The Phylogeny of Lance Lacewings (Neuroptera: Osmylidae)

Systematic Entomology (2017), 42, 555–574 DOI: 10.1111/syen.12231

The phylogeny of lance lacewings (Neuroptera: Osmylidae)

SHAUN L. WINTERTON1,JINGZHAO2, IVONNE J. GARZÓN-ORDUÑA1, YONGJIE WANG3 andZHIQI LIU2

1California State Collection of Arthropods, California Department of Food & Agriculture, Sacramento, CA, U.S.A., 2Department of Entomology, China Agricultural University, Beijing, China and 3College of Life Sciences, Capital Normal University, Beijing, China

Abstract. The first phylogeny of the lacewing family Osmylidae is presented here based on a total evidence analysis of DNA sequences for multiple gene loci and morphology for representatives of almost all extant genera. Our phylogeny shows a basal dichotomy in the family, with subfamilies Protosmylinae, Spilosmylinae and Gumilli- nae comprising one lineage, and the other lineage including Osmylinae, Porisminae, Eidoporisminae, Kempyninae and Stenosmylinae. The status of Paryphosmylus Krüger and Lysmus Navás as members of Protosmylinae is affirmed as well as the placement of Gumillinae near Protosmylinae and Spilosmylinae. Our results suggest that Poris- minae, Eidoporisminae and Stenosmylinae evolved from a common ancestor, and their relationships, including likely paraphyly of Stenosmylinae, requires further assessment. Divergence time analysis revealed that the family originated during the Late Permian before the break-up of the supercontinent Pangaea and that present generic distributions are not due to Gondwanan biogeographic events. All major subfamily-level lineages were present by the end of the Triassic, in agreement with the rich Mesozoic-aged fossil record for the family.

Introduction recently described from South America and Asia (Wang & Liu, 2009, 2010; Ardila-Camacho & Noriega, 2014; Martins et al., The lance lacewings (Neuroptera: Osmylidae) are a small 2016; Winterton & Wang, 2016). family of charismatic insects distributed today in all major The phylogenetic position of Osmylidae has traditionally biogeographical regions except the Nearctic (New, 1989a). been highly contentious. The family has been allied with They are medium to large lacewings with cryptically patterned various families within Neuroptera, but most recent authors wings and reticulated venation (Figs 1, 2A, B). The highly agree on a close relationship among Osmylidae, Nevrorthidae distinctive larvae (Fig. 2C–E) are generalist predators that have and Sisyridae, with all three placed towards the base of the spectacularly elongated, lance-like jaws, which they use to neuropteran phylogeny (Withycombe, 1925; Haring & Aspöck, impale their prey. Larvae of many species are in riparian habitats 2004; Aspöck & Aspöck, 2008; Winterton et al., 2010; Yang near freshwater streams, although members of some subfamilies et al., 2012; Wang et al., 2016). Based on mounting evidence (i.e. Stenosmylinae, Porisminae) are distinctly terrestrial and are from quantitative analyses of morphology, DNA sequences and typically found under bark in much drier habitats. Presently examples in the fossil record, it is clear that these three families there are at least 225 extant species described in around 26 arose early in lacewing evolution during the Late Permian genera (Kimmins, 1940; New, 1986a,1986b,1986c; Wang & and Early Triassic, although the actual relationships among Liu, 2009; S.L. Winterton, unpublished data), although the them are still unclear (Winterton et al., 2010; Yang et al., 2012; number of species is expected to increase, with new species Wang et al., 2016). Some authors have proposed Nevrorthidae present in collections. For instance, new species have been as the sister family to the rest of the order (e.g. Aspöck et al., 2001; Haring & Aspöck, 2004; Aspöck & Aspöck, 2008), but compelling evidence to the contrary has been forwarded using Correspondence: Shaun L. Winterton, California State Collection total evidence analyses of morphology and DNA sequence of Arthropods, California Department of Food & Agriculture, 3294 Meadowview Rd., Sacramento, California 95832-1148, U.S.A. data. These analyses suggest that the family Coniopterygidae E-mail: [email protected] are instead the sister family to the rest of the order, with

Fig. 1. Examples of living Osmylidae adults. (A) Gryposmylus pennyi Winterton & Wang (Protosmylinae) (Vietnam) (photograph © Stephen D. Gaimari); (B) Isostenosmylus sp. (Stenosmylinae) (Peru) (photograph © Steve Marshall); (C) Phymatosmylus caprorum Adams (Stenosmylinae) (Chile) (photograph © Steve Marshall); (D) Heterosmylus processus Dong, Xu, Wang, Jia & Liu (Protosmylinae) (China) (photograph © Jishen Wang); (E) Osmylus fulvicephalus Stephens (Osmylinae) (Spain) (photograph used under Creative Commons); (F) Oedosmylus latipennis Kimmins (Stenosmylinae) (Australia) (photograph © Shaun L. Winterton); (G) Porismus strigatus Burmister (Porisminae) (Australia) (photograph © Shaun L. Winterton).

Fig. 2. Examples of living Osmylidae adults and larvae. (A) Kempynus incisus (McLachlan) (Kempyninae) (New Zealand) (photograph © Gil Wizen); (B) Thyridosmylus paralangi Wang, Winterton et Liu (Spilosmylinae) (China) (photograph © Shaun L. Winterton); (C) Kempynus sp. (larva) (Kempyninae) (Australia) (photograph © Kristi Ellington); (D) Isostenosmylus sp. (larva) (Stenosmylinae) (Brazil) (photograph © Enio Branco); (E) unidentified larvae (Stenosmylinae) (Australia) (photograph © Kristi Ellington).

