2008). In Sweden, large areas of key habitats for the bumblebees, such as hayfields and pastures, have during the past decades often been made into arable land or forest, and even semi natural landscape elements such as dikes and edges of fields have lessened in extension.
That is believed to be the main causes of the observed decrease in several bumblebee species in Sweden (Bommarco et al. 2010; Williams et al. 2010).
The purpose of my study was to investigate whether a connection existed in northwestern Scania between the number of flowering angiosperms and the number of bumblebee species in locations of different environments, though mainly agricultural. Is it possible to know, from a location’s number of flowering angiosperms, how many bumblebee species there will be present?
I have also investigated whether gene flow occur among bumblebees of the species B.
terrestris , B. hortorum, B. sylvarum, B. hypnorum, B. lapidarius, B. pascuorum and B.
locorum collected from four different locations in northwestern Scania.
5 1.1 Restrictions
In this study I have only identified social bumblebees and no cuckoo bumblebees, and social bumblebees is what I am referring to when using the word bumblebees.
When investigating the flowering angiosperms of the locations, I excluded all grasses.
2. Materials and Methods 2.1 Study area
The study took place in northwest of Scania. Scania is a province in the most southern part of Sweden. The study area I chose is on the border between the landscape of large fields and the landscape of forests with small fields incorporated.
2.2 Locations
In the summer of 2012 eight locations were selected in northwestern Scania and were named in the order they were visited, location 1 being the first location visited and location 8 being the last. In May and June year 2013 the locations 1, 3 and 7 were visited again to examine bumblebees’ ability to spread between the mentioned locations. A new location was also added as a null point between the other three locations, and given the name 0. The positions of the locations are shown in Figure 1. Between the locations 0, 1, 3 and 7 and between locations 2 and 4, the distance was measured with Distance Meter version 4.2.2, an android application.
Figure 1, map of the northwestern parts of Scania showing the locations visited.
6 2.3 Netting and examination of the locations
During the summer of 2012 eight locations, location 1-8 showed in Figure 1, were visited when the weather was calm with only moderate wind and it was sunny to partly cloudy. Still the locations were visited twice to counteract any influence of the current weather.
On site, an inventory along a 40 meter transect was made. The flowering angiosperms along the transect and within 0.5 meter from the transect were noted.
Bumblebees were caught and after half an hour of netting along the transect the captured bumblebees were species identified and released. The species identification was made using Humlor i Sverige – 40 arter att älska och förundras över (Cederberg 2012) and Humlor: Alla Sveriges arter – Så känner du igen dem i naturen och i trädgården (Holmström 2007). Each location was netted twice, the two times being on different days.
For each location a Shannon’s Diversity Index was calculated based on the bumblebee data from the location. The diversity index reaches from 0 and up, 0 being no diversity for the location whatsoever. The higher Shannon’s Diversity Index, the higher diversity.
To investigate whether it is possible to know the number of bumblebee species based on the location’s number of flowering angiosperms, a correlation between the two variables was made. Correlation was also used to see if Fabaceae is as important as thought to be for bumblebees (Prys-Jones 1982; Rasmont 1988; Carvell 2002; Goulson & Darvill 2004;
Goulson & Hanley 2004; Goulson et al. 2005; Goulson et al. 2006) at my locations and to see if the diversity index showed any significance to any other variables.
Tongue length is thought to be an important factor describing why some bumblebee species manage well in the agricultural landscape of today and some species don’t (Goulson et al. 2005), and therefore a correlation was made between the approximate tongue length of a species, based on data from Goulson et al. (2005), and how abundant the bumblebee species was at my locations.
All statistics were conducted in the R environment version 3.0.0 (R Development Core Team, 2013) and I used Spearman Rank Correlation since the data was not normally distributed.
2.4 Netting and capture of bumblebees at four different locations
Twelve bumblebees were collected respectively from four different locations, location 0, 1, 3 and 7, and put in 100% EtOH (May,2013). During the early summer 36 additional
bumblebees of the species B. sylvarum were collected, put in 100% EtOH and used along with
the other collected bumblebees to examine gene flow between the four locations.
