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Genomic variation in montane bumblebees in Scandinavia: High levels of intraspecific diversity despite population vulnerability

December 2023
Molecular Ecology 33(4)

December 2023
33(4)

DOI:10.1111/mec.17251

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CC BY-NC 4.0

Authors:

Yuanzhen Liu

Aarhus University

Anna Olsson

Uppsala University

Tuuli Larva

Uppsala University

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Populations of many bumblebee species are declining, with distributions shifting northwards to track suitable climates. Climate change is considered a major contributing factor. Arctic species are particularly vulnerable as they cannot shift further north, making assessment of their population viability important. Analysis of levels of whole‐genome variation is a powerful way to analyse population declines and fragmentation. Here, we use genome sequencing to analyse genetic variation in seven species of bumblebee from the Scandinavian mountains, including two classified as vulnerable. We sequenced 333 samples from across the ranges of these species in Sweden. Estimates of effective population size ( N E ) vary from ~55,000 for species with restricted high alpine distributions to 220,000 for more widespread species. Population fragmentation is generally very low or undetectable over large distances in the mountains, suggesting an absence of barriers to gene flow. The relatively high N E and low population structure indicate that none of the species are at immediate risk of negative genetic effects caused by high levels of genetic drift. However, reconstruction of historical fluctuations in N E indicates that the arctic specialist species Bombus hyperboreus has experienced population declines since the last ice age and we detected one highly inbred diploid male of this species close to the southern limit of its range, potentially indicating elevated genetic load. Although the levels of genetic variation in montane bumblebee populations are currently relatively high, their ranges are predicted to shrink drastically due to the effects of climate change and monitoring is essential to detect future population declines.

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Molecular Ecology. 2023;00:e17251.  

1 of 18

https://doi.org/10.1111/mec.17251

wileyonlinelibrary.com/journal/mec

Received:4October2023

Revised:24November2023

Accepted:11December2023

DOI :10.1111/mec.17251

ORIGINAL ARTICLE

Genomic variation in montane bumblebees in Scandinavia:

High levels of intraspecific diversity despite population

vulnerability

Yuanzhen Liu1 | Anna Olsson1 | Tuuli Larva1 | Aoife Cantwell- Jones2 |

Richard J. Gill2 | Björn Cederberg3 | Matthew T. Webster1

This is an open access article under the terms of the CreativeCommonsAttribution-NonCommercial License, which permits use, distribution and reproduction

in any medium, provided the original work is properly cited and is not used for commercial purposes.

©2023TheAuthors.Molecular Ecology published by John Wiley & Sons Ltd.

1DepartmentofMedicalBiochemistryand

Microbiology,ScienceforLifeLaboratory,

Uppsala University, Uppsala, Sweden

2Depar tmentofLifeSciences,Georgina

MaceCentreforTheLivingPlanet,

ImperialCollegeLondon,Ascot,UK

3SwedishUniversityofAgricultural

Sciences,SwedishSpeciesInformation

Centre, Uppsala, Sweden

Correspondence

MatthewT.Webster,IMBIM,BMCD11:3,

Box582,UppsalaUniversity,75123

Uppsala, Sweden.

Email: matthew.webster@imbim.uu.se

Funding information

Vetenskapsrådet,Grant/AwardNumber:

2018-05973;Naturvårdsverket,Grant/

AwardNumber:225-21-003

Handling Editor:SeanD.Schoville

Abstract

Populations of many bumblebee species are declining, with distributions shift-

ing northwards to track suitable climates. Climate change is considered a major

contributingfactor.Arcticspeciesareparticularlyvulnerableastheycannot shift

furthernorth,makingassessmentoftheirpopulationviabilityimportant.Analysis

oflevelsofwhole-genome variation is a powerful way to analyse population de-

clines and fragmentation. Here, we use genome sequencing to analyse genetic vari-

ation in seven species of bumblebee from the Scandinavian mountains, including

two classified as vulnerable. We sequenced 333 samples from across the ranges

of these species in Sweden. Estimates of effective population size (NE) vary from

~55,000 for species with restricted high alpine distributions to 220,000 for more

widespreadspecies.Populationfragmentationisgenerallyveryloworundetect-

able over large distances in the mountains, suggesting an absence of barriers to

gene flow. The relatively high NE and low population structure indicate that none of

the species are at immediate risk of negative genetic effects caused by high levels

of genetic drift. However, reconstruction of historical fluctuations in NE indicates

that the arctic specialist species Bombus hyperboreushasexperiencedpopulation

declines since the last ice age and we detected one highly inbred diploid male of

this species close to the southern limit of its range, potentially indicating elevated

geneticload.Althoughthelevelsofgeneticvariationinmontanebumblebeepopu-

lations are currently relatively high, their ranges are predicted to shrink drastically

due to the effects of climate change and monitoring is essential to detect future

population declines.

KEYWORDS

bumblebees, climate change, conservation genetics, genomics/proteomics, population

dynamics, population genetics – empirical

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LIU et al.

1 | INTRODUCTION

Bumblebees are important pollinators, particularly in temperate

regions of the northern hemisphere. However, declines in species

abundance and distribution have been reported globally in recent

decades (Cameron et al., 2011; Cameron & Sadd, 2020; Williams

et al., 2009).AccordingtotheIUCNRedList,nearlyone-quarterof

the bumblebee species that have been assessed are declining(De

Meulemeesteretal.,2016).Asbumblebeesaremainlycoldadapted,

they are particularly vulnerable to climate change, which is predicted

to have a major negative impact on their populations (Cameron &

Sadd, 2020). Climatic niche modelling of future suitable habitats on

the basis of multiple future climate change scenarios has shown that

the majority of species are predicted to lose a significant proportion

of their current ranges within this century. Up to 36% of species are

predic ted to lose more t han 80% of thei r current ran ge (Ghisbain

et al., 2023; Rasmont et al., 2015).

ThedistributionsofmanyNorthAmericanand European bum-

blebee species have shifted northwards in recent decades (Kerr

et al., 2015; Mar tinet et al., 2015; Soroye et al., 2020). However,

although the southern edges of their distributions have retreated

rapidly,expansionsofthenorthernedgesarereportedtobeoccur-

ring muchmore slowly.Climate change is the mostlikely explana-

tion for these changes, and in particular the increased occurrence

of extreme weather events that limit sur vival. Bumblebee popu-

lations in the arctic and alpine regions are particularly threatened

by climate change because it is impossible for them to shift their

distributions furthernorth (Kerretal.,2015; Rasmont et al., 2015;

Soroye et al., 2020; Williams & Osborne, 2009). The assessment of

the viability of arctic and mountain specialist bumblebee species is,

therefore, high priority.

Analysi s of genetic diver sity is a power ful way to assess p op-

ulation viability and inform conservation strategies (Allendorf

et al., 2010; Hohenlohe et al., 2021). Rarer species of bumblebees

inEuropeandNorthAmericahavebeenfoundtoexhibitthelower

levels of genetic diversity and the elevated levels of genetic dif-

ferentiation (Cameron et al., 2011; Charman et al., 2010; Darvill

et al., 2006; Ellis et al., 2006;Kentetal., 2018; Lozier et al., 2011;

Lozier & Cameron, 2009), which suggests that an analysis of genetic

variation could be an important way to assess conservation status of

bumblebees.Analysisofgeneticdiversityonawhole-genomescale

enables a range of more powerful analyses than traditional conser-

vation genetic analyses based on limited numbers of loci (Webster

et al., 2023). These include reconstructing population history and

quantifying levels of inbreeding and genetic load in natural pop-

ulations ( Allendor f et al., 2010; Hohenlohe et al., 2021; Supple &

Shapiro, 2018; Webster et al., 2023).

The main focus of this study is to provide an assessment of

populationhistoryandvulnerabilitybasedongenome-widevaria-

tion in bumblebee species found in mountain habitats in Sweden,

which are among the most threatened by climate change (Rasmont

et al., 2015).Wefocuson sixspeciesthatarerestrictedtoarctic

and mountain environments, and one species (Bombus pascuo-

rum) that is present in the Swedish mountains but is also wide-

spread in multiple habitats throughout Europe (Table 1; Figure 1).

Of the montane species, Bombus balteatus, Bombus hyperboreus,

Bombus pyrrhopygus and Bombus alpinus belong to the subgenus

Alpinobombus(Cameronetal.,2007; Williams et al., 2008). B. bal-

teatus is relatively common in the Swedish mountains, whereas the

other three have a more restricted range, inhabiting higher lati-

tudes and elevations (Rasmont et al., 2015; Williams et al., 2019).

Bombus lapponicus and Bombus monticola belong to the subgenus

Pyrobombus. Both of these species are found throughout the

Swedish mou ntains (Rasmont e t al., 2015). I n a d dit i o n t o t h e irran g e

in Scandinavia, two of the species are also present in mountain en-

vironments further south in Europe. B. monticola is found in high

elevationsat manylocations across Europe including the British

Isles,Pyrenees,AlpsandBalkans.B. alpinus occurs at the highest

elevationsin the Alps inadditiontothe Scandinavianmountains.

The other montane species B. lapponicus, B. pyrrhopygus, B. baltea-

tus and B. hyperboreusarenotpresentsouthofScandinavia.Allof

these species apart from B. alpinus and B. monticola are also found

further east in Siberia (Rasmont et al., 2015; Williams et al., 2019).

Incontrasttotheotherspecies,B. hyperboreus is a parasitic spe-

cies, which lays eggs in the nests of other bumblebee species

(Ødegaard et al., 2015).

TAB LE 1 Speciesofbumblebeesanalysedinthisstudy.

Bombus sp. Subgenus English Swedish

IUCN red list

(3.1)

Climate

riskaDistribution in Europe

alpinus Alpinobombus Alpinebumblebee Alphumla Vulnerable HHR EuropeanhighlandsandArctic

pyrrhopygus Alpinobombus Polarbumblebee Polarhumla Least concern HHHR Arctic

balteatus Alpinobombus Golden-beltedbumblebee Fjällhumla Least concern HHR ScandinavianhighlandsandArctic

hyperboreus Alpinobombus High-Arcticbumblebee Tundrahumla Vulnerable HHHR Arctic

lapponicus Pyrobombus Lapland bumblebee Lapphumla Least concern HHR ScandinavianhighlandsandArctic

monticola Pyrobombus Mountainbumblebee Berghumla Least concern HHR EuropeanhighlandsandArctic

pascuorum Thoracobombus Common carder bee Åkerhumla Least concern RCommon

aClimateriskcategories:HHHR,extremelyhighclimatechangerisk,lossof>95%ofrangeby2100;HHR,veryhighclimatechangerisk,lossof>85%

of range by 2100, HR, high climate change risk, loss of >70% of range by 2100, R, climate change risk, loss of >50% of range by 2100, LR, lower

climate change risk, <50% loss of range by 2100 (Rasmont et al., 2015).

