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\begin{document}

\title{Distinguishing white-tailed bumblebees in the Netherlands: morphology,
ecology and DNA-barcoding}



\author[1]{Lucas Alferink}%
\author[1]{Leon Marshall}%
\author[2]{Roland De Jonghe}%
\author[1]{Jacobus Biesmeijer}%
\affil[1]{Naturalis Biodiversity Center}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
White-tailed bumblebee species, \emph{Bombus cryptarum}, \emph{B.
lucorum}, \emph{B. magnus} and \emph{B. terrestris} are known to be very
similar in their morphological characters across the majority of their
ranges. This hampers assessment of their status and trends because
reliable identification is difficult. In this study, we use a
combination of characters and methods to assess how ecologists and
citizen scientists can reliably and quickly separate these four species
occurring in the Netherlands. Bumblebees (queens, workers and males)
were sampled from 10 locations across the Netherlands and specimens were
identified based on COI sequence data. Next, the same specimens where
scored for morphological traits. We show that a combination of easy to
recognise characteristics can separate some specimens of the species
depending on caste and sex. \emph{Bombus magnus} males and queens and
\emph{B. lucorum} males were most reliably separated from the other
species using morphological characters. Workers of all four species
cannot be separated completely using morphological characters alone.
This is the first time standard morphological characters and ecological
data has been used to study the differences between the white-tailed
bumblebees in the Netherlands. Based on our findings we need to conclude
that the status of these bumblebee species in the Netherlands is
uncertain due to possible misidentifications in the past and present.
People who wish to work with these species should be careful in species
identification based on morphology.%
\end{abstract}%



\sloppy


\textbf{Concise cover letter}

We attempt to answer the question if easily recognizable characteristics
can be used by non-experts to reliably identify bumblebees of
the\emph{lucorum} complex and \emph{Bombus terrestris} in the
Netherlands.

\textbf{Introduction}

Bumblebees are one of the most important groups of pollinators, both in
their natural environment as well as in the pollination of crops
(Velthuis and Doorn, 2006). Due to their ecological significance, the
necessity to conserve them is high, but in order to do so it is vital to
understand the distribution ranges of separate species, as bumblebee
species have different habitat requirements and respond differently to
environmental stressors (Goulson et al., 2008; Marshall et al., 2018).
Over the last century, bumblebees have declined in abundance and
richness in Western Europe (Biesmeijer et al. 2006; Carvalheiro et al.
2013) and North America (Cameron et al. 2011), due to habitat loss and a
decrease in floral resources (Goulson et al. 2008). Data from the
Netherlands predicts a 19\% decrease in bumblebee species richness from
1945 to 2018 (van Dooren et al. 2019). To understand changing patterns
in biodiversity and its significance to agriculture and society, it is
essential that species are correctly identified. However, the difficulty
with species identification in bumblebees is that many share a similar
morphology. In the Netherlands the most difficult species to identify
belong to the subgenus \emph{Bombus sensu stricto} , namely \emph{Bombus
terrestris} , \emph{B. cryptarum} , \emph{B. lucorum} and \emph{B.
magnus} , the latter three being the \emph{lucorum} complex. They are
cryptic species, separate species which are difficult to distinguish by
morphology. In the past, these three species were grouped under the
name\emph{B. lucorum} . However, blends of male labial gland secretions
(Bertsch et al. 1997; Bertsch et al. 2004; Bertsch et al. 2005),
together with DNA sequencing of the mitochondrial COI gene (Pedersen,
2002; Bertsch et al. 2005; Murray et al. 2008), revealed that \emph{B.
cryptarum} , \emph{B. lucorum} and \emph{B. magnus} were distinct
species. In addition, interbreeding experiments have resulted in no
offspring (De Jonghe and Rasmont, 1983), confirming that they are
separate species. \emph{Bombus terrestris} is another white-tailed
bumblebee from the subgenus \emph{Bombus sensu stricto} . On continental
Europe, the subspecies \emph{B. terrestris terrestris} occurs (Rasmont
et al. 2013), of which the different castes and sexes are
morphologically similar to those of the \emph{lucorum} complex species
(Wolf et al. 2010; Bossert, 2015).

In the past, many different characters have been used to distinguish
the\emph{lucorum} complex species. The key features often used are the
colour patterns in the collar of queens, such as an S-shape line of
black hairs and the extent of the yellow collar downwards (Figure 1;
Rasmont, 1984; Bertsch et al. 2004; Bertsch et al. 2005; Carolan et al.
2012). These characteristics can be helpful in species identification,
but are hampered by overlap between species and geographic variation
within species (Bertsch et al. 2004; Bertsch et al. 2005; Carolan et al.
2012). Waters et al. (2011) assessed the extension of the yellow collar
in workers of the \emph{lucorum} complex in the Western Isles of
Scotland. Although they found that this character can be diagnostic
for\emph{B. lucorum} and \emph{B. magnus} , there was much variation
and\emph{B. cryptarum} was not properly distinguishable from the other
two species. According to Murray et al. (2008) Irish workers of
the\emph{lucorum} complex are morphologically indistinguishable in the
field.

Most authors agree that males of the \emph{lucorum} complex are either
morphologically indistinguishable (Waters et al. 2011; Murray et al.
2008) or that identifications based on morphology are unreliable
(Bertsch, 2009). Male genitalia are identical for the species of the
complex (Rasmont et al. 1986). Characters used to identify males are
often based on the colour of the facial hair (Rasmont et al. 1986), but
the reliability of these characters has not been tested so far.

