Molecular Phylogenetics and Evolution 107 (2017) 103–116
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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
A first higher-level time-calibrated phylogeny of antlions (Neuroptera: Myrmeleontidae) ⇑ ⇑ Bruno Michel a, , Anne-Laure Clamens b, Olivier Béthoux c,d, Gael J. Kergoat b,1, Fabien L. Condamine e, ,1 a CIRAD, UMR 1062 CBGP (INRA, IRD, CIRAD, Montpellier SupAgro), 755 Avenue du Campus Agropolis, 34988 Montferrier-sur-Lez, France b INRA, UMR 1062 CBGP (INRA, IRD, CIRAD, Montpellier SupAgro), 755 Avenue du Campus Agropolis, 34988 Montferrier-sur-Lez, France c Sorbonne Universités, UPMC Univ Paris 06, MNHN, CNRS, Centre de Recherche sur la Paléobiodiversité et les Paléoenvironnements (CR2P), Paris, France d Muséum National d’Histoire Naturelle, 57 rue Cuvier, CP38, F-75005 Paris, France e CNRS, UMR 5554 Institut des Sciences de l’Evolution (Université de Montpellier), Place Eugène Bataillon, 34095 Montpellier, France article info abstract
Article history: In this study, we reconstruct the first time-calibrated phylogeny of the iconic antlion family, the Received 25 May 2016 Myrmeleontidae (Neuroptera: Myrmeleontiformia). We use maximum likelihood and Bayesian inference Revised 20 October 2016 to analyse a molecular dataset based on seven mitochondrial and nuclear gene markers. The dataset Accepted 21 October 2016 encompasses 106 species of Neuroptera, including 94 antlion species. The resulting phylogenetic frame- Available online 22 October 2016 work provides support for a myrmeleontid classification distinguishing four subfamilies: Acanthaclisinae, Myrmeleontinae, Palparinae, and Stilbopteryginae. Within Myrmeleontinae, Myrmecaelurini and Keywords: Nemoleontini are recovered as monophyletic clades; Gepini also appears as a valid tribe, distinct from Early Cretaceous Myrmecaelurini whereas Myrmecaelurini and Nesoleontini on one hand and Brachynemurini and Fossil calibrations Higher-level phylogeny Dendroleontini on the other hand, appear closely related. Some preliminary information related to gen- Molecular dating eric and specific levels are also implied from our results, such as the paraphyly of several genera. Dating Myrmeleontidae analyses based on thoroughly evaluated fossil calibrations indicate that the antlion family likely origi- Stilbopteryginae nated in the Cretaceous, between 135 and 138 million years ago (depending on the set of fossil calibra- tions), and that all higher-level lineages appeared during the Early Cretaceous. This first phylogenetic hypothesis will provide a valuable basis to further expand the taxonomic coverage and molecular sam- pling, and to lay the foundations of future systematic revisions. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Within Neuroptera, antlions (Myrmeleontiformia: Myrmeleon- tidae) constitute the most species-rich family. In the most recent The order Neuroptera (ca. 6000 species) consists of small to catalogue, Stange (2004) listed 1522 extant species and about 13 large holometabolous insects that are considered, together with fossil species classified in 14 tribes and 201 genera. Since the pub- Megaloptera + Raphidioptera, as a sister group of Coleopterida lication of Stange’s catalogue, numerous new species have been (i.e. Coleoptera + Strepsiptera) (Misof et al., 2014). Neuroptera are described, and the world antlion fauna is currently estimated at commonly divided into three sub-orders (Hemerobiiformia, ca. 2000 species (Acevedo et al., 2013). Myrmeleontidae occur in Myrmeleontiformia and Nevrorthiformia) and 17 families most temperate and tropical regions of the world, with the greatest (Aspöck, 1992, 1993; Grimaldi and Engel, 2005). The higher-level diversity being found in the intertropical area (see Fig. 1 for an phylogenetic relationships among these groups, as well as the excerpt of antlion diversity). Most antlions are psammophylous family-level hierarchy of Neuroptera, have been extensively dis- and live in arid or semiarid environments (Mansell, 1996). Most cussed based on both morphological and molecular data, and is adults are crepuscular or nocturnal and rest on vegetation during now rather satisfactorily resolved (Aspöck et al., 2001, 2012; day, but some species are more typically diurnal. Adults in most Aspöck, 2002; Haring and Aspöck, 2004; Aspöck and Aspöck, species retain a predatory diet, which is potentially complemented 2008; Winterton and Makarkin, 2010; Winterton et al., 2010). by pollen in a few species (Stelzl and Gepp, 1990). Although most myrmeleontid species are ambush-hunters (see Miller and Stange,
⇑ Corresponding authors. 1985; Stange et al., 2003; Stange, 2004; Badano and Pantaleoni, E-mail addresses: [email protected] (B. Michel), fabien.condamine@gmail. 2014 for an overview), antlions are well known for the behavior com (F.L. Condamine). of the species that construct pitfall traps in sandy soil to catch 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ympev.2016.10.014 1055-7903/Ó 2016 Elsevier Inc. All rights reserved. 104 B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116
Fig. 1. Illustration of antlion diversity. (A) Adult of Macronemurus appendiculatus (Latreille) (Myrmeleontinae); (B) Adult of Palparellus spectrum (Rambur) (Palparinae); (C) Adult of Centroclisis sp. (Acanthaclisinae); (D) Adult of Stilbopteryx napoleo (Lefèbvre) (Stilbopteryginae); (E) Unidentified antlion larva; (F) Sand pits of Myrmeleon inconspicuus Rambur (Myrmeleontinae). prey; the pit construction and the pit-dwelling larva behavior have Larvae of Myrmeleontidae exhibit several combinations of char- been described in detail (e.g. New, 1986; Mansell, 1988; Devetak acters (see Badano and Pantaleoni, 2014 for a very detailed et al., 2005; Mencinger-Vracˇko and Devetak, 2008; Hollis et al., description of antlion larvae). The buccal cavity is closed anteriorly 2011, 2015; Lambert et al., 2011; Scharf et al., 2011). Ambush- with each mandible and maxillary lacinia elongate and closely uni- hunter larvae live on cliffs, under rocks, on trees or in trunk cavi- ted to form complex piercing and sucking tubes, the mid-gut is ties; their biology is far less well known (Badano and Pantaleoni, closed posteriorly and disconnected of the hind-gut, and the Mal- 2014). Some species, such as the Mediterranean species Myrme- pighian tubes produce silk to make the cocoon that surround the caelurus trigrammus (Pallas), are also facultative pit-builders, either pupa. They are also flattened dorso-ventrally and covered with ambushing their prey at surface or constructing pitfall traps dolichasters representing modified setae. Their elongate and api- (Devetak et al., 2013). Antlions larvae have effective toxins, which cally curved jaws bear generally three mandibular teeth of variable are derived from both the insect and bacterial symbionts (Dunn length, but additional teeth are observed in certain species and a and Stabb, 2005). They also possess potent digestive enzymes, few genera have a reduced number, and the head capsule is heavily which are used to liquefy the internal components of their prey sclerotized with a convex posterior margin (Aspöck, 1992). Adults (Griffiths, 1980, 1982; Van Zyl et al., 1998). In all species with are characterized by short and slightly enlarged antennae (clubbed known biology, pupation occurs in a silk cocoon spun by the last in Stilbopteryginae). Wing venation varies between sub-families, larval instar in the same habitat. Overall, reproduction biology and includes a long hypostigmatic cell (except in Stilbopteryginae) has been poorly investigated. Sexual communication relies on located under the pterostigma. Legs are well developed and vari- pheromones (Baeckström et al., 1989; Bergström et al., 1992; ably equipped with stout setae. In some species claws can be Yasseri et al., 1996; Bergström, 2008) and few studies have inves- folded on the ventral surface of the last tarsomere. Configurations tigated in details the morphological structures that are involved in of male and female genitalia exhibit also differential characters pheromone perception (but see Zhang et al., 2015). Copulation has (Stange, 1970, 1994; Aspöck and Aspöck, 2008). The phylogenetic rarely been observed and it is assumed that in most cases eggs are significance of external and internal morphological characters laid separately on the ground (New, 1986; Miller, 1990; Yasseri was discussed in detail by Aspöck et al. (2001), Aspöck and and Parzefall, 1996; Yasseri et al., 1996). Aspöck (2008) and Zimmermann et al. (2011). B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116 105
