A revision of the genus Armillipora Quate (Diptera: Psychodidae) with the descriptions of two new species

. The genus Armillipora Quate is recorded for the first time in Ecuador, with a new geographical record for Armillipora selvica Quate, 1996 and the descriptions of two new species, namely Armillipora muyu sp. nov. and Armillipora imitata sp. nov., doubling the total number of species in the genus. In addition, we make available the first DNA barcodes for the genus, providing a sequence of the 5´-end of the cytochrome c oxidase subunit I (COI) gene for A. imitata , A. muyu , and, A. selvica. Moreover, we describe the second known female of the genus and we provide a taxonomical key for the known males of the world. Finally, we build Species Distribution Models and discuss the potential distribution of the genus in the Neotropical region.


Introduction
The moth fly genus Armillipora Quate, 1996 (Diptera: Psychodidae) has only been recorded in the Neotropical region (Quate 1996(Quate , 1999;;Ježek et al. 2020).For many years a single species was known, namely Armillipora selvica Quate, 1996.This species was described from Costa Rica (Quate 1996) and recorded in Panama three years later (Quate 1999).More recently, Ježek et al. (2020) described a second species from Bolivia, Armillipora suapiensis Ježek, Oboňa & Le Pont, 2020, breaking the monotypy of the genus and adding a new geographical record to A. selvica from Nicaragua.Up to now, A. selvica has been recorded from Central America (Nicaragua, Costa Rica, Panama) and A. suapiensis is known only from Bolivia.Besides its scattered known distribution, we do not have information about the larval stages or the adult biology of this genus.
In the present study, we report for the first time the genus Armillipora from Ecuador, and we describe two new species based on morphological and molecular characters, bringing the total number of species of Armillipora to four.We also describe the second known female of this genus, which belongs to Armillipora muyu sp.nov., and provide a new geographical record for Armillipora selvica from Ecuador.Moreover, we make available the first DNA barcodes (the sequences of the 5´-end of the cytochrome c oxidase subunit I or COI gene) for A. selvica, A. imitata sp.nov., and A. muyu and provide an identification key for the world males of Armillipora.Finally, we discuss the potential distribution of the genus in the Neotropical region based on our species distribution model.

Study area
The Cantón Pedro Vicente Maldonado is located in the Pichincha Province in the northern part of Ecuador, (0°10′00″ N, 79°00′00″ W) with an average altitude of 600 m a.s.l.The climate is warmhumid with an average annual temperature of 24.5°C and an annual precipitation of 4341 mm.The main vegetation is characterized as pre-mountain rainforest (HPPC 2015).

Collection and preparation of specimens
Specimens were collected using a Malaise trap, euthanized and preserved in 96% ethanol, and later stored at -20°C.Specimen preparation was done following the protocol explained by Jaume-Schinkel & Kvifte (2022), with the modification of using the whole specimen for DNA extraction instead of just the thorax.
In the material examined section and at the end of each record the holding institution is stated, and between square brackets ([ ]) the number of the specimen is indicated.The abbreviations used for holding institutions and their equivalents are given below: INABIO = Instituto Nacional de Biodiversidad, Quito, Ecuador.LACM = Natural History Museum of Los Angeles County, Los Angeles, California, USA.ZFMK = Museum Koenig, Leibniz-Institut zur Analyse des Biodiversitätswandels (previously known as Zoologisches Forschungsmuseum Alexander Koenig), Bonn, Germany.

Genetics
A non-destructive methodology from complete specimens was performed in the facilities of ZFMK with the following workflow: A Qiagen (Hilden, Germany) BioSprint 96 magnetic bead extractor, and the corresponding kits were used following the manufacturers' specifications.We amplified from the 5´-end of the cytochrome c oxidase subunit I (COI) gene using the primers HCO2198-JJ (forward) and LCO1490-JJ (reverse) (Astrin & Stüben 2008).PCR was carried out using a TouchDown PCR (TD-PCR) as proposed by Korbie & Mattick (2008) using a QIAGEN Multiplex PCR Kit.Later the PCR products were shipped to Beijing Genomic Institute (BGI) (China, Hong Kong) for bidirectional sequencing.DNA sequences were assembled, aligned, and cleaned using Geneious Prime ver.2022.1.1 (Biomatters, Auckland, New Zealand).The total sequence length was set to 658 bp.
We downloaded all the available sequences of the tribe Maruinini from BOLD and we used Geneious Prime ver.2023 to perform a distance-based neighbor-joining (NJ) analysis using the Jukes-Cantor model.All of the sequences generated for this study can be accessed in BOLD under the Dataset DS-ARMI (available at: https://doi.org/10.5883/DS-ARMI).Bootstrap support (BS) values were estimated from 1000 replicates as calculated in Geneious.

Species Distribution Models
We built a distribution model for the genus using the software MaxEnt ver.3.4.4 (Phillips et al. 2023) with the species' geographical records to infer the potential distribution in the Americas.In MaxEnt ver.3.4.4we used all the records of the genus as a single biological entity to evaluate the distribution model for the genus, instead of using each species separately.The resulting map was trimmed to North America, Central America, and South America.Climate variables were obtained from WorldClim (Fick & Hijmans 2017).Geographic coordinates used for the analysis were extracted from the localities reported in the literature and museum collection databases.Localities are summarized in Table 1.For the records without exact coordinates in the literature, we used Google® Earth to search for the reported locality and obtain proxy coordinates and compared them with the coordinates in the collection data base.After comparison, if coordinates did not match, we adjusted them to have at most a difference of ± 50 meters between both.

Differential diagnosis
The genus Armillipora has been placed in the tribe Maruinini Enderlein, 1937 based on the presence in the wing of a radial fork being basal to the medial fork and both forks located basally on the wing, as well as the broad and dorsally flattened shape of the ejaculatory apodeme (Quate 1996;Kvifte 2018;Ježek et al. 2020) ).But species of Armillipora can be easily differentiated using characteristics of the male genitalia as follows: the characteristic shape of the irregularly-asymmetrical epandrial appendage (not irregularly asymmetrical in Alepia and Platyplastinx), the long accessory tenacula (long in Alepia, but short in Platyplastinx) with a group of short cylindrical tip-folded tenacula (not present in Alepia and Platyplastinx), with the absence of apical tenacula (usually none, one or more apical tenacula in Alepia and Platyplastinx), the lack of gonostyli and the gonocoxites fused in Armillipora (gonostyli present and gonocoxites usually not fused in Alepia and Platyplastinx), and the absence of the aedeagal sheath (present in Alepia, but absent in Platyplastinx) (see Ježek et al. 2020).
Females of Armillipora can be differentiated of those of Alepia and Platyplastinx by the following characters: antennal flagellomeres with double circle of teardrop-shaped pores in the center, although less conspicuous than males they are present in Armillipora (absent in Alepia and Platyplastinx); Armillipora with the subgenital plate longer than wide (subgenital plate length variable in Alepia, usually about the same length as its width in Platyplastinx), with apical lobes separated by a broad concavity (concavity is broader than twice the length of the apical lobe) with a pair of long spines JAUME-SCHINKEL S. & MENGUAL X., Revision of Armillipora (Diptera, Psychodidae) on apical margin of concavity (apical lobes not separated by broad concavity (concavity being less than the length of apical lobe) and pair of long spines on apical margin of concavity absent in Alepia and Platyplastinx).

Biology
To date, nothing is known about the immature stages and the biology of the species of Armillipora.
Given the known information about the tribe Maruinini, it is expected that larvae of Armillipora breed in some aquatic or semi-aquatic environment.

Differential diagnosis
Armillipora imitata sp.nov. is very similar to A. muyu sp.nov.and A. selvica but the three can be differentiated as follows: in A. selvica the interocular suture has a short posterior spur (posterior spur absent in A. imitata and in A. muyu); one conical apical and one spiniform tooth at the apex of labella in A. imitata (only one conical apical teeth in A. selvica; one preapical spiniform and one apical clawshaped in A. muyu); A. imitata has six apical setae at the apex of gonocoxites (three to four setae placed on a preapical lump in A. muyu, and three to four preapical setae in A. selvica); the gonocoxal condyles is not triangular and not protruding beyond the base of the ejaculatory apodeme in A. imitata and A. muyu (the sclerite is triangular and protruding beyond the base of ejaculatory apodeme in A. selvica).

Etymology
The species epithet 'imitata' derives from the Latin word 'imitātus' (feminine 'imitāta') referring to its similarity with other species.It is to be treated as an adjective.

Male
Head.About 1.10 times as wide as long; eye bridge separated by l facet's diameter, with four rows of facets, interocular suture as sclerotized, almost straight line; frontal patch of alveoli divided.Antennal scape about 1.5 times as long as pedicel, almost cylindrical; pedicel spherical, smaller than scape; flagellomeres fusiform and longer than scape, with scattered setae on surface, setae almost as long as flagellomere bearing them, each flagellomere with two rings of teardrop-shaped pores, apical flagellomeres absent in examined material, maximum number of flagellomeres present: five; ascoids indistinguishable in examined material.Palpal segments cylindrical, palpal proportions: 1.0:1.1:1.1:1.8, last palpal segment corrugated; labium without any strong sclerite; labella elongated and irregularly shaped scattered setae on surface, with one apical spiniform tooth on each.
tHorax.Without allurement organs.With single patch of alveoli in paraterguite and antepronotum; all coxae with stripe of one to two rows of alveoli.Wing length about two times its width; wing membrane brown-infuscated, with lightened spots in between apex of longitudinal veins, and with light triangularshaped spot between origin of R 1 and R 2+3 (Fig. 1B); subcostal vein short ending beyond origin of R 5 ; junction of R 2+3 basal to junction of M 1+2 , not joining R 4 , origin of M 1+2 basal to origin of R 2+3 ; R 5 ending at wing apex; CuA 2 faintly ending at wing margin.D, 2B-E).Hypandrium in dorsal view V-shaped, sclerotized and joining base of gonocoxites, in lateral view hypandrium looks membranous (Figs 1C, 2B, E) with sclerotized margin; gonocoxites joining at apex forming U-shaped sclerite, with concavity at lower margin.Gonocoxite sclerite placed above aedeagal complex.On each side of sclerite is a preapical cluster of six setae (Figs 1C, 2B); gonostyli absent; aedeagus in dorsal view straight, as single sclerite, no discernible parameres.In lateral view, aedeagus apex curved towards epandrial appendage (Figs 1D, 2E); ejaculatory apodeme about half length of aedeagus, in dorsal view basal margin rounded and slightly concave in middle, in lateral view, ejaculatory apodeme looks like half-circle, with basal margin convex; gonocoxal condyles fitting in concavity on underside of ejaculatory apodeme, not triangular-shaped and not protruding beyond base of ejaculatory apodeme; epandrium rectangular, wider than long, with more sclerotization at margins, anterior and posterior margins with medial concavity; hypoproct tongue-shaped (Fig. 1C), shorter than epandrium and covered with small setulae, epiproct not visible in examined material; epandrial appendage barely hemispherical, prolonged and tapering distally, covered with small setae; epandrial appendage lacking apical tenacula.In dorsal view (Figs 1C, 2B), line of five short and cylindrical tenacula, with folded tips; in lateral view, (Figs 1D, 2E) first four tenacula close to each other, last tenaculum separated and located in projection of epandrial appendage, this projection not visible in dorsal view; epandrial appendage possesses additional patch of long accessory tenacula basally concentrated in darkened patch, these accessory tenacula being as long as or longer than epandrium (Figs 1C, 2B).

Female
Unknown.

Distribution
Only known from the type locality in Ecuador.

Differential diagnosis
Male: see differential diagnosis under A. imitata sp.nov.

Etymology
The species epithet 'muyu' derives from the Quechuan word 'muyu', meaning circle and referring to the circular shape of the base of the ejaculatory apodeme.It is to be treated as a name in apposition.
tHorax.Allurement organs absent, with single patch of alveoli in paraterguite and antepronotum; all coxae with stripe of one to two rows of alveoli.Wing length about two times its width; wing membrane brown-infuscated, with darkened spots on apex of longitudinal veins (Fig. 1B); subcostal vein short ending beyond origin of R 5 ; junction of R 2+3 basal to junction of M 1+2 , not joining R 4 , origin of M 1+2 basal to origin of R 2+3 ; R 5 ending at wing apex; CuA 2 faintly ending at wing margin.
terMinalia (Figs 3C-D, 4B-C, E-G).Hypandrium in dorsal view U-shaped, and sclerotized, joining base of gonocoxites.In lateral view hypandrium looks membranous (Fig. 3D) with sclerotized margin; gonocoxites joining at apex forming V-shaped sclerite placed above aedeagal complex, each with preapical lateral lump with cluster of three to four setae (Figs 3C, 4B), gonostyli absent; aedeagus in dorsal view straight, as single sclerite, no discernible parameres.In lateral view, aedeagus has curved apex towards hypandrium (Fig. 4G); ejaculatory apodeme about same length as aedeagus, in dorsal view, basal margin rounded and slightly concave in middle, in lateral view, ejaculatory apodeme looks like half-circle, with basal margin concave; gonocoxal condyles fitting in concavity on underside of ejaculatory apodeme, not triangular-shaped and not protruding beyond base of ejaculatory apodeme; epandrium rectangular, slightly wider than long, with more sclerotization at margins, lateral margins with slight concavity in middle; hypoproct tongue-shaped, shorter than epandrium and covered with small setulae, epiproct shorter than hypoproct; epandrial appendage barely hemispherical, prolonged and tapering distally, covered with small setae, lacking apical tenacula but with line of five short and cylindrical tenacula, with folded tips, and additional patch of long accessory tenacula basally concentrated in darkened patch, these accessory tenacula being as long as or longer than epandrium (Figs 3C,4C).4D, 5A-C) Similar to male except for following characteristics: two rings of teardrop-shaped pores in flagellomeres not as well defined as in males being more scattered and smaller, flagellomeres smaller than male flagellomeres.Wing length equals 2.55 times its width.Subgenital plate long, lateral margins concave in middle, and apical margin has rectangular concavity, with two setae at margin of concavity in addition to scattered setae on surface (Figs 4D, 5A, C); cerci about 1.5 times as long as subgenital plate (Fig. 5A), each with scattered setae on basal surface; genital chamber appears asymmetrical; however, this might be due to bad slide preparation, nonetheless, structures can be seen in Figs 4D, 5B.Female of Armillipora muyu sp.nov.can be easily differentiated from female of Armillipora selvica by following characters: apical concavity in subgenital plate rectangular (rounded in A. selvica); genital chamber with two anterior lobes in Armillipora muyu as in Figs 4D, 5B (genital chamber quadrate without anterior lobes in A. selvica, see Quate 1996: fig.11d).

Female (Figs
Egg (Fig. 5D) Female specimen contained eggs inside abdomen, shape of eggs long-ovoid, being five times as long as wide; general appearance of membrane corrugated, with irregular folds across entire surface; anterior pole of eggs has semi-circular small projection.JAUME-SCHINKEL S. & MENGUAL X., Revision of Armillipora (Diptera, Psychodidae)

Remarks
In the paratype ZFMK-DIP-00081667, the palpal segments are missing; the thorax and right wing were used for DNA extraction and are not present in the slide.

Distribution
Only known from the type locality in Ecuador.

Genetics
Six specimens were successfully sequenced (ZFMK-TIS-2636967, ZFMK-TIS-636968, ZFMK-TIS-2636969, ZFMK-TIS-2636973, ZFMK-TIS-2637146, ZFMK-TIS-2629905).The maximum intraspecific uncorrected pairwise distance for COI sequences was 3.04 % or 20 bp.Genbank accession numbers are: OQ706383, OQ706381, OQ706385, OQ706386, OQ706378, OQ706376.Quate, 1996 Fig. 6 Armillipora selvica Quate, 1996: 29 (description  results.In other words, we think that morphological determination in combination with DNA barcodes is a good way to determine species of the genus Armillipora.In the Neotropical region, however, there is a gap in the number of known species (Linnean shortfall) in combination with a poorly documented geographic distribution (Wallacean shortfall).Both shortfalls with the combination of an absent DNA barcode reference library and only a handful of DNA barcodes generated from the Neotropical region lead to a lack of important information for an integrative taxonomy approach, and future species delimitation techniques could provide valuable information for the genus and for the subfamily Psychodinae in the Neotropics, in general.

Armillipora selvica
Species distribution models do well in predicting the occurrence of many species (Lee-Yaw et al. 2021), even dealing at genus level (Stas et al. 2020), and our inferred distribution model (Fig. 7) shows that Armillipora could be found in several other countries than the ones from which the genus is currently reported (e.g., Brazil, Colombia, French Guyana, Guyana, Peru, Surinam, Venezuela).Following the proposed biogeographic regionalization of Morrone (2014: fig.12) it can be expected to find the genus in the Mexican Transition Zone, the Antillean subregion, Brazilian subregion, Mesoamerican dominion, Pacific dominion, Boreal Brazilian dominion, South Brazilian dominion and the Chacoan subregion, with a potential distribution mainly restricted to the Neotropics.There is no doubt that the known range of the species lacks information and further records will be found.As more information about the immature stages of Psychodinae is known it will become easier to find specific habitats to find new records and new species (e.g., searching for specific microhabitats).
Biodiversity loss in the Neotropical region is mainly due to the high fragmentation of the habitats, and many species are doomed to disappear (Antonelli 2021).Hence, the importance to fill the distribution gaps in order to increase our understanding of the natural environments and the relationships with the species inhabiting them (Santos & Hoppe 2018).Moreover, these knowledge gaps are usually encumbered by insufficient taxonomical information (e.g., lack of taxonomists, lack of funding, lack of species surveys, and taxonomic impediments) and highlight the importance of both the need for taxonomical works and the usual apathy of governments to support taxonomical initiatives.This is why projects such as "Diversidad de moscas florícolas (Insecta: Diptera) del Ecuador", together with international collaborations, are crucial to fill the taxonomical gaps in the Neotropical region aiming for a more complete species list and their distribution in the Neotropics.