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LIU et al.

Allofthese montane bumblebeespecies arethreatenedbycli-

mate change. Those that are judged to be most at risk are the ones

restricted to the highest elevations and latitudes: B. pyrrhopygus, B.

alpinus and B. hyperboreus. Of these, B. alpinus and B. hyperboreus

are classified as vulnerable in the species red list whereas all the

other species studied here are considered least concern (Table 1)

(IUCN,2022). However, these risk assessments have been shown

to be unreflective of projected population trends that model future

scenarios of climate change (Ghisbain et al., 2023). Species have

been classified into climate risk categories based on species distri-

bution modelling (Rasmont et al., 2015). Two of the species under

study here, B. pyrrhopygus and B. hyperboreus, are assigned the most

extremeriskclass,HHHR,indicatingthat>95%oftheirinhabitable

range is predicted to be lost by 2100 under a realistic future climate

change scenario. The other four montane species, B. alpinus, B.

balteatus, B. lapponicus and B. monticola, are assigned the category

HHR, indicating a loss of >85%.

We also included the samples of Bombus pascuorum in the study,

whichisamemberofthesubgenusThoracobombus.Incontrasttothe

other spe cies, it is common all ove r Europe, present in a r ange of habitats

fromtheArctictotheMediterranean.Itisthemostabundantbumble-

bee species in Europe and is relatively unthreatened by climate change

compared to other species (Rasmont et al., 2015).Itthereforeserves

asauseful comparisontoother species as it is expectedtohaverela-

tively high levels of genetic variation. Bombus pascuorum is a highly poly-

typic species with 24 described subspecies found throughout Europe

(Lecocq et al., 2015). Four of these subspecies are found in Sweden:

B. p. smithianus north of the Arctic circle, B. p. sparreanus in northern

SwedensouthoftheArcticcircle,B. p. pallidofacies in central and south-

ern Sweden, B. p. gotlandicusonGotland.B. pascuorum is the most wide-

spread and abundant bumblebee species in Europe and can survive in

urban and suburban areas where other bumblebee species are scarce

(Rasmont et al., 2015). B. pascuorum is assigned the lower climate risk

category R, indicating >50% predicted loss in a similar time.

FIGURE 1 Distributionofstudy

speciesinSweden.Pointsonthe

map represent observations since

1997reportedinSwedishSpecies

Observation System (N is the number of

observations). B. lapponicus, B. monticola

and B. balteatus are found widely in the

SwedishmountainsandintheArctic.B.

alpinus, B. pyrrhopygus and B. hyperboreus

are restricted to higher elevations and

latitudes in these areas. B. pascuorum is

a common bumblebee in the whole of

Europe,distributedfromtheArcticto

theMediterranean.MapsfromSwedish

Species Observation System (artportalen.

se).

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LIU et al.

Here,weperformedwhole-genomesequencingon333samples

of the seven bumblebee species collected throughout their ranges

in Sweden. The data were used to estimate a range of population

genetic parameters for each species to inform about their conserva-

tion status and population viability. We first generated phylogenetic

trees of all the samples in order to confirm evolutionary relationship

between species and search for the presence of any previously un-

knowncrypticspecies.Wenextperformedananalysisofpopulation

structure in each species to detect the presence of subpopulations

and barriers to gene flow across their ranges. We estimated the lev-

els of genetic variation across the genome and used these values

to estimate effective population size (NE) for each species. We also

performed a reconstruction of historical variation in NE using the

programmeSMC++, which analyses variation in coalescent time and

the site frequency spectrum of genetic variation across the genome

(Terhorst et al., 2017). Finally, we analysed the length distribution of

runs of homozygosity (ROH) in samples of each species to infer the

prevalence of inbreeding (Ceballos et al., 2018).

2 | METHODS

2.1 | Sampling and DNA extraction

Specimens of seven bumblebee species were collected during the

summer of 2021 from several locations in Sweden. This involved

field trips to high elevation locations in Lapland, Härjedalen and

Jämtland to collect montane species and additional collections of the

widespread species B. pascuorum in locations throughout Sweden.

Sampling locations are displayed in Figure 2, a summary of collec-

tions is found in Table 2 and a full list of samples and associated

data is presented in Table S1. Samples were captured with sweeping

hand nets and kept temporarily in Falcon tubes accompanied with

ice packs. Species and sex/caste of each sample were identified

using a guide (Ødegaard et al., 2015). The identity of each sample

was confirmed by the genetic analysis, which also enabled a subset

of samples for which field identification was ambiguous or incorrect

tobecorrectlyidentified (see below).Sampleswere storedin95%

ethanolat−20°C.TheDNAwasextractedusingtheQiagenDNeasy

Blood&TissueKitfromamiddlelegandthoraxmuscletakenfrom

thecoxalcavityofthemiddleleg.

2.2 | Sequencing and variant calling

All species from the Pyrobombus and Alpinobombus subgen-

era studied here are closely related to a single species from their

subgenus for which a high-quality genome assembly is available.

It is, ther efore, possible to use one genome assembl y from each

subgenus to map reads from all of the other species of that sub-

genus. We used a genome assembly of B. lapponicus sylvicola

(ASM1967717v1;PRJNA646847) to map Pyrobombusspeciesand

a genome assembly of B. kirbiellus(ASM1920181v1;PRJNA704506)

to map Alpinobombus species. Bothof these genome assemblies

FIGURE 2 Mapofbumblebeesample

collection for this study. The solid dots

in different colours denote the sampling

sites for each species across Sweden.

Numberofmales(M)andfemales(F;

workers and queens) collected are given.

MapfromStamendesign(CCBY4.0).

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LIU et al.

arederivedfromsamplesfromtheRockyMountains,UnitedStates

(Christmas et al., 2021, 2022). B. lapponicus sylvicola in United States

isan extremelycloserelative ofB. lapponicus in Sweden. The most

recent ta xonomic revis ion considers t hem to be the sa me species

(B. lapponicus) although they were previously considered to be two

species, B. sylvicola (North America) and B. lapponicus (Eurasia)

(Mart inet et al., 2019). A simil ar situation ex ists for B. kirbiellus in

NorthAmerica and B. balteatus in Eurasia. In this case, these two

species were previously both considered to be B. balteatus, but the

mostrecenttaxonomicrevisionhassplitB. balteatus into two species

B. kirbiellus in North A merica and B. balteatus in Eurasia (Williams

et al., 2019). The genome assemblies that are available are therefore

from species currently known as B. lapponicus sylvicola (previously B.

sylvicola) and B. kirbiellus (previously B. balteatus), which are closely

related to the study species B. lapponicus and B. balteatus found in

Sweden. Bombus pascuorum is distantly related to the other study

species and weused apubliclyavailable genomeassembly (iyBom-

Pasc1.1;PRJEB43481)tomapreadsfromthisspecies.

Paired-end sequencinglibrarieswerepreparedwith the Nextera

DNAFlexLibraryPreparationKitandsamplesweresequencedonan

IlluminaNovaSeq6000with2 × 150 bpreads.Thereaddataforeach

species were mapped to their corresponding reference genomes. B.

monticola and B. lapponicus were mapped to B. lapponicus sylvicola

(Pyrobombus).B. balteatus, B. alpinus, B. hyperboreas and B. pyrrhopygus

were mapped to B. kirbiellus (previously B. balteatus,Alpinobombus),

and B. pascuorum was mapped to B. pascuorum (Thoracobombus).

Mappingwasperformed using the BWA-MEMalgorithm (Li, 2013).

Mappingsweresortedandwrittentobamfilesusingsamtools.Picard

was used to add read groups and mark duplicate reads in the bam files

thatwerethenindexedinsamtools.Thevariantswerecalledfollowing

theGATKbestpractices fornon-modelspecies (Poplinetal.,2018).

HaplotypeCaller was run with each bam fileswith flag ‘-ploidy’ to

generate individual-specific gVCF files. All gVCF files were merged

on a per contig basis byGenomicsDBImport and called variants by

GenotypeGVCFs.The resulting variant data sets were hard-filtered

followingtheGATKrecommendedthresholds.

Samples were initially all called as diploids, which allowed us to

confidentlyconfirmthesex ofeach sample bythe absenceofhet-

erozygoussitesinhaploidmales.Samplesforwhichthesexassigned

fromidentificationinthefieldwasincongruentwithploidywereex-

aminedmorphologicallyinmoredetailtoconfirmsex.Heterozygous

variants that were detected when HaplotypeCaller was run with

‘-ploidy 2’ for haploid sampleswereconsideredunreliableandfil-

teredoutforeachcohortdataset.Allsampleswereassigned their

correct ploidy to infer genotypes for the final data set.

AdditionalSNP filters wereappliedwith bcftools(Li, 2011): (1)

only including biallelic SNPswith minimum minorallele countof2

orgreaterandexcluding SNPs withreaddepthandgenotypequal-

itypersiteineachsampleunder3 and20,respectively(-vsnps-m

2 -M 2 --min-ac 2:minor -e ‘FMT/DP < 3 | FMT/GQ < 20’); (2) ex-

cluding sites with missing data exceeding 0.5, that is, ‘F_MISSING

> 0.5’; (3) excluding sites with highmean depth across all samples

andexcessiveheterozygosity(INFO/DP > X|INFO/ExcessHet> 25),

whereX = 1000forbothB. pascuorumandAlpinobombusdatasets

andX = 3134forPyrobombustoexcludesiteswithdepthinthetop

5% of the distr ibution. Th e additional f ilters INFO/DP and IN FO/

ExcessHetallowedustoremovesitesduetoalignmentstomultiple

regionsandvariant callingerrors.ThreeSNPdatasetsweregener-

ated, corresponding to sets of samples mapping to each of the three

reference genomes, ready for the downstream analyses.

In order to directly compare the levels of genetic variation

in B. lapponicus and B. balteatus with their sis ter species in N orth

America,the gVCFfilesgeneratedbyHaplotypeCallerwerecom-

bined with a similar data set of samples collected previously in the

UnitedStatesRockyMountains(Christmasetal.,2021, 2022)(NCBI

PRJNA646847and PRJNA704506).The variantswere thengeno-

typed and filtered for the two combined sets of gVCF files follow-

ing the same steps and filters as above. For the combined data sets,

‘INFO/DP> 25000’wasused.Thiscut-offwaschosenbyexamining

theINFO/DPdistributionsineachdataset.

