A genetic and morphological survey to trace the origin of Melipona beecheii (Apidae: Meliponini) from Cuba William de Jesús May-Itzá, Walberto Lóriga Peña, Pilar de la Rúa, José Javier G. Quezada-Eúan To cite this version: William de Jesús May-Itzá, Walberto Lóriga Peña, Pilar de la Rúa, José Javier G. Quezada-Eúan. A genetic and morphological survey to trace the origin of Melipona beecheii (Apidae: Meliponini) from Cuba. Apidologie, 2019, 50 (6), pp.859-870. 10.1007/s13592-019-00696-7. hal-02985195 HAL Id: hal-02985195 https://hal.archives-ouvertes.fr/hal-02985195 Submitted on 2 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2019) 50:859–870 Original article * INRA, DIB and Springer-Verlag France SAS, part of Springer Nature, 2019 DOI: 10.1007/s13592-019-00696-7 A genetic and morphological survey to trace the origin of Melipona beecheii (Apidae: Meliponini) from Cuba 1 2 3 William de Jesús MAY-ITZÁ , Walberto Lóriga PEÑA , Pilar DELARÚA , 1 José Javier G. QUEZADA-EÚAN 1Departamento de Apicultura, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Mérida Yucatán México 2Departamento de Prevención, Facultad de Medicina Veterinaria, Universidad Agraria de la Habana, Havana Cuba 3Departamento de Zoología y Antropología Física, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia Spain Received 26 February 2019 – Revised 5 September 2019 – Accepted 2 October 2019 Abstract – The stingless bee Melipona beecheii is extensively distributed across Mexico and Central America and the only Melipona found in the Greater Antilles. The aim of this work was to establish possible continental affinities of M. beecheii from the Antilles. We compared populations from the main island (Cuba) with two clearly separated genetic lineages of M. beecheii on mainland, namely the Yucatán peninsula (Mexico) and Costa Rica. We used morphometrics, the degree of cephalic maculation, and microsatellite variation. Results indicate a greater morpho- logical and genetic similarity between populations from Cuba and the Yucatán peninsula with respect to those of Costa Rica. Based on our findings, we conclude that the origin of M. beecheii from Cuba is more likely the Yucatán peninsula (Mexico). We found evidence that isolation has not been large enough to result in a new genetic lineage. Melipona / morphology / microsatellites / Antilles / Mesoamerica 1. INTRODUCTION deed, European honey bees were not introduced into the Yucatán until the early part of the twenti- Stingless bees have been important for Meso- eth century because M. beecheii produced honey american civilizations as sources of food, medi- and cerumen in large quantities in this area cine, and craft (Crane 1992;Quezada-Euánetal. (González-Acereto 2008). 2001). The main species in Mesoamerica before The widely dispersed M. becheeii species coexists European colonization was Melipona beecheii with Melipona variegatipes Gribodo, 1893 in the Bennett, 1831 (Quezada-Euán et al. 2018). Inter- Antilles archipelago (Schwarz 1932) but showing a estingly, in contrast with other areas of Mexico different distribution: while M. beecheii is presently and Central America, this species continued being reported in two of the Greater Antilles (Cuba, Jamai- the most important bee for honey production in ca) (Genaro and Lóriga 2018), the other species is the Yucatán peninsula of Mexico even after the found in the Lesser Antilles (Schwarz 1932; Camargo introduction of Apis mellifera by European colo- et al. 1988). Previous comparison of a few Melipona nizers (Michener 1979, 1982; Genaro 2008). In- specimens from Cuba and the Yucatán peninsula led Schwarz (1932) to propose they belonged to the species M. becheeii due to morphological similarities. Corresponding author: W. May-Itzá, Given that colonies of the genus Melipona seem [email protected] capable of rafting in their tree nests, a plausible Manuscript editor: Klaus Hartfelder hypothesis of the presence of M. becheeii on Cuba 860 W. de Jesús May-Itzá et al. could be a continental origin given rise to a founding Mexico (Yucatán peninsula) and Costa Rica using population on this oceanic island (Roubik and morphometric characters, the degree of cephalic mac- Camargo 2012). Another hypothesis explaining the ulation, and molecular markers (microsatellites loci). presence of M. beecheii in the Greater Antilles is that If island populations have a more recent continental colonies could have been introduced to Cuba during origin, we predict more morphometrical similarity the Spanish rule of this area due to its economic between them and continental populations and lower importance in Mesoamerica (Michener 1979; allelic richness and heterozygosity in Cuban Camargo et al. 1988). Both of those hypotheses populations. wouldimplyarecentoriginofM. beecheii in Cuba and a lack of substantial differentiation