Suzanna Persson

Degree project for Master of Science (120 credits)

Biodiversity and Systematics

60 hec

Department of Biological and Environmental Sciences

University of Gothenburg

June 2015

Examiner: Bengt Oxelman

Department of Biological and Environmental Sciences

University of Gothenburg

Supervisor: Urban Olsson

Department of Biological and Environmental Sciences

University of Gothenburg

Phylogeny and taxonomy of the subfamily

Vespinae (Hymenoptera: Vespidae),

based on five molecular markers

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Table of Content

1 Introduction……………………………………………………………………2

1.1 General background………………………………………………………..2

1.1.1 Phylogenetic inference………………………………………………….3

1.1.2 Vespinae………………………………………………………………...4

1.2 Introduction to the thesis…………………………………………………...9

2 Material and methods………………………………………………………...10

2.1 Taxon sampling…………………………………………………………...10

2.2 DNA extraction, amplification and sequencing…………………………..10

2.3 Sequence alignment, data partitioning, and model selection……………..11

2.4 Phylogenetic inference……………………………………………………15

3 Results………………………………………………………………………..15

4 Discussion……………………………………………………………………27

5 Conclusions…………………………………………………………………..30

6 Acknowledgements…………………………………………………………..31

7 References……………………………………………………………………32

8 Appendix……………………………………………………………………..37

Abstract

Sixty-four species have been recognized in Vespinae and divided into four or five genera. Based

on Bayesian inference and multi-species coalescent we found that the branching pattern among

the genera are unresolved and needs further study, except the clade Vespula + Paravespula

where the support was high. We recovered five well supported clades corresponding to named

taxa. In the case Vespula rufa and Vespula intermedia respectively Dolichovespula norwegica

and Dolichovespula albida our data does not support that they have diverged. We found

taxonomically unrecognized divergence within Provespa barthelemyi, Provespa anomala,

Paravespula flaviceps and Vespa bicolor. This is also the case in Dolichovespula pacifica where

there is also indication of introgression. Paravespula vulgaris also shows indication of

introgression.

Key words: Wasps, Provespa, Vespa, Vespula, Paravespula, Dolichovespula, Evolution,

Bayesian inference, Multi-species coalescent.

1. Introduction

1.1 General background

“On the ordinary view of each species having been independently created, we gain no scientific

explanation.” – Charles Darwin.

When Carl von Linné in 1735 published his book Systema Naturae, he started the work of

classifying (categorized based on morphology) all the animal and plant species in the world. By

the year 1749 he realised that he was way in over his head with that project and that it was too

much for one person to handle alone (Winston, 1999), and he was right. Now, more than two

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hundred years later, and with about 1.4 million species described, millions of species remain

undescribed (Winston, 1999). In 1809, Jean Baptiste de Lamarck published his book

Philosophie Zoologique and promoted Aristoteles concept Scala Naturae or the “ladder of life.”

He argued that some organisms were higher up on this ladder, and the higher up, the more

complex and also more superior the organisms were. This view of thinking implied that

organisms had independent origin and that they had evolved at different times. It implies that

evolution has a goal to produce high evolved species. He believed that the higher organisms

had evolved earlier and therefore had had a longer time to evolve (Baum & Smith, 2013).

Charles Darwin, however, in his book On the Origin of Species, argued that all species derived

from a common ancestor. Instead of the ladder thinking, Darwin preferred tree thinking to

describe the evolution of species. Darwin argued that a common ancestor is an indication and

prerequisite that the evolution took place (Darwin, 1859).

1.1.1 Phylogenetic inference

In 1953, an event took place that would change the study of molecular evolution - the discovery

of the double helix, the structure of DNA (Watson & Crick, 1953). This was the key piece that

revealed how the DNA molecule could be the carrier of information from generation to

generation and that the information determined how organisms functioned and developed (Page

& Holmes, 1998). A DNA sequence consists of four bases, adenine (A), cytosine (C), guanine

(G) and thymine (T), where A and G are purines and C and T are pyrimidines. At every

nucleotide position, one of these four bases occur. The DNA molecule is a double helix and the

two strains are complements. Because the bases have different structures, A always binds to T,

and C always binds to G. In the evolutionary process, these bases are continuously substituted

by mutations (Baum & Smith, 2013). In time, the bases will either become fixed (fixed

mutations) or lost, depending on which base will be inherited by the next generation (Nielsen

& Slatkin, 2013). You may calculate the substitutions by mutations in terms of number of

substitutions at every nucleotide position in order to get an indication of how long ago two taxa

shared a common ancestor. This is called the evolutionary distance or mutation rate (Baum &

Smith, 2013). To calculate this we use different kinds of substitution models. The simplest of

the models is the Jukes-Cantor model (JC model) which assumes that the bases are equally

likely to occur and that the rate of the substitutions is equal (Jukes and Cantor, 1969). But in

reality, the bases do not usually occur at the same frequency, and some substitutions occur at a

higher rate than others. Purines and pyrimidines have different chemical structure so transitions

(substitutions from one purine to another purine or one pyrimidine to another pyrimidine)

usually occur at a different rate than transversions (substitutions from one purine to one

pyrimidine, or one pyrimidine to one purine). Transitions usually occur at a higher rate (Baum

& Smith, 2013).

There are several different methods for phylogenetic inference. One of them is maximum

parsimony. The parsimony criterion states that the tree that shows the least amount of character

changes is the one we choose. Another method is maximum likelihood. The maximum

likelihood criterion tries to find the tree that is the most probable that the evolution has made

for the observed data. Further, there is Bayesian inference. The Bayesian inference produces

trees with the help of prior knowledge and models, and based on the posterior probability (the

likelihood of the data and priors), finds the tree that is most probably true (Baum & Smith,

2013).

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1.1.2 Vespinae

Insects have existed for more than 400 million years, which makes them one of the earliest

terrestrial groups. Wasps belong to the order Hymenoptera (Fig 1) and the earliest

Hymenopteran that have been recognized, due to their distinctive wing venation, are from the

Triassic period about 230 million years ago (MYA). The suborder Apocrita evolved about 195

MYA and the infraorder Aculeata about 155 MYA (Grimaldi & Engel, 2005).

Fig 1. Phylogeny of the insect orders. Redrawn from Wheeler et al. (2001).

One of the characteristic morphological traits of the suborder Apocrita (wasps, bees and ants)

(Fig 2) is the “wasp waist,” which is the constriction between the metasoma and mesosoma (Fig

3). This allows for more manoeuvrability in order to control a long ovipositor (Grimaldi &

Engel, 2005). Yellowjackets, hornets and Provespa, however, belong to the infraorder Aculeata

where the ovipositor has developed into a sting, which injects a venom, for offensive and

protective usage. Thousands of other insects mimic wasps that are in their near existence, called

mimicry, which reflects the value and success of the sting (Grimaldi & Engel, 2005).

The family Vespidae (Fig 4) is the second most well studied among the vespoid aculeates, after

the ants (Formicidae). The Vespidae family consist of approximately 4,500 species (Grimaldi

& Engel, 2005). Vespidae are recognized by the kidney-shaped eyes, with a distinct inward

bend (ocular sinus), folded wings lengthwise while at rest and the forewings with an elongated

first sub marginal cell (Douwes et al., 2012). Vespidae is divided into three subfamilies:

Eumeninae (potter wasps), Polistinae (paper wasps) and Vespinae (social wasps). The

subfamily Vespinae is defined by the following characteristics: abruptly narrow waist called

petiole, the mid tibia with two spical spurs, the straight clypeus at the apical margin, and the

triangular and serrated mandibles (Fig 5, 6) (Douwes et al., 2012). Archer (1989) recognized

five genera in the subfamily Vespinae and 64 species (the number of species is in brackets):

Provespa (3), Vespula (10), Vespa (23), Dolichovespula (18) and Paravespula (10). Vespula

and Paravespula are sometimes merged as Vespula with species groups, the Vespula vulgaris

species group (Vespula sensu Archer, 1989; Archer, 2008) and the Vespula rufa species group

(Paravespula sensu Archer, 1989; Archer, 2008).