Problems with morphological identification have not refrained authors to
draw conclusions on the distribution of the individual species. For
example, \emph{Bombus lucorum} is considered widespread and abundant
across Europe (Banaszak and Rasmont 1994; Urbanov\selectlanguage{ngerman}á et al. 2001; Murray
et al. 2008; Vesterlund et al. 2014; Scriven et al. 2015; Bossert et al.
2016) as is \emph{B. terrestris} (Banaszak and Rasmont 1994; Peeters et
al. 2012; Bossert et al. 2016). DNA-based studies in Great-Britain and
Ireland (Murray et al. 2008; Waters et al. 2011; Scriven et al. 2015)
have shown that species of the \emph{lucorum} complex co-occur at many
sites and that much of their distribution overlaps. In
addition,\emph{Bombus lucorum, B. terrestris} and \emph{B. cryptarum}
are polylectic in the British Isles (they visit plant species from
multiple plant families; Waters et al. 2011; Scriven et al. 2015),
although in the Western Isles of Scotland \emph{B. lucorum} was observed
mostly feeding on Ericaceae and Apiaceae (Waters et al. 2011).
\emph{Bombus magnus} was found to be associated with heather, in
particular with\emph{Calluna vulgaris} (Waters et al. 2011; Scriven et
al. 2015; Scriven et al. 2016), \emph{Erica cinerea} (Scriven et al.
2015; Scriven et al. 2016) and \emph{Erica tetralix} (Scriven et al.
2015; Scriven et al. 2016).

The difficulty in separating these four bumblebee species has important
consequences for studies investigating distribution and ecology. The
chances of misidentification are high and can result in cascading
effects which ultimately may lead to incorrect conclusions regarding
ecosystem functioning and species trends over time (Bortolus, 2008). For
example, many misidentifications occur in natural history collections
(Bertsch, 2009) and these historical records are often used in studies
regarding long-term species trends, leading to incorrect outcomes. Many
of these species are also regularly reported on citizen science websites
and in ecological scientific studies and due to the different ecology of
the species it then is important to make an accurate distinction.

In this study, the goal was to investigate to what extent easily
recognizable characteristics can be used by non-experts to reliably
identify species of the \emph{lucorum} complex and \emph{B.
terrestris}in the Netherlands. We expected that these four white-tailed
bumblebee species are similar in most of their morphology as they are in
other parts of continental Europe. We hypothesise that reliable
identification will only be possible for all four species using COI
sequences.

\textbf{Materials and Methods}

In total, 287 specimens were sampled from nine different locations
across the Netherlands and one in Belgium (Table 1) between
April-August, 2017. The ten sample locations were selected because of
the likely occurrence of all four species in many of these sites based
on historical collection data (that may have been identified
incorrectly, but were used as a starting point). Bumblebees were sampled
randomly with an insect net or collection tube from 10:30 am until 04:00
pm at each site. A further 152 bumblebees from 13 self-bred colonies
(containing \emph{B. cryptarum} , \emph{B. lucorum} , \emph{B.
terrestris}and \emph{B. magnus} ) were added to the study (Table 2),
because more individuals of especially \emph{B. magnus} were needed. Two
bumblebees collected from a box of \emph{B.} terrestris marketed for
crop pollination by Koppert Biological Systems were also added to the
dataset (Table 2), because some of them showed the S-shape line, which
is described as a characteristic for \emph{B. cryptarum} queens
(Rasmont, 1984; Bertsch et al. 2004; Bertsch et al. 2005; Carolan et al.
2012), while they are claimed to be the subspecies \emph{B. terrestris
dalmatinus} . This resulted in a total of 451 individual bumblebees. The
number of specimens that was used in the different analyses is lower,
because only a DNA sample was taken from spring queens in the field in
order to not to disturb the establishment of new colonies. Only one
specimen from each self-bred colony was sequenced, because this allowed
assignment of the whole colony to species level.

\emph{Morphology analysis}

Both qualitative and quantitative characters were used in this analysis.
Some of the qualitative characters have previously been used to
distinguish queens of the four species (Rasmont, 1984; Bertsch et al.
2004; Bertsch et al. 2005; Bertsch, 2009; Carolan et al. 2012). Other
qualitative characters are associated with the males, such as facial
hair colour. An overview of the colour characteristics used by Rasmont
to separate the males of the \emph{lucorum} complex and \emph{B.
terrestris} can be found in Table 3. We did not look at characters that
need examination with a stereomicroscope, such as the absence of
punctures and microsculpture on the hind margin of tergite 2 to
distinguish \emph{B. terrestris} queens from the other species (Rasmont,
1984; Amiet, 1996), because we wanted to test to what extent easily
recognizable characteristics can be used by non-experts. The following
morphological characters have been included in the analysis: S-shape
right and S-shape left (ssr, ssl; Figure 1A), melanisation of the collar
(mc; Figure 1B), extension of the collar right and extension of the
collar left (ecr, ecl; Figure 1C), brown-yellow bands (byb; Figure 1D),
yellow hairs on scutellum (yhs; Figure 1E), yellow facial hair (yh;
Figure 2B) and yellow moustache (ym; Figure 2C). The character
``brown-yellow bands'' was included because this coloration of the
collar and tergite 2 is often associated with \emph{B. terrestris} (see
Figure 1; Peeters et al. 2012; Falk, 2017). An overview with photographs
of the qualitative characteristics used in this analysis can be seen in
Figures 1 and 2.

The quantitative characters were measured on each individual by using
Axiovision x64 version 4.9.0.0. Focus was given to measurements
concerning the yellow collar. The following measurements have been
included in the analysis: intertegular distance (the length between the
bases of the wings, a proxy for body size; itd; Figure 2D), collar
breadth dorsal side (cb; Figure 2D), length of collar below tegula right
(lcr; Figure 2E), breadth of collar below tegula right (bcr; Figure 2E),
length of collar below tegula left (lcl; Figure 2F) and breadth of
collar below tegula left (bcl; Figure 2F). An overview of the
measurements included with photographs is provided in Figure 2.
Photographs in Figure 1 and 2 were made with a Zeiss Discovery.v20
Photomicroscope and Axiovision x64 version 4.9.0.0 software and flipped,
so that images from both the right and left side were comparable. Figure
1 and 2 were made in Adobe Photoshop CS6 (version 13.0; 1990-2012 Adobe
Systems Incorporated). The same person took all measurements.

