Molecular evolution and genetic diversity of defective chorion 1 in Anastrepha fraterculus and Anastrepha obliqua (Diptera, Tephritidae)

doi:10.21203/rs.3.rs-4327320/v1

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Molecular evolution and genetic diversity of defective chorion 1 in Anastrepha fraterculus and Anastrepha obliqua (Diptera, Tephritidae)

https://doi.org/10.21203/rs.3.rs-4327320/v1

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The family Tephritidae comprises numerous fruit fly species, some of which are economically significant, such as several in the genus Anastrepha. Most pest species in this genus belong to the fraterculus group, characterized by closely related species that are difficult to differentiate due to recent divergence and gene flow. Identifying genetic markers for their study is paramount for understanding the group’s evolution and eventual phytosanitary control. Because there is variation in eggshell morphology among species in the genus, we studied the rapidly evolving defective chorion 1 (dec-1) gene, which is crucial for chorion formation and reproduction. Fragments of dec-1 from two of the most important pest species in the genus, Anastrepha fraterculus and Anastrepha obliqua, were amplified and compared to transcriptome sequences, as well as dec-1 sequences from Anastrepha ludens, which was used for structure prediction. These data revealed three alternative transcripts in A. fraterculus and A. obliqua, consistent with patterns found in other Tephritidae; we obtained orthologous sequences for these other tephritids from NCBI to investigate patterns of selection affecting this gene at different hierarchical levels using different methods. These analyses show that purifying selection has had an important role across the whole gene and throughout its history, but there was evidence of positive and episodic diversifying selection at different levels. Different parts of the gene have shown distinct evolutionary rates, potentially correlating with the diverse proproteins produced by posttranslational changes. This correlation appears evident in certain lineages, including the branch that separates Anastrepha, as well as other internal branches that differentiate species within the genus. Our data showed that this gene shows remarkable variation across its different exons, which has proven to be informative at different evolutionary levels. These changes hold promise not only for studying differentiation in Anastrepha but also for the eventual management of selected pest species.

dec-1

Anastrepha

chorionic genes

differentiation

molecular evolution

The dipteran family Tephritidae harbors approximately 4,930 described species divided into 500 genera distributed worldwide (Norrbom, 2004; Schutze et al., 2017). Among these, a few fruit fly species, such as Anastrepha, Bactrocera, Ceratitis, and Rhagoletis, are of significant interest because they are among the most important agricultural pests (White, 2006; Raza et al., 2020) (Zucchi et al., 2004). Anastrepha, the largest genus in the tribe Toxotrypanini(Norrbom et al., 2012), is composed of 330 species in 23 taxonomic groups distributed across the tropics and subtropics of the veAmericas(Norrbom et al., 2018; Norrbom et al., 2021). The fraterculus species group in Anastrepha, which comprises 34 species that have been formally described to date (Norrbom et al., 2012), includes two widely distributed and closely related species: the South American fruit fly Anastrepha fraterculus (Wiedmann 1830) and the West Indies fruit fly Anastrepha obliqua (Macquart 1835). These species are known for infesting several host fruits across a broad geographical range, though the former tends to prefer Myrtaceae, whereas the latter favors Anacardiaceae (Solferini and Morgante, 1987; Zucchi, 2000).

Due to their host diversity and extensive spatial distribution, A. fraterculus and A. obliqua may experience genetic and ecological forces that promote differentiation across their distribution. Understanding these forces is paramount for investigating the processes behind species formation in this group, but it has also become increasingly important for planning and implementing population control strategies (Ruiz-Montoya et al., 2020). It has been suggested that reproductive isolation is an essential factor leading to speciation (Mayr, 1959; Rabosky, 2016), so much so that it has been commonly employed as a yardstick for defining and delineating species. Others have suggested that species are formed by a combination of forces that define separately evolving metapopulation lineages (De Queiroz, 2007). While there is evidence of pre- and postzygotic isolation among some sympatric and allopatric populations of Anastrepha species (Selivon et al., 2005; Vera et al., 2006; Rull et al., 2018), hybridization between A. fraterculus and A. obliqua has successfully produced viable offspring in the laboratory, though following Haldane’s rule (Santos et al., 2001). This finding has been corroborated by population studies that indicate gene flow between these species, despite the presence of reproductive barriers (Díaz et al., 2018; Scully et al., 2018; Congrains et al., 2021; Congrains et al., 2023). In this scenario, it is possible that different parts of the genome may introgress differently, so genes with specific reproductive biases may play a crucial role in differentiation and speciation, making their study of particular relevance for understanding the underlying forces separating Anastrepha species.

Genes associated with egg formation have been suggested to play important roles in the differentiation of some groups, such as Drosophilidae (Eickbush and Izzo, 1995) and even Anastrepha (Gonçalves et al., 2013). In the Drosophila melanogaster subgroup, these genes have been implicated in adaptive processes to the environment, with chorionic proteins evolving more rapidly, especially those in the outer membrane (chorion), showing strong positive selection compared to the inner (vitelline) membrane proteins (Jagadeeshan and Singh, 2007), a pattern that was also found among Anastrepha species (Gonçalves et al., 2013). This selective pressure may foster rapid gene diversification and reproductive incompatibilities (Findlay and Swanson, 2010), similar to sexual selection, sexual conflict, immunity, and protein recognition (Swanson and Vacquier, 2002). The importance of studying egg formation genes among Anastrepha species is reinforced by the fact that chorion morphology is a diagnostic tool that exhibits significant variability among Anastrepha species, particularly in distinguishing morphotypes of A. fraterculus (Selivon and Perondini, 1998; Selivon et al., 2004; Figueiredo et al., 2013; Figueiredo et al., 2017).

The defective chorion 1 (dec-1) gene has been extensively studied in Drosophila melanogaster, where it encodes follicular proteins (fcl) (Andersson and Lambertsson, 1991) and plays a crucial role in several stages of choriogenesis (Waring et al., 1990). The DEC-1 protein and its derivatives are essential for the formation of insect eggshells, and mutations in this gene can result in gross eggshell deformities during morphogenesis, as well as in sterile females (Bauer and Waring, 1987). This gene produces at least three alternatively spliced mRNAs with different isoforms in Drosophila that encode the posttranslationally modified proproteins FC106, FC125, and FC177 (Waring et al., 1990; Noguerón et al., 2000; Badciong et al., 2001; Mauzy-Melitz and Waring, 2003; Fakhouri et al., 2006). FC106 is the most abundant proprotein (Mauzy-Melitz and Waring, 2003) and is present in all eggshell layers, with a protein derivative located in the inner chorionic layer (ICL) (Noguerón et al., 2000). This process is related to several mechanisms of oogenesis, such as organization (Noguerón et al., 2000; Badciong et al., 2001), stability (Waring, 2000), and physicochemical protection of the egg (Badciong et al., 2001).

