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Research Article
A new perspective on the phylogeny of Polietina Schnabl & Dziedzicki (Diptera: Muscidae): integrating morphological and molecular evidence
expand article infoKirstern Lica Follmann Haseyama‡, Claudio José Barros de Carvalho§, Elaine Della Giustina Soares|, Silvio Shigueo Nihei
‡ Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
§ Universidade Federal do Paraná, Curitiba, Brazil
| Universidade Federal da Integração Latino-Americana, Foz do Iguaçu, Brazil
¶ Universidade de São Paulo, São Paulo, Brazil

Corresponding author: Kirstern Lica Follmann Haseyama ( licahaseyama@gmail.com )

Academic editor: Arianna Thomas-Cabianca
© 2025 Kirstern Lica Follmann Haseyama, Claudio José Barros de Carvalho, Elaine Della Giustina Soares, Silvio Shigueo Nihei.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
ZooBank: urn:lsid:zoobank.org:pub:79296BC8-424B-40B7-A663-419442205F6E
Open Access

Abstract

Polietina is a genus of Muscidae found in the New World, particularly in the Neotropical region. The genus is currently classified within the subfamily Muscinae and consists of 15 species whose phylogenetic relationships have been previously studied using morphological data. In our study, we conducted a phylogenetic analysis based on 37 morphological characters sourced from the literature and eight molecular markers (12S, 16S, 18S, 28S, cytochrome C oxidase subunit I, cytochrome b, region 4 of carbamoyl-phosphate synthetase-aspartate transcarbamoylase dihydroorotase, and elongation factor 1-ɑ), totaling 5366 characters. We obtained molecular data for eight species of the genus and four outgroup taxa from original sequencing and public repositories. We used Bayesian posterior probabilities to estimate the topology, as follows: (P. flavidicincta ((P. rubella (P. concina (P. wulpi, P. orbitalis))) (P. steini ((P. flavithorax (P. ponti, Polietina sp.)) (P. major (P. prima (P. bicolor (P. minor (P. univittata, Polietina sp.))))))))). The reciprocal monophyly of all species for which more than one identified sequence was available is supported by our results. Our analysis largely supports the previously published hypotheses regarding the phylogeny of Polietina. However, major differences were observed between the locations of P. flavidicincta and P. flavithorax. Additionally, we discuss the identity of unidentified Polietina specimens with sequences published in GenBank or new sequences produced as part of our study.

Key words

Bayesian posterior probabilities, biodiversity, COI, Genbank, house flies, Muscinae, Neotropical region

1. Introduction

Polietina Schnabl and Dziedzicki, 1911 is a genus of flies primarily found in the tropical forests of Central and South America, with three species also occurring in Mexico and the Nearctic region (Löwenberg-Neto and de Carvalho 2013). This genus belongs to Muscinae subfamily (Diptera, Muscidae) (Couri and de Carvalho 1997; Nihei and de Carvalho 2007b; Haseyama et al. 2015) and is considered monophyletic based on both morphological and molecular evidence (Nihei and de Carvalho 2007a, b; Haseyama et al. 2015; de Carvalho and Haseyama 2018). Polietina has been revised twice over the last 30 years (Couri and de Carvalho 1997; Nihei and de Carvalho 2007a) with a single new species described since the last revision (Polietina ponti de Carvalho and Haseyama, 2018). Additionally, de Carvalho and Haseyama (2018) proposed a synonymy between Polietina prima (Couri and Machado, 1990) and Polietina nigra (Couri and de Carvalho, 1996), resulting in a genus currently comprising 15 species.

The phylogenetic relationships of Polietina were first examined using morphological characteristics by Nihei and de Carvalho (2007a), who analyzed 13 of the 15 recognized species at the time due to the unavailability of specimens of Polietina basicincta (Stein, 1904) and Polietina mellina (Stein, 1904). Their dataset included 39 characters and was analyzed using parsimony with equal, implied, and successive character weightings. De Carvalho and Haseyama (2018) reanalyzed this dataset with the inclusion of P. ponti using equal weighting parsimony, reaffirming previously published relationships (Nihei and de Carvalho 2007a), except for the addition of the new species. They also examined the cytochrome c oxidase subunit I (COI) sequences from GenBank for five Polietina species, confirming the reciprocal monophyly of two species with multiple sequences available (P. prima and Polietina orbitalis Stein, 1904).