7 2.5 DNA extraction
One leg from each bumblebee individual was placed in a separate tube, after which 100 μl lysis buffer (0.1M TRIS, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, pH 8.5) and 5 μl proteinase K was added. The ingredients in the tubes were thoroughly mixed where upon the tubes got incubated in a water bath of 56˚C. After an hour they were mixed before an additional 12 hours in the water bath. After the 12 hours they got mixed again and then centrifuged in ten minutes at 10 000 rpm.
In new tubes 10 μl 3M NaA was added, after which the supernatant was transferred to the new tubes. 220 μl ice cold 99% ethanol was added to the tubes. The contents were then inverted a few times.
Thereafter the tubes were centrifuged in ten minutes at 10 000 rpm. The supernatant was removed without disturbing the pellet and 100 μl ice cold 70% ethanol was added in order to wash the pellet. The tubes were centrifuged for another five minutes at 10 000 rpm. After removing the supernatant the samples with the DNA pellet were set to dry without lids for 13 hours. 50 μl 1xTE buffer was added in order to solve the pellet.
2.6 PCR (Polymerase Chain Reaction), sequencing reaction and analysis
Mitochondrial DNA sequences (mtDNA) for bumblebee cytochrome C from NCBI (www.ncbi.nlm.nih.gov) was used to design primers (forward primer 5’-TGA ATT AAY AAT GAT CAA ATT TAT-3’ and the reverse primer 5’-GGT ATT AAA ATT TCG ATC AA-3’).
A polymerase chain reaction (PCR) was done according to the AmpliTaq polymerase kit protocol to amplify the cytochrome C gene. The samples were run on a 2% agarose gel using electrophoresis. A sequencing reaction was made using BigDye direct Cycle sequencing kit.
Sequences were received from an ABI PRISM 3130 genetic analyzer (Applied biosystems, New Jersey, US).
The sequences were aligned in the program BioEdit version 7.2.3. Neighbor joining trees
were constructed in Mega version 5.2.2. 1000 replications were used to form bootstraps. As
outgroup a mtDNA sequence for cytochrome C of a honeybee, Apis mellifera, from NCBI
(www.ncbi.nlm.nih.gov) was used.
8 3. Results
3.1 Results when examining WKHSRVVLEOHFRUUHODWLRQEHWZHHQDQJLRVSHUPVDQGEXPEOHEHHVSHFHLV A total of 140 bumblebees were netted and altogether 8 different species were identified
during the summer of 2012 while visiting the eight locations. For the single locations, the number of species varied between zero and seven. Number of individuals at the locations varied between zero and twenty-nine, and the diversity index did not rise above 0.675, which was obtained at location 1. B. pascuorum was the most abundant species in total and B.
soroeensis the rarest, with only one individual found at location 1 (Table 1).
Table 1. Bumblebee species and number of individuals of each species detected at locations 1-8 during the summer of 2012. The bumblebees were captured by netting along a 40 meter long transect. The locations are named after the order in which they were visited, location 1 as the first location visited.
Of the flowering angiosperms, Achillea millefolium (Figure 2 to the left) was the only species found on all locations. The second most common species was Ranunculus acris that was abundant on five locations. The most Fabaceae (Figure 2, middle and right) species rich location was location 1, with six species of Fabaceae. Two locations, location 6 and 8,
contained no Fabaceae species at all. The garden location, location 4, had the highest number of flowering angiosperms only abundant at one location. It also had the highest number of flowering angiosperms in total, while location 6 had the lowest number (Table 2).
Figure 2, showing Achillea millefolium, Trifolium repens and Trifolium pratense. Photographs from http://www.linnaeus.nrm.se/flora.
B.
hortorum B.
lucorum B.
terrestris B.
lapidarius B.
pascuorum B.
sylvarum B.
soroeensis B.
pratorum
Total specimens
number
Total species number
Shannon’s diversity
index
Location 1 4 1 2 5 10 6 1 29 7 0.675
Location 2 8 4 12 2 0.265
Location 3 4 1 6 7 5 24 6 0.576
Location 4 2 9 10 3 24 4 0.508
Location 5 3 8 3 4 18 4 0.430
Location 6 0 0 0
Location 7 11 4 2 17 3 0.362
Location 8 8 2 6 16 3 0.359
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Table 2. The locations visited during the summer of 2012, named after the order visited in, location 1 as the first location visited. In the column "Type of land" the location is shortly described and in the column "Flowering angiosperm species" the angiosperms at the location found at the 40 meter long transect is recounted in order of first appearance.