Vespinae belong to the social insects and there are different forms of social behaviour: e.g.,

subsocial, communal, semisocial and eusocial behaviour (Grimaldi & Engel, 2005). Subsocial

behaviour is the simplest form of social behaviour, simply meaning that the brood is cared for

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during a limited time (Grimaldi & Engel, 2005). Communal behaviour is when females share a

nest structure, but each female cares for its brood separately (Grimaldi & Engel, 2005; Gadau

et al., 2009). Semisocial behaviour is when the same generation together take care of the brood,

but there are no overlapping generations (Grimaldi & Engel, 2005). Eusociality is when

overlapping generations together takes care of the brood, and the brood is produced by one

female alone or a few related females, and there is a sterile caste of workers (Carpenter, 1991;

Grimaldi & Engel, 2005). Eusocial behaviour is thought to have evolved independently in

insects several times, and twice in vespid wasps (Hines, 2007). There are two traits required for

the evolution of advanced societies in Hymenoptera (semi- and eucocial behaviour): close

genetic relatedness and nest living. Living in a nest provides among other things, cooperative

protection and the ability to collectively care for the brood (Grimaldi & Engel, 2005). Eusocial

wasps are characterized by a division in labour where the queen is the only one reproducing,

and the workers (females) are sterile (however, they still have their ovaries) and help the queen

to build the nest and care for the brood. The queen maintains her reproductive dominance by

aggressive behaviour or by the help of pheromones, which leads to suppression of workers and

the maintenance of their continued sterility (van Zweden et al., 2013).

Fig 2. Phylogeny of Apocrita. Redrawn from Johnson et al. (2013).

Fig 3. Wasp anatomy. Redrawn from Fred Miranda (2011).

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One derived trait that all hymenopterans share is that they are haplodiploid, which means that

all females of the order are diploid (i.e., they have two sets of chromosomes) and all males are

haploid (i.e., they have one set of chromosomes) (Grimaldi & Engel, 2005). This means that

males pass on 100% of their genes to their daughters, while the females pass on 50% of their

genes. Due to this, female siblings have a high relatedness, 0.75 (they share 75% of their genes)

while in the typical diploid species it’s 0.50 between siblings (they share 50% of their genes)

(Hamilton, 1964; Grimaldi & Engel, 2005). This is believed to be the main reason why the

evolution of eusociality has evolved several times in Hymenoptera (Grimaldi & Engel, 2005).

In each generation, the females contribute more to the gene pool than the males, and as long as

the number of males does not decrease too much, females are more profitable to produce than

males (Hamilton, 1964). Egg laying by workers are known from all of the social groups, wasps,

bees and ants. The male-egg production does not require mating by the workers (hence, all eggs

that are laid by the workers are unfertilized and become males) (Hamilton, 1964).

Wasps create their nests by chewing on different plant fibers, which in combination with the

saliva becomes a paper pulp. The pulp is then chewed to a flat strain that dries quickly when it

is in place. The nests consist of combs with hexagonal cells and an outer layer that protects and

isolates (Douwes et al., 2012) and it can vary from having just a few cells, to having up to over

a hundred thousand cells (Kimsey & Carpenter, 2012). Vespa, Vespula and Paravespula place

the nests either underground or in other cavities above ground, while Dolichovespula nests are

more exposed (Archer, 2006; Archer, 2007; Archer, 2008a; Archer, 2008b; Douwes et al.,

2012).

Social nest parasitism (when a queen invades and takes over another nest) has evolved several

times in wasps, bees and ants (Carpenter & Perera, 2006). Among the yellowjackets (Vespula

and Dolichovespula) three species are inquilines (obligate social parasites), Vespula austriaca

(hosts: V. acadica and V. rufa), Dolichovespula adulterina (hosts: D. alpicola, D. saxonica and

D. arenaria) and Dolichovespula omissa (host: D. sylvestris), which means that they lack a

worker caste and they don’t build nests. Instead they invade another nest, kill the host queen

and use the workers to rear their own brood. (Carpenter & Perera, 2006; Buck et al., 2008). Some yellowjackets are facultative temporary social parasites and invade the nest of another

species and use the nest in order to rear for their own brood. They have their own caste and the

usurpation leads to a colony that is mixed with host workers and parasite workers. Facultative

social parasites can, however, build their own nests (e.g., Vespula squamosa and Vespa

dybowskii) (Reed & Akre, 1983; Carpenter & Perera, 2006; Buck et al., 2008).

Most colonies are annual (they have one year life cycles) and the castes in the nest differ in

feeding behaviour. In spring, the queen feeds on nectar and is an important pollinator for some

plant species (Douwes et al., 2012). In the early nest life, the queen takes care of the brood;

however, when the workers become adults, they take over the brood care. The workers bring

proteins (i.e., prey of other insects) to the larvae (Douwes et al., 2012) and in exchange, they

get droplets of secretion (Douwes et al., 2012; Ishay & Ikan, 1968), which is based on

carbohydrates and amino acids which the workers are unable to produce themselves (Douwes

et al., 2012). This exchange is called trophallaxis (Douwes et al., 2012; Ishay & Ikan, 1968).

In the genus Paravespula, species have in most places an annual life cycle that is monogynous

(i.e., with only one queen), but in places where the weather is mild, colonies that are perennial

polygynous (i.e., longer than one year and with more than one queen) can sometimes develop.

These colonies are characterized by having an enormous amount of workers (Gambino, 1986).

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Fig 4. Subfamilies in Vespidae. Redrawn from Johnson et al. (2013).

Fig 5. Wasp anatomy. Redrawn from Richard Bartz (2007).

Fig 6. Wasp face anatomy. Redrawn from Omid Golzar (2012).

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Species Distribution Habitat Nesting

Provespa barthelemyi Southeast Asia Nocturnal, probably a

forest species

Probably aerial nesting

Provespa anomala Southeast Asia Nocturnal, lowland

habitats

Usually aerial nesting

Provespa nocturna Southeast Asia Nocturnal, lowland

habitats

Usually aerial nesting

Vespa orientalis Southwest Asia,

Northeast Africa,

Madagascar, Southern

Europe

Various habitats Usually subterranean

nesting

Vespa basalis Asia Forest species Usually aerial nesting

Vespa crabro Europe, Asia, Canada,

USA

Forest species Usually aerial nesting

in cavities

Vespa simillima North-eastern China,

Korea, Japan, Russia

Various habitats Subterranean and

covered aerial nesting

Vespa bicolor North-eastern India,

Central and Southern

China, Burma,

Thailand, Vietnam

Various habitats Both aerial and

subterranean nesting

Vespa velutina Asia Forest species Aerial nesting

Vespa soror South East Asia Mountainous regions No nesting

information

Dolichovespula albida North America Various habitats Usually subterranean

nesting

Dolichovespula

arenaria

Canada, USA Various habitats Usually aerial nesting.