Historically, the family Myrmeleontidae was created by (Huang et al., 2016); as acknowledged by the authors, this fossil Latreille (1802) who regrouped the genera Ascalaphus Fabricius also cannot be assigned with certainty to Myrmeleontidae and and Myrmeleon Linnaeus without providing any supra-generic clas- was placed in a distinct neuropteran group (yAraripeneuridae). sification. This conception was followed by most authors of the Only two fossils from the Cenozoic are assignable to Myrmeleonti- 19th century (e.g. Lamarck, 1817; Burmeister, 1839). Walker dae. The first one is the well-described yPorrerus dominicanus Poi- (1853) added the genus Stilbopteryx Newman, previously classified nar and Stange, the single definitive myrmeleontid species in the family Stilboptericidae (sic) by Newman (1853). Rambur known from amber deposits of the Dominican Republic (Poinar (1842) divided the family into Ascalaphidaes and Myrmeleonides and Stange, 1996; Engel and Grimaldi, 2007). The second is yDen- in which he considers four genera (Acanthaclisis Rambur, Megisto- droleon septemmontanus Statz, the only Cenozoic compression fos- pus Rambur, Myrmeleon, and Palpares Rambur). Later on, Hagen sil of a myrmeleontid from the late Oligocene-early Miocene (Statz, (1866) split up antlion species into 16 genera and made 1936); the assignation of this fossil is however potentially ques- Myrmeleontidae a subfamily of Hemerobidae (sic), in the same tionable because the fossil only consists of the impression of the way as Ascalaphidae, Chrysopidae, Coniopterygidae, Hemerobidae, apical half of the posterior wing (Poinar and Stange, 1996). It is also Mantispidae, and Nemopteridae. Banks (1899) differentiated for worth highlighting that many fossil genera previously assigned to the first time two tribes, Dendroleoni and Myrmeleoni, on the basis Myrmeleontidae (e.g. yCratopteryx Martin-Neto and Vulcano, of differences in wing venation. During the 20th century, several yDiegopteryx Martin-Neto, yNeurastenyx Martin-Neto and Vulcano, authors proposed various classifications of Myrmeleontidae as yPalaeoleon Rice, ySamsonileon Ponomarenko) have been trans- reported by Mansell (1985, 1999). It is worth mentioning the sig- ferred to distinct neuropteran families (yAraripeneuridae, yPalae- nificant contributions made by: (i) Banks (1911, 1927, 1943), oleontidae) (Martins-Neto and Vulcano, 1989a,b, 1997; Martins- who studied the African and the North American faunas and pro- Neto and Rodrigues, 2010), which likely constitute stem lineages posed arrangements into subfamilies and tribes; (ii) Tillyard to Myrmeleontidae (Rice, 1969; Martins-Neto, 2000; Ren, 2002; (1916), who provided a ‘cladogram’ for the Australian Myrmeleon- Huang et al., 2016). These two families were first considered as tidae with two subfamilies: Dendroleontinae and Myrmeleontinae subfamilies of Myrmeleontidae (Martins-Neto, 1992b; Stange, (with three tribes); (iii) Markl (1954), who classified the 2004) but are now generally considered as distinct families Myrmeleontidae into 23 tribes on the bases of adult morphological (Dobruskina et al., 1997; Heads et al., 2005; Menon and characters but without providing subfamily ranks; and (iv) Stange Makarkin, 2008; Martins-Neto and Rodrigues, 2010). Altogether, and Miller (1990), who established a classification on the basis of the myrmeleontid fossil record brings little evidence regarding larval characters. It is only twenty years ago that Stange (1994) the tempo and mode of diversification of antlions, highlighting conducted the first cladistic study of the superfamily Myrmeleon- the need for a time-calibrated phylogenetic framework. toidea, and also provided a tribal classification for Myrmeleonti- In this study, we present a first higher-level phylogenetic dae, on the basis of adult and larval characters. Despite all these framework for the family Myrmeleontidae based on a molecular studies, the supra-generic classification of Myrmeleontidae is still matrix comprising seven genes (five mitochondrial and two insufficiently resolved, which explains the lack of consensus nuclear genes) and 106 species using maximum likelihood and between researchers working on this family. Several authors (e.g. Bayesian inference. The molecular phylogenetic analyses allow Stange and Miller, 1990; Stange, 2004) consider three extant sub- the assessment of the monophyly of some subfamilies and investi- families: (i) Myrmeleontinae (ca. 1800 species, including the tribe gate the status and relationships of several major tribes. The result- Acanthaclisini); (ii) Palparinae (ca. 130 species); and (iii) Stil- ing molecular phylogeny is also used to estimate the age of the bopteryginae (seven species). Others, such as Oswald and Penny clade as well as the divergence times of major groups using up (1991) or New (1985, 2003) do not recognize the acanthaclisines to four thoroughly evaluated fossil calibrations and a relaxed as a myrmeleontine tribe, and instead consider them as a fourth molecular-clock approach inferred within a Bayesian framework. subfamily, the Acanthaclisinae (ca. 100 species). It is also worth mentioning the work of Hölzel (1972), who created the subfamily Echthromyrmicinae that only includes the genus Echthromyrmex 2. Material and methods McLachlan, and the work of Krivokhastky (2011), who classified Myrmeleontidae into seven subfamilies: Acanthaclisinae, Den- 2.1. Taxon sampling droleontinae, Glenurinae, Myrmecaelurinae, Myrmeleontinae, Nemoleontinae, and Palparinae. All these discrepancies highlight For the family Myrmeleontidae, new DNA sequences were gen- the need for molecular studies in antlions, especially because of erated for 61 antlion species (see Table S1 for a detailed list of spec- the fact that the family Myrmeleontidae is still lacking even an ini- imens). Identification of specimens was conducted by Michel, who tial molecular phylogeny, unlike other neuropteran families (e.g. has expertise in antlion taxonomy (e.g. Michel and Mansell, 2010; Chrysopidae; Winterton and de Freitas, 2006; Haruyama et al., Michel and Akoudjin, 2011; Michel, 2014). We also relied on Gen- 2008; Mantispidae; Liu et al., 2015; Nymphidae; Shi et al., 2015; Bank data for 33 antlion taxa; the corresponding data is generally and Ithonidae and Polystoechotidae; Winterton and Makarkin, associated with studies focusing on antlions (e.g. Wilson et al., 2010). 