Location Type of
land Flowering angiosperm species Number of
angiosperm species
1
Meadow close to
urban areas, forest and agricultural
landscape.
Trifolium repens, Ranunculus acris, Hieracium sect. Vulgata, Knautia arvensis, Bellis perennis, Platanthera bifolia, Vicia cracca, Galium album, Campanula rotundifolia, Achillea millefolium, Lathyrus linifolius, Medicago lupulina, Pilosella aurantiaca, Vicia tetrasperma, Trifolium pratense, Stellaria graminea, Veronica chamaedrys, Hypericum maculatum, Senecio jacobaea, Galium verum, Prunella vulgaris, Sambucus nigra, Rubus fruticosus, Epilobium angustifolium.
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2 Meadow in forest.
Anthriscus sylvestris, Stellaria graminea, Cirsium arvense, Achillea millefolium, Galium boreale, Melampyrum sylvaticum, Melampyrum pratense, Vicia cracca, Hypericum maculatum, Ranunculus acris, Prunella vulgaris, Potentilla erecta, Knautia arvensis, Hieracium sect.
Vulgata, Lamium album, Epilobium angustifolium, Veronica chamaedrys, Lamium purpureum, Heracleum sphondylium.
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3
Edge of field close
to urban areas.
Chamomilla recutita, Achillea millefolium, Matricaria matricarioides, Myosotis sp, Veronica chamaedrys, Trifolium repens, Cirsium arvense, Lotus corniculatus, Ranunculus acris, Trifolium arvense, Lamium purpureum.
11
4 Garden in forest landscape.
Lavandula angustifolia, Salvia nemorosa, Leucanthemum vulgare, Prunella vulgaris, Campanula rotundifolia, Stellaria graminea, Melampyrum pratense, Trifolium repens, Achillea millefolium, Tagetes sp, Ranunculus acris, Myosotis sp, Verbascum olympicum, Digitalis sp, Pilosella aurantiaca, Hypericum maculatum, Hydrangea arborescens, Rosa sp, Thymus vulgaris, Origanum vulgare, Papaver radicatum, Heuchera sp, Tanacetum parthenium, Mentha sp, Viola tricolor.
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5
Edge of field close
to urban areas.
Achillea millefolium, Trifolium repens, Matricaria matricarioides, Trifolium campestre, Myosotis sp, Chamomilla recutita, Hieracium sect.
Vulgata, Calystegia sepium, Cirsium vulgare, Cirsium arvense. 10
6 Edge of
field. Chamomilla recutita, Achillea millefolium, Hieracium sect. Vulgata,
Ranunculus acris. 4
7 Edge of
field close to gardens.
Campanula rotundifolia, Achillea millefolium, Hypericum perforatum, Trifolium repens, Trifolium pratense, Cirsium arvense, Epilobium
angustifolium, Echium vulgare, Trifolium arvense. 9 8 Meadow in
forest. Calluna vulgaris, Campanula rotundifolia, Potentilla erecta, Achillea
millefolium, Jasione montana, Knautia arvensis. 6
The correlation showed a non-significant correlation coefficient between number of bumblebee species found at the locations and the number of flowering angiosperm species found at the locations (Table 3), suggesting that bumblebee species and angiosperm species of a location do not co-vary.
However, between angiosperm species and bumblebee individuals found at the locations, a
significant correlation was found, suggesting that those two variables might be linked (Table 3).
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Number of bumblebee species of the locations was positively and significantly correlated to the location’s number of Fabaceae species. Number of Fabaceae species correlated with bumblebee individuals of a location was non-significant.
