Molecular Analysis of Parasitoid Flies Tachinidae

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Introduction

Tachinidae are characterized as among the most diverse families of Diptera in characters, colors and behavior, in addition to the hosts. This family counts about 10 000 species [1] and is composed of four subfamilies (Phasiinae, Dexiinae, Tachininae and Exoristinae) including nearly 60 tribes [2, 3]. At least 15 orders of Arthropoda are hosts by all known tachinid species that considered as internal parasitoids of insects or other arthropods [1]. However, the most of tachinid species parasitize holometabolous insect larvae (Lepidoptera, Coleoptera, and Hymenoptera) or adult beetles, as well as true bugs [1, 2]. As enemies of these primarily phytophagous groups, tachinids play important role in biological control.

Materials and methods

Tachinids were obtained by hand collecting and malaise trap from Russia and Egypt, and one species from Panama. Tachinidae were sampled to provide representative specimens (A total of 120 specimens were analyzed) of the four subfamilies (Dexiinae, Exoristinae, Phasinae, Tachininae), and 30 genera of 20 tribes, and out-group based on previous works we selected the studied taxa [2, 4–7].

We collected nearly all the sequences of COI gene (mDNA) for tachinid species that opposite the genera which we brought from Russia and Egypt. They were composed of 73 nucleotide sequences collected from NCBI with Accession numbers (table 1). We used COI gene in this study to compare its ability as a traditional marker for solving the cladistic relationships within the radiative family Tachinidae with the recent studies that used new markers, which revealed more of these relationships for this family. COI gene was used only once but very few species of Tachinidae family were used for phylogeny. Alignment Sequences and analysis were performed by MEGAX program [8].

Table 1
Nucleotide sequences that collected from NCBI ^[1] and analyzed in this study

Species	Accession number	Number of base pairs	Link (URL)
Estheria petiolata	KX844428	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844428.1/
Rondania nr. dimidiata	KX843735	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843735
Athrycia cinerea	KR 395891	585 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 395891
Thelaira nigripes	KX844342	622 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844342
Thelaira americana	HM417100	654 bp	https://www.ncbi.nlm.nih.gov/nuccore/HM417100
Thelaira solivaga	KX844207	620 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844207
Voria erasmocoronadoi	MF325369	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/MF325369
Voria sp.	KR 386101	579 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 386101
Admontia degeerioides	JN 302070	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/JN 302070
Admontia grandicornis	KR 621021	555 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 621021
Bactromyia aurulenta	MG475545	516 bp	https://www.ncbi.nlm.nih.gov/nuccore/MG475545
Prooppia crassiseta	HQ581756	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HQ581756
Drino sp.	HM882180	521 bp	https://www.ncbi.nlm.nih.gov/nuccore/HM882180
Elodia ambulatoria	KX844551	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844551
Cylindromyia bicolor	MN 868900	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/MN 868900
Cylindromyia rufipes	MN 868879	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/MN 868879
Cylindromyia intermedia	MK660720	312 bp	https://www.ncbi.nlm.nih.gov/nuccore/MK660720
Cylindromyia fumipennis	HQ945071	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HQ945071
Gymnosoma nudifrons	KP044778	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KP044778
Ectophasia crassipennis	MN 868783	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/MN 868783
Phasia aurulans	JN 310367	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/JN 310367
Phasia mesnili	KX844068	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844068
Phasia pusilla	MN 868790	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/MN 868790
Phasia punctigera	HM417303	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HM417303
Phasia aurulans	KM571524	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KM571524
Phasia obesa	JN 310368	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/JN 310368
Leucostoma sp.	KP047351	593 bp	https://www.ncbi.nlm.nih.gov/nuccore/KP047351
Leucostoma simplex	KX843880	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843880
Leucostoma tetraptera	KX843764	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843880
Leucostoma gravipes	KR 520627	629 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 520627
Leucostoma meridianum	KX843930	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843930
Macquartia nudigena	KX844477	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844477
Macquartia dispar	JN 298651	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/JN 298651
Macquartia tessellum	KY 846615	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KY 846615
Macquartia viridana	KX844333	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844333
Mintho rufiventris	KX843818	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843818
Nemoraea pellucida	KX844529	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844529
Peribaea setinervis	KY 421538	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KY 421538
Peribaea hertingi	KX844508	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844508
Peribaea setinervis	KX844049	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844049
Peribaea tibialis	KX843900	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843900
Siphona grandistylum	KX844528	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844528.1/
Siphona hokkaidensis	HM431957	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HM431957
Siphona sonorensis	JF871072	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/JF871072
Siphona plusiae	HM417418	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HM417418
Actia nr. cinerea	JF271139	657 bp	https://www.ncbi.nlm.nih.gov/nuccore/JF271139
Actia interrupta	KR 395397	588 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 395397
Actia diffidens	KR 394266	549 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 394266
Loewia sp.	KR 393520	562 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 393520
Loewia foeda	KR 667561	589 bp	https://www.ncbi.nlm.nih.gov/nuccore/KR 667561
Loewia erecta	KX844484	630 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844484
Loewia brevifrons	KX844315	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844315
Loewia adjuncta	KX843701	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843701
Synactia parvula	KX844364	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844364
Bithia modesta	MG968012	532 bp	https://www.ncbi.nlm.nih.gov/nuccore/MG968012
Bithia acanthophora	KX844149	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844149
Bithia spreta	KX843739	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843739
Strongygaster celer	KP046934	635 bp	https://www.ncbi.nlm.nih.gov/nuccore/KP046934
Strongygaster sp.	JF867537	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/JF867537
Strongygaster triangulifera	HM412619	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HM412619
Nowickia alpina	KX843975	590 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX843975
Nowickia ferox	KX844164	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844164
Nowickia marklini	MF836056	588 bp	https://www.ncbi.nlm.nih.gov/nuccore/MF836056
Nowickia sp.	KM571428	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KM571428
Dexiosoma caninum	JN 310385	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/JN 310385
Microphthalma obsoleta	HQ945084	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HQ945084
Microphthalma disjuncta	HQ583135	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/HQ583135
Microphthalma europaea	KX844102	658 bp	https://www.ncbi.nlm.nih.gov/nuccore/KX844102
Calliphora vomitoria (outgroup)	AY 536643	414 bp	https://www.ncbi.nlm.nih.gov/nuccore/AY 536643