Together with FC125 and FC177, the proprotein FC106 has been shown to be essential for functional egg formation and female fertility (Noguerón et al., 2000; Mauzy-Melitz and Waring, 2003). The “FC” in their names denotes that they are synthetized and secreted by follicular cells and subsequently stored in the vitelline membrane and chorion layers, where they are cleaved into at least five polypeptide products (Noguerón et al., 2000). In Drosophila, dec-1 contains five tandem copies of a 78 bp-long sequence followed by seven repeats of varying lengths and identities (Waring et al., 1990; Escher and Lambertsson, 1996). The structural complexity and distinct spatial distributions of dec-1 products, which are involved in egg structure and function (Mauzy-Melitz and Waring, 2003), are a reflection of several gene pre- and posttranslational changes that lead to different DEC-1 protein variants (Hawley and Waring, 1988), providing evidence that this gene is rapidly evolving (Badciong et al., 2001). This gene has already been subject to phylogenetic studies demonstrating a high degree of conservation of its organization among Drosophila species, with partially conserved, as well as extensive, divergence in their coding regions (Badciong et al., 2001). Mutagenesis experiments targeting conserved regions have demonstrated the importance of this gene in comparative evolutionary analyses (Hanna-Rose et al., 1997; Takahashi et al., 1999), supported by the presence of conserved transcribed regions both within and between species, as observed in Drosophila (Andersson and Lambertsson, 1991).

Despite its general conservation, understanding the underlying genetic and evolutionary mechanisms that control the presence of multiple expressed DEC isoforms within and between individuals, as observed in Drosophila species, remains a significant challenge. The primary objective of this study was to expand the knowledge on the dec-1 gene in Anastrepha and Tephritidae, particularly regarding its role in their differentiation. To address this knowledge gap, we investigated whether there is variation in this gene in Anastrepha, and our findings provide insight into Anastrepha diversification and systematics. In this context, we investigated the dec-1 structure and phylogenetic signals by checking for potential sites of positive and purifying selection across different evolutionary levels. This investigation extends beyond the scope of the studied fruit flies to include Diptera taxa for which there is information on the dec-1 gene. By exploring potentially influential genes involved in fundamental biological processes, such as oogenesis and reproduction, in a phylogenetic framework, this research provides relevant insights not only into Anastrepha differentiation but also into broader-scale evolutionary processes affecting this gene.

2.1 Identification of the defective chorion 1 gene in Anastrepha

The Drosophila melanogaster dec-1 sequence (Dmdec-1) (Flybase FBgn0000427) was used as a query to identify putatively homologous transcripts in the A. fraterculus and A. obliqua reproductive and head transcriptomes previously produced in our laboratory (Gonçalves et al., 2013; Rezende et al., 2016; Congrains et al., 2018; Congrains et al., 2021).

The genomic sequence of dec-1 was obtained by mining a previous draft of the A. ludens genome, generously shared by Dr. Scott Geib, which was masked for repeats and low-complexity regions (Dupuis et al., 2019); this sequence is now published (Sim et al., 2024) and available in GenBank (GCA_028408465.1). Genome prediction and annotation were performed using Funannotate (v. 1.8.9) (Palmer and Stajich, 2019), which performs ab initio prediction with Augustus (v. 3.3.3) (Keller et al., 2011), Snap (v. 2006-07-28) (Korf, 2004), and GlimmerHMM (v. 3.0.4) (Majoros et al., 2004). Augustus was trained using protein sequences from Rhagoletis zephyria, another Tephritidae species, along with Diptera BUSCO models (Odb10) (Manni et al., 2021) to enhance the identification of protein-coding regions. Following gene model prediction, protein-coding sequences in this pipeline were annotated by querying curated databases such as UniProt (UniProt_Consortium, 2019), with a cutoff value of 1e10^− 3, and Gene Ontology (Ashburner et al., 2000) for protein function and domain identification. These putative gene models derived from the A. ludens genome were then used to identify and manually mine genomic sequences putatively orthologous to dec-1 (Aluddec-1). The A. obliqua genome has also recently been made available in GenBank (NCBI accession number GCA_027943255.1) (Sim et al., 2023), and its annotation indicated two dec-1 (GenAoblidec-1) isoforms, fc106 and fc125 (XM_054883939.1 and XM_054883939.1, respectively).

2.2 Characterization and molecular structure of dec-1

Putative dec-1 sequences and potential splicing variants were identified using A. ludens gene sequences as queries in BLAST searches (Altschul and Koonin, 1998) against the NCBI nr and sra databases. We used R. zephyria dec-1 sequences (NCBI gene ID: 108362233) as templates to identify dec-1 exons in A. fraterculus and A. obliqua obtained from transcriptomes produced in our laboratory. The inferred nucleotide coding sequences of A. ludens, A. fraterculus, and A. obliqua (Aluddec-1, Afradec-1, and Aobldec-1, respectively) were translated into amino acid sequences using the ExPASy Translate tool (https://web.expasy.org/translate/). DNA sequences were aligned using MAFFT (Katoh and Standley, 2013) implemented on BioEdit (Hall, 2004). Motif identification was performed using the MOTIF server (http://www.genome.jp/tools/motif/) with Pfam domain settings. Finally, motif similarity between Afradec-1 and its putative orthologs was estimated using the SMS tool (https://www.bioinformatics.org/sms2/ident_sim.html).

2.3 Molecular procedures

2.3.1 Population samples and fly stocks

Specimens of A. fraterculus and A. obliqua were derived from fruits collected at eight sampling points distributed across Brazil (Table 1), which were maintained in plastic cages (21 x 13 x 13 cm) with vermiculite until pupation occurred. The cages were maintained under laboratory-controlled conditions of temperature (26°C ± 5°C), 65% relative humidity, and natural lighting. Adult flies were allowed to emerge and mature sexually for at least 10–15 days in a new cage with food (yeast, sugar, honey, and hydrolyzed protein) and water ad libitum. Mature adults were identified following the criteria outlined by Malavasi et al. (2000). Some specimens were subsequently stored at -80°C for molecular analyses. Total DNA extraction was performed with five females from each original population (Table 1) following the standard acid guanidinium thiocyanate-phenol‒chloroform protocol (Nelson and Krawetz, 1992).