Dolichovespula

sylvestris

Europe, Asia, North

Africa

Various habitats Both aerial and

subterranean nesting

Dolichovespula

pacifica

Europe, Asia No habitat information Both aerial and

subterranean nesting

Dolichovespula media Europe, Asia Various habitats Aerial nesting

Dolichovespula

maculata

Canada, USA Forest and urban

habitats

Aerial nesting

Dolichovespula

saxonica

Europe, Asia Various habitats Usually aerial nesting

Dolichovespula omissa Europe, Turkey, Iran Various habitats Obligate parasite of D.

sylvestris

Dolichovespula

adulterina

Europe, Asia, Canada,

USA

Various habitats Obligate parasite of D.

arenaria, D. alpicola

and D. saxonica

Dolichovespula

norwegica

Europe, Asia, Canada,

USA

Various habitats Usually aerial nesting

Dolichovespula

alpicola

Canada, USA Mountainous regions Usually aerial nesting

Dolichovespula

norvegicoides

Canada, USA Closed coniferous

forest

Usually aerial nesting

Vespula intermedia Eastern Asia, Canada,

USA

Rural areas Usually subterranean

and cavity nesting

Vespula consobrina Canada, USA Mountain forests Usually subterranean

nesting

Vespula acadica Canada, USA Closed forest Nesting at ground

level in hollow

decaying logs

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Vespula squamosa Canada, USA, Mexico,

Guatemala

Forest species Facultative parasite of

P. maculifrons and P.

flavopilosa

Vespula vidua Canada, USA Forests species Usually subterranean

nesting

Vespula austriaca Europe, Northern

Asia, Canada, USA

Various habitats Obligate parasite of V.

acadica and V. rufa

Vespula rufa Europe, Northern and

Western Asia

Various habitats Both aerial and

subterranean nesting

Paravespula

flavopilosa

Canada, USA Disturbed areas near

forest mountain

regions

Usually underground

nesting

Paravespula

pensylvanica

Canada, USA, Mexico,

Hawaii

Various habitats Usually underground

nesting

Paravespula flaviceps Asian Palearctic,

Oriental regions

Lowland and mountain

regions

Usually subterranean

nesting

Paravespula

germanica

Cosmopolitan Urban and rural areas Both aerial and

subterranean nesting

Paravespula

maculifrons

Canada, USA, Mexico Various habitats Usually subterranean

nesting

Paravespula vulgaris Cosmopolitan Various habitats Both aerial and

subterranean nesting

Paravespula structor Asian Palaearctic and

Oriental regions

Indication of

mountainous regions

No nesting

information

Rugovespula orbata Asian Pa1aearctic and

Oriental regions

No habitat information No nesting

information

Table 1. Distribution, habitat and nesting characteristics of the 38 Vespinae species in our study

(Archer, 1989; Archer, 2006; Archer, 2007; Archer, 2008a; Archer, 2008b; Kimsey &

Carpenter, 2012).

1.2 Introduction to the thesis

The aim of this paper is to determine the phylogenetic relationship of the Vespinae subfamily.

Some earlier studies have been based on morphological characters e.g., Yamane (1976);

Matsuura & Yamane (1984); Carpenter (1987); Carpenter & Perera (2006). Other studies have

been based on molecular data e.g., Pantera et al. (2003); Collins & Cardner (2006); Hines et al.

(2007); Landolt et al. (2010); Pickett & Carpenter (2010); Saito & Kojima (2011) and most

recently Lopez-Osario et al. (2014) conducted a study where they used nine loci to investigate

the relationships among Vespula, Paravespula and Dolichovespula. Their analysis shows

strong support for monophyly within the clades of Vespula (which they divided as the V.

vulgaris species group and the V. rufa species group) and Dolichovespula. But their results

show low or no support for a Vespula and Dolichovespula clade. A previous hypothesis of the

relationships between the genera are shown in fig 7 and a previous hypothesis of the

relationships between the species groups are shown in fig 8.

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Fig 7. Hypothesis of the phylogeny of the genera (Pickett & Carpenter, 2010).

Fig 8. Hypothesis of the phylogeny of the species groups (Carpenter, 1987).

2. Materials and methods

2.1 Taxon sampling

We sequenced 68 specimens for five loci: 28S, COI, EF1a, Pol II and WG from Europe, North

America and Asia (Fig 9). In addition, COI was sequenced for a further 97 specimens. We also

downloaded all sequences available for Vespinae on Genbank (Table 3 for Genbank accession

number). In total we downloaded 346 sequences from Genbank (Benson et al., 2009); 45 for

28S, 219 for COI, 24 for EF1a, 25 for Pol II, and 23 for WG. Of these 346 sequences, 119 were

from the study of Lopez-Osorio et al. (2014) and included 22 specimens (22 species). Ten

sequences were also downloaded from two Polistes specimens as outgroup species (Table 3).

In this study, we follow the taxonomy of Archer (1989).

2.2 DNA extraction, amplification and sequencing

One leg of each voucher specimen was removed. Each leg was cut open with sterile knife

blades. The DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) with an

incubation period of about 3 h at 56°C in Buffer ATL and Proteinase K, and for the rest of the

procedure the manufacturer’s instructions were followed. Five loci were used in this study, both

nuclear and mitochondrial. The selected loci for the study are cytochrome oxidase I (COI),

wingless (WG), RNA polymerase II (Pol II), elongation factor 1 a (EF1a) and 28S ribosomal

DNA (28S) (Table 2). The loci were then amplified using the Polymerase Chain Reaction

(PCR) on an MJ Mini Thermal Cycler and an Eppendorf Mastercycler Thermal Cycler. Each

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PCR consisted of 10 µl red taq mastermix, 0,5 µl forward primer, 0,5 µl reverse primers, 1 µl

dH20 and 0,5 µl DNA. A typical PCR program started with 4 min of initial denaturation at

94°C, followed by 35-40 cycles of 30 s at 94°C, 45 s of annealing at 45-58°C, and 45 s of

elongation at 72°C, and ended with a 6 min period of final elongation at 72°C. The PCR product

was visualized on a 1% agarose/TAE electrophoresis gel.

2.3 Sequence alignment, data partitioning, and model selection

All loci were aligned independently using MAFFT v.7 (Katoh et al., 2002) with a gap opening

penalty default value of 1.53 and a gap extension penalty default value of 0.123. All the loci

were then manually adjusted and trimmed in Geneious v.6.1.8 created by Biomatters.

The appropriate substitution model for respective data sets was determined using the HIV

database (http://www.hiv.lanl.gov/).

Primer Sequence PCR temperature (°C)

COI 45

LCO1490 5’-GGT CAA CAA ATC ATA AAG ATA TTG G-

3’

HCO2198 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-

3’

WG 58

beewgFor 5’-TGC CAN GTS AAG ACC TGY TGG ATG AG-

3’

Lepwg2a 5’-ACT CGC ARC ACC ART GGA ATG TRC A-3’

Pol II 52

polfor2a 5’-AAY AAR CCV GTY ATG GGT ATT GTR CA-

3’

polrev2a 5’-AGR TAN GAR TTC TCR ACG AAT CCT CT-

3’

EF1a 57

F2-557F 5’-GAA CGT GAA CGT GGT ATY ACS AT-3’

F2-1118R 5’-TTA CCT GAA GGG GAA GAC GRA G-3’

28S 48-52

For28SVesp 5’-AGA GAG AGT TCA AGA GTA CGT G-3’

Rev28SVesp 5’-GGA ACC AGC TAC TAG ATG G-3’

Table 2. List of primer sequences used for PCR amplification and sequencing.