In total 337 specimens (39 queens, 201 workers and 97 males) were
included in this analysis. Caste and sex were analysed separately
because of the morphological differences between the sexes. Principle
component analyses (PCAs) were carried out in order to visualize the
variation among the specimens and to see if specimens from the same
species are clustered together due to certain morphological
characteristics. The qualitative characteristics were also analysed with
a chi-square test to see which characters had a positive relation to
certain species. Quantitative characteristics were analysed with a
one-way ANOVA test and pairwise comparisons, with either Tukey HSD test
or Wilcoxon rank sum test and were performed to see if species differ
significantly based on certain quantitative characters. Boxplots were
made to visualize the difference in quantitative characteristics and
residual plots were made to show the relationship between qualitative
characteristics and species. All analyses were performed in \emph{R}
and\emph{RStudio} (\emph{R} version 3.4.2 and \emph{RStudio} version
1.1.383; R Core Team, 2017).

\emph{DNA sequencing}

In order to correctly identify the specimens to species level DNA
sequencing was used. Sequencing was done after morphological
characteristics were measured to remove any bias. The gene that was
sequenced is the cytochrome c oxidase subunit I (COI), which in
bumblebees has been used to separate morphologically identical species
(Williams et al. 2012) and has been used in many studies concerning the
four white-tailed bumblebees (Pedersen, 2002; Bertsch et al. 2005;
Murray et al. 2008; Waters et al. 2011; Carolan et al. 2012; Scriven et
al. 2015; Bossert et al. 2016).

Of 346 specimens the pretarsus and part of the tarsus were taken for DNA
extraction. Spring queens were put in queen marking tubes in order to
extract a tarsus in the field. The tarsi were put in 200 \selectlanguage{greek}µ\selectlanguage{english}l T1-Lysis
buffer and a scalpel was used to cut the tarsi in several pieces so that
the DNA was accessible. Twenty-five \selectlanguage{greek}µ\selectlanguage{english}l Proteinase K was added to each
sample and extraction was done according to a standard protocol with the
use of a KingFisher DNA extraction robot, program:
Machery\_Nagel\_Tissue\_96 KingFisher Flex. Polymerase chain reactions
(PCRs) were performed by using the universal primers LCO1490 and HC02198
(Folmer et al. 1994) with each a pipetting volume of 1,00 \selectlanguage{greek}µ\selectlanguage{english}l (10pMol/\selectlanguage{greek}µ\selectlanguage{english}l)
per sample. The PCR mix per sample further contained 18,8 \selectlanguage{greek}µ\selectlanguage{english}l mQ
(Ultrapure), 2,50 \selectlanguage{greek}µ\selectlanguage{english}l buffer CL (10x) (Qiagen), 0,50 \selectlanguage{greek}µ\selectlanguage{english}l dNTP (2,50 mM),
0,25 \selectlanguage{greek}µ\selectlanguage{english}l Taq polymerase (5,00 U/\selectlanguage{greek}µ\selectlanguage{english}l) (Qiagen) and 1,00 \selectlanguage{greek}µ\selectlanguage{english}l template DNA,
resulting in a total volume of 25,0 \selectlanguage{greek}µ\selectlanguage{english}l. The PCR program was perfomed
with a Bio-Rad C1000 Touch\textsuperscript{TM} Thermal Cycler and
consisted of initial denature at 94\selectlanguage{ngerman}°C for 3 minutes, followed by 40
cycles of denature at 94°C for 15s, annealing at 50°C for 30s and
extension at 72°C for 40s. Final extension was performed at 72°C for 5
minutes followed by an infinite pause at 12°C. To estimate the success
of the PCRs, 3 \selectlanguage{greek}µ\selectlanguage{english}l of each PCR product was run on a 96 Agarose Gel, 2\%
(E-Gel\selectlanguage{ngerman}®). Photograph of the gel was taken with a
Red\textsuperscript{TM}Imaging System (Cell Biosciences) and edited by
using E-Editor\textsuperscript{TM} version 2.0.2. Successful PCR
products were sent to BaseClear (BaseClear B.V. Leiden) for Sanger
sequencing. Raw sequence reads were quality controlled, edited and
stored in Geneious® (version 8.1.8; 2005-2015 Biomatters Ltd). Alignment
was done with the plugin MAFFT Multiple Alignment version 1.3.3. Edited
reads were exported as FASTA files and compared with the NCBI GenBank
BLAST page (Altschul et al. 1990) to identify the individual reads.
Specimens which were not successfully sequenced were excluded from
further analyses. A maximum likelihood tree was made in Geneious® by
using the plugin PHYML (Guindon and Gascuel, 2003) and edited in FigTree
(version 1.4.3; Rambaut, 2006-2016). The bee \emph{Macropis europaea}
was added as outgroup (Schmidt et al. 2015; GenBank accession number:
KJ839299.1). The tree was modified in Adobe Illustrator CS6 (version
16.0.0; 1987-2012 Adobe Systems Incorporated).

\textbf{Results}

The residual plot of the qualitative characteristics shows a strong
positive relationship between \emph{B. magnus} and the extension of the
yellow collar and to a lesser extent a positive relationship
between\emph{B. cryptarum} and the S-shape line (Figure 3). This is in
agreement with the presence/absence scores of the qualitative
characteristics (Table 4). Almost all queens of \emph{B. cryptarum} show
the presence of an S-shape (11/12) and all queens of \emph{B.
magnus}show extension of the yellow collar (4/4).

The results from the pairwise comparisons of the quantitative characters
show that in queens, \emph{B. terrestris} has a significantly larger
intertegular distance compared to \emph{B. cryptarum} (0.01\selectlanguage{english}[?]p\selectlanguage{english}[?]0.001,
Table 5; Figure 4). However, there was overlap between all species
within this characteristic. \emph{Bombus cryptarum} also has a
significantly smaller breadth of the yellow collar at the dorsal side
than the other three species (p\textless{}0.001, Table 5; Figure
4).\emph{Bombus magnus} has a significantly longer yellow collar below
the tegula on both the right and left side compared to the other species
(p\textless{}0.001, Table 5; Figure 4). There were no significant
differences in the breadth of the collar below the tegula on both the
right and left side and there was much overlap within these
characteristics (Table 5; Figure 4).