B. hortorum is the species in this study with the longest tongue (Goulson et al. 2005).
Species with long tongues is thought to be more connected to Fabaceae than species with shorter tongues (Goulson et al. 2005; Carvell et al. 2006; Goulson et al. 2008). However, B.
hortorum showed not as strong correlation to Fabaceae as B. sylvarum and B. lapidarius, who both had significant correlation coefficients (Table 3). B. sylvarum also showed a quite strong and significant correlation to abundance of Trifolium repens.
Abundance of Trifolium repens at the locations showed a strong and significant correlation to the diversity index of the locations as well, while Trifolium pratense did not. Even so, Fabaceae as a whole showed a strong and significant correlation to the diversity index of bumblebees. Significantly correlated to the diversity index of the location was also the numbers of B. hortorum and B. sylvarum (Table 3).
Table 3. Correlation between two variables of data collected in the summer of 2012 using Spearman Rank Correlation. The Shannon’s Diversity Index was made on the bumblebee data for each location (Table 1). The r value describes how strong the correlation is, 1 being as strong as can be and 0 being no correlation whatsoever.
The p value describes how significant the r value is, where values up to 0.05 are significant.
Variable 1 Variable 2 r p
Number of bumblebee species Number of angiosperm species 0.61 0.10 Number of bumblebee ind Number of angiosperm species 0.74 0.04 Number of bumblebee species Number of Fabaceae species 0.71 0.05
Number of bumblebee ind Number of Fabaceae species 0.68 0.06 Number of B. hortorum Number of Fabaceae species 0.58 0.16
Number of B. sylvarum Number of Fabaceae species 0.84 0.004 Number of B. lapidarius Number of Fabaceae species 0.72 0.04
Number of B. sylvarum Abundance of Trifolium repens 0.72 0.04 Diversity index of bumblebee Number of Fabaceae species 0.75 0.03
Diversity index of bumblebee Abundance of Trifolium repens 0.84 0.008 Diversity index of bumblebee Abundance of Trifolium pratense 0.38 0.36
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Diversity index of bumblebee Number of B. hortorum 0.86 0.006
Diversity index of bumblebee Number of B. sylvarum 0.78 0.02
When the approximate tongue length from Goulson et al. (2005) was correlated to how abundant every species was in a total at the locations, the result was non-significant (t=0.83, df=6, r=0.32, p=0.43, Figure 3).
Figure 3, the species found at the locations 1-8 during the summer of 2012, with approximate tongue length from Goulson et al. (2005) on the x-axis and total abundance of the bumblebee species at locations 1-8 on the y-axis.
3.2 Results when examining possible gene flow
During the summer of 2013, 84 bumblebees of the species B. terrestris, B. hortorum, B.
sylvarum , B. hypnorum, B. lapidarius, B. pascuorum and B. lucorum were collected from location 0, 1, 3 and 7 and successfully sequenced for cytochrome C. The gene sequences were used to form neighbor joining trees for each species. None of the species showed clear
isolation in any of the locations. The neighbor joining tree of B. sylvarum, the species that had the most collected individuals, is shown in Figure 4, and the trees of B. terrestris, B.
hortorum , and B. lapidarius can be viewed in the supplementary material. The phylogenetic tree of B. sylvarum shows that individuals from different locations grouped together in the same clusters. The bootstrap values are however mostly quite low, making this result a little uncertain. But there are some clusters with high bootstrap values that diverge early from the rest: one group of two individuals from location 0 and one from location 1, one group of four
B. pascuorum
B. lucorum B. terrestris B. lapidarius
B. sylvarum
B. hortorum
B. pratorum B. soroeensis 0
5 10 15 20 25 30 35
6 7 8 9 10 11 12 13 Tongue length (mm)
Number of specimens at location 1-8
12
individuals from location 0 and one group of one individual from location 0 and one from location 3. Among the clusters with low bootstrap values, there is one cluster with individuals just from location 7 and one cluster with five individuals from location 3 and one individual from location 1, indicating on separate populations at these locations. But since not all of the individuals from these locations are placed within the clusters but are more genetically alike other individuals, it suggests that gene flow still occurs.
