Increased levels of introgression evident in Irish honey bees

Abstract

Ireland is home to a significant population of the black bee, Apis mellifera mellifera, however, in recent years the numbers of imported queens have increased 10 fold and beekeepers are observing more bees that are not totally black. Here we confirm an increase in the proportion of introgressed honey bees in Ireland via multiple approaches. The use of colour and wing morphometrics is becoming less reliable in Ireland to identify native honey bees due to the increased presence of various hybrid phenotypes and genotypes.

Keywords:

• Apis mellifera mellifera
• free-living
• Ireland
• Introgression
• imports
• morphometrics
• molecular
• colour

Apis mellifera mellifera L is native to Ireland with a distributional range covering most of Northern Europe. Its decline throughout its range is well known since the transhumance of other subspecies became common after the development of hives with moveable frames and A. m. ligustica and A. m. carnica subspecies and the commercial Buckfast strain became more popular in parts of Europe for honey production (e.g. De la Rúa et al., 2009; Pinto et al., 2014; Ruttner, 1988). In Ireland, it was shown by Hassett et al. (2018) that A. m. mellifera was still widespread throughout Ireland and the amount of introgressed bees amongst those sampled was very low (2%). Browne et al. (2021) in their study of 76 free-living colonies sampled across Ireland in 2016/17 confirmed that 95% of the free-living bee cohort had a high probability of assignment to the M lineage. However, Irish beekeepers regularly report problems with trying to maintain black bees in certain places across Ireland due to the increased numbers of imports of honey bees from abroad since 2012. In 2012 102 queens were imported into Ireland from abroad, which increased to 1053 in 2020 and 1279 in 2021. Imported bees are a threat to locally adapted bees due to disruption to, and potential loss of, locally adapted traits (De la Rúa et al., 2009). Cooper (1986) describes a number of traits unique to A. m. mellifera in the UK and Ireland, including mating behaviours that allow mating in poor weather conditions in summer. These traits, including apiary vicinity mating and supercedure, may have contributed to the ability of this subspecies to persist in high numbers in Ireland and if lost could have long-term negative impacts on beekeeping in Ireland. Investigation of free-living honey bees provides a good picture of what variants are circulating in the population as it does not target any specific beekeeping cohort. Here we apply multiple approaches (wing morphometrics, colour assessment, mitochondrial DNA sequencing, and microsatellite analyses) to investigate potential introgression in free-living honey bee colonies sampled in 2019/2020 to help determine if an increase in introgression is being detected that confirm beekeeper observations.

Individuals from 36 free-living A. mellifera colonies were collected from around Ireland including Galway, Clare, Limerick, Fermanagh, Waterford, Roscommon, Dublin, Wicklow and Wexford from records submitted to the online reporting tool at the National Biodiversity Data Centre (https://biodiversityireland.ie/surveys/wild-honey-bee-study/) or sent by beekeepers. Seven of the colonies were collected in Autumn 2019 and 29 were collected in summer 2020 which included samples from four colonies that were previously sampled in 2016 (F13R, F27G, F72G, F47G). DNA was extracted from the hindleg from one representative per colony and the mitochondrial COI to COII intergenic region was amplified and sequenced as described in Browne et al. (2021). Bees were assigned to their subspecies according to the presence or absence of the P and Q elements (Cornuet & Garnery, 1991). Individual bees were also genotyped using 11 microsatellites and analysed in Structure (Pritchard et al., 2000) as previously described in Browne et al. (2021) using the same reference data. Right, forewings from ten workers from each colony were mounted on photographic slides, scanned, trimmed and submitted to DrawWing and MorphPlot (https://bibba.com/downloads; Tolfiski, 2004). Subsequently, wing images were also uploaded into the new DeepWings software (Rodriguez et al., 2022) for assessment of lineage. The colour of each bee was assessed by eye and assigned to one colour group: BLK (black), SPT (spot), B1T (1 banded tergite on gaster) or B2T (2 or more banded tergites on gaster).

