1This is a postrprint of the paper published in the journal research

2https://www.sciencedirect.com/science/article/pii/S019566711730229X and its posted here in

3accordance with the journal’s self-archiving policies

4A new of Buchonomyiinae (Diptera, ) from Late Cretaceous Burmese

5amber, with the phylogeny of the subfamily revisited.

6Viktor Baranov1,2*, Tomasz Goral3 and Andrew Ross4

7

81Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310,12587,

9Berlin, Germany

102Humboldt University of Berlin, Faculty of Mathematics and Natural Sciences, Geography

11Department, Rudower Chaussee 16, 12489 Berlin, Germany

123Imaging and Analysis Centre, Natural History Museum, London, United Kingdom

134 Department of Natural Sciences, Chambers St., National Museum of Scotland, Edinburgh,

14EH1 1JF, UK

15*present address: Senckenberg Research Institute and Natural History Museum Frankfurt,

16Department of River Ecology and Conservation, Clamecystrasse 12, 63571 Gelnhausen,

17Germany.

18Viktor Baranov is corresponding author.

19Highlights

20  Here we are presenting the oldest record of the Chironomidae subfamily

21 Buchonomyiinae

22  This subfamily is a sister group to the rest of the family 23  This discovery is representing a second genus in the previously monotypic group

24  New genus is shading a light on the evolution of the subfamily

25Abstract

26Among the eleven modern subfamilies of non-biting (Diptera: Chironomidae),

27Buchonomyiinae are the most primitive and considered to be the sister group to the rest of the

28chironomids. The subfamily is monotypic with a single genus Buchonomyia, including three

29Recent species from Europe, South-East Asia and Central America, and a single fossil

30species, B. succinea Seredszus and Wichard, 2002, from Baltic . The elusive nature of

31the larvae and pupae, who develop as parasites or are commensal of larvae, means

32that records of recent Buchonomyiinae extremely rare. From the latest dated phylogeny of the

33Chironomidae, the Buchonomyiinae branched from the rest of the Chironomidae in the Early

34to Middle . Here we present the oldest record of the subfamily Buchonomyiinae, from

35Late Cretaceous . The record is represented by a new genus

36Furcobuchonomyia, with a single species F. saetheri sp. nov. Bayesian analysis firmly places

37the new genus as the sister group to the rest of the Buchonomyiinae. Thus the discovery of

38this genus is sheding new light on the origin of the most basal group of the Chironomidae. A

39species level key to the subfamily is included.

40Introduction

41With almost 6000 described species, non-biting midges (Diptera: Chironomidae) are among

42the most diverse and abundant aquatic in the world (Ferrington, 2008). Non-biting

43midges are widely used as indicators of ecological status in freshwaters, due to the close and

44specific association of the preimaginal stages with their habitat (Armitage, Cranston, &

45Pinder, 1995; Walker, 2001). The family has also left a rich fossil record dating back to the

46Triassic, with numerous extant genera known from the Paleogene (Ansorge, 1999; Kalugina, 471980; Krzemiński & Jarzembowski, 1999; Seredszus & Wichard, 2007; Zakrzewska,

48Krzeminski, & Gilka, 2016, Doitteau & Nel, 2007). Chironomids are one of the most

49common taxa in amber, sometimes comprising up to 70% of all inclusions (Doitteau

50& Nel, 2007; Wichard, Gröhn, & Seredszus, 2009). Their abundance in amber, together with

51knowledge about the ecological requirements of modern representatives, allows conclusions

52on palaeoenviromental conditions to be made (Grund, 2005; Seredszus & Wichard, 2007).

54Among the 11 extant subfamilies of chironomids, Buchonomyiinae has a controversial

55taxonomic history (Murray & Ashe, 1985). The first known species of the subfamily –

56Buchonomyia thienemanni Fittkau, 1955 was described by E.J. Fittkau based on a single male

57from Hesse, Germany in 1955. It was classified as a strange representative of subfamily

58Podonominae (Fittkau, 1955). Later, Fittkau suggested placement of the genus into the

59subfamily Orthocladiinae, as a highly plesiomorphic representative (Brundin & Sæther, 1978;

60B. D. A. Murray & Ashe, 1985). It was not until 1978, when both an adult male and a female