Figure 4, neighbor joining tree of B. sylvarum. The specimens were collected in summer 2013. The names 0, 1, 3 or 7 of the individuals tell which location the specimen was collected in. The locations are shown in Figure 1.
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A neighbor joining tree was constructed with one individual of each species found in 2013 (Figure 5). It shows a possible relationship of the different bumblebee species.
B. lucorum, the species in this phylogenetic tree that diverges first from the rest, is the one of the species collected in the summer 2013 with the shortest tongue, while B. hortorum and B. sylvarum , which is the last of diverging from the rest, is the two species with the longest tongues (Goulson 2005). It is therefore likely that the ancestor of these bumblebee species was short tongued, and that a longer tongue has been evolved over time.
It was a bit surprising that B. pascuorum and B. lapidarius are so genetically alike, but although they are not very morphologically alike they are both small species with quite similar tongue length. However, B. lucorum and B. terrestris are very close to one another morphologically and have just about the same tongue length, which could indicate on that B.
terrestris is misplaced; the divergence of B. terrestris do have the lowest bootstrap value in the phylogenetic tree.
Figure 5, neighbor joining tree of one individual from all Bombus species collected in summer 2013.
14 4. Discussion
The answer whether it is possible to tell from a location’s number of flowering angiosperm species how many bumblebee species that will be abundant is, based on my results, no. The correlation between the two variables gave a medium strong non-significant correlation coefficient at 0.61 (Table 3). Number of flowering angiosperm species could definitely be a factor influencing bumblebee species abundance of a location, but it is most likely not the only factor. For example, Mänd et al. (2002) found in their study that the changed species composition of plants is a greater cause than abundance of forage flowers to why species richness in bumblebees has declined. However, it might be possible to tell from a location’s number of flowering angiosperms how many bumblebee individuals there will be, since the correlation coefficient between those two variables was strong and significant.
Fabaceae is one group of plants that has disappeared a lot as the agriculture has become more intense all over Europe (Goulson 2003). Fabaceae, and especially Trifolium pratense, has been identified as an important key nectar and pollen source for bumblebees (Prys-Jones 1982; Rasmont 1988; Carvell 2002; Goulson & Darvill 2004; Goulson & Hanley 2004;
Goulson et al. 2005; Goulson et al. 2006). This concurs with my results, as a quite strong positive correlation coefficient of 0.71 was found between the number of Fabaceae species of a location and the number of bumblebee species of a location. Significant and positive
correlations was also found between diversity index of the location and number of Fabaceae species abundant (Table 3). That indicates that if a location contains a plentiful of Fabaceae species, the bumblebee species will also be rich and the total of bumblebees numerous, and vice versa. It also suggests that the number of Fabaceae species of a location is more
important than the total number of flowering angiosperms, at least for species richness, since the correlation coefficient was higher with Fabaceae when correlated to the number of bumblebee species.
My data showed a strong correlation between abundance of Trifolium repens on a location
and the location’s diversity index of bumblebees (0.84) whilst not between diversity index
and Trifolium pratense (0.38). That may be because Trifolium repens was quite abundant in
my locations, while Trifolium pratense only was abundant in two of the locations (Table 2)
and in one of those locations very sparsely. The location that contained high abundance of
Trifolium pratense , location 1, also had the highest diversity index.
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Location 1 was also the most Fabaceae species rich location in total (Table 2). The
location contained the highest number of bumblebee species, one of them being B. soroeensis which only was found in that location. This indeed indicates the importance of Fabaceae.
Fabaceae is thought to be extra important to bumblebee species with long tongues
(Goulson et al. 2005; Carvell et al. 2006; Goulson et al. 2008) since they are more specialized in collecting pollen from flowers with deep corolla, which many Fabaceae species have (Goulson et al. 2005). The most long tongued species I found is B. hortorum with
approximately 12.5 millimeters in tongue length (Goulson et al. 2005). B. hortorum showed some correlation, though non-significant, with the abundance of Fabaceae species (0.58), however not as strongly as B. sylvarum who showed the strongest and most significant correlation to Fabaceae (0.84), while B. lapidarius showed the second strongest correlation (0.72). B. sylvarum is the one of my species after B. hortorum with the longest tongue, and B. lapidarius is as well one of the longer tongued species in my study, though neither of these two species are counted as a long tongued bumblebees in general (Goulson et al 2005). My results thereby almost concur with the theory of long tongued species’ special relationship to Fabaceae.