Recent phylogenetic analyses of the Muscidae at the subfamily or family level have predominantly utilized morphological (de Carvalho 1989; Couri and Pont 2000; Couri and de Carvalho 2003; Savage and Wheeler 2004; Nihei and de Carvalho 2007b; de Carvalho et al. 2019; Sorokina and Ovtshinikova 2020; Gomes et al. 2020) or molecular evidence (Schuehli et al. 2007; Kutty et al. 2008, 2010, 2014; Haseyama et al. 2015; Ren et al. 2019; Grzywacz et al. 2021; Li et al. 2023; Walczak et al. 2023). Conversely, studies focusing on species-level phylogenetic inference have primarily relied on morphological data (de Carvalho 1999; de Carvalho and Couri 2002; Soares and de Carvalho 2005; Schuehli and de Carvalho 2005; de Carvalho and Pont 2006; Couri et al. 2007; Nihei and de Carvalho 2007a, 2011; Couri and de Carvalho 2008; Haseyama and de Carvalho 2012; de Carvalho and Haseyama 2018; Gomes et al. 2018a, 2018b; Patitucci et al. 2023; Pérez et al. 2023). Examples of species-level studies based on molecular evidence include those on Hydrotaea Robineau-Desvoidy, 1830 (Grzywacz et al. 2017), the Hydrotaea dentipes species group (Vikhrev 2024), Lispe Latreille, 1796 (Gao et al. 2022; Walczak et al. 2025), and Stomoxys Geoffroy, 1762 (Dsouli et al. 2011). To our knowledge, Savage et al. (2004) were the only to propose a phylogenetic analysis using combined morphological and molecular evidence for species relationships within a muscid genus, specifically for Thricops Rondani, 1856.

In this study, we conducted the first combined analysis of morphological and molecular evidence to explore species relationships within Polietina.

2. Materials and Methods

2.1. Dataset design and data acquisition

The species studied here included four outgroups and all Polietina species, except P. basicincta and P. mellina, due to the loss of their type material and the absence of recognized specimens (Nihei and de Carvalho 2007a) (Table S1). We utilized the morphological dataset published by Nihei and de Carvalho (2007a) with modifications proposed by de Carvalho and Haseyama (2018). The character list follows Nihei and de Carvalho (2007a, pages 500–501), excluding characters 19 and 24. Remaining characters were renumbered subsequently. Outgroup terminals were combinations of different species based on the availability of molecular data: Fannia bahiensis Albuquerque, 1957 was combined with F. canicularis (Linnaeus, 1761) sequences, Delia platura (Meigen, 1826) with D. radicum (Linnaeus, 1758); Cyrtoneuropsis multomaculta (Stein, 1904) with C. maculipennis (Macquart, 1843); Morellia xanthoptera Pamplona, 1986 with M. nigricosta Hough, 1900. For in-group analysis, individual specimens were used as terminals when multiple specimens were available for the same species. In these cases, the individuals were specified using voucher numbers; in cases where this was not available, we used the initials of the authors, as specified in GenBank. Specimens used for the acquisition of molecular data were identified using the key provided by Nihei and de Carvalho (2007a) and the original species descriptions.

We amplified eight molecular markers for Polietina and outgroup species, including protein-coding and ribosomal genes. For nuclear markers, we amplified: region 4 of carbamoyl-phosphate synthetase-aspartate transcarbamoylase dihydroorotase (CAD), elongation factor 1-ɑ (EF1-ɑ), 18S rRNA, and 28S rRNA. As mitochondrial markers, we targeted cytochrome C oxidase subunit I (COI), cytochrome b (Cytb), 12S rRNA, and 16S rRNA. We complemented our data with sequences from GenBank, primarily from the studies by Schuehli et al. (2007) and Haseyama et al. (2015). We used all available sequences for the target markers, even if the voucher was not identified at the species level. The only exceptions were the sequences of P. orbitalis associated with voucher DZUP459957 (AJ879599, AJ871209) and P. steini associated with voucher DZUP459958 (AJ879598, AJ871208); these sequences were excluded due to the uncertainty of their identities (de Carvalho and Haseyama 2018).

For specimens collected for DNA extraction, three legs from each specimen were separated, placed in a vial, preserved in absolute ethanol, and frozen. Depending on the specimen size, one to three legs were dried, macerated, and incubated overnight in 300 μL of sodium dodecyl sulfate lysis solution (Sigma-Aldrich, Merck) with 5 μL of proteinase K 20 mg/mL (Thermo Fisher Scientific Inc.) at 55 ºC. Protein and DNA were precipitated using ammonium acetate (Sigma-Aldrich, Merck) and isopropyl alcohol (Labsynth), respectively. The final DNA was eluted in 50 μL of Tris-HCl–EDTA buffer.