2010; Pantaleoni and Badano, 2012; Cheng et al., 2015). The ratio- Regarding their origin, the family Myrmeleontidae is seen as nale here was to favour taxa that were unambiguously identified potentially originating in the Late Jurassic, and definitively by the by specialists of the group (e.g. Badano, Pantaleoni, and Schiaffino). Early Cretaceous (Grimaldi and Engel, 2005; Engel and Grimaldi, Whenever possible, we also tried minimizing the amount of miss- 2007; Winterton et al., 2010). Several compression fossils of ing data by selecting specimens with the maximum number of Myrmeleontoidea are known from the Early Cretaceous deposits available molecular markers. However, for some sparsely sampled of the Crato Formation in Brazil (e.g. Martins-Neto and Vulcano, groups we had no choice but to include taxa with a large amount of 1989a,b; Grimaldi, 1990; Martins-Neto, 1994, 1998) and the Yixian missing data in order to maximize taxonomic coverage. This strat- Formation in China (e.g. Ren and Engel, 2008); however none of egy was motivated by the results of several studies (Wiens, 2005; them are unambiguously assignable to Myrmeleontidae because Crête-Lafrenière et al., 2012; Wiens and Tiu, 2012) which indicate they are mostly defined by plesiomorphies (Huang et al., 2016). that adding incomplete taxa is generally beneficial to phylogenetic Recently one fossil of Myrmeleontoidea, yBurmaneura minuta accuracy, especially when taxon sampling is sparse. A denser sam- Huang et al., was described from Burmese Cretaceous amber pling was also achieved for two species-rich genera, Myrmeleon 106 B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116 and Palpares, whose monophyly is often questioned. Within extant positions. The combination of the eight gene fragments resulted in Myrmeleontidae, and adopting the classification of Stange (2004), a matrix of 106 taxa and 3942 aligned nucleotides. our sampling encompasses 94 antlion species (61 + 33), which rep- resent all three subfamilies and eight of 14 tribes. The tribes that 2.3. Phylogenetic analyses are absent from our sampling are the following: (i) Gnopholeontini (four genera), Lemolemini (seven genera), and Maulini (two gen- Phylogenetic analyses were conducted in both a maximum like- era), within Myrmeleontinae; and (ii) Dimarini (three genera), Pal- lihood (ML) and a Bayesian framework using both the online com- paridiini (one genus), and Pseudimarini (one genus), within puter cluster CIPRES Science Gateway (Miller et al., 2015; Palparinae. www.phylo.org) and the HPC cluster hosted in the Centre de Biolo- On the basis of recent higher-level molecular phylogenies for gie pour la Gestion des Populations (CBGP) in Montferrier-sur-Lez. Neuroptera (Aspöck et al., 2001, 2012; Aspöck, 2002; Aspöck and First, the molecular dataset was divided into codon partitions for Aspöck, 2008; Haruyama et al., 2008; Winterton et al., 2010), we protein-coding genes (COI, COIII, Cyt b), and by genes for the rRNA selected as outgroups encompassing representatives from the four genes (12S, 16S, 18S, and 28S). To determine the best partitioning other extant families of the suborder Myrmeleontiformia: scheme for the dataset, we used PartitionFinder 1.1.1 (Lanfear Ascalaphidae, Nemopteridae, Nymphidae, and Psychopsidae. Six et al., 2012). The greedy algorithm was set, along with the linked species were included for the family Ascalaphidae, which consti- option, and the Bayesian information criterion (BIC) was used to tutes the sister family of Myrmeleontidae (Ascaloptynx appendicu- compare partitioning scheme and corresponding substitution lata (Fabricius), Ascalohybris subjacens (Walker), Libelloides models (Ripplinger and Sullivan, 2008). macaronius (Scopoli), Libelloides rhomboideus (Schneider), Melam- Maximum likelihood analyses were carried out using the brotus papio Tjeder, and Neomelambrotus molestus Tjeder. As more recently developed IQ-TREE (Nguyen et al., 2015) as implemented distinctly related outgroups (Winterton et al., 2010), we also on the multicore version of IQ-TREE 1.3.5 (also available on the included three species belonging to the family Nemopteridae web server at: http://iqtree.cibiv.univie.ac.at/). IQ-TREE has been (two from subfamily Crocinae: Austrocroce attenuata (Froggatt) shown to outperform RAxML or PhyML (Nguyen et al., 2015). It and Laurhervasia setacea (Klug), and one from subfamily optimises the ML search by focusing on local optima and compar- Nemopterinae: Lertha barbara (Klug)) and two species belonging ing them to find the best ML tree (Nguyen et al., 2015). The con- to the family Nymphidae (one from subfamily Myiodactylinae: catenated dataset was partitioned as determined by Myiodactylus osmyloides Brauer, and another from subfamily Nym- PartitionFinder. The corresponding models of substitutions were phinae: Nymphes myrmeleonoides Leach). The phylogeny was searched using the Auto function on the IQ-TREE web server based rooted using Psychopsis margarita Tillyard, a representative of the on the corrected Akaike information criterion (AICc). We per- family Psychopsidae, which was recovered as sister to all other formed 1000 ultrafast bootstrap replicates (Minh et al., 2013)to extant myrmeleontiform families in the study of Winterton et al. investigate nodal support across the topology considering boot- (2010). Data for these twelve outgroup species was recovered from strap values (BV) P 70 as strongly supported (Hillis and Bull, GenBank. 1993). Bayesian inference (BI) was performed with MrBayes 3.2.6 (Ronquist et al., 2012). We used the partitioning scheme and 2.2. DNA extraction, sequencing, and molecular matrix among-site rate variation suggested by PartitionFinder, but instead of selecting one substitution model a priori, we used reversible- DNA was extracted following the non-invasive protocol of jump Markov Chain Monte Carlo (rj-MCMC) to allow sampling extraction of Gilbert et al. (2007). Five mitochondrial gene frag- across the entire substitution rate model space (Huelsenbeck ments were sequenced: (i) cytochrome oxidase I (COI); (ii) cyto- et al., 2004). Analyses were performed with two separate runs, a chrome oxidase III (COIII); (iii) cytochrome b (Cyt b); (iv) 12S random starting tree, and eight rj-MCMC running for 50 million ribosomal RNA (12S); (v) 16S ribosomal RNA (16S). Two nuclear generations with sampling every 5000th tree (resulting in 10,000 fragments (comprising parts of two genes) were sequenced: (i) trees). We also specified: (i) a uniform prior probability of phyloge- the domain D2-D3 of the 28S ribosomal DNA (28SD2-D3); (ii) nies (i.e. all possible trees are considered a priori equally probable), 18S ribosomal DNA (18S). All these gene fragments were chosen and (ii) a uniform prior probability distribution on branch lengths. because they are known to be informative in phylogenetic analyses The convergence of the runs was assessed by checking the poten- of Neuroptera at various levels (Haring and Aspöck, 2004; tial scale reduction factor (PSRF) values of each parameter in Winterton et al., 2010). Polymerase chain reaction (PCR) amplifica- MrBayes and the Effective Sample Size (ESS) values of each param- tions were performed with standard settings for primer sequences eter in Tracer 1.6 (Rambaut et al., 2015). Values of PSRF close to and thermocycler procedures (see Table S2 for details). The PCR 1.00 were considered as good indicator of convergence (see the products were processed by the French sequencing