Nevrorthidae placed in a subsequent clade near the base of the Plethosmylus Krüger, Porismus McLachlan, Spilosmylus Kolbe, lacewing tree (Winterton et al., 2010; Yang et al., 2012; Wang Stenolysmus Kimmins, Stenosmylus McLachlan, Thaumatosmy- et al., 2016). lus Krüger and Thyridosmylus Krüger. Specimens suitable for Osmylidae is divided into nine subfamilies – Eidoporisminae, DNA sequencing were unavailable for Gumilla Navás, Eido- Gumillinae, Kempyninae, Mesosmylininae, Osmylinae, Poris- porismus Esben-Petersen, Sinosmylus Yang, Paryphosmylus minae, Protosmylinae, Spilosmylinae and Stenosmylinae Krüger, Clydosmylus New, Euporismus Tillyard and Euosmy- (Krüger, 1912–1915; New, 1989a) – although Mesosmylininae lus Krüger, although they were scored for morphology based are only known from fossils. Mesosmylininae comprise at least on pinned specimens and included in some analyses. The genus seven genera known from the Late Triassic to Mid-Cretaceous Lahulus Navás is known only from the original description and (e.g. Jepson et al., 2009, 2012; Khramov, 2014a,2014b). The was not included here. Little is discernible about this genus from extinct subfamily Epiosmylinae is considered a junior synonym the description by Navás (1930), although he did state a clear of Gumillinae (Lambkin, 1988; Wang et al., 2009a,2009b; similarity with the genus Hyposmylus Krüger, a genus subse- Khramov, 2014a,2014b). The putative sister family to Osmyl- quently found to be a synonym of Osmylus Latreille. The other idae is Archeosmylidae (Late Permian to Early Triassic) and genera known from India are Spilosmylus Kolbe, Thyridosmy- is differentiated based on several wing venation features, such lus Krüger, Lysmus Navás and Osmylus. We cannot immediately as a nonpectinately branched forewing CuP (Khramov, 2014a). deduce that Lahulus as a synonym of Osmylus as the male gen- Saucrosmylidae Ren & Yin (2003) was originally included italic drawing by Navás (1930) is unlike any other found in the as a subfamily in Osmylidae, but recently was elevated to family and it will require further examination to determine its family rank by Fang et al. (2015) as a possible sister family status as a valid genus. The genus Glenosmylus Krüger is consid- to Osmylidae (cf. Yang et al., 2012). Similarly, the subfam- ered a synonym of Thaumatosmylus Krüger, while Mesosmylus ily Cratosmylinae (comprising only the genus Cratosmylus Krüger and Grandosmylus Makarkin are considered synonyms Myskowiak et al.) was recently described in Osmylidae by of Osmylus and Parosmylus Needham, respectively. Myskowiak et al. (2015), but we consider that this subfamily Outgroups were selected based on recent phylogenetic esti- is more suitably placed in Nymphidae based on the same wing mates of the Neuroptera using morphology and DNA sequence venation characters presented by the authors. data (Winterton et al., 2010; Yang et al., 2012), including Few synoptic works on world Osmylidae have been published mitochondrial genomes and gene arrangement (Wang et al., since the important series of papers on the family by Krüger 2016); we selected representatives from the closely related (1912–1915), and those few treatments have focused only families Nevrorthidae (Austroneurorthus Nakahara, Nevrorthus on provincial faunas (e.g. Tjeder, 1957; New, 1983a,1983b, Costa), Sisyridae (Climacia McLachlan, Sisyra Burmeister) 1986a,1986b,1986c, 1988, 1991; Wang, 2010; Martins et al., and Coniopterygidae (Conwentzia Enderlein, Cryptoscenea 2016) or particular subfamilies and genera (Kimmins, 1940, Enderlein). 1942; Adams, 1969; Wang & Liu, 2009; Wang et al., 2011a; Ardila-Camacho & Noriega, 2014; Winterton & Wang, 2016). The proliferation of genera based on spurious characters by Morphology and terminology Krüger (1912–1915) and the rarity of specimens in collections mean that there are few publications on the family, and none Neuroptera has a wide variety of terms applied by vari- with an emphasis on the entire world fauna. We present the ous authors to genitalic structures, especially in Osmylidae. first phylogenetic study of Osmylidae, here using total evidence The enormous disparity in morphology across families makes analysis of DNA sequence data (18S and 16S rDNA, COI and hypotheses of homology tenuous, often argued by authors on CAD) and morphology. Representatives of almost all genera weak grounds and generous interpretation. The terminology were studied and included in the analyses. We also present of morphological structures used here follows Tjeder (1957); divergence time estimations and propose scenarios of evolution Adams (1969) and Wang et al. (2011a) with modifications fol- of particular characters within the family. lowing Winterton & Wang (2016). Terms used for particular structures are based on comparative examination of multiple exemplars from all lineages in each family, with strong pref- Materials and methods erence for those where position and functional homology are clearly evident in each case. Redundant or unnecessary novel Taxon sampling terms were avoided considering the large number of terms already in use in neuropteran taxonomy. Figure 3 depicts var- Twenty-five genera of Osmylidae were included in the anal- ious male genitalic structures (colour-coded) exhibited in sub- ysis, representing almost all recognized genera in the family. families of Osmylidae. We follow Adams (1969) in the use In most cases, multiple species were included to represent each of mediuncus (pink colour) for the main intromittent organ, genus. DNA sequences were obtained for 18 ingroup genera, which is equivalent to the parameres of some authors (e.g. including Australysmus Kimmins, Carinosmylus New, Grypos- Tjeder, 1957; New, 1986b), or more recently the gonocoxite mylus Krüger, Heterosmylus Krüger, Isostenosmylus Krüger, 10 complex of Aspöck & Aspöck (2008) and Martins et al. Kempynus Navás, Lysmus Navás, Oedosmylus Krüger, Osmy- (2016), or gonocoxites by Ardila-Camacho & Noriega (2014). lus Latreille, Parosmylus Needham, Phymatosmylus Adams, The baculum (sensu Tjeder, 1957) is the anterior arm of the

Fig. 3. Male genitalic sclerite structure in Osmylidae: (A) Gryposmylus pennyi Winterton & Wang dorsal view; (B) Osmylus fulvicephalus (Scopoli), dorsal view; (C) Gryposmylus pennyi oblique view; (D) same, lateral view; (E) Porismus strigatus (Burmeister), lateral view; (F) Oedosmylus pallidus (McLachlan), lateral view; (G) Parosmylus liupanshanensis Wang & Liu, lateral view; (H) Kempynus incisus (McLachlan), lateral view. Colour code of structures: gonarcus including baculum (red); hypandrium internum (orange); entoprocesses (blue); parameres (green); mediuncus (pink). Scale line = 0.2 mm. gonarcus (red colour) and is sometimes articulated anteriorly either flanking the mediuncus (Osmylinae) or cradling from (e.g. Osmylinae) (Fig. 3G) or absent (e.g. Kempyninae, Poris- below or immediately anterior to it (Protosmylinae and Spi- minae) (Fig. 3E, H). The parameres (green colour) herein are losmylinae). In Osmylinae the parameres are separate, paired, equivalent to the subarcus of Tjeder (1957) and are present in rod-shaped sclerites (Fig. 3B, G), while in Protosmylinae and only three subfamilies (i.e. Osmylinae, Spilosmylinae and Pro- Spilosmylinae they are fused medially as a single arched scle- tosmylinae). The presence or absence of the parameres in dif- rite (Fig. 3A, C, D). It is unclear if the parameres are present ferent subfamilies of Osmylidae has apparently fostered this in Gumillinae (see Martins et al., 2016), but at this stage we confusion in osmylid genitalic terminology, but a comparison of conclude that they are not. The gonarcus (red colour) is vari- all major groups together in light of the phylogeny herein pro- able in form depending on the subfamily. It is relatively narrow vides a novel opportunity to understand these complexities. The and lacking setae in Protosmylinae and Spilosmylinae, while in parameres are always closely associated with the mediuncus, Porisminae, Stenosmylinae, Kempyninae, Eidoporisminae and