Results

This analysis includes 73 nucleotide sequences. 683 positions were the total of the final dataset. Phylogenetic trees were obtained by different methods (Maximum Likelihood «ML» and Neighbor Joining «NJ»).

The both analyses (ML and NJ) using COI gene showed that all subfamilies were polyparaphyletic except Phasiinae; most tribes used in this study (three from four tribes) were monophyletic; Cylindromyiini tribe and Gymnosoma nudifrons species were outside of the phasiin clade. Unlike all previous phylogenetic studies, we found that the Phasiinae were grouped as sisters to the Tachininae (in part). And all other subfamilies relationships contained diversity.

According to the ML analysis, dividing Tachinidae into two big clades was observed:

The first clade includes Tachininae (Siphonini + Nemoraeini + Macquartini + Tachinini) and Phasiinae (Phasiini + Gymnosomatini + (Leucostomatini + Proopia crassiseta (Aldrich & Webber, 1924))).

The second clade includes the subfamilies (remaining Tachininae, Dexiinae and Exoristinae) in addition to genus Cylindromyia Meigen, 1803 and species Gymnosoma nudifrons.

The first clade is divided into two groups:

1)    Siphonini ((Peribaea Robineau-Desvoidy, 1863 + Goniocera Brauer & Bergenstamm, 1891) + Siphona Meigen, 1803);
2)    Siphonini ((Peribaea + Goniocera) + Siphona) are monophyletic group except Actia cinerea (Macquart, 1834) and Peribaea tibialis (Robineau-Desvoidy, 1851), where the first is clustered with Exoristinae genera (BACTROMYIA Brauer & Bergenstamm, 1891 and Admontia Brauer & Bergenstamm, 1889) and Dexinae, while the second is placed as a sister to Nemoraea pellucida (Meigen, 1824).

Phasiinae is divided into two groups:

The first genus composed of Leucostoma (Leucostomatini) + the exoristin species Proopia crassiseta (Erycini) that is originated within Phasiinae.
The second includes Ectophasia crassipennis (Fabricius, 1794) (Gymnosomatini) + genus Phasia Latreille, 1804 (Phasini).