2.3 | Population structure

Reconstructionofneighbour-joining(NJ)trees(Saitou&Nei,1987), prin-

cipalcomponentanalyses(PCA)andadmixture(Alexanderetal.,2009)

analyses were performed to reveal the genetic diversity of each geo-

graphicalpopulation using the SNPdatasets. Fortheseanalyses, only

TAB LE 2 Numberandlocationofbumblebeesamplesusedinthisstudy.

Bombus sp.

Province

Tot alLappland Jämtland Härjedalen Västerbotten Uppland Dalarna Östergötland Västergötland Gotland

alpinus 12 0 0 0 0 0 0 0 0 12

pyrrhopygus 5 1 0 0 0 0 0 0 0 6

balteatus 42 0 1 0 0 0 0 0 0 43

hyperboreus 91 0 0 0 0 0 0 0 10

lapponicus 109 16 3 0 0 0 0 0 0 128

monticola 63 115 0 0 0 0 0 0 79

pascuorum 23 0 4 82 2 4 4 855

Tot al 263 19 23 82 2 4 4 8333

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SNPswithminorallelefrequencygreaterthan5%wereincluded.Scripts

providedbySimonMartinwereusedtocalculatedistancematrixforNJ

treestaking intoaccountmixed ploidyofsamples (https:// github. com/

simonhmartin/genomics_general). The trees were made and visualized

withFastMe(Guindon&Gascuel,2003) and FigTree v1.4.4 (http:// tree.

bio. ed. ac. uk/ softw are/ figtr ee/ ), respectively.

PCA analyses for each species were implemented in PLINK

v1.9(Chang etal., 2015). To better present the geographical clus-

tering, one sample(DA_2)of B. pascuorum overlapped with sample

DA_03 wasremoveddue totheir closerelatedness, probably from

thesame nest. TheAdmixtureprogramwas usedforeight runsof

population structure by specifying the level of ploidy for each sam-

ple (Alexander et al., 2009). The best-supported values ofK were

inferred byselectingtheonewiththelowestcross-validationerror

byadding‘--cv’flaginthisprogram.

2.4 | Confirmation of species identification

TheNJtreeswereusedinconjunctionwithdetailedmorphological

analysis to correctly identify samples where species identification in

the field was incongruent with genetic analysis. This was particularly

important for species with similar colouration patterns, which is the

case for B. lapponicus and B. monticola, and for B. alpinus and B. pyr-

rhopygus.AWILDM5-47147stereomicroscopeforpreparationwas

used for this purpose.

AllspecimensofB. lapponicus in this study had yellow hairs on

the last two tergites in contrast to samples of B. monticola which had

orange red hairsonthelasttergites.Detailedanalysisofthischar-

acter was particular important for populations of B. lapponicus in the

southern part of the mountain chain, which are darker than northern

populations and difficult to distinguish from B. monticola in the same

area (Ødegaard et al., 2015).

Agood diagnostic field character to separate B. alpinus and B.

pyrrhopygus proved to be the colour of hairs on the two first metaso-

malsegments.InB. alpinus, there is a distinct limit between the black

hairsonthefirsttergiteandtheredhairsonthesecondtergite.In

B. pyrrhopygus, the colour transition is much more diffuse and does

notnecessarilyfollow the tergitelimitation. Inaddition,the cuticle

in B. pyrrhopygus is densely shagreened and dull while in B. alpinus,

it has a smoother, shinier surface. This can preferably be seen on

the outer s urface of the h ind tibiae. Af ter detaile d morphologi cal

analysis of all samples for which genetic analysis was incongruent

with field identification, we were able to confidently determine the

species for each sample.

2.5 | Estimation of effective population size

Effective population size (NE) for each species was estimated using an

estimate of population mutation rate, Watterson's estimator (θW), in-

volving two equations:

𝜃

, where K is the number of segregating

sites in the species and an is the (n–1)th harmonic number, n denoting

the number of chromosome sets. NE can be obtained using an estimate

of θW per base and an estimate of the mutation rate per base μ based

on θ = 2pNEμ, where p is the ploidy of the species (p = 1.5forhaplodip-

loidbumblebees)(Ferretti&Ramos-Onsins,2015; Fu, 1994). For these

analyses, we used a direct estimate of mutation rate from B. terrestris

(μ = 3.6 × 10−9) (Liu et al., 2017).

2.6 | Detection of runs of homozygosity (ROH)

The segments of ROHs for females from each species were assayed

usingthe‘--homozyg’flaginPLINKv1.9(Changetal.,2015), which

uses a sliding window approach to scan each individual's genotype at

each marker position across the genome. The following criteria were

defined to present the length of ROHs: (1) a minimum ROH length of

10 kbcontainingatleast50SNPs;(2)amaximumgapbetweencon-

secutiveSNPsof1 Mb;(3)aminimumdensityofoneSNPin50 kb;(4)

aslidingwindowof50SNPsacrossthegenomewithoneSNPstep;

(5)notoleranceofheterozygoussitesandamaximumthreemissing

genotypes; (6) a default window threshold of 0.05. The lengths of

ROHweredividedintothreeclasses: 0.01–0.5 Mb, 0.5–1 Mb,1–1.5

(detectedmaximumlength)Mb.

2.7 | Inference of population demographic history

SMC++ (Terhorst et al., 2017) was used to infer past fluctuations in

NE. This method uses the inferred distribution of coalescence times at

lociacrossthegenomeandthesitefrequencydistributionofSNPsto

infer temporal variation in NE (Terhorst et al., 2017).To ensure the reli-

abilityofSNPcalling,onlyfemalesampleswithreadcoveragegreater

than 13×were selected forrunningSMC++, in line with other stud-

ies (Wang et al., 2021).None of the samples included in this analy-

sisshowedhighrelatednessaccordingtothePCAanalysis.Themean

depthperindividualwasestimatedusingthe‘--depth’flaginVCFtools

(Daneceket al.,2011). The previously filtered VCF files were used as

inputs by keeping only these samples. Eventually, 49 samples were

used for the demographic inference, including seven B. pascuorum, 10

B. monticola and 11 B. lapponicus, as well as 12 B. balteatus and three for

B. alpinus, B. hyperboreus and B. pyrrhopygus, respectively. We used the

‘-d’optiontospecifybetween3and12differentsamplesperspeciesas

‘distinguishedindividuals’acrossrunsinordertogenerateacomposite

likelihood, which should result in improved estimates of population size

history (Terhorst et al., 2017). EstimationsfromSMC++ were scaled

using a generation time (g)of1 yearandamutation rate(μ)3.6 × 10−9

substitutions per site per year (see above).

3 | RESULTS

3.1 | Field sampling of seven bumblebee species

The samples of the study species were collected during the summer

of2021.Samplecollection in Abisko and thevicinity yielded sam-

plesofallsevenspecies(263samplesintotal).Additionalcollections

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LIU et al.

were also made in Jämtland (19samples) and Härjedalen (23 sam-

ples). Samples of the widespread species B. pascuorum were col-

lected from throughout its range in Sweden, including the island of

Gotland.Thisincorporatedsixmaingeographicareasinwhichfour

different subspecies and hybrids between them were located.

The final data sets consisted of 333 samples (Webster, 2022).

Three of the species—B. alpinus, B. pyrrhopygus and B. hyperboreus—

have a more res tricted distribution at higher elevations and latitudes.

These species were found in lower numbers than the other, more

widespread, montane species and were only present in a subset of

localities. The sampling locations for all seven species are displayed

in Figure 2.Asummaryofsamplescollectedineachlocationispre-

sented in Table 2. A full list of sa mples that wer e sequenced an d

associated metadata is presented in Table S1.

3.2 | Whole- genome sequencing and

bioinformatic analysis

All samples were subjected to Illumina whole-genome sequenc-

ing. Raw sequence reads from each sample were aligned to the

appropriate reference genome to generate three separate multi-

speciesalignments:(1)The207Pyrobombussamples(B. lapponicus

and B. monticola) were aligned to B. lapponicus sylvicola (Christmas

et al., 2021). (2) The 71 Alp inobombus s amples (B. alpinus, B. pyr-

rhopygus, B. balteatus, B. hyperboreus) were aligned to the B. kirbiellus

reference assembly (Christmas et al., 2022). (3) The 55 B. pascuorum

samples aligned to the B. pascuorum reference genome assembly.

Meansequencingdepth was 10.5xperindividualanddidnotvary

substantially between species (Table S1).

We first determined ploidy of each sample by detecting the pres-

ence of heterozygous sites. This analysis confirmed whether samples

were male (haploid) or female (diploid). One sample of B. hyperboreus

was identified morphologically as a male, but was found to be diploid

on the basis of the genetic analysis, indicating that it is a diploid male

(see below for additional analysis). We updated sample classifications

in cases of i ncorrect classif ication in the fi eld. Species ide ntity was then

confirmedbyconstructingNJtreesforeachofthethreemultispecies

alignments. The seven different species were clearly distinguishable

from NJ trees. A small number of samples thathad beenincorrectly

classified were identified from NJ trees and correctly classified. In

addition, the identity of samples of the two species B. alpinus and B.

pyrrhopygus, which are difficult to identify due to their convergence in

morphology, was confirmed by a combination of genetic clustering in

NJtreesanddetailedmorphologicalanalysis.The numbersinTable 2

refer to correctly classified samples.

3.3 | Levels of genetic variation

We estimated the levels of genetic variation in all species using

Watterson's theta (θW) and nucleotide diversity (average number of

pairwise differences, π) (Watterson, 1975) (Table 3). Estimates of θW

TAB LE 3 GeneticvariationinsevenSwedishbumblebeespecies.

Bombus sp. Male Female

Haploid genomes

sampledaNo. of SNPs

Watterson's theta per

base (θW) (%)

Nucleotide diversity

(π) (%)

Effective population size

(NE)

Observed

heterozygosity (HOBS)

alpinus 12 24 570,149 0.061 0.08 56,534 0.333

pyrrhopygus 612 1,390,927 0.184 0.236 170,545 0.371

balteatus 17 26 69 2,370,054 0.197 0.24 182,673 0.283

hyperboreus 10 20 660,899 0.074 0.099 68,978 0.306

lapponicus 62 66 194 3,095,046 0.21 0.227 194,583 0.280

monticola 28 48 124 1, 085,476 0.08 0.091 73,814 0.298

pascuorum 14 41 96 3,80 0,079 0. 241 0.273 222,742 0.218

aSum of haploid genomes found in both haploid and diploid individuals.

