Revision of the birch-associated genus Massalongia (Diptera, Cecidomyiidae), with description of a new species from Japan and a taxonomic key to worldwide species

Abstract Betula (Betulaceae), or birch, is a Holarctic genus of trees and shrubs whose species have ornamental, industrial, and medical importance. Gall midges of the genus Massalongia (Diptera: Cecidomyiidae: Cecidomyiidi) are exclusively associated with birches in the Palearctic region. In 2018, an undescribed Massalongia species was discovered forming leaf galls on the midveins of B. grossa on Mount Tara, Saga Prefecture, Kyushu, Japan. In this study the species is described as M. nakamuratetsui Elsayed & Tokuda, sp. nov., and a DNA barcode provided for it. The other known species of Massalongia are redescribed because the original descriptions are outdated and insufficient. A lectotype is designated for M. bachmaieri. In addition, the monotypic genus Apagodiplosis, containing A. papyriferae associated with B. papyrifera in the Nearctic region, is synonymized here under Massalongia, resulting in M. papyriferaecomb. nov., rendering Massalongia a Holarctic genus with six species. Comparing the sequence data of M. nakamuratetsui with all sequences available in The Barcode of Life Data (BOLD) system supports the occurrence of Massalongia in the Nearctic region and suggest that more species could be discovered there. Massalongia species form leaf or bud galls, and their mature larvae drop to the ground in autumn and overwinter in characteristic waterproof bottle-like cocoons, which is possibly a protective adaptation for pupation in wet and snowy lands. A taxonomic key to all Massalongia species is provided.

were dissected under a stereoscopic microscope and larvae were preserved in 75% ethanol for morphological examinations and 99.5% ethanol for molecular analysis.
Mature larvae inside their cocoons (Figs 2, 3) were collected from leaf litter under the galled tree in the same location as the earlier leaf collection. Some cocoons were cut open to retrieve larvae and preserve them in 75% ethanol. Remaining cocoons were transferred to plastic cups containing a mixture of peat moss and sand following Elsayed et al. (2018a). The cups were half buried in the soil and maintained until the beginning of March 2019 in a research farm of Faculty of Agriculture, Saga University, Saga Prefecture (elevation 5.5 m a.s.l.). After the cups were brought back to the laboratory, gall midge adults emerged in late March 2019. Adults were preserved in 75% ethanol and pupal exuviae were preserved in 99.5% ethanol.
Morphological examination and terminology. Gall midge specimens of the newly described species and M. bachmaieri Möhn, 1958 were mounted on microscope slides in Canada balsam following the technique outlined in Gagné (1994), except for the clearing step for the larval and adult specimens following Elsayed et al. (2018b). The slide-mounted specimens were examined under a bright-field and phase-contrast microscope (CX43, Olympus, Tokyo) and line illustrations were made with a mechanical pencil with the aid of a drawing tube. These illustrations were scanned and inked using Apple Pencil 2 and the application Procreate (version 5.0.3) on iPad Pro 2018 (Apple Inc., California). Photomicrographs were taken with a digital camera (DP22, Olympus, Tokyo) attached to a semi-motorized fluorescence microscope (BX53, Olympus, Tokyo).
Morphological terminology mainly follows Kirk-Spriggs and Sinclair (2017) for adults. Larval and pupal terminology follow Gagné (1994). All types of the newly described species are deposited in the collection of Entomological Laboratory, Faculty of Agriculture, Kyushu University, Japan (ELKU).
The ethanol-preserved adults, pupal exuviae and larvae of M. bachmaieri were borrowed from the collection of Staatliches Museum für Naturkunde, Stuttgart (SMNS) The holotype and paratypes of M. betulifolia Harris, 1974 adults were borrowed from the Natural History Museum in London, United Kingdom (BMNH). The ethanolpreserved larvae of M. rubra (Kieffer, 1890) were obtained from the collection of Marcela Skuhravá and mounted on slides following the technique mentioned above.
DNA extraction, sequencing, and alignment. The total DNA was extracted from the whole body of three second instars and one third instar of the Japanese species using the NucleoSpin Tissue kit (Macherey Nagel, Germany) following the manufacturer's protocol. Fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified using a TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan) and following set of primers: J-1718 (5'-GGA GGA TTT GGA AAT TGA TTA GTT CC-3') (Simon et al. 1994) and COIA (5'-CCC GGT AAA ATT AAA ATA TAA ACT TC-3') (Funk et al. 1995). The PCR products were purified using ExoSAP-IT reagent (Affymetrix Inc., USB products, Ohio, USA). The sequencing reaction was performed using the BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA, USA). Ethanol precipitation was used for post-reaction cleanup, and an ABI 3130 sequencer (Applied Biosystems) was used for sequence determination. The obtained sequences were aligned using the software MEGA (ver. 6.0) (Tamura et al. 2013), and were deposited in the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank (http://www.ncbi.nlm.nih.gov/genbank).