Phasiinae are represented here by 5 genera (4 tribes), 3 tribes (Phasiini, Leucostomatini and Gymnosomatini by genus Ectophasia Townsend, 1912) clustered as monophyletic group. While the Cylindromyia (Cylindromyiini) is placed as a sister with (Synactia Villeneuve, 1915 and Loewia Egger, 1856) and Gymnosoma Meigen, 1803 is sister with Exoristinae (Admontia Brauer & Bergenstamm, 1889 and Elodia Robineau-Desvoidy, 1863). Genus Phasia (Phasini), Leucostoma Meigen, 1803 (Leucostomatini) and Cylindromyia (Cylindromyiini) are recovered (reconstructed) as monophyletic groups.

Tachininae (part) include:

1) Group genus Nemoraea (Nemoraeini) + Siphonin species Peribaea tibialis.
2) Group Nowickia Wachtl, 1894 (Tachinini) + Macquartia Robineau-Desvoidy, 1830 (Macquartini).

The genera Macquartia and Nowickia are weakly supported as sisters (29 %) and this cluster is placed as a sister to (Nemoraea pellucida+ Peribaea tibialis).

The second variable clade splits into two groups.

The first group includes two assemblages:

Strongygaster Macquart, 1834 (Strongegasterini) + ((Gymnosoma nudifrons (Phasiinae) + Admontia blanda (Fallén, 1820)) + Admontia egeerioides (Coquillett, 1895)) + Elodia ambulatoria (Meigen, 1824)) the latter three Exoristinae.

Tachininae (part) (Mintho Robineau-Desvoidy, 1830 (Minthoini)) + (Bithia RobineauDesvoidy, 1863 (Leskini)), where Estheria petiolata (Bonsdorff, 1866) (Dexiinae) and Voria erasmocoronadoi Fleming & Wood, 2017 (Dexiinae) are sisters to Bithia.

Gymnosoma nudifrons (Phasiinae) (this species is a sister with Admontia blanda) is originated within the exoristin two genera Admontia and Elodia and both (Gymnosoma nudifrons, Admontia and Elodia) are clustered as a sister group to Strongegaster (Strongegasterini) that is placed in unstable subfamily, where morphologically in our study was a sister with Phasiinae as in Cerreti 2014. While that in [3] was positioned within Tachininae.

The Tachininae genus Mintho rufiventris (Fallén, 1817) is a sister with the cluster [Voria erasmocoronadoi (Voriini) + (Estheria petiolata (Dexiini) + Bithia)].

The second group is composed of:

A-    [Cylindromyia (Phasiinae) + (Loewia (Loewini) + Synactia (Ernestini)] latter two belong to Tachininae) and cluster is a sister to the next assemblage.

B-    The two species of Thelaira Robineau-Desvoidy, 1830 (voriini, Dexiinae) as a sister to the following two clades:

First: Thelaira americana Brooks, 1945 sister to [(Voria sp. + Athrycia cinerea (Coquillett, 1895)) (voriini, Dexiinae) + [(Bactromyia aurulenta (Meigen, 1824) + Admontia grandiconis (Zetterstedt, 1849) (the two Exoristinae)) + (Nowickia marklini Zetterstedt, 1838 + Actia cinerea)]].

Second: [Microphthalma europaea Egger, 1860 + (Microphthalma Macquart, 1844 (remaining species) + Dexiosoma)] this clade belongs to tribe Megaprosopini (Tachininae).

Cylindromyia (Cylindromyini) is a sister with the two genera Loewia and Synactia belonging to subfamily Tachininae. And, this assemblage is a sister to Dexiinae, Exoristinae and Tachininae taxa.

Voriini tribe taxa, Thelaira Americana sister to (Voria sp. + Athrycia cineria) are clustered in one group with the two Exoristinae species (Bactromyia aurulenta + Admontia grandiconis) and (Bactromyia aurulenta (Eryciini) + Admontia grandiconis (Blondellini)) and Tachininae species (Nowickia marklini (Tachinini) + Actia cinerea (Siphonini)).