In workers, the residual plot shows again a strong positive relation
between \emph{B. magnus} and the extension of the collar but also a
negative relation with brown-yellow bands (Figure 5). \emph{Bombus
cryptarum} shows positive relations with the S-shape line as well as
with melanisation of the collar (Figure 5). \emph{Bombus terrestris} has
a positive relation with brown-yellow bands and a negative relation with
the extension of the collar (Figure 5). On the contrary, the
presence/absence data shows that only 25/41 of \emph{B. magnus} workers
have extension of the collar (Table 4). In \emph{B. cryptarum} , only
3/10 of the workers show the S-shape and 1/10 show melanisation (Table
4).

Results from the pairwise comparisons show that in workers, \emph{B.
terrestris} and \emph{B. lucorum} have significantly larger intertegular
distances than \emph{B. cryptarum} and \emph{B. magnus} respectively
(ter-cry: 0.001\selectlanguage{english}[?]p\selectlanguage{english}[?]0.01, ter-mag: p\textless{}0.001, luc-cry:
0.01\selectlanguage{english}[?]p\selectlanguage{english}[?]0.05, luc-mag: 0.001\selectlanguage{english}[?]p\selectlanguage{english}[?]0.01, Table 5; Figure 6). \emph{Bombus
cryptarum} has a significantly smaller breadth of the yellow collar at
the dorsal side than \emph{B. lucorum} and \emph{B.
magnus}(0.001\selectlanguage{english}[?]p\selectlanguage{english}[?]0.01, Table 5; Figure 6) and \emph{B. lucorum} has a
significantly larger breadth of the dorsal collar compared to the other
three species (0.001\selectlanguage{english}[?]p\selectlanguage{english}[?]0.01, Table 5; Figure 6). However, both the
intertegular distance as well as the breadth of the dorsal collar show
overlap between all species. Again, \emph{B. magnus} shows a
significantly longer yellow collar below the tegula on both the right
and left side than the other species (p\textless{}0.001, Table 5; Figure
6), but at the lower margin there is also overlap between all three
other species. When considering the breadth of the yellow collar below
the tegula, \emph{B. magnus} has a significantly larger breadth compared
to \emph{B. terrestris} on the right and left side (p\textless{}0.001,
Table 5; Figure 6) and compared to \emph{B. cryptarum} on the left side
(0.001\selectlanguage{english}[?]p\selectlanguage{english}[?]0.01, Table 5; Figure 6). However, all the species show overlap
within these characteristics.

The residual plot for males shows strong positive relations
between\emph{B. cryptarum} and melanisation of the collar and between
\emph{B. lucorum} and yellow facial hair (Figure 7). \emph{Bombus
magnus} has positive relations with both yellow hairs on the scutellum
and the yellow moustache and again a negative relation with brown-yellow
bands (Figure 7). \emph{Bombus terrestris} males show a positive
relation with brown-yellow bands (Figure 7). The presence/absence table
shows something similar to the residual plot. Almost all males of
\emph{B. lucorum} possess yellow facial hair (7/8) and almost all males
of\emph{B. magnus} show the presence of a yellow moustache (18/19) and
yellow hairs on the scutellum (17/19; Table 4). Noteworthy is the high
amount of \emph{B. terrestris} males with brown-yellow bands (42/59;
Table 4).

According to the results from the pairwise comparisons, \emph{B.
terrestris} males have a significantly larger intertegular distance than
the other three species (p\textless{}0.001, Table 5; Figure 8).
Considering the breadth of the yellow collar at the dorsal side,\emph{B.
lucorum} and \emph{B. magnus} have a significantly larger breadth than
\emph{B. terrestris} (luc-ter: 0.01\selectlanguage{english}[?]p\selectlanguage{english}[?]0.05, mag-ter: 0.001\selectlanguage{english}[?]p\selectlanguage{english}[?]0.01, Table
5; Figure 8). However, again these characteristics show overlap between
all species. \emph{Bombus lucorum} and \emph{B. magnus} also have a
significantly longer yellow collar below the tegula than \emph{B.
cryptarum} and \emph{B. terrestris} on both the right and left side
(p\textless{}0.001, Table 5; Figure 8), but \emph{B. magnus}shows
overlap between \emph{B. cryptarum} and \emph{B. terrestris} at the
lower margin of these characteristics. \emph{Bombus magnus} has a
significantly larger breadth of the yellow collar compared to the other
three species on both the right (p\textless{}0.001, Table 5; Figure 8)
and left side (mag-cry: p\textless{}0.001, mag-luc: 0.001\selectlanguage{english}[?]p\selectlanguage{english}[?]0.01,
mag-ter: p\textless{}0.001, Table 5; Figure 8), but at the lower margin
there is also overlap with the other three species.

\textbf{Discussion}

Overall, this study shows that distinction of the four white-tailed
bumblebee species in the Netherlands by means of easily identifiable
morphological characteristics is difficult. There is overlap in
morphological traits, especially in workers and males and supports the
results observed in other areas were these four species co-occur
(Rasmont, 1984; Rasmont et al. 1986; Murray et al. 2008; Bertsch, 2009;
Waters et al. 2011). In every combination of species and caste/sex there
was at least one specimen that overlapped (in 2-dimensional space) with
other species' characteristics, except for \emph{B. magnus} queens. DNA
sequencing was the only method that clearly separated all four species,
as shown by other studies (Murray et al. 2008; Carolan et al. 2012). Not
using DNA-barcoding for species identification can lead to potential
errors.

The S-shape in \emph{B. cryptarum} and the collar extension in \emph{B.
magnus} are often mentioned as reasonably useful in queens, because they
are present in most cases (Bertsch et al. 2004; Bertsch et al. 2005;
Carolan et al. 2012). However, even though Carolan et al. (2012) used a
larger sample size from Ireland and Denmark, they found much overlap of
these two characters with the other species (Carolan et al. 2012), which
is in contrast to the present study in which the S-shape was only
present in \emph{B. cryptarum} queens and one queen of \emph{B.
terrestris dalmatinus} (used for crop pollination in greenhouses and a
non-native subspecies) and extension of the collar only present
in\emph{B. magnus} queens. This suggests that at least for the
Netherlands these are simple and useful characteristics that can even be
used in the field. The fact that queens showed the clearest separation
is in concordance with Bertsch, who states that spring queens are not
difficult to identify based on morphology (Bertsch et al. 2004; Bertsch
et al. 2005; Bertsch 2009).