centre Geno- MrBayes manual for details). A 25% burn-in (2500 trees) was scope using a BigDye 3.1 sequencing kit and Applied 3730xl applied after checking for convergence and stationarity, and a con- sequencers. The resulting sequences of complementary strands sensus tree was built. All analyses were run twice, each beginning were further edited and reconciled using Geneious 5.1 (www.ge- with a different random starting tree. Posterior probabilities (PP) neious.com). All of the sequences generated in this study were that were P0.95 were considered as strongly supported (Erixon deposited in GenBank (see Table S1 for the accession numbers). et al., 2003). For all protein-coding genes, we used Mesquite 3.03 (Maddison and Maddison, 2015) to check the coding frame for possible errors 2.4. Evaluation of suitable fossil calibrations or stop codons. The sequences of six ribosomal RNA genes (i.e. 12S, 16S, 28SD2-D3, and 18S) contained some variations in length; their We carried out a critical evaluation of fossil material suitable to alignment was accomplished using MAFFT 7.245 (Katoh and provide minimum age constraints for specific nodes, assessing the Standley, 2013) with default settings. For the nuclear ribosomal identity of a number of fossils formerly considered suitable for cal- sequences, ambiguously aligned regions were identified and ibration and some additional ones. Based on the presence of excluded using Gblock 0.91b (Castresana, 2000; Talavera and synapomorphies linking the fossils to extant clades, unambiguous Castresana, 2007), with parameters allowing for smaller final fossils were used as minimum age constraints for specific nodes. To blocks, gap positions within the final blocks and less strict flanking do so we mostly relied on wing venation homologies, using abbre- B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116 107 viations following the scheme provided by Shi et al. (2012): poste- tional analysis reflects the fact that we think the corresponding rior Subcosta (ScP); anterior Radius (RA); posterior Radius (RP); fossil assignation is not entirely unambiguous. Media (M); anterior Media (MA); posterior Media (MP); anterior For the two analyses, Markov Chain Monte Carlo (MCMC) were branch of MP (MP1); posterior branch of MP (MP2); Cubitus run for 100 million generations and sampled every 10,000 genera- (Cu); anterior Cubitus (CuA); anterior branch of CuA (CuA1); poste- tions, resulting in 10,000 trees in the posterior distribution; we dis- rior branch of CuA (CuA2); posterior Cubitus (CuP); and first ante- carded the first 2500 trees as burn-in. We used BEAGLE (Ayres rior Analis (AA1). et al., 2012), a library for high-performance statistical phylogenetic As for the ages, fossils are typically assigned to a stratigraphic inference, with default parameters. This allowed a faster and more interval. We followed a conservative option such that the ages pre- accurate computation of likelihood models within our BEAST anal- sented below always correspond to the upper boundaries of strati- yses. All BEAST analyses were performed on the computer cluster graphic intervals within which fossils were found (the fossil will CIPRES Science Gateway 3.3 (Miller et al., 2015; www.phylo.org). actually be older than this age designation), or of the correspond- Tracer was used to assess graphically the convergence of runs, ing locality. The corresponding ages were taken from Gradstein and to check the ESS for all parameters (indicated by ESS above et al. (2012). 200, following the BEAST manual). For each analysis, we conducted two independent runs to ensure convergence of the MCMC. Post 2.5. Bayesian relaxed-clock analyses burn-in trees from the two distinct runs (7500 trees for each run) were further combined to build the maximum clade credibil- For all dating analyses, we used a Bayesian relaxed-clock ity tree using TreeAnnotator 1.8.2. approach as implemented in BEAST 1.8.2 (Drummond et al., Using the time-calibrated phylogeny, lineages-through-time 2012). BEAUti 1.8.2 was used to create the BEAST input file. We (LTT) plots were reconstructed to graphically visualize the overall implemented partitioned relaxed-clock models with an uncorre- pattern of diversification rates through time. We did not aim at lated lognormal clock model that assumes an underlying uncorre- unveiling an accurate pattern, given available species sampling, lated lognormal distribution (UCLD) of the evolutionary rates but instead we focussed on showing the early evolution of the (Drummond et al., 2006), which is more likely to yield accurate group. estimates than the uncorrelated relaxed clock model that assumes an exponential distribution of the evolutionary rates (Baele et al., 2013). We used the nucleotide substitution models as determined 3. Results by PartitionFinder analyses (greedy algorithm, linked branch lengths, BEAST models, BIC). For the tree speciation model we used 3.1. Phylogeny of Myrmeleontidae a birth-death process to better account for extinct and missing lin- eages. We used as a guide tree the most robust phylogenetic The 106-taxa molecular matrix contains 1174 parsimony infor- hypothesis, which corresponds to the results of the ML analyses. mative sites (1478 sites are variable). The best-fit partitioning The ‘tree operators’ of the BEAST tree model were disabled to keep scheme recovered by PartitionFinder includes five partitions: one the topology fixed so that only branch lengths were optimized. for the first codon positions of the mtDNA genes (p1), one for the This procedure ensures better convergence of the dating analyses second codon positions of the mtDNA genes (p2), one for the third by greatly reducing the space of parameters. Other parameters codon positions of the mtDNA genes (p3), one for the mtDNA rRNA were kept to their default prior distribution or were indirectly genes (12S and 16S) (p4), and one for the nucDNA rRNA genes (18S specified through other parameters. and 28S) (p5). The best-fitting substitution models according to BIC Fossil calibrations were set using a uniform distribution, which were as follows: p1 = SYM + C4, p2 = HKY + I + C4, p3 = GTR + I is more conservative than normal, lognormal or exponential distri- + C4, p4 = HKY + I + C4, and p5 = TVM + I + C4 (where C4 means butions, and has been recommended when there is little informa- four gamma categories). tion on the fossil record (Ho and Phillips, 2009). We also used the Both ML and BI analyses with MrBayes were performed multi- uniform prior in this study because of the scarcity of the fossil ple times using different seeds to ensure a good exploration of tree record in the Cenozoic, suggesting that more ancient fossils could space and parameter values. The best log-likelihood of the best ML be found for Myrmeleontidae. The uniform distributions were tree was 53486.47. For BI analyses, average standard deviation of bounded by the fossil ages conferring a minimum age (as explained split frequencies were 0.022 at 12.5 million generations (burn-in above) and a conservative maximum age corresponding to the threshold) and 0.007 at the end of the runs. The BI and ML analyses minimum age of the root. Based on our assessment of the fossil resulted in very similar trees (see Fig. S1 for the BI tree); however record we used either three or four primary fossil calibrations the support for the ML tree is way higher than those of the BI tree (see the Results section for more detailed information on fossil (84 of the 112 nodes are