61of the second species – B. burmanica were described by Brundin & Sæther (1978), that the

62subfamily Buchonomyiinae was established, mostly based on female characters (Brundin &

63Sæther, 1978). Since the erection of the subfamily, it’s position in the family tree of the

64Chironomidae was disputed, Brundin and Sæther (1978) and Sæther (1979, 2000) argued that

65Buchonomyiinae should be placed within the clade

66Chironominae+Orthocladiinae+Diamesinae+Prodiamesinae. Later, after the description of

67the 1st instar larvae, 4th instar larvae and pupa, the position of the subfamily within a more

68basal clade including Tanypodinae+Podonomiinae+Aphroteninae was decided (Murray &

69Ashe, 1985). The dated molecular phylogeny of the family (Cranston et al., 2012)

70unexpectedly and robustly placed the subfamily as the sister group to the rest of the

71Chironomidae, suggesting an Late /Early Jurassic origin of the subfamily, though still 72being closer to clade Tanypodinae+Podonomiinae+Aphroteninae. The controversial position

73of the subfamily is partially due to its rarity, as only a handful of specimens of each species

74have ever been caught (Ashe, O’Connor & Murray, 2015), due to the elusive nature of larvae

75and adults. Rarity seems to be a result of it’s specialised larval biology, involving parasitism

76of caddisfly larvae belonging to the family Psychomyiidae (Ashe et al., 2015). It is assumed

77that Buchonomyiinae larvae live as parasites of the Psychomyiidae, pupating within the

78caddisfly larval case. In Ireland the distribution of B. thienemanni seems to closely follow

79that of pusilla (Fabricius, 1781) (Ashe et al., 2015). It is difficult however to

80associate Buchonomyia larvae exclusively with Psychomyiidae, as while modern species of

81Buchonomyia co-exist with numerous Psychomiidae species in Europe and Burma (Wichard,

82Ross, & Ross, 2011), and B. succinea was co-inhabiting the Baltic amber forest with at least

835 species of Psychomyiidae (Wichard, Gröhn, & Seredszus, 2009), there are no records of

84this family from Costa Rica, thus B. brundini probably has a different host or a different

85lifestyle (Holzenthal & Calor, 2017)

86Such unusual biology of the larvae makes records of Buchonomyiinae extremely rare, with

87only B. thienemanni known from more than the single (type) locality (Andersen & Sæther,

881995; Ashe et al., 2015; Brundin & Sæther, 1978; Fittkau, 1955). Also the abundance of

89plesiomorphic characters in the genus Buchonomyia has made placement based on the

90morphology alone extremely difficult.

91The subfamily was monotypic with a single genus Buchonomyia, including three Recent

92species from Europe, South-East Asia and Central America, and a single fossil species B.

93succinea Seredszus & Wichard, 2002, from Baltic amber. Currently Buchonomyiinae are

94known from Western and Central Europe, Northern Africa (B. thienemanni), Northern

95Myanmar (B. burmanica), Costa-Rican mountains (B. brundini), Baltic Amber (B. succinea)

96(Murray, Langton, O’Connor & Ashe, 2013) and now from Burmese amber. 97Due to the extreme rarity of the subfamily Buchonomyiinae in the fossil record and its basal

98position in the Chironomidae evolutionary tree, any new fossil of the Buchonomyiinae is of

99utmost interest. Here we describe a new genus and species of the subfamily Buchonomyiinae

100from Late Cretaceous Burmese amber. This new genus bears a close resemblance to

101Buchonomyia, while possessing a number of specialized traits in the structure of the

102hypopygium and legs. This new record is the oldest known for the subfamily.

103

104Geological context

105Burmese amber, from Myanmar, is proving to be the most interesting of all the Cretaceous

106ambers. Until relatively recently it was regarded as rare and difficult to get hold of but over

107the past few years new mines have opened up and it is now freely available. As a result there

108has been a proliferation of papers describing new species, demonstrating the high diversity

109that has been preserved in this amber. By the end of April 2017 481 species of insects had

110been named (Ross, 2017). Although 141 specimens of Chironomidae were listed by Grimaldi

111et al. (2002), demonstrating they are relatively common in Burmese amber, no species have

112been named until now.