Osmylinae, the gonarcus is large and observable externally, with drawing in New (1986c: figs 29–30) of S. obliquus New. Unfor- distinct setal pile evident. Entoprocesses (blue colour) are fused tunately the actual wing venation in the former species is laterally on the gonarcus (sensu Tjeder, 1957) and are equiv- blurred, and examination of specimens of these species shows alent to gonocoxite 9 (sensu Adams, 1969). They are narrow that the HW MP2 clearly originates on a common stem with and curved (e.g. Spilosmylinae, Protosmylinae) (Fig. 3C, D), MP1 and is not associated with CuA at all (see Fig. 4E). More- broad and subtriangular (e.g. Porisminae, Kempyninae, Stenos- over, examination of specimens of multiple Spilosmylus species mylinae) (Fig. 3E, F, H) or reduced to a rounded process (e.g. shows that the wing venation presented by Shi et al. (2012) Osmylinae) (Fig. 3G). Aspöck & Aspöck (2008) and Martins apparently misinterpreted HW CuP as A1 (see Fig. 4D). The et al. (2016) interpreted the gonarcus and entoprocessus as a form of HW CuP is highly variable in species of Spilosmy- complex of gonocoxite 9 and gonopophyses 9, respectively. The lus, exhibited in the form of a small loop, joining back to CuA hypandrium internum (orange colour) is of typical shape for neu- approximately two-thirds the way along the length, a small loop ropterans, although it is unusually large compared with other plus a faint distal vein to the wing margin, or as a small vein families. closely proximal to A1 and joining to the wing margin. Simi- In the female genitalia (Fig. 8), the structure of the female larly, Cousin & Béthoux (2015) proposed a similar mechanism sclerites is also variable among subfamilies. The enlarged of vein fusion between MP and CuA in species of Stenosmyli- gonocoxite 9 (= gonopophyses lateralis) is closely associated nae. Here the fusion occurs more distally and there are examples anteriorly with gonopophysis 9 (= sternite 9 of Wang et al., of specimens of Stenosmylus and Oedosmylus where a diagonal 2011a) in Protosmylinae, Spilosmylinae and Osmylinae, with crossvein between the forewing MP and CuA could be inter- two separate sclerites clearly visible. By contrast, in more preted as fusion between longitudinal veins in the distal part of derived subfamilies Porisminae, Kempyninae, Eidoporisminae the wing. Unfortunately, similar to Shi et al. (2012), while the and Stenosmylinae, gonopophyses 9 is not closely associated authors correctly identify forewing veins (using the terminology as a regular sclerite with gonocoxite 9, but instead is more of Kukalová-Peck & Lawrence, 2004), in the hindwing they mis- elaborately shaped (i.e. enlarged with multiple lobes) as a single takenly identify the posterior branch of M (i.e. MP) as CuA and articulating sclerite which acts upon sternite 8. In all subfamilies thus there is an erroneous shift anteriorly in the identity of the there is a terminal stylus (gonostylus 9). Sternite 8 (sensu New, wing veins (i.e. CuA as CuP, CuP as A3, etc.). 1986b) has also been referred to as the subgenitale (sensu Tjeder, Genitalia were macerated in 10% KOH to remove soft tissue, 1957), or as a fusion of gonocoxites 8+ gonopophyses 8 by then rinsed in distilled water and dilute glacial acetic acid, Aspöck & Aspöck (2008) and Martins et al. (2016). The shape dissected in 80% ethanol and subsequently stained with a and position of this sclerite are highly variable. In Osmylinae, solution of Chlorazol Black in 40% ethanol. The dissected Porisminae, Kempyninae, Eidoporisminae and Stenosmylinae, genitalia were placed in glycerine in a genitalia vial mounted it is a plate-like sclerite immediately posterior to sternite 7. on the pin beneath the specimen. Seventy-two adult and larval In Kempyninae and Stenosmylinae it is frequently modified morphological characters were scored for all taxa and were all into a bowl-like structure. In Protosmylinae and Spilosmylinae, equally weighted and unordered (Tables S3–S4). sternite 8 is much smaller in size and located posteriorly as a small knob-like process. Neuroptera wing venation, and especially Osmylidae wing DNA extraction and sequencing venation, has been the subject of much confusion regarding identity and homology of wing veins. Based on recent unpublished Specimen data and GenBank accession numbers are presented studies using mature adult wing venation and tracheation (L. in Table S1. Adult specimens were collected at light sheet or in Breitkreuz, personal communication) we disregard the assump- Malaise traps. Individuals were placed directly into 95–100% tion that the MA vein is fused anteriorly with R (Fig. 4) and ethanol and stored at −80∘C. Methods and references used in the that it is represented in both wings as the posterior-most vein identification of exemplars are also listed in Table S1. Sequenc- of the Rs field (sensu Kukalová-Peck & Lawrence, 2004). Con- ing conditions are the same as those in Winterton et al. (2010). sequently, we do not consider the sigmoid vein as the rem- Genomic DNA was extracted from thoracic muscle tissue using nant of vein MA. Winterton & Makarkin (2010) identified an the DNeasy (Qiagen, Germantown, MD, U.S.A.) or TIANamp example of an Ithonidae wing that did not confirm to the ter- (Tiangen Biotech, Beijing, China) genomic DNA kits as per the minology proposed by Kukalová-Peck & Lawrence (2004) and manufacturer’s instructions except that specimens were incu- consequently simply identified the vein as a basal radial vein bated in the extraction buffer/proteinase-K mixture for 24–48 h. instead of MA. Difficulties in determining homology of wing Two ribosomal genes were sequenced (16S rDNA and 18S veins in fossil Neuroptera in particular, including some Osmyl- rDNA) along with two protein-encoding genes [cytochrome oxi- idae, have led some authors to propose basal fusions of various dase I (COI) and the CPSase region of carbamoyl-phosphate wing veins. For example, Shi et al. (2012): p. 623) suggested a synthetase-aspartate transcarbamoylase-dihydroorotase (CAD)] basal fusion of CuA and MP2 in the hindwing of Osmylidae, (see Winterton et al., 2010 for details). Primer sequences with a ‘(presumed) translocation of MP2 onto CuA in the hind are included in Table S2. Specimen vouchers are deposited wing’ in Phymatosmylus. As stated in their figure caption, Shi in the entomology collections of China Agricultural Univer- et al. (2012) based this interpretation on an image of the wings sity (Beijing) and California State Collection of Arthropods of Phymatosmylus by Adams (1969): fig. 1A) and an inaccurate (Sacramento).

Fig. 4. Wing venation in Osmylidae. (A) Gumilla longicornis (Walker) (Gumillinae); (B) Porismus strigatus (Burmeister) (Porisminae); (C) Gryposmylus pennyi Winterton & Wang (Protosmylinae); (D) Spilosmylus sp. (Spilosmylinae); (E) Phymatosmylus caprorum Adams (Stenosmylinae). Wings not drawn to scale. D–E modified after Shi et al. (2012).

Table 1. Fossils used to determine age constraints on nodes in chronogram.

Higher or sister grouping Node Taxon Estimated age constraint Constraint type References

Stem Neuropterida A Nanosialis bashkuevi 296 million years (Ma) (Early Maximum Shcherbakov (2013) Shcherbakov (Nanosialidae) Permian) Archeosmylidae B Babykamenia eskovi 248 to 241 Ma (Early Minimum Riek (1955), Lambkin Ponomarenko & Shcherbakov Triassic) (1988) and Ponomarenko Lithosmylidia Riek & Shcherbakov (2004) Protosmylinae C Juraheterosmylus antiquatus 170 Ma (Mid-Jurassic) Minimum Wang et al. (2010a) Wang, Liu, Ren & Shih Gryposmylus + Lysmus D Jurosmylus parvulus Khramov 165 to 160 Ma (Late Jurassic) Minimum Khramov (2014b) Spilosmylinae E Palaeothyridosmylus 170 Ma (Mid-Jurassic) Minimum Wang et al. (2009a) septemaculatus Wang, Liu & Ren Thyridosmylus F Ensiosmylus acutus Khramov 165 to 160 Ma (Mid-Jurassic) Minimum Khramov (2014b) India–Madagascar split G Secondary calibration of split of Approximately 100 Ma Minimum India from Madagascar Osmylinae + Kempyninae H Archaeosmylidia fusca Makarkin, 168 Ma (Mid-Jurassic) Minimum Makarkin et al. (2014) + Stenosmylinae et al. Yang & Ren Kempyninae + I Sauktangida aenigmatica 165 to 160 Ma (Mid-Jurassic) Minimum Khramov (2014a) Stenosmylinae et al. Khramov Kempyninae J Jurakempynus sinensis Wang, 170 Ma (Mid-Jurassic) Minimum Wang et al. (2011b) Liu, Ren & Shih Kempynus Navás K Arbusella bella Khramov 165 to 160 Ma (Mid-Jurassic) Minimum Khramov (2014a)

Nodes refer to those identified in Fig. 7.