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LIU et al.

are 0.06%–0.25% (Wallberg et al., 2014), which we converted into

estimates of long-term NE based on a direct estimate of mutation

rate in the bumblebee Bombus terrestris (see Section 2). Levels of NE

vary between species. The highest estimates of NEareexhibitedby

B. pascuorum (~220,000; Table 3). This is consis tent with the fact t hat

it is a widespread species in Europe, found in many habitats, which

maybeexpandinginnumbers(Rasmontetal.,2015). Two other spe-

cies that are relatively widespread in the Swedish mountains, B. lap-

ponicus and B. balteatus,alsoexhibitrelativelyhighNE (~195,000and

~180,000,respectively).B. pyrrhopygusalsoexhibitedrelativelyhigh

NE (~170,000), which is surprising considering that it restricted to

high elevations and was the species observed the fewest number

of times during collection. Conversely, those species with the most

restricted distributions had significantly lower levels of genetic di-

versity. Estimates of NE for B. alpinus and B. hyperboreus were both

in the vicinity of 60,000 (Table 3). Surprisingly, B. monticola also has

an NE in this range, despite being one of the more abundant montane

species. In general, however,differences in levels of long-term NE

betweenspecieswereconsistentwithexpectationsbasedon spe-

cies abundance and range size.

3.4 | Population structure

We estimated population structure and species relationships of all

species using three methods: Neighbour-joining(NJ)trees (Saitou &

Nei,1987),principlecomponentanalysis(PCA)andadmixtureanalyses

(Alexanderetal.,2009).TheNJ treeofB. lapponicus and B. monticola

reveals that the samples cluster by species with no additional struc-

turing apparent (Figure 3a). Samples of B. lapponicus from Lappland,

Jämtland and Härjedalen showed no evidence of population substruc-

tureorcluste ringbyge og raphyinthePCA(Figure S1). This was further

supportedbytheadmixtureanalysis,inwhichasinglepopulationwas

thebest supportedmodelbycross-validation (Figure 3b). Samples of

B. monticolafromAbiskoformedclustersaccordingtoPCA(Figure S1).

However, the three samples that form a separate cluster were col-

lected inexactlythesamelocationand themost likely reasonforthe

clusteringis thereforethattheyarerelated.Admixtureanalysisfor B.

monticola also supports a single panmictic population (Figure 3b).

NJ trees f or the species of sub genus Alpinobu mbus—B. bal-

teatus, B. alpinus, B. pyrrhopygus and B. hyperboreus—reflect the

evolutionary relationships between these species (Figure 4a).

Tree topologies are the same as those published based on anal-

yses of phenotypic characters and molecular barcodes (Williams

et al., 2019), which place B. alpinus and B. pyrrhopygus as sis-

ter taxa. No significant evidence of clustering either within or

among geographical regions was observed for any of the four

AlpinobombusspeciesusingPCA(Figure S1).Asinglepopulation

was the be st-suppor ted model by cross-valid ation using admix-

ture for all species (Figure 4b). However, it should be noted that

the sample sizes for B. alpinus, B. pyrrhopygus and B. hyperboreus

were all relatively low (6–12 individuals per species), which results

in low power to detect population structure. B. alpinus was only

sample d in Abisko, which me ans it is impossibl e to identify ge-

netic structuring among geographic regions for this species. For

B. hyperboreus, a single sample collected in Jämtland did not clus-

terwiththeothersamplesfromAbisko,whichcouldindicatethe

presence of geographical structure in the species although more

samples would be required to confirm or refute this.

In contrast to the species already discussed, B. pascuorum is a

polytypic species found in a wide range of habitats across Europe

(Lecocq et al., 2015). The sampling strategy for this species incorpo-

rated the four subspecies present in Sweden, in addition to samples

identified as hybrids between pairs of subspecies collected in hy-

bridzones. TheNJtreeofthese samples revealedlimited levelsof

clustering (Figure 5a).ThesamplescollectedfromGotland,fromthe

subspecies, B. p. gotlandicus, were clearly separated from the other

samples.Alimitedamountofseparationwasalsoobservedforsam-

plesfromAbisko,consisting ofthesubspecies B. p. smithianus. The

remaining samples, consisting of B. p. sparreanus, B. p. pallidofacies

and hybrids between subspecies, collected over a wide geographical

area,donotshowevidenceofsubstructure.ThePCAplotrevealsa

similarpattern,withsamplesfromGotlandandAbiskoformingsep-

arate clusters from the remaining samples from mainland Sweden.

These remaining samples do not show evidence for geographic

structuring (Figure S1). The a dmixture anal ysis also revealed t hat

samplesfromAbiskoandGotlandformedseparateclustersfromthe

remaining samples (Figure 5b). Cross-valida tion analysis indic ated

that K = 2hadbestsupport,implyingtwomajorpopulations,oneon

GotlandandoneonmainlandSweden,althoughtheadmixtureplots

alsoshowsomedegreeofdistinctnessoftheAbiskopopulation.

Taken together, these analyses indicate that the six montane

bumblebee species studied here do not exhibit obvious popula-

tion subs tructure a cross their ra nges in Sweden. By co ntrast, th e

widespread polytypic species B. pascuorumexhibitsadegreeofsub-

structure.ApopulationonGotlandappearstobeisolatedfromthe

mainlandpopulation.Furthermore,samplescollectedinAbiskoap-

pear to have a degree of isolation from other mainland populations

further south, which display different colouration patterns and are

described as belonging to different subspecies (Lecocq et al., 2015).

3.5 | Inference of historical fluctuations in NE using

SMC++

Weusedthe program SMC++ to infer historical fluctuations in NE

in all seven of the study species (Figure 6). We infer that popula-

tions of B. hyperboreus have declined since ~104 years ago (the end

of the last ice age). The analysis suggests that current NE could be

less than 5000, although historically it has been more than 10 times

higher (Table 3, ~70,000). The species B. pyrrhopygus also shows

signs of decline in this period, which could indicate a contemporary

NE around 25,000 compared to a value of ~75,000 estimated from

θW. The other montane species under investigation do not show the

signals of majorpopulation expansions or declines and have been

relativelystableduring theHolocene.Incontrast to the arctic and

9 of 18

LIU et al.

montane species, there are signs that B. pascuorum has undergone a

populationexpansionduringthisperiod.

3.6 | Runs of homozygosity

We scanned the genomes of all diploid samples (typically females) for

runs of homozygosity (ROH) (Table 4; Figures S2–S5). Figure 7 shows

the proportion of the genome of each sample that falls into ROH

>0.1 Mb, grouped byspecies. The most striking finding was a single

individual of B. hyperboreus(WO_001)with>55% of the genome lying

inROHlongerthan0.1 Mb(Figure 7). This pattern is predicted to result

from a sib-mating.This sample was also classified as a diploid male,

whichindicatesthatitishomozygousatthesex-determininglocus.The

sample is the only B. hyperboreuscollected from Jämtland.All of the

othersamplesofthisspecieswerefoundinthevicinityofAbisko.

FIGURE 3 (a)Neighbour-joiningtreeforsamplesofB. lapponicus and B. monticolageneratedusingwhole-genomesequencedata.Both

speciesareinthesubgenusPyrobombus.Eachexternalbranchrepresentsanindividualsample.(b)Admixtureanalysesforallgeographical

populations of B. lapponicus (top) and B. monticola(bottom).NosignificantstructurewasdetectedforeitherspeciesandK = 1wasthebest

supported parameter value for both species.

