﻿Phylogeography and ecology of bumble bees on Kolguev Island, a remote European Arctic landmass

﻿Abstract The bumble bee fauna of the Russian Arctic is rather poorly known. Kolguev Island, a remote insular territory in the Barents Sea, is one of the deficiently studied areas. In this study, material on Kolguev’s bumble bees is re-examined, phylogeographic data analysed, putative scenarios explaining the origin of the bumble bee fauna on the island discussed, and the biology and phenology of these insular populations described. Five bumble bee species, i.e., Bombusflavidus, B.lapponicus, B.jonellus, B.pyrrhopygus, and B.balteatus, were recorded on this island. All of these species are widespread throughout the Eurasian Arctic. Bumble bee populations on Kolguev Island are characterised by a low level of molecular divergence from mainland populations. Based on paleogeographic reconstructions and phylogeographic patterns, it is hypothesised that the bumble bees appeared on this island in the Early Holocene. The lack of rodents (lemmings and voles) sharply decreases the number of available nesting places for bumble bees on Kolguev Island.


Introduction
Kolguev Island is a remote insular territory on the continental shelf in the south-eastern part of the Barents Sea, with a total area of 5130 km 2 (Lavrinenko and Lavrinenko 2014). This island is composed of Quaternary sediments and is located approximately 70 km north of the coast of Eurasia (Lavrinenko and Lavrinenko 2014). Most of its area is occupied by an accumulative plain, with an average altitude of 20-30 m. However, the southern part of the island is covered by low-elevation wet tundra and peat bogs, while hilly landscapes with average elevations of 80-100 m prevail in its central part.
In the Late Pleistocene, during the period of maximum development of the Scandinavian Ice Sheet, Kolguev Island was a part of the continent due to lower sea levels (Velichko 2002). Available paleogeographic reconstructions reveal that the island area was covered by Arctic deserts (Velichko 2002) or even by massive ice sheets (Svendsen et al. 2004;Hughes et al. 2016). In the Early Holocene, Kolguev Island was isolated from the mainland due to rising sea levels and intense coastal erosion (Velichko 2002).
A review of published literature indicates that the insect fauna of the island may have originated in the Late Pleistocene or Early Holocene (Bolotov 2011). The species diversity of insects on Kolguev Island is rather poorly known, and most available works deal with Lepidoptera and Coleoptera (Buturlin 1903;Semenov 1904;Bolotov 2011;Kullberg et al. 2019;Spitsyn and Bolotov 2020;Potapov et al. 2021b). However, a few publications report on the fauna and species richness of bumble bees collected on Kolguev Island (Berezin 1995a;Potapov 2010, 2011;Potapov et al. 2014;Paukkunen and Kozlov 2020). None of the published entomological works contains data on the phylogeography and biogeographic affinities of insects from Kolguev, including bumble bees.
Bumble bees (genus Bombus Latreille) are well adapted to the harsh climatic conditions of the Arctic compared with other groups of bees (Panfilov 1968). The high adaptive capabilities of high-latitude bumble bees could be linked to more effective thermoregulation and shorter life cycle (Berezin 1990(Berezin , 1995aRadchenko and Pesenko 1994;Goulson 2010). In general, the bumble bee fauna of the Arctic is well studied, especially in Northern Europe and North America (Williams et al. 2014;Rasmont et al. 2021). Conversely, several remote, hard-to-reach areas of the European and Asian Arctic remain poorly studied, including Kolguev Island.
The bumble bee fauna of the Eurasian Arctic can be separated into three distinct groups of species: (1) the High Arctic taxa; (2) the Lower Arctic taxa; and (3) boreal species (Chernov 1978;Chernov and Matveeva 2002). The High Arctic species group contains Bombus hyperboreus Schönherr, 1809, B. pyrrhopygus Friese, 1902, and the polar relict species B. glacialis Friese, 1902(Richards 1973Chernov and Matveeva 2002;Williams et al. 2019;Potapov et al. 2021a). The latter species is endemic to the Novaya Zemlya Archipelago and Wrangel Island (Potapov et al. 2021a). The Lower Arctic group consists of a number of species such as B. lapponicus (Fabricius, 1793), B. jonellus (Kirby, 1802), B. cingulatus Wahlberg, 1854, and B. balteatus Dahlbom, 1832(Chernov 1978Chernov and Matveeva 2002). The expansion of boreal bumble bee species associated with species-rich flowering plant associations through river valleys is a common means of enrichment of the Arctic fauna. The boreal species that may colonise the Arctic by this manner are B. distinguendus Morawitz, 1869, B. hortorum (Linnaeus, 1761), B. flavidus Eversmann, 1852, and others (Kolosova and Potapov 2011Potapov et al. 2019). In general, the species richness of bumble bees in the Arctic and Subarctic regions ranges from two or three on islands to 12-14 at mainland sites (Potapov et al. 2014).
Most of the bumble bee species mentioned above have Palearctic distributions, with the exception of B. jonellus, B. distinguendus, and B. flavidus (Williams 1998 Curtis, 1835, B. kirbiellus Curtis, 1835, and B. natvigi Richards, 1931, respectively (Williams et al. 2015. However, a number of scholars consider that the Nearctic B. polaris and the Palearctic B. pyrrhopygus are conspecific and that the older name B. polaris should be used for this circumpolar taxon (Rasmont et al. 2021). At the same time, there are different opinions on the distribution of species belonging to the Bombus (Pyrobombus) lapponicus-complex (Sheffield et al. 2020).
This paper aims to (1) re-examine material on Kolguev's bumble bees using newly collected samples; (2) analyse phylogeographic data and discuss putative scenarios explaining the origin of the bumble bee fauna on Kolguev Island; and (3) describe ecological and phenological patterns for these insular populations.

