4. Discussion
We employed a geometric morphometrics framework to elucidate the
intraspecific phenotypic variability of the two subspecies of G.
morsitans that occur in Zambia. Population-level variability in
centroid size (CS) and wing morphology can serve as a useful proxy for
assessing the extent of divergence between conspecific populations
(Ostwald et al., 2023) and may further provide preliminary data for the
diagnosis of isolated populations (Dujardin, 2008). This information has
important implications for the area-wide integrated vector management
(AW-IVM) of G. morsitans in Zambia and further provides insights
into the population differentiation status in its entire geographical
range. Broadly, these results provide evidence for microevolutionary
change in both CS and wing morphology
in G. m. centralis andG. m. morsitans populations in Zambia.
Our results are consistent with the long-held observation that size
sexual dimorphism is well established in tsetse as female G.
morsitans were found to be larger than male flies. The estimated CS
difference between the two sexes (nine percent) was similar to that
reported by Hargrove et al. (2019), who found wings of female G.
morsitans to be eight percent longer than those of males. This
observation provides further evidence that size studies based on wing
measurements as described by Hargrove et al. (2019) and CS generated by
geometric morphometric analysis, produce comparable results. Therefore,
both measures are reliable estimators of mean wing size inGlossina spp .
This study has demonstrated that the mean wing size of G. m.
centralis is larger than G. m. morsitans . It has been reported
that the size of tsetse is largely dependent on the nutritional state
(Bursell, 1966) and temperature (Hargrove, 2001) experienced by the
female. High temperatures exceeding 32°C result in tsetse entering
cooler dark refuges such as rot holes in trees and antbear holes in the
ground (Vale, 1971), a behaviour that reduces their metabolic rate but
also reduces feeding opportunities (Lord et al., 2018). As such, female
tsetse have reduced fat levels and produce progressively smaller pupae
as temperature increases (English et al., 2016). Hargrove et al. (2018)
showed that small pupae have lower fat reserves which results in the
emergence of smaller-sized adults. Thus, the smaller fly size ofG. m. morsitans may be an adaptation to its occupation of a
hotter environment than that of G. m. centralis as reported by
Evison and Kathuria (1982) and Muyobela et al. (2023) and reaffirmed by
our results. Location differences in mean wing size were observed in
both subspecies’ ranges and temperature is again implicated as the major
source of fly size variation.
We postulate that the observed environmentally driven fly size variation
between the two subspecies may be explained by the hypotheses of
phenotypic plasticity and genetic assimilation (Dujardin, 2011).
Phenotypic plasticity is defined as the occurrence of phenotypic
variation of a single genotype interacting with different environments
(Pigliucci et al., 2006). The observed within species differences in fly
size are probably adaptive to the different ecotopes where G.
morsitans occurs, with plastic responses facilitating the enlargement
of its ecological range. Consequently, phenotypic plasticity may have
aided G. morsitans to survive in both warm (G. m.
morsitans ) and cooler (G. m. centralis ) environments within its
range, by providing both small and large-sized flies upon which natural
selection has acted. It is conceivable that selection has resulted in
fly size being genetically determined at the subspecies level through
the process of genetic assimilation (Flatt, 2005), and has now become a
heritable trait. Heritability for insect size has been demonstrated inAnopheles mosquitoes (Lehmann et al., 2006) and its
transgenerational effects were shown in G. f. fuscipes (Mbewe et
al., 2018).
Although fly size differences within the subspecies G. m.
morsitans are known to occur (Bursell, 1966) and are reported in this
study, it is unlikely that these within subspecies differences are
heritable. This is because temperature variability within a subspecies
range is expected to be less variable than across the subspecies range.
Therefore, other factors that affect size variability such as host
availability affecting the nutritional state of females, ovarian age,
and capture month and year (Hargrove et al., 2019) are likely to be more
important. Since these factors are highly variable within the subspecies
range and consequently do not exert selection in any specific direction,
fly size change concerning these factors is unlikely to result in
heritable change (Jirakanjanakit et al., 2007). As such, size is
expected to be a poor discriminator of G. morsitans subspecies
population structure.
Our results showed that allometry and environmental variability
accounted for 11.6% and 10.7% of shape variation in G.
morsitans . As such, we estimate that 77.7% of wing shape variation
could be attributed to genetic effects, a finding in support of the
suggestion by Patterson and Klingenberg (2007) that shape exhibits high
genetic determinism. The low contribution of environmental variability
to allometry-free wing shape variation suggests that G. morsitanswing shape exhibits high environmental canalization, in agreement with
results from other Diptera, sand flies (Dujardin and Le Pont, 2004) and
mosquitoes (Henry et al., 2010).
