Sexual Dimorphism in Wing Shape and Its Impact on Conspecific Identification of Neotropical Fannia Species (Diptera: Fanniidae)

Abstract

Neotropical species of the genus Fannia remain poorly known despite their potential collection as biological evidence in criminal investigations. This is partly due to taxonomic difficulties and a lack of specialists. Identifying neotropical species of Fannia relies mainly on the classical morphological characters of adult males, as females show high similarity between species. Here, landmark-based geometric morphometrics of the wing could constitute an additional tool for associating adult females with conspecifics in this genus. In this study, we used a reference dataset of males belonging to ten putative species of Fannia from Colombia and molecular data to test this hypothesis. We found a strong wing shape sexual dimorphism, resulting in an almost perfect sex assignment based solely on this trait. However, the differences in wing shape between sexes were greater than those between species, making conspecific identification difficult. Our data show that wing shape could only feasibly be used for identifying adult males and females of F. lamosca, and males of F. dorsomaculata and F. pseudoconstricta. Low discrimination scores among remaining species may be partly explained by high intraspecific variation, slight wing shape differences among closely related species, or sampling bias. Although this study provides the first wing size and shape comparison among neotropical Fannia species, more samples and species are needed to validate these findings and identify the potential factors influencing this trait. Furthermore, the wing shape sexual dimorphism across Fannia species suggests different life-history strategies between sexes and possible genetic canalization mechanisms.

1. Introduction

The “little house fly” genus, Fannia Robineau-Desvoidy, 1830 (Diptera: Fanniidae) has forensic relevance since some species have been found on decaying organic matter, including human cadavers [1,2,3,4,5,6,7]. Currently, the identification of Fannia species mostly relies on the recognition of morphological characters by trained staff. However, this task is challenging due to the lack of morphological descriptions or keys for all life stages, as well as few molecular reference data for all species, particularly in the neotropics [8]. Further, though rearing larvae to adults is a typical practice in forensic entomology to achieve species identification as adults [9], high interspecific morphological similarity among adult females of this genus constrains the taxonomic utility. Thus, morphological identification of the Fannia species depends mostly on adult males.

Wing shape, a multivariate trait analyzed through geometric morphometrics [10], could help address the challenges of identifying species. This trait has a polygenic basis, and its variation is more constrained than wing size [11], making it a potential trait for species identification. Recent morphometric studies have proven the utility of the wing shape for identifying necrophagous flies of Calliphoridae [12], Muscidae [13], Sarcophagidae [14], and Piophilidae [15]. However, its applicability to species of the Fanniidae family has not been tested to date.

Here, we aimed to assess the wing shape differences of adult males of ten putative Fannia species and the potential association of adult females collected from the same study area with their conspecifics based on this trait.

2. Materials and Methods

2.1. Specimens

Specimens deposited in the Colección Entomológica Tecnológico de Antioquia—CETdeA in Medellín, Colombia—were selected for morphometric analysis (Figure 1 and Table S1). They were collected during field collections between 2010 and 2019 using Van Someren–Rydon traps baited with decomposing chicken viscera and fish heads following a standardized protocol [16] under the biodiversity collection permit for non-commercial scientific research issued by ANLA (Autoridad Nacional de Licencias Ambientales, Colombia) in Resolution 0839 on 25 July 2014. Adult male Fannia specimens were identified at the species level based on external morphological characters and genitalia [17,18,19]. For adult females, genus confirmation was accomplished using a morphological key [20], and seven morphotypes were defined based on wing patterns and body coloration, named here as F. sp followed by a number (i.e., F. sp1–F. sp7) (Table S2 [21]). We also included some Fannia pusio females obtained from laboratory rearing experiments. In total, we chose 149 specimens with 45 belonging to 10 putative species of Fannia males (3–6 individuals per species depending on availability) and 104 from Fannia females morphotypes (

Table 1. Samples used in the geometric morphometric analysis of Fannia.