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Species Voucher Locality COI 28S EF1a Pol II WG

Vespa orientalis KMP59 Cairo, Egypt KJ147245 KF981707 KF981681 KF981654 KF955654

Vespa basalis KMP147 Ha Giam, Vietnam KJ147243 KF981705 KF981679 KF981652 KF955652

Vespa crabro KMP400 Georgia, USA KJ147244 KF981706 KF981680 KF981653 KF955653

Provespa barthelemyi KMP61 Trang Pr, Thailand KJ147241 KF981704 KF981677 KF981651 KF955650

Provespa anomala KMP62 Trang Pr, Thailand KJ147240 KF981703 KF981676 KF981650 KF955649

Dolichovespula albida KMP111 Alaska, USA KJ147229 KF981692 KF981666 KF981641 KF955639

Dolichovespula arenaria KMP126 Washington, USA KJ147230 KF981693 KF981667 KF981642 KF955640

Dolichovespula sylvestris KMP141 Talloires, France KJ147235 KF981698 KF981672 KF981647 KF955645

Dolichovespula pacifica KMP268 Mt. Fuji, Japan KJ147233 KF981696 KF981670 KF981645 KF955643

Dolichovespula media KMP321 Berkshire, UK KJ147232 KF981695 KF981669 KF981644 KF955642

Dolichovespula maculata KMP365 New York, USA KJ147231 KF981694 KF981668 KF981643 KF955641

Dolichovespula saxonica KMP428 Hungary KJ147234 KF981697 KF981671 KF981646 KF955644

Vespula intermedia KMP112 Alaska, USA KJ147252 KF981713 KF981687 KF981660 KF955660

Vespula consobrina KMP125 Washington, USA KJ147248 KF981709 KF981684 KF981657 KF955657

Vespula acadica KMP131 Washington, USA KJ147246 KF981708 KF981682 KF981655 KF955655

Vespula vidua KMP369 Vermont, USA KJ147256 KF981717 KF981691 KF981664 KF955664

Vespula squamosa VSPL3 Arkansas, USA KJ147255 KF981716 KF981690 KF981663 KF955663

Paravespula flavopilosa KMP113 - KJ147250 KF981711 KF981685 KF981658 KF955658

Paravespula alascensis KMP114 Alaska, USA KJ147247 - KF981683 KF981656 KF955656

Paravespula pensylvanica KMP128 Washington, USA KJ147254 KF981715 KF981689 KF981662 KF955662

Paravespula germanica KMP366 Vermont, USA KJ147251 KF981712 KF981686 KF981659 KF955659

Paravespula maculifrons VSPL2 New York, USA KJ147253 KF981714 KF981688 KF981661 KF955661

Polistes fuscatus KMP285 New York, USA KJ147238 KF981701 KF981674 KF981665 KF955665

Polistes metricus KMP286 Arkansas, USA KJ147239 KF981702 KF981675 KF981649 KF955648

Vespa crabro U412 Falkenberg, Sweden Own Own Own Own Own

Vespa crabro U1758 Falkenberg, Sweden Own Own Own Own Own

Vespa simillima U2213 Japan Own Own Own Own Own

Vespa bicolor U3481 Yunnan, China Own Own Own Own Own

Vespa bicolor U3674 Shanghai, China Own Own Own Own Own

Vespa basalis U3483 Yunnan, China Own Own Own Own Own

Vespa velutina U3484 Yunnan, China Own Own Own Failed Own

Vespa velutina U3670 Yunnan, China Own Own Own Own Own

Vespa soror U3485 Yunnan, China Own Own Own Own Own

Provespa anomala U3399 Borneo, Malaysia Own Own Own Own Own

Provespa anomala U3402 Borneo, Malaysia Own Own Own Own Own

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Provespa nocturna U3400 Borneo, Malaysia Own Own Own Own Own

Provespa nocturna U3401 Borneo, Malaysia Own Own Own Own Own

Provespa barthelemyi U3658 Yunnan, China Own Own Own Own Own

Provespa barthelemyi U3662 Yunnan, China Own Own Own Own Own

Provespa barthelemyi U3665 Yunnan, China Own Failed Own Own Own

Provespa barthelemyi U3666 Yunnan, China Own Own Own Own Own

Dolichovespula saxonica U1731 Falkenberg, Sweden Own Own Own Own Own

Dolichovespula saxonica U2932 Falkenberg, Sweden Own Own Own Own Own

Dolichovespula omissa U1734 Luleå, Sweden Own Own Own Own Own

Dolichovespula omissa U1924 Luleå, Sweden Own Own Own Own Own

Dolichovespula adulterina U1735 Luleå, Sweden Own Own Own Failed Own

Dolichovespula adulterina U2902 Manitoba, Canada Own Own Own Own Own

Dolichovespula sylvestris U1739 Kristineberg, Sweden Own Own Own Own Own

Dolichovespula sylvestris U1926 Luleå, Sweden Own Own Own Own Own

Dolichovespula pacifica U1742 Luleå, Sweden Own Own Own Own Own

Dolichovespula pacifica U1922 Luleå, Sweden Own Own Own Failed Own

Dolichovespula pacifica U3924 Mt. Fuji, Japan Own Own Own Own Own

Dolichovespula pacifica U3926 Mt. Fuji, Japan Own Own Own Own Own

Dolichovespula pacifica U3928 Mt. Fuji, Japan Own Own Own Own Own

Dolichovespula pacifica U3937 Mt. Fuji, Japan Own Own Own Own Own

Dolichovespula norwegica U1745 Luleå, Sweden Own Own Own Own Own

Dolichovespula norwegica U1919 Luleå, Sweden Own Own Own Own Own

Dolichovespula norwegica U1921 Luleå, Sweden Own Own Own Own Own

Dolichovespula media U1763 Falkenberg, Sweden Own Own Own Own Own

Dolichovespula media U1764 Falkenberg, Sweden Own Own Own Failed Own

Dolichovespula alpicola U2826 Canada Own Own Own Own Own

Dolichovespula alpicola U2833 Canada Own Own Own Own Own

Dolichovespula maculata U2832 Canada Own Own Own Own Own

Dolichovespula maculata U2900 Manitoba, Canada Own Own Own Own Own

Dolichovespula arenaria U2915 Manitoba, Canada Own Own Own Own Own

Dolichovespula arenaria U2917 Manitoba, Canada Own Own Own Own Own

Dolichovespula norvegicoides U2922 Manitoba, Canada Own Own Own Own Own

Dolichovespula norvegicoides U2930 Manitoba, Canada Own Own Own Own Own

Vespula austriaca U1736 Luleå, Sweden Own Own Own Own Own

Vespula austriaca U1929 Luleå, Sweden Own Own Own Own Own

Vespula austriaca U3933 Mt. Fuji, Japan Own Own Failed Own Own

14

Vespula rufa U2214 Hokkaido, Japan Own Own Own Own Own

Vespula rufa U3258 Göteborg, Sweden Own Own Own Own Own

Vespula acadica U2904 Manitoba, Canada Own Own Own Own Own

Vespula acadica U2910 Manitoba, Canada Own Own Own Own Own

Vespula consobrina U2906 Manitoba, Canada Own Own Own Own Own

Vespula consobrina U2907 Manitoba, Canada Own Own Own Own Own

Paravespula vulgaris U416 Falkenberg, Sweden Own Own Own Own Own

Paravespula vulgaris U1729 Falkenberg, Sweden Own Own Own Own Own

Paravespula vulgaris U3934 Mt. Fuji, Japan Own Own Own Own Own

Paravespula germanica U1759 Falkenberg, Sweden Own Own Own Own Own

Paravespula germanica U2931 Falkenberg, Sweden Own Own Own Own Own

Paravespula alascensis U2830 Canada Own Own Own Own Own

Paravespula alascensis U2842 Canada Own Own Own Own Own

Paravespula flavopilosa U2912 Manitoba, Canada Own Own Own Own Own

Paravespula flavopilosa U2914 Manitoba, Canada Own Own Own Own Own

Paravespula structor U3254 China Own Own Own Own Own

Paravespula flaviceps U3678 Yunnan, China Own Own Own Own Own

Paravespula flaviceps U3936 Mt. Fuji, Japan Own Own Own Own Own

Paravespula pensylvanica U3944 USA Own Own Own Own Own

Rugovespula orbata U3482 Yunnan, China Own Own Own Own Own

Rugovespula orbata U3676 Yunnan, China Own Own Own Own Own

Table 3. All specimens included in the species tree analyses, with voucher numbers and Genbank accession numbers.

