The scuttle flies (Diptera: Phoridae) of Iran with the description of Mahabadphora aesthesphora as a new genus and species

Scuttle flies (Diptera: Phoridae) are mega-diverse and often synanthropic insects that play superb roles in various ecosystems. Identification of this group of insects is challenging due to their small size, morphological identification difficulties, niche diversity, and lack of taxonomic keys. To pave the way, an in-depth investigation was directed toward the scuttle flies in Iran using morphological and molecular data. A dichotomous key was also developed to identify the genus and species of the phorids reported in the country. The faunistic findings revealed the presence of about 22,000 (13,903 male and 8,097 female) phorid materials organized into 11 genera. Megaselia species (n = 13768), made up about 99% of the specimens studied. Moreover, 71 morphologically defined species belonging to nine genera were molecularly characterized using COI, 28S rRNA, and Arginine kinase datasets. Excluding four Megaselia Rondani, 1856 species, our results specified that morphologically delimited species were in agreement with the molecular analyses inferred from the COI/28S rRNA and COI/Arginine kinase sequences with genetic distances and phylogenetic trees. According to the results of the present study and previously published data, the Phoridae recorded for Iran are a total of 97 species that are ordered in 13 genera and three subfamilies, including Chonocephalinae, Metopininae and Phorinae. By comparing the known world phorid genera, a new monotypic genus of scuttle flies, Mahabadphora aesthesphora gen. nov., sp. nov., was identified based on its morphological and molecular characteristics and included in an updated key. Our results could comprehensively determine the taxonomic status of scuttle flies in Iran, scrutinize their phylogenetic structures and facilitate their identification.


Introduction
Scuttle flies (Diptera: Phoridae) are considered as one of the most abundant and diverse families of flies [1]. The adults resembling fruit flies can simply be recognized through the morphological (hump-backed outwards and reduced wing venations) and behavioral ( study genetic diversity in a single mode and also to reconstruct phylogenetic trees in the combined modes (S1 Table). The quality of raw sequences obtained herein was proofread using the Chromas 2.6.6 (Technelysium Pty Ltd., South Brisbane, Australia). The BLAST (Basic Local Alignment Search Tool) search was employed to compare under-investigated sequences. Multiple sequence alignments were conducted by Clustal Omega software [56]. The basic sequence statistics, including polymorphic and parsimony-informative sites, were analyzed by the aid of MEGA X software [57]. Interspecific and inter/intrageneric divergences for the studied gene sets were estimated with the suite of molecular genetic programs embedded in MEGA X using the Kimura two-parameter (K2P) distance model [58]. The combination of COI-28S and COI-AK gene sets was exploited to infer relationships. Phylogenetic relationships were examined using maximum likelihood (ML) and neighbour joining (NJ) algorithms with K2P correction models embedded in MEGA X software. Confidence of internal nodes was assessed by bootstrap analysis with 1,000 replicates. Sequences of the target genes in brachyceran fly species Drosophila melanogaster Meigen, 1830, Glossina morsitans Westwood, 1851 and Musca domestica Linnaeus, 1758 were designated as outgroups. All sequences achieved in this study were deposited in the GenBank database (S1 Table). Literature review and providing an identification key. An extensive literature review was conducted based on a search of online scientific databases (Scientific Information Database, PubMed and Google Scholar) to find published reports on phorid flies in Iran before 30 th June 2020. Searches were performed in titles, abstracts, keywords, and full texts. Keywords for the search were Phorids AND fauna AND Iran, Iran AND Phoridae, and Iran AND scuttle flies. An updated dichotomous key to all known species of Phoridae in Iran, including specimens from the current study, was subsequently generated.

Nomenclatural acts
The electronic edition of this paper follows to the necessities of the adjusted International Code of Zoological Nomenclature, and therefore the new names included herein are accessible under that Code from the electronic edition of this article. This published paper and the nomenclatural acts it comprises have been registered in ZooBank, the online registration organization for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org: pub:9145941B-10BF-4B27-8B4C-D90006A857B5. The LSIDs of the all publications and species mentioned in the present project are available in the supplementary materials (S2 Table). Table 1. Details of the primer sequences and thermal profile used for the amplification of target genes.