113The specimen studied here was purchased by National Museums Scotland from Scott

114Anderson in 2010. It came from the Noije Bum Amber Mine, Kachin State, Myanmar,

115described by Cruickshank & Ko (2003). The age of Burmese amber has been controversial.

116Originally considered to be Miocene, it is now accepted as Cretaceous (see Ross et al., 2010),

117however the exact age has been under dispute. Palynomorph dating suggested an Albian\

118Cenomanian age, and a Mortoniceras ammonite suggested it was late Albian (Cruickshank &

119Ko, 2003). However radioactive zircons provided an age of 98.79 +/- 0.62 Ma, thus early

120Cenomanian (Shi et al., 2012). This provided a minimum age for the bed and not the amber 121as it could have taken time for the amber to be deposited, particularly as many of the pieces

122had been bored by pholadid bivalves. It has recently been established that although the

123outside of the resin must have been hard enough to be bored, the inclusion of bivalves within

124the amber demonstrate that the cores of those resin pieces had been soft when bored, so were

125contemporaneous with the age of the bed (Smith & Ross, in press). Thus an early

126Cenomanian age is most likely for Burmese amber from Noije Bum. What is not known is

127whether the new mines are harvesting amber from the same horizon and this needs to be

128investigated.

129

130Materials and Methods

131The unique holotype is deposited in the Palaeobiology Collection of National Museums

132Scotland (NMS). The species was described based on a single adult male, collection number

133NMS G.2010.20.26 (Scott Anderson Collection). The sample was prepared as a thin (2.5

134mm) square piece of amber and polished. All photographs were taken using an Leica

135DMRBE Research microscope and Leica MC 170 HDcamera (IGB, Berlin). Stacking of the

136images was conducted using Helicon focus software v. 6.7.1

137The specimen was imaged using micro-CT and Confocal Laser Scanning Microscopy

138(CLSM) at The Natural History Museum (London). Micro-CT was carried out using a Nikon

139HMX ST 225 (Nikon Metrology, Tring, UK) with a molybdenum target, at 140kV and

140yielding a resolution of 5 μm. The scan was reconstructed using CT Pro (Nikon Metrology)

141and the data provided good contrast between the specimen and surrounding amber, but lacked

142the resolution necessary to resolve fine details. Confocal images were acquired with a Nikon

143A1-Si confocal microscope using 10x and 20x objectives (numerical apertures of 0.3 or 0.5,

144respectively). 145The character matrix for the phylogenetic analysis was built using nexus data editor v.0.5.0

146(Page, 2001). The character matrix was complied using data from (Andersen & Sæther, 1995;

147Brundin & Sæther, 1978; Cranston, Hardy, & Morse, 2012; Fittkau, 1955; Seredszus &

148Wichard, 2002). Only adult male characters were included in the analysis, as adult females

149are known only for two recent species (B. thienemanni and B. burmanica) and preimaginal

150stages only for B. thienemanni (Andersen & Sæther, 1995; Ashe, 1985; Brundin & Sæther,

1511978; Fittkau, 1955). The full data set comprised 6 taxa and 26 characters, (see Appendices 1,

1522). Austroconops mcmillani Wirth and Lee, 1958 was chosen as an outgroup based on the

153modern hypotheses on the phylogeny of (Borkent, 2012; Borkent & Craig,

1542004)

155We conducted a small-scale phylogenetic analysis, to evaluate the relationships between the

156new genus and other known Buchonomyiinae. The phylogenetic analyses was carried out in

157PAUP*4 for parsimony analyses, and in mrbayes 3.2.2. (Ronquist, Huelsenbeck, &

158Teslenko, 2011). PAUP trees were obtained using the ‘heuristic search’ with default settings

159applied, robustness of the trees was tested using 100 replications of bootstrap. Bayesian

160analysis for the morphological data was conducted as described in Baranov et al. (2016).

161We have plotted the consensus trees showing all compatible groups and 50% compatible

162groups from mrbayes output. Morphological terminology, abbreviations and measurements

163follow Sæther (Sæther, 1980).