supported by BV P 70% under ML selection). The first constraint is provided with yTriassopsychops whereas only 31 nodes are supported by PP P 0.95 under BI). Max- superbus Tillyard, which is used to set a minimum age for the root. imum Likelihood and BI analyses differ only in the position of the The tree root age was given a uniform distribution between 228.4 Stilbopteryginae: under ML the genus Stilbopteryx (Stilbopterygi- Myrs and a maximum age of 300 Myrs (early Permian). The latter nae) is the first lineage to branch off, sister to the remaining was set to explore (putative) ancient origin of the clades and also myrmeleontids, whereas it is recovered sister to the sampled because the molecular and fossil data in hand did not evidence Ascalaphidae under BI, but without significant support (PP of any myrmeleontiforms as ancient as the Triassic (e.g. Grimaldi 0.77). The ML consensus tree reconstructed from 10,000 ultrafast and Engel, 2005; Engel and Grimaldi, 2007; Winterton et al., bootstrap trees is represented in Fig. 2. This tree supported the 2010). The second constraint is provided by yRoesleriana exotica monophyly of Myrmeleontidae with BV = 85. The subfamily Martins-Neto and Vulcano, which is used to set a minimum age Myrmeleontinae is recovered paraphyletic if the acanthaclisines for crown Nemopteridae (minimum age of 116.6 Myrs). The third are considered a myrmeleontine tribe; they are recovered sister constraint is provided by yPorrerus dominicanus, which is used to to a clade comprising the tribe Palparini (as the sole representa- set a minimum age for crown Myrmeleontini (minimum age of tives of the subfamily Palparinae, which also consists of three other 15.97 Myrs). A fourth constraint was also used in an additional tribes that are species poor) (BV = 99) plus the rest of Myrmeleon- analysis, which corresponds with the use of yChoromyrmeleon tinae, which forms a monophyletic group (BV = 83). All tribes were spp. to set a minimum age for stem Myrmeleontidae. This addi- recovered as monophyletic: Acanthaclisini (BV = 100), Brachyne- 108 B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116
Fig. 2. Maximum likelihood phylogeny of Myrmeleontidae. The tree is rooted with the representative of the family Psychopsidae. The tree shows support for a four-subfamily classification, with Stilbopteryginae found as sister to all remaining myrmeleontid species. Acanthaclisinae are sister to a clade composed of Myrmeleontinae + Palparinae. Contrasting colors are used to highlight major lineages in the tree. Nodal supports are shown as maximum likelihood bootstrap values. See Fig. S1 for more details on the phylogenetic analyses made with Bayesian inference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116 109 murini (BV = 86), Gepini (BV = 97), Myrmecaelurini (BV = 92), Huang et al., 2016 for a more conservative opinion). In any case Myrmeleontini (BV = 99), Nemoleontini (BV = 83), Nesoleontini the earliest yAraripeneurinae are from the same locality as yRoesle- (BV = 99), and Palparini (BV = 99). The tribe Dendroleontini was riana exotica and yCratonemopteryx robusta. Our molecular dataset represented by only one taxon, hence its monophyly was not includes representatives from the two distinct nemopterid subfam- assessed. In addition, five myrmeleontid genera (Centroclisis Navás, ilies (Crocinae and Nemopterinae), thus allowing us to estimate the Distoleon Banks, Myrmeleon, Neuroleon Navás and Palpares) were age of the most recent common ancestor of the family. Here we used consistently recovered paraphyletic in all analyses. the upper boundary of the Aptian (112.6 Myrs ago) to enforce a min- imum age for the corresponding node. 3.2. Evaluation of suitable fossil calibrations 3.2.4. Myrmeleontiformia: yPalaeoleontidae 3.2.1. Myrmeleontiformia: Psychopsidae Taxa assigned to the fossil family yPalaeoleontidae (likely para- The oldest known fossil representative of the family is yTrias- phyletic) are close relatives of the clade including the Nemopteri- sopsychops superbus from the Late Triassic Blackstone formation dae, Ascalaphidae, Stilbopteryginae and Myrmeleontidae (below, of Australia. This species exhibits several apomorphies that are ‘NASM clade’; Menon and Makarkin, 2008; Shi et al., 2012; only found in the Psychopsidae (Peng et al., 2011; Lambkin, Myskowiak and Nel, 2015), owing to the fusion of MP2 with 2014), especially the presence of vena triplica, which corresponds CuA1 in the forewing, substantiated by a visible, oblique section to the fusion of ScP, RA and RP in the hindwing. This fossil thus of MP2 directed towards CuA1. The earliest representative of the appears to be an unequivocal representative of this family. Our ‘Palaeoleontidae assemblage’ is yGuyiling jianboni Shi et al. from molecular dataset only includes one representative for this family, the Yixian Formation (ca. 125 Myrs ago ± 4 Myrs; Chang et al., which is used to root the tree. Consequently we can use this fossil 2009). However these insects can be readily excluded from the as a stem calibration for Psychopsidae, which allows us to set a NASM clade as they retain distinct main stems of MP2 and CuA1 minimum age for the root of the tree. The fossil was found in the in the forewing (as opposed to a full fusion of CuA1 main stem with Denmark Hill insect bed, which is a Carnian lacustrine siltstone MP2). in the Blackstone Formation of Australia. We thus used the upper boundary of the Carnian age i.e. 228.4 million years (Myrs) ago 3.2.5. Myrmeleontiformia: Ascalaphidae to enforce a minimum age to the root with a uniform distribution. For Ascalaphidae, several fossils were originally considered clo- sely related, such as yKarenina breviptera Martins-Neto and yMesas- 3.2.2. Myrmeleontiformia: Nymphidae calaphus yangi Ren (Martins-Neto, 1997; Ren et al., 1995). However As demonstrated by Myskowiak et al. (2016) the placement of several authors assigned these species to the fossil family supposed ancient Nymphidae fossil species by Shi et al. (2015) yMesochrysopidae, which is more closely related to the Chrysopi- was supported only by larval characters undocumented in current dae than to any other extant family (Makarkin and Menon, 2005; fossils. In other words, Shi et al. (2015) attributed states to the fos- Nel et al., 2005), i.e. it is only remotely related to our ingroup. sil species based on the a priori assumption that they belonged to The fossil species yCratoascalapha electroneura Martins-Neto and Nymphidae, an approach that cannot be followed. Therefore we Vulcano (Crato Formation) is known from a single, incomplete refrained from using any nymphid Mesozoic fossil for phylogenetic forewing (Martins-Neto and Vulcano, 1997). There are legitimate calibration. For instance we did not use the Cretaceous fossils ySpi- doubts regarding the unusually long stem of RP (distal to the emer- lonymphes major Shi et al., yBaissoleon cretaceus Makarkin and gence of MA from RP + MA), as the beginning of a banksian line yBaissoleon similis Shi et al. that are phylogenetically recovered as could have been misinterpreted as MA. Moreover this species is stem lineages of Nymphinae and Myiodactylinae, respectively very similar to (putative synonymy) yParaneurastenyx ascalaphix (Shi et al., 2015). As an unequivocal nymphid fossil we preferred Martins-Neto, from the same locality, but of ‘nemopterid type’. to use the comparatively recent yNymphes georgei Archibald et al. Additionally yCratoascalapha electroneura exhibits a simple MA, from