with respect to 2. MATERIALS AND METHODS continental populations. Alternatively, as the original Cuban bee fauna appears to have its origin during the 2.1. Sampling sites and collecting bees Proto Antilles period millions of years ago (Genaro 2008), it could be that populations of M. beecheii Samples of worker bees were collected from may have an ancestral origin and, thus, exhibit sub- colonies of M. beecheii distributedinCuba(30 stantial differentiation from continental populations. colonies), Mexico (hereinafter referred to as Indeed, Roubik and Camargo (2012) suggested that Yucatán) (10 colonies), and Costa Rica (nine col- M. variegatipes found on Guadeloupe and Dominica onies) (Figure 1, Table I). Each sample consisted may have arrived there from South America during of 10–20 young workers collected directly from the last glaciations. the brood chamber of managed colonies, which Presently, M. beecheii has a distribution rang- were preserved in absolute ethanol at − 20 °C. ing from the Pacific and Gulf coasts of Mexico down to Costa Rica (Schwarz 1932;Ayala1999; May-Itzá et al. 2012). Differentiation within the 2.2. Morphometric and cephalic maculation species M. becheeii is considered relatively high analyses (Roubik and Camargo 2012) and molecular stud- ies have revealed at least two clear genetic line- From 10 to 20 worker bees per colony were ages of M. beecheii , one distributed in the used for morphometric analysis. From each bee, Yucatán peninsula and Northern Chiapas in Mex- the head, right forewing, the right hindwing, and ico and the other from central Guatemala down to the right posterior leg were dissected and mounted Costa Rica (May-Itzá et al. 2009, 2012). Given the on slides. Eight morphometric characters (fore- existence of genetic lineages, it is important to wing length (FWL); forewing width (FWW); analyze the genetic profile of the populations from hindwing length (HWL); hindwing width Cuba to discern which ones are present in Cuba (HWW); femur length (FL); tibia length (TL); and if isolation events have given rise to substan- tibia width (TW) and basitarsus width (BW)) were tial differentiation from continental populations. measured in accordance with the methodology of Because of the mode of reproduction, stingless Quezada-Euán et al. (2007) bees have strong dispersal limitations The same samples were discriminated accord- (Rasmussen and Cameron 2010), and are there- ing to their degree of cephalic maculation by fore of particular interest for the study of relation- determining the area covered with yellow color ships between islands and continents (Roubik and on the clypeus and supraclypeal malar areas using Camargo 2012). The study of bee populations a scale of three levels: (1) scarce, yellow marks located on islands is also of interest to understand covering less than 10%; (2) medium, yellow aspects related to extinction risks, possible bottle- marks between 10 and 50%; and (3) intense, neck effects, gene drift, and inbreeding in haplo- yellow marks covering more than 50% as in diploid Hymenoptera (Alves et al. 2011; Quezada-Euán et al. (2007). Images for morpho- Francisco et al. 2016, 2017;Soroetal.2017). metric and maculations were taken with a camera, To trace the origin of M. beecheii from Cuba, we Canon EOS 60D, connected to a microscope have analyzed M. beecheii populations from Cuba, Leica S8APO and the software ImageJ V.1.50i. Genetic survey of Melipona beecheii of Cuba 861 Figure 1. Locations and distance (~ Km) between collection sites of M. beecheii in Cuba, Yucatán, and Costa Rica. 2.3. Molecular analysis first reaction) or 57 °C (for the second reaction), 30 sec. at 72 °C, and a final extension of 10 min. at Given the different number of colonies sam- 72 °C. Amplified fragments (= microsatellite al- pled in each country for the molecular analysis, leles) were detected in an ABI PRISM 3100 se- one worker bee per colony from Cuba and five quencer (Applied Biosystem) and analyzed with worker bees per colony (as in May-Itzá et al. the program Genemapper v3.7 (Applied 2010) from Yucatán and Costa Rica were used. Biosystem). Total DNA was extracted from three legs per worker using the DNeasy tissue kit (QIAGEN) 2.4. Data analysis and following manufacturer instructions. Two mi- μ croliters out of a final dilution of 100 l was used Morphometric data were analyzed using for microsatellite amplification. ANOVAto compare population (= all the colonies Seven microsatellite loci were amplified by settled in one country) means for each measured two multiplex reactions. In the first reaction loci, character. Measure variation was studied using T4-171 and T7-5 were amplified (Paxton et al. principal component analysis (PCA) as a reduc- 1999) and in a second reaction loci, Mbi28, tion method to analyze body size globally.