Species/Morphotype	Sex	n
F. aburrae, Durango and Ramírez-Mora, 2019	Males	4
F. colazorrensis, Durango and Ramírez-Mora, 2013		4
F. dodgei, Seago, 1954		3
F. dorsomaculata, Grisales, Wolff and Carvalho, 2012		4
F. isa, Durango and Ramírez-Mora, 2019		5
F. laclara, Durango and Ramírez-Mora, 2013		6
F. lamosca, Grisales, Wolff and Carvalho, 2012		4
F. pseudoconstricta, Durango and Ramírez-Mora, 2019		5
F. pusio, Wiedemann, 1830		5
F. spinosa, Durango and Ramírez-Mora, 2019		5
F. sp1	Females	16
F. sp2		16
F. sp3		16
F. sp4		16
F. sp5		16
F. sp6		16
F. sp7		8

and Table S1).

2.2. Slide Preparation and Landmark Digitization

Both wings per specimen were carefully dissected and mounted dry between coverslip and slide, and the margin of the coverslip was sealed with adhesive tape. The wings were photographed with a millimetric scale using a digital camera OPTIKAM Pro3 (OPTIKA^®, Ponteranica, Italy) adapted to a trinocular stereomicroscope Nikon SMZ745T (Nikon Corp., Tokyo, Japan). Twelve landmarks previously reported [13,22] were digitized by the same person (YD) using the TpsDig2 v. 2.32 software [23] (Figure 2). We evaluated the repeatability for centroid size and landmarks with a subset of thirty wings using the repeatability index [24]. Further, the robustness of wing shape results was explored using LaSEC [25] and testing different landmark numbers and varying sample sizes per group to determine the potential impact of unbalanced sample size among groups.

2.3. Molecular Reference Data

As a reference dataset, we used the adult male Fannia specimens whose morphological and molecular identification was previously confirmed by Durango-Manrique et al. [26]. Then, we randomly selected around 3 specimens per female morphotype for molecular comparison with the reference dataset. For these samples, the protocols previously described for DNA extraction and PCR of a fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) [26] were used. Females with an identity higher than 97% when compared to any of the species of the molecular reference dataset were confirmed as the same species.

2.4. Landmark-Based Geometric Morphometrics

Landmark-based geometric morphometric analyzes were conducted independently by sex and pooling both sexes in one dataset to assess the potential species-specific association in the morphospace. Fluctuating asymmetry was tested using Procrustes ANOVA [27]. For each dataset, we applied a generalized Procrustes analysis to generate centroid size and shape variables. Centroid size (CS) differences were evaluated using the Mann–Whitney test or the Kruskal–Wallis one-way ANOVA with post-hoc testing using Dunn’s multiple comparisons test. The shape variation associated with size (allometry) was computed using a multivariate regression of CS onto shape variables, and the differences in the allometric slopes among groups were tested under a multivariate analysis of covariance (MANCOVA) using 10.000 permutations [28]. Sexual dimorphism and species/morphotype distinctiveness were assessed using discriminant analysis (DA) or canonical variates analysis (CVA), respectively. The Mahalanobis distances (Md) and their statistical significance after 10.000 permutations are reported. Additionally, we carried out DA with cross-validation to assess whether groups can be distinguished reliably based on wing shape. Morphometric analyses were performed using MorphoJ v. 1.08.02 [29], XYOM v. 3.0 [30] and PAST v. 3.0 [31].

3. Results

We had acceptable repeatability values higher than 80% for each landmark and 98% for CS. The LaSEC analyses evidenced that 9 landmarks are enough to capture ≥90% of wing shape variation in all datasets (median fit ≥ 0.9) (Figure S1). In addition, the mean wing shape for each group was slightly affected by the unbalanced sample size among groups following subset comparisons. The left and right wings did not differ significantly in CS and shape (ANOVA, p > 0.05).

3.1. Sexual Dimorphism

The CS comparisons revealed no significant sexual dimorphism at either the overall or species-specific levels from those females where species assignment was possible based on molecular data (F. dorsomaculata and F. lamosca) (Mann–Whitney U, p > 0.05). In contrast, the full dataset found a strong sexual dimorphism in wing shape (Md = 6.5) (Figure 3A), with a DA clearly allowing discrimination between the sexes (>99%) (Figure 3B). An intersex comparison of mean wing shape revealed the greatest displacement of landmarks 1, 2, 7, and 12 at the wing border (Figure 3C).

3.2. Interspecific Differences in Wing Size and Shape in Males

For the 10 putative species, wing CS significant differences were found in only 13.3% of the pairwise comparisons (Kruskal–Wallis 36.05, p < 0.0001) (

Table 2. Wing centroid size (CS) values in male specimens of Fannia species analyzed in this study.