In workers, the large breadth of the collar at the dorsal side
separated\emph{B. lucorum} and the length and extension of the collar
slightly separated \emph{B. magnus} from the other species, which was
also shown by Waters who crudely distinguished \emph{B. magnus} from
\emph{B. lucorum} based on the length of the collar (Waters et al.
2011). However, the fact that not all specimens were correctly
identified and\emph{B. cryptarum} workers could not be separated in
their study indicates the difficulty in species identification of
workers of the\emph{lucorum} complex. This follows the conclusion from
the present study that workers of the four species do not show clear
groups even if several characters and measurements are used. This is in
concordance with Murray et al. (2008), who states that workers of the
four species are difficult to identify based on morphology. This study
also showed that workers possess great intraspecific variation, which
makes morphological species identification even more difficult.

Facial hair characteristics are of significance in separating males of
the four species, particularly the many yellow hairs on the face
of\emph{B. lucorum} and the presence of a yellow moustache in \emph{B.
magnus} . This is partly in line with Rasmont, who saw on average more
yellow hairs on the face of \emph{B. lucorum} compared to \emph{B.
magnus} in Belgian bumblebees (Rasmont et al. 1986) and this was also
mentioned by Roland De Jonghe (R. De Jonghe personal communication).
Other characters which play an important role in distinguishing \emph{B.
magnus} males are the presence of yellow hairs on the scutellum and the
large breadth of the yellow collar. Again, this is partially in
accordance with Rasmont et al. 1986. He describes that both \emph{B.
lucorum} and \emph{B. magnus} possess yellow hairs on the scutellum,
although more prominent in \emph{B. magnus} . On the other hand, he too
mentions a broad yellow collar in \emph{B. magnus} . It must be said
that extremely pale males of \emph{B. lucorum} exists with yellow hairs
on most of the thorax and abdomen (Falk, 2017). Moreover, \emph{Bombus
lucorum} males tend to have pale hairs in the black areas on the thorax
and abdomen, which gives them a more greyish appearance (Rasmont et al.
1986; Amiet, 1996).

Bumblebees are hairy insects and for that reason, distance measurements
are difficult. It is not always clear what the borders are of a
particular hair patch, such as the yellow collar. Therefore, anyone
using these measurements has the possibility to make mistakes, which
makes them less useful as characteristics.

What is remarkable is that many studies do not describe accurately in
what way the breadth and length of the collar were measured (Williams
2000; Carolan et al. 2012). Then the problem arises that measurements
are done in different ways by different authors, which makes their work
less comparable to each other and also less useful for researchers
wishing to identify the four white-tailed species. In studies of the
British Isles, \emph{B. terrestris} is not included because it is easily
distinguishable from the \emph{lucorum} species complex based on colour
pattern. However, for the Netherlands this distinction is not clear, as
illustrated above. Because of this similarity, the danger often arises
that the \emph{lucorum} species complex together with \emph{B.
terrestris} is combined under the name \emph{terrestris/lucorum} . The
species status of \emph{B. cryptarum} , \emph{B. lucorum} and \emph{B.
magnus} is then unclear, due to the much greater abundance of \emph{B.
terrestris} .

Correct identification of species is vital for biodiversity
conservation. At present, the problem is that for many species it is not
known if they are present in a certain area or even constitute a
separate species, because of the occurrence of morphologically similar
species. Even in a well-studied area like Western-Europe, this is the
case for the bumblebees \emph{Bombus cryptarum} , \emph{B. lucorum}
,\emph{B. magnus} and \emph{B. terrestris} . It is likely that there are
many misidentifications in natural history collections, which was
demonstrated by Bertsch (Bertsch, 2009), and nowadays these species are
often lumped together as \emph{terrestris} -complex. These errors can
lead to incorrect conclusions about the distribution and long-term
trends of these species (Bortolus, 2008). Next, these incorrect
conclusions can have serious consequences, such as the unnoticed
extinction of one of these bumblebee species in the Netherlands
(Bortolus, 2008). Given that most bumblebees are highly susceptible to
climate warming (Marshall et al. 2018) and with rising mean temperatures
in the Netherlands, this scenario can become reality. This highlights
the importance of giving these species the correct name. Many bumblebees
of the \emph{terrestris} -complex are also often used in ecological
experiments and citizen science projects. Identifying them incorrect to
species level can result in different conclusions, as we know that at
least \emph{B. magnus} has a different ecology than the other species.

As we have shown, DNA-barcoding is the only method that clearly
separates all three castes of all four species. Of course, DNA-barcoding
is not feasible for everyone who wants to study the four white-tailed
bumblebee species. Therefore, a recommendation was made to help with the
identification of these species. The first step in the identification
process is to determine the caste/sex of the individual bumblebee. If it
is a queen and it has an extension of the yellow collar far below the
tegula, it is \emph{B. magnus} . If the queen has an S-shape line of
black hairs in the yellow collar, it is \emph{B. cryptarum} . If none of
these characteristics are met, DNA-barcoding is the final solution. For
workers we recommend the use of DNA-barcoding to identify the species.

If it is a male with a yellow moustache and yellow hairs on the
scutellum, it is probably \emph{B. magnus} . If a male has many yellow
hairs on the face, it is most likely \emph{B. lucorum} . If these
characteristics are not met, DNA-barcoding is the final solution.

