Molecular Phylogenetics and Evolution 43 (2007) 808–832 www.elsevier.com/locate/ympev

Phylogeny and Bayesian divergence time estimations of small-headed Xies (Diptera: ) using multiple molecular markers

Shaun L. Winterton a,¤, Brian M. Wiegmann a, Evert I. Schlinger b

a Department of Entomology, North Carolina State University, Raleigh, NC, USA b The World Parasitoid Laboratory, Santa Ynez, CA, USA

Received 16 May 2006; revised 19 July 2006; accepted 13 August 2006 Available online 24 August 2006

Abstract

The Wrst formal analysis of phylogenetic relationships among small-headed Xies (Acroceridae) is presented based on DNA sequence data from two ribosomal (16S and 28S) and two protein-encoding genes: carbomoylphosphate synthase (CPS) domain of CAD (i.e., rudi- mentary locus) and cytochrome oxidase I (COI). DNA sequences from 40 species in 22 genera of Acroceridae (representing all three sub- families) were compared with outgroup exemplars from , , Tabanidae, and . Parsimony and Bayesian simultaneous analyses of the full data set recover a well-resolved and strongly supported hypothesis of phylogenetic relation- ships for major lineages within the family. Molecular evidence supports the monophyly of traditionally recognised subfamilies Philopoti- nae and Panopinae, but Acrocerinae are polyphyletic. Panopinae, sometimes considered “primitive” based on morphology and host-use, are always placed in a more derived position in the current study. Furthermore, these data support emerging morphological evidence that the type Acrocera Meigen, and its sister genus Sphaerops, are atypical acrocerids, comprising a sister lineage to all other Acroceri- dae. Based on the phylogeny generated in the simultaneous analysis, historical divergence times were estimated using Bayesian methodol- ogy constrained with fossil data. These estimates indicate Acroceridae likely evolved during the late but did not diversify greatly until the . © 2006 Elsevier Inc. All rights reserved.

Keywords: Parasitoid; Diptera; Phylogeny; Bayesian; Spider; Divergence times

1. Introduction the only host-restricted endoparasitoids of (Sch- linger, 1987). Spiders (Araneae) are parasitized by a variety of ecto- Of more than 150 families of Diptera, 21 contain parasit- parasitic and endoparasitic nematodes and . Most oid species (Eggleton and Belshaw, 1992). With an esti- are parasitoids, i.e., organisms that during development, mated 16,000 described species (or 16% of ordinal diversity) live in or on the body of a single host individual, usually as parasitoids it is hypothesized that this life history has killing it ultimately. Spider ectoparasites include ichne- evolved over 100 times throughout the order (Eggleton and umonid wasps, e.g., Hymenoepimecis Viereck, Trychosis Belshaw, 1992; reviewed by Feener and Brown, 1997). Förster (Eberhard, 2000; Morse, 1994), whilst endopara- Whilst some families contain only individual sub-clades of sites include merminthid nematodes (Poinar, 1987), bris- parasitoids (e.g., and ), other Xy tle-Xies () (Vincent, 1985), and small-headed families have become completely specialized as parasitoids Xies (Acroceridae) (Schlinger, 1981). Acrocerid Xies are (e.g., , Tachinidae, , and Conopi- dae). Acroceridae and their hypothesized sister group,

* Nemestrinidae, are entirely parasitoids; Acroceridae are Corresponding author. Present address: California Department of host-restricted parasitoids of spiders, while Nemestrinidae Food and Agriculture, California State Collection of , Sacramento, CA, USA. Fax: +1 916 262 1190. are parasitoids of beetles and grasshoppers. Both families E-mail address: [email protected] (S.L. Winterton). constitute the , a poorly deWned group of