Table 1

Samples of *Anastrepha obliqua* and *Anastrepha fraterculus* collected in different locations and used for population analysis.
Species	Locality	Coordinates		Samples
	Capanema/PR	53º 49' 04"W	25º 39' 49S	Ao_Cap1 Ao_Cap2
	Marialva/PR	51º 46' 53"W	23º 29' 48"S	Ao_Mari1 Ao_Mari2 Ao_Mari3
A. obliqua	Caetité/BA	42º 29' 33"W	14º 03' 50"S	Ao_Cae1 Ao_Cae2 Ao_Cae3 Ao_Cae4 Ao_Cae5
	Passos/MG	46º 37' 04"W	20º 43' 45"S	Ao_Pas1 Ao_Pas2 Ao_Pas3 Ao_Pas4
	São Thomé das Letras/MG	45º 01' 27"W	21º 43' 13"S	Af_Tho1 Af_Tho2 Af_Tho3 Af_Tho4 Af_Tho5
A. fraterculus	Pinheiros/ES	40º 12' 47"W	18º 24' 28"S	Af_Pin1 Af_Pin2 Af_Pin3 Af_Pin4
	Espírito Santo do Turvo/SP	49º 25' 54"W	22º 41' 30"S	Af_Tur1 Af_Tur2
	Criciúma/SC	49º 23' 47"W	28º 40' 17"S	Af_Cri1

2.3.2 Sequence analysis and phylogenetic sets

Given that several isoforms are produced by alternative splicing of dec-1 across different insect species, we selected a specific region on exon 3 that is common to all isoforms to investigate intraspecific variation and patterns of selection across different individuals and populations. To do so, primers (dc1_2371F: 5’- ATG TCT ATG GAT AGG CAA ATG − 3’/dc1_3236R: 5’- GGA GYG GCC AAG CCT AGC ATA TC -3’) were used to amplify this region in 27 flies from four populations of A. fraterculus and A. obliqua (Table 1). Amplifications were carried out using a Bio-Rad T100™ Thermocycler programmed for 35 cycles, including an initial denaturation step of 3’ at 94°C, followed by 34 cycles of 1’ at 94°C, 90” at 54.2°C, and 1’ at 72°C, with a final extension step of 72°C for 3’. The amplification products were checked on a 1% agarose gel containing ethidium bromide under UV illumination. Positive PCR products were PEG-purified (polyethylene glycol 8000) (Lis and Schleif, 1975) and cloned and inserted into E. coli DH5α using an InsTAclone kit (Fermentas) according to the manufacturer’s instructions. Positive clones were then amplified with standard M13 primers (M13F: 5’- GTA AAA CGA CGG CCA GT − 3’/M13R: 5’- GGA AAC AGC TAT GAC CAT G − 3’) following a protocol involving 1’ at 94°C, 34 cycles of 30” at 94°C, 30” at 56°C, and 60” at 72°C, with a final extension step of 3’ at 72°C. The products were subsequently subjected to PEG purification, and the resulting clones were Sanger sequenced (approximately 3 sequenced clones per sample) at Macrogen, Inc. (Seoul, Korea).

High-quality sequences derived from amplified clones were visually inspected for each sample. Because of the short size of the amplified product, we sequenced only the M13 forward primer. When there was variation among clones per sample (most often point changes), we retained only the one with the highest overall chromatographic quality.

To investigate molecular evolution and selection at different levels of taxonomic divergence, we analyzed two separate datasets, one that investigated levels of intra- and interspecific variation in two of the most important Anastrepha fraterculus group species and another that investigated patterns of molecular evolution of dec-1 across Cyclorrhapha (Diptera: Muscomorpha). Both datasets were independently aligned using MAFFT software, employing the L-INS-i method (Katoh et al., 2019).

The “Anastrepha” dataset was composed of 31 sequences derived from fragments of the third exon of dec-1 amplified from natural populations of A. obliqua and A. fraterculus (Table 1), which belong to the two most important branches in the fraterculus group (Congrains et al., 2023), as well as dec-1 sequences from A. ludens and A. grandis. The latter was included as an outgroup. The reason we included A. ludens here is not only because it was derived from the best curated genome available at the time but also because A. fraterculus has been considered a species complex, some of which are more related to A. ludens than to other A. fraterculus (Hernández-Ortiz et al., 2012; Hernández-Ortiz et al., 2015; Congrains et al., 2021; Congrains et al., 2023).

The “Cyclorrhapha” dataset includes complete dec-1 sequences derived from the transcriptomes of A. obliqua, A. fraterculus, and A. grandis, as well as the dec-1 genomic sequences of A. ludens annotated and curated here. The shorter dec-1 isoform (referred to as X3 here but representing the FC106 proprotein) was used in this dataset because it was the predominant isoform obtained from GenBank, since several species indicated below lack alternative isoforms. This dataset included not only putative dec-1 orthologs from Anastrepha and other Tephritidae available in GenBank, including Rhagoletis zephyria, Rhagoletis pomonella, Zeugodacus curcubitae, Bactrocera tryoni, and Ceratitis capitata but also from a few Drosophilidae, such as Drosophila melanogaster, D. pseudoobscura, and D. albomicans, as well as from Diopsidae Teleopsis dalmanni, treated as an outgroup within Cyclorrhapha (Table s1).

2.4 Phylogenetic and selection tests

The use of two distinct datasets enables the examination of diverse selection patterns over different evolutionary periods and divergence levels. These analyses started by investigating saturation levels among putative homologous sequences of each dataset using Xia's saturation test (Xia and Li, 1998) on DAMBE 7.3.11, using the GTR evolutionary model to assess genetic distances by comparing transition and transversion rates. This test takes into account saturation levels and transition/transversion rates, aiming to determine whether there was an underestimation of synonymous substitution rates (dS) with increasing evolutionary distance.

Phylogenetic relationships using maximum likelihood were inferred in PhyML v.3.0 (Guindon, 2010), with the best evolutionary model of nucleotide substitution for each set estimated using ModelTest-ng (Darriba et al., 2019), considering the Bayesian Information Criterion (BIC) (Schwarz, 1978). The topologies of each dataset were visualized with FigTree v.1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/). These phylogenetic inferences were used to investigate patterns of molecular evolution in dec-1 under two frameworks: CODEML and BEB implemented in PAML software (Yang, 1997; Chen et al., 2009) and the HyPhy package in DataMonkey (Weaver et al., 2018; Kosakovsky Pond et al., 2019). For all tested models (CODEML, BEB, and HyPhy), two categories of analysis were employed: a global estimate of gene evolution (i.e., across all taxa within the phylogeny) and a branch-site test, both of which investigate the ratio of nonsynonymous (dN) to synonymous (dS) substitutions. If this dN/dS ratio (or ω) is less than 1, the region is under purifying selection; if it is close to 1, it is neutral, and it is considered to be under positive selection when it is greater than 1.

The test for positive selection using the CODEML model considered the framework of the inferred ML topology for each set to contrast different evolutionary models using a hierarchical series of likelihood ratio tests (LRTs). The first test involves contrasting a null model (M0 null) of evolution that does not allow for sites to have ω > 1 (i.e., those that are experiencing positive selection) against an alternative model (MA) that allows for such occurrences. Other tests that make more explicit inferences about the model are hierarchically contrasted: M0 vs. M3, M1a vs. M2a, M7 vs. M8, and M8a vs. M8. These comparisons explore different assumptions about the ω ratio, including constant ω ratios in M0 (Goldman and Yang, 1994; Yang et al., 1998), discrete site classes in M3 (Yang et al., 2000), and positive selection in M2a (Nielsen and Yang, 1998; Yang et al., 2005). Further analyses were performed with M7 assuming a beta distribution of ω below 1 and M8 adding a category with ω > 1, suggesting potential positive selection (Anisimova et al., 2001). In the final comparison, M8a vs. M8 contrasted the presence of an extra codon class with ω = 1 against an additional category with ω > 1, providing a comprehensive assessment of positive selection likelihoods (Swanson et al., 2003; Wong et al., 2004).