164Results

165Phylogeny

166Both methods of the phylogenetic analysis have yielded a very weak signal in the available

167data matrix. Only six characters were parsimoniously informative. A heuristic search has

168yielded 41 most parsimonius trees, with both strict and majority rule consensus trees 169collapsing all the Buchonomyinae taxa in the single polytomic comb, thus the results of the

170parsimony analysis are not given here. Bayesian analysis has yielded better results, with

171higher posterior probabilities support for most nodes, in comparison to maximum parsimony

172(Fig 1). As expected the bayesian phylogeny has placed Furcobuchonomyia as the sister

173group to the genus Buchonomyia. Bayesian analysis also have supported the phylogenetic

174hypothesis of Seredszus & Wichard (2002), stating that B. thienemanni is closer to B.

175brundini, and B. succinea is closer to B. burmanica.

176

177Fig. 1. Phylogeny of recent and fossil Buchonomyiinae, Bayesian analysis, consensus tree

178showing frequencies of all observed bipartitions (‘allcompat’ tree in mrbayes; see text for

179details). New taxa are marked by arrows.

180 181Systematic palaeontology

182Family Chironomidae Newmann, 1834

183Subfamily Buchonomyiinae Brundin & Sæther, 1978

184The subfamily comprises two genera: Buchonomyia with three extant and one extinct species,

185and the extinct Furcobuchonomyia gen. nov. with one new species.

186

187Furcobuchonomyia gen. nov.

188Diagnosis. The new genus is erected based on the following unique combination of the adult

189male’s characters. The male of Furcobuchonomyia has a distinctive bifurcated dorsal lobe of

190the gonostylus, with strong setae inserted midlength on the dorsal branch. The volsella of the

191gonocoxite is distinctively sickle-shaped, as opposed to the rode-like volsella of the

192Buchonomyia. In contrast to the Buchonomyia spp., the new genus has a bare postnotum. A

193single tibial spur present on the hind tibia is highly vestigial. A pseudospur of Ta1 (hind leg)

194is very long and strong (~40 µm). The Ta1 of the foreleg bears two unusual pseudospur-like

195structures at the base of the tarsomere instead of the apex. The distance between MCu and

196RM is more than 1/3 of the wing length. R2+3 is present, vestigial. Auxiliary sclerite bears at

197least 3 setae. Brachiolum is setose.

198Etymology: from latin “Furca” – a fork, to signify the peculiar shape of gonostylus’s dorsal

199lobe and “Buchonomyia” – nominal genus of the subfamily Buchonomyinae 200

201Fig. 2. A) Habitus of the of Furcobuchonomyia saetheri male, side view; B) Wing of F.

202saetheri male.; C) Hind tibia spur; D) Pseudospur on the Ta1 of F. saetheri male; E) Ta5 and

203tarsal claws of the hind leg; Scalebar is 100 µm long.

204

205Furcobuchonomyia saetheri sp. nov.

206Holotype. NMS G.2010.20.26, Noije Bum Amber Mine, Kachin State, Myanmar. Scott

207Anderson Collection.

208Diagnosis: as for genus. 209Etymology: Named “saetheri” after prominent Chironomidae taxonomist and ecologist Ole

210Anton Sæther (1936-2013), in recognition of his role in erection of the subfamily

211Buchonomyiinae.

212Material: Male imago (n=1, holotype)

213Total length:1.95 mm. Wing length: 1.35 mm. Ratio of total length/wing length1.44. Thorax,

214abdomen, legs and head reddish-brown; postnotum and anterior half of first abdominal

215tergite yellow. Longitudinal yellow stripes on scutum (Fig 2A,B).

216Head. AR= 0.53. Ultimate flagellomere 73 µm, penultimate flagellomere123 µm. No visible

217setae on pedicellus. Inner and outer verticals numerous, postorbitals and temporals setae

218present but hard to count due to obscured frontal view. At least 6 clypears. Palpomers length

219(in µm): MP2 83; MP3 77; MP4 143; MP5 150. Eyes bare, with shallow wedge-like

220extension.

221Thorax. Six anteropronotals; 65 dorsocentrals; 20 strong, erect acrostichals ; 7 preallars; at

222least 50 scutellars. Postnotum bare, with small median heel.