the Klondike Mountain Formation (Ypresian, 52 Myrs while all extant Ascalaphidae possess a MA with many branches, ago ± 4 Myrs), which is closely related to the extant species Nym- adding doubts on its actual affinities. In summary we failed to find phes aperta New. This fossil can be confidently assigned to the sub- grounds for the assignment of this species to the family Ascalaphi- family Nymphinae because it possesses several derived characters dae. We could have selected yAscaloptynx oligocenicus Nel, from the only found in this subfamily such as the characteristic triangular Oligocene of Southern France (Rupelian stage; ca. 31 Myrs ago ± 3 area of the hind wing MP space (for a complete list see Archibald Myrs). Its placement at the stem node of the genus Ascaloptynx is et al., 2009). Its hind wing venation is also indistinguishable from well ascertained (Nel, 1990); however this fossil is comparatively that of extant Nymphes species (Archibald et al., 2009). Despite recent; therefore we did not use it as to calibrate the sampled its recent age, this fossil could constitute a useful primary calibra- Ascalaphidae to avoid underestimations of clade ages. tion to constrain either the crown or the stem node of the genus Nymphes Leach. For our study such option was not followed 3.2.6. Myrmeleontiformia: Myrmeleontidae because of the limited sampling we had for Nymphidae (two spe- The two known species of the genus yChoromyrmeleon, namely cies from two distinct subfamilies, including one representative of yChoromyrmeleon othneius Ren and Guo and yChoromyrmeleon the genus Nymphes). aspoeckorum Ren and Engel, were both recovered from the Yixian Formation (Ren and Guo, 1996; Ren and Engel, 2008), in the area 3.2.3. Myrmeleontiformia: Nemopteridae of the Chaomidian village (China; ca. 125 Myrs ago; Chang et al., Nemopteridae are distinctive in their spoon- or filament-shaped 2009). Ren and Guo (1996) first assigned this genus to Myrmeleon- hindwings. The earliest fossils exhibiting this condition are yRoesle- tinae, while Ren and Engel (2008) later assigned it to Myrmeleon- riana exotica, and yCratonemopteryx robusta Martins-Neto, both tidae. The available data shows that the two species are very recovered from the Crato Formation (Martins-Neto, 1992a, 1997) similar. The occurrence of a long hypostigmatic cell is a strong indi- (late Aptian; Batten, 2007). We noted that several species assigned cation of their affinities with Myrmeleontidae, but this trait occurs to the fossil taxon yAraripeneurinae possess a comparatively narrow also in some crown- and stem-Nemopteridae (such as yRoesleriana hind wing (such as yCaririneura microcephala Martins-Neto and Vul- exotica), suggesting that the character is homoplasic (see also cano; Martins-Neto and Vulcano, 1989b), suggesting that this taxon Stange, 1994). Unfortunately, in the absence of well-preserved is composed, at least in part, of remote stem-Nemopteridae (see forewing bases, it is impossible to determine the length and course 110 B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116 of CuP (the free part of CuP is very short before it fuses with AA in 3.3. Estimation of divergence times Myrmeleontinae). In addition to these uncertainties we noted that yChoromyrmeleon othneius has the oblique vein (the free part of Convergence of the two dating analyses was ensured by ESS val- MP2) located opposite the first fork of CuA, while it is located distal ues above 200 for all parameters (with many >1000) for the post to it in extant Myrmeleontini (the position of the free stem of MP2 burn-in trees and Tracer plots indicated that BEAST runs reached in yChoromyrmeleon aspoeckorum is not evident, but is probably convergence before the burn-in threshold. The choice of fossil cal- basal to the first fork of CuA). Moreover, in their hind wings, both ibrations dataset had no significant effect on age estimates (Fig. 3). species have the first fork of MP2 + CuA in a very distal position The dating analyses set with the birth-death model as branching (with respect to the forewing), while in extant Myrmeleontidae process prior showed that the parameter ‘relative extinction rate’ both wings are sub-equal in that respect. It cannot be excluded was estimated at 0.04 for the median (ranging from 0.001 to 0.18 that MP2 is not fused with CuA, in which case the placement of for the 95% HPD). The dating analyses also indicated that there these species should be deeply reconsidered. In summary the are no inferred age differences resulting from the inclusion or placement of yChoromyrmeleon spp. as stem of Myrmeleontidae exclusion of the fossil calibration at the stem of Myrmeleontidae raises a number of unresolved issues. Given that the (Fig. 3), suggesting that the use of yChoromyrmeleon spp. as a fossil yChoromyrmeleon-like Early Cretaceous species yBlittersdorfia pul- calibration is valid. cherina Martins-Neto and Vulcano is known from a single, isolated The dating analyses showed very similar divergence time esti- wing for which the base is incompletely described, the morphology mates across all nodes. For simplicity, we present the results of of its hind wing cannot be assessed, and its position with respect to the dating analysis relying on the full fossil dataset (four calibra- Myrmeleontidae is equally problematic. As a consequence, we per- tions, including the one at the stem of Myrmeleontidae) and the formed dating analyses with and without this fossil calibration to birth-death model as the tree prior (Fig. 3). We recovered a Late assess its reliability for calibration of Myrmeleontidae. Both speci- Permian origin (ca. 254.6 Myrs ago, 95% HPD 228.0–293.5 Myrs mens (yChoromyrmeleon othneius GMCB LB95013, and ago) of Myrmeleontiformia. Three of the families diversified in yChoromyrmeleon aspoeckorum CNU LB20003) were found in a Bar- the Early Cretaceous: Nemopteridae originated 141.1 Myrs ago remian lacustrine environment of the Yixian Formation. On the (95% HPD 112.6–178.1 Myrs ago), Ascalaphidae appeared 119.8 basis of the Barremian stage duration, which ranges from 129.4 Myrs ago (95% HPD 87.7–158.1 Myrs ago), and Myrmeleontidae to 125.0 Myrs ago, we tentatively used 125.0 Myrs as a minimum diversified 138.2 Myrs ago (95% HPD 114.9–170.8 Myrs ago). Nym- age for the stem age of Myrmeleontidae (see below for more phidae originated 79.1 Myrs ago (95% HPD 34.4–132.5 Myrs ago). details on the corresponding analysis). This age was also used as The time-calibrated trees resulting from the dating analyses are a conservative maximum age for the fossil calibration within presented in the appendices (Figs. S2 and S3), with nodal age and Myrmeleontidae (see below). It is worth mentioning that this fossil 95% HPD for each node. age is a good match with the molecular estimate obtained by Using the time-calibrated trees dated with three and four fossil Winterton et al. (2010) who found the divergence time of calibrations and the birth-death model as tree prior, LTT plots were Myrmeleontidae at 134 Myrs ago. However in this study, reconstructed (Fig. 4). The overall shape of the LTT plots shows a Ascalaphidae are paraphyletic making it difficult to place the stem rapid, early diversification of the group in the Early Cretaceous, age of Myrmeleontidae, and they calibrated yChoromyrmeleon as a corresponding to the divergence of extant subfamilies, which con- minimum age of 150.0 Myrs, which has now been revised to 125.0 tinues in the Late Cretaceous with the divergence of extant