Posterior probabilities that sites are under positive or purifying selection were estimated using the Bayes Empirical Bayes (BEB) method to provide additional evidence regarding potential regions and codons of dec-1 evolving in response to selection (Yang et al., 2005). A Likelihood Ratio Test (LRT) estimated the posterior probability that a nucleotide site evolved under positive selection (significant if P > 0.95).

The HyPhy package also identifies signatures of positive selection or purifying selection by investigating the dN/dS ratio but allows different branches to have distinct ω values. Multiple methods were employed, specifically FEL (Kosakovsky Pond and Frost, 2005) and FUBAR (Murrell et al., 2013), which assume constant selection pressure per site across the phylogeny to infer ω; MEME, which investigates ω across individual sites on a proportion of branches using a mixed-effects maximum likelihood approach (Murrell et al., 2012); BUSTED, which tests whether at least one branch has experienced positive selection in specific sites (Murrell et al., 2015); and RELAX, which tests for the relaxation or strengthening of natural selection in specific branches (Wertheim et al., 2014). Amino acid replacements were further analyzed by TreeSAAP v.3.2, which investigates whether amino acid changes lead to radical physicochemical changes at the protein level (Woolley et al., 2003) on topologies generated using baseml on PAML (Yang, 1997). Methods by Xia and Li (1998) and McClellan and McCracken (2001) were applied to infer amino acid substitution patterns, focusing on significant positive z scores in categories six to eight, indicating the most extreme changes in structure or function. All conceptual references on physicochemical properties were extracted from Gromiha and Selvaraj (1997), Gromiha et al. (1999), Woolley et al. (2003), Novotny et al. (2007), and Beckstead et al. (2009). Departures from neutrality impact the distribution of radical/nonradical changes in tip and interior branches (younger and older lineages, respectively, following the expectations under neutrality and coalescence) the same way as it is for synonymous and nonsynonymous substitutions (Takahata, 1990; Yang, 2002). We tested the difference in this distribution using Fisher's exact test on a 2×2 contingency table (Templeton, 1996), which is a more refined version of the (McDonald and Kreitman, 1991) used to study intra- and interspecific polymorphisms. We expected that under neutrality the ratio of radical to nonradical changes should be the same in tip haplotypes (younger haplotypes) as in interior haplotypes (older haplotypes). Under purifying selection, we would expect to see fewer radical changes in interior branches, whereas when there were recurrent events of directional positive selection or balancing selection, we would expect more radical changes in the interior rather than on younger nodes, similar to patterns expected for synonymous/nonsynonymous substitutions (Fay and Wu, 2005) (Sobrinho and de Brito, 2012). The same contingency tests were used to contrast rates across different portions of the dec-1 gene to investigate whether they would show different rates of radical to nonradical amino acid changes.

3.1 Structural characterization of A. fraterculus and A. obliqua dec-1

Genomic and transcriptomic data, as well as direct sequencing of PCR products, were employed to determine the dec-1 genomic sequence in A. fraterculus and A. obliqua. In the absence of genomic information for these species at the time these experiments were performed, we used genomic sequences from A. ludens to validate the transcriptome and sequencing data of dec-1 exons in A. obliqua and A. fraterculus. Comparative analysis and alignment of the A. ludens genomic data with those of the other two species enabled inference of the presence of six exons in these species, one more than that described for D. melanogaster.

Figure 1 provides a graphical representation of the dec-1 structure, including exon lengths for Anastrepha fraterculus. Exon lengths, coding sequences (CDSs), and putative proteins show slight size differences among the three Anastrepha species (Table 2). We identified three distinct dec-1 isoforms in A. fraterculus and A. obliqua, designated as X1, X2, and X3, following the designations for putatively homologous isoforms of this gene from other Tephritidae flies, such as R. zephyria (Gene ID 108362233), R. pomonella (Gene ID: 118735351), and C. capitata (Gene ID: 101457983). These three isoforms are closely aligned with isoforms A, B, and C of the six currently described for D. melanogaster (Flybase: FBgn0000427).

The first four exons in the 5′-upstream region, along with the canonical AUG initiation codon on exon-1, are shared by Anastrepha isoforms. Differences arise in the arrangement of the last two exons in the 3' downstream region, particularly at exon 5, which exhibits variations in length due to alternative internal splicing. The putative isoforms generated by alternative splicing in A. fraterculus measure 1472 aa for X1, 1192 aa for X2, and 955 aa for the shorter X3 isoform and correspond to the fc125, fc177, and fc106 proproteins, respectively. In A. obliqua, the corresponding values were 1526 aa for X1, 1181 aa for X2, and 950 aa for X3 (Table 2). These isoform labels lack a clear rule, and we followed those of R. zephyria (Gene ID 108362233) and R. pomonella (Gene ID 118735351). It should be noted that the A. obliqua dec-1 isoforms in GenBank are named differently since their X1 is what we name here X2, with 1191 aa, whereas the GenBank X2 isoform has 954 aa and probably corresponds to what we refer to as the X3 isoform (fc106).

Table 2

Arrangement of exons and CDS (Coding DNA Sequence), and respective inferred proteins (amino acid sequence) of the three isoforms (X3, X2, and X1) detected in *dec-1* of *Anastrepha obliqua* fruit flies and *Anastrepha fraterculus*. The letters a, b, and c in exon 5 represent different splice points.
	Isoforms
	A. obliqua				A. fraterculus
		X3	X2	X1			X3	X2	X1
Exon 1	9	✓	✓	✓		9	✓	✓	✓
Exon 2	189	✓	✓	✓		189	✓	✓	✓
Exon 3	2286	✓	✓	✓		2340	✓	✓	✓
Exon 4	348	✓	✓	✓		309	✓	✓	✓
Exon 5(a)	18	✓				18	✓
Exon 5(b)	194		✓	✓		197		✓	✓
Exon 5(c)	1552			✓		1372			✓
Exon 6	517		✓			532		✓
CDS size	-	2850	3543	4578		-	2865	3576	4416
Protein size	-	950	1181	1526		-	955	1192	1472

Two primary motifs were identified in the putative DEC-1 proteins using the Pfam database (with P value < 0.05), denoted in D. melanogaster as the N-terminal region (Pfam ID: DEC-1 N; Description: PF04625) and the C-terminal region (Pfam ID: DEC-1 C; Description: PF04626). In contrast to D. melanogaster DEC-1, Anastrepha species did not have a motif in nine repeat regions (Pfam ID: Dec-1, Description: PF04624), whereas a novel RNAse P Rpr2/Rpp21/SNM1 subunit domain motif (Pfam ID: Rpr2, Description: PF04032) was identified on the longer isoform (X1) of A. obliqua (Figure S1). A comparative analysis of motifs detected in A. fraterculus and putative orthologs from various species revealed different degrees of conservation. The N-terminal motif displayed identity percentages ranging from ~ 27% (Drosophila melanogaster) to ~ 97% (Anastrepha ludens), while the C-terminal motif showed percentages ranging from ~ 17% (Teleopsis dalmanni) to 75% (A. obliqua) (Table 3). Both motifs are highly conserved in the genus Anastrepha, with identities close to or above 90% in the N-terminal motif. However, their similarity to motifs from other flies is much lower, including those from other Tephritidae, a pattern that is more pronounced in the C-terminal region. The repeat region motif, exclusive to Drosophila, varied in the number of copies across different species, with D. pseudoobscura being more similar to D. melanogaster but having only two repeat regions, whereas D. albomicans lacks the repeat region altogether.