In spite of Voriini was clustered in one clade (first clade), it is not monophyletic group (graduated), where Thelaira solivaga (Harris, 1780) and Thelaira nigripes (Fabricius, 1794) are sisters with remaining voriini, Exoristinae and Tachininae taxa and Voria erasmocoronadoi are placed in another clade.

The tribe Megaprosopini is moderately supported (72 %) as monophyletic and represented here by two genera Microphthalma and Dexiosoma Rondani, 1856, where Microphthalma europea is a sister with Microphthalma (remaining species) + Dexiosoma caninum (Fabricius, 1781), where Microphthalma is not clustered as monophyletic group.

NJ analysis method showed that the cluster [Siphonini + (Mintho rufiventris + (Admontia + Gymnosoma nudifrons)] is a sister to all rest of Tachinidae. Strongygaster + (Thelaria and Elodia) are grouped as a sister with Phasiinae and both are clustered as a sister group with the variable clade [Tachininae (Ernitini, Loewiini, Megaprosopini, Actia cinerea and Nowickia marklini)], [Exoristinae (Bactromyia aurulenta and Admontia grandicornis)], [Dexiinae (Athrycia cinerea, Thelaira americana and Voria sp.)] and [genus Cylindromyia]. This big group is placed as a sister with the remaining Tachininae where the two Dexiinae species (Estheria petiolata and Voria erasmocoronadoi) are originated within it.

Discussion

It has long been proposed that the Tachinidae are a monophyletic clade depending on larval and adult characters [2, 9] and the current molecular studies nearly entirely reported this view [3, 5, 10, 15]. However, the works so far contain few outgroup taxa [4], unable to solve this issue.

The big split of subfamilies in recent studies grouping Phasiinae + Dexiinae and Exoristinae + Tachininae was strongly supported whether using morphological data in [2] or molecular by [3, 10, 15]. This hypothesis, first proposed by [10], reports other one differs from previous that clustered subfamilies containing (Phasiinae + Exoristinae) and (Dexiinae + Tachininae, [11]).

Our (molecular) dendograms (figs. 2 and 3 show that all the subfamilies are polyparaphyletic except Phasiinae that is grouped as monophyletic group and confirmed by [2] and molecular by [1, 3, 10], exclusive placement of Cylindromyia (Fig. 2) is positioned as a sister to Tachininae taxa, weakly supported (17 % ML; 20 % NJ) and agrees with [10] in outside of this genus from Phasiinae. It is reported in [10] that the suggestion that Cylindromyiini may not be included in Phasiinae, or that the subfamily itself may not be monophyletic group is novel. However, using greater taxon sampling in [2, 3, 15] revealed the monophyly of Phasiinae with Cylindromyini.

A potential solution would be to split the subfamily Phasiinae along these statistical lines and create a monophyletic Phasiinae and a new monophyletic subfamily Cylindromyiinae. However, Cylindromyiini shares many traits with Dexiinae and several dexiines have historically been placed in Cylindromyiini (notably Epigrimyia Townsend, 1891 and Beskia Brauer & Bergenstamm, 1889). Before Cylindromyiini can be elevated to subfamily status, the status of Dexiinae and its relationship to Phasiinae needs to be solidified [15].

We placed the enigmatic genus Strongygaster (clustered with Thelaria and Elodia) with very weak confidence as a sister to Phasiinae (NJ analysis) and originated within Tachininae and Exoristinae (ML analysis), where it had been previously placed by [13] and others [3, 15].

However, some authors [3, 14], reported Strongygastrini to be joined with Tachininae, Strongygaster is apparently similar to Phasia and some other phasiines, with large compound eyes and few bristles, but the biology does not couple with Phasiinae, and phallic reduction increases the confusion relationships. The morphological and molecular phylogenetic analyses [2, 15] respectively, has also been supported the position of Strongygaster within Phasiinae that correspond with our morphological study (fig. 1).

Relationships among the Dexiinae are among the most poorly supported in our analyses. In analyses of less informative COI gene, the few included dexiine taxa in ML are grouped in a cluster as sisters to each other and with other Tachininae and Exoristinae taxa, but not in monophyletic clade. In addition, Estheria petiolata and Voria erasmocoronadoi are clustered as sisters out of this clade. While in NJ they are widely dispersed across the tree and in both cases the relationships among them (Dexiinae taxa) are unclear. This result about Dexiinae relationships also was observed by [8] using the same marker COI but a very few Tachinidae taxa were used. However, in [3] the Dexiinae comprised a well-supported monophyletic Dexiinae, relationships among tribes within the subfamily exhibited more uncertainty.