Fig 9. Distribution of specimens. Red shows specimens from our collection and black shows specimens from Genbank.

15

2.4 Phylogenetic inference

Bayesian analyses were estimated in MrBayes 3.2, both as single locus analyses (SLAs) and a

concatenated data set of all loci. All SLAs were set to run for 50 M generations with sampling

every 100 000 generations and the concatenated analyses were run for 50 M generations with

sampling every 50 000 generations. We used the default settings except for the models that

were according to the model test, which was GTR + gamma model (nst = 6 rates = gamma) for

COI, EF1a, Pol II and 28S and HKY + gamma model (nst = 2 rates = gamma) for WG.

A species tree based on the multi-species coalescent was generated in *BEAST (BEAST

v.1.8.0.; Heled & Drummond, 2010). The analyses contained all individuals that had all five

loci successfully sequenced. An xml file was created using BEAUTi v.1.8.0 (Drummond et al.,

2012) where substitution models, tree models and clock models were unlinked across all the

loci. Uncorrelated lognormal relaxed clock was fixed to 1 for COI and was estimated for the

other loci. The substitution model was GTR + gamma for COI, 28S, EF1a and Pol II, and HKY

+ gamma for WG, and the base frequencies for all loci was estimated. Species tree prior was

set to Yule Process, the Population Size Model was set to Piecewise linear & constant root. The

ploidy type was set to autosomal nuclear for 28S, EF1a, Pol II and WG and mitochondrial for

COI and a random starting tree was used. The species population mean and the species yule

birth rate was set to lognormal distribution with log(mean) = 0 and log(Stdev) = 1. For the

relaxed clock the uniform prior was used with upper = 1.0, lower = 0.0 and initial value = 0.5.

All other priors were set to default settings. The analysis was run in BEAST v.1.8.0 with 500

M generations and a sampling every 100 000 generations.

A species delimitation analyser was estimated in DISSECT (Jones et al., 2015). An xml file

was created using BEAUTi v.1.8.2 (Drummond et al., 2012) with the same settings as for the

*BEAST analysis except for the species tree prior that was set to Birth-Death process. The xml

file was then altered according to Jones (2014). The analysis was run in BEAST v.1.8.2

(Drummond et al., 2012) with 100M generations and a sampling every 10 000 generations. The

output file from the analyses was then run in Species Delimitation Analyser (Jones et al., 2015).

We used the R code in Jones (2014) in order to create a similarity matrix in the program R (R

Core Team, 2013).

3. Results

Out of 340 possible sequences, 334 were successfully sequenced and 6 failed (Table 3).

In the Bayesian species tree analysis (Fig 10), all currently recognized genera form

monophyletic clades with high posterior probability (PP). The clade Vespula + Paravespula

has high support (PP = 1.00). However, the clade Provespa + Vespa has low support (PP =

0.68) as have Dolichovespula + Provespa and Vespa (PP = 0.53). Vespula squamosa is

recovered as sister to Vespula, with high support (PP = 1.00). Rugovespula orbata is recovered

as sister to Paravespula, with high support (PP = 0.98). Dolichovespula maculata and

Dolichovespula media are recovered as sisters to Dolichovespula, with high support (PP =

1.00).

In the SLAs of the loci 28S, COI and Pol II, the clade Paravespula + Vespula receives high

support (PP = 1.00). The clade Vespa + Provespa is only supported in the EF1a SLA (PP =

0.99). In the COI SLA, Vespa is recovered as sister group to all other clades, with high support

16

(PP = 1.00). In the 28S and Pol II SLAs, Provespa is recovered as sister group to all other clades

(PP = 1.00). In the COI SLA, Provespa is recovered as sister group with the clade D. maculata

+ D. media (PP = 1.00), Dolichovespula is recovered as sister group with the clades Vespula +

Paravespula and Provespa + D. maculata and D. media (PP = 0.99). In the WG SLA,

Dolichovespula is recovered as sister group to all other clades (PP = 1.00). In the EF1a and Pol

II SLAs, D. maculata and D. media are recovered as sisters to Dolichovespula (PP = 1.00). This

is also the case in the WG SLA, however, the support is not that high (PP = 0.94). In the EF1a,

Pol II and WG SLAs, Vespula squamosa is recovered as sister to Vespula (PP = 1.00, 0.99,

1.00). This is also the case in the COI SLA; however, the support is not that high (PP = 0.94).

In the Pol II SLA, R. orbata is recovered as sister to Paravespula, with high support (PP =

0.95). This is also the case in the 28S SLA; however, the support is not that high (PP = 0.93).

In the COI SLA, R. orbata is, however, recovered as sister to Vespula with support which is

not that high (PP = 0.94).

In the multi species coalescent tree (Fig 11), the clades Vespula + Paravespula and Vespa +

Provespa have high support (PP = 0.96, 0.99). D. maculata and D. media are recovered as

sisters to Dolichovespula (PP = 1.00). There is also divergence between the two P. vulgaris

specimens from Sweden and Japan, the two P. flaviceps specimens from China and Japan, and

the P. barthelemyi specimens from China and Thailand. The data shows no evidence of

divergence between D. albida and D. norwegica and between V. intermedia and V. rufa.

17

Fig 10. Bayesian phylogeny, inferred using MrBayes, of the species tree analysis. Values at

nodes are posterior probability (PP).

18

Fig 11. Multi-species coalescent tree, inferred using *BEAST. Values at nodes are posterior

probability (PP).

19

There is no divergence between Vespula rufa and Vespula intermedia in our species tree

analysis (Fig 12) or in the COI SLA (Fig 13).

Fig 12. Part of Bayesian species tree showing V. rufa and V. intermedia. Values at nodes are

posterior probability (PP).

Fig 13. Part of COI single locus analysis (SLA) tree showing V. rufa and V. intermedia. Values

at nodes are posterior probabilities (PP).

There is a divergence between the two Vespa bicolor specimens in our species tree analysis

(Fig 14) and in the COI SLA (Fig 15). We have two morphs that have both been determined to

be V. bicolor using Archer (1989). We have one morph (V. cf bicolor U3481) that matches the

morphology description for a V. bicolor perfectly, and one morph (V. cf bicolor U3674) that

diverges from the description but still matches the description for us to determine it to be a V.

bicolor. V. cf bicolor U3481 found no match in BOLD systems (Ratnasingham & Hebert, 2007)

and only 94% identity to V. bicolor in BLAST (Benson et al., 2009). V. cf bicolor U3674 found

99% identity to V. bicolor in both BOLD systems and BLAST. The morphology for the V. cf

bicolor U3674 specimen is that the size is the same but they have two yellow stripes at the

upper mesoscutum, and the scutellum is parted by a large black stripe. Second and third gastral

tergum has large black stripes.

