Notas / Notes


DNA barcode confirms the distribution of Bombus magnus (Vogt, 1911) (Hymenoptera: Apidae) in the Iberian Peninsula

Nuria Blasco-Lavilla1, Concepción Ornosa2 & Pilar De la Rúa3

1Departamento de Zoología y Antropología Física, Facultad de Veterinaria, Universidad de Murcia, 30100, Murcia, Spain. Email: — ORCID iD:

2Departamento de Biodiversidad, Ecología y Evolución, Facultad de Ciencias Biológicas, Universidad Complutense, 28040, Madrid, Spain. Email: — ORCID iD:

3Departamento de Zoología y Antropología Física, Facultad de Veterinaria, Universidad de Murcia, 30100, Murcia, Spain. Email: — ORCID iD:



Bombus magnus (Vogt, 1911) (Hymenoptera: Apidae) is one of the three cryptic species belonging to the lucorum complex besides B. lucorum (Linnaeus, 1761) and B. cryptarum (Fabricius, 1775). In the Iberian Peninsula, only B. lucorum and B. magnus are present but the presence of this last species south of the Pyrenees has not yet been confirmed. Given their morphological similarity, we used the DNA barcode region for the identification of 113 individuals of this species complex in an Iberian sampling. Results confirm the presence of B. magnus in the Pyrenees and extend its current distribution to the Northern Iberian Plateau. Given these results, we suggest that the distribution and conservation status of this species in the Iberian Peninsula should be revised.

Key words: Bumblebees; Bombus magnus; B. lucorum complex; Apidae; Hymenoptera; DNA barcode; cox1.



El código de barras de ADN confirma la distribución de Bombus magnus (Vogt, 1911) (Hymenoptera: Apidae) en la península Ibérica

Bombus magnus (Vogt, 1911) (Hymenoptera: Apidae) es una de las tres especies crípticas pertenecientes al complejo lucorum junto con B. lucorum (Linnaeus, 1761) y B. cryptarum (Fabricius, 1775). En la península Ibérica solo se encuentran B. lurocum y B. magnus pero la presencia de esta última no ha sido confirmada al sur de los Pirineos. Dada su similitud morfológica, usamos la región del código de barras de ADN para identificar 113 individuos de este complejo de especies en un muestreo ibérico. Los resultados confirman la presencia de B. magnus en los Pirineos y amplían su distribución actual hacia la meseta Norte ibérica. Dados estos resultados, sugerimos que ha de revisarse su distribución y el estado de conservación de esta especie en la península Ibérica.

Palabras clave: Abejorros; Bombus magnus; complejo B. lucorum; Apidae; Hymenoptera; código de barras de ADN; cox1.


Recibido/Received: 27/04/2018; Aceptado/Accepted: 12/12/2018; Publicado en línea/Published online: 19/02/2019

Cómo citar este artículo/Citation: Blasco-Lavilla, N., Ornosa, C. & De la Rúa, P. 2019. DNA barcode confirms the distribution of Bombus magnus (Vogt, 1911) (Hymenoptera: Apidae) in the Iberian Peninsula. Graellsia, 75(1): e083.

Copyright: © 2019 SAM & CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License.



Bumblebee species (Bombus Latreille, 1802) were initially identified by their colour patterns. However, different species can show convergent colour patterns and furthermore, one given species can display a high intraspecific variation in different geographical locations (Williams, 2007). This results in the presence of cryptic species very similar or identical in morphology which cannot be readily distinguished (Rasmont, 1984). To overcome this, a molecular approach based on the sequence variation of a region of the mitochondrial cytochrome c oxidase subunit I (cox1) gene known as DNA barcode, has proved useful for revealing species identical to those recognised by traditional methods (Schmidt et al., 2015). DNA barcodes are included together with other data in an integrative taxonomy approach to delimit, discover and identify meaningful, natural species and taxa at all levels (Will et al., 2005), and have been widely used in bumblebee identification (Williams et al., 2012).