Species/Morphotype	n	Median CS	IQR	CV
F. aburrae	4	10.12	9.77–10.90	6.00%
F. colazorrensis	4	6.62	6.07–6.76	6.20%
F. dodgei	3	6.3	5.98–6.53	4.40%
F. dorsomaculata *	4	9.77	8.99–10.54	8.20%
F. isa	5	9.49	8.90–10.45	9.30%
F. laclara	6	9.59	9.25–9.87	5.70%
F. lamosca *	4	12.04	10.86–12.61	7.90%
F. pseudoconstricta	5	10.25	9.84–10.54	3.60%
F. pusio	5	5.73	5.46–7.60	26%
F. spinosa	5	11.2	10.77–11.56	3.60%

CV = Coefficient of variation. IQR = Interquartile range (25–75%). n = number of specimens. * Molecular association based on the COI region does not discriminate between these species.

and Figure S2). Fannia colazorrensis (6.6 mm), F. pusio (5.7 mm), and F. dodgei (6.3 mm) were significantly different from F. lamosca (12.0 mm) and F. spinosa (11.2 mm). The latter two species and F. aburrae (10.1 mm), had the largest wing CS, whereas the smallest were F. pusio, F. dodgei, and F. colazorrensis (6.6 mm). A significant allometric effect was found (p < 0.0001), but it was negligible (18.9%). The mean wing shape revealed differences for all pairwise comparisons between species (

Table 3. Differences in wing shape for males of Fannia species expressed as Mahalanobis distances.

Species	abu	col	dod	dor	isa	lac	lam	pse	pus
col	18.43
dod	16.15	7.20
dor	8.39	16.68	14.88
isa	7.81	14.62	13.03	7.74
lac	4.45	17.68	14.71	8.49	8.18
lam	11.86	18.96	17.99	6.61	10.62	12.46
pse	6.24	19.76	17.47	10.27	6.92	8.05	12.46
pus	20.49	4.96	7.55	18.03	15.94	19.34	19.58	21.15
spi	4.71	17.96	16.51	7.86	8.21	6.03	10.85	8.32	20.03

Fannia aburrae-abu; F. colazorrensis-col; F. dodgei-dod; F. dorsomaculata-dor; F. isa-isa; F. laclara-lac; F. lamosca-lam; F. pseudoconstricta-pse; F. pusio-pus; F. spinosa-spi. All pairwise comparisons were statistically significant (p value < 0.001).

and Figure 4). The CVA and the DA analyses allowed correct assignments between 75 and 100% after cross-validation for F. lamosca, F. pseudoconstricta, and F. dorsomaculata (Table S3). In contrast, the assignment based on wing shape for the remaining species was low, with an overall correct assignment rate of 46.67%.

3.3. Differences in Wing Size and Shape in Females

We obtained twenty COI sequences from 7 female morphotypes and compared them with the reference molecular dataset. Using the molecular data, we were only able to assign the morphotypes F. sp4 to F. lamosca, and F. sp6 to F. dorsomaculata. We found significant differences in wing CS (Kruskal–Wallis 74.27, p < 0.0001) for 80% of the species/morphotypes pairwise comparisons. Additionally, we detected an allometric effect (p < 0.0001), but it was negligible (12.92%). Wing shape comparisons indicated differences for all pairwise comparisons between species/morphotypes (

Table 4. Differences in wing shape for female morphotypes of Fannia species expressed as Mahalanobis distances.

Morphotype	F. sp1	F. sp2	F. sp3	F. sp4	F. sp5	F. sp6
F. sp2	7.95
F. sp3	7.29	3.44
F. sp4 *	8.61	9.54	8.44
F. sp5	8.55	3.21	4.92	9.33
F. sp6 *	5.75	7.00	5.98	5.17	7.31
F. sp7	7.03	3.79	3.66	7.95	3.97	5.36

* Females assigned to F. sp4 and F. sp6 morphotypes were molecularly confirmed as Fannia lamosca and F. dorsomaculata, respectively. All pairwise comparisons were statistically significant (p value < 0.0001).

and Figure 5). The overall cross validation rate of correct assignment for females was of 75%, with correct assignments rates between 75 and 100% for F. sp1, F. sp4 (F. lamosca), and F. sp5 (Table S3).