Alignment and phylogenetic analyses of its most frequent contradictory group. Accordingly, it is possible for a clade with low frequency that is never contradicted to The total length of the aligned gene sequences was 2591 bp have a higher GC value than a group with moderate support but (CAD, 713 bp; COI, 659 bp; 16S, 447 bp; 18 s, 769 bp). All that is often contradicted. GC values are reported from −100 to alignments were done manually using mesquite 3.02 (Mad- 100 in tnt: GC = 100 indicates that the group is never contra- dison & Maddison, 2015) and were relatively straightforward, dicted; GC = 0 indicates ambiguity, as the group is as frequently with few ambiguous regions present in the ribosomal sequence supported as it is contradicted; and GC =−100 indicates that the data and no introns in the protein encoding genes (PCGs). CAD group is more frequently contradicted. and COI were aligned with reference to translated amino acid Bayesian analyses were performed using mrbayes 3.2.3 (Ron- sequences. As with previous studies using these loci to inves- quist & Huelsenbeck, 2003) on the combined morphological tigate Neuroptera phylogeny, third positions for PCGs were and molecular data. The data were partitioned by type (DNA excluded in all analyses, as inclusion resulted in violation of sequence, morphology), locus and codon positions for each monophyly of major lineages (e.g. families and subfamilies) protein-coding locus. A separate GTR + 𝛾 nucleotide substitu- already widely accepted as monophyletic based on extraneous tion model was applied to each DNA partition. For the mor- morphological evidence (Winterton et al., 2010). All analyses phological partition, a gamma distribution was used with ratepr were done with a total evidence approach combining all avail- commands set to variable. In all cases, all the parameters in the able data in simultaneous analyses when available. Parsimony model were unlinked. Each analysis consisted of four Markov analyses were conducted with tnt (Goloboff et al., 2008) using chain Monte Carlo chains run simultaneously for 10 million a heuristic search that included 500 replicates of random addi- generations, although the chains always converged long before tion sequence, holding ten trees per replication after tree bisec- reaching this number. Trees were sampled every 1000th gen- tion and reconnection for branch swapping and 90 iterations of eration and the burn-in fraction was set to 0.25 (25%). A ratchet. Branch support was calculated from 1000 pseudorepli- majority-rule consensus tree was calculated with posterior prob- cates of resampled datasets using symmetrical resampling (or abilities (PPs) for each node. symmetrical jackknife) in tnt, which uses the same probability Divergence times were estimated on the total evidence topol- for character deletion and character inclusion (Goloboff et al., ogy, which was calibrated using ten fossils as minimum ages for 2003). The results of resampling are expressed in differences of particular clades and a 296 Ma secondary calibration (maximum group frequencies (GC values, for group present/contradictory; age) placed at the root (Table 1). Empirically, maximum age Goloboff et al., 2003) rather than straight group frequencies; GC secondary calibration is necessary in lacewing dating estimates offers the advantage (over straight frequencies) of reporting the to prevent the divergence times at the root being overestimated support for groups with less than 50% frequency that would oth- to about 100–200 Ma older than the oldest known lacewings erwise be collapsed and expressing the support of a group in rela- (typically placing root divergences during the Devonian) (see tionship not only to its own frequency but also to the frequency Winterton et al., 2010; Wang et al., 2016). In phylobayes

3.3 (Lartillot et al., 2009) two chains of an autocorrelated resolved except for a sister-group relationship between Poris- log-normal (Thorne et al., 1998) relaxed clock model (LN) were minae and Eidoporisminae. Within Kempyninae, Australysmus run for 17 000 cycles and assuming a birth–death speciation was placed as sister to the rest of the subfamily. Euosmylus was model on the divergence times. The chronogram was obtained found as sister to a paraphyletic Kempynus containing the genus after discarding the first 3000 saved cycles as burn-in. Fossil Clydosmylus. Within Stenosmylinae, relationships among the age calibrations presented in Table 1 are based on a wealth genera were not resolved, except placement of Carinosmylus as of fossil osmylids and related families dating from the Per- sister to Euporismus in a clade sister to Stenosmylus and Oedos- mian to the Tertiary (e.g. Cockerell, 1908; Carpenter, 1943; mylus. In all analyses Stenosmylus was rendered paraphyletic Wang et al., 2009a,2009b, 2010b; Shcherbakov, 2013; Khramov, with respect to Oedosmylus. 2014a,2014b; Makarkin et al., 2014).

Divergence time estimations Results The results of the divergence time estimation indicate signif- Phylogenetic relationships icant antiquity of Osmylidae, with the family arising from a common ancestor with Nevrorthidae during the Mid-Permian The results of the total-evidence Bayesian analysis of all extant (247 Ma) (Fig. 7). All major lineages of Osmylidae had subse- taxa where we had both DNA sequence data and morphology quent origins during the Late Permian and throughout the Trias- are presented in Fig. 5. We recovered a well-resolved phylogeny sic, with all subfamilies present by the beginning of the Jurassic with relatively high statistical support throughout, especially and before the final break-up of the pangaean landmass (ca. along the backbone nodes; 75% of all nodes had branch support 180 Ma). Protosmylinae diverged from Spilosmylinae during values above 0.9 Bayesian PP and parsimony jackknife group the Mid-Triassic (223 Ma) with all genera present in both sub- frequency values above 80% (Figure S1). Branch lengths were families by the beginning of the Jurassic. Similarly, Osmylinae relatively consistent throughout the tree, with slightly longer (clade B) diverged from the rest of the family (clade C) during branches only for representatives of Coniopterygidae, Clima- the Jurassic (244 Ma), although relationships among the gen- cia and one species of Heterosmylus. At no time did we recover era are weakly supported and lack monophyly in all cases, and Sisyridae as sister to Nevrorthidae in any analysis, and in all estimated divergences have relatively large ranges. Kempyninae cases Nevrorthidae was recovered as the sister to Osmylidae diverged from Stenosmylinae during the Mid-Triassic (229 Ma) with high support. Osmylidae was recovered as monophyletic with Australysmus diverging from Kempynus during the Trias- with a distinct basal dichotomy of major lineages of osmylid sic. Relationships among Stenosmylinae, Porisminae and Eido- subfamilies. Protosmylinae and Spilosmylinae (clade A) were porisminae are weakly supported, with Stenosmylinae rendered found as sister subfamilies in one lineage, with the remain- paraphyletic by the other two subfamilies. ing subfamilies Osmylinae (clade B), Stenosmylinae, Eidoporis- minae, Porisminae and Kempyninae (clade C) comprising the other lineage. All subfamilies were recovered as monophyletic Discussion with significant statistical support, except for Stenosmylinae, which was rendered paraphyletic by Porisminae in some anal- Osmylidae have attracted relatively little attention with regard to yses, or placed in a polytomy in others. Additional extant their taxonomy or evolutionary relationships. The relative rarity genera were also included in the analysis based on morpho- of members of the family, the diverse morphology among sub- logical scoring only (i.e. lacking DNA sequence data), such families and the unwieldy taxonomy proposed for the family by as Gumilla, Paryphosmylus, Sinosmylus, Euosmylus, Clydos- Krüger (1913a) has hampered a comprehensive investigation of mylus, Eidoporismus and Euporismus (Fig. 6). Again, over- the group until now. The combined phylogeny presented here all support throughout the phylogeny was relatively high, with is surprisingly well supported statistically throughout and pro- loss of resolution in just a few nodes, specifically at the base vides important insights into the wing and genitalic morphology, of Protosmylinae, among Osmylinae genera and Stenosmyli- as well as validating the previously established classification for nae. Gumillinae was recovered in a polytomy with protosmy- the family. line genera. Paryphosmylus was recovered with Heterosmylus while Lysmus was placed as the sister to Gryposmylus. In Spilos- mylinae, all genera were recovered as monophyletic, with Thau- Phylogenetic position of Osmylidae and early evolution matosmylus placed as sister to Spilosmylus and Thyridosmylus. of major lineages In the other main lineage, Osmylinae (clade B) was placed as sister to the remaining subfamilies, with Osmylus rendered Osmylidae have been placed in multiple differing positions in paraphyletic in all cases by Plethosmylus, Parosmylus and the lacewing phylogeny by various authors, including, notably, Sinosmylus, suggesting that most genera in this subfamily are as sister to Nymphidae (as Myiodactylidae) (Handlirsch, not monophyletic. In clade C, Kempyninae was recovered as sis- 1906–1908), Sisyridae (Withycombe, 1925) or Nevrorthidae ter to the remaining subfamilies, although relationships among (Yang et al., 2012). Recent estimates based on morphology Eidoporisminae, Porisminae and Stenosmylinae were not and/or DNA sequence data indicate a close relationship among