Locus
Gene & Primer code
Their favoring substrates include decaying coconuts [91], ripe bananas [92] and dead rabbits [1]. However, they were reported from human corpses [93] and near dirty floor-drains and mausoleums [94]. This species is recognized as an important pest of the button mushrooms in Karaj mushroom houses [49]. Also, it invades insect cultures [90] and parasites asilid species and honey bee colonies [51] in Iran. M. scalaris is reported to be a cause of myiasis on humans and animals [24,95,96].  Etymology. Named after it being strange (Greek aesthes). Ecological data. The climate at the type locality is temperate with very cold winters and hot summers. The sampling site is located within the valley, which has a seasonal river running until the end of July. Herbaceous and woody vegetation plants in the area comprises of Glycyrrhiza glabra L. (Fabaceae), Achillea millefolium L. (Asteraceae), Peganum harmala L. (Zygophyllaceae), Convolvulus arvensis L. (Convolvulaceae), and Salix alba L.) Salicaceae (etc. In general, the region has previously been quite untouched and pristine, but recently, it has been modified or influenced by human activities (Fig 3).

Molecular surveys
Sequence analysis. Two COI and 28S genes for all studied specimens and three COI, 28S and AK genes for the new genus were successfully amplified and sequenced (S1 Table). In total, 720, 512-549, and 543 base pairs were sequenced for the COI, 28S, and AK genes of the
Relationship analysis. The ML method provided more reliable phylogenetic trees than NJ method (S1-S3 Figs). Consequently, three ML phylogenetic trees were constructed using combined sequences of COI-28S (1200-1269 bp) and COI-AK (1263 bp) datasets (Figs 4-6). The first one was drawn based on COI-28S dataset for the Megaselia spp. determined in this study along with the sequences of M. scalaris (KF974742-KC177721), D. melanogaster (KY559392-NR133562), Glossina morsitans (KC192971-KC177834) and Musca domestica (AB479529-AJ551427) from the Genbank (Fig 4). The tree divided the Megaselia species into six groups. Sixteen similar morphospecies clustered together in the clade I. Morphological parsing showed that in all species grouped in this clade, except for M. styloprocta, the mesopleuron was bare, and dorsal face of epandrium was longer than or equal to the length of the anal tube. The M. styloprocta was joined to M. minuta and M. subnudipennis branch as a sister group. Our study found four out of the 10 members of the M. brevior complex, all of which were correctly classified in the clade I.
Clade    In clade V, there were 11 species that shared similar morphological properties, e.g. the hairs of left side of epandrium were, at most, only as robust as those of cerci. Megaselia ardabilensis was morphologically very similar to M. khoyensis but could be distinguished by the relative size of hairs below basal half of hind femur, as compared to those of anteroventral row of outer half. Moreover, their hypandrial lobes were clearly different.
The clade VI comprising of M. evogliensis / M. longiseta was established as paraphilitic group of other megaselias in the phylogenetic tree. The M. evogliensis and M. longiseta species had definite dissimilarities (see the key).
The second tree covered the COI-28S sequences of other 11 species found in this study, along with those of 17 other species retrieved from the Genbank (Fig 5). The tree divided the studied taxa into two subfamilies: Phorinae (six clades) and Metopininae (three clades). Taxonomically challenging species Triphleba intermedia and Conicera tibialis were arranged in clades II and III of Phorinae, respectively. The newly described taxon, Mahabadphora aesthesphora, and the genus Phora were classified in the clade IV under the Phorinae subfamily. They shared some morphological characteristics e.g. vein Rs without hairs along the dorsal face, at least two differentiated dorsal or near-dorsal bristles in basal two-thirds of mid tibia and hind tibia without longitudinal hair palisade. However, unlike Phora, the Vein 3 in Mahabadphora, was forked. The Diplonevra and Dohrniphora genera were organized in the same expected clade (VI) as a sister group of other species of subfamily Phorinae.
The third ML consensus tree, in support of the second one, recovered from 16 pairs of COI-AK sequences confirmed that Mahabadphora aesthesphora was a genetically quite distant clade from Metopininae, clearly representing valid taxon in the Phorinae scuttle flies (Fig 6).
Checklist of scuttle flies occurring in Iran and their identification key. An inventory of 97 scuttle fly species known from Iran, together with their collection data, hierarchical classification, and synopsis of their life history is summarized in Table 2. These flies were distributed to several locations of 12 Provinces of the country, namely Alborz, Ardabil, East Azerbaijan, Fars, Golestan, Kermanshah, Markazi, Mazandaran, Razavi Khorasan, Tehran, West