PCR reactions were performed with GoTaq™ G2 Flexi DNA Polymerase (Promega Corporation) according to the manufacturer’s protocol. The primers and annealing temperatures used are listed in Table S2. All PCR reactions consisted of an initial step of 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, annealing temperature for 30 sec and 72°C for 1 min, then a final step of 72°C for 7 min and hold at 4°C. Amplified products were purified using Ampure XP (Beckman Coulter). Sequencing reactions utilized BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc.) and were precipitated with 3 M sodium acetate (Labsynth). The samples were sequenced on a 3730 sequencing machine (Thermo Fisher Scientific Inc.) at GateLab (https://gatelab.ib.usp.br), and chromatograms were assembled using CodonCode Aligner software (https://www.codoncode.com). All contigs were analyzed using the BLASTn suite on the NCBI website to identify potential contamination. Additionally, all coding genes (CAD, COI, Cytb, and EF1-ɑ) were translated into proteins to check for stop codons and possible errors. No issues were detected in either analysis. Finally, the sequences were deposited in GenBank (Table S1).

2.2. Data analysis

The sequences were aligned using the MAFFT 7 online server (Katoh et al. 2019) with default settings. Each marker´s alignment was subsequently trimmed to minimize missing data using TRIMAL 1.3 (Capella-Gutiérrez et al. 2009) via PHYLEMON 2.0 (Sánchez et al. 2011) with the “automated1” option. Individual alignments were then concatenated using SEQUENCEMATRIX v.1.7.8 (Vaidya et al. 2010). For individuals that were not identified to the species level, their morphological datasets were completed with questionnaires.

Initially, we analyzed the molecular data using the maximum likelihood optimality criterion implemented in IQTREE v1.6.12 (Nguyen et al. 2015). We considered the entire sequence of ribosomal genes and individual codon positions of protein-coding genes as starting partitions. Then we used MODELFINDER within IQTREE (Kalyaanamoorthy et al. 2017) with the “-m MFP+MERGE” option to determine the best partitioning scheme. The best-fit model of nucleotide evolution for each partition was determined using MODELFINDER with default settings. We also analyzed the data without partitioning, and since both yielded identical topologies, a combined analysis (morphological and molecular data) was accomplished without partitioning the molecular data.

For analysis of the combined data, Bayesian posterior probabilities were chosen as the optimality criterion, and the analysis was conducted using MRBAYES 3.2.5 (Ronquist et al. 2012) with the Metropolis-coupled Markov Chain Monte Carlo algorithm. We performed two simultaneous runs, each with four chains, for 10 million generations, saving samples every 1000 generations. Convergence was assessed using the average standard deviation of the split frequencies (below 0.01), and the effective sample size of the analysis parameters was verified using TRACER 1.7.2 (Rambaut et al. 2018). For morphological partitioning, we used the MRBAYES model for standard discrete data based on Lewis (2001), specifying the following options: coding = variable coding = informative rates = gamma ngammacat = 8. For molecular partitioning, we utilized the GTR+G+I model suggested by MODELFINDER. The consensus type was set to add all compatible groups with all other options set to default (File S1). The resulting tree was visualized using FIGTREE 1.4 (Rambaut and Drummond 2012), with Fannia used to root the tree. WINCLADA (Nixon 2002) was used to optimize the synapomorphies in the combined tree.

We estimated pairwise genetic distances using MEGA XI (Tamura et al. 2021) and Kimura-2P with default settings based on COI sequences.

3. Results

We obtained new data from 10 muscid specimens, eight of which belonged to six Polietina species (Table S1). When combined with the data from GenBank, we obtained molecular information for eight out of the 15 species within the genus. Five of these species were sequenced for the first time. Five specimens could not be identified at the species level. The combined final matrix included 37 morphological characters and 5329 nucleotide characters for 17 species and five unidentified specimens.

Bayesian posterior probability analysis of the combined molecular and morphological data produced a tree that recovered Polietina monophyletic and divided it into two main clades, A and B, with clade C nested within clade B (Fig. 1). However, P. flavidicincta is a sister group of all other Polietina species. Our findings support the reciprocal monophyly of all species for which information is available. The divergence values varied from 0.0 to 0.1446 within the in-group (Table S3).