223Wing. VR 0.51. Anal lobe weak. Extension of Costa difficult to measure due to the poor state

224of the wing membrane. Numerous microtrichia at the wing tip. First auxiliary sclerite with 35

225setae. Scale like setae present on R1, Costa, Subcosta and Brachiolum. Costa with at least 30

226setae. Squama with 33 setae. First auxiliary sclerite with 3 setae. Legs. Front tibia without

227spur, base of the of the front Ta1 with two prominent pseudospurs about 10 µm. Mid tibia

228without spur. No pseudospurs on midtarsi, hind tibia with the vestigial spur. Spur slim, setae-

229like, without denticles 16 µm (Fig 2C). Apex of the hind Ta1 with long pseudospur 40 µm

230(Fig 2D). Legs covered with the scale -like setae (Fig 2E, 3C). Pulvilli absent, claws simple

231(Fig 2E). Length and 1xproportions of the legs are in the table 1 (in µm).

232 Fe Ti Ta1 Ta2 Ta3 Ta4 Ta5 LR BV SV

P1 510 600 310 190 130 110 100 0.51 2.67 3.58 P2 675 610 375 215 160 150 105 0.61 2.63 3.42 P3 550 700 410 275 210 160 125 0.58 2.15 3.05 233

234Table 1. Furcobuchonomyia saetheri leg segments lengths and proportions (in µm).

235

236Fig. 2. A) Thorax of F.saetheri male, side view (confocal microscope); B) Head of the F.

237saetheri male (confocal microscope); C) Thorax and head of the F. saetheri male (optical

238microscope); D) Hypopigium of F. saetheri male, side view (confocal microscope).

239 240Hypopygium (Fig 3D, 4A). Tergite IX with 52 setae. Aedeagus obscured by gonocoxites and

241gonostyles, and unavailable for inspection. Gonostylus completely split into the dorsal and

242ventral lobe. Ventral lobe club-shaped covered in numerous micro and microtrichia, 115 µm

243long. Dorsal lobe bilobed, with basal trunk. Trunk length to bifurcation point – 46 µm ,

244ventral branch of the dorsal lobe is thumb-shaped, 24 µm long, with strong apical setae, 10

245µm long. Dorsal branch rode-like, 88.4 µm long, carrying long, strong setae midleghnt (79

246µm). Volsella long, robust, somewhat sickle – shaped, broadest midlenght (length - 69 µm,

247width -25 µm). Gonocoxite oval, 116 µm long. HR (Gonocoxite length/ gonostylus ventral

248lobe length) - 1, HV -1.69.

249

250Fig. 4. A) Hypopygium of F. saetheri male, sideview; B)Antenna of the F. aetheri male; C)

251Wing of the F. saetheri male. Scalebar is 100 µm long. 252

253Key to fossil and extant species of Buchonomiinae Brundin and Sæther, 1979 (adult

254males)

2551.Dorsal lobe of gonostylus forked (Fig. 4A)...... F. saetheri†

256Dorsal lobe of gonostylus simple cylindrical...... 2

2572 (1). VR ≥ 0.9……………………………………………………………………………...... 3

258- VR ≤ 0.9 ...... 4

2593 (2) Ventral lobe of gonostylus is club-shaped...... B. burmanica

260- Ventral lobe of gonostylus cylindricalelongated...... B.succinea†

2614 (2).Pseudospur on P1 present...... B.

262brundini

263Pseudospur on P1 pabsent...... B.

264thienemanni

265

266Discussion

267Furcobuchonomyia from Burmese amber was placed in the subfamily Buchonomyiinae based

268on the combination of the following characters: male macropterous, with distinct wing, cross-

269vein MCu present on wing, R2+3 vestigial, almost indistinguishable, space between R1 and R4+5

270is wide, scale like setae are present on the wing veins and auxiliary sclerite 1. It differs from

271the other Buchonomyiinae by the absence of the developed tibial spurs, sickle-shaped

272volsella, forked dorsal lobe of gonocoxite, higher distance between MCu and RM, absence of

273setae on postnotum. It is extremely difficult to pinpoint the relationships between the new 274genus, Buchonomyia and the rest of the Chironomidae, based only on the single male

275available, since females and larvae are crucial for determining Buchonomyiinae phylogenetic

276relationships, and adult males are essentially “collections of plesiomorphies” (Brundin &

277Sæther, 1978; Murray & Ashe, 1985). Genital characters are too specialised and variable