Of mitochondrial sequences generated of sufficient quality from 34 colonies, nine (26.5%) were assigned to the C lineage, missing as they did the entire P region. Two such colonies were ones that had been resampled, F13R in 2016 and F72G in 2017. Their mitochondrial sequence in the previous period (Browne et al., 2021) was M lineage and here showed as C lineage indicating that there was a change of colony at the nest site in the intervening period. One colony (F95D) contained the full P region including Po, characteristic of the A lineage and was most similar to available Buckfast sequences from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). All above colonies were assigned to M lineage via microsatellites with a probability >0.9 indicating that they were colonies originally headed up by non-native queens but having significantly backcrossed with native drones over a number of generations. This is the first report of this genotypic pattern in Irish free-living colonies. Of 32 newly sampled free-living colonies examined via microsatellites, four had a < 0.9 probability of being M lineage and thus identified as being introgressed (12.5%). All four colonies (F79D, F86Ce, F91D, F104G) were M lineage via mitochondrial DNA. These cases indicate native A. m. mellifera queens mating with drones from imported colonies. The four resampled colonies (2016/17 and 2019/20) were all M lineage via microsatellite data. Overall this is a significant increase in levels of introgression in comparison to Browne et al who identified <5% of free-living colonies sampled in 2016/2017 as being introgressed.

Thirty two colonies yielded useful morphometric data of which only one was clearly defined as A. m. mellifera via Drawing & Morphplot. By contrast, all colonies were identified as M lineage via DeepWings with high probability (examples in Table 1). Support has been discontinued for Drawing by its author and while DeepWing shows a very good correlation between its output and genetic data for bees that are clearly one subspecies or another its ability to identify hybrids is weaker, as confirmed here (García et al., 2022). While most colonies sampled were black in colour two had significant banding and one had a small orange spot on either side of the upper abdomen (Table 1). One colony (F85Ce, Table 1) originating from a non-native C lineage queen still had significant colour banding while assigned to A. m. mellifera via microsatellite analysis. Two further colonies (F89Ce and F93Ce) also headed originally by non-native queens, in contrast, were black in colour and had also been backcrossed with native drones so that microsatellite data indicated M lineage. Two colonies (F86Ce and F104G), originating from native A. m. mellifera queens exhibited signs of colour banding and also showed evidence of hybridization in the microsatellite data indicating some introgression from non-native drones in these colonies. All mitochondrial sequence data are available from McCormack on request.

Table 1. Data from the colonies where all methods could be applied.

Colony No.	Location	Year	mtDNA	Microsatellites	DrawWing %M	Deepwing	Colour
			Lineage
F85Ce377	Roof, Whitegate, Clare	2020	C	M (0.997)	40%	M	B2T
F101G373	Roof, Gort, Galway	2020	C	M (0.998)	70%	M	BLK
F89Ce381	Roof, Ennis, Clare	2020	C	M (0.983)	50%	M	BLK
F13R380	Roof, Athleague, Roscommon	2020	C	M: (0.997)	60%	M	SPT
F72G374	Tree, Bell Harbour, Galway	2020	C	M: (0.945)	90%	M	SPT
F93Ce375	Tree, Inagh, Clare	2020	C	M (0.980)	60%	M	BLK
F103F349	Roof, Enniskillen, Fermanagh	2020	C	M: (0.996)	50%	M	BLK
F96F353	Roof, Tempo, Fermanagh	2020	C	M (0.997)	50%	M	BLK
F94Ce350	Roof, Turk Head, Cork	2020	C	M (0.996)	NT	NT	BLK
F107L365	Roof, Mount Mellick, Laois	2020	M	M (0.997)	80%	NT	BLK
F97R370	Tree, Ballintubber, Roscommon	2020	M	M: (0.996)	50%	M	BLK
F110L367	Roof, Mount Mellick, Laois	2020	M	M: (0.952)	70%	M	BLK
F86Ce376	Roof, Whitegate, Clare	2020	M	C (0.861)	60%	M	B2T
F102G372	Roof, Gort, Galway	2020	M	M: (0.996)	70%	M	BLK
F104G529	Roof, Furbo, Galway	2020	M	C: (0.791)	70%	M	B2T
F109R360	Roof, Mount Mellick, Laois	2020	M	M: (0.996)	70%	M	BLK
F91Ce384	Roof, Inagh, Clare	2020	M	C: (0.872)	70%	NT	BLK
F106R358	Roof, Mount Mellick, Laois	2020	M	M (0.997)	67%	M	BLK
F99G533	Roof, Killeeneneen, Galway	2020	M	M: (0.993)	100%	M	BLK
F96F368	Roof, Tempo, Fermanagh	2020	M	M (0.998)	50%	M	BLK
F107R359	Roof, Mount Mellick, Laois	2020	M	M: (0.979)	50%	M	BLK
F92aCe382	Roof, Inagh, Clare	2020	M	M: (0.992)	70%	NT	BLK
F95D354	Tree, Oldtown, Dublin	2020	A	M (0.973)	70%	M	BLK