Figure 1. 

Bayesian molecular phylogenetic hypothesis of 13 Polietina species (25 specimens) and four outgroup taxa, based on Bayesian posterior probabilities derived from eight molecular markers (12S rRNA, 16S rRNA, 18S rRNA, 28S rRNA, cytochrome C oxidase subunit I, cytochrome b, region 4 of carbamoyl-phosphate synthetase-aspartate transcarbamoylase dihydroorotase, and elongation factor 1-ɑ; 5329 bp, model = GTR+I+G) and 37 discrete morphological characters (model = GTR+G). Specimens with molecular data have voucher numbers associated with its name, except for Fannia canicularis and Delia radicum (please refer to Table S1 for details). Numbers represent Bayesian posterior probability values.

4. Discussion

The Bayesian posterior probability analysis of the combined molecular and morphological data generated a tree (Fig. 1), which was largely consistent with two previous studies on Polietina phylogeny (Nihei and de Carvalho 2007a; de Carvalho and Haseyama 2018), with notable exceptions regarding the position P. flavidicincta and P. flavithorax. In our current analysis, P. flavidicincta emerged as the sister group to all other Polietina species, whereas in previous analyses, it was positioned as the sister group of Clade A, which included (P. rubella (P. concina (P. wulpi, P. orbitalis))) (Fig. 1). Since there were no molecular data for P. flavidicincta, we believe that this difference was due to the chosen analysis method. Females of this species are the only females in the genus without median anterodorsal setae on the foretibia, whereas P. orbitalis and P. bicolor females are polymorphic in this character (Fig. S1 [character 29]). The structure of Clade A was consistent with that in the literature. Notably, molecular data were available only for P. orbitalis within this clade. Consequently, the topology of Clade A was mainly based on morphological characters, resulting in relationships that were nearly identical to those previously reported.

In Clade B (Fig. 1), all species had molecular data for at least one specimen, except for P. minor. Polietina steini was recovered as a sister group to other species, which is consistent with the previous hypotheses. Polietina prima was estimated to be a sister group of (P. bicolor (P. univittata and P. minor)). This topology aligns with previously published hypotheses, which had a lower resolution for most of Clade B (Nihei and de Carvalho 2007a; de Carvalho and Haseyama 2018). However, our tree strongly supports P. major as the sister group of the remaining Clade C, whereas previous hypotheses had placed this species as the sister group of P. flavithorax. According to de Carvalho and Haseyama (2018), P. major and P. flavithorax share three unique traits within the genus, all related to hind tibia chaetotaxy: five or more anteroventral setae on male hind tibia; four anteroventral setae on female hind tibia; and a series of posteroventral setae on male hind tibia. In contrast, the new grouping of Clade C had no morphological support in the combined analysis (Figs S1–S3). Polietina flavithorax was recovered as a sister group to the clade containing P. ponti and unidentified specimens of Polietina (Polietina sp. QCAZI samples), in contrast to the results of de Carvalho and Haseyama (2018), in which P. ponti was in an uncertain position within Clade B. According to our combined analysis, P. flavithorax shares with P. ponti one homoplastic synapomorphy, three postsutural dorsocentral setae (Fig. S2 [character 7]). WINCLADA also pointed to the presence of four anteroventral setae on the female hind tibia as a synapomorphy for the clade; however, P. ponti is known only in males. Therefore, the character was coded as an unknown state for P. ponti, leading to errors in the program. Polietina ponti was found in a clade with unidentified Polietina specimens (sequences from GenBank). The COI sequences for specimens QCAZI_212190 and QCAZI_122048 were identical to P. ponti, whereas QCAZI_121842 had a single-variable site. Considering the evidence provided by this single marker, as no other sequences were available, these three specimens were very likely P. ponti. Another unidentified specimen, QCAZI_122047, could also be P. ponti, although it had a longer branch and its position had low support. Its genetic distance from P. ponti was 0.014, which was very similar to the distance found within the most divergent pair of P. orbitalis (0.010, BIOUG24749-C03, and M192). These unidentified specimens were collected in Ecuador, whereas the only known P. ponti specimens were from the type series from Peru.