278within the Chironomidae to make a solid prediction about polarity of these characters in fossil

279Chironomidae. Both the shape of the gonocoxite volsella and gonostylus seem to be

280autapomorphies of the genus, since they are unique among the Chironomidae, though some

281Podonominae (especially Podochlus) and Propsilocerus and some other Orthocladiinae

282possess the dorsal lobe of the gonostylus (Brundin, 1965; P. Cranston & Barley, 2011; Tang,

283Guo, Wang, & Sæther, 2004). This lobe is however, protruding from the body of the

284gonostylus and never connected to the gonocoxite directly, as in Buchonomyiinae (Brundin,

2851965). Furthermore, none of the other Chironomidae, whether Buchonomyiinae or not, do not

286possessing additional branching of the dorsal lobe of the gonostylus (Andersen & Sæther,

2871995; Armitage et al., 1995).

288The reduction of the tibial spurs appears to be secondary, a similar reduction is characteristic

289for some small Podonomiinae (i.e. Podnomopsis discoceros Brundin, 1965) and the

290subfamily Aphroteniinae (except the fossil Electrotenia brundini Kalugina, 1980, which

291possesses small tibial spurs). It is notable that all these Chironomidae that lack developed

292tibial spurs are small, less then 2mm total length (Brundin, 1965; Cranston & Edward, 1992;

293Kalugina, 1980). Most of the other Chironomidae and do have tibial spurs

294(Armitage et al., 1995; Azar, Veltz, & Nel, 2008; Borkent, 2008)

295Furcobuchonomyia appears to be a specialized, rather than basal representative of the

296subfamily, which is consistent with Cranston’s et al. (2012) Jurassic dating of the origin of

297the subfamily. 298In our putative phylogeny, Furcobuchonomyia was recovered as the sister group to the rest of

299the Buchonomyiinae species (Fig 1). Currently Buchonomyiinae are divided into two genera

300with 5 species. The genus Buchonomyia in our phylogeny was divided into two clades – one

301consisting of two species which were considered more basal by Seredszus & Wichard (2002)

302– B. burmanica and B. succinea. Those two species are both more robust, sharing a similarly

303high VR (0.9) and arrangement of the tibial pseudospurs. The other clade includes B.

304brundini and B. thienemanni, which is again consistent with existing phylogenetic hypothesis

305(Seredszus & Wichard, 2002).

306

307While the ecology of the new genus is difficult to speculate on, it is notable that the

308trichopteran genus Palerasnitsynus of the family Psychomiidae was described from Burmese

309amber (Wichard et al., 2011), suggesting the possibility of commensal/parasitic relationships

310between Buchonomyiinae and Psychomyiidae existing back in the Late Cretaceous.

311Concluding remarks

312The new taxon described in this paper, Furcobuchonomyia saetheri, based on one specimen

313in Late Cretaceous Burmese amber, is the oldest known genus and species to be described

314belonging to the basal chironomid subfamily Buchonomyiinae. A phylogenetic analysis

315placed this genus as the sister group to the rest of subfamily, represented by the extant genus

316Buchonomyia. The presence of Buchonomyiinae midges and Psychomyiidae in

317Burmese amber may suggest a long lasting association between these two groups, as

318demonstrated by modern taxa whereby Buchonomyia larvae are parasites of larval

319psychomyiids.

320Acknowledgments 321Work of the first author was funded by the European Union’s Seventh Framework Program

322for research, technological development and demonstration under grant agreement no.

323607150 (FP7-PEOPLE-2013-ITN - Ecohydrological interfaces as critical hotspots for

324transformations of ecosystem exchange fluxes and biogeochemical cycling).

325The first author is grateful to Martin Spies (ZSM, Münich) for his help in obtaining relevant

326literature, and Valery Korneyev (IZAN) for his helpful comments on the drawings. The first

327author also wants to express his gratitude to Stig Walsh (National Museums Scotland,

328Edinburgh) for his assistance in the screening of the material in the National Museums

329Scotland collection during the 7th International Conference on Fossils Insects, and

330Amber. Authors are grateful to “Cretaceous research” editor - Eduardo Koutsoukos and two

331anonymous reviewers for their help in improving the manuscript.

332

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