Fig. 5. Combined phylogeny of Osmylidae, including taxa with both DNA sequences and morphology available. Nodes with posterior probability values of 0.9 and bootstrap values above 80% are indicated by a black circle.

Fig. 6. Combined phylogeny of Osmylidae based on DNA sequences and morphology, with additional taxa scored only for morphology. Nodes with posterior probability values of 0.9 and bootstrap values above 80% are indicated by a black circle.

Osmylidae, Sisyridae and Nevrorthidae near the base of the lacewings of the Jurassic (Ren & Yin, 2003; Wang et al., 2010b) lacewing tree, along with Dilaridae and Coniopterygidae and was originally placed as a subfamily in Osmylidae; recent (Winterton et al., 2010; Wang et al., 2016). Among the fossil studies by Fang et al. (2015) have elevated the group to family families it is well established that the Permian-Triassic family level and suggest a sister-group relationship with Osmyli- Archeosmylidae (comprising the genera Archeosmylus Riek, dae, although the relationship to Archaeosmylidae remains Babykamenia Ponomarenko & Shcherbakov and Lithosmylidia uncertain. Within Osmylidae, the putative sister to the rest Riek) probably represent important stem osmylids precedent to of the family is the extinct subfamily Mesosmylininae, with the modern lineages (Riek, 1976; Lambkin, 1988; Yang et al., representatives in various deposits ranging in age from the 2012; Makarkin et al., 2014). The charismatic extinct family Late Triassic to the Early Cretaceous (Khramov, 2014a,2014b; Saucrosmylidae was similarly described as ‘osmylid’-like Makarkin et al., 2014). It is clear based on the rapidly increasing

Fig. 7. Chronogram of estimated divergence times amongst Osmylidae genera. Ages of nodes are presented on each node along with distribution of ages as green bars. Age constraints for particular nodes are presented as primary minimum age constraints (yellow diamonds) or secondary age constraints (orange diamonds).

© 2017 The Royal Entomological Society, Systematic Entomology, 42, 555–574 Lance lacewing phylogeny 567 knowledge of fossil Osmylidae that the Jurassic was proba- Protosmylinae and Spilosmylinae are closely related and are bly an explosive period of diversification for the family, with united by a series of morphological synapomorphies, includ- numerous fossils already described, as well as large amounts of ing female spermathecal duct greatly elongate and coiled (44: undescribed material in collections from Jurassic-aged deposits 2), sternite 8 modified into small knob-like process proximal (Makarkin et al., 2014). to gonocoxite 9 (45: 2; 46: 1), male genitalia with narrow The monophyly of Osmylidae is strongly supported in all anal- arch-like gonarcus (54: 2), parameres present and ends fused yses here and has never been disputed previously. Characters medially into an arched structure (60: 1) (Fig. 3A, C). Proto- that support the monophyly of the family (unambiguous under smylinae and Gumillinae diverged from Spilosmylinae during accelerated transformation optimization) include adult dorsal the Mid-Triassic (223 Ma). tentorial arms weakly developed (character 65: state 1) (Beu- Gumillinae is represented by the extant South America tel et al., 2010; Randolf et al., 2013, 2014; Zimmermann et al., genus (Gumilla) containing two species known from just a 2009, 2011), forewing CuP pectinate or, more rarely, dichoto- few specimens (Navás, 1912; Martins et al., 2016). There mously branched (character 33: states 1, 2), and female gono- are nine additional genera known from Mid-Jurassic to Early coxite 9 (gx9) (gonopophyses lateralis) with terminal stylus (50: Cretaceous-aged deposits in Europe, Asia, and North and South 1). Osmylid wings usually have the forewing radial area with America (Menon & Makarkin, 2008; Wang et al., 2009b; Yang veins curved posteriorly, frequently sinuously (14: 1, 2). The et al., 2010; Khramov, 2014b; Myskowiak et al., 2016). Gumil- presence of ocelli has been used as diagnostic for the family, linae have multiple wing venation features (Fig. 4A) that sup- but while the presence of ocelli is clearly present as a plesiomor- port their placement in clade A, including relative scarcity of phic condition throughout the family, they are secondarily absent crossveins (7: 1) and simple hindwing CuP (35: 0), although in Gumilla and some species of Spilosmylus and reduced in the male genitalia of Gumilla are unusually disparate in mor- Paryphosmylus (2: 1). The placement of Nevrorthidae as the phology from other members of this clade (see Martins et al., sister to Osmylidae in this analysis is novel, as other authors 2016). Our analyses placed Gumilla as a very long branch in have placed Nevrorthidae as either sister to all other Neuroptera a polytomy with protosmyline genera. Affinities of Gumilli- (e.g. Aspöck et al., 2001) or as sister to Sisyridae (e.g. Winterton nae have been unclear previously, with Navás (1912) proposing et al., 2010; Wang et al., 2016). the tribe Gumillini and Lambkin (1988) raising it to subfamily. Higher-level relationships among the subfamilies of Osmyli- Our results strongly support its close relationship with Proto- dae were strongly supported in our combined analyses of mor- smylinae, but not as far as confirming its status as a separate phology and DNA sequences, with a basal dichotomy into two subfamily. Khramov (2014b) and Menon & Makarkin (2008) clades: one comprising Protosmylinae, Gumillinae and Spi- described various fossil genera in this subfamily and it is clear losmylinae (clade A) and the other comprising Osmylinae, that the group was far more diverse during the Mesozoic than Kempyninae, Eidoporisminae and Stenosmylinae (clades B, C). it is. Krüger (1913a, 1913b, 1913c) similarly identified two major Protosmylinae comprise four extant genera: three from the lineages within the family as Nomosmylidae, comprising Pro- Oriental region (Lysmus, Heterosmylus and Gryposmylus)and tosmylinae, Osmylinae and Spilosmylinae, and Anomosmyl- one monotypic genus from Ecuador (Paryphosmylus). Heteros- idae containing Kempyninae (as Kalosmylinae), Stenosmyli- mylus (Fig. 1D) contains nine species and was recently revised nae and Porisminae. The main difference between our result by Dong et al. (2016), while Gryposmylus (Fig. 1A), with two and Krüger’s classification is his placement of Osmylinae and species, was revised by Winterton & Wang (2016). Five addi- absence of Gumilla, the latter only being described in the previ- tional fossil genera are known from Jurassic- (Juraheterosmylus ous year by Navás (1912). Wang, Liu, Ren & Shih, Jurosmylus Makarkin & Archibald), The divergence of the major osmylid clades occurred dur- Cretaceous- (Protosmylina Jepson, Makarkin & Jarzembowski) ing the Early Triassic (247 Ma) during a time when Pan- and Tertiary-aged (Osmylidia Cockerell, Protosmylus Krüger) gaea would be the dominant landmass for at least another deposits throughout the Holarctic region, and, as in other sub- 70 Ma (Fig. 7). Indeed, all subfamilies were probably present families, is suggestive of a broader historical distribution for the throughout Pangaea during this time, based on our divergence subfamily during the Mesozoic. Protosmylinae are recognized estimates, and there are numerous fossil representatives on sepa- only by their wing venation, with relatively few radial veins and rate continents of Gumillinae (Wang et al., 2009b), Mesosmylin- distinctive female spermathecal shape (43: 4); it is otherwise dif- inae (Khramov, 2014a), Protosmylinae (Wang et al., 2010a), ficult to differentiate them from Spilosmylinae. Presently, the Kempyninae (Wang et al., 2011b), Osmylinae (Makarkin et al., main diagnostic character separating the two subfamilies is over- 2014) and Spilosmylinae (Wang et al., 2009a) dating before and all appearance and density of crossveins in the wing venation. after the brea- up of the super continent during the Mid-Jurassic. Lysmus was placed by Krüger (1913b, 1913c) in Spilosmylinae and subsequently treated as a synonym of Spilosmylus by Kim- mins (1942), but our results strongly support placement in Pro- Clade A: Protosmylinae, Spilosmylinae and Gumillinae tosmylinae in a sister-group relationship with Gryposmylus.The two genera diverged during the Jurassic (179 Ma) and are very This group of three subfamilies is very strongly supported sta- similar in appearance except for some wing venation characters tistically and easily identified by morphological characters such (Winterton & Wang, 2016). The confusion in subfamilial place- as hindwing CuP short and not pectinately branched (35: 0). ment of Lysmus underscores the ambiguity and lack of distinct