K =2

K =3

Abisko/Kiruna

Jämtland

Härjedalen

Populations of B. lapponicus

K =2

K =3

Abisko/Kiruna

Jämtland

Härjedal

Populations of B. monticola

0.05

AC-028

M_L018

AC-012

M_L005

M_L223

M_L077

M_L121

M_L085

M_L012

BH_02

M_L144

M_L215

M_L004

M_L007

AC-017

M_L081

M_L150

M_L205

WO_111

4 5 1 L _ M

M_L021

AC-141

M_L199

M_L056

M_L130

M_L122

BH_06

AC-034

M_L185

M_L139

8 4 1 L _ M

M_L143

M_L140

M_L184

M_L054

WO_358

M_L030

BH_19

M_L003

M_L138

190

-C

M_L027

M_L169

M_L220

M_L166

500_

WO_006

M_L187

M_L171

BH_09

M_L180

AC-111

WO_815

M_L216

AC-021

M_L055

AC-061

M_L115

AC-043

M_L110

M_L174

AC-118

BH_14

AC-129

M_L076

M_L206

M_L124

M_L114

M_L218

AC-038

M_L167

M_L203

M_L060

M_L153

WO_007

AC-073

WO_448

BH_01

M_L211

M_L192

M_L176

M_L175

M_L109

M_L146

AC-082

M_L113

M_L172

M_L147

M_L071

M_L194

AC-135

AC-014

M_L009

AC-115

M_L075

AC-049

M_L183

M_L089

M_L200

WO_449

M_L152

M_L083

M_L043

M_L112

WO_002

BH_08

M_L136

M_L125

AC-138

M_L160

M_L157

AC-133

M_L017

M_L090

M_L086

AC-080

M_L091

M_L226

M_L099

M_L092

AC-006

BH_18

M_L100

M_L186

M_L133

M_L097

M_L159

WO_067

M_L189

M_L028

M_L074

M_L013

M_L219

BH_05

M_L008

BH_15

WO_269

721L_ M

M_L178

BH_11

M_L120

M_L224

AC-136

M_L117

AC-105

M_L082

M_L072

AC-137

M_L111

M_L029

M_L123

AC-071

M_L093

M_L191

AC-087

AC-069

M_L145

WO_703

M_L025

M_L095

BH_03

M_L135

M_L014

AC-046

AC-023

M_L096

M_L214

M_L193

BH_07

BH_10

AC-067

WO_004

4 1

L _

M_L087

M_L221

WO_383

AC-106

WO_331

M_L057

BH_04

M_L210

M_L213

WO_003

M_L119

M_L002

M_L088

BH_16

M_L131

M_L046

M_L084

M_L116

M_L173

AC-072

BH_17

AC-150

M_L061

AC-047

M_L197

M_L018

AC-012

M_L005

M_L077

M_L121

M_L085

M_L012

M_L144

M_L215

AC-017

M_L081

WO_111

5 1

M_L021

AC-141

M_L199

M_L122

M_L185

M_L139

M_L184

WO_358

M_L030

M_L003

M_L138

WO_006

M_L187

M_L171

M_L180

WO_815

M_L216

M_L115

AC-043

M_L110

M_L174

M_L076

M_L124

M_L114

C-038

M_L060

WO_007

AC-073

WO_448

M_L192

M_L176

M_L175

M_L109

AC-082

M_L113

M_L071

M_L194

AC-135

M_L009

AC-115

M_L183

M_L089

M_L200

WO_449

M_L083

M_L043

M_L112

M_L125

AC-138

M_L157

M_L090

M_L086

C-080

M_L091

M_L226

M_L099

M_L092

AC-006

M_L100

M_L186

M_L097

WO_067

M_L189

M_L028

M_L074

M_L008

WO_269

7 2

M_L178

BH_11

M_L120

C-136

M_L117

AC-105

M_L082

M_L072

C-137

M_L111

M_L123

AC-071

AC-087

AC-069

M_L145

WO_703

M_L095

M_L135

M_L014

M_L096

M_L193

BH_10

AC-067

WO_004

M_L087

WO_383

WO_331

M_L057

WO_003

M_L002

M_L088

M_L046

M_L084

M_L116

M_L173

AC-072

AC-150

M_L061

AC-028

M_L223

BH_02

M_L004

M_L007

M_L150

M_L205

M_L056

M_L130

BH_06

AC-034

M_L143

M_L140

M_L054

BH_19

M_L027

M_L169

M_L220

M_L166

BH_09

AC-111

AC-021

M_L055

AC-061

AC-118

BH_14

AC-129

M_L206

M_L218

M_L167

M_L203

M_L153

BH_01

M_L211

ML22

M_L146

M_L172

M_L147

AC-014

M_L075

AC-049

M_L152

WO_002

BH_08

BH_09

M_L136

M_L160

AC-133

M_L017

H_18

M_L133

M_L159

L166

M_L013

M_L219

BH_05

H_15

M_L224

M_L029

M_L093

M_L191

M_L025

BH_03

AC-046

AC-023

M_L214

M_L224

BH_07

M_L221

AC-106

BH_04

ML210

ML211

M_L213

ML075

M_L119

BH_16

M_L131

BH_17

AC-047

M_L197

Bombus lapponicusBombus monticola

M_L168

AC-020

M_L202

(a)

(b)

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LIU et al.

Populations of B. balteatus

Populations of B. alpinus

Populations of B. pyrrhopygus

Populations of B. hyperboreus

K =2

Abisko/Kiruna_ML

Abisko/Kiruna_AC

K =2

Abisko/Kiruna

Jämtland

K =2

Abisko/Kiruna

Härjedalen

Abisko/Kiru

Jämtland

0.05

AC-152

AC-029

WO_456

AC-022

M_L179

M_L155

M_L137

AC-090

M_L022

M_L020

M_L129

AC-027

M_L033

M_L188

AC-045

M_L019

M_L011

M_L126

M_L102

M_L106

AC-121

M_L195

AC-007

M_L149

M_L026

M_L062

M_L053

M_L032

AC-016

M_L098

AC-041

M_L151

WO_001

AC-003

M_L177

M_L198

AC-153

M_L024

AC-025

AC-019

AC-004

M_L196

AC-008

M_L103

M_L181

M_L006

AC-126

M_L058

M_L010

AC-156

AC-002

M_L142

AC-078

AC-015

AC-033

M_L015

BH_13

M_L105

M_L049

M_L107

M_L108

AC-113

M_L101

AC-018

M_L190

AC-112

AC-011

AC-036

M_L182

AC_026

M_L020

AC-027

M_L062

M_L053

AC-041

WO_001

M_L058

AC-015

AC-018

AC-036

Bombus hyperboreus

AC-152

-029

022

M_L179

M_L155

M_L137

090

M_L129

M_L033

M_L188

M_L126

M_L102

M_L106

AC-121

M_L195

M_L149

M_L032

AC-016

M_L098

M_L151

AC-003

M_L177

M_L198

M_L024

AC-004

M_L196

M_L103

M_L181

AC-126

AC-156

AC-002

M_L142

AC-078

BH_13

M_L105

M_L049

M_L107

M_L108

AC-113

M_L101

M_L190

AC-011

M_L182

Bombus balteatus

WO_456

M_L026

025

M_L015

C-112

AC_026

Bombus pyrrhopygus

M_L022

AC-045

M_L019

M_L022

M_L

M_L011

AC-007

AC-153

AC-019

AC-008

M_L006

M_L010

M_L

AC-033

M_L

Bombus alpinus

K =2

(a)

(b)

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LIU et al.

AsmallnumberofindividualsofB. pyrrhopygus, B. pascuorum

and B. monticola were also identified with 5%–20% of the genome

covered by ROH bl ocks longer than 0.1 Mb indicating that in-

breeding occurs sporadically in these species as well (Figure 7).

Outliers with long ROH tracts were not detected in the other spe-

cies, but may still occur at low frequencies. Only a small number

of sample s contained any ROH s longer than 1 Mb, an d for the

majority of species, none of samples had any such long ROHs

(Table 4). The mean coverage of the genome by ROH is <5% for all

species apart from B. hyperboreus, where the presence of the one

highly inbred diploid male individual strongly influences this mea-

sure. Average levelsofROH in B. pascuorum are high compared

FIGURE 4 (a)Neighbour-joiningtreeforB. alpinus, B. pyrrhopygus, B. balteatus and B. hyperboreus.Allspeciesareinthesubgenus

Alpinobombus.Eachexternalbranchrepresentsanindividualsample.(b)AdmixtureanalysesforallgeographicalpopulationsofB. alpinus, B.

pyrrhopygus, B. balteatus and B. hyperboreus.NosignificantstructurewasdetectedforanyofthespeciesandK = 1wasthebestsupported

parameter value for all species.

FIGURE 5 (a)Neighbour-joiningtreeforB. pascuorum. Several subspecies of B. pascuorumarepresentinSweden.Eachexternalbranch

representsanindividualsample.Samplinglocationsandsubspeciesareindicated,includingseveralfromhybridzones.(b)Admixtureruns

for all geographical populations of B. pascuorum. The best supported parameter value is K = 2,whichindicatesthepresenceofadistinct

subpopulationonGotland(B. pascuorum gotlandicus).

K = 2

K = 3

K = 4

Abisko

Härjedalen/Ume

Ammarnäs

Västergötland

Uppsala/Dalar

Gotland

0.02

NS_02

M_L068

SS_07

SL_04

M_L204

GT_01

SS_02

GT_05

M_L201

GT_02

M_L064

NS_01

GT_04

M_L209

SS_08

M_L132

M_L065

M_L134

GT_08

M_L208

M_L041

M_L066

DA_04

SL_01

DA_02

NS_07

SS_05

M_L207

M_L070

M_L067

SS_01

GT_03

M_L039

M_L170

DA_03

M_L051

M_L063

SS_03

GT_06

SS_04

NS_08

NS_05

M_L050

NS_03

SS_06

M_L069

GT_07

NS_04

M_L016

M_L001

M_L217

NS_06

SL_02

SL_03

DA_01

B. pascuorum smitheanus

Abisko

B. pascuorum gotlandicus

Gotland

B. pascuorum sparreanus/smitheanus

Ammarnäs

B. pascuorum sparreanus

Härjedalen/Umeå

B. pascuorum pallidofacies

Västergötland

B. pascuorum pallidofacies/sparreanus

UppsalaDalarna

(a)

(b) Populations of B. pascuorum

12 of 18

LIU et al.

to the other species, with ROH covering ~5% of the genome on

average (Table 4).

3.7 | Comparison of levels of genetic variation with

North American montane bumblebee species

Sister species of B. lapponicus and B. balteatus are found in mountain

andarcticlocationsinNorthAmerica.Thesespeciesarehighlysimi-

lar in morphology and recently evolutionarily diverged from their

European counterparts. The sister species of B. lapponicusinNorth

AmericaisalsoknownasB. lapponicus (subspecies sylvicola). The sis-

ter species of B. balteatusinNorthAmericaisB. kirbiellus. We com-

pared our results to a data set of 214 samples of B. lapponicus and

299 sampl es of B. kirbiellusf rom the Rock y Mountains, C olorado,

United States (Christmas et al., 2021, 2022). The data set generated

here also permitted comparisons of genetic diversity in this popula-

tion (Table 5). To do this, we jointly called genetic variation in both

data sets simultaneously in order to directly compare the levels of

genetic variation. For the two populations of B. lapponicus(USAand

Sweden), we find that the levels of nucleotide diversity and NE are

extremelysimilar(~160,000). We find that B. kirbiellus(USA)hassig-

nificantly higher NE compared to B. balteatus (Sweden) (NE ≈ 310,000

compared to 170,000).

4 | DISCUSSION

Weusedwhole-genomesequencingtosurveygeneticvariationand

analyse population history and structure of seven bumblebee spe-

ciesfoundinSweden:sixspeciesrestrictedtoarcticandmountain

environmentsandonewidespreadspecies.Ingeneral,wefindalack

of population structure in all of the montane species, and compara-

ble levels of NE in both montane and widespread species. We iden-

tify some indications of declines and elevated genetic load in the

rarer montane species. The results are useful to evaluate the conser-

vation status of each species and to inform management decisions.

4.1 | Relatively high NE and low population

structure in montane bumblebees

Forall of the species under study here, we estimate long-term NE

from levels of genetic variation to be greater than 50,000. Variation

in estimates of NE among species reflects differences in their range

and abundance. B. pyrrhopygus, B. balteatus and B. lapponicus have

NE close to that estimated for the widespread species B. pascuorum

(~200,000), which indicates their range and abundance are also

relatively large. These levels of NE are similar to other widespread

social bees such as honeybees (Wallberg et al., 2014).Bycontrast,

FIGURE 6 EstimationofhistoricalvariationinNEusingtheSMC++ method. The x-axisdenotesyearsbeforepresent.Strongsignalsof

population decline are inferred for B. hyperboreus, whereas population size increases are inferred for B. pascuorum.

Bombus pascuorum

Bombus monticola

Bombus lapponicus

Bombus balteatus

Bombus alpinus

Bombus hyperboreus

Bombus pyrrhopygus

13 of 18

LIU et al.

