Geometric Morphometric Characterization of Isolated Colonies of Honeybees (Apis mellifera intermissa) in Northern Algeria ^†

Abstract

This study investigates morpho-geometric variations in wing conformations among honeybee (Apis mellifera L.) populations in Algeria, focusing on Apis mellifera intermissa in the northern zone. It addresses the threats posed by human beekeeping practices and hybridization. The analysis involves collecting and digitizing 445 honeybee specimens from nine localities in northern Algeria: Batna, Blida, Constantine, Cheffa, Jijel, Mila, Setif, Skikda, and Tipaza. Statistical assessments reveal significant wing anatomy variations across these zones. Principal Component Analysis identifies distinct shapes within populations, with notable differences in specific regions. Discriminant Analysis classifies samples into three groups, showcasing the model’s effectiveness. The Mahalanobis D distances provides insights into population similarities and differences, emphasizing the potential of morpho-geometric analysis in differentiating bee populations. The study concludes by highlighting the significance of size-independent data, offering recommendations for capturing honeybee diversity in different climatic zones of northern Algeria. This research advances our understanding of honeybee morphology in relation to environmental dynamics, providing valuable insights for preserving indigenous bee races and exploring biodiversity.

1. Introduction

Different subspecies of honeybee are present in Algeria [1], with Apis mellifera intermissa [2] residing in the north. However, this natural heritage faces an escalating threat due to human beekeeping practices spreading at an alarming pace [3]. The pursuit of high economic performance in bee colonies, coupled with desirable behavioral characteristics, have induced significant changes that jeopardize regional races and ecotypes through hybridization [4,5]. Currently beekeepers and biologists are engaged in a dual effort to sustainably preserve the two indigenous subspecies and explore biodiversity thoroughly. This characterization involves the utilization of powerful morphometry and molecular genetics methods. Wing venation may exhibit subtler patterns of variation oppositely to traditional observation methods. Geometric morphometrics proves promising in quantifying variation in complex morphological traits, particularly in wings due to their high heritability of shape [6,7]. Our objective is to assess the level of similarity among structures deduced from the forewings of domestic honeybee specimens (particularly those of females) in different parts of the country. This assessment is based on a well-established theory of shape [8] and adheres to the protocol outlined by Barour in 2012 [9,10,11]. This methodology employs the coordinates of 19 points, called landmarks, which are superimposed by translation, scaling, and rotation. After superposition, the landmark configurations differ only in shape and can be analyzed by multivariate statistical methods [11]. This approach has successfully discriminated patterns of insect wing venation among various species and even within species. Geometric morphometrics holds the potential to assess the extent of phenotypic divergence among discrete insect populations by quantifying variation in this highly conserved trait. Our aim with this paper is to provide recommendations and detailed descriptions of the geometric morphometric method for a more coherent capture of the diversity of honeybees in different climatic zones of northern Algeria.

2. Materials and Methods

Geo-Morphometry Approach

The investigation involved the random sampling of worker honeybees (30 per colony, totaling 445) during expeditions to nine distinct locations in northern Algeria, each characterized by different altitudes (

Table 1. The geographical and climatic situation of sample collection sites.

Locality	Altitude (m)	Latitude	Longitude	Bioclimat	Country	Temperature (°C)
Bejaia	298	36.3419	4.5635	Humid	Algeria	16.9
Blida	114	36.3153	2.5546	Sub-humid	Algeria	17.1
Jijel	74	36.4104	6.1628	Humid	Algeria	18.1
Mila	753	36.2116	5.5138	Sub-humid	Algeria	14.5
Setif	955	35.4913	5.3026	Semi-arid	Algeria	14
Skikda	236	36.5324	7.0532	Humid	Algeria	17.2
Tipaza	186	36.2805	2.261	Sub-humid	Algeria	18
Constantine	640	36.3613	6.6296	Sub-humid	Algeria	15.6
Batna	1048	35.5527	6.1506	Semi-arid	Algeria	13.5

), conducted between March and May 2021. The collected samples are preserved in 50 mL vials with 90° absolute ethanol at a very low temperature (−20 °C) until laboratory processing [12]. Subsequently, the right forewing of each specimen is dissected and digitized using a binocular microscope (10- to 100-fold magnification) equipped with a camera and Image Focus 4.0 image analyzer software.

To discern conformational differences in shape parameters, including size, we aligned a set of 19 homologous wing venation landmarks of type I [9] based on the definitions of Bookstein in 1991 [10,11,12,13]. This alignment process utilizes TPS software: TpsUtil32 version 1.76 and TpsDig232 version 1.31 [14], alongside dedicated software tools R version 4.1.0 (i386) [15] and Morpho J version 1.06 [16], exclusively designed for the modeling and visualization of honeybee wing structures.

3. Results

Global Trends in Wing Shape

Principal Component Analysis (PCA) identified significant variations in wing anatomy across geographic zones. Wings from Jijel, Sétif, Skikda, Tipaza, and Mila showed a complex structure with variability in the upper wing veins. Batna, Bejaia, Blida, and Constantine had wings with branched veins in the central part and contractions in the upper sections. Cheffa wings were elongated with slender veins and less developed anatomy (Figure 1).

Examining deformations with variations in inertia values, the first PCA component (PC1) controls contractions and relaxations in the upper wing veins. The second component (PC2) governs the central wing veins and the wing’s concavity or convexity. The third component (PC3) is influenced by itinerary points, binding points between veins, and histological boundaries. The conditioning table (Figure 2) visualizes deformations as variations unfold within the inertia values indicated by PCA.

The Discriminant Analysis revealed that the first two dimensions (Axis 1 and Axis 2) accounted for 59.45% of the inertia, identifying three distinct groups. Group 1 included samples from Sétif, Mila, Tipaza, and Skikda. Group 2 comprised specimens from Bejaia, Constantine, Cheffa, and Blida, while Group 3 exclusively featured specimens from Batna (Figure 3).

To gain further insight into the similarity and dissimilarity rates between population samples, Mahalanobis D distances [17] were computed. Overall, populations did not exhibit significant differences in geometric shapes, except for specific points. The most notable statistical dissimilarity was between the Batna and Jijel regions, with a distance score of 4.28. Conversely, the closest populations were Mila and Skikda, with a score of 1.77 (

Table 2. Results for Mahalanobis distances between groups.

	Bejaia	Blida	Batna	Cheffa	Constantine	Jijel	Mila	Skikda	Sétif
Bejaia	2.26
Batna	3.12	3.17
Cheffa	2.66	2.50	3.51
Constantine	2.45	1.86	3.66	2.32
Jijel	3.43	3.05	4.29	3.11	3.28
Mila	3.42	3.50	4.10	3.35	3.55	2.80
Skikda	2.75	2.89	3.90	2.70	2.96	1.91	1.78
Sétif	2.81	2.80	3.00	2.74	3.16	1.98	2.31	1.90
Tipaza	3.51	2.98	3.95	2.83	3.43	2.13	2.62	2.26	2.02

).

4. Discussion

The morpho-geometric analysis of wing conformations emerges as a dependable tool for discriminating among bee subspecies, potentially surpassing classical morphometry. In 2011, Kandemir et al. highlighted the utility of morpho-geometric analysis in studying the biodiversity of honeybees through wing venation patterns [18], while Rattanawannee et al. already proposed wing morpho-geometry as a preliminary analysis step, reserving molecular analysis for cases of uncertainty in 2010 [19]. The significance of employing size-independent data to characterize biogeographic races of Apis mellifera is emphasized by the insights from the research of Ruttner and Barour [20,21]. Our study incorporated a range of univariate and multivariate analyses, with Principal Component Analysis aimed at identifying distinct shapes within the population. Specifically, the regions of Jijel, Sétif, Skikda, Tipaza, and Mila exhibited a more intricate wing anatomy compared to other regions, with notable variability in the upper wing veins, inclining upwards on lateral sides. Remarkably, the Cheffa zone showcased a distinctively elongated wing shape, while bees from Batna, Bejaia, Blida, and Constantine exhibited branched veins in the central part. The classification model indicated the greatest statistical dissimilarity between the Mila and Skikda regions (score: 1.77), whereas the closest populations were Batna and Jijel (distance score: 4.28). Overall, the populations did not exhibit significant differences in geometric shapes. The morpho-geometric results signify the potential for distinguishing regional bee populations based on the wing venation conformations of worker bees, an observation not previously reported [9].

This study conclusively delved into the realm of morpho-geometric analysis as a powerful tool for discerning intricate variations in wing conformations among regional populations of Apis mellifera. Supported by appropriate statistical analyses, the investigation illuminated significant variations in the shape and size of the parameters under scrutiny. These variations bear relevance to a multitude of biological, ecological, and social factors, including the local vegetation and climate, the diverse ages of bees, genetic heterogeneity, and the tolerance of foreign bees to accessing different hives. Integrating these findings enhances our understanding of the intricate relationships between honeybee morphology and environmental dynamics. The observed variations underscore the complex interplay that shapes bee populations and highlights the dynamic factors contributing to morphological diversity. This study not only advances the methodology of morpho-geometric analysis but also enriches our broader comprehension of the nuanced interdependencies governing honeybee populations. As we navigate the intricate landscape of bee biodiversity, the insights gained from this research pave the way for more nuanced and context-specific approaches to studying Algerian bee subspecies in their natural biotope.