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\textbf{Tables}

\textbf{Table 1. Sample sites and number of specimens per site.} Five
individual bumblebees were added by John Smit from Dwingelderveld.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Sample site} & \textbf{Number of sampling days} & \textbf{Dates
(2017)} & \textbf{Number of specimens}\tabularnewline
\midrule
\endhead
Dwingelderveld & 1 & 26-07 & 25\tabularnewline
Kalmthoutse heide (BE) & 1 & 24-08 & 25\tabularnewline
Lunterse bos & 2 & 24-05, 14-06 & 7\tabularnewline
Meijendel & 2 & 04-04, 31-05 & 31\tabularnewline
Slikken van de Heen & 1 & 24-08 & 20\tabularnewline
Terschelling & 6 & 09/11-05, 02/04-08 & 66\tabularnewline
Tiengemeten & 1 & 26-05 & 29\tabularnewline
Vliegbasis Soesterberg & 2 & 16-05, 26-06 & 37\tabularnewline
Wijlre, Zuid-Limburg & 1 & 13-07 & 20\tabularnewline
Zuid-Kennemerland & 2 & 08-05, 18-07 & 37\tabularnewline
& & & \textbf{297}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2. Additional specimens}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Specimens from} & \textbf{Number of specimens}\tabularnewline
\midrule
\endhead
Koppert Biological systems & 2\tabularnewline
Roland De Jonghe (self-bred) & 152\tabularnewline
& \textbf{154}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3. Overview male characteristics used by Rasmont (Rasmont
et al. 1986)}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& \emph{B. cryptarum} & \emph{B. lucorum} & \emph{B. magnus} & \emph{B.
terrestris}\tabularnewline
\midrule
\endhead
Facial hair & Black hairs on face, on vertex few light hairs. Sometimes
light hairs on both sides of clypeus & Light hairs on face and vertex,
but with few black hairs on top of vertex. Many black hairs possible,
but clypeus and vertex almost always light haired & Light hairs on face
and mixed with black hairs on vertex. Clypeus always light. Rest of face
black. & Black hairs on face, on vertex sometimes mixed with light
hairs. Sometimes light hairs on both sides of clypeus\tabularnewline
Yellow collar & Collar below tegula, longer than \emph{terrestris} &
Collar extends far below tegula & Collar extends far below tegula,
longer than \emph{lucorum}. Also very broad. & Collar below
tegula\tabularnewline
Thorax colour & Few yellow hairs on scutellum, but this is rare. Rest of
thorax black & Always with few yellow hairs on scutellum. All black
hairs with greyish extremities & Always yellow hairs on apex of
scutellum. Few black hairs with greyish extremities &
black\tabularnewline
Abdomen colour & Tergite 1 with few yellow hairs in centre & Tergite 1
sometimes completely black, sometimes completely yellow. Always with
yellow hairs in the middle of tergite 1. Black hairs always with light
extremities Tergite 3 can be dark or light & Tergite 1 yellow with few
black hairs. Rare black hairs never with light extremities Tergite 3
sometimes with few light hairs in centre and on both sites. & Tergite 1
with yellow hairs in centre\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 4. Presence/absence of qualitative characteristics in
queens, workers and males.}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllll@{}}
\toprule
& Queens & Queens & Queens & Queens & Workers & Workers & Workers &
Workers & Males & Males & Males & Males\tabularnewline
\midrule
\endhead
& \emph{cry.} & \emph{luc.} & \emph{mag.} & \emph{ter.} & \emph{cry.} &
\emph{luc.} & \emph{mag.} & \emph{ter.} & \emph{cry.} & \emph{luc.} &
\emph{mag.} & \emph{ter.}\tabularnewline
S-shape & 11/12 & 0/10 & 0/4 & 1/13 & 3/10 & 0/11 & 0/41 & 9/139 & 0/11
& 0/8 & 0/19 & 4/59\tabularnewline
Melanisation & 2/12 & 0/10 & 0/4 & 1/13 & 1/10 & 0/11 & 0/41 & 2/139 &
5/11 & 0/8 & 0/19 & 1/59\tabularnewline
Extension & 0/12 & 0/10 & 4/4 & 0/13 & 0/10 & 0/11 & 25/41 & 5/139 &
6/11 & 8/8 & 19/19 & 30/59\tabularnewline
Brown-yellow bands & 8/12 & 9/10 & 0/4 & 13/13 & 4/10 & 6/11 & 3/41 &
95/139 & 2/11 & 0/8 & 0/19 & 42/59\tabularnewline
Yellow moustache & 0/12 & 0/10 & 0/4 & 0/13 & 0/10 & 0/11 & 0/41 & 0/139
& 1/11 & 1/8 & 18/19 & 0/59\tabularnewline
Yellow facial hair & 0/12 & 0/10 & 0/4 & 0/13 & 0/10 & 0/11 & 0/41 &
0/139 & 0/11 & 7/8 & 1/19 & 0/59\tabularnewline
Yellow hair scutellum & 0/12 & 0/10 & 0/4 & 0/13 & 0/10 & 0/11 & 0/41 &
0/139 & 0/11 & 1/8 & 17/19 & 0/59\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 5. Results from the pairwise comparisons of the
quantitative characteristics.} Significance level: p\textgreater{}0.05 =
non-significant (NS), 0.01[?]p[?]0.05 = *, 0.001[?] p [?]0.01 = ** and
p\textless{}0.001 = ***\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Comparison} & \textbf{Character} & \textbf{Caste/sex} &
\textbf{Significance}\tabularnewline
\midrule
\endhead
\emph{lucorum-cryptarum} & intertegular distance & queen &
NS\tabularnewline
\emph{magnus-cryptarum} & intertegular distance & queen &
NS\tabularnewline
\emph{terrestris-cryptarum} & intertegular distance & queen &
**\tabularnewline
\emph{magnus-lucorum} & intertegular distance & queen &
NS\tabularnewline
\emph{terrestris-lucorum} & intertegular distance & queen &
NS\tabularnewline
\emph{terrestris-magnus} & intertegular distance & queen &
NS\tabularnewline
\emph{lucorum-cryptarum} & collar breadth & queen & ***\tabularnewline
\emph{magnus-cryptarum} & collar breadth & queen & ***\tabularnewline
\emph{terrestris-cryptarum} & collar breadth & queen &
***\tabularnewline
\emph{magnus-lucorum} & collar breadth & queen & NS\tabularnewline
\emph{terrestris-lucorum} & collar breadth & queen & NS\tabularnewline
\emph{terrestris-magnus} & collar breadth & queen & NS\tabularnewline
\emph{lucorum-cryptarum} & length collar right & queen &
NS\tabularnewline
\emph{magnus-cryptarum} & length collar right & queen &
***\tabularnewline
\emph{terrestris-cryptarum} & length collar right & queen &
NS\tabularnewline
\emph{magnus-lucorum} & length collar right & queen & ***\tabularnewline
\emph{terrestris-lucorum} & length collar right & queen &
NS\tabularnewline
\emph{terrestris-magnus} & length collar right & queen &
***\tabularnewline
\emph{lucorum-cryptarum} & breadth collar right & queen &
NS\tabularnewline
\emph{magnus-cryptarum} & breadth collar right & queen &
NS\tabularnewline
\emph{terrestris-cryptarum} & breadth collar right & queen &
NS\tabularnewline
\emph{magnus-lucorum} & breadth collar right & queen & NS\tabularnewline
\emph{terrestris-lucorum} & breadth collar right & queen &
NS\tabularnewline
\emph{terrestris-magnus} & breadth collar right & queen &
NS\tabularnewline
\emph{lucorum-cryptarum} & length collar left & queen &
NS\tabularnewline
\emph{magnus-cryptarum} & length collar left & queen &