Within Bombus, the subgenus Bombus s. str. has presented more problems than other subgenera when it comes to species identification (Williams et al., 2012). In Western Europe, only two species were widely accepted until the mid-twentieth century: B. terrestris (Linnaeus, 1758) and B. lucorum (Linnaeus, 1761). However, a detailed review of the species B. lucorum based on morphological methods led to the confirmation of the species B. magnus (Vogt, 1911) by Krüger (1954) and B. cryptarum (Fabricius, 1775) by Rasmont (1984). Their status as species was supported by cross-breeding experiments (De Jonghe & Rasmont, 1983) and mating observations that indicated reproductive isolation between them (Bučánková et al., 2011). Later, molecular data and male labial gland secretions contributed enough evidence to accept all three as separate species (Bossert, 2015; McKendrick et al., 2017). This grouping is now known as the B. lucorum complex.

Nevertheless, there are not enough morphological characteristics to discriminate the species reliably. The first collar may be the only characteristic that allows discrimination of the three species (Williams, 2000; Bossert, 2015), but it is only useful for queens. Ecological data cannot distinguish the species as they live sympatrically, and there are no comparative studies about their habitat preferences over a wide geographic area (Bossert, 2015). Consequently, Rasmont et al. (2015) has considered B. magnus as a taxonomically problematic species.

In the Iberian Peninsula, only two species from this complex are known to be present: B. lucorum and B. magnus. Bombus magnus in particular has been located in the northern half of the Iberian Peninsula: between altitudes of 100 and 2000 m in the Cantabrian Range, the Pyrenees, Sierra de Guadarrama and provinces of Guadalajara and Teruel (Rasmont, 1984; Castro, 1996; Ornosa et al., 2017). However, these reports have not been confirmed with molecular assays and have been the topic of further discussion (Williams et al., 2012; Ornosa et al., 2017). Only one report from a specimen collected in 1965 in Soria (also in the northern half of the Iberian Peninsula) has been confirmed with DNA barcoding (Williams et al., 2012).

The lack of certainty in the B. magnus records from the Iberian Peninsula south of the Pyrenees (Bossert, 2015; Ornosa et al., 2017) reveals the need for further sampling to clarify the distribution of this species in the Iberian Peninsula. In addition, specimens from the Iberian Peninsula are of particular importance since there are indications that queens of B. lucorum exhibit a yellow thoracic collar coloration similar to B. magnus queens in central Spain (Bertsch, 2009). Bombus lucorum queens in Spain can also be exceptionally large and this could lead to wrong identifications as B. magnus queens are supposedly larger, although specimens with medium and small size have been also observed (Bertsch, com. pers., 2017). In this study, we used the DNA barcode to molecularly identify individuals of this species complex. Although this species is not considered to be threatened in the IUCN Red List of European Bees (Rasmont et al., 2015), distribution models project a reduction of suitable areas by 2050, therefore an update of the distribution of B. magnus in the Iberian Peninsula is needed to propose conservation measures.

Bumblebees were sampled from different areas of the northern half of the Iberian Peninsula between the summers of 2013 and 2017 (Table 1). Individuals (N = 113) of different castes (Seven queens; 47 workers; 59 males) were preserved in absolute ethanol until analysed.