Genus Massalongia Kieffer, 1897
Massalongia Kieffer, 1897: 12. Type species, Hormomyia rubra Kieffer by original designation. Apagodiplosis Gagné, 1973: 862. Type species, Oligotrophus papyriferae Gagné, comb. nov. Diagnosis. Massalongia differs from other genera of the supertribe Cecidomyiidi in the following combination of characters: antennal flagellomeres are cylindrical in both sexes; male flagellomeres possess three sets of short-looped circumfila that appear interconnected at least in some flagellomeres of each specimen; the reduced abdominal setation; the unmodified female tergite VIII; the presence of dorsal pigmentation on the protrusible part of ovipositor; the massive gonocoxites and mediobasal lobes; the habit of mature larvae to pupate in the soil inside hyaline bottle-shaped cocoons. The following diagnosis lists the attributes shared by known species and can serve as a checklist for future species descriptions.
Pupa (Figs 19-20). Exuviae not pigmented except antennal horns and prothoracic spiracles. Two asetose and 2 setose cephalic papillae present. Prothoracic spiracle long, slightly curved. Abdominal spiracles present on segments II-VI. Abdominal segments I-VII each with 6 dorsal papillae. Dorsal and lateral parts of abdominal segments covered evenly with pointed spinules, diminishing gradually in length and width, except on posterior third.
Etymology. The species is named in honor of the late Japanese physician Dr. Tetsu Nakamura in recognition to his lifelong dedication to supporting poor people and his significant contributions to the development of Afghanistan. Dr. T. Nakamura was fatally shot by extremists on 4 December 2019 in Afghanistan, exactly on the date when we prepared the first draft of this paper and were considering what to name the species. In this way, we wish to immortalize his contributions to humanity.   (Fig. 1). One leaf can bear several galls and some galls become fused with larvae occupying separate chambers. Galls are 1.52-3.10 mm in diameter and 6.46- 18.03 mm long. Galls collected at the end of August contained white first instars. Larvae develop to second and mature larvae by the end of September. In late October, the mature larvae leave the galls to overwinter in the ground, where they spin hyaline, bottle-shaped cocoons on leaf litter (Figs 2, 3). The cocoon of M. nakamuratetsui is waterproof and does not allow water to reach the overwintering larva (Suppl. material 1: Video S1). Adults emerge between the end of March and the beginning of April.
Male abdomen. Tergite VIII with posterior row of setae. Terminalia (Figs 33-35): gonostylus with pointed denticles; cerci base with setae; cerci with setae on apical margin; hypoproct entire, slightly notched, narrowed after basal third; aedeagus shorter than cerci and hypoproct, cylindrical in dorsoventral view, wide basally in lateral view.  Pupa (Figs 36-38). Head and thorax of exuviae slightly pigmented; abdomen not pigmented. Antennal horns with short, acute, apical protuberances. Two setose lower facial papillae present; 1 asetose and 1 setose lateral facial papillae present on each side. Prothoracic spiracle long, ca. 210 μm, with trachea extending to just before tip. Abdominal segment VIII with 2 setose dorsal papillae. Abdominal terga II-VIII with 2-3 median rows of wider and longer spinules than surrounding ones. Distribution. Europe: Germany and Russia (Gagné and Jaschhof 2017). Gall and life history. Massalongia bachmaieri induces parenchymal leaf galls on B. nana (Fig. 42). Mature larvae leave the galls and drop to the ground in mid to late October. They overwinter in cocoons that are spun on the fallen leaves. This species has one generation a year (Möhn 1958;Bachimaier 1965).
Remarks. Möhn (1958) designated a male specimen as a holotype of M. bachmaieri and two males and a female as paratypes. When we requested the types for this study, we found that all specimens deposited in SMNS were preserved in alcohol. Möhn probably prepared his illustrations of the species from temporary slide mounts and then put the specimens back in alcohol with the others. Because it was not possible to determine Möhn's holotype and paratypes among these ethanol-preserved specimens, we designated a lectotype and paralectotypes from the permeant slide-mounted specimens we prepared.
Distribution. Europe: England and Norway (Gagné and Jaschhof 2017). Gall and life history. Massalongia betulifolia forms blister-like leaf galls on B. pendula and B. pubescens. Galls are formed usually between or on veins and are 2.5-3.0 mm wide and 5.0-6.0 mm long. Mature larvae drop to the ground to overwinter in cocoons. Adults emerge probably in May and June, and the galls can be found on the trees between June to October (Harris 1974;Askew and Ruse 1974).