tribes. Myrs. Finally, it is also worth highlighting that no fossil species has ever been assigned to the following three subfamilies: Acantha- 4. Discussion clisinae, Palparinae and Stilbopteryginae. y In the subfamily Myrmeleontinae, the fossil species Porrerus The different classifications of Myrmeleontidae are mostly dominicanus was described based on a fossil adult (Poinar and based on adult morphological characters (particularly wing vena- Stange, 1996). A fossil larva from the same fossil formation was tion), and seldom rely on larval characters (e.g. Banks, 1911; further tentatively assigned to the same species by Engel and Navás, 1912; Markl, 1954; Stange and Miller, 1985, 1990; Stange, Grimaldi (2007); this fossil larva could be selected for calibration 1994; Badano and Pantaleoni, 2014). This study provides the first as it clearly belongs to the tribe Myrmeleontini based on the com- comprehensive phylogenetic study of Myrmeleontidae using DNA bination of elongate mandibles, elongate mandibular setae, sessile sequences. Although the resulting phylogenetic framework encom- mesothoracic spiracle, presence of submedian teeth on S8, and passes a fraction of the known antlions species diversity (91 spe- absence of bladelike digging setae on S9. However, immatures of cies), the main supra-generic subfamilies and tribes commonly myrmeleontines are relatively homogeneous and the characters recognized within Myrmeleontidae are represented, and we pro- separating the genera are presently not well defined, which pose that this work is an important step towards a better under- impedes a confident assignment to an extant genus (Engel and standing of antlion systematics and evolution. Given the limited Grimaldi, 2007). The holotype specimen (adult MACT-1220, number of species included, however this study provides little Poinar and Stange, 1996) and other specimens (adults: MACT- information relevant to the classification of Myrmeleontinae at 1170, MACT-1414, and MACT-3496; larva: MACT-1282; Engel the generic and specific levels, except for the fact that five genera and Grimaldi, 2007) were all found in the Miocene amber of the are recovered paraphyletic. Dominican Republic, which ranges from the Oligocene to the A long controversy has emerged regarding whether Stilboptery- mid-Miocene (Iturralde-Vinent and MacPhee, 1996; Poinar and ginae belong to Ascalaphidae, Myrmeleontidae or forms their own Poinar, 1999). In a conservative way, we used the minimum age family (Tillyard, 1916; Navás, 1921; Kimmins, 1940; Stange, 2004). of the mid-Miocene (i.e. 15.97 Myrs ago) to set a constraint for For instance, Aspöck et al. (1980) considered it a family including the crown age of the tribe Myrmeleontini. We choose to not the Australian species and the Neotropical genus Albardia Weele, y include Dendroleon septemmontanus as a potential calibration whereas New (1982), on the basis of genitalic features of both point because: (i) its assignation is questionable because of the sexes, considered Stilbopterygidae a subfamily of Myrmeleontidae fragmentary nature of the wing impression used by Statz (1936) restricted to Australia, and transferred the genus Albardia into to assign it to an extant myrmeleontid genus; and (ii) the author Ascalaphidae, erecting the subfamily Albardiinae. The results of did not provide any justification in the description of the fossil. the ML phylogenetic analyses support the hypothesis that this .Mce ta./MlclrPyoeeisadEouin17(07 103–116 (2017) 107 Evolution and Phylogenetics Molecular / al. et Michel B.
Fig. 3. Time-calibrated trees of Myrmeleontidae. (A) Chronogram corresponding to the Bayesian relaxed-clock analysis relying on three fossil calibrations. (B) Chronogram corresponding to the Bayesian relaxed-clock analysis relying on four fossil calibrations. For both chronograms grey-filled circles denote the subfamilial taxonomic rank within Myrmeleontidae, except for Stilbopteryginae (which has a single lineage here); the placement of fossils is also illustrated with colored circles and detailed in left box legend. The larva fossil of yPorrerus dominicanus is
portrayed in the bottom right. The last geological period, Q, is the Quaternary. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of 111 this article.) 112 B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116
Fig. 4. Lineages-through-time (LTT) plot for Myrmeleontidae (outgroups removed). (A) Accumulation of lineages over time as inferred with trees resulting from the Bayesian dating analysis relying on three fossil calibrations. (B) Accumulation of lineages over time as inferred with trees resulting from the Bayesian dating analysis relying on four fossil calibrations. The red curve indicates the maximum clade credibility tree with median ages, and the black curves represent the 95% confidence interval. The LTT shows the rapid early evolution of Myrmeleontidae during the Early Cretaceous. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
clade is sister to all remaining antlions, a placement compatible Our phylogeny strongly supports the monophyly of Palparini as with either a subfamilial status within Myrmeleontidae or a family defined by Markl (1954). The monophyly of this tribe corroborates following Aspöck et al. (1980), sister to Myrmeleontidae. Interest- the opinions of Mansell (1990, 1992) and Stange and Miller (1990). ingly, the same result was recently recovered by Badano et al. The subdivision of Palparinae into tribes is still discussed and sev- (2016), who carried out phylogenetic analyses of a morphological eral tribal arrangements have been proposed (e.g. Hölzel, 1972; dataset of Myrmeleontiformia. Here, for the sake of stability, we Stange and Miller, 1990; Stange, 2004). Given that no taxa from chose to follow the opinion of previous authors (e.g. Navás, the other tribes of Palparinae have been included here, our study 1921; Kimmins, 1940; New, 1982), and consider Stilbopteryginae does not provide information on the classification of the subfamily as a subfamily of Myrmeleontidae. at tribal level. The grouping of species, however, shows that the Acanthaclisids are well defined by both larval and adult mor- systematics of Palparini is unresolved and that the genus Palpares phological characters and constitute a homogeneous clade is clearly polyphyletic. This confirms the necessity of a revision (Markl, 1954; Hölzel, 1972; Stange and Miller, 1985, 1990; Insom of the genus and its separation into new genera (Insom and Carfì, and Carfì, 1992). Depending on authors, it is considered either a 1988; Mansell, 1992, 2004). tribe or a subfamily of Myrmeleontidae (New, 1985, 2003; All other species considered in this study belong to the subfam- Stange and Miller, 1990; Oswald and Penny, 1991; Stange, 2004). ily Myrmeleontinae. Concerning the classification of this subfamily Stange (1994) provided results supporting the relationship of our results provide some information related to the tribe ranking. Acanthaclisini sister to a clade comprised of Myrmecaelurini The Gepini are represented in our study by the genera Gepus + Nesoleontini, but our results are not congruent with this hypoth- Navás and Solter Navás, and appears to be a monophyletic group. esis. In our phylogenetic analyses, this clade is not recovered; These genera are commonly classified into the tribe Myrme- instead Acanthaclisini is sister to a large clade encompassing rep- caelurini whose generic limits are, however, unclear (Stange, resentatives of the subfamilies Myrmeleontinae and Palparinae. 2004). The tribe Gepini was erected by Markl (1954) for the genera The later supports the proposal of acanthaclisids as a monophyletic Furgella Markl, Gepus and Solter to which Hölzel (1969) added the subfamily of Myrmeleontidae. genus Gepella Hölzel. This tribe is now commonly included in the B. Michel et al. / Molecular Phylogenetics and Evolution 107 (2017) 103–116 113