Table 1.— Samples of the B. lucorum-complex collected between the summers of 2013 and 2017 in the Iberian Peninsula. The samples of B. magnus are shaded in grey (a.s.l.= above sea level; M= male, W= worker, Q= queen).
Tabla 1.— Muestras del complejo B. lucorum recogidas entre los veranos de 2013 y 2017 en la península Ibérica. Sombreado en gris se señalan las muestras de B. magnus (a.s.l.= sobre el nivel del mar; M= macho, W= obrera, Q= reina).
Species Latitude/ Longitude Collection date Locality/Province Elevation (m a.s.l.) Caste
B. lucorum 42.63683
10/07/2013 Parque Nacional de Ordesa, Huesca 1720 1 W
B. lucorum 42.63483
11/07/2013 Parque Nacional de Ordesa, Huesca 1950 2 Q, 1 W
B. lucorum 42.61313
12/07/2013 Linas de Broto, Huesca 1420 1 M
B. lucorum 40.75388
20/08/2013 Cercedilla, Madrid 1323 3 M
B. lucorum 42.63896
08/08/2014 Torla, Huesca 1390 2 W
B. lucorum 42.63582
08/08/2014 Torla, Huesca 1407 1 M
B. lucorum 42.63527
08/08/2014 Torla, Huesca 1665 2 M, 1 W
B. lucorum 42.63519
08/08/2014 Torla, Huesca 1649 1 W
B. lucorum 42.63500
08/08/2014 Torla, Huesca 1648 1 M
B. lucorum 42.63767
08/08/2014 Torla, Huesca 1673 1 Q
B. lucorum 42.69796
09/08/2014 San Nicolás de Bujaruelo, Huesca 1477 1 W
B. lucorum 42.69847
09/08/2014 San Nicolás de Bujaruelo, Huesca 1591 2 M
B. magnus 42.69847
09/08/2014 San Nicolás de Bujaruelo, Huesca 1591 1 M
B. lucorum 42.70055
09/08/2014 San Nicolás de Bujaruelo, Huesca 1381 1 M
B. lucorum 42.63033
10/08/2014 Biescas, Huesca 914 1 M
B. lucorum 42.74025
11/08/2014 Ansó, Huesca 1053 2 M, 4 W
B. lucorum 42.86482
11/08/2014 Ansó, Huesca 1216 1 M
B. lucorum 42.58361
07/07/2014 Parque Nacional Aigüestortes, Lérida 1548 1 W
B. lucorum 42.58750
08/07/2014 Parque Nacional Aigüestortes, Lérida 2035 1 W
B. lucorum 42.58194
08/07/2014 Parque Nacional Aigüestortes, Lérida 2035 1 W
B. lucorum 42.58916
08/07/2014 Parque Nacional Aigüestortes, Lérida 2152 4 W
B. lucorum 42.58111
10/07/2014 Jou, Lérida 1369 1 W
B. lucorum 42.69388
10/07/2014 Ruda, Lérida 1440 1 W
B. lucorum 42.66666
10/07/2014 Colomers, Lérida 1589 1 W
B. lucorum 40.79194
29/07/2014 Puerto de la Fuenfría, Madrid 1797 4 M, 1 W
B. lucorum 40.78611
29/07/2014 Puerto de la Fuenfría, Madrid 1750 3 M
B. lucorum 40.75388
02/08/2014 Cercedilla, Madrid 1323 1 M
B. lucorum 40.82305
08/08/2014 Parque Natural de Peñalara, Madrid 1830 1 M
B. lucorum 40.77666
21/08/2014 Puerto de la Fuenfría, Madrid 1696 2 M
B. lucorum 40.79194
26/08/2014 Puerto de la Fuenfría, Madrid 1797 3 M
B. lucorum 40.78611
26/08/2014 Puerto de la Fuenfría, Madrid 1750 1 M
B. lucorum 42.58900
02/09/2015 Clots de Rialba, Lérida 2174 2 W
B. lucorum 42.45116
04/09/2015 Llessuí, Lérida 1405 1 Q
B. lucorum 42.42666
08/07/2015 Valle de Camprodón, Gerona 2175 1 W
B. lucorum 40.75388
01/05/20155 Cercedilla, Madrid 1260 1 Q
B. lucorum 42.38472
09/07/2015 Estación La Molina, Gerona 1730 1 Q, 4 W
B. lucorum 42.32027
09/07/2015 Estación La Molina, Gerona 1800 5 W
B. lucorum 40.79194
20/07/2015 Puerto de la Fuenfría, Madrid 1797 6 M, 3 W
B. lucorum 40.78611
04/08/2015 Fuente de Antón Velasco, Madrid 1750 16 M, 1 W
B. lucorum 40.79194
20/07/2015 Puerto de la Fuenfría, Madrid 1797 2 W
B. lucorum 40.79194
26/08/2016 Puerto de la Fuenfría, Madrid 1797 1 W
B. lucorum 40.75388
18/06/2017 Cercedilla, Madrid 1230 1 M
B. magnus 42.44222
20/06/2017 Naveros de Pisuerga, Palencia 800 1 W
B. lucorum 43.04138
21/06/2017 Piedrasluengas, Palencia 1355 1 W
B. lucorum 40.75388
25/06/2017 Cercedilla, Madrid 1230 1 W
B. lucorum 40.79194
26/07/2017 Puerto de la Fuenfría, Madrid 1797 1Q, 4M, 3W
B. lucorum 40.79194
01/09/2017 Puerto de la Fuenfría, Madrid 1797 1 M