Materials and methods
Data sampling, morphological study, and statistical tests Samples of bumble bees from Kolguev Island were collected by Boris Yu. Filippov, Natalia A. Zubrii, Vitaly M. Spitsyn, Alisa A. Zheludkova, Aleksey G. Ardeev, and Grigory S. Potapov in 2009Potapov in , 2018Potapov in , and 2020 (total N = 287 specimens) (Suppl. material 1: Table S1). The bumble bees were collected in the southern part of the island, i.e., near the village of Bugrino (68.7819°N, 49.3087°E) and along a route from this village to Lake Krivoe (69.0194°N, 48.7211°E) (Fig. 1, Suppl. material 1: Table S1). In most cases, one sample represents a daily sampling effort of a single collector along a walked route of approximately 5 km.
The bumble bees were collected with an entomological net. In some cases, foraging plants of bumble bees were not accurately recorded or bumble bees were caught in flight. For this reason, it is impossible to give a detailed range of the bumble bee foraging resources on Kolguev Island. Our field research was carried out from July to August, allowing us to study phenological patterns for the most abundant species of bumble bees. However, the exact dates of the beginning of the bumble bee flight season on Kolguev Island are unknown because sampling was not possible in May and June. Specimens of bumble bees from Kolguev Island are deposited in the Russian Museum of the Biodiversity Hotspots (RMBH) of the N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences (Arkhangelsk, Russia). Additional material from the Chukotka and Yamal peninsulas were used for a comparative phylogeographic analysis (Suppl. material 1: Table S1). Only two specimens of bumble bees were found in historical samples from Kolguev Island collected by Buturlin's (1903) expedition (Suppl. material 1: Table S1). These two specimens were examined in the Zoological Institute of the Russian Academy of Sciences, St. Petersburg (ZIN).
Statistical procedures (Kolmogorov-Smirnov test for normality and Mann-Whitney test) for the analysis of relative abundance were performed with Statistica v. 13.3 (Stat Soft Inc., USA). We compared the parameters of relative abundance between Kolguev Island and the Novaya Zemlya Archipelago. Novaya Zemlya is the closest insular land to Kolguev Island (approximately 210 km) but differs by having a much larger total area (82,000 km 2 ) and harsher environmental conditions (Isachenko 1995).

Laboratory protocols
We generated new sequences of the cytochrome c oxidase subunit I (COI) gene from 15 bumble bee specimens (Table 1). The laboratory protocols followed those described in Potapov et al. (2018aPotapov et al. ( , 2018b. Resulting COI gene sequences were checked manually using a sequence alignment editor (BioEdit v. 7.2.5;Hall 1999). The sequencing was carried out at the Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences (Moscow, Russia) using the ABI PRISM BigDye Terminator v. 3.1 reagent kit.