20

Fig 14. Part of Bayesian species tree showing V. bicolor. V. bicolor is highlighted with blue.

Values at nodes are posterior probability (PP).

Fig 15. Part of COI single locus analysis (SLA) tree showing V. bicolor. Values at nodes are

posterior probabilities (PP). V. cf bicolor U3481 from China is highlighted with blue.

There is a divergence between the Paravespula flaviceps specimens from China and Japan in

both our species tree analysis (Fig 16) and in the COI SLA (Fig 17). All the specimens have

been determined to be P. flaviceps using Archer (1989). The specimen from Japan, U3936,

shares all characters described in Archer (1989) for a P. flaviceps. The specimen from China,

U3678, however, diverges from the description but has still been determined as a P. flaviceps.

The size is the same; however, the stripes are pale yellow rather than ivory white. Scutellum

has two large yellow spots; otherwise, it has the same markings as P. flaviceps. Other specimens

from China share the same characters as U3678. The divergence in the morphology is supported

by the phylogeny.

Fig 16. Part of Bayesian species tree showing P. flaviceps. Values at nodes are posterior

probability (PP). P. flaviceps highlighted with blue.

Fig 17. Part of COI single locus analysis (SLA) tree showing P. flaviceps. Values at nodes are

posterior probabilities (PP). P. flaviceps from China highlighted with blue.

21

Paravespula vulgaris and Paravespula alascensis diverge from each other in both our species

tree analysis (Fig 18) and in the COI SLA (Fig 19).

There is a divergence between the Paravespula vulgaris specimens from Sweden and Japan.

They separate in two clades, one with only specimens from Japan (U3934, U3935) and one

with specimens from Sweden (U416, U1729) and from Japan (U3940, U3841). There is,

however, no morphological difference between these specimens. This is supported in both the

species tree analysis (Fig 18) and in the COI SLA (Fig 19).

Fig 18. Part of Bayesian species tree showing P. vulgaris. Values at nodes are posterior

probability (PP). The P. vulgaris from Japan that diverge is highlighted with blue.

Fig 19. Part of COI single locus analysis (SLA) tree showing P. vulgaris. Values at nodes are

posterior probabilities (PP). The specimens P. vulgaris (highlighted with yellow) in the P.

alascensis clade has been named P. vulgaris in Genbank since P. alascensis has been

considered as a synonym for P. vulgaris. P. vulgaris from Japan that diverge is highlighted with

blue.

There is a divergence between the Provespa barthelemyi specimens from different localities in

the species tree analysis (Fig 20). The specimens from China, U3658, U3662, U3665 and

U3666, diverge from the specimen KMP61 from Thailand. The same divergence is also shown

in the COI SLA (Fig 21). The morphology for the specimens from China match the description

in (Archer, 1989), although there are some colour variations.

There is a divergence between the Provespa anomala specimens from different localities in our

species tree analysis (Fig 20). The P. anomala from Borneo are sisters to both P. anomala from

Thailand and P. nocturna from Borneo. The morphology for the specimens from Borneo match

the description in Archer (1989) for P. anomala.

22

Fig 20. Part of Bayesian species tree showing the Provespa clade. Values at nodes are posterior

probability (PP). P. anomala from Borneo is highlighted with yellow and P. barthelemyi from

China is highlighted with blue.

Fig 21. Part of COI single locus analysis (SLA) tree showing the Provespa clade. Values at

nodes are posterior probabilities (PP). P. anomala from Borneo is highlighted with yellow and

P. barthelemyi from China are highlighted with blue.

Dolichovespula pacifica and Dolichovespula norvegicoides diverge from each other in both our

species tree analysis (Fig 22) and in the COI SLA (Fig 23).

The Dolichovespula pacifica specimens in our study separate in three clades, one with

specimens from Sweden and two with specimens from Japan. The morphology for the

specimens from Japan are the same and we have not been able to divide these using

morphological characters. They do, however, have a different morphology than the specimens

from Sweden. The specimens from Japan are all black with pale yellow stripes on the abdomen.

The specimens from Sweden are black with broader and darker yellow stripes on the abdomen.

The Swedish specimens, U1742 and U1922 are sisters to U3926 and U3937 from Japan. The

third clade with the Japanese specimens U3924, U3928 and KMP268 are sisters to

Dolichovespula saxonica. This divergence is supported in both the species tree analysis (Fig

22) and in the COI SLA (Fig 23).

23

Fig 22. Part of Bayesian species tree showing D. pacifica. Values at nodes are posterior

probability (PP). The three separate D. pacifica groups are highlighted with blue, red and

yellow.

Fig 23. Part of COI single locus analysis (SLA) tree showing D. pacifica. Values at nodes are

posterior probabilities (PP). The three separate D. pacifica groups are highlighted with red,

yellow and blue.

24

There is no divergence between Dolichovespula albida and Dolichovespula norwegica in our

species tree analysis (Fig 24) or in the COI SLA (Fig 25).

Fig 24. Part of Bayesian species tree showing D. albida and D. norwegica. Values at nodes are

posterior probability (PP). D. albida and D. norwegica highlighted with blue.

25

Fig 25. Part of COI single locus analysis (SLA) tree showing D. albida and D. norwegica.

Values at nodes are posterior probabilities (PP). Numbers at the nodes are posterior probability

values. D. norwegica highlighted with blue.

26

Fig 26. Similarity matrix inferred by DISSECT and Species Delimitation Analyser.

27

The similarity matrix from the DISSECT analysis (Fig 26) supports our other data. It supports

that Vespula rufa and Vespula intermedia do not diverge, and that Dolichovespula norwegica

and Dolichovespula albida do not diverge. It also supports that there is a divergence between

the Paravespula flaviceps specimens. The dark grey squares indicate that they are similar, but

not identical. The same goes for the Paravespula vulgaris specimens. It also supports that there

is some gene flow between P. vulgaris and Paravespula alascensis, and also between Vespula

austriaca and Vespula vidua. It supports that the molecular divergences between the

Dolichovespula pacifica specimens are substantial. The D. pacifica from Sweden (in the matrix

labelled as Dolichovespula pacifica Sweden) and the specimens U3926 and U3937 from Japan

(in the matrix labelled as Dolichovespula pacifica Japan B) have some gene flow with each

other and with Dolichovespula omissa, but no gene flow with the other D. pacifica group from

Japan (in the matrix labelled as Dolichovespula pacifica Japan A). Similar difference is also

supported for the two Vespa cf bicolor morphs. It also supports that there is a divergence

between the two Provespa barthelemyi specimens and the two Provespa anomala specimens,

respectively.

4. Discussion

Our Bayesian species analysis support the taxonomic view of Archer (1989), who divides the

group into five genera, Vespa, Provespa, Dolichovespula, Vespula and Paravespula. All clades

in this study are monophyletic (Fig 10). While our species trees support this view, the SLAs

have some variations, particularly in how the genera form groups. All the SLAs have different

topologies and all except COI and WG recover all genera as monophyletic. In the COI SLA,

Dolichovespula is not monophyletic since Dolichovespula maculata and Dolichovespula media

form a sister group with Provespa (PP = 1.00). In the WG SLA, Paravespula is not

monophyletic since the specimen U3678 Paravespula flaviceps is in the Vespula group and not

in the Paravespula group (PP = 1.00).