Table 3

Identity of putative *dec-1* domains (fc106, which corresponds to the X3 isoform) of *Anastrepha fraterculus* sequences and other putative orthologs in Cyclorrhapha.
Species	Family	Protein size	Motif N-terminal	Motif C-terminal
Anastrepha obliqua	Tephritidae	950	88.69	75.00
Anastrepha ludens	Tephritidae	984	97.86	71.26
Anastrepha grandis	Tephritidae	972	91.42	64.20
Rhagoletis zephyria	Tephritidae	1071	64.82	48.68
Rhagoletis pomonella	Tephritidae	1071	64.82	48.68
Zeugodacus cucurbitae	Tephritidae	1106	64.74	46.24
Ceratitis capitata	Tephritidae	1120	63.86	46.11
Bactrocera tryoni	Tephritidae	1125	64.88	39.43
Drosophila melanogaster	Drosophilidae	950	27.07	29.01
Drosophila albomicans	Drosophilidae	921	30.57	27.10
Drosophila pseudoobscura	Drosophilidae	949	29.32	24.84
Teleopsis dalmanni	Diopsidae	1347	29.27	17.71

3.2 Phylogenetic tests

The results from Xia's saturation test suggest that there is no significant underestimation of dS rates with increasing evolutionary distance at the levels of the contrasts investigated here. The maximum likelihood phylogenetic inference of the “Anastrepha dataset” limited to the amplified portion of the 3rd exon using the HKY + G4 substitution model placed all the A. obliqua samples together, while the A. fraterculus samples were divided into three clusters (Fig. 2A). The most internal branch included all A. obliqua and two samples of A. fraterculus from central Brazil, which we will refer to as the A. fraterculus Clade VII branch. We will refer to these different A. fraterculus clades by names that loosely correspond to branches that seem to match the phylogenetic and geographic location of said branches in samples from another study (Congrains et al., 2023). This group was sister to other specimens that form the A. obliqua branch, which is generally distributed in southern and central Brazil and is related to A. ludens. There is also an external branch with samples from the Brazilian coast that forms the A. fraterculus Clade VI branch and a branch that connects different species in the fraterculus species group, dubbed Multispecies branch. The maximum likelihood tree for the "Cyclorrhapha dataset” using the TPM3uf + I + G4 substitution model was based on the whole dec-1 gene and confirmed the monophyly of Anastrepha, with A. grandis as basal to the other species in the fraterculus group (Fig. 2B). This topology places Anastrepha closer to Rhagoletis than to the other Tephritidae investigated here, Ceratitis and Bactrocera (Fig. 2B); however, in this inference, Ceratitis is basal to Tephritidae, which is different from the standard phylogeny for these taxa (Segura et al., 2006).

3.3 Selection tests

3.3.1 Global analysis of dec-1

ML phylogenetic trees were inferred to investigate patterns of molecular evolution and positive selection on the dec-1 gene. The phylogenetic tree previously inferred allows for the proper contrast of the likelihood of different evolutionary models. The significant contrast between the M0 model (which assumes a uniform rate of evolution) and the M3 model (which incorporates discrete substitution rates and allows for positive selection) (M0 vs M3) across both the “Anastrepha dataset” and "Cyclorrhapha dataset" suggests that there is evolutionary rate heterogeneity along the dec-1 gene (p values < 0.0001). On the other hand, we failed to detect significant differences between the M1a and M2a evolutionary models for either set, which indicates insufficient evidence of positive selection affecting the whole gene. Although the contrast between M7 and M8 for the more closely related “Anastrepha” dataset was borderline significant (p value = 0.075), it was highly significant for the "Cyclorrhapha dataset” (p value = 0.00), suggesting that there are specific sites under positive selection in dec-1.

The results of the BEB analysis revealed 7 possible codons with greater probabilities of being under positive selection in the “Anastrepha dataset” (Fig. 3), although a single codon, 523G (ω = 1.27), retained a significant 95% posterior probability.

As revealed by the BEB analysis, the global analysis performed on the “Anastrepha dataset” conducted by the FUBAR test in HyPhy, which estimates dN/dS substitution rates per site, identified the same codons under positive selection (523, 560, 656, 743, and 761), with posterior probabilities of 97.6%, 91.3%, 93.9%, 92.5%, and 94.2%, respectively, but it also identified codon 617 (with 93.5% posterior probability). The BUSTED test suggested that 12.7% of the branches were evolving under neutral selection, while 87.3% were under purifying selection, and the majority of codons on exon 3 were under purifying selection (Fig. 3), particularly those associated with the proprotein s20, which was confirmed by FEL results that suggested no constant positive selection pressure along the gene. Despite these results, the MEME test suggested that codons 617, 656 and 724 underwent episodic or diversifying selection, and the two former codons were also identified as being under positive selection by FUBAR. This suggests that positive selection may have impacted these codons at specific evolutionary times and episodically without affecting the entire gene region.

A global analysis by PAML on the “Cyclorrhapha dataset”, which investigates more distantly related taxa, identified codons 1309G and 1314 V as being under positive selection, with posterior probabilities of 96% and 98.2%, respectively. These findings were further supported by FUBAR analysis, which showed a probability of 96.7% for codon 1314 V, and by FEL, which identified an additional seven sites under diversifying selection. Additionally, MEME revealed 13 sites under episodic diversifying selection, affecting either the coding regions for s25 or s60 proproteins but not s20, which are basically the same sites shown in the BEB graph (Fig. 3); however, in this test, these sites are possibly evolving under positive selection but fail to reach posterior probabilities greater than 95%.

3.3.2 Branch selection analysis

The branch-site selection analyses of the “Anastrepha dataset” dataset identified selection episodes in the A. obliqua branch, the A. fraterculus Clade VII branch, and in the branch that separates A. fraterculus Clade VI and the other A. fraterculus species (Multispecies branch). It should be noted that A. fraterculus was paraphyletic in our analysis, which corroborates other analyses that suggest that A. fraterculus is a species complex (Selivon and Perondini, 1998; Selivon et al., 2004; Hernández-Ortiz et al., 2012; Congrains et al., 2021; Selivon et al., 2022; Congrains et al., 2023). The overall ω value of 0.1280 indicated that most branches in the “Anastrepha dataset” dataset were under purifying selection with a probability of 97%. The RELAX test indicated that the A. fraterculus Clade VII branch was under intensified selection (K = 2.03), while the Multispecies branch was under relaxed selection (K = 0.49), whereas the A. obliqua branch had K = 0, indicating an absence of evidence for relaxation or intensification of selection. Despite that, this branch showed a significant ΔLRT (4.27685; p = 0.03863) on the branch-site test, with codon 724 having a 95% posterior probability of being under positive selection. On the other hand, the A. fraterculus Clade VII branch and the Multispecies branch showed no signs of positive selection.