Remarks. See Remarks under M. bachmaieri and M. nakamuratetsui.
Distribution. Widespread in Europe and west Asia (Gagné and Jaschhof 2017). Gall and life history. Massalongia rubra induces barely noticeable midrib leaf galls on Betula pubescens Ehrh. and other Betula spp. (Gagné and Jaschhof 2017;Kieffer 1913b). The females lay eggs on young leaves in May, and most mature larvae leave the galls to overwinter in the ground in October, but some hibernate in the galls (Skuhravá and Skuhravý 1973).
Remarks. The larval specimens we described here were collected from similar galls to those described by Kieffer (1913b) for M. rubra and the larval morphology fits Kieffer's description and illustrations, thus we believe they indeed belong to M. rubra. Types of M. rubra, like most of Kieffer's types, are considered lost (Gagné and Jaschhof 2017). We considered using one larva for designating a neotype for the species, but because no adults were reared from these larvae, we cannot be completely certain about their identity and decided to refrain from doing so. Kieffer (1913b) provided an illustration of male terminalia showing that the species is distinctive from the other known species of Massalongia by its long and apically enlarged aedeagus (Fig. 52). Because this illustration is important for separating species and it was drawn from the type speci-men, future designation of a neotype for the species must rely on reared adults that will enable to compare characters of the male terminalia. (Gagné, 1967), comb. nov.
Gall and life history. Massalongia papyriferae forms bud galls on the paper birch, B. papyrifera. The mature larva drops to the leaf litter to overwinter in a bottle-shaped cocoon. Adults emerge in spring (Gagné 1967).
Gall and life history. Massalongia altaica form barely visible swellings, 5-7 mm long, on the leaves of Betula nana var. rotundifolia (Spach) Regel. (B. rotundifolia in the original description). The mature larva leaves the gall through an opening on the lower side of the leaf and overwinters in the ground (Fedotova 1991).
Remarks. Massalongia altaica was described from adult specimens reared from larvae that emerged from leaf galls on B. rotundifolia, which is currently known as a variety of B. nana, the same host plant of M. bachmaieri (The Plant List 2013). The illustration of M. altaica galls provided in its original description (Fedotova 1991) is quite similar to the galls of M. bachmaieri (Fig. 43). Morphologically, the adults of M. altaica are closest to M. bachmaieri and differ from them only in the shape of the aedeagus and male hypoproct and the relative length of cerci to male hypoproct, but these differences are based on the original description of M. altaica (Fedotova 1991). Because the type specimens of M. altaica were not available to us, we could not verify the differences between M. altaica and M. bachmaieri. A future examination of M. altaica types and its immature stages may result in synonymizing it under M. bachmaieri.

Discussion
Massalongia has been considered so far a Palearctic genus (Gagné and Jaschhof 2017), but in the present study we synonymized the Nearctic Apagodiplosis under Massalongia, thus the distribution of Massalongia corresponds now to that of its Holarctic host plant, Betula (Shaw et al. 2014). Comparing the sequence data of M. nakamuratetsui with all sequences available in The Barcode of Life Data (BOLD) system revealed several sequences with interspecific similarity of up to 96.85% (Ratnasingham and Hebert 2007), all from Canada (Hebert et al. 2016). The profile of one of these cecidomyiids (sequence ID: CNPKE263-14) included a photo of a female specimen that resembles Massalongia and the interspecific similarity was 95.3%. This strongly supports Massalongia as a Holarctic genus and suggests that more Massalongia species can be discovered in the Nearctic region.
Larvae of many gall midge species that drop to the ground are known to spin cocoons in which they overwinter and eventually pupate (Gagné 1989). Bakhshi and Grover (1976) studied cocoons of various gall midge taxa and concluded that the cocoon shape is specific to genus. The bottle-shaped cocoon of Massalongia has never been reported from other gall midge taxa and thus it appears to be a unique characteristic of the genus. Cocoons of many insects that overwinter in the soil provide mechanical protection against unfavorable surrounding conditions (Danks 2004). Because the cocoon of M. nakamuratetsui is waterproof, the bottle-like cocoons of Massalongia possibly represent a protective adaptation for pupation in wet and snowy lands. Further research on these cocoons is necessary in order to understand the nature of its texture and other roles of its bottle-like shape.
ing on an early draft of the manuscript. We thank Duncan Sivell (Natural History Museum of London, UK) for arranging the loan of type specimens of M. betulifolia, and Daniel Whitmore and Hans-Peter Tschorsnig (Staatliches Museum für Naturkunde, Stuttgart, Germany) for arranging the loan of type specimens of M. bachmaieri. Ayman K. Elsayed is JSPS International Research Fellow (Graduate School of Science, The University of Tokyo, Japan).