4. Discussion

The Fannia species analyzed in this study evidenced a strong wing shape sexual dimorphism, enabling almost precise sex assignment based solely on this anatomical structure. However, the classification at the species level proved challenging for males and females, with the highest overall accurate classification rate observed for females and good results for some species. Here, conspecific association based on wing shape was hindered by strong sexual dimorphism.

Wing shape sexual dimorphism has also been detected in calyptrate flies of the Calliphoridae [12,32,33], Muscidae [34,35,36], Sarcophagidae [14], and Piophilidae [37] families. However, it is worth noting that it is the first study evidencing wing shape sexual dimorphism in the Fanniidae, particularly in the Fannia genus in the neotropics. Most intersexual wing shape differences were located at the wing border, as evidenced in other morphometric studies [12,35]. These intersexual differences in wing shape may be correlated with sexual dimorphism of wingbeat frequency and its potential role in sexual communication, as previously suggested [38]. In addition, wing shape sexual dimorphism across species suggests possible genetic canalization mechanisms between sexes [11,39].

Despite potential environmental stressors during development, we did not detect significant deviations of wing symmetry in the genus Fannia. We hypothesize genetic canalization mechanisms for wing shape, which may buffer the effects of environmental stressors during the growth and development of immature stages and a strong natural selection pressure to maintain wing symmetry [40,41]. Since our analysis was based on field-collected samples, further controlled experiments are necessary to evaluate the potential impact of specific environmental stressors on wing shape. Wing symmetry in Fannia species could facilitate future morphometric comparisons, especially for specimens with missing or damaged wings where either wing may be used without potential bias. The molecular reference data provided us with a baseline to confirm or assign morphological species or morphotypes (i.e., females) to Fannia species for morphometric analyses, with some limitations in differentiating F. isa/F. pseudoconstricta and F. dodgei/F. colazorrensis, as previously reported [26]. Further, the available molecular data had limitations in associating some COI sequences from some females to species. This was likely due to the absence of molecular reference data for other Fannia species not included in the initial study and the public databases. Thus, we highlight the relevance of conducting additional field studies to capture and rear specimens of the genus Fannia, and associate female morphotypes to specific species for use as a reference in future molecular and morphometric comparisons.

Pairwise comparisons of wing shape revealed significant interspecific differences, but species assignment based on this trait was generally low in accuracy, except for males of F. dorsomaculata and F. pseudoconstricta, and F. lamosca in both sexes. The low assignment rates could be attributed to several factors, including high intraspecific variation, slight wing shape differences among phylogenetically closely related species, or sampling bias. Based on the current knowledge, molecular data remains a better tool for accurately identifying Fannia species.

Within the Fannia genus, species have been categorized into groups and subgroups based on their external morphological characteristics and the structures of the male genitalia [42,43]. Interestingly, analysis of wing shape data supports the closest relationship in the morphospace among species belonging to those proposed groups and subgroups. This is also consistent with their genetic distances [26]. Therefore, we hypothesize that there is a phylogenetic signal in the wing shape data of Fannia species.

Previous studies have shown that wing shape in muscid flies such as Polietina orbitalis (Stein, 1904), [44] and Stomoxys calcitrans (Linnaeus, 1758) [45] have a plastic response to local environmental conditions leading to variation within the species, which may also impact Fannia species. Additionally, similarities in wing shape might also be linked to the close phylogenetic relationship among species [46]. However, there is currently no phylogeny incorporating most of the Fannia species of this study for more definitive conclusions. Furthermore, previous research has pointed out the impact of sampling size on the mean wing shape in geometric morphometric studies [47,48], and the influence of the selected landmarks [49] or the morphometric approach [34,50]. Although we implemented methods to address potential issues with sampling size, our results should be interpreted with caution. Further studies are necessary to confirm the wing shape findings and identify the potential factors influencing this trait in this genus.

This study shows that identifying adult females solely through wing shape is only feasible for F. lamosca. Hence, we propose delving into alternative anatomical structures, particularly the head, as it has exhibited potential for analyzing intraspecific variation in F. pusio [51]. However, it has yet to be explored at the macroevolutionary level. In addition, wing image analysis using machine learning automated approaches, as those recently used in other Calyptratae [52] could exploit other relevant data to discriminate Fannia species.