Phylogeographic analyses
We used a median-joining network approach using Network v. 5.0.0.1 with default settings (Bandelt et al. 1999). Additional available COI sequences of B. lapponicus, B. pyrrhopygus, and B. balteatus were obtained from the BOLD (the Barcode of Life Data System; Ratnasingham and Hebert 2007) and GenBank databases (N = 35; Table  1). Each COI sequence of the aligned datasets was trimmed, leaving a 425-bp fragment for B. lapponicus, 455-bp fragment for B. pyrrhopygus, and 627-bp fragment for B. balteatus. The alignment of COI sequences was performed using the ClustalW algorithm implemented in MEGA7 (Kumar et al. 2016).

Species richness
Five species of bumble bees were recorded on Kolguev Island, B. flavidus, B. lapponicus, B. jonellus, B. pyrrhopygus, and B. balteatus (Suppl. material 1: Table S1). During our studies in 2009, 2018, and 2020, B. pyrrhopygus and B. lapponicus were the most common and widespread taxa on the island (N = 124 and 123 specimens, respectively), while B. balteatus was observed less frequently (N = 43 specimens). Six specimens of B. flavidus were collected on this island and only one specimen of B. jonellus was sampled in 2009.
Relative abundance of bumble bees on Kolguev Island is 6.27±1.05 specimens per sample (mean ± s.e.; N = 48; in most cases, one sample represents a daily sampling effort of a single collector along a walked route of approximately 5 km), which is two times higher than that on Novaya Zemlya, with 3.11±0.46 specimens per sample (mean ± s.e.; N = 44; data from Potapov et al. 2019: table 1). The differences between these insular areas in relation to their relative bumble bee abundances are highly significant (Mann-Whitney test: U = 711, P = 0.0058). The mean number of recorded species per sample (± s.e.) on Kolguev Island and Novaya Zemlya is 1.94±0.13 (N = 48) and 1.43±0.10 (N = 44), respectively. This parameter is also higher on the first island (Mann-Whitney test: U = 707, P = 0.0029).

Phylogeography
We found that the sequenced B. lapponicus specimens from Kolguev Island belong to a single COI lineage (haplotype LP-1) that also occurs in the population from Yamal (Table 1, Fig. 2). Bombus pyrrhopygus shares two COI haplotypes on Kolguev, one of which is also known to occur in Norway, Novaya Zemlya, Chukotka, and Kamchatka (haplotype PY1). In contrast, the second haplotype of this species (PY10) was not recorded anywhere else but is genetically close to the first lineage. Bombus balteatus from Kolguev Island also shares two COI haplotypes (BL2 and BL3), which were not recorded anywhere else (Table 1, Fig. 2).

Colour variations
Bombus pyrrhopygus and B. balteatus are highly variable in their colour patterns (Løken 1973;Williams et al. 2019;Rasmont et al. 2021). The dark form of B. pyrrhopygus, which is known to occur in Scandinavia and the Kola Peninsula, was not recorded on Kolguev Island. Specimens of this species collected on Kolguev Island share a colour variation of tergites T4-T6. It ranges from a black coloration without ferruginous to a quite distinct ferruginous colouration. Regarding B. balteatus, the colour variation of T4-T6 from yellowish white to whitish occur in a series of specimens from Kolguev Island.

Bumble bee habitats, foraging resources, and phenological patterns
The main places of aggregation of foraging bumble bee individuals in the southern part of Kolguev Island are river valleys, where foraging resources and appropriate nesting places are concentrated (Fig. 3). Bumble bees rarely occur beyond these areas because   continuous wet tundra and peat bog landscapes between river valleys are unfavourable for foraging and the establishment of colonies. Different species of bumble bees do not vary in their habitat preferences on Kolguev Island. Bumble bees have been recorded on water avens (Geum rivale), whorled lousewort (Pedicularis verticillata), hairy lousewort (Pedicularis hirsuta), tall Jacob's ladder (Polemonium acutiflorum), candle spur (Delphinium elatum var. hirsutum), cloudberry (Rubus chamaemorus), and on different willow species (Salix spp.).
Queens of bumble bees were recorded from early July to late August (Fig. 4). The majority of workers were collected in late July and early August, whereas males were caught in August. The final date when a bumble bee (B. pyrrhopygus) was recorded on Kolguev Island was 30 August (Suppl. material 1: Table S1).