B. alpinus, B. monticola and B. hyperboreus have lower NE than the

other montane bumblebee species. B. alpinus and B. hyperboreus

have a more restricted distribution in at higher elevations and lati-

tudes (Figure 1) (Ødegaard et al., 2015; Rasmont et al., 2015) and are

thereforeexpectedtohavesmallerpopulationsizes. Avalue of NE

for a population less than 500 is commonly used to suggest it is vul-

nerableandlikelytoexperiencethenegativeeffectsofgeneticdrift

and inbreeding (Allendorf etal., 2022). Levels of NE in the species

studied here are substantially higher than this.

The six mon tane species of bu mblebees stu died here did not

show evidence for population structure among the montane loca-

tions whe re they were collec ted in Lapland , Jämtland and Här jedalen.

This indicates that there are no substantial barriers to gene flow and

that the populations have historically been continuous across the

Swedish mountain range. Studies of genetic structure B. kirbiellus

and B. lapponicus sylvicolafromNorthAmer icathataresiste rs pecies

oftwo of the species studied here (Martinet etal., 2019; Williams

et al., 2019) do not show evidence for geographical substructure

within the Rocky Mountains, Colorado, United States (Christmas

et al., 2022). Other studies have, however, found evidence for ge-

netic differentiation of these populations on different mountain

rangesinNorthAmerica(Kochetal.,2017; Whitley, 2018). We find

that B. lapponicus samples collected from populations in Sweden and

Colorado have similar levels of genetic variation, whereas popula-

tions of B. kirbiellus from Colorado have significantly higher levels

of genetic variation compared to its sister species B. balteatus in

Sweden. This indicates that B. kirbiellus may be relatively more abun-

dantinNorthAmerica,whichisconsistentwithobservationsfrom

field collections (Christmas et al., 2022).

Ingeneral,studiesinbothEuropeandNorthAmericahavefound

that relatively abundant bumblebee species such as B. terrestris do

notexhibitpopulationstructureoverthousandsofkilometres(Colgan

et al., 2022; Ghisbain et al., 2020; Heraghty et al., 2023; Lozier

et al., 2011), whereas very rare and declining species (e.g. B. musco-

rum)mayshowstructureonascaleofonly10 km(Darvilletal.,2006).

Geneticdifferentiationofpopulationscanoccurinmountainrangesif

species are restricted to high elevations, as with B. vancouverensis in

the southern part of its range in the western United States (Jackson

et al., 2018; Lozier et al., 2023). The results presented here are there-

fore consistent with observations from other studies indicating that

bumblebeesdonotexhibitpopulationstructureintheabsenceofgeo-

graphical barriers unless they have particularly low abundance.

We analysed population structure in four subspecies of B. pas-

cuorum, which are geographically separated and differ in coloura-

tion (Lecocq et al., 2015). The subspecies B. p. gotlandicus, which is

restricted to the island of Gotland,was clearly genetically distinct

fromtheother subspecies,consistentwithisolationin allopatry.In

addition, the subspecies B. p. smithianus, present in the far north of

Sweden, was genetically separable from other subspecies (Figure 5).

Incontrast, however,the twosubspecies B. p. sparreanus and B. p.

pallidofacies were indistinguishable genetically, indicating that there

are unlikely to be any barriers to gene flow between these two

subspecies.

TAB LE 4 Summarystatisticsofrunsofhomozygosity(ROH).

Bombus sp. Female no.

Total no. of ROH of each length

Mean total length of ROH per sample (Mb) Mean genome coverage by ROH per sample (%)0.01–0.1 Mb 0.1–1 Mb >1 Mb

alpinus 12 301 48 01.72 0.69

pyrrhopygus 6290 87 05.17 2.07

balteatus 26 4797 55 04. 51 1.80

hyperboreus 10 1844 804 125.47 10.18

lapponicus 53 7511 771 05.45 2.16

monticola 49 3799 971 26.99 2.77

pascuorum 41 12,329 1325 314.42 4.69

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LIU et al.

4.2 | Evidence of inbreeding and

population declines

Haplodiploid organisms such as bumblebees and other Hymenoptera

are particularly vulnerable to inbreeding because it can lead to ho-

mozygosityat the sex locus, whichproduces diploid males, which

are unable to successfully fertilize females. The presence of diploid

males can therefore severely reduce the fitness of a population.

When populations become small and inbreeding is common, this ef-

fect can leadto a particularly extremeextinctionvortex (Lozier &

Zayed, 2017; Zayed & Packer,2005). Diploid male production has

been observed in populations of rare and declining bumblebee spe-

ciesandisanindicatorofhighvulnerability(Darvilletal.,2006; Ellis

et al., 2006).

Inouranalysis,weidentifiedoneexampleofadiploidmale—aB.

hyperboreus collected in Jämtland. This sample also had the highest

degree of inbreeding detected in any sample: >50% of its genome

consistedof ROH greater than 100 kbpin length.It wasthe only

B. hyperboreus collect ed in Jämtland. It is n oteworthy that thi s is

the only observation of B. hyperboreus this far south reported in

Swedish Species Observation System, which has records over the

last25 years,withalloftheother143reportsrestrictedtoLapland

(themajorityinthevicinityofAbisko).Thisindicatesthatthissample

was found at or close to the southern limit of the species distribu-

tion in the Swedish mountains. This finding indicates that inbreeding

is occurring in the population of B. hyperboreus, particularly on the

edges of its range where it is most likely to be influenced by the

effects of climate change, although it is not possible to determine

theextentofinbreedinginapopulationbasedontheobservationof

a single diploid male. We do not find evidence for inbreeding in any

other species. Relatively high average levels of ROH are found in B.

pascuorum.AsB. pascuorumisanextremelywidespreadbumblebee,

itisunlikelythatitisexperiencinginbreeding.Itisthereforeunlikely

that the average levels of ROH observed in any of the species here

FIGURE 7 Proportionofgenomeof

eachsamplecomprisingROH > 0.1 Mbp.

Outliers are labelled.

AC−029

M_L198

WO_001

WO_456

AC−028

AC−111

BH_09

M_L169

GT_02

GT_03

GT_05

GT_06

GT_07

B. alpinus

B. balteatus

B. hyperboreus

B. lapponicus

B. monticola

B. pascuorum

B. pyrrhopygus

020 40

Proportion of genomewith ROH greater than 0.1 Mb (%)

Species

TABLE 5 ComparisonofgeneticvariationinrelatedspeciesfromNorthernEuropeandNorthAmerica.

Species

Haploid genomes

sampledcNo. of SNPs

Watterson's theta per

base (θw) (%)

Nucleotide diversity

(π) (%)

Effective

population size (NE)

lapponicus cluster

B. lapponicus (SWE) 194 2,514,341 0.171 0.161 158,075

B. lapponicusa(USA) 428 3,010,424 0.18 0.219 166,652

balteatus cluster

B. balteatus (SWE) 69 2, 250,496 0.187 0.207 173,458

B. kirbiellusb(USA) 598 5,824,609 0.334 0.271 3 0 9,42 7

aThespeciesinNorthAmericacurrentlynamedBombus lapponicus (subspecies sylvicola) was previously considered to be a separate species named

Bombus sylvicola(Martinetetal.,2019).

bThe species Bombus kirbiellusinNorthAmericawaspreviouslyknownasBombus balteatus (Williams et al., 2019).

cSum of haploid genomes found in both haploid and diploid individuals.

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LIU et al.

have been influenced by inbreeding as they do not differ markedly

from those observed in B. pascuorum.

AlthoughweestimatethatNE is relatively high in all of the study

species, it is important to note that our estimate of NE from θW re-

flects the harmonic mean over evolutionary time since the coales-

cence of the sequences in the sample, rather than being an estimate

of contemporary NE(Nadachowska-Brzyskaet al., 2022). We also

performedanSMC++ analysis to estimate fluctuations in NE over

the evolutionary history of the populations (Terhorst et al., 2017).

Asignificantfindingof thisanalysis was thatB. hyperboreus and to

alesserextent B. pyrrhopygus showed signals of population decline

during the current postglacial period in the last ~10,000 years.Both

of these species have restricted arctic distributions, but may have

been more widespread earlier in the current post-glacial period

when conditions were more favourable in a greater part of Europe.

Itis therefore possiblethat their populations have declined in the

current post-glacial period. This could suggest that the range of

these species has shrunk and that current NE is much smaller than

predicted from overall levels of genetic variation. The observation of

inbred individuals in these species from the ROH analysis also sup-

portsthissuggestion.Bycontrast,B. pascuorum is currently the mos t

widespread and abundant bumblebee in Europe and is able to sur-

vive in a range of modern habitats (Lecocq et al., 2015).TheSMC++

analysisindicatesthatthisspeciesmayhaveexpandedsincethelast

ice age, although it is possible that the weak population structure

observed in this species may have influenced this result.

Acontrastingpatternofinferred population history usingSMC

approachesisexhibitedbythemontanebumblebeeB. vancouveren-

sis, which shows greater degree of population fragmentation along

anorth–south gradientin western North America. In this species,

more isolated southern montane populations show lower genetic

variation and population declines (Lozier et al., 2023). This could

reflect a greater degree of habitat fragmentation, whereby sub-

populations of this species have become geographically isolated

athighelevationmountainpeaks.By contrast, thecommon North

AmericanbumblebeeB. vosnesenskii shows low population structure

and no evidence of decline (Heraghty et al., 2023), similar to our ob-

servations in B. pascuorum.

4.3 | Absence of cryptic species

Asmentionedabove,themontanebumblebeespeciesstudiedhere

havesister speciesinNorthAmericaandtheir subgenera have cir-

cumpolardistributions(Martinetetal.,2019; Williams et al., 2019).

Previous studies have used whole-genome sequencing to assess

genetic variation and population structure in B. lapponicus sylvicola

(sister species of B. lapponicus) and B. kirbiellus (sister species of B.

balteatus) in several high-elevation locations in Colorado, United

States (Christmas et al., 2021, 2022). One significant finding was the

detection of the presence of a previously unknown cryptic species,

which was indistinguishable from B. lapponicus sylvicola on the basis

ofstandardmorphological characters:67of281samplesoriginally

identif ied as B. l. sylvicola formed a distin ct cluster of samp les, termed

‘incognitus’,thatwerehighlydivergentfromtheremainingsamples

(Christmas et al., 2021). However, we did not observe any distinct

clusters related to B . lapponicusin our dat ase tof129s a mpl es.Thisis 

consistentwiththehypothesisthat‘incognitus’isrestrictedtoNorth

America,andwhetheritoccursoutsideofColoradoisunknown.