We found that wing shape in G. morsitans varies according to
gender, subspecies, and location of origin. The detection of
allometric-free shape sexual dimorphism indicates that the phenotypic
expression of wing shape in this tsetse is sex-specific. Shape sexual
dimorphism has been reported in other Dipteran families such as
Drosophilidae (Gilchrist et al., 2000) and Culicinae (Virginio et al.,
2015). Gilchrist et al. (2000) suggest that the gender regulation of
shape in the Diptera represents a developmental constraint during
morphogenesis rather than adaptive change. Tsetse biology appears to
support this view as female flies reproduce by adenotrophic viviparity
(Vreysen et al., 2013) which may present a different aerial dynamic
challenge to pregnant females compared to males, hence the need for
female wings to be designed differently. Evidence of strong genetic
determinism of wing-shape sexual dimorphism in the Diptera has been
presented by Cowley et al. (1986).
Subspecies wing shape variation in G. morsitans may be an
adaptive trait as G. m. centralis and G. m. morsitans in
different habitats with different aerodynamic conditions due to
temperature differences. Temperature is known to significantly affect
aerodynamic lift (Jun et al., 2015). As air temperature increases, its
density decreases leading to a decrease in the amount of lift generated
by the wings. Therefore, selection may be acting on the wing phenotypes
of the two subspecies differently as G. m. centralis occupies a
cooler environment than G. m. morsitans , thereby producing wing
shapes aerodynamically suitable for their specific environments. Ray et
al. (2016) showed that selective pressure resulting in large and small
changes in the wing shape of Drosophila can lead to significant
changes in key flight performance metrics, leading to improved
manoeuvrability and agility.
Significant wing shape variation was also observed within the subspecies
ranges of both G. m. centralis and G. m. morsitans . As
environmental conditions within the specific subspecies range are
similar, it is unlikely that aerobatic capability selection is the
primary cause of this variation. Since shape is known to be the output
of polygenic genes (Patterson and Klingenberg, 2007), it is more likely
that this within subspecies variation is non-adaptive and a result of
random genetic drift. Several field studies have implicated genetic
drift as a source of shape variation among geographic isolates of
conspecific populations (Dujardin, 2011; Henry et al., 2010; Kaba et
al., 2012a; Ravel et al., 2006). Shape change due to genetic drift has
also been demonstrated in the laboratory (Jirakanjanakit et al., 2007).
Based on the allometry-free shape variation data, our results suggest
that both G. m. centralis and G. m. morsitans populations
in Zambia are highly structured and exhibit significant morphological
divergence. Within the G. m. centralis range, structuring is
probably the result of the physical separation of KNP, KSP, and SNP
populations (Fig 1) by large areas of unsuitable habitat (Muyobela et
al., 2023). Such geographic isolation in natural conditions tends to
quickly generate wing shape changes due to genetic drift. Physical
separation between the two G. m. centralis locations in KNP and
all sample locations within the G. m. morsitans does not occur
(Fig 1). The observed population structuring at these locations could be
attributed to the notion that tsetse is essentially a local insect and
the interchange of individuals between contiguous parts of the general
population is limited (Bursell, 1966).
The observed structuring of G. morsitans populations in Zambia
suggests that the implementation of tsetse population management
technologies that target an entire isolated population may be
technically feasible. However, to categorically designate populations as
isolated, it is essential to estimate the number of migrants per
generation or the levels of gene flow between them (Bouyer et al.,
2007), and methods using morphometric variation are not suited for these
tasks. Therefore, the results presented in this study only provide
preliminary information justifying further investigation using molecular
techniques to conclusively identify genetically isolated populations
(Dujardin, 2008). This is particularly crucial in the G. m.
morsitans range where physical separation of sample location was not
apparent. It should be noted however, that some authors have suggested
that results from geometric morphometric studies are comparable to those
of molecular studies using microsatellite markers (Bouyer et al., 2010,
2007; Solano et al., 1999).
We conclude that G.
morsitans populations in Zambia exhibit significant population-level
variation in body size and allometry-free wing shape. This variation
suggests high levels of population structuring that may be indicative of
population isolation. Molecular studies to estimate the levels of gene
flow between these populations and determine their levels of genetic
isolation will be able to shed even more on G. morsitanspopulation structure in Zambia and its under lying drivers.