The posterior left leg was removed to extract the DNA with the Chelex method (Walsh et al., 1991). A fragment of the mitochondrial gene cox1 of 614 bp was amplified with MyTaq™ Red Mix (Bioline) using two different methods. For the samplings between 2013 and 2016, we used the primers LCO/HCO (Folmer et al., 1994) and the following program: initial denaturation at 94 °C for 3 min; 40 cycles of 94 °C for 30 s, 48/50 °C for 30 s, 72 °C for 1 min, and a final extension step at 72 °C for 10 min. For the sampling of 2017, we used the mini-barcode primers Barbee and MtD9 (Françoso & Arias, 2013) and the following program: initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 1 min, 46 °C for 1 min 20 s, 64 °C for 2 min, and a final extension step at 64 °C for 10 min.

PCR products were sequenced in Secugen (Madrid). Sequences were edited with Geneious 7.1 and species identification was performed by BLAST (

Both amplification methods amplified the same region of the gene cox1. DNA barcodes of two individuals showed an identity of 100% with the sequences GU705915 (B. magnus from Germany) and JN872621 (B. magnus from Denmark) from Genbank, therefore of 113 individuals sampled, 111 were found to be B. lucorum and two individuals firstly identified as B. lucorum by morphometrical data were found to be B. magnus. One of the individuals was a male caught in San Nicolás de Bujaruelo (Huesca) in the Pyrenees, at 1591 m above sea level, 9 VIII 2014 (P. De la Rúa leg.). The second individual was a worker sampled in Naveros de Pisuerga (Palencia), at 800 m above sea level, 20 VI 2017 (C. Ornosa leg.), foraging on Rubus ulmifolius Schott (Fig. 1).

Fig. 1.— Known distribution range of B. magnus (Vogt, 1911) and B. lucorum (Linnaeus, 1761) according to Bossert (2015) and Ornosa & Ortiz-Sánchez (2004), respectively, and sampling sites of the present study. A blue square indicates a site where only B. magnus was found, a pink triangle indicates sites where only B. lucorum was found and a purple star indicates a site where both species were found.
Fig. 1.— Rango de distribución conocido de B. magnus (Vogt, 1911) y B. lucorum (Linnaeus, 1761) según Bossert (2015) y Ornosa y Ortiz-Sánchez (2004) respectivamente, y localidades de muestreo de este estudio. El cuadrado azul indica la localidad donde se encontró sólo B. magnus, un triángulo rosa indica las localidades donde sólo se encontró B. lucorum y una estrella morada indica la localidad donde se encontraron ambas especies.


These new reports of B. magnus confirm the current distribution of the species in the Iberian Northern Plateau. The fact that only two individuals were sampled in four years strongly suggests that the species is rare and much less abundant than B. lucorum. Although both species are more widely distributed in northern Europe, previous works have already reported that B. lucorum is more frequent than B. magnus in mainland Europe (Bossert, 2015).

These reports agree with previous studies, which have pointed to a patchy distribution for B. magnus, with an association to heathlands with low diversity of plant species in Europe (Bossert, 2015) and other Mediterranean landscapes in the Iberian Peninsula, where it was previously reported based on morphological data. These results highlight the need for sampling more individuals in the Iberian Peninsula, and the utility of the DNA barcode region for identifying them. Obtaining more samples could help pinpoint both the period of the year when B. magnus nests are active and their habitat, facilitating the discrimination of the species from B. lucorum. It could also help to assess its conservation status in Spain which is unknown at present.



This study was supported by the projects E-RTA2014-00003-C03 (Spanish National Institute for Agricultural and Food Research and Technology, and European Regional Development Fund) and 19908/GERM/2015 of Regional Excellence (Seneca Foundation, CARM). Sampling permissions were obtained from the corresponding authorities (PPNN Aigüestortes i Estany de Sant Maurici, Ordesa y Monte Perdido y Sierra de Guadarrama). N. B-L is supported by the grant FPU14/05189. The authors wish to thank Dr. Andreas Bertsch and two anonymous reviewers for their kind comments and suggestions, to Ana Isabel Asensio for her technical support and José Herrera for English edition.



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