We investigated three branches in the “Cyclorrhapha dataset”: one that defines the genus Anastrepha (Anastrepha branch), which showed a single codon, 526N (96.8%), with a significant probability of being under positive selection (ΔLRT = 9.3923, p = 0.0022). There was no evidence of selection on the Trypetinae Branch, whereas 20% of the codons showed evidence of positive selection (ΔLRT = 36.539; p = 0.0000) in the Tephritidae Branch, including codons 4A (98,6%), 52L (99.6%), 100T (95,7%), 127Q (96,3%), 172N (99.5%), 182L (99.6%), 211 H (95,7%), 277S (99.4%), 297 (96,7%), 310I (99.2%), 318P (95,0%), 419V (98,4%), 448M (99.8%) and 523K (98,7%), although the RELAX test failed to find significant evidence of relaxation or intensification of selection in any of the three branches analyzed at this level.

3.3.3 Measurement of the physicochemical magnitude of amino acid substitutions

We found evidence that radical amino acid changes potentially altered some physicochemical properties (global z values equal to or greater than six) at different evolutionary levels. In the “Anastrepha dataset”, 22 codons exhibited significant z scores (with z = 6 and 7, p < 0.01), most of which affected the dec-1 region that codes for s20 and only 7 affecting the regions that code for s60. Furthermore, radical mutations on s20 are more frequent on interior nodes than on tips, contrary to nonradical mutations on s20 (Fisher’s exact test P = 0.0214) and any mutations on s60 (Table 4). The difference in the distribution of radical mutations in tips and interiors between s20 and s60 was also significant (Fisher’s exact test P = 0.0030), since the latter showed no radical mutations on internal branches (Table 4). This pattern is different from what is observed in the "Cyclorrhapha dataset”, which showed more than 400 sites with radical physicochemical alterations and more than 1100 radical mutations (Table 5). Although the latter dataset showed a greater percentage of radical mutations on tips than on internal branches, there was no significant difference in these relative rates across different regions of dec-1. However, there are different rates of mutation across these regions, with pro-protein s60 showing significant radical physicochemical alterations in close to 60% of the codons (Table 5). However, there was a significant contrast in the pattern of radical mutations for the s20 proprotein region when we compared the “Anastrepha dataset” and the “Cyclorrhapha dataset”, since the latter has many more radical changes on interior branches (Chi-Square 15.1, P = 0.001733), which was not observed on s60.

Table 4

Distribution of radical and nonradical changes, defined on TreeSAAP, across different parts of *dec-1* using the *Anastrepha dataset*, separated by interior branches and tip branches.
	Radical		Non-Radical
	tip	interior	tip	interior
s20	10	13	7	0
s60	24	7	16	11
Total	34	20	23	11

Table 5

Distribution of radical and nonradical changes, defined on TreeSAAP, across different parts of *dec-1* using the Cyclorrhapha *dataset*, separated by interior branches and tip branches.
	Radical		Non-Radical
	Tip	interior	tip	interior
Signal peptide	11	11	34	30
s25	86	62	239	199
s20	149	138	363	293
s60	378	275	776	580
Total	624	486	1412	1102

Protein-coding genes experience a myriad of selection pressures. Deleterious mutations may be expected, whereas those enhancing fitness can become fixed and traced through evolutionary signals of positive selection (Sémon and Wolfe, 2007; Conant and Wolfe, 2008; Studer and Robinson-Rechavi, 2009). The characterization of the chorionic gene, defective chorion-1 (dec-1), in Anastrepha and its comparison to other Tephritidae and Diptera allowed us to investigate which forces have shaped its evolution at different evolutionary scales and identify conserved gene regions, as well as some possibly subjected to positive selection events.

Structural analysis of the dec-1 gene revealed 6 exons and three isoforms in Anastrepha fraterculus and A. obliqua, a pattern similar to what has been shown for Drosophilidae, such as D. melanogaster, D. yakuba, and D. virilis. Transcriptomic and sequencing data from laboratory samples of A. fraterculus and A. obliqua provided a more detailed characterization of the dec-1 gene, improving our understanding of gene boundaries and expressed isoforms in this genus. Previous studies have shown the structural conservation of dec-1, so many transgenic studies have indicated its functional interchangeability across distant Drosophilidae species with regard to fertility and wild-type eggshell morphology (Badciong et al., 2001). Here, we show that despite amino acid sequence divergence, especially among more distantly related species, such as Drosophilidae and Tephritidae, there is evidence of preserved protein functionality throughout evolution across Cyclorrhapha (Badciong et al., 2001). This is supported by evidence of purifying selection acting across its length at different evolutionary levels. Despite the overall evidence of selective constraint, especially in some portions of the gene associated with some proproteins, occasional episodes of positive selection suggest the impact of dynamic evolutionary processes, shedding new light on the adaptive mechanisms of dec-1.

The overall pattern detected by global tests of selection on dec-1 across different evolutionary hierarchies indicates that purifying selection seems to be the driving force along its five protein-coding exons throughout most of its history, although there is evidence of positive selection in some portions of the gene and at different points in time. This was shown in PAML, as well as on the global BUSTED test, which indicates that overall synonymous substitution rates are higher than nonsynonymous rates (dN/dS < 1) and that most sites are evolving under purifying selection, although some sites have evidence of evolving under neutral conditions. Considering the functional role of dec-1, its isoforms, and protein derivatives in oogenesis (Noguerón et al., 2000), particularly in the formation of the chorion and vitelline membrane (Bauer and Waring, 1987), it is reasonable to expect a prevalence of purifying selection across its length and history. This becomes even more apparent when we consider that mutations in exon 3, which may affect all three isoforms, can lead to sterility or egg anomalies (Waring et al., 1990; Kim et al., 2002; Spangenberg and Waring, 2007), underscoring the biological significance of the products of this gene. Nonetheless, the results from MEME, as well as the contrast between the M7 and M8 models in PAML, suggest that some sites are evolving under positive selection.