Key to the genera and species of the Phoridae known from Iran
The following key is based on the male insects of phorid spp.

Discussion
The phorid flies are very diverse in terms of species number and lifestyle but are poorly known. During this study, the phorids captured from three northwestern provinces of Iran were investigated via morphological and molecular methods, and subsequently, a genus/species-level morphological identification key was developed for male flies reported throughout the country. By comparing the known world phorid genera maintained in UCMZ, we proposed a new monotypic genus of scuttle flies, Mahabadphora aesthesphora gen. nov., sp. nov. The faunistic findings revealed the presence of 13,903 males and 8,097 females during this project. All male (and some female) flies were morphologicaly identified and organized into 11 genera. Megaselia species (n = 13768), made up about 99% of the specimens studied (Table 2). In bulk collections of other studies, the genus Megaselia constitutes the most frequently captured flies [70,[112][113][114][115][116].
The specimens of the present study were gathered from relatively restricted localities in the mountainous cold areas. Hence, with the expansion of sampling to the areas with temperate and tropical climates, we can anticipate the precise reflections of the phorid's distribution since they are very responsive to microclimatic/habitat alterations [72,117,118].
We were able to include only 71 paratypes in our molecular experiments as the type specimens were archived in UCMZ and ICHMM collections after identity verifications. The mithocondorial COI and nuclear 28S/AK markers were preferred to other targets because they have been proved to be informative for species-level and genus-level analyses, as indicated in a large number of resources regarding evolutionary associations in insects [e.g., [37][38][39][40]44,46,119,120].
Based on the single gene datasets, the preliminary molecular analysis of this study resulted in trees with less resolution and support; owing to fewer included characters (data are not shown). Mitochondrial markers are also more variable than nuclear ones. The mitochondrial genes help to solve the more recent divergences and nuclear ones better resolver deeper divergences, hence, the combinations of COI-28S and COI-AK datasets were applied to describe the studied paratypes, as well as to determine their relationships with known taxa. The reason for using combined analyses is that they may reliably resolve disagreements between the individual genes analyzes, enhance phylogenetic resolutions, and be more consistent with morphological data [121]. We also tried to include sequences from the same specimens in combined analyses whenever possible.
Excluding two pairs of Megaselia species, M. hirticaudata / M. shabestarensis and M. khoyensis / M. ardabilensis, our results specified that morphologically delimited species were congruent with the molecular analysis inferred from the COI-28S and COI-AK sequences with genetic distances and phylogenetic trees. Broadly speaking, the failure of the target genes to discriminate above-mentioned pair species is controversial. Although, belonging to the M. sulphuripes species group, M. hirticaudata and M. shabestarensis are morphologically distinct. This dissimilarity is also true for the species of M. ardabilensis and M. khoyensis. The discrepancy in morphological and molecular analysis could be a consequence of conspecificity, misidentification, or inefficiency of target genes in differentiating these species. However, the original data and photographs in combination with the quality of the sequences were carefully examined, and none of the aforesaid matters were resolved. Molecular investigations were repeated even in the case where syntypes were available, though the results did not change. According to the literature, even when two COI sequences are the same, there is still a chance that they belong to different taxa [122]. Therefore, to solve inconsistency like this, we suggest using supplementary loci or sequencing of the mitochondrial / nuclear genomes if possible.
Phylogenetic relationships of the understudy sequences were first examined using the NJ (S1-S3 Figs) and then by the ML method (Figs 4-6), but the second one showed more agreement with the morphological classifications. The fact that ML or Bayesian methods are more efficient than the NJ method in obtaining the true tree has been indicated in other studies [123,124].
Herein, the results of the relationship analysis were offered through three ML phylogenetic trees; the first and second trees with relying on COI-28S datasets for the Megaselia spp. and non-Megaselia species, respectively and the third tree, in support of the second one, using COI-AK sequences, to confirm the position of the newly described species within Phorinae. We reported six major clades for Megaselia species with low bootstrap values. Low bootstrap values may indicate that there are conflicting or little signals in the data set. Most genera within the Phoridae were monophyletic taxa with relatively a few species; however, Megaselia with remarkable radiation comprised of about 1,700 described species, presumably accounting for the largest genus in the animal kingdom [46,125]. Initially, the genus Megaselia was morphologically divided into two Megaselia and Aphiochaeta subgenera, and subsequently into further divisions and series [103,[126][127][128][129]. Later, a new species (the lucifrons) group in Megaselia was introduced, using two COI and 28S molecular markers [46]. Recently, 22 informal species groups have been proposed for this species-rich genus based on nuclear (28S rDNA) and mitochondrial (ND1, COI, and 16S) markers [130]. The topology obtained for Megaselia sequences in this study, in agreement with other studies, represents a monophyletic lineage for this challenging genus [46,130]. Genome-scale phylogenetics is necessary to infer true monophyly and radiation of Megaselia species.
The last consensus tree, in support of the second one, verified Mahabadphora aesthesphora gen. nov., sp. nov. as a valid new taxon in the Phorinae subfamily. Both morphological and molecular analyses specified M. aesthesphora gen. nov., sp. nov. as sister taxon to Phora spp. Two specimens of this species were collected from West Azerbaijan, Mahabad City, which the first specimen was deposited in the UCMZ, and the second one was used for molecular analysis. This species may have a wider distribution in Iran and other areas with this type of habitat, which requires further sampling.
Literature review revealed that the phorids fauna in 12 provinces of Iran comprises of three subfamilies, 13 genera, and 97 species (Table 2). However, information on other species in the remaining 19 provinces is largely unavailable. Among 87 species offered during the current project, two new genera (Mahabadphora g. nov. and Iranphora Namaki-Khameneh & Disney, 2021) and 32 species represented new records for the world, and four genera (Conicera, Dohrniphora, Gymnophora, and Triphleba) and 47 species were new reports from Iran. Moreover, 10 species of the current study have previously been reported [49,65,69,79,100].
As a most evolutionarily successful group of macro-organisms, true flies (Diptera) can exploit almost all terrestrial and aquatic ecosystems on the earth. Indeed, Diptera is divided into families with regard to the habits (nutrition) and habitats (environment) of adults and larvae [131]. In this respect, phorid flies display the greatest diversity among all the dipterous families. The life histories of most scuttle flies are rarely documented in Iran, and limited studies have focused on only renowned species that act as the pest of edible mushrooms [49,50], invade honey bee colonies [51], or cause myiasis in humans [24]. A synopsis of bio-ecological information of 97 phorid species reported in this study was assembled from various sources and is shown in Table 2. Due to the fact that the way of life of most species is unknown, this information could expand our knowledge on the bionomics of scuttle flies in terms of environmental, agricultural, medical, and forensic prospectives.

Conclusion
The present study is the most extensive sampling of Phoridae in Iran and the first study that utilizes the molecular characters for the identification of specimens to address morphological identification problems. Obviously, our research work has limitations in terms of sample size and sampling locations. Despite these downsides, we believe our results can comprehensively determine the taxonomic status of scuttle flies in Iran, scrutinize their phylogenetic structures, facilitate their identification and introduce a new monotypic genus.