© 2017 The Royal Entomological Society, Systematic Entomology, 42, 555–574 568 S. L. Winterton et al. morphological characters separating Spilosmylinae and Proto- the branching pattern of M in the wing venation, the presence of smylinae. short and broadly rounded entoprocesses on the male gonarcus Spilosmylinae is represented by three extant genera (Spilosmy- (55: 3) appears to unite the group, although short and rounded lus Kolbe, Thaumatosmylus Krüger and Thyridosmylus Krüger) entoprocesses are also found in Gumilla. The elongate, paired distributed throughout the Oriental region and parts of the Aus- parameres also support this clade. The difficulty in defining tralasian and Afrotropical regions. This subfamily is poorly rep- the subfamily extends into defining the constituent genera, with resented in the fossil record, with only two genera described most being paraphyletic in our phylogeny (Figs 5, 6). The from the Mid-Jurassic of China (Palaeothyridosmylus Wang, number of genera recognized in the subfamily varies among Liu & Ren) (Wang et al., 2009a) and Late Jurassic of Kaza- authors, with some being considered merely subgenera of khstan (Ensiosmylus Khramov) (Khramov, 2014b). Synapomor- Osmylus by some; the distinction of most genera in Osmylinae phies characterizing this subfamily include the presence of a is based on just a few rather spurious characters in the wing basal sclerotized arm of the mediuncus (58: 1) in the male venation or genitalia (Wang & Liu, 2009). The monotypic genitalia (sensu Wang et al., 2011a) and the hindwing with a genus Sinosmylus is exceptional among Osmylidae in lacking spur vein originating basally on the MP vein (40: 1) (Fig. 4D). most crossveins in the wings (i.e. only the two gradate series Thaumatosmylus is a small genus of eight species differentiated are present), but this is clearly autapomorphic for the genus from Spilosmylus and Thyridosmylus based on the presence of and it could not be recovered as separate from the other forewing M-Cu crossveins basally. The distinction of Spilosmy- osmyline genera in the combined analyses. Some authors have lus from Thyridosmylus is more problematic and the supposed already cast doubts on some genera in Osmylinae, and even diagnostic features of each genus are absent in at least a few the status of various subgenera seems unlikely to withstand species of both (Wang et al., 2011a; Zhao et al., 2013), although scrutiny. Synonymies of various genera and/or subgenera, as our results show each genus as monophyletic. Thyridosmylus well as varied assignments of species to assorted genera, have comprises 20 species while Spilosmylus is by far the largest been proposed for almost all taxa in this subfamily (Nakahara, genus in the family, with at least 85 described species. Spilosmy- 1914; Makarkin, 1985; Wang & Liu, 2009, 2010; Sekimoto lus and Thyridosmylus show an interesting distribution pattern, & Yoshizawa, 2011; Xu et al., 2016). Based on the results as both predominantly Oriental genera have a few putative basal presented here, the status of all genera relative to the nominal species present in the Afrotropical region, principally Madagas- Osmylus is questionable and further detailed examination is car. This supports the conclusion that the genera were diverged required. during at least the Late Cretaceous and that ancestors of both Surprisingly, no definitive fossils of Osmylinae are described rafted on India during the Early Cenozoic as the continent moved as yet, although there are some undescribed Jurassic-aged northwards to collide with Asia to then form the basis for a sub- taxa present in collections. The fossil genus Archaeosmylidia sequent Oriental radiation (Wang et al., 2011a). Our divergence Makarkin, Yang & Ren was described as the putative link time estimates place the divergence of these two genera during between Permo-Triassic Archeosmylidae and modern osmylids the Mid-Jurassic (177 Ma). that originated during the Triassic (Makarkin et al., 2014). This argument was based on supposed plesiomorphic characters exhibited by this new genus. When included in the simultaneous Clades B + C analysis, though, we recovered Archaeosmylidia as sister to the rest of Osmylinae, not as sister to the rest of the family The remaining subfamilies of Osmylidae contained in this (Figure S2). clade are strongly supported statistically. This clade comprises Osmylinae (clade B) as sister to the remaining subfamilies in clade C, estimated to have diverged during the Early Triassic Clade C: Kempyninae, Eidoporisminae, Porisminae (244 Ma); this branch is supported by a series of morphological and Stenosmylinae characters including wing crossveins typically numerous (7: 2), hindwing CuP elongate and pectinately branched (35: 1), female Some species in each of these four subfamilies are among the sternite 8 immediate posterior to sternite 7 (46: 0) and gonarcus most spectacular living osmylids, often with large wings and enlarged, setose and visible externally (53: 1; 54: 1). dramatic contrasting wing patterns (Figs 1B, C, F, G, 2A). The close relationship among Kempyninae, Eidoporisminae, Poris- minae and Stenosmylinae has been noted by previous authors Clade B: Osmylinae based on wing venation and genitalic characters (Krüger, 1913a; Esben-Petersen, 1917; Kimmins, 1940; New, 1983a). Multiple Clade B comprises only Osmylinae, a subfamily of six genera morphological characters are exhibited by this clade, includ- distributed principally throughout the temperate parts of the ing forewing CuA with predominantly dichotomous branch- Palaearctic and Oriental regions. Osmylus (Fig. 1E) is the ing pattern (31: 1), female sternite 8 modified into a concave largest genus in this clade. Members of this subfamily are receptacle for the enlarged articulating gonopophyses 9 (45: well supported as a monophyletic group, but poorly defined 2; 47: 3), broad entoprocesses (55: 2) and loss of parameres morphologically with respect to other subfamilies, as most (60: 2), adding further support for the monophyly of this characters supporting the clade are homoplasious. Aside from derived clade.