Our Bayesian inference data supports that between five and eight genera may be justified. It

has been suggested that Rugovespula could be treated as a genus in its own right rather than

being a subgenus to Paravespula; our data is compatible with either of these treatments. In the

species tree analysis and in the Pol II SLA, there is a strong support for a divergence between

Rugovespula and Paravespula (PP = 0.98 and 0.95). V. squamosa is sister to Vespula in most

of the SLAs by a deep divergence. Such deep divergence could also be an argument for having

V. squamosa in its own genus, instead of being in the genus Vespula. The support for the

divergence between V. squamosa and Vespula are for the species tree analysis PP = 1.00. For

the SLAs, the support is for COI the PP = 0.94, for EF1a the PP = 0.99, for Pol II the PP = 1.00

and for WG the PP = 1.00. Also, a D. maculata and D. media clade is sister to Dolichovespula

in most of the SLAs, and the support for that is a PP = 1.00. That could also be an argument for

placing those in an independent genus rather than in the genus Dolichovespula. Whether we

consider all of these to be their own genera or part of the existing genera is a matter of opinion

and the phylogenies are consistent with both.

The loci 28S, EF1a, Pol II and WG are all nuclear conservative loci which evolve slowly and

cannot therefore be used singularly on species level. They are not variable enough for species

limit determination in recently evolved species.

Carpenter et al. (2012) concluded that Dolichovespula albida and Dolichovespula norwegica

are two different species based on morphological characteristics of the paramere (side parts of

28

the male external reproductive organs). They stated that the different shapes of the parameres

had been overlooked and that it is a difference that is species-specific. Our data, however, does

not support this theory. There is no phylogenetic divergence between these specimens that

supports them being two different species (Fig 24, 25). This is also supported in the multi-

species coalescent tree (Fig 11) and in the species delimitation analyser (Fig 26). We suggest

that D. albida (Sladen, 1918) should continue being treated as a synonym of D. norwegica

(Fabricius, 1781). The reason that they are morphologically different but genetically

inseparable might be that they are in a very early stage of speciation or a recent mitochondrial

introgression. How species are being separated is dependent upon the criteria being used for

species delimitation and how important certain characters are. Is it enough with morphological

differences when there is no molecular difference?

Kimsey & Carpenter (2012) suggested that Vespula intermedia (du Buysson, 1905) should be

a valid species and not a synonym of Vespula rufa (Linnaeus, 1758) based on colour variation.

Our Bayesian inference data does not support this theory (Fig 12, 13). The divergence is small

or non-existent, depending on locus, which means that V. rufa and V. intermedia appear to have

been separated only a short time period, if at all. The species delimitation is only based on

colour variation and we therefore rather suggest that V. intermedia should continue to be

considered a synonym of V. rufa. This is also supported in the multi-species coalescent tree (Fig

11) and the species delimitation analyser (Fig 26).

The results for our Vespa bicolor (Fabricius, 1787) specimens are quite confusing and

problematic (Fig 14, 15). We have several individuals that match each other morphologically

and one individual that diverges. The specimen that diverges (U3481) matches the description

in Archer (1989) for a V. bicolor. However, this specimen does not match other V. bicolor on

either BOLD systems or BLAST. The other individuals, however, (U3480, U3656, U3657,

U3673, U3674, U3675) that are morphologically similar do not match the description in Archer

(1989) for a V. bicolor, or anything else, but the closest match is for a V. bicolor. But they found

99% identity match to other V. bicolor on both BOLD systems and BLAST. This divergence is

also supported in the species delimitation analyser (Fig 26). All specimens are from China, but

the localities are unknown and we do not know if there are natural barriers that divide these

populations. That fact that there is a divergence between these specimens is obvious, but which

specimen represents the actual V. bicolor is still unclear.

Two specimens that were determined to be Paravespula flaviceps (Smith, 1870), one from

China and one from Japan, based on morphological characters, appear to represent two different

evolutionary lineages (Fig 16, 17). This is supported in the Bayesian inference species tree, the

COI SLA, the multi-species coalescent tree (Fig 11) and in the species delimitation analyser

(Fig 26). The specimen U3936 from Japan matches the morphology described in Archer (1989)

for a P. flaviceps. The sample found no match in BOLD systems but 99% identity to P. flaviceps

in BLAST, among them the specimen from the Lopez-Osorio et al. (2014) study (also from

Japan). The specimen U3678 from China has a slightly different morphology, but was still

determined to be P. flaviceps. The sample found no match in BOLD systems and only 95%

identity to P. flaviceps in BLAST. This match is to the P. flaviceps specimen in the Lopez-

Osorio et al. (2014) study. Dong et al. (2002) described a new species from Yunnan, China that

they named Vespula yulongensis, the characters of which were very similar to P. flaviceps.

Carpenter et al. (2011) suggested that V. yulongensis is a synonym of P. flaviceps and not a

valid species. However, our data supports that there is a divergence between the specimen from

Japan and the specimen from China, and since there is a described species from that area, it

29

might be the same as our specimen. However, we need to compare their specimen with our

specimen before we can make any suggestions about that.

Carpenter & Glare (2010) conducted a morphological and molecular study in order to

investigate if the North American species Vespula alascensis (Packard, 1870) was a synonym

of the European Paravespula vulgaris (Linnaeus, 1758) or a species sui generis. Specimens

from North America and Europe were used, and they concluded, based on the male genetalia

and molecular divergence, that they were in fact two separate species. Our data corroborates

this theory (Fig 18, 19, 26).

Four Japanese specimens that were determined to be Paravespula vulgaris based on

morphological characters appear to represent two different evolutionary lineages. This is

supported in the Bayesian inference species tree (Fig 18), the COI SLA (Fig 19), the multi-

species coalescent tree (Fig 11) and in the species delimitation analyser (Fig 26). Two of them

(U3940 and U3941) are part of the same unresolved clade that contains our samples of P.

vulgaris from Europe (Sweden and UK) and New Zealand, whereas the other two (U3934 and

U3935) form a sister group to these. The samples U3934 and U3935 (U3935 was only

sequenced for COI) found no match in BOLD systems, and only 97% identity to P. vulgaris in

BLAST. The reason for the existence of two separate mitochondrial lineages in Japan may be

introgression from Europe. It could be an unintentional introduction from Europe or New

Zealand, or it could even be a natural immigration from Europe via Siberia.

Four Chinese specimens that were determined to be Provespa barthelemyi based on

morphological characters appear to represent their own evolutionary lineage diverged from a

P. barthelemyi specimen from Thailand. This is supported in the Bayesian inference species

tree (Fig 20), the COI SLA (Fig 21), the multi-species coalescent (Fig 11) and in the species

delimitation analyser (Fig 26). The samples from China found no match in BOLD systems and

only 93% identity to P. barthelemyi in BLAST. This divergence is an indication that there are

two different species. P. barthelemyi was described by du Buysson (1905) with a type specimen

from India, so we cannot say with certainty if any of the specimens from Thailand and China

belong to the same population as the type.

All of our Provespa anomala specimens from Borneo conform to the description in Archer

(1989). However, they diverge from the P. anomala specimen from Thailand in our Bayesian

inference species tree (Fig 20), the COI SLA (Fig 21), the multi-species coalescent tree (Fig

11) and in the species delimitation analyser (Fig 26). The samples U3399 and U3402 found

99% identity to another P. anomala from Borneo but only 92% identity to P. anomala from

Thailand, which is the P. anomala specimen from the Lopez-Osorio et al. (2014) study. The

result in BLAST is about the same. The divergence is an indication that there might be two

different species. P. anomala was described by de Saussure (1854) with a type specimen from

Indonesia (Java), so we cannot say for sure if the Thai and Bornean populations belong to the

same lineage as the type.