An investigation of different rates of evolution across the gene using the Bayes Empirical Bayes model (BEB) in PAML (Yang, 1997), along with various tests conducted using HyPhy (Kosakovsky Pond et al., 2019), revealed that even though the majority of codons are evolving under purifying or neutral evolution, there are codons that experienced positive selection or episodic diversifying selection. A better understanding of the potential impact of changes across dec-1 should consider the different proproteins and derivatives produced by the three isoforms normally found at different developmental times, which may have different functions. For instance, along with the former, fc106 is much more abundant than are fc125 and fc177 and is found in oogenesis stages 9 and 10. On the other hand, fc177 expression increases in stages 11 and 12 (Hawley and Waring, 1988; Mauzy-Melitz, and Waring, 2003). The fc106 product is cleaved into the s25 and s80 proproteins during late oogenesis stage 10, but s80 is cleaved later into s60 and s20, so much so that s25, s60, and s85 are deposited in the egg layers, whereas s20 does not seem to be a structural component of the eggshell, although it is detected in vesicles within the oocyte (Spangenberg and Waring, 2007). This might be one of the reasons why s20 seems to be more conserved than the other dec-1 proproteins.

Analyses such as BEB (Fig. 3) suggest that evidence of positive selection between more distantly related taxa is restricted to the beginning of proprotein s25, corresponding to exon 2, and the middle portion of exon 3, which putatively encodes the proprotein s60 but is also part of the s95 proprotein. Significant positively selected sites were detected only for the putative s20 proprotein in the more closely related Anastrepha dataset. Similar results, which in general point to the same sites under selection, were detected by the FUBAR and MEME tests. A similar pattern was revealed by TreeSAAP, which identified the same codons, 523 and 656, in the “Anastrepha dataset” and codon 1314 in the "Cyclorrhapha dataset" group, among other codons that showed evidence of positive selection.

Physicochemical changes in codons 523 and 656 could affect all three dec-1 isoforms since they all share sequences from exon 3, but each derivative proprotein would be differentially affected since only the s20 derivative (produced from FC106), s85, and the initial regions of the s95 (FC125) derivatives would retain these domains. In contrast, the 1314 V codon found in the “Cyclorrhapha dataset”, located at the end of exon 5 (equivalent to exon 4 in D. melanogaster), impacts the C-terminal region of DEC-1, a part of the s80 derivative, s60 and s85 proproteins. Changes in the s20 derivative could disrupt the organization of early protein interactions in the egg assembly process (Badciong et al., 2001) and induce female sterility, similar to alterations in the s95 derivative (Mauzy-Melitz and Waring, 2003). Furthermore, changes in the function of the s85 derivative may destabilize the vitelline membrane (Waring et al., 1990) and the tripartite endochorion, as proper aggregation of chorion proteins to form the pillars, floor and reticulated roof does not occur (Mauzy-Melitz and Waring, 2003). Overall, the identified mutations occur in regions that might compromise egg formation and viability (Hawley and Waring, 1988).

Different rates of synonymous and nonsynonymous changes potentially indicate regions subject to different selection pressures, but it is not known whether these changes impact fitness. Another way to look at these changes is to evaluate whether inferred amino acid substitutions produce potentially radical physicochemical changes, which are more likely to have a fitness impact, since they are more likely to be selected against (Smith, 2003), particularly in large populations (Weber and Whelan, 2019). In general, regions that are under purifying selection are less likely to undergo radical amino acid changes since they are more likely to alter protein structure and, potentially, function, whereas positive selection might favor radical amino acid changes. Therefore, in conserved regions, radical changes are more likely to be found on the tips of trees, which would indicate that they are evolutionarily recent, whereas nonradical changes could be found in internal branches just as likely as on the tips (Pupko et al., 2003).

Overall, the dec-1 results indicate that radical changes are more likely to be found on tree tips than on the interior, and this finding is consistent across the whole gene. The one distinction we observed is related to the part of the gene that codes for the s20 proprotein, since there are several internal amino acid changes segregating in the “Anastrepha dataset”, but not for s60, whereas that pattern is not observed when we compare more distantly related taxa. To explain these differences, we should consider the impact of selection on the underlying genetic variation. Positive selection may lead to the sweep of other variation in the population, but the speed with which that happens depends on whether it is a hard or a soft sweep, and it is not necessarily trivial to distinguish them (Schneider et al., 2021). On the other hand, purifying or background selection does not remove neutral variation, or even deleterious mutations at the same rate as with positive selection, showing larger variance (Cvijović et al., 2018). In this case, we would expect that regions that are subject to positive selection might not have any segregating radical changes when we compare closely related specimens but rather greater rates of evolution when comparing more distantly related specimens. In regions under purifying selection, however, we would still see segregating variation between closely related species, but that would not be reflected in higher rates of evolution; rather, we would not see that reflected when comparing more distantly related taxa because that would not be favored by selection.

Another important aspect to consider when using rates of synonymous and nonsynonymous changes to investigate selection in recently diverged lineages is that dN/dS is a measure of selective pressure that contrasts rates of fixed substitution between two homologous sequences (Kimura, 1977), and as such, it was designed for analyzing more divergent lineages; otherwise, it might suffer from the stochasticity of the mutation/selection process. The comparison of these changes between more closely related taxa, generally by Ka/Ks, may be influenced by other factors that are not directly associated with selection intensity, such as population expansion or contraction, and other evolutionary forces that may even affect neighboring regions (Kryazhimskiy and Plotkin, 2008). Therefore, it is relevant to contrast patterns of evolution considering adequate evolutionary models that could differentiate evidence of selection from its relaxation. In this framework, the RELAX analysis indicated that clade 2 in the “Anastrepha dataset” has an intensification of selection (K = 2.03), and no clades in the "Cyclorrhapha dataset" showed evidence of intensification or relaxation of selection. Intensified selection pressures can push ω values away from neutrality (ω = 1), while relaxed selection allows for the accumulation of neutral or slightly deleterious genetic variations, promoting genetic diversity within populations. This relaxation of selection potentially influences differentiation by providing raw material for adaptation to new environments and conditions (Templeton, 2008).

Global analysis of selection detected potential sites experiencing positive and/or episodic diversifying selection, but because the average ω ratio is rarely above 1, detecting positive selection in specific lineages considering a phylogenetic framework may be more effective, as it targets those lineages specifically (Álvarez-Carretero et al., 2023). Branch-site tests in PAML identified specific branches with evidence of positive selection, such as the lineage separating A. obliqua and A. fraterculus in the “Anastrepha dataset” and all three tested branches in the Cyclorrhapha dataset. In all cases, we have evidence of a few lineages experiencing episodic positive selection in a background of purifying selection.

The pattern of purifying selection with a few events of positive or diversifying selection identified in the dec-1 gene is also observed in genes directly and indirectly related to reproduction and sex determination, such as transformer (tra) and fruitless (fru) in Drosophila (McAllister and McVean, 2000; Kulathinal et al., 2003; Parker et al., 2014) and even in Anastrepha (Sobrinho and de Brito, 2010; Sobrinho and de Brito, 2012). More importantly, this trend has also been observed for several choriogenic genes investigated in Anastrepha, such as Cp15, Cp16, Cp19, and Cp38, whereas vitelline membrane genes, in general, have been experiencing only purifying selection, with the possible exception of a recently duplicated gene (Vm26Ab and Vm26Aa’) in the genus Anastrepha (Gonçalves et al., 2013).