Kempyninae diverged early from this clade during the Early to data do not recover these relationships among these taxa with Mid-Triassic (229 Ma). This subfamily contains four extant gen- any certainty, even between genera on different continents (Figs era and seven extinct genera predominantly from Jurassic-aged 5, 6); Phymatosmylus and Isostenosmylus occur in South Amer- deposits (Kimmins, 1940; New, 1983a; Wang et al., 2011b; ica while the remaining genera occur in Australia (New, 1986a; Khramov, 2014b). All extant Kempyninae are found in the Ardila-Camacho & Noriega, 2014; Martins et al., 2016). One southern hemisphere, with some authors inferring a Gondwanan clade that was recovered consistently comprised Carinosmylus, vicariance scenario of evolution for this group, especially for Stenosmylus, Oedosmylus and Euporismus (Fig. 6). Carinosmy- Kempynus, with species in Australia, New Zealand and Chile lus and Euporismus were placed in a clade sister to Stenosmy- (New, 1983a; Oswald, 1994). Our results suggest otherwise, that lus and Oedosmylus. We also recovered Stenosmylus as para- diversification among kempynine genera, and even species of phyletic with respect to Oedosmylus.BothCarinosmylus and Kempynus, occurred during the Jurassic long before Gondwana Euporismus are monotypic genera with highly modified male began to break up during the Cretaceous. In his evaluation of genitalia in the former and spectacularly marked wings in the Lithosmylus Carpenter, Lambkin (1988) noted that contrary to latter. Conversely, Stenosmylus and Oedosmylus have a more Krüger (1913a) the basic wing venation of Kempyninae and uniform morphology across the combined ten species in these Osmylinae was quite similar, and this close relationship was genera. Various authors have identified a likely close relation- also recovered here. Members of Kempyninae also share with ship among some or all of these genera (Tillyard, 1916; Kim- Osmylinae the presence in the male of eversible scent glands mins, 1940; Adams, 1969; New, 1986a, 1989b) and in some between abdominal tergites 8 and 9 (51: 1); these are lost in more cases noted the difficulty in adequately defining generic lim- derived subfamilies Porisminae, Eidoporisminae and Stenos- its beyond the autapomorphies exhibited by Carinosmylus and mylinae. The genus Australysmus wasrecoveredasthesister Euporismus (New, 1986a). A re-examination of the generic lim- genus to the rest of Kempyninae, followed by the diminutive its of all genera within Stenosmylinae (inclusive of Porisminae New Zealand endemic Euosmylus. Kempynus was rendered as and Eidoporisminae) is warranted. paraphyletic by the Australian genus Clydosmylus.Indeed,New (1983a) noted that Clydosmylus had more elaborate and exten- sive wing venation than Kempynus, but otherwise was very simi- Osmylidae larval habitats lar. Based on these results, Clydosmylus should be synonymized with Kempynus. Although lacewing larvae display a variety of larval habi- The three subfamilies Eidoporisminae, Porisminae and tats, including arboreal (e.g. Coniopterygidae, Chrysopidae), Stenosmylinae form a well-supported clade differentiated from fossorial (e.g. Myrmeleontidae, Ithonidae) and full aquatic (e.g. other osmylids by morphological features such as forewing Sisyridae, Nevrorthidae), traditionally the plesiomorphic condi- M vein forked at, or beyond, the mid-length of the wing (26: tion of the Neuroptera larvae has been assumed to be terrestrial. 1, 2), and male abdominal tergites 8 and 9 fused with scent Megaloptera have larvae that are fully aquatic and are, accord- glands secondarily lost (42: 1). The close relationship among ing to our results, the sister group to Neuroptera; this and the these subfamilies was previously identified by authors such as close position of Sisyridae and Nevrorthidae (both with fully Krüger (1913a), Esben-Petersen (1917) and New (1983b). Eido- aquatic larvae) to the base of the Neuroptera tree (relative to the porisminae and Porisminae each comprise a single monotypic other mainly terrestrial families) raise the interesting possibil- genus endemic to eastern Australia (Krüger, 1913a, 1913b; ity that the larva of the ancestral lacewing was indeed aquatic, Esben-Petersen, 1917; New, 1983b), the latter being the charis- suggesting that the arboreal habitats of Coniopterygidae are a matic pied lacewing (Fig. 1G). Stenosmylinae, by contrast, is novelty (autapomorphy) of this clade, only adopted later in the relatively species-rich, with seven genera present in Australia evolution of lacewings (Wang et al., 2016). Like Sisyridae and and South America (Kimmins, 1940; New, 1986a; Martins Nevrorthidae, Osmylidae are also closer to the base of the Neu- et al., 2016). Our phylogeny recovered Eidoporisminae as roptera tree of life than to other exclusively terrestrial lacewings sister to Porisminae and rendered Stenosmylinae paraphyletic (Winterton et al., 2010; Wang et al., 2016) and some osmylid (Figs 5–7). Both Eidoporismus pulchellus Esben-Petersen subfamilies live along the riparian zone of freshwater streams and Porismus strigatus Burmeister are highly autapomorphic (Wichard et al., 2009). Osmylid larvae have greatly elongate species that also exhibit the more generalized characters of jaws (Fig. 2C–E) that they presumably use to probe the moist Stenosmylinae, such as an elongate prothorax and forewing soil for prey. Larval stages are known for most subfamilies of medial vein forking in the distal part of the wing. They differ Osmylidae and not all are found in the riparian zone of lotic habi- only in that the proximal branch of forewing Rs branches in the tats. Subfamilies that do have larvae that live in riparian zones middle part of wing rather than close to the base of the wing. include Kempyninae, Osmylinae and Spilosmylinae, while lar- Consequently, the status of Eidoporisminae and Porisminae as vae of the subfamilies Porisminae and Stenosmylinae live in separate subfamilies from Stenosmylinae is unsupported here much drier habitats under bark and leaf litter. The larval stages but requires more detailed examination with regard to their are unknown for Gumillinae, Protosmylinae and Eidoporismi- status as separate subfamilies. nae. Larvae of the genera Kempynus (Fig. 2C) and Euosmy- Our divergence date estimates suggest that the crown age of lus (Kempyninae) are known from the moist riparian habitat genera in Eidoporisminae, Porisminae and Stenosmylinae date along lotic (often montane) streams in Australia (S.L. Winter- back to the Early to Mid-Jurassic (Fig. 7). Unfortunately our ton, unpublished observations), New Zealand (Hudson, 1904)