4.4 | Assessment of species vulnerability

ArcticandmountainousregionsofScandinaviaharbourbumblebee

species that are most threatened by climate change and are also

projected to become refugia for more European bumblebee species

infuture(Ghisbainetal.,2023). Habitat loss due to agriculture or

urbanization is not currently prevalent in these regions, although

could potentially increase in future, which could have negative ef-

fects on bumblebees (Rasmont et al., 2015). Three of the montane

species studied here, B. lapponicus, B. monticola and B. balteatus, are

found relatively commonly in the Swedish mountains. Of these, B.

monticolais alsofound at higherelevationsinthe UnitedKingdom

and in mountain ranges in mainland Europe, whereas the other spe-

ciesarerestrictedtotheScandinavianmountains.Accordingtothe

IUCN RedList, all these speciesare categorized as ‘least concern’

(IUCN,2022). However, they also fall in the climate risk category

HHR (very high risk), indicating they are predicted to lose >85%of

their current range by 2100 (Rasmont et al., 2015). This implies that

all of these species would occupy a significantly reduced range in the

Scandinavian mountains, and B. monticola would additionally lose

muchofitsrangeelsewhereinEuropeexceptthehighAlps.

Our analysis of genome variation in these three species did not

uncover any evidence for depleted levels of genetic variation that

wouldsuggestthey lie in the immediate riskzoneforexperiencing

negative genetic effects due to inbreeding or genetic drift. We find

the levels of v ariation to be lower tha n expected in B. monticola

based on its relative abundance, but no indications of population de-

cline or inbreeding. This suggests that the populations are currently

healthy, despite the prediction that they will be strongly threatened

by climate change in future.

The other three montane species, B. alpinus, B. pyrrhopy gus and B.

hyperboreus, have a much more restricted dis tribution in the Swedish

mountains, occurring at higher elevations and latitudes (Rasmont

et al., 2015). Of these, B. alpinus is also present at higher altitudes

intheAlps,whereastheothersarerestricted toScandinavia. B. al-

pinus and B. hyperboreus are catego rized as ‘vulne rable’ accord ing

to the red list, whereas B. pyrrhopygus isconsidered‘leastconcern’

(IUCN,2022). B. hyperboreus and B. pyrrhopygus are placed in the

HHHRclimateriskcategory(extremelyhighrisk),indicatingtheyare

predicted to lose >95%oftheirrangeby2100whereasB. alpinus is

placed in category HHR (very high risk) (Rasmont et al., 2015).

Amongthesethreespecies, wefindevidenceofpopulationde-

clines or inbreeding over evolutionary time. B. hyperboreus is a less

abundant species with a very restricted range and, in contrast to the

other species studied here, it has a specialized parasitic lifestyle. Our

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LIU et al.

analysis indicated that this species has become much less abundant

in the current postglacial period, and we uncovered one highly in-

bred diploid male individual among the 10 samples we collected.

This speciesis considered to be oneofthe mostvulnerable to ex-

tinction in Europe (Rasmont et al., 2015) and our analysis suggests

it may be suffering negative genetic effects of population declines,

particularly in the southern limit of its range. We also find evidence

that B. alpinus and B. pyrrhopygus exhibitlowerordecliningNE rel-

ative to the other species, consistent with their relatively restricted

abundance and distribution.

Climatechangeisthemainthreattothesixmontanebumblebee

species studied here (Ghisbainet al., 2023). Our analysis suggests

their current ranges in the Swedish mountains would be sufficient

to support healthy populations in future. However, these ranges

are predicted to shrink drastically under future scenarios of climate

change (Rasmont et al., 2015), which could lead to population bottle-

necks and fragmentation and eventually loss of all suitable habitat.

Monitoring of these p opulations using both genetic and conven-

tional methods would be advisable in order to understand how their

populations are affected by changing climate in future.

AUTHOR CONTRIBUTIONS

MatthewT.Websterdesignedtheresearch.AoifeCantwell-Jones,

Richard J. G ill, Björn Cederberg and M atthew Webster collected

samplesand performedspeciesidentification.Yuanzhen Liu, Anna

Olsson andTuuli Larva performedtheDNA extractions. Yuanzhen

Liu analy sed the data . Matthew T. Webster w rote the manusc ript

withfeedbackfromallco-authors.

ACKNO WLE DGE MENTS

This project was mainly funded by the Swedish Environmental

Protect ion Agenc y (agreement nr. 225-21-003). The pr oject made

use of facilitiesat theAbisko Scientific Research Station, which is

connectedtoSITES(SwedishInfrastructureforEcosystemScience),

INTERACT (International Network for Terrestrial Research and

MonitoringintheArctic)andSLU(SwedishUniversityofAgricultural

Sciences). Aoife Cantwell-Jones is funded by the NERC Science

andSolutionsforaChangingPlanet Doctoral Training Programme,

ImperialCollegeLondon(NE/S007415/1).Collectionswerealsosup-

portedbygrantsfromINTERACT(H2020—agreementno.730938)

an d Q u e k e t t M icroscop i c a l C l u b awardedt o R i c h a r dGill.T h e c o m p u -

tational analysis was enabled by resources provided by the Swedish

National InfrastructureforComputing(SNIC) at UPPMAX partially

funded by the Swedish Research Council through grant agreement

no.2018-05973. We alsoacknowledge supportfrom the National

Genomi cs Infrastr ucture in Stockh olm funded by Sci ence for Life

Laboratory, the Knut and Alice Wallenberg Foundation and the

Swedish Research Council. We thank the following people for help

with sample collection: Johan Lindell (Lappland), Willhelm Osterman

(Jämtland), Peter Nolbrant (Västergötland), Niklas Johansson

(Östergötland), Sven Hellqvist (Västerbotten). Turid Everitt and

MatthewChristmasassistedwithdataanalysis.

CONFLICT OF INTEREST STATEMENT

There are no conflicts of interest.

DATA AVAIL AB ILI T Y STATEMEN T

All sequence data generated by the study have been deposited at

theNationalCenterforBiotechnologyInformation(NCBI)together

withassociatedmetadata underBioProject PRJNA890771.Scripts

for bioinformatic analysis are available at GitHub: https:// github.

c o m / y z l i u 0 1 / S w e d i s h _ m o u n t a i n _ b u m b l e b e e .

ORCID

Yuanzhen Liu https://orcid.org/0000-0003-0212-0674

Matthew T. Webster https://orcid.org/0000-0003-1141-2863

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SUPPORTING INFORMATION

Additional supporting information can be found online in the

SupportingInformationsectionattheendofthisarticle.

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Distinct genome architecture underlies fine-scale population differentiation in two common European bumblebees ( Bombus pascuorum and Bombus lapidarius )

Preprint

Full-text available

May 2024

Bumblebees are keystone pollinators which facilitate the reproduction of a wide range of wild and agricultural plants. Their abundance and diversity have been severely reduced by anthropogenic stressors such as land-use change and widespread habitat fragmentation. However, we lack a comprehensive understanding of bumblebee population structure and local adaptation in response to human-altered landscapes. We here discover surprisingly fine-scaled population structure (e.g. ~300km) within two widely occurring bumblebee species, Bombus lapidarius and Bombus pascuorum, by analysing whole genome data of 106 specimens from 7 sites in Northern Europe. Our sample range encompasses a mosaic of land-use types with varying levels of habitat fragmentation and natural oceanic barriers. While the observed population structure is largely associated with reduced gene flow across natural barriers, we also detect significant divergence between populations sampled from more fragmented, agricultural landscapes. Furthermore, we identify species-specific patterns of population structure which are underpinned by distinct genomic architecture. Whereas genetic divergence in B. lapidarius is spread relatively evenly across the genome, divergence in B. pascuorum is concentrated within several megabase-sized genomic regions with significantly elevated differentiation, including a putative chromosomal inversion, which may underlie well-known colour polymorphisms across its range. Our observations reveal unexpectedly high levels of inter- and intraspecific genomic diversity within the bumblebee genus, and highlight the necessity of increasing our understanding of bumblebee population structure and connectivity to design optimal bumblebee conservation strategies.

Projected decline in European bumblebee populations in the twenty-first century

Article

Full-text available

Sep 2023
NATURE

Habitat degradation and climate change are globally acting as pivotal drivers of wildlife collapse, with mounting evidence that this erosion of biodiversity will accelerate in the following decades1–3. Here, we quantify the past, present and future ecological suitability of Europe for bumblebees, a threatened group of pollinators ranked among the highest contributors to crop production value in the northern hemisphere4–8. We demonstrate coherent declines of bumblebee populations since 1900 over most of Europe and identify future large-scale range contractions and species extirpations under all future climate and land use change scenarios. Around 38–76% of studied European bumblebee species currently classified as ‘Least Concern’ are projected to undergo losses of at least 30% of ecologically suitable territory by 2061–2080 compared to 2000–2014. All scenarios highlight that parts of Scandinavia will become potential refugia for European bumblebees; it is however uncertain whether these areas will remain clear of additional anthropogenic stressors not accounted for in present models. Our results underline the critical role of global change mitigation policies as effective levers to protect bumblebees from manmade transformation of the biosphere.

Whole genome demographic models indicate divergent effective population size histories shape contemporary genetic diversity gradients in a montane bumble bee

Article

Full-text available

Jan 2023

Understanding historical range shifts and population size variation provides an important context for interpreting contemporary genetic diversity. Methods to predict changes in species distributions and model changes in effective population size (Ne) using whole genomes make it feasible to examine how temporal dynamics influence diversity across populations. We investigate Ne variation and climate‐associated range shifts to examine the origins of a previously observed latitudinal heterozygosity gradient in the bumble bee Bombus vancouverensis Cresson (Hymenoptera: Apidae: Bombus Latreille) in western North America. We analyze whole genomes from a latitude‐elevation cline using sequentially Markovian coalescent models of Ne through time to test whether relatively low diversity in southern high‐elevation populations is a result of long‐term differences in Ne. We use Maxent models of the species range over the last 130,000 years to evaluate range shifts and stability. Ne fluctuates with climate across populations, but more genetically diverse northern populations have maintained greater Ne over the late Pleistocene and experienced larger expansions with climatically favorable time periods. Northern populations also experienced larger bottlenecks during the last glacial period, which matched the loss of range area near these sites; however, bottlenecks were not sufficient to erode diversity maintained during periods of large Ne. A genome sampled from an island population indicated a severe postglacial bottleneck, indicating that large recent postglacial declines are detectable if they have occurred. Genetic diversity was not related to niche stability or glacial‐period bottleneck size. Instead, spatial expansions and increased connectivity during favorable climates likely maintain diversity in the north while restriction to high elevations maintains relatively low diversity despite greater stability in southern regions. Results suggest genetic diversity gradients reflect long‐term differences in Ne dynamics and also emphasize the unique effects of isolation on insular habitats for bumble bees. Patterns are discussed in the context of conservation under climate change. We use whole genome demographic modeling to demonstrate that existing patterns of genetic diversity across latitudes in a montane bumble bee are shaped by historical differences in population size dynamics. High‐diversity populations have a history of larger effective population sizes through time, even with larger losses in habitat suitability during glacial periods resulting in larger Pleistocene bottlenecks. Results suggest that southern high‐elevation restricted populations have a reduced maximum population size through time than northern low‐elevation populations despite greater niche stability, which has implications for conserving genetic diversity in these more isolated regions under climate change.