Bumble bee fauna of Kolguev Island
The fauna of bumble bees on Kolguev Island with five species (B. flavidus, B. lapponicus, B. jonellus, B. pyrrhopygus, and B. balteatus) is similar to other species-poor Arctic faunas, dominated by cold-adapted bumble bee species (Potapov et al. 2019). The bumble bee fauna of Vaygach Island (the total area 3400 km 2 , geographic distance approximately 340 km from Kolguev) is the most similar to the studied fauna. In particular, five bumble bee species occur on Vaygach Island, i.e., B. flavidus, B. lapponicus, B. pyrrhopygus, B. balteatus, and B. hyperboreus (see Potapov et al. 2017).
We have no reliable records of B. hyperboreus from Kolguev Island because earlier references to the existence of this species on the island (Kolosova and Potapov 2010Potapov , 2011Rasmont et al. 2021) were incorrect. However, future records of B. hyperboreus on Kolguev Island can be expected, mainly in the central and northern part of this island, where the species richness of insects (e.g., Lepidoptera: Bolotov 2011) is higher due to the prevalence of hilly landscapes with richer plant diversity (Potapov et al. 2021b).
Only one specimen (queen) of B. jonellus was found on Kolguev Island in 2009. However, in subsequent studies, this species was not rediscovered there. As in the case of B. hyperboreus, additional research is needed in the central and northern parts of Kolguev Island. It is also possible that our solitary record of B. jonellus reflects an unsuccessful colonisation event because no workers and males of this species were recorded on Kolguev Island. It is well known that queens of bumble bees may migrate for quite considerable distances and that they are not deterred by larger water barriers (Fijen 2020). In the case of Kolguev Island, located 70 km from the mainland, the possibility of an accidental dispersal of a B. jonellus female to the island cannot be excluded. Earlier, two migrant butterfly species were recorded on Kolguev Island (Bolotov et al. 2021). A few specimens of B. flavidus were recorded on Kolguev Island, i.e., one female and five males. This cuckoo bumble bee is known as a social parasite of B. lapponicus (see Lhomme and Hines 2019), which is a common and widespread species on the island. Earlier reference to the record of B. norvegicus, another cuckoo bumble bee species, on Kolguev Island Potapov 2010, 2011;Rasmont et al. 2021) is incorrect. It was based on a misidentification of a B. flavidus female. Hence, Kolguev's fauna contains only one species of cuckoo bumble bee, B. flavidus.
Finally, records of B. glacialis Friese, 1902 on Kolguev Island, mentioned by earlier scholars (Pittioni 1943), were not confirmed by our recent surveys (Potapov et al. 2021a). The latter species is a Pleistocene glacial relict and is endemic to Novaya Zemlya and Wrangel Island (Potapov et al. 2018a(Potapov et al. , 2019(Potapov et al. , 2021a. It is unlikely that B. glacialis is present on Kolguev Island due to the significant environmental differences between Kolguev and Novaya Zemlya.

Phylogeographic pattern in the populations of Kolguev's bumble bees and a prospective scenario of their expansion to this island
We analysed the COI sequences of three widespread species of bumble bees on Kolguev Island and found that they belong to common Northern Eurasian lineages. Kolguev's B. lapponicus reveals a single COI haplotype that also occurs in a population from Yamal and is close to the Norwegian lineage. Bombus pyrrhopygus shares two haplotypes, which are also known to occur in Norway, Novaya Zemlya, Chukotka, and Kamchatka. Bombus balteatus from Kolguev Island have two haplotypes, which were not recorded anywhere yet, but they are close to the COI lineage from Chukotka, Kamchatka, and Siberia. In summary, all three species from Kolguev share a low level of molecular divergence from mainland populations, which aligns with the results of earlier phylogeographic research on B. hyperboreus and B. pyrrhopygus from Novaya Zemlya (Potapov et al. 2019).
We hypothesise that B. lapponicus, B. pyrrhopygus, and B. balteatus spread across the emerged Eurasian shelf margin in the Late Pleistocene, with subsequent fragmentation of their continuous ranges in the Holocene. Taking into account the geological history of the region (Velichko 2002) and our data on bumble bee phylogeography, we conclude that the bumble bees appeared on Kolguev Island no earlier than the Early Holocene, as did some other animal species such as a tiger moth (Bolotov et al. 2015) and a freshwater fish (Artamonova et al. 2020). During the Last Glacial Maximum, Kolguev Island was covered by Arctic deserts or the ice sheet. After the Holocene Climate Optimum, the vegetation cover on the island shifted to tundra ecosystems (Velichko 2002), which are more suitable as habitats for cold-tolerant Arctic bumble bees.