© 2017 The Royal Entomological Society, Systematic Entomology, 42, 555–574 570 S. L. Winterton et al. and Chile (O. Flint, unpublished observations in Oswald, 1994). similarly enlarged and subtriangular entoprocesses. The Larvae of Osmylus (Osmylinae) are well known as living in the parameres are secondarily absent in clade C. As mentioned air–water riparian interface in the Palaearctic (Ward, 1965). Lar- previously, eversible scent glands are present dorsally between vae of Spilosmylus (Spilosmylinae) are also known from this tergite 8 and 9 in the males of Kempyninae and Osmylinae. In type of habitat in Japan (Kawashima, 1957). Of the osmylid Stenosmylinae, Eidoporisminae and Porisminae, the eversible larvae that live in drier habitats, Porismus lives under the bark scent glands are lost and tergites 8 and 9 are fused either of Eucalyptus trees in dry sclerophyll forests in eastern Aus- partially or completely as a single sclerite. tralia (New, 1986a; S.L. Winterton, unpublished observations). In the female the arrangement and shape of tergite 9 and In Stenosmylinae, larvae have been collected and observed in gonocoxite 9 are similar between Nevrorthidae and Osmylidae. similarly dry habitats. Specific examples include under Eucalyp- In clade A of Osmylidae, though, a distinct gonopophysis 9 is tus bark (Oedosmylus sp.) (S.L. Winterton, unpublished obser- present as a separate sclerite at the base of gonocoxite 9 and vations), in soil litter (Fig. 2E) and on foliage (Isostenosmylus articulates with the posteroventral margin of tergite 9. Sternite sp.) (Fig. 2D). New (1974) proposed a subcortical habitat for 8 (subgenitale of some authors) is a small knob-like sclerite in the larva of Stenosmylus when describing larval stages. A uni- Protosmylinae and Spilosmylinae that acts against a depression versal feature of osmylid larvae in riparian habitats is that they in the intersegmental membrane immediately anterior to sternite have a dark brownish-black colouration (Fig. 2C), while larvae 7 during copulation. This position of sternite 8 in clade A in drier habitats are typically of much lighter colouration with is a derived condition, with the plesiomorphic condition (in light brown sclerites (Fig. 2D, E); the functional significance of lacewings) of sternite 8 typically adjacent to sternite 7 being this is unknown. present in clades B and C. Moreover, the female genitalia of Osmylinae may represent an intermediate in shape and sclerite complement between clades A and C. In this case, Functional morphology shifts in genitalic sclerites gonopophyses 9 is elongated anteriorly and during copulation acts upon a flattened sternite 8 immediately posterior to sternite Genitalic morphology is highly varied among lineages of 7. In the females of clade C there is remarkable modification osmylids, and structural homology of sclerites has been prob- of gonopophyses 9 into an enlarged, often multilobed process lematic in the past, largely due to basing investigations of geni- that acts upon sternite 8, which itself is often modified into a talic structure on single lineages in isolation instead of broader large concave sclerite. The most extreme forms of this can be investigations across the entire family compared with outgroups. found in various species of Kempyninae. In some females (e.g. This study presented a rare opportunity to undertake such a com- Carinosmylus, Australysmus) sternite 7 is also modified with a parative analysis of the male and female genitalic sclerites and posteromedial process along the margin; a similar modification explore the origin of such variability in a phylogenetic context. also occurs independently in some species of Osmylus. We found that throughout the evolutionary history of Osmylidae, Basedonthisobservedvariationinthemaleandfemalegeni- there has been a series of dramatic modifications to the struc- talic sclerites, it is difficult to definitively assign directionality to tural and functional organization of genitalic sclerites among the the transformations. This is because both clades A and B display major clades, particularly in the female (Fig. 8). a mixture of plesiomorphic and derived character states when In clade A, the male genitalia are in some respects probably compared with the conditions displayed in the outgroup taxa. closer to the plesiomorphic condition than are those of clades Detailed examination of Cretaceous-aged amber fossils, where B and C. In Protosmylinae and Spilosmylinae the genitalic externally visible genitalic structures may be observable, may sclerites are contained within the ectoproct and sternite 9, the provide insights into the condition of these states in stem taxa entoprocesses appear unmodified and parameres are present and provide further insights into character polarities. (although fused medially into an arched structure). The mediuncus and hypandrium internum are very conserved in structure throughout Osmylidae, varying little among subfamilies. Conclusions Gumillinae appears to have derived and highly reduced male genitalic sclerites (Martins et al., 2016), although with a pilose It is hoped that this first-ever phylogenetic framework for lance gonarcus and reduced entoprocesses similar to Osmylinae. lacewings will be useful to other workers for further exploration In clades B and C, the male genitalia have an enlarged and of the morphology and taxonomy of the family. The apparent broad gonarcus visible externally and covered with a setal pile rarity of the family and the confusing taxonomic classification (Fig. 3). In Osmylinae (clade B), the male entoprocesses are erected for the genera have limited progress on the group since reduced to small knob-like processes laterally on the gonarcus, the works of Krüger (1912–1915). Stem Osmylidae diversity with the parameres present as paired elongate sclerites flanking dates back to the Permian, with significant radiations of modern the mediuncus. Interestingly, this paired arrangement of the subfamilial lineages in the Jurassic and Cretaceous, leading to parameres exhibited by Osmylinae is most similar to the ple- the disparate diversity found in most major biogeographical siomorphic condition for this structure. In clade C, comprising regions of the world today. Our estimate of phylogeny supports Kempyninae, Eidoporisminae, Porisminae and Stenosmyli- in large part the subfamilial classification previously proposed nae, the male genitalic sclerites are relatively uniform with for the family and highlights potential areas where the taxonomy an enlarged subtriangular gonarcus with setal pile, and and classification require further evaluation, including potential

Fig. 8. Phylogeny recovered in Bayesian analysis with distribution of female genitalic sclerites indicated. Sclerite colour code: yellow, sternite 8; green, gonopophysis 9; blue, tergite 9; orange, gonocoxite 9.

© 2017 The Royal Entomological Society, Systematic Entomology, 42, 555–574 572 S. L. Winterton et al. subfamilial and generic synonyms. The phylogeny also places (Insecta: Holometabola: Neuropterida: Neuroptera). Systematic Ento- into evolutionary context the apparently disparate morphology mology, 26, 73–86. in the genitalia amongst osmylid subfamilies and posits a Beutel, R.G., Zimmermann, D., Krauss, M., Randolf, S. & Wipfler, sequence of evolution for character systems such as wing B. (2010) Head morphology of Osmylus fulvicephalus (Osmylidae, Neuroptera) and its phylogenetic implications. Organisms, Diversity venation and male and female genitalia. and Evolution, 10, 311–329. Carpenter, F.M. (1943) Osmylidae of the Florissant shales, Colorado (Insecta-Neuroptera). American Journal of Science, 241, 753–760. Supporting Information Cockerell, T.D.A. (1908) Fossil Osmylidae (Neuroptera) in America. Canadian Entomologist, 40, 341–342. 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