Previous studies have shown that P. bicolor, P. minor and P. univittata form an unresolved clade (Nihei and de Carvalho 2007a; de Carvalho and Haseyama 2018). Here, we found the same clade, but P. minor was the sister-group to the clade P. univittata + Polietina sp. M060, although there are no molecular data for P. minor. The molecular data indicated that P. univittata M194 and Polietina sp. M060 is probably the same species since the COI sequences of M194 and M060 are identical, whereas P. univittata M190 has a single-variable site. The female specimen, M060 (Polietina sp.), was previously identified as P. minor. Males of P. minor and P. univittata closely resemble each other and are distinguished only when the lower spinning processes in the cercal plate are examined. Every published key characterizes P. minor as having upwardly oriented spines and P. univittata as having downwardly oriented spines (Couri and de Carvalho 1997; Nihei and de Carvalho 2007a; de Carvalho and Haseyama 2018). Polietina minor is supposedly the only species of the genus with upwardly oriented spines; however, a recent examination of a paratype brings new light to the original description provided by Albuquerque (1956). Dr. Márcia Couri examined the genitalia of a paratype deposited at the Museu Nacional (Rio de Janeiro) and confirmed that P. minor spines were actually oriented downward, similar to other Polietina species. Additionally, Dr. Couri recognized the lower spinned process of P. univittata with three spines subequal in length (despite one being stronger), while P. minor has one very long and strong spine, and two shorter and weaker ones. Therefore, despite the inaccuracies of previous studies, males of the two species could be distinguished through genital examination. Females of P. minor and P. univittata can be distinguished by a brown infuscation on the apex of R1 and Sc (absent in P. minor and present in P. univittata) and the number of anteroventral setae on the hind tibia (three in P. minor and two or three in P. univittata). As the latter trait varies in P. univittata, there is a single characteristic that can be used in all cases to distinguish females. Besides, we must also consider that the conspecific association of males and females under a valid name is not a simple and objective task in Muscidae species, as well as in Polietina species. Therefore, it is likely that females of P. minor and P. univittata are not well defined or described. Further studies are required to distinguish the females of P. univittata and P. minor properly. Therefore, we opted not to assert the species identity of Polietina sp. M060.

5. Declarations

Competing interests. The authors have declared that no competing interests exist.

Funding. CJBC thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, grant #307959/2021–9. EDGS thanks PRPPG/UNILA for their financial support (137/2018, 80/2019). SSN thanks FAPESP (grants 2007/50836-7 and 2013/05131-6) and CNPq (grant 409847/2021-6 and fellowship 310630/2021-5) for their financial support.

6. Acknowledgements

We are grateful to Márcia S. Couri (MNRJ) for revising the male genitalia of the type specimens of P. minor and P. univittata deposited at the MNRJ. We thank two anonymous reviewers for their helpful suggestions that improved the manuscript. Wiley Services has revised the manuscript.

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Supplementary materials

Supplementary material 1 

Figures S1–S3

Haseyama KLF, de Carvalho CJB, Soares EDG, Nihei SS (2025)

Data type: .pdf

Explanation notes: Figure S1. Bayesian molecular phylogenetic hypothesis of 13 Polietina species (25 specimens) and four outgroup taxa with synapomorphies plotted using fast optimization. — Figure S2. Bayesian molecular phylogenetic hypothesis of 13 Polietina species (25 specimens) and four outgroup taxa with synapomorphies plotted using slow optimization. — Figure S3. Bayesian molecular phylogenetic hypothesis of 13 Polietina species (25 specimens) and four outgroup taxa with synapomorphies plotted using unambiguous optimization.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (87.01 kb)
Supplementary material 2 

Tables S1–S3

Haseyama KLF, de Carvalho CJB, Soares EDG, Nihei SS (2025)

Data type: .zip

Explanation notes: Table S1. List of species used in this study, including voucher ID, GenBank number, voucher repository, sex of the specimen, and data collection (only for original sequences). — Table S2. List of primers used in this study, with annealing temperatures. — Table S3. Pairwise sequence distances between Polietina specimens and outgroup taxa estimated by MEGA XI (model = Kimura 2P).

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
https://doi.org/10.3897/asp.83.e144844.suppl2
Download file (42.82 kb)
Supplementary material 3 

File S1

Haseyama KLF, de Carvalho CJB, Soares EDG, Nihei SS (2025)

Data type: .nex

Explanation notes: List Data matrix and options used to run MrBayes.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
https://doi.org/10.3897/asp.83.e144844.suppl3
Download file (158.83 kb)
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