Colonies are labelled by; F for free-living, a number code that refers to the nest site, a letter code to denote the county in Ireland that they were sampled, (Ce = Clare, G = Galway etc) and finally a number code that refers to the specific honey bee DNA sample used in the molecular work. Geographical locations of each nest site and colony are provided and whether the colony resided in a tree or roof space. Exact locations are not provided due to personal data protection (many colonies were sampled from people’s houses) and nest site protection but detailed locations are held by McCormack and colonies continue to be being monitored. F13R and F72G, for example, were also sampled in Browne et al. (2021) where the colonies present were M lineage but are now C indicating a change in colony occupation. Bees were assigned to M or C lineage by mtDNA and Microsatellite data (probability of assignment to lineage), two morphometric approaches and by colour. In the column for DrawWing/Morphplot are the % of bees that fell within the cut off values for A. m. mellifera. B2T = two tergites on the gaster with orange colouring, BLK = black, SPT = a small spot of colour on either side of the first tergite of the gaster.

In conclusion, despite the small sample size, these data show a significant increase in introgression (>12%) in Irish honey bees compared to previous studies of Irish honey bees (Browne et al., 2021; Hassett et al., 2018). In Browne et al. (2021) all wild honey bee colonies sampled were headed by queens originating from the M lineage (mtDNA) and the 5% introgression evident was via breeding with non-native drones. This current data shows the presence (>26%) of sampled free-living lineages descended from non-native queens that show significant back crossing with native drones, meaning that imported bees have swarmed into the environment and are perpetuating for a number of generations at least. Henriques et al. (2022) showed significant cytonuclear introgression from C lineage into the M native range in managed colonies from mainland France (Avignon), but not in the island population on Ouessant confirming the refuge nature of the Ouessant population. Similarly, Ireland as an Island with a high proportion of pure A. m. mellifera present is an important refuge for this threatened subspecies. However, hybrid offspring and continued imports of non-native queens and packets of bees may pose significant risks to the A. m. mellifera native population over time. Our results indicate that the use of colour or wing venation to identify subspecies status in colonies in Ireland is becoming less reliable as the numbers of hybrids in the population increase. This, plus the increase in imports into Ireland in recent years, is worrying because some hybridisation is invisible to the beekeepers who are helping to conserve native bees and who are trying to make a living from selling honey and raising native queens for onward sale.

Acknowledgements

We particularly thank the recorders of the free-living honey bee colonies and the National Biodiversity Data Centre for their assistance. Funding was gratefully received from the Eva Crane Trust [grant number ECTA20160303] and The Native Irish Honey Bee Society. Thanks also to Eoin MacLoughlin for help in sampling colonies.

Disclosure statement

The authors report there are no competing interests to declare.

Additional information

Funding

Eva Crane Trust [grant number ECTA20160303] and The Native Irish Honey Bee Society.