***\tabularnewline
\emph{terrestris-cryptarum} & length collar left & queen &
NS\tabularnewline
\emph{magnus-lucorum} & length collar left & queen & ***\tabularnewline
\emph{terrestris-lucorum} & length collar left & queen &
NS\tabularnewline
\emph{terrestris-magnus} & length collar left & queen &
***\tabularnewline
\emph{lucorum-cryptarum} & breadth collar left & queen &
NS\tabularnewline
\emph{magnus-cryptarum} & breadth collar left & queen &
NS\tabularnewline
\emph{terrestris-cryptarum} & breadth collar left & queen &
NS\tabularnewline
\emph{magnus-lucorum} & breadth collar left & queen & NS\tabularnewline
\emph{terrestris-lucorum} & breadth collar left & queen &
NS\tabularnewline
\emph{terrestris-magnus} & breadth collar left & queen &
NS\tabularnewline
\emph{lucorum-cryptarum} & intertegular distance & worker &
*\tabularnewline
\emph{magnus-cryptarum} & intertegular distance & worker &
NS\tabularnewline
\emph{terrestris-cryptarum} & intertegular distance & worker &
**\tabularnewline
\emph{magnus-lucorum} & intertegular distance & worker &
**\tabularnewline
\emph{terrestris-lucorum} & intertegular distance & worker &
NS\tabularnewline
\emph{terrestris-magnus} & intertegular distance & worker &
***\tabularnewline
\emph{lucorum-cryptarum} & collar breadth & worker & **\tabularnewline
\emph{magnus-cryptarum} & collar breadth & worker & **\tabularnewline
\emph{terrestris-cryptarum} & collar breadth & worker &
NS\tabularnewline
\emph{magnus-lucorum} & collar breadth & worker & **\tabularnewline
\emph{terrestris-lucorum} & collar breadth & worker & **\tabularnewline
\emph{terrestris-magnus} & collar breadth & worker & *\tabularnewline
\emph{lucorum-cryptarum} & length collar right & worker &
NS\tabularnewline
\emph{magnus-cryptarum} & length collar right & worker &
***\tabularnewline
\emph{terrestris-cryptarum} & length collar right & worker &
NS\tabularnewline
\emph{magnus-lucorum} & length collar right & worker &
***\tabularnewline
\emph{terrestris-lucorum} & length collar right & worker &
NS\tabularnewline
\emph{terrestris-magnus} & length collar right & worker &
***\tabularnewline
\emph{lucorum-cryptarum} & breadth collar right & worker &
NS\tabularnewline
\emph{magnus-cryptarum} & breadth collar right & worker &
NS\tabularnewline
\emph{terrestris-cryptarum} & breadth collar right & worker &
NS\tabularnewline
\emph{magnus-lucorum} & breadth collar right & worker &
NS\tabularnewline
\emph{terrestris-lucorum} & breadth collar right & worker &
NS\tabularnewline
\emph{terrestris-magnus} & breadth collar right & worker &
***\tabularnewline
\emph{lucorum-cryptarum} & length collar left & worker &
NS\tabularnewline
\emph{magnus-cryptarum} & length collar left & worker &
***\tabularnewline
\emph{terrestris-cryptarum} & length collar left & worker &
NS\tabularnewline
\emph{magnus-lucorum} & length collar left & worker & ***\tabularnewline
\emph{terrestris-lucorum} & length collar left & worker &
NS\tabularnewline
\emph{terrestris-magnus} & length collar left & worker &
***\tabularnewline
\emph{lucorum-cryptarum} & breadth collar left & worker &
NS\tabularnewline
\emph{magnus-cryptarum} & breadth collar left & worker &
**\tabularnewline
\emph{terrestris-cryptarum} & breadth collar left & worker &
NS\tabularnewline
\emph{magnus-lucorum} & breadth collar left & worker & NS\tabularnewline
\emph{terrestris-lucorum} & breadth collar left & worker &
NS\tabularnewline
\emph{terrestris-magnus} & breadth collar left & worker &
***\tabularnewline
\emph{lucorum-cryptarum} & intertegular distance & males &
NS\tabularnewline
\emph{magnus-cryptarum} & intertegular distance & males &
NS\tabularnewline
\emph{terrestris-cryptarum} & intertegular distance & males &
***\tabularnewline
\emph{magnus-lucorum} & intertegular distance & males &
NS\tabularnewline
\emph{terrestris-lucorum} & intertegular distance & males &
***\tabularnewline
\emph{terrestris-magnus} & intertegular distance & males &
***\tabularnewline
\emph{lucorum-cryptarum} & collar breadth & males & NS\tabularnewline
\emph{magnus-cryptarum} & collar breadth & males & NS\tabularnewline
\emph{terrestris-cryptarum} & collar breadth & males & NS\tabularnewline
\emph{magnus-lucorum} & collar breadth & males & NS\tabularnewline
\emph{terrestris-lucorum} & collar breadth & males & *\tabularnewline
\emph{terrestris-magnus} & collar breadth & males & **\tabularnewline
\emph{lucorum-cryptarum} & length collar right & males &
***\tabularnewline
\emph{magnus-cryptarum} & length collar right & males &
***\tabularnewline
\emph{terrestris-cryptarum} & length collar right & males &
NS\tabularnewline
\emph{magnus-lucorum} & length collar right & males & NS\tabularnewline
\emph{terrestris-lucorum} & length collar right & males &
***\tabularnewline
\emph{terrestris-magnus} & length collar right & males &
***\tabularnewline
\emph{lucorum-cryptarum} & breadth collar right & males &
NS\tabularnewline
\emph{magnus-cryptarum} & breadth collar right & males &
***\tabularnewline
\emph{terrestris-cryptarum} & breadth collar right & males &
NS\tabularnewline
\emph{magnus-lucorum} & breadth collar right & males &
***\tabularnewline
\emph{terrestris-lucorum} & breadth collar right & males &
NS\tabularnewline
\emph{terrestris-magnus} & breadth collar right & males &
***\tabularnewline
\emph{lucorum-cryptarum} & length collar left & males &
***\tabularnewline
\emph{magnus-cryptarum} & length collar left & males &
***\tabularnewline
\emph{terrestris-cryptarum} & length collar left & males &
NS\tabularnewline
\emph{magnus-lucorum} & length collar left & males & NS\tabularnewline
\emph{terrestris-lucorum} & length collar left & males &
***\tabularnewline
\emph{terrestris-magnus} & length collar left & males &
***\tabularnewline
\emph{lucorum-cryptarum} & breadth collar left & males &
NS\tabularnewline
\emph{magnus-cryptarum} & breadth collar left & males &
***\tabularnewline
\emph{terrestris-cryptarum} & breadth collar left & males &
NS\tabularnewline
\emph{magnus-lucorum} & breadth collar left & males & **\tabularnewline
\emph{terrestris-lucorum} & breadth collar left & males &
NS\tabularnewline
\emph{terrestris-magnus} & breadth collar left & males &
***\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Figure 1. Qualitative characters used in colour pattern
analysis.} Abbreviations of variables are in brackets. With (A) S-shape
right and left (ssr, ssl; white arrow; \emph{Bombus cryptarum} queen),
(B) melanisation of the collar (mc; \emph{Bombus cryptarum} queen), (C)
extension of the collar right and left (ecr, ecl; white
arrow;\emph{Bombus magnus} queen), (D) brown-yellow bands (byb;
\emph{Bombus terrestris} queen) and (F) yellow hairs on the scutellum
(yhs; white arrow; \emph{Bombus magnus} male).