Genomic Signatures of Recent Adaptation in a Wild Bumblebee

Article

Full-text available

Feb 2022
MOL BIOL EVOL

Environmental changes threaten insect pollinators, creating risks for agriculture and ecosystem stability. Despite their importance, we know little about how wild insects respond to environmental pressures. To understand the genomic bases of adaptation in an ecologically important pollinator, we analyzed genomes of Bombus terrestris bumblebees collected across Great Britain. We reveal extensive genetic diversity within this population, and strong signatures of recent adaptation throughout the genome affecting key processes including neurobiology and wing development. We also discover unusual features of the genome, including a region containing 53 genes that lacks genetic diversity in many bee species, and a horizontal gene transfer from a Wolbachia bacteria. Overall, the genetic diversity we observe and how it is distributed throughout the genome and the population should support the resilience of this important pollinator species to ongoing and future selective pressures. Applying our approach to more species should help understand how they can differ in their adaptive potential, and to develop conservation strategies for those most at risk.

A genomic and morphometric analysis of alpine bumblebees: Ongoing reductions in tongue length but no clear genetic component

Article

Full-text available

Nov 2021

Over the last six decades, populations of the bumblebees Bombus sylvicola and Bombus balteatus in Colorado have experienced decreases in tongue length, a trait important for plant‐pollinator mutualisms. It has been hypothesized that this observation reflects selection resulting from shifts in floral composition under climate change. Here we used morphometrics and population genomics to determine whether morphological change is ongoing, investigate the genetic basis of morphological variation, and analyze population structure in these populations. We generated a genome assembly of B. balteatus. We then analyzed whole‐genome sequencing data and morphometric measurements of 580 samples of both species from seven high‐altitude localities. Out of 281 samples originally identified as B. sylvicola, 67 formed a separate genetic cluster comprising a newly‐discovered cryptic species (“incognitus”). However, an absence of genetic structure within species suggests that gene flow is common between mountains. We found a significant decrease in tongue length between bees collected between 2012‐2014 and in 2017, indicating that morphological shifts are ongoing. We did not discover any genetic associations with tongue length, but a SNP related to production of a proteolytic digestive enzyme was implicated in body size variation. We identified evidence of covariance between kinship and both tongue length and body size, which is suggestive of a genetic component of these traits, although it is possible that shared environmental effects between colonies are responsible. Our results provide evidence for ongoing modification of a morphological trait important for pollination and indicate that this trait likely has a complex genetic and environmental basis.

Navigating the temporal continuum of effective population size

Article

Full-text available

Oct 2021
Methods Ecol. Evol.

Effective population size, Ne, is a key evolutionary parameter that determines the levels of genetic variation and efficacy of selection. Estimation and interpretation of Ne are essential in diverse areas of evolutionary and conservation biology, ranging from assessing the evolutionary potential or extinction risk to empowering research on the genomic basis of adaptation. The diverse applications of the Ne concept resulted in largely independent methodological developments using different Ne definitions and focusing on the estimation of either contemporary or long‐term Ne. Recently, several novel approaches appeared that allow the estimation of temporal Ne trends at various parts of the temporal Ne continuum. Here, we provide an overview of the temporal aspects of Ne, asking whether the existing methods and available data provide Ne estimates for the entire temporal continuum. We outline the apparent division into contemporary and long‐term Ne existing in the literature, review Ne estimation methods across the temporal continuum and navigate the reader through it pointing to the important biological and methodological limitations inherent to the estimation procedures. Finally, we provide examples of recent research and future perspectives. We conclude that the entire temporal Ne continuum can now be inferred using several complementary approaches, bringing together contemporary and long‐term perspectives. Further rapid progress in the field is expected from the wide adoption of machine learning and transition to haplotype‐resolved population genomic data. The expected progress notwithstanding, interpretational difficulties are likely to remain, emphasising the need for a thorough understanding of the Ne theory behind the methods, their assumptions and inherent limitations.

Large-scale genomic analysis reveals the genetic cost of chicken domestication

Article

Full-text available

Jun 2021
BMC BIOL

Background Species domestication is generally characterized by the exploitation of high-impact mutations through processes that involve complex shifting demographics of domesticated species. These include not only inbreeding and artificial selection that may lead to the emergence of evolutionary bottlenecks, but also post-divergence gene flow and introgression. Although domestication potentially affects the occurrence of both desired and undesired mutations, the way wild relatives of domesticated species evolve and how expensive the genetic cost underlying domestication is remain poorly understood. Here, we investigated the demographic history and genetic load of chicken domestication. Results We analyzed a dataset comprising over 800 whole genomes from both indigenous chickens and wild jungle fowls. We show that despite having a higher genetic diversity than their wild counterparts (average π, 0.00326 vs. 0.00316), the red jungle fowls, the present-day domestic chickens experienced a dramatic population size decline during their early domestication. Our analyses suggest that the concomitant bottleneck induced 2.95% more deleterious mutations across chicken genomes compared with red jungle fowls, supporting the “cost of domestication” hypothesis. Particularly, we find that 62.4% of deleterious SNPs in domestic chickens are maintained in heterozygous states and masked as recessive alleles, challenging the power of modern breeding programs to effectively eliminate these genetic loads. Finally, we suggest that positive selection decreases the incidence but increases the frequency of deleterious SNPs in domestic chicken genomes. Conclusion This study reveals a new landscape of demographic history and genomic changes associated with chicken domestication and provides insight into the evolutionary genomic profiles of domesticated animals managed under modern human selection.

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics

Article

Full-text available

Apr 2021
MOL BIOL EVOL

Evidence is accumulating that gene flow commonly occurs between recently-diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that co-localize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely-related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (dXY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.

Whole genome analyses reveal weak signatures of population structure and environmentally associated local adaptation in an important North American pollinator, the bumble bee Bombus vosnesenskii

Article

Sep 2023

Studies of species that experience environmental heterogeneity across their distributions have become an important tool for understanding mechanisms of adaptation and predicting responses to climate change. We examine population structure, demographic history and environmentally associated genomic variation in Bombus vosnesenskii , a common bumble bee in the western USA, using whole genome resequencing of populations distributed across a broad range of latitudes and elevations. We find that B. vosnesenskii exhibits minimal population structure and weak isolation by distance, confirming results from previous studies using other molecular marker types. Similarly, demographic analyses with Sequentially Markovian Coalescent models suggest that minimal population structure may have persisted since the last interglacial period, with genomes from different parts of the species range showing similar historical effective population size trajectories and relatively small fluctuations through time. Redundancy analysis revealed a small amount of genomic variation explained by bioclimatic variables. Environmental association analysis with latent factor mixed modelling (LFMM2) identified few outlier loci that were sparsely distributed throughout the genome and although a few putative signatures of selective sweeps were identified, none encompassed particularly large numbers of loci. Some outlier loci were in genes with known regulatory relationships, suggesting the possibility of weak selection, although compared with other species examined with similar approaches, evidence for extensive local adaptation signatures in the genome was relatively weak. Overall, results indicate B. vosnesenskii is an example of a generalist with a high degree of flexibility in its environmental requirements that may ultimately benefit the species under periods of climate change.

Population Genomics for Insect Conservation

Article

Nov 2022

Insects constitute vital components of ecosystems. There is alarming evidence for global declines in insect species diversity, abundance, and biomass caused by anthropogenic drivers such as habitat degradation or loss, agricultural practices, climate change, and environmental pollution. This raises important concerns about human food security and ecosystem functionality and calls for more research to assess insect population trends and identify threatened species and the causes of declines to inform conservation strategies. Analysis of genetic diversity is a powerful tool to address these goals, but so far animal conservation genetics research has focused strongly on endangered vertebrates, devoting less attention to invertebrates, such as insects, that constitute most biodiversity. Insects’ shorter generation times and larger population sizes likely necessitate different analytical methods and management strategies. The availability of high-quality reference genome assemblies enables population genomics to address several key issues. These include precise inference of past demographic fluctuations and recent declines, measurement of genetic load levels, delineation of evolutionarily significant units and cryptic species, and analysis of genetic adaptation to stressors. This enables identification of populations that are particularly vulnerable to future threats, considering their potential to adapt and evolve. We review the application of population genomics to insect conservation and the outlook for averting insect declines. Expected final online publication date for the Annual Review of Animal Biosciences, Volume 11 is February 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Conservation and the Genomics of Populations

Book

Feb 2022

Loss of biodiversity is among the greatest problems facing the world today. Conservation and the Genomics of Populations gives a comprehensive overview of the essential background, concepts, and tools needed to understand how genetic information can be used to conserve species threatened with extinction, and to manage species of ecological or commercial importance. New molecular techniques, statistical methods, and computer programs, genetic principles, and methods are becoming increasingly useful in the conservation of biological diversity. Using a balance of data and theory, coupled with basic and applied research examples, this book examines genetic and phenotypic variation in natural populations, the principles and mechanisms of evolutionary change, the interpretation of genetic data from natural populations, and how these can be applied to conservation. The book includes examples from plants, animals, and microbes in wild and captive populations. This third edition has been thoroughly revised to include advances in genomics and contains new chapters on population genomics, genetic monitoring, and conservation genetics in practice, as well as new sections on climate change, emerging diseases, metagenomics, and more. More than one-third of the references in this edition were published after the previous edition. Each of the 24 chapters and the Appendix end with a Guest Box written by an expert who provides an example of the principles presented in the chapter from their own work. This book is essential for advanced undergraduate and graduate students of conservation genetics, natural resource management, and conservation biology, as well as professional conservation biologists and policy-makers working for wildlife and habitat management agencies. Much of the book will also interest nonprofessionals who are curious about the role of genetics in conservation and management of wild and captive populations.

Genomic variation in montane bumblebees in Scandinavia: High levels of intraspecific diversity despite population vulnerability

Abstract

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