1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.08.015 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 809 lower whose monophyly has not been unequiv- spiracle to breath outside air (Schlinger, 1981, 1987; Nart- ocally determined (Woodley, 1989; Yeates, 2002). The sis- shuk, 1997). Once attached, the Wrst instar may enter a state ter-group relationship of Acroceridae and Nemestrinidae is of diapause for several months (Acrocerinae) to 10 years based on the shared parasitoid lifestyle of the larva. Mono- (Panopinae) (Schlinger, 1987). Upon cessation of diapause, phyly of Acroceridae has never been doubted and is based the active feeding larva completes its life cycle relatively on autapomorphic characters such as an antennal Xagellum quickly (days to weeks), undergoing four instars (Schlinger, composed of a single segment, enlarged lower calypter and 1987). The larva typically consumes the entire contents of obligate parasitoid life history on spiders (Woodley, 1989). the host spider, leaving an empty, unbroken exoskeleton Adult Acroceridae are distinctive Xies, either with small before emerging to pupate (Nartshuk, 1997). heads and inXated bodies, or slender, hunch-backed bodies There are presently more than 500 described species of where the head may be close to the abdomen (Fig. 1) (Sch- acrocerids worldwide, divided into about 50 genera linger, 1987). They may be covered with dense setal pile or amongst three subfamilies: Panopinae, Acrocerinae, and smooth and shiny, sometimes with metallic coloration. Philopotinae (Schlinger, 2003). Schlinger (1981, 1987, Adults of some species mimic bees or wasps and are often 2003) considered Panopinae to be the most “primitive” specialised Xower pollinators with long mouthparts (e.g., subfamily, Acrocerinae the most “derived” and Philopoti- Wiedemann), although some species have no mouth- nae an intermediate between the two. Host records for parts (e.g., Turbopsebius Schlinger) (Schlinger, 1981, 1987). Acrocerids parasitizing spiders are known for at least 61 Larval acrocerids are specialized internal parasitoids of species, recorded from 23 of over 100 spider families (Sch- juvenile spiders. Adult females lay large numbers of micro- linger, 2003). Panopinae attack mygalomorph spiders and type eggs, either scattered during Xight or deposited on are recorded from families Ctenizidae, , branches and foliage (Schlinger, 1987). First instar acroce- Migidae, Dipluridae, and Theraphosidae (Schlinger, rid larvae (i.e., maggots) are free-living planidia, which 1987). Acrocerinae and Philopotinae only attack aran- actively seek out a spider host by crawling, looping or eomorph spiders (Schlinger, 1987, 2003). Most rearing jumping, often with the aid of well-developed setae, spines, records for these latter two subfamilies are for the genera and a caudal suction disk (Nartshuk, 1997). Larval Acro- Gray, Acrocera, and Latreille (Acro- cera Meigen are distinct from other Acroceridae by having cerinae), which have particularly broad host ranges across reduced setae and vestiture and a diVerent mode of locomo- multiple spider families (Schlinger, 2003). Various Philo- tion (Schlinger, 1981). Larvae of most species also quest, potinae genera have been reared from Amaurobiidae, waiting for a host spider to pass, whereby the minute larvae Miturgidae, Phyxelididae, and Salticidae spider hosts board the spider (Schlinger, 1981). In the case of Ogcodes (Schlinger, 2003). adaptatus (Schlinger), the boarding larva only moves when Acrocerids are rarely collected and are represented in the spider is moving, so not to alert the spider to its pres- most collections by few specimens. Over the last few years, ence (Schlinger, 1960c, 1987). Larval acrocerids enter the dedicated collecting and rearing of acrocerids by EIS has spider directly through the cuticle of the cephalothorax, yielded a wealth of new ecological and biological informa- opisthsoma or leg joint. All larvae eventually locate in the tion, including numerous undescribed species. Based opisthsoma and attach to the book lung via the posterior largely on this new material, we present the Wrst compre- hensive quantitative analysis of Acroceridae phylogenetic relationships based on DNA sequence data. Nucleotide evidence from multiple independent gene regions is used to test and further deWne the composition and monophyly of the three subfamilies of Acroceridae. Philopotinae are well deWned morphologically by their bizarre hunch-back body shape and with postpronotal lobes strongly devel- oped into an anterior shield or collar on the anterior part of the thorax. However, the key character diVerentiating Panopinae from Acrocerinae is the presence of an apical tibial spur in Panopinae (Schlinger, 1981). This character applies to most genera except Pterodontia. Pterodontia has been placed in Panopinae by Schlinger (1981, 1987) and Nartshuk (1997), but in Acrocerinae by other authors as it lacks tibial spurs (Cole, 1919; Nartshuk, 1988; Bar- raclough, 1991). Acrocera the type genus of Acrocerinae, has unique larval morphology and behavior compared with other genera of the family (Schlinger, 1981). The phylogenetic position of Acrocera and Pterodontia rela- Fig. 1. Dimacrocolus pauliani Schlinger (Acroceridae: Philotopinae), adult tive to other Acroceridae are discussed in light of the habitus. Original painting by Jenny Spreckles. Acroceridae phylogeny and historical divergence times 810 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 presented herein. Our new sequence-based phylogenetic homogenization protocol of Chirgwin et al. (1979) as modi- hypothesis is intended both to Wnd corroborative support Wed by Cho et al. (1995) or the SDS/proteinase K-based for the traditional morphology-based classiWcation and to protocol of Moulton and Wiegmann (2004). The latter pro- provide new evidence on the position of enigmatic acroce- tocol involves samples being homogenised in lysis buVer rid taxa (Schlinger, 1960a, 1981, 1987, 2003; Nartshuk, and proteinase K solution and heated at 55 °C for 6–24 h. 1997). Moreover, this hypothesis serves as a template to Extraction of DNA was accomplished with phenol/chloro- estimate times of origin and diversiWcation of major form/isoamylalcohol mixture (25:24:1), followed by chloro- Acroceridae clades in a geological time frame. While the form/isoamyl alcohol mixture (24:1). DNA precipitation divergence time of Acroceridae was established by Wieg- was done using 3 M sodium acetate and chilled (¡20 °C) mann et al. (2003) at approximately 225–175 MYA, the isopropanol and microcentrifuged. Seventy to 90% EtOH chronology of divergence patterns in Acroceridae is was used to wash the DNA pellet, then air-dried, and resus- unknown and is explored further herein. pended in 50–150 l of TE buVer. Genomic extractions were stored at ¡80 °C. 2. Materials and methods Four genes were targeted for ampliWcation and sequencing: two ribosomal genes, 16S rDNA and 28S 2.1. Taxon sampling rDNA and two protein-encoding genes, cytochrome oxi- dase I (COI) and the CPSase region of CAD. 16S and COI Exemplars were obtained for 40 species of Acroceridae are mitochondrial genes while 28S and CAD are nuclear from 22 genera representing all three subfamilies (Table 1). genes. Both 16S and 28S ribosomal genes and COI have Eight genera (16 species) of Panopinae were sampled, most been widely used in molecular systematics (see from the New World, along with a single Palaearctic genus Simon et al., 1994; Brower and DeSalle, 1994; Caterino (Astomella Lamarck) and one from New Zealand ( et al., 2000), especially in Diptera (Winterton, 2002), but Westwood). Seven genera (10 species) of Philopotinae were CAD has only recently been identiWed as useful for recov- sampled including Wiedemann, four species of ering insect phylogeny (Moulton and Wiegmann, 2004; the metallic colored genus Thyllis Erichson from Madagas- Danforth et al., 2006; Winterton and deFreitas, 2006). car, Terphis Erichson from Costa Rica and a species repre- Carbamoyl-phosphate synthetase-aspartate transcarba- senting an undescribed genus from New Caledonia. Seven moylase-dihydroorotase (CAD) is a large multifunