Cladistic analysis of the genus Bruggmanniella Tavares (Diptera, Cecicomyiidae, Asphondyliini) with evolutionary inferences on the gall inducer-host plant association and description of a new Brazilian species

In this study, we present a phylogenetic analysis of the genus Bruggmanniella Tavares based on morphological features. Cladistic analyses were conducted using 57 characters from 26 species. All species of Bruggmanniella except for B. byrsonimae were selected as ingroup and the genera Asphondylia Loew, Bruggmannia Tavares, Illiciomyia Tokuda, Parazalepidota Maia, Pseudasphondylia Monzen, Schizomyia Kieffer, and Lopesia Rübsaamen as outgroup. We used characters from larvae, pupae, adults, and galls. The results of this study supported Bruggmanniella as the sister group of Pseudasphondylia. Bruggmanniella actinodaphnes Tokuda and Yukawa and B. cinnamomi Tokuda and Yukawa have been moved to genus Pseudasphondylia (Pseudasphondylia actinodaphnes (Tokuda and Yukawa) comb. nov. and Pseudasphondylia cinnamomi (Tokuda and Yukawa) comb. nov.). The new genus Odontokeros gen. nov. has been erected for the single species Odontokeros brevipes (Lin, Yang & Tokuda) comb. nov. In addition, we described a new Brazilian species, Bruggmanniella miconia Garcia, Lamas and Urso-Guimarães sp. nov. Identification keys to the New World species of Bruggmanniella are presented.


Introduction
The family Cecidomyiidae is one of the most diversified families of Diptera, with more than 6,600 described species worldwide [1]. Estimates indicate thousands [2,3] to more than a million [4] of undescribed species. The family belongs to the Sciaroidea within the infraorder Bibionomorpha [5,6] and the monophyly is recognized without controversy [7,8]. The phylogenetic analyses of Sikora et al. [9] and Dorchin et al. [10] corroborate the classification of the main branches of Cecidomyiidae outlined in Gagné and Jaschhof [1]. PLOS  stereomicroscope to obtain immature instars (larva and pupa). The obtained specimens were mounted on slides according to the methodology outlined in Gagné [11]. Additional material was stored in 100% ethanol for DNA extraction. Morphological terminology for adults follows Cumming and Wood [29] and immature terminology follows Gagné [11]. Types of the new species are deposited in the Museu de Zoologia of the Universidade de São Paulo, São Paulo, Brazil (MZSP). Voucher specimens of the host plant species are deposited in the Herbarium of the Universidade Federal de São Carlos, campus Sorocaba, Brazil.

DNA extraction
DNA was extracted from two whole pupae using DNeasy Blood & Tissue Kit (Quiagen) following the manufacturer's instructions. The mitochondrial cytochrome oxidase subunit I (COI) barcoding region was amplified by polymerase chain reaction (PCR) using the following primers: LCO1490 (forward) and HCO2198 (reverse) [30]. The PCR products were purified using Exo/SAP and the COI sequences were assembled and aligned using the Geneious Prime 2020 software [31]. The COI-alignment was constructed using the MUSCLE algorithm in Geneious with eight iterations. The obtained sequences were deposited in the GenBank under the accession number MN686345.

Selection of taxa
For the phylogenetic study, a total of 26 species were selected and examined. Fifteen species were selected as outgroups, including the genera Asphondylia Loew, Bruggmannia Tavares, Illiciomyia Tokuda, Parazalepidota Maia, Pseudasphondylia Monzen, Schizomyia Kieffer (Asphondyliini), and Lopesia Rübsaamen (Lopesiini). The ingroups were represented for all the species within the genus Bruggmanniella, except for Bruggmanniella byrsonimae (Maia and Couri) due to its unknown larval stage. The list of species examined for the analysis can be found in the S1 Table. Most of the studied material was already preserved on mounted slides and were borrowed from the following institutions: Museu Nacional of Rio de Janeiro, Rio de Janeiro, Brazil (MNRJ), Museu de Zoologia of the Universidade de São Paulo, São Paulo, Brazil (MZSP), and National Museum of Natural History, Washington, DC, USA (NMNH). Literature data and photographs of the types unavailable for examination were used to aid in the character coding states. These photographs were received from the Collection of the Entomological Laboratory of the Kyushu University, Japan (KUEC).

Character sampling
Morphological data of third-instar larva, pupa and adults were coded in a matrix. Our sampling resulted in 55 discrete characters and two continuous characters, totaling 57 characters. Of these, seven characters were coded from larvae, 17 from pupae (two continuous and 15 discrete), 33 from adults (25 of sex-specific characters) and one from galls. Continuous characters were used as it was not possible to categorize certain numeric features, nor to establish a range of values, avoiding arbitrary coding. These characters were treated according to unity-based normalization methods outlined in Thiele [32] and Goloboff et al. [33] (normalized data can be found in S2 Table). Nonapplicable and unknown data were entered in the matrix as '-' and '?', respectively (S3 Table). See S1 File for input matrix script.

Phylogenetic analysis
The cladistic analysis was performed under the parsimony criterion with implicit weighting in TNT v1.1 (Willi Hennig Society Edition) [34]. Equal and implied weighting were implemented, the latter with values of k ranging from 3 to 20. All characters were treated as unordered. Tree searches were conducted using 'Traditional Search' with 1,500 replicas of Random Addition Sequence (RAS) combined with the branch-swapping: Tree Bisection and Reconnection (TBR) algorithm, saving 50 trees per replica. These values were chosen to reach the number of hits greater than 50 and to avoid overflow, so the most parsimonious trees could be chosen.
Relative Bremer support was calculated in TNT, retaining suboptimal trees by 10 steps, under implied weighting [35]. The suboptimal trees were obtained from a 'traditional search', using the 'trees from RAM' setting. The numbers of relative support are given by TNT in percentage, ranging from 0 to 100%. The performance of the characters was verified by the consistency index (CI) [36] and retention index (RI) [37]. Cladograms were rooted with Lopesia andirae Garcia, Lima, Calado and Urso-Guimarães (outgroup). The resulting tree was displayed in Adobe Illustrator CC software (17.0).

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system 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:90AF24-F9-AEAD-458D-99DB-FDA1B12F9642. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Continuous characters
0. Length of pupal antennal horn (ci = 0.31; ri = 0.48; fit = 0.73). Pupal antennal horn length is measured from the base of the antennal sheath to the tip of the apical horn. In Bruggmanniella the length ranges from 0.13 mm (B. oblita Tavares) to 0.6 mm (B. duguetiae Urso-Guimarães and Amorim).
4. Relative size of apical teeth; (0) same size, (1) outer larger than inner, (2) inner larger than outer (ci = 0.33; ri = 0.5; fit = 0.6). Bruggmanniella species are known for having prothoracic spatula with inner teeth larger than the outer ones. However, B. brevipes presents a spatula with the outer tooth larger than the inner ones. Bruggmanniella actinodaphnes and B. cinnamomi have equally sized teeth.

Phylogenetic analysis
The phylogenetic analysis under equal and implied weights produced a single parsimonious tree (S1 and S2 Figs), with both analyses showing Bruggmanniella as the sister group of Pseudasphondylia. The tree found for k = 6-20 seems to be stable with the resulting topology for the ingroup taxa with no changes in k values. The MPT for k = 6 (fit = 35.91, length = 175 steps, CI = 0.41, RI = 0.56) is shown in Fig 1. In this study, three synapomorphies support the monophyly of the genus Bruggmanniella: Tokuda and Yukawa [21,22] state that the similarity between Bruggmanniella and Pseudasphondylia is based on the presence of the two separated teeth in the gonostylus and in the presence of parameres. One of the synapomorphies of Bruggmanniella resultant of this analysis is the absence of parameres, which is present in all Schizomyiina species, but missing in the most genera of Asphondyliina. Bruggmanniella brevipes Lin, Yang and Tokuda appears in our results in the base of (Bruggmanniella + Pseudasphondylia). Bruggmanniella brevipes shares with Pseudasphondylia and the other Bruggmanniella species, the two separated teeth of the gonostylus in the male and the absence of frontal horns on pupa. However, B. brevipes differs by having prothoracic larval spatula with four teeth with the outer teeth larger than the inner ones. Also, B. brevipes has shorter legs than its congeners and induces bud galls on Neolitsea parvigemma (Lauraceae), which is endemic in Taiwan.
The results of this study indicate that B. brevipes does not belong to any described genus of Asphondyliina, so as a consequence of this analysis we propose the erection of a new genus named Odontokeros Garcia, Lamas and Urso-Guimarães gen. nov., to which this species will be transferred based on the following synapomorphies: presence of prothoracic spatula with four teeth in larva, with the outer teeth larger than the inner ones, and shorter legs in adults. Odontokeros brevipes (Lin, Yang and Tokuda) comb. nov. is a monotypic genus with Bruggmanniella brevipes as the type species by present designation.
Illiciomyia Tokuda is recovered as a monotypic genus, as the sister group of the clade formed by (Odontokeros gen. nov. + (Bruggmanniella + Pseudasphondylia)). Moreover, these four genera share the two separated teeth of the gonostylus and the absence of frontal horns on pupa. Tokuda [25] argued that Illiciomyia yukawai is phylogenetically diverging from Bruggmanniella and Pseudasphondylia based on the shallow constriction of male flagellomeres, less appressed male circumfila, absence of apical spur on the first tarsomere of all legs, and larva with five lateral papillae. Our analysis supports these arguments and additionally points out the difference in the consistency and presence/absence of parameres, which are absent in Bruggmanniella, membranous in Pseudasphondylia and in Odontokeros gen. nov., and sclerotized in Illiciomyia.
Remarks on geographical distribution and gall maker-host plant association. The result of this cladistic analysis allows us to discuss some aspects of the gall inducer-host plant association of Bruggmanniella, Pseudasphondylia, Illiciomyia, and Odontokeros gen. nov. Information on host plant families and geographical location data for each species were added to the resulting topology (Fig 2). The genus Pseudasphondylia has its distribution restricted to the Australian, Oriental, and Palearctic regions, while Bruggmanniella is now restricted to the New World, mostly to the Neotropics. Most Bruggmanniella species have their distribution concentrated in the South-Eastern Region of Brazil. The same is true to Pseudaphondylia, Illiciomyia, and Odontokeros gen. nov. in which most species are recorded in Japan. This may reflect the lack of sampling in other areas/regions, as well as the concentration of experts, and consequently of collection efforts, in these specific regions.
Galls on stems are much more frequent in Bruggmanniella (60%) than in its sister group Pseudasphondylia (25%-including B. actinodaphines and B. cinnamomi). Fifty percent of Pseudasphondylia species, the monotypic genera Illiciomyia and Odontokerus gen. nov. induce galls on leaves. The induction on stems appears as a homoplastic synapomorphy to the genus Bruggmanniella. In addition, the clade P. neolitseae + (P. actinodaphnes + P. cinnamomi) induces galls on species of the same family of the host plant, Lauraceae, in the geographic distribution in Japan, giving consistency to the proposed hypothesis.
Tokuda and Yukawa [22] also argued that B. perseae seems to be morphologically close to the congeners of the Neotropical Region than those of the Oriental and Palearctic regions, due to the presence of prothoracic larval spatula with four teeth and male cerci basally fused. Our analysis corroborates this perception. Bruggmanniella perseae is positioned among the Neotropical species, which reinforces the hypothesis that the induction of galls on Lauraceae arose independently in the Neotropical and Oriental/Palearctic regions.
Our results do not show patterns of coevolution or co-speciation among gall inducers and families of host plants in New World species. Each of the ten Bruggmanniella species induces galls on different families of plants. Möhn [38] had already stated that there was no "parallelism" between the evolution of "Asphondyliidi" and their host plants. Also, the morphocline of growing development of the antennal horn in pupae indicates a preference of inducing gall on stems instead of leaves, most commonly in Oriental and Eastern Palearctic Pseudasphondylia species.
Hypotheses about when and where these species initially diverged are difficult to state. Although most Asphondyliini species are distributed throughout the Neotropical Region, there is no information about when the association of these species with their host plants has begun. Biological data on life cycle strategies, behavior, host interactions and physiology for Bruggmanniella species are still unknown or incomplete. More complex analyses on biogeography and co-speciation could offer a better understanding of these issues.

Taxonomy
We present below the new taxonomic treatment for B. actinodaphnes, B. cinnamomi and B. brevipes; the description of the new species and the identification keys.
Pseudasphondylia actinodaphnes ( Diagnosis. Prothoracic spatula with 4-teeth, outer teeth larger than the inner ones; pupa with deeply toothed antennal horns and prothoracic spiracles well developed, upper and frontal horns absent, presence of thickening on pupal cephalic margin; antenna with 12 flagellomeres, male genitalia with two well-developed gonostylar teeth, hypoproct deeply bilobed, parameres present; cerci-like lobes, short female legs [2]. Etymology. The name Odontokeros is a reference to the pupal antennal horns, which have the outer edge deeply toothed (Gr. Odonto, tooth; keros, horn).
Bruggmanniella miconia Garcia, Lamas and Urso-Guimarães sp. nov. urn:lsid:zoobank.org:act:80866883-69C9-46F2-8361-52F60D69A584 (Figs 3-6) Diagnosis. The new species can be easily segregated from the congeners by the following combination of characters: prothoracic spatula with well-spaced inner teeth, terminal segment with absence of papillae; right triangle-shaped antennal horns and edge minutely serrate, cephalic setae long, abdominal segments with conspicuous spines; flagellomeres short and rounded with two appressed circumfila, connected to one another, on both sexes; subcoastal vein convex; tarsal claws simple and longer than empodium; hypoproct deeply bilobed with apically rounded lobes, cercus slightly bilobed, twice as wide as hypoproct, aedeagus narrow at base and pointed at apex; needle-like ovipositor with cercilike lobes present.
Female. Brown. Length: 1.8 mm (n = 1) (ovipositor not extruded). Head: Frons, clypeus, labellum, and palpus as in male. Antenna with 12 flagellomeres; scape and pedicel globoid and setose; flagellomeres with two appressed circumfila connected to each other ( Fig 3C); flagellomeres 9-12 subequal in length (Fig 3D). Thorax: Scutum and scutellum dark brown; anepimeron setose; other pleura bare. Legs with dense blackish scales; first tarsomere of all legs with apical spur; tarsal claw simple, similar in size in all legs, empodium shorter than claw but reaching its curvature, pulvilli absent (Fig 3F). Wings length 2.1 mm and 1.08 mm in width. Venation as in Fig 3G, subcostal convex. Abdomen (Fig 4B): First through seventh tergites rectangular and sclerotized; First through sixth sternites rectangular and sclerotized, seventh sternite longer than the others and strongly striated; tergites and sternites without trichoid sensilla. Ovipositor (Fig 4B and 4C): protrusible, basal portion membranous and striated (0.7 mm length), apical portion sclerotized and aciculate (0.6 mm length); cercilike lobes present at eight abdominal tergites.   Pupa (Fig 5A and 5B). Integument light brown. Length: 2.4-2.8 mm (n = 10). Cephalic setae long (0.06 mm). Antennal horns elongate (0.3 mm length), well-sclerotized, right triangle-shaped, outer edges minutely serrate and inner edges smooth. Upper facial margin not thickened, two pairs of lower facial papillae (one setose and one bare), two pairs of lower lateral setose papillae. Prothoracic spiracles with 0.2 mm length. Leaflets reaching 1/3 of the fifth abdominal segment; all pairs of legs reaching the sixth abdominal segment. Abdominal tergites of the second through eighth segments with dorsal rows of conspicuous spines mesally positioned and different in size; four dorsal papillae with setae situated below the most posterior row of spines. Eighth tergite with fewer spines than the seventh tergite. Abdominal spiracles of the first, seventh and eighth segments sessile. Those of the second through sixth segments well-developed and spiniform.
Larva (Fig 5C and 5D). Yellowish. Integument delicate and hyaline, entirely covered by micro spicules. Length: 1.8-2.5 mm (n = 10). Prothoracic larval spatula (0.3 mm length) with four teeth, inner teeth well-spaced and longer than outer ones. Cervical and sternal papillae with short setae. Three lateral papillae present on each side of the spatula, all setose (one internal and two external). Ventral anus. Terminal segment with papillae not distinguishable from spicules.
Galls and biology. Globoid, glabrous; reddish when young and brown when mature. Induced on stems of Miconia cf. cinnamomifolia (Melastomataceae) (Fig 6A). The galls are unilocular and individualized, occurring in clusters ranging from 10 to more than 100 galls per branch (Fig 6B). The larva pupates in the gall and the adults emerge from a small hole positioned at the apex carved by the antennal horns of the pupa (Fig 6C). Non-identified pupa and female of Schizomyia Kieffer were also found in the galls of the new species. This is the first record of Schizomyia on Melastomataceae and as inquiline in Bruggmanniella galls.
Etymology. The specific name "miconia" is in apposition, referring to the name of the host plant genus. Remarks. Bruggmanniella miconia sp. nov. is configured as a new species belonging to the genus Bruggmanniella by the following characters: prothoracic larval spatula with four teeth, with inner teeth larger than the outer ones; pupal antennal horns strongly sclerotized and welldeveloped; gonostylus teeth completely divided, parameres absent, needle-like ovipositor with cercilike lobes present and subcoastal vein convex.
The new species differs from all its congeners by the unique shape of the male and female flagellomeres, which are shorter and rounded, with two appressed circumfila connected to one another. Bruggmanniella miconia sp. nov. also differs from B. perseae, B. oblita and B. braziliensis by the absence of pulvilli (present in these species) and the cerci shorter than hypoproct. Bruggmanniella perseae and B. braziliensis have cerci longer than hypoproct. The subcoastal vein of the new species is convex, resembling Zalepidota piperis Rübsaamen (Asphondyliini). Bruggmanniella byrsonimae, B. maytenuse, and B. perseae have a straight subcoastal vein and a concavity between Sc and R 1 . Both B. duguetiae and B. ingae have a slightly convexity on Sc, but they have CuA 1 and CuA 2 veins complete, while the wing of B. miconia sp. nov. has CuA 1 and CuA 2 veins incomplete.
The pupae of B. miconia sp. nov. is distinguishable from those of B. perseae and B. duguetiae by the antennal horn straight laterally except for serrations on the outer edge. Bruggmanniella duguetiae has an antennal horn which is concaved laterally, while B. perseae has a convex antennal horn. Also, B. duguetiae has projections present in the eighth abdominal segment, as well as B. bysonimae, which are absent in B. miconia sp. nov. Bruggmanniella maytenuse has a laterally straight antennal horn, but this species has the upper facial margin thickened while the pupa of B. miconia sp. nov. has no thickening on the upper facial margin.
The larvae of the new species and of B. doliocarpi shares the prothoracic spatula with wellspaced inner teeth, but B. miconia sp. nov. differs from B. doliocarpi by having inner and outer teeth with rounded apices, and a narrow long stalk. The terminal papillae are also absent on B. miconia sp. nov., B. oblita and B. perseae. However, the prothoracic spatula of B. oblita has three teeth (the new species has four teeth) and B. perseae has inner and outer teeth of the same size.
Identification keys to the new world species of Bruggmanniella. The identification keys presented here are based on that of Gagné et al. [42]. Three keys are presented below, each for a different life stage, including all New World species of Bruggmanniella. Data on galls and distribution were also included in the adult key. It was not possible to key females of all species since they are very similar to each other.
Identification key to the third instar of the known larvae of Bruggmanniella 3a. Antennal horn twice as long as the widest diameter, convex laterally ( Fig 8D); nonthickened upper facial margin; prothoracic spiracle elongated (0.6 mm length); dorsal spines conspicuous and shattered; projections present in the eighth abdominal segment (Fig 8F) .

Conclusion
This is the first phylogenetic study of the genus Bruggmanniella. The results corroborated previous ideas about the relationship among Bruggmanniella, Pseudasphondylia, and Illicomyia. Some new nomenclatural combinations were proposed and supported by the results of our cladistic analyses: Pseudasphondylia actinodaphnes, Pseudasphondylia cinnamomi, and Odontokeros gen. nov. Bruggmanniella is a monophyletic genus with ten species distributed in the New World, including Bruggmanniella byrsonimae which was not added to the analysis. Further studies and sampling efforts should be conducted in order to find the missing stages of B. byrsonimae and test its placement among the genus. As we have indicated, studies on coevolution also need to be conducted to investigate the association of these genera with their host plants, as well as a biogeographic approach of the tribe Asphondyliini is still lacking.
Supporting information S1