Tongue length has often been suggested as one of the traits determining how well the bumblebee species adapt to the intense agricultural landscape of nowadays (Goulson et al.
2005), since some of the bumblebee species still are very abundant and common while most bumblebee species are declining and might even be locally extinct (Goulson 2003). Tongue length has been strongly negatively correlated with diet breadth in plants with pollen (Goulson et al. 2008), and therefore is thought to determine how specialized the bumblebee species are in collecting pollen. However, tongue length varies a lot even within a species, with great individual differences that creates specialization among nest mates and thereby broadens the possible forage plants of the colony, but also increases the niche overlap between species (Goulson et al. 2002; Peat & Goulson 2005; Peat et al. 2005). Even so the different tongue length has long been thought to be an important factor dividing the resources among the species, thereby determining how bumblebee species, which otherwise are very similar in biology and functioning to one another, can coexist (Barrow & Pickard 1984;
Johnson 1986; Graham & Jones 1996). However, several studies in Europe have not been able to find any relationship between tongue length and coexisting (Ranta & Vepsäläinen 1981;
Ranta 1982; Ranta & Tiainen 1982; Ranta 1983; Williams 1985; Williams 1988); instead it is common to find several short tongued species at the same location and just a few with
medium or long tongue. As was the case of my study, were B. hortorum was the only long
tongued bumblebee species (Goulson et al. 2005). B. hortorum is quite special since it is the
16
only long tongued species that has fared well in general with the modern agriculture, and that despite the fact that it is a species thought to have a strong relationship to Fabaceae (Goulson et al. 2005; Goulson et al 2008). Goulson et al. (2005) has a theory that it may be so that the few remaining flowers with deep corollas are just enough to maintain one bumblebee species with long tongue, and that species happens to be B. hortorum.
For long it has been suggested that long tongued species is the ones that have declined most (Rasmont 1988; Prys-Jones & Corbet 1991; Rasmont et al. 1993), which is somewhat alarming since they are irreplaceable pollinators of flowers with long corollas (Heinrich 1976;
Corbet et al. 1991; Rasmont et al. 1993); when some short tongued bumblebee species visit deep flowers they tend to rob nectar from the flower without pollinating it (McGregor 1976;
Diekötter et al. 2005). My results show no correlation between bumblebee species tongue length (Goulson 2004) and how abundant the species was in my locations, which might be because of the abundance of B. hortorum. Moreover, tongue length is most likely not the only factor deciding how well the species cope in today’s landscape; other traits with impact has been thought to be body size of workers (Westphal et al. 2006), colony size (Rundlöf et al.
2008; Westphal et al. 2006), if the nest is above or below ground (Williams et al. 2010), emergence time (Fitzpatrick et al. 2007), the geographical range of the species (Williams 2005) and how close to the edge of that range the species is located (Williams et al. 2009;
Williams et al. 2007). Both foraging distances (Benton 2006) and queen dispersal (Darvill et al. 2010) is thought to be important as well. It has been shown that species very specialized in their diets often are rare, while the still abundant species are generalists (Goulson and Darvill 2004; Goulson et al. 2005) and that species that both emerge early in spring, have short reproductive cycles and which colonies are individual rich, seem to be able to reproduce even in landscapes with great homogeneity (Persson 2011).
There are six species of bumblebees that are still widespread and common (Goulson et al.
2005), and all those species are generalists in their diets and five of the six species have queens emerging early in spring. All of the six species were abundant in my study, the species being B. lucorum, B. pratorum, B. terrestris, B. pascuorum, B. lapidarius and B. hortorum.
This means that in my eight locations during the summer of 2012, I only found two species that are a bit rarer, B. sylvarum and B. soroeensis, of which only B. sylvarum was abundant in more than one location. Both B. sylvarum and B. soroeensis are species declining over Europe (Benton 2006).
