Phylogeny and taxonomy of the subfamily Vespinae (Hymenoptera: Vespidae), based on five molecular markers

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1 Phylogeny and taxonomy of the subfamily Vespinae (Hymenoptera: Vespidae), based on five molecular markers 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

2 Table of Content 1 Introduction General background Phylogenetic inference Vespinae Introduction to the thesis Material and methods Taxon sampling DNA extraction, amplification and sequencing Sequence alignment, data partitioning, and model selection Phylogenetic inference 15 3 Results Discussion 27 5 Conclusions Acknowledgements 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 2

3 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) 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). 3

4 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 4

5 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). 5

6 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). 6

7 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). 7

8 Species Distribution Habitat Nesting Provespa barthelemyi Southeast Asia Nocturnal, probably a Probably aerial nesting forest species Provespa anomala Southeast Asia Nocturnal, lowland Usually aerial nesting habitats Provespa nocturna Southeast Asia Nocturnal, lowland Usually aerial nesting habitats Vespa orientalis Southwest Asia, Northeast Africa, Various habitats Usually subterranean nesting Madagascar, Southern Europe 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, Various habitats Subterranean and Vespa bicolor Korea, Japan, Russia North-eastern India, Central and Southern China, Burma, Thailand, Vietnam 8 Various habitats covered aerial nesting 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 Canada, USA Various habitats Usually aerial nesting. arenaria 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 Canada, USA Forest and urban Aerial nesting maculata habitats 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 Dolichovespula norwegica Dolichovespula alpicola Dolichovespula norvegicoides Vespula intermedia and D. saxonica Europe, Asia, Canada, Various habitats Usually aerial nesting USA Canada, USA Mountainous regions Usually aerial nesting Canada, USA Closed coniferous forest Rural areas Usually aerial nesting Eastern Asia, Canada, USA 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

9 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 Various habitats Both aerial and Paravespula flavopilosa Western Asia Canada, USA Disturbed areas near forest mountain regions Various habitats subterranean nesting Usually underground nesting Paravespula pensylvanica Canada, USA, Mexico, Hawaii Usually underground nesting Paravespula flaviceps Asian Palearctic, Lowland and mountain Usually subterranean Oriental regions regions nesting Paravespula Cosmopolitan Urban and rural areas Both aerial and germanica subterranean nesting Paravespula Canada, USA, Mexico Various habitats Usually subterranean maculifrons nesting Paravespula vulgaris Cosmopolitan Various habitats Both aerial and subterranean nesting Paravespula structor Asian Palaearctic and Indication of No nesting Oriental regions mountainous regions information Rugovespula orbata Asian Pa1aearctic and No habitat information No nesting Oriental regions 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. 9

10 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 10

11 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 cycles of 30 s at 94 C, 45 s of annealing at 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 All the loci were then manually adjusted and trimmed in Geneious v created by Biomatters. The appropriate substitution model for respective data sets was determined using the HIV database ( Primer Sequence PCR temperature ( C) COI 45 LCO GGT CAA CAA ATC ATA AAG ATA TTG G- HCO TAA ACT TCA GGG TGA CCA AAA AAT CA- 3 WG 58 beewgfor 5 -TGC CAN GTS AAG ACC TGY TGG ATG AG ACT CGC ARC ACC ART GGA ATG TRC A-3 Lepwg2a Pol II 52 polfor2a 5 -AAY AAR CCV GTY ATG GGT ATT GTR CA- polrev2a 3 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 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. 11

12 Species Voucher Locality COI 28S EF1a Pol II WG Vespa orientalis KMP59 Cairo, Egypt KJ KF KF KF KF Vespa basalis KMP147 Ha Giam, Vietnam KJ KF KF KF KF Vespa crabro KMP400 Georgia, USA KJ KF KF KF KF Provespa barthelemyi KMP61 Trang Pr, Thailand KJ KF KF KF KF Provespa anomala KMP62 Trang Pr, Thailand KJ KF KF KF KF Dolichovespula albida KMP111 Alaska, USA KJ KF KF KF KF Dolichovespula arenaria KMP126 Washington, USA KJ KF KF KF KF Dolichovespula sylvestris KMP141 Talloires, France KJ KF KF KF KF Dolichovespula pacifica KMP268 Mt. Fuji, Japan KJ KF KF KF KF Dolichovespula media KMP321 Berkshire, UK KJ KF KF KF KF Dolichovespula maculata KMP365 New York, USA KJ KF KF KF KF Dolichovespula saxonica KMP428 Hungary KJ KF KF KF KF Vespula intermedia KMP112 Alaska, USA KJ KF KF KF KF Vespula consobrina KMP125 Washington, USA KJ KF KF KF KF Vespula acadica KMP131 Washington, USA KJ KF KF KF KF Vespula vidua KMP369 Vermont, USA KJ KF KF KF KF Vespula squamosa VSPL3 Arkansas, USA KJ KF KF KF KF Paravespula flavopilosa KMP113 - KJ KF KF KF KF Paravespula alascensis KMP114 Alaska, USA KJ KF KF KF Paravespula pensylvanica KMP128 Washington, USA KJ KF KF KF KF Paravespula germanica KMP366 Vermont, USA KJ KF KF KF KF Paravespula maculifrons VSPL2 New York, USA KJ KF KF KF KF Polistes fuscatus KMP285 New York, USA KJ KF KF KF KF Polistes metricus KMP286 Arkansas, USA KJ KF KF KF KF 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 12

13 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 13

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. 14

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 generations and the concatenated analyses were run for 50 M generations with sampling every 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 ; Heled & Drummond, 2010). The analyses contained all individuals that had all five loci successfully sequenced. An xml file was created using BEAUTi v (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 with 500 M generations and a sampling every generations. A species delimitation analyser was estimated in DISSECT (Jones et al., 2015). An xml file was created using BEAUTi v (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 (Drummond et al., 2012) with 100M generations and a sampling every 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 15

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. 16

17 Fig 10. Bayesian phylogeny, inferred using MrBayes, of the species tree analysis. Values at nodes are posterior probability (PP). 17

18 Fig 11. Multi-species coalescent tree, inferred using *BEAST. Values at nodes are posterior probability (PP). 18

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. 19

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. 20

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. 21

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). 22

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. 23

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. 24

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. 25

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

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 = 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 = 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 = 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 27

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 multispecies 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 28

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 multispecies 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). 29