Similar results of vitelline membrane genes under purifying selection and chorionic genes under positive selection were also described for Drosophila, which has been attributed to the fact that vitelline membranes are internal to the chorion and therefore more subject to purifying selection, whereas chorionic proteins may have important roles with regard to desiccation and oxygen transport that could be subject to positive selection depending on environmental cues (Jagadeeshan and Singh, 2007). Notably, portions of dec-1 associated with s25, which are expressed earlier in egg development, seem to be more conserved than portions of the gene related to the s60 proprotein, which is produced later and seems to be deposited in more outer chorionic layers. However, most of the positive sites detected in the branches that define Tephritidae, as well as the genus Anastrepha, are associated with the s25 proprotein, which might be relevant when we consider the great variation across species of Anastrepha in terms of chorion morphology (Selivon and Perondini, 1998; Selivon et al., 2004; Figueiredo et al., 2013).

Despite the general pattern of purifying selection, the evidence of positive selection on dec-1 in Tephritidae, particularly among different Anastrepha species, is relevant because of the great diversity of the inner chorion patterns among Anastrepha species, which are much more diverse than those found in other Tephritidae (Murillo and Jirón, 1994; Selivon and Perondini, 1998; Dutra et al., 2011; Figueiredo et al., 2017). Although rapidly evolving genes in general have been associated with sexually selected traits or sexual conflict across different taxa (Swanson and Vacquier, 2002; Clark et al., 2006; Panhuis and Swanson, 2006; Turner and Hoekstra, 2006), adaptive responses to environmental cues can also play an important role (Jagadeeshan and Singh, 2007).

It is important to emphasize that phylogenetic inferences derived from dec-1 are not meant to help elucidate relationships among the taxa studied here, which are generally well established, at least at higher levels. Rather, they are used to establish a framework that enables the investigation of molecular evolutionary patterns. Nonetheless, we should mention that the relationships depicted among the more diverse taxa in general reflect what has been indicated in the literature, with a closer relationship between Anastrepha and Rhagoletis, although the phylogenetic tree here produced placed Ceratitis basal to Tephritidae rather than on a clade with Bactrocera and Zeugodacus (Virgilio et al., 2015), a more accepted relationship among Toxotrypanini (Mengual et al., 2017).

The overall phylogenetic pattern observed, with short internal branches even between more distant groups, provides a crucial backdrop for understanding the broader evolutionary context of the dec-1 gene and its potential functional implications, especially for more closely related lineages, such as the inferences among Anastrepha specimens. Our results indicated that the samples of A. fraterculus we investigated seemed to be separated into three different lineages. It has been suggested that A. fraterculus is a species complex with several cryptic species, with at least three in Brazil that can be identified via morphometric methods (Hernández-Ortiz et al., 2012; Hernández-Ortiz et al., 2015). Although we did not investigate different morphotypes in this study that would enable us to compare these taxa to the ones described elsewhere (Hernández-Ortiz et al., 2015; Prezotto et al., 2019), the topology we found is remarkably similar to what has been described using transcriptomic and genomic data (Congrains et al., 2021; Congrains et al., 2023), which identified three distinct branches for A. fraterculus while supporting a single A. obliqua branch. These diverse A. fraterculus lineages may be subject to distinct selective pressures (Smith-Caldas et al., 2001; Manni et al., 2015), despite their recent divergence. This detailed phylogenetic context sets the stage for understanding the evolutionary dynamics of the dec-1 gene within specific Anastrepha lineages, although evidence of intraspecific gene flow among these lineages (Díaz et al., 2018; Congrains et al., 2023) may limit our ability to investigate that adequately.

Shifting focus to broader implications, a quest for mutations fixed by positive selection events can help identify novel functions and differentiate species (Sémon and Wolfe, 2007; Conant and Wolfe, 2008). In the context of a structural gene related to egg and chorion development, these changes may not be readily discernible or confer adaptive advantages, especially due to the complex interactions between genotype, phenotype and environmental influences. Therefore, comprehending how reproductive genes are influenced by selective forces through selection analyses enables the identification of this genotypic and potentially phenotypic variation in the genome. Furthermore, these genes can serve as markers in the context of species differentiation amid phylogenetic uncertainties. Remarkably, dec-1 produces adequate phylogenetic signals at different evolutionary levels across Tephritidae, but it seems to distinguish among different lineages of A. fraterculus for which there are only a handful of informative markers, despite the plethora of genes investigated (Congrains et al., 2021; Congrains et al., 2023). Understanding its structure, isoforms, and evolutionary patterns across broader sampling also sets the stage for its establishment as a viable adequate marker to study variation across Anastrepha. It also provides insight into reproductive biology, which could foster the development of genetically modified females through transgenes, similar to what has been done in Drosophila, which could be important for population management, particularly in pest insects such as those of the Tephritidae family.

Funding: The authors have been supported by grants and fellowships from the following Brazilian research founding agencies: Coordination for the Improvement of Higher Education Personnel - CAPES to M. C. B., S.C.-E., and R.A. B. (PVE 056/13); State of São Paulo Research Foundation - FAPESP (2018/06611-5, 2022/12583-0) and CNPq (313504/2017-2) grants to R. A. B. and CNPq fellowships to E. M. S. and R. A. B.

Competing interests: The authors have no competing interests to declare that are relevant to the content of this article.

Author Contributions: Bisio, M. de C. and de Brito, R. A. designed the experiments. Bisio, M. de C., Santos, E. M. dos, Chahad-Ehlers, S., Santos. C. A., and de Brito, R. A. wrote the manuscript. Bisio, M. de C., de Brito, R. A., Santos, E. M. dos and Santos, C. A. performed different data analyses. All the authors have read and agreed to the final version of the manuscript. Bisio, M. de C. and Santos, E. M. dos, share the first authorship of this manuscript.

Authorship: Bisio, M. de C. and Santos, E. M. dos, share the first authorship of this manuscript.

Acknowledgments: We are grateful to Dr. Scott Geib for the data shared with us and with people from the Population Genetics and Evolution Lab, UFSCar, and Dr. Julian Dupuis, Univ. of Kentucky, for invaluable comments on a previous version of this manuscript.

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Molecular evolution and genetic diversity of defective chorion 1 in Anastrepha fraterculus and Anastrepha obliqua (Diptera, Tephritidae)

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Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Identification of the defective chorion 1 gene in Anastrepha

2.2 Characterization and molecular structure of dec-1

2.3 Molecular procedures

2.3.1 Population samples and fly stocks

2.3.2 Sequence analysis and phylogenetic sets

2.4 Phylogenetic and selection tests

3. Results

3.1 Structural characterization of A. fraterculus and A. obliqua dec-1

3.2 Phylogenetic tests

3.3 Selection tests

3.3.1 Global analysis of dec-1

3.3.2 Branch selection analysis

3.3.3 Measurement of the physicochemical magnitude of amino acid substitutions

4. Discussion

Declarations

References

Additional Declarations

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Version 1