Ranta et al. (1981) and Ranta and Vepsäläinen (1981) found in the early 1980’s that
bumblebee communities most often consist of six to eleven species in North and Central
17
Europe. I found totally eight species during the summer of 2012, and on the different locations the species number varied between 0 and 7, the mean value being 3.625 (Table 1).
This indicates that the richness and well-being of the bumblebee community in this region of northwestern Scania might be poor, and locally very poor. Hopefully there are species at the locations that I missed in my field work, perhaps species that are active either very early or very late in the year; otherwise the situation of pollination in this region is in critical condition.
Diversity is important in pollinator communities, since species composition and abundance varies a lot from year to year in bumblebee communities, and a low diversity community cannot buffer these changes like a high diversity community can; with a low diversity community the functioning of pollination too will vary from year to year (Lawton & Brown 1993; Tilman et al. 1997; Naeem 1998; Kremen et al. 2002; Balvanera et al. 2006; Cardinale et al. 2006). The mean diversity of my locations was 0.40, which unfortunately is quite low.
It has been shown that gardens have a positive effect on bumblebee populations since gardens often contain a lot of different plant species and is flowering for a long period (Fussel
& Corbet 1992; Comba et al. 1999a; Comba et al. 1999b; Goulson et al. 2002; Goulson 2010;
Stelzer et al. 2010; Persson 2011). Quite recently the importance of gardens in rural areas both for bumblebees (Osborne et al. 2008a; Goulson et al. 2010; Persson 2011) and for the
pollination of wild plants (Cussans et al. 2010; Goddard et al. 2010; Persson 2011) was discovered; it is thought that gardens can serve as refuges for bumblebees. Maybe that is true for gardens in forests as well, since the garden in my study, location 4, had the double amount of both bumblebee species and bumblebee specimens (Table 1) compared to location 2, a forest meadow just 0.46 kilometers away from location 4.
One may think that urban areas would be very beneficial for bumblebees because of the many gardens and parks, and some studies have indeed found that bumblebee species richness is higher in the heterogeneity of urbanization than in the surrounding natural landscape
(Goulson et al. 2002; McFrederick & Le Buhn 2006; Winfree et al. 2007; Matteson &
Langellotto 2009), but if urbanization is measured by percentage of impervious surface, it is clear that increasing urbanization affects species richness and diversity of bumblebees negatively (Ahrné 2008; Ahrné et al. 2009). But however the effect, urbanization alters species composition (Grimm et al. 2008; Matteson et al. 2008; Kadlec et al. 2009) and is thought to favor generalists the most since gardens and parks often is dominated by exotic plants and typical garden plants not found in the natural landscape (Thompson et al. 2003;
Frankie et al. 2005; Gaston et al. 2005) and generalist pollinators do more easily adapt to the
18
new forage plants (Darvill et al. 2004; Cane 2005a; Eremeeva & Sushchev 2005) with the consequence of floral specialists remaining scarce in urban areas (Cane 2005b; Frankie et al.
2005; Cane et al. 2006; McFrederick & Le Buhn 2006). In my study location 1, 3 and 5 was close to urbanization and location 7 close to gardens. Since the locations was on the border between urbanization and surrounding landscape it should be just beneficial for the
bumblebees, since the percentage of impervious surfaces was low but the closeness to gardens high. And indeed these locations along with location 4, the garden in the forest, are the
locations richest in bumblebee species, bumblebee specimens and bumblebee diversity (Table 1). It was on location 1, 3, 5 and 7 the somewhat rarer B. sylvarum was found, as with the more common B. lapidarius, and this indicates that these borders between urban areas and rural areas may be important in their richness of forage plants and closeness to both natural and horticultural flowers.