\textbf{Figure 2. Qualitative characters and distances measured for
colour pattern analysis.} Abbreviations of variables are in brackets.
With (A) black facial hairs (\emph{Bombus terrestris} male, (B) yellow
facial hairs (yh; \emph{Bombus lucorum} male), (C) yellow moustache (ym;
white arrow; \emph{Bombus magnus} male), (D) intertegular distance
(itd;\emph{Bombus terrestris} queen) and collar breadth dorsal side
(cb;\emph{Bombus terrestris} queen), (E) length of collar and breadth of
collar below tegula right (lcr, bcr; \emph{Bombus terrestris} male) and
(F) length of collar and breadth of collar below tegula left (lcl,
bcl;\emph{Bombus terrestris} male).

\textbf{Figure 3. Residual plot showing the relationship of the
qualitative characteristics and queens of \emph{B. cryptarum}, \emph{B.
lucorum}, \emph{B. magnus} and \emph{B. terrestris}.} Byb: brown-yellow
bands, Extension: extension of the collar, Melanisation: melanisation of
the collar and S-shape: S-shape line of black hairs in the collar.

\textbf{Figure 4. Boxplots showing the variation in the quantitative
characteristics for queens.} The significant stars are the results of
the pairwise comparisons. With A) intertegular distance, B) collar
breadth, C) length of collar right, D) length of collar left, E) breadth
of collar right and F) breadth of collar left.

\textbf{Figure 5. Residual plot showing the relationship of the
qualitative characteristics and workers of \emph{B. cryptarum}, \emph{B.
lucorum}, \emph{B. magnus} and \emph{B. terrestris}.} Byb: brown-yellow
bands, Extension: extension of the collar, Melanisation: melanisation of
the collar and S-shape: S-shape line of black hairs in the collar.

\textbf{Figure 6. Boxplots showing the variation in the quantitative
characteristics for workers.} The significant stars are the results of
the pairwise comparisons. With A) intertegular distance, B) collar
breadth, C) length of collar right, D) length of collar left, E) breadth
of collar right and F) breadth of collar left.

\textbf{Figure 7. Residual plot showing the relationship of the
qualitative characteristics and males of \emph{B. cryptarum}, \emph{B.
lucorum}, \emph{B. magnus} and \emph{B. terrestris}.} Yh: yellow hairs
on the face, Yhs: yellow hairs on the scutellum and Ym: yellow
moustache.

\textbf{Figure 8. Boxplots showing the variation in the quantitative
characteristics for males.} The significant stars are the results of the
pairwise comparisons. With A) intertegular distance, B) collar breadth,
C) length of collar right, D) length of collar left, E) breadth of
collar right and F) breadth of collar left.

\textbf{Data accessibility statement}

DNA-sequences will be uploaded to Genbank once our manuscript has been
accepted to Ecology and Evolution. Species coordinates have been
transferred to the EIS database (http://www.eis-nederland.nl), which
will be uploaded to www.waarneming.nl. Species coordinates will also be
uploaded to Dryad once our manuscript has been accepted to Ecology and
Evolution.

\textbf{Competing interests' statement}

None declared.

\textbf{Author contributions}

L.A., K.B. and L.M. conceived and designed the project. L.A. conducted
the DNA-barcoding. All authors carried out fieldwork and supplied
specimens. L.A. and L.M. conducted the statistical analyses. L.A. took
the lead in writing the manuscript. All authors provided critical
feedback and helped shape the research, analysis and manuscript.

\textbf{Acknowledgements}

We would like to thank Roland But\selectlanguage{romanian}ȏt and Frank Stokvis of the Naturalis
DNA barcoding facility for helping with the DNA sequencing workflow; to
John T. Smit for providing his bumblebee specimens and Lisette van
Kolfschotten for help with the fieldwork. We would also like to thank
Werner de Gier for helping with data visualizations.\selectlanguage{english}
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