Myrmecaelurini (Stange, 2004). Nevertheless, our results suggest a 5. Conclusion valid tribal status for Gepini that probably could be confirmed by adult morphological characters and genitalic features (Markl, The classification of Myrmeleontidae is still contentious and no 1953, 1954; Hölzel, 1968, 1969, 1983; Badano et al., 2014; consensus has yet arisen from examination and interpretation of Michel, 2014). morphological characters. Numerous subfamilial rankings have Stange and Miller (1990), on the basis of examination of larval been proposed, based mainly on wing venation, and more rarely characters, considered the sub-tribe Nesoleontina included in on genitalic features or larval characters, but, to date, no study using Myrmecaelurini. But the cladistic analysis performed by Stange DNA sequences has been carried out. This study provides the first (1994), taking into account adult and larval characters, confirmed comprehensive molecular phylogenetic study of Myrmeleontidae. the Nesoleontini as a sister group of Myrmecaelurini. Such a close Our results support the monophyly of Myrmeleontidae and its sub- relationship was proposed by Markl (1954). Although each tribe is division into four subfamilies, Acanthaclisinae, Myrmeleontinae, represented in our study by only two genera, Cueta Navás and Palparinae and Stilbopteryginae, as proposed by some authors on Nesoleon Banks (Nesoleontini), and Myrmecaelurus Costa and Lope- the basis of morphological characters. Even if the definition of the zus Navás (Myrmecaelurini), our result is congruent with the opin- tribes and their phylogenetic relationships remain unresolved, our ion of Stange (1994). study represents a valuable step towards the comprehension of The tribe Nemoleontini was created by Banks (1911) to antlion classification and evolution. Our study establishes a phylo- incorporate the genera Formicaleon Banks, Macronemurus Costa, genetic framework for future analyses on Myrmeleontidae; addi- and Nemoleon Navás plus the species Echthromyrmex fasciapennis tional sampling and information on species’ biology would be Banks, which is now assigned to the genus Palparidius Péringuey useful for tackling large-scale biogeographical patterns, or the evo- (Palparinae, Palparidiini). Hölzel (1987) considered the tribe lution of ecological traits such as pit building. Dendroleonini to include all the genera currently in the tribe Nemoleontini, except Echthromyrmex McLachlan. The tribe Acknowledgments Nemoleontini is the largest group in the Myrmeleontidae encom- passing 63 genera and 640 species (Stange, 2004). Although the We are grateful to the associate editor, Stephen Cameron, and to subtribal classification is not well established (Stange, 2004), the two anonymous reviewers for their constructive and insightful tribe appears to be monophyletic as shown also by Stange comments. We also thank Shaun Winterton for many interesting (1994). At the genus level, our analyses indicate that at least two comments and suggestions on an earlier version of this study. genera (Distoleon and Neuroleon) are paraphyletic, stressing the Financial support was provided by the INRA and by the program need for more studies in the future. ‘‘Bibliothèque du Vivant” (Project ‘Identification des COmmunautés Stange (1970) included the Brachynemurini in the subfamily de NÉvroptères des régions soudano-sahélienne’; ICONE) sup- Dendroleontinae, but noted doubts about this assignation. Later ported by a joint CNRS, INRA and MNHN consortium. Laboratory on, Stange (1994) split the Brachynemurini into three tribes, facilities were provided by the UMR CBGP in Montferrier-sur-Lez Brachynemurini, Gnopholeontini, and Lemolemini, and his results (France). The authors also thank A. Dehne Garcia for his help on indicated a close relationship between Dendroleontini and Gnop- the CBGP HPC computational facility. We thank Y. Wang (Capital holeontini. Although we had sequences for only one species of Normal University, Beijing) for providing photographs of the spec- Dendroleontini, Epacanthaclisis banksi Krivokhatsky, this species imen LB20003 (holotype of yChoromyrmeleon aspoeckorum). appears to be closely related to the Brachynemurini. More investi- gation, including more species, is definitely needed to assess the real relationships between these tribes. Appendix A. Supplementary material The tribe Myrmeleontini is well characterized by adult and lar- val morphological characters (Stange and Miller, 1990; Stange, Supplementary data associated with this article can be found, in 1994; Badano and Pantaleoni, 2014). In the cladograms obtained the online version, at http://dx.doi.org/10.1016/j.ympev.2016.10. by Stange (1994) this tribe was the sister group of all other 014. Myrmeleontinae including Acanthaclisini. Our result shows a close relation between Myrmeleontini and the clade comprising Brachynemurini + Dendroleontini, and is not congruent with the References result of Stange (1994). The genus Myrmeleon is also recovered Acevedo, F., Monserrat, V.J., Badano, D., 2013. Comparative description of larvae of polyphyletic, highlighting the need for more studies to clarify its the European species of Distoleon Banks: D. annulatus (Klug, 1834) and D. status. tetragrammicus (Fabricius, 1798) (Neuroptera, Myrmeleontidae). Zootaxa 3721, Our dating of the origin of Myrmeleontiformia to the 488–494. Archibald, J.M., Makarkin, V.N., Ansorge, J., 2009. New fossil species of Nymphidae Permian (ca. 254 Myrs ago, 95% HPD 228–293 Myrs ago) is con- (Neuroptera) from the Eocene of North America and Europe. Zootaxa 2157, 59–68. sistent with the estimation of Misof et al. (2014) who recovered Aspöck, H., Aspöck, U., Hözel, H., 1980. Die Neuropteren Europas, vol. I. Goecke and an early Permian to Early Triassic origin for Neuropterida. Evers, Krefeld, Germany. Aspöck, U., 1992. Crucial points in the phylogeny of the Neuroptera (Insecta). In: Interestingly, our age estimates for Myrmeleontidae to the Early Canard, M., Aspöck, H., Mansell, M.W. (Eds.), Current Research in Cretaceous (ca. 138 Myrs ago; 95% HPD 115–171 Myrs ago) Neuropterology. Proceedings of the Fourth International Symposium on predate the extant antlion fossil record by about 20 Myrs, which Neuropterology. Bagnères-de-Luchon, France, Toulouse, France, pp. 63–73. suggests that older antlion fossils are likely to be discovered in Aspöck, U., 1993. Geklärtes und ungeklärtes im system der Neuroptera (Insecta: Holometabola). Mitt. Dtsch. Ges. Allg. Angew. Entomol. 8, 451–456. the coming years. Our age estimates also indicate that major Aspöck, U., 2002. Phylogeny of the Neuropterida (Insecta: Holometabola). Zool. Scr. antlions lineages (subfamilies) originated in the Early Creta- 31, 51–55. ceous, and the age of most tribes (for which we have sampled Aspöck, U., Aspöck, H., 2008. Phylogenetic relevance of the genital sclerites of Neuropterida (Insecta: Holometabola). Syst. Entomol. 33, 97–127. more than one species) predates the K-Pg boundary (Fig. 4). Aspöck, U., Haring, E., Aspöck, H., 2012. 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