Fig. 1. Dendogram was constructed using the Maximum Parsimony (MP) method. Pollenia sp. and Calliphora sp were used as out groups. On the branches — white circles are homoplastic (plesiomorphic) characters, and black circles are autapomorphic characters

Fig. 2. Maximum likelihood phylogeny based on COI gene analysis. ML bootstrap values are shown above

Fig. 3. The evolutionary history was reconstructed using the Neighbor-Joining method

Monophyly of the subfamily Exoristinae has been supported by all six major phylogenetic analyses of Tachinidae [2–5, 15], and it is supported here as well (in our morphological tree), but it was not supported in our molecular analysis, where Exoristinae taxa are scattered on the tree. Prooppia Townsend, 1926, is within Phasiinae, while Bacteromyia aurulenta and Admontia grandicornis are originated within tachinin and dexiin taxa. Admontia (remaining speices) and Elodia ambulatoria are sisters with Strongygaster.

Tachininae are the most morphologically heterogeneous subfamily of Tachinidae, lacking clear morphological synapomorphies [12]. In the recent morphological analysis, [2] found the Tachininae to be polyphyletic, with clades of Myiophasiini + Palpostomatini, Macquartini and Ormiini and in molecular [3] Myiophasiini + Macquartini (none of which is represented here except Macquartia) were placed at the base of Tachinidae, and the subfamily being paraphyletic with respect to the Exoristinae as in our study, Tachininae is a paraphyletic group and scattered along the tree. Here, some Tachininae (Tachinini, Macquartini, Nemoraeini and Siphonini) are sisters with Phasiinae clade, while Ernestini and Loewini are sisters with Cylindromyini. In addition, Megaprosopini is a sister with Actia cinerea and Nowickia marklini with Dexiinae taxa. Moreover, Minthioini and Leskini are sisters with Dexiinae and Exoristinae. The Monophyly of Siphonini was supported in previous studies [2, 3], and it is in agreement with our study but in our analysis there is exception, where genus Actia and Peribia tibialis are out of this tribe.

In the morphological and molecular analyses (figs. 2 and 3, the relationships of some Tachininae taxa are nearly similar as following:

Macquartia (Macquartini) is a sister to Nowickia (Tachini) and both are clustered with Nemoraea (Nemoraeini).

Morphologically, Loewia and Synactia are positioned in the base of the tree, where, in contrast to molecular analysis, but with the same relationship, they are grouped as sisters.

In addition, Monophyly of Megaprosopini (Microphthalma and Dexiosoma) is moderately supported in both analyses. Moreover, Mintho-leskiine is clustered in ML (COI) and morphological analyses but it is dispersed (separated) in NJ analysis.

Conclusion

We can conclude that additional taxa sampling and using informative markers (genes) will be necessary to resolve some questionable taxa specially the higher taxa.

However, we used few taxa for each genus and each tribe, but observed that using COI gene does not have ability to recover (reveal) the subfamilies as monophyletic and reveal its problems. While, the same marker (COI) revealed that the most of the genera and tribes are monophyletic groups:

Tribes: Siphonini, Megaprosopini, Strongygasterini, Leucostomatini, Macquartini, Tachinini (Nowickia), Phasiini, Cylindromyini.

Genera: Macquartia, Nowickia, Siphona, Peribaea, Leucostoma, Cylindromyia, Phasia, Microphthalma, Strongygaster, Admontia, Bithia, Leowia.

COI gene used in the study was observed to be very poor informative for solving (could not solve) relationships in the higher taxonomic tachinid taxa.

 

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About the authors

El-Sayed El-Hashash Arafa

Peoples’ Friendship University of Russia; Plant Protection Research Institute, Agricultural Research center

Author for correspondence.
Email: Arafa.elhashash@yahoo.com
ORCID iD: 0000-0001-5210-5515

postgraduate student

8/2, Miklukho-Maklaya st., Moscow, 117198, Russian Federation

References

Supplementary files