How far bumblebees can travel has long been a question of discussion, but most studies trying to give an answer have been focusing on foraging ranges. Of those studies it is clear that different species have different ranges (Free & Butler 1959; Walther-Hellwig & Frankl 2000a; Chapman et al. 2003; Knight et al. 2005), where B. terrestris can travel several hundred meters from the nest (Dramstad 1996; Osborne et al. 1999), some say even
kilometers (Kreyer et al . 2004; Osborne et al. 2008b), while other species are thought to be
”doorstep foragers” that not readily travel longer than a few hundred meters from the nest. Of the species I found, B. pascuorum (Walther-Hellwig & Frankl 2000b; Darvill et al. 2004;
Knight et al. 2005) and B. sylvarum (Witte et al. 1989; Hedtke 1996) are thought to be doorstep foragers. Foraging ranges of the worker bumblebees does not have any impact on gene flow, but the one study made on queen dispersal found results that indicate that queens travel distances exceeding that of foraging distances of workers (Lepais et al. 2010). In B.
pascuorum , a species thought to be a doorstep forager, the study showed that queens disperse at least 3 kilometers in total when counting all the different steps in her lifetime. For B.
lapidarius , a species thought to travel quite well (Knight et al. 2005), Lepais et al. (2010) found that the queens probably can disperse up to 5 kilometers. No matter species, from their birth nest to their own nest, bumblebee queens seemed able to disperse a few kilometers.
As with the dispersal distances, little is known of how bumblebees cope with barriers in the landscape. It is known that animal movement can be restricted as the landscape becomes fragmented by roads, railroads, buildings etc. (Mader 1984; Didham et al. 1996; Forman &
Alexander 1998) and it is also known that bumblebees can cross these barriers if necessary
(Bhattacharya et al. 2003). However, Bhattacharya et al. (2003) also saw that the barriers may
19
constrain or discourage bumblebees, at least if resources are enough already where the bumblebees are.
Of the four locations from which I collected specimens to investigate gene flow, location 3 was separated from the other three locations by both railroad and a highly trafficked road.
Location 7 had a distance of between 2.8 and 3.6 kilometers to all other locations. But neither distance nor barriers prevented gene flow between the locations in my study, since none of the species showed sign of isolation at any location. The neighbor joining tree in this report shows the specimens collected of B. sylvarum (Figure 4), which is thought to be a doorstep forager (Witte et al. 1989; Hedtke 1996). Even so, no isolation shows, except for four individuals from location 0 forming a cluster of their own, and two clusters with low
bootstrap values containing individuals from location 3 and 7 respectively. This indicates that there are some tendencies of isolation at location 3, separated from the other locations by a road and railroad, and location 7, separated by a distance of 2.8-3.6 kilometers from the other locations. So maybe these factors, i.e. roads and high distances, constrain gene flow to some extent, at least in this species thought to be a doorstep forager, but still not completely since other individuals from location 3 and 7 are placed in clusters with specimens from other locations. In the case of the four individuals from location 0 forming a cluster of their own, location 0 is somewhat of a null point between the other locations and the most likely location not to be isolated; therefore I believe that these four individuals are nest mates and very genetically alike, and that is why they are forming a cluster of their own.
In the other clusters with high bootstrap values, the individuals are collected in different locations. This indicates on good gene flow, even though the gene flow mostly seems to occur to and from location 0. The exception is the four individuals from location 1 that form clusters with individuals from all other locations but not with each other. Location 1 was the most Fabaceae species rich location, had the highest Shannon’s Diversity Index of bumblebees and was the most bumblebee individual rich location, which all suggests that bumblebees are able to travel considerable distances, at least several kilometers, to find these favorable places hidden in the landscape, and that they do it as well.
It is truly hopeful that bumblebee populations in this region of Scania show no signs of
clear isolation. It indicates that if only areas with forage plants, including Fabaceae species as
a suggestion, are restored in the landscape, bumblebees, and not only the highly mobile
generalist species, have a high ability to reclaim the land they lost. And it is something to be
glad about, when all else points toward a future without wild pollinators.
20 Acknowledgements
A big THANK YOU to my mentor, Maria Karlsson. Without your help and good advices I would still be struggling in the dark with this study; you truly have enlightened me.
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27 Supplementary material
Supplementary figure 1, neighbor joining tree of B. terrestris collected in summer 2013.
Supplementary figure 2, neighbor joining tree of B. hortorum collected in summer 2013.
Supplementary Figure 3, neighbor joining tree of B. lapidarius collected in summer 2013.
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