The life cycle and ecology of the Kolguev's bumble bees
The three most common species of the insular fauna (B. lapponicus, B. pyrrhopygus, and B. balteatus) are widespread throughout Kolguev Island but B. balteatus occurs less frequently. Obviously, the flight activity of bumble bees is dependent on weather conditions. Their flight season is typical for the Arctic territories with the maximum abundance of individuals in the warmest period. On Kolguev Island this period lasts from the second half of July to the first half of August and is characterised by a mean air temperature of 8 °C (Potapov et al. 2019(Potapov et al. , 2021a. We have no exact dates of the earliest emergence of bumble bee queens on Kolguev Island. As in the case of other Arctic islands, it should be sometime between mid-May and mid-June (Potapov et al. 2019(Potapov et al. , 2021a. No bumble bee nests have been recorded on Kolguev Island prior to our recent discovery of a nest of B. lapponicus, described herein. This nest was found in mid-August, when the life cycle of B. lapponicus on Kolguev Island enters its final stage. Hence, we did not have the opportunity to examine several aspects of the species' development such as the emergence of the first-brood adults, behaviour of workers in the nest, and the emergence of males. From available data, we can only conclude that the nest on Kolguev is typical for this species (Martinet et al. 2022). The number of individuals in the colony was quite small, which is typical for bumble bee colonies from Arctic territories (Berezin 1990).
The complete absence of rodents (e.g., lemmings and voles) is a unique feature of Kolguev Island that influences the animal life of the island in several ways, especially by switching the Arctic predators from rodents to other prey resources (Pokrovsky et al. 2015). It is unknown how exactly the absence of lemmings affects the bumble bees of Kolguev Island, but in the Arctic, bumble bees frequently use lemming burrows as nesting sites. Hence, the abundance of bumble bees increases in areas with higher concentrations of lemmings and their burrows (Berezin 1990(Berezin , 1995bPotapov et al. 2021a). The lack of lemming burrows considerably limits the nesting places of bumble bees on Kolguev Island. It seems that bumble bees use every available resource such as tussocks, edges of river terraces, and human buildings to establish a nesting colony on the island.
The abundance of bumble bees on Kolguev Island is rather low but the mean value (number of specimens per sampling effort) is two times higher than that on Novaya Zemlya (Potapov et al. 2019(Potapov et al. , 2021a. The total and mean species richness of bumble bees on Kolguev Island is also higher compared with those on Novaya Zemlya. These differences could be explained by specific landscape and climatic features of Kolguev Island, which is a plain insular landmass, taking a more southern geographic position compared with that of the mountainous Novaya Zemlya Archipelago (Potapov et al. 2019(Potapov et al. , 2021a. However, a possible role of the interannual variability in weather conditions (sampling in the two areas was made in different years) may also be considered there.
guev Island. Special thanks go to Aleksey G. Ardeev for his assistance in the collection of material. We thank to Dr. Elena Y. Churakova (FCIARctic) for providing help in identification of flowering plant species during the field research. We would like to thank Dr. Juho Paukkunen (Finnish Museum of Natural History, University of Helsinki, Finland) for his help with literature, valuable discussions on the Arctic bumble bees, and access to the collection of the Museum for the purposes of this research. Special thanks go to Sergey A. Rybalkin (Russia) for material from Chukotka and Dr. Ilya V. Vikhrev (FCIARctic) and Dr. Manuel Lopes-Lima (University of Porto, Portugal) for their assistance in the collection of material. Additionally, we are grateful to the staff of the Zoological Museum of the Moscow State University and the Zoological Institute of the Russian Academy of Sciences (St. Petersburg, Russia) for the opportunity to examine bumble bees in their collections. This study was supported by the Ministry of Science and Higher Education of the Russian Federation (project no. 122011400384-2).