Pekkarinen (1995) concluded that Dolichovespula pacifica was a synonym of Dolichovespula

norvegicoides based on morphological characters. Archer (1989) however, regarded them as

two separate species based on morphological characters. When Sladen (1918) described D.

norvegicoides, he used a type specimen collected in Canada. When Birula (1930) later

described D. pacifica, he used a type specimen collected in Eastern Siberia. Our data supports

the theory that they should be regarded as two different species and that D. pacifica (Birula,

1930) is a valid name (Fig 22, 23).

30

All of our Dolichovespula pacifica specimens were determined to be D. pacifica based on

morphological characters; however, they appear to represent three different evolutionary

lineages. Two of these lineages consist of all our Swedish specimens that form a sister clade

with a subset of our Japanese specimens. The third lineage, consisting of a subset of our

Japanese samples, forms a sister group to our samples of D. saxonica from Sweden. This is

supported in the Bayesian inference species tree (Fig 22), the COI SLA (Fig 23), the multi-

species coalescent tree (Fig 11) and in the species delimitation analyser (Fig 26). The specimens

U1742 and U1922 from Sweden found 100% identity to Dolichovespula norvegicoides (early

release, with no information for country of collection) in BOLD systems. The closest match

after that was 96% identity to D. albida and D. norwegica. The closest match in BLAST was

96% identity to D. albida. The specimens U3926 and U3937 from Japan form a sister group

with the Swedish specimens. The specimens found 97.8% identity to D. norvegicoides in BOLD

systems. The closest match after that was 96.9% identity to D. norwegica. The specimens found

97% identity to D. albida as the closest match in BLAST. The specimens U3924 and U3928

from Japan are sister to D. saxonica. This group also contains the Japanese specimen KMP268

from the Lopez-Osorio et al. (2014). The specimens found 100% identity to KMP268, 98%

identity to D. saxonica in BOLD systems. The results are the same in BLAST. The result in

BLAST and BOLD systems are interesting since the specimens get closer identity match to D.

saxonica and D. albida than to other specimens of D. pacifica. One possible explanation of this

could be that Dolichovespula is a young and unresolved clade with species that are very closely

related, and that the divergence of the species happened recently and during a short period of

time. From Japan we only have males from a single location, so it is unknown to what extent

this represents the Japanese population as a whole. The unexpected sister relationship between

some D. pacifica and D. saxonica may be due to haplotypes retained from past hybridization

or ongoing introgression.

5. Conclusion

Our conclusions are that all currently recognized genera form monophyletic clades. Vespula

and Paravespula are sisters and are most closely related. The relationships between the other

genera are inconclusive. We have low support for internal branching patterns for all other

clades. More research is therefore needed. Vespula squamosa is sister to Vespula and might be

considered its own genus. The subgenus Rugovespula is sister to Paravespula and could also

be considered an independent genus on its own. The same goes for Dolichovespula maculata

and Dolichovespula media. They are sisters to Dolichovespula, but could also be considered a

genus of their own. More research on this is needed. Our data supports a treatment of Vespula

rufa and Vespula intermedia as the same species, and V. intermedia as a junior synonym of V.

rufa (Table 4). Our data also supports that Dolichovespula norwegica and Dolichovespula

albida may be regarded as the same species, with D. albida as a junior synonym of D.

norwegica (Table 4). Our data supports that Dolichovespula norvegicoides and Dolichovespula

pacifica should be regarded as two different species (Table 4). Our data also supports that

Paravespula vulgaris and Paravespula alascensis may be regarded as two different species

(Table 4). Our Vespa bicolor specimens appear to represent two separate evolutionary lineages,

but we do not have access to material for morphological comparison to make any conclusions.

Further research is therefore needed. Our Paravespula flaviceps specimens from China diverge

from the specimens from Japan. These may be Vespula yulongensis described by Dong et al.

(2002) but available information on these is lacking. The divergence of the Paravespula

vulgaris specimen from Japan from other P. vulgaris specimens needs further research. Our

data indicates that the genus Provespa may contain five species rather than three. We suggest

31

that Provespa barthelemyi and Provespa anomala may each be divided into two species.

Further research is also needed for Dolichovespula pacifica since our specimens cluster in three

separate groups.

Species according

Archer (1989)

Previously changes in

taxonomy

Suggested taxonomic changes

in this study

Vespula rufa as a species

and not Vespula

intermedia.

Kimsey & Carpenter (2012)

recognized two species, one

European Vespula rufa and

one North American,

Vespula intermedia.

We propose to recognize one

species, Vespula rufa with

Vespula intermedia as a junior

synonym.

Dolichovespula

norwegica as a species

and not Dolichovespula

intermedia.

Carpenter et al. (2012)

recognized two species, one

European Dolichovespula

norwegica and one North

American, Dolichovespula

albida.

We propose to recognize one

species, Dolichovespula

norwegica with Dolichovespula

albida as a junior synonym.

Two species,

Dolichovespula pacifica

and Dolichovespula

norvegicoides.

Pekkarinen (1995) did not

recognize Dolichovespula

pacifica as an own species,

only as a synonym to D.

norvegicoides.

We propose to recognize two

species, Dolichovespula pacifica

and Dolichovespula

norvegicoides.

Paravespula vulgaris

and not Paravespula

alascensis.

Carpenter & Glare (2010)

recognized two species, one

European Vespula vulgaris

and one North American

Vespula alascensis.

We propose to recognize two

species, Paravespula vulgaris

and Paravespula alascensis.

Paravespula orbata Vespula orbata in Carpenter

& Kojima (1997)

Rugovespula orbata

Table 4. Recommended taxonomic changes compared to Archer (1989), Pekkarinen (1995),

Carpenter & Kojima (1997), Carpenter & Glare (2010), Carpenter et al. (2012) and Kimsey &

Carpenter (2012), based on the results of this study.

6. Acknowledgements

I especially want to thank my supervisor Urban Olsson for all the help and guidance. Svante

Martinsson for all the valuable help throughout the study. Bengt Oxelman for all the valuable

help with DISSECT. Mats Töpel for all the valuable help with Albiorix. Britt Anderson for

kindly improving my English. Mårten Eriksson, Tobias Hoffman, Allison Perrigo for valuable

inputs during the study. Tina Persson for valuable comments on the introduction.

32

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8. Appendix

Fig 27. Bayesian phylogeny, inferred using MrBayes, of locus 28S with sequences from the

species tree analysis. Numbers at the nodes are posterior probability values.

38

Fig 28. Bayesian phylogeny, inferred using MrBayes, of locus 28S with complete set of

sequences. Numbers at the nodes are posterior probability values.

39

Fig 29. Bayesian phylogeny, inferred using MrBayes, of locus COI with sequences from the

species tree analysis. Numbers at the nodes are posterior probability values.

40

Fig 30. Bayesian phylogeny, inferred using MrBayes, of locus COI with complete set of

sequences. Numbers at the nodes are posterior probability values.

41

Fig 31. Bayesian phylogeny, inferred using MrBayes, of locus EF1a with sequences from the

species tree analysis. Numbers at the nodes are posterior probability values.

42

Fig 32. Bayesian phylogeny, inferred using MrBayes, of locus Pol II with sequences from the

species tree analysis. Numbers at the nodes are posterior probability values.

43

Fig 33. Bayesian phylogeny, inferred using MrBayes, of locus WG with sequences from the

species tree analysis. Numbers at the nodes are posterior probability values.

44

Fig 34. Bayesian phylogeny, inferred using MrBayes, of locus WG with complete set of

sequences. Numbers at the nodes are posterior probability values.