1. Introduction
Glossina morsitans (Diptera: Glossinidae) is a savannah tsetse
species of the subgenus Glossina (morsitans group) whose
distribution is restricted to savannah woodlands (Leak et al., 2008) and
is correlated with that of wildlife (Vreysen et al., 2013). Three
allopatric subspecies occur, namely, G. m. submorsitansNewstead, G. m. centralis Machado, and G. m. morsitansWestwood (Jordan, 1993), all of which are efficient vectors of
trypanosomes (Kinetoplastida: Trypanosomatidae), which cause human and
animal trypanosomiasis in sub-Saharan Africa (Rogers, 2000). The
geographical distribution of G. m. submorsitans is from Western
to Central Africa, while G. m. centralis and G. m.
morsitans occur in Eastern, Central, and Southern Africa (Rogers and
Robinson, 2004). In Zambia, G. m. centralis and G. m.
morsitans are predicted to occupy 151,353 km2 or 20%
of the land mass (Muyobela et al., 2023).
In conformity with most insect species, the distribution of G.
morsitans within its geographic range is generally discontinuous
(Krafsur, 2009; Muyobela et al., 2023), being strategically arranged
based on the availability of food sources, reproductive needs, dispersal
capacity, and adaptation to local environmental conditions (Dujardin,
2008). Due to this spatial arrangement, adaptation to local conditions
can give rise to population-level phenotypic variation that may result
in the structuring of populations into biogeographical islands or
subpopulations (Dujardin and Le Pont, 2004; Getahun et al., 2014; Mbewe
et al., 2018). In the presence of significant barriers to the exchange
of individuals between them, these subpopulations become isolated and
can undergo rapid evolutionary changes in morphological traits due to
phenotypic plasticity, founder effects, and genetic drift (Dujardin,
2011; Ostwald et al., 2023). The identification of isolated tsetse
populations has been deemed crucial for the successful and sustainable
implementation of area-wide integrated vector management (AW-IVM)
(Bouyer et al., 2010; Kgori et al., 2006), guiding the decision whether
to undertake suppression or elimination campaigns (Bouyer et al., 2007).
A relatively low-cost approach for investigating tsetse population
structure is the use of landmark-based geometric morphometrics (GM),
defined as the statistical analysis of shape variation and its
covariation with other variables (Rohlf and Bookstein, 2003). Unlike
traditional morphometrics, GM is a powerful technique that captures the
geometry of the morphological structure under study and retains this
information throughout the analysis (Zelditch et al., 2004). The
procedure is accomplished through the Procrustes paradigm (Adams et al.,
2013) in which a set of two-dimensional landmark coordinates recording
the relative positions of homologous anatomical points are obtained and
then subjected to generalised Procrustes analysis (GPA) (Rohlf and
Slice, 1990). This least-squares superimposition technique produces a
set of shape variables whose geometric dissimilarity is expressed as the
Procrustes distance between the homologous points of two configurations
(Zelditch et al., 2004) and whose pattern of variation can be visualised
by graphical methods (Baken et al., 2021). An additional output of this
analysis is centroid size (CS), defined as the square root of the summed
squared distance of each landmark from the centroid of the form (Tatsuta
et al., 2018). This isometric measure of size is used as an estimator of
the global size of the form under study in GM studies (Dujardin, 2008).
Conspecific size variability within and among insect populations is
generally known to be an environmentally induced and reversible
character (Jirakanjanakit et al., 2007). In G. morsitans , size
variability has been attributed to seasonal effects (Hargrove et al.,
2019) with temperature being the major source of variation (Glasgow,
1961; Phelps and Clarke, 1974). High heritability values for insect size
have however been reported (Lehmann et al., 2006) and the
transgenerational effects of size among the Glossina spp have
been demonstrated (Mbewe et al., 2018). Therefore, heritable size
variation can be used to discriminate populations. Size-corrected or
allometry-free shape is known to be a polygenic character and strong
evidence of its genetic determinism has been provided (Klingenberg and
Leamy, 2001; Patterson and Klingenberg, 2007). Allometry-free shape has
also been shown to be a powerful discriminator of groups (Dujardin,
2008) and is, therefore, a very useful tool in taxonomic studies
(Klingenberg, 2016).
The insect body part most subjected to
GM studies is the wing (Tatsuta
et al., 2018). This is due to several reasons. Firstly, insect wings are
almost entirely two-dimensional structures, a fact that greatly reduces
digitisation errors (Dujardin, 2008). Secondly, the arrangement and
branching patterns of insect wing veins contain taxonomic information
that has been used to construct classification schemes, infer phylogeny
(Bybee et al., 2008), elucidate evolutionary patterns (Debat et al.,
2003), and evaluate fluctuating asymmetry – deviations from perfect
symmetry that indicate developmental noise (Klingenberg et al., 2001).
Lastly, the geometric shape of insect wings has been shown to exhibit
high environmental canalisation – the ability of a genotype’s phenotype
to remain relatively invariant when exposed to different environments
(Henry et al., 2010). These attributes, therefore, make the geometric
shape of insect wings, a suitable phenotypic character to distinguish
conspecific populations and species using GM (Dujardin, 2011). Insect
wing shape is captured by placing homologous landmarks on the
intersection of wing veins.
Geometric morphometrics has been used to study natural population
variation in several species of Glossina . These include G.
palpalis gambiensis (Bouyer et al., 2007; Solano et al., 1999),G. p. palpalis (Ebhodaghe et al., 2017; Kaba et al., 2012b),G. m. submorsitans (Achukwi et al., 2013), G. pallidipes(Getahun et al., 2014), G. austeni (De Beer et al., 2019),G. fuscipes fuscipes (Mbewe et al., 2018), G. tachinoides(Mustapha et al., 2018) and G. brevipalpis (De Beer et al.,
2019). However, phenotypic variation in natural populations of G.
m. centralis and G. m. morsitans has not been investigated.
Therefore, this study aimed to use landmark-based wing geometric
morphometrics to investigate phenotypic variation and determine the
level of population structuring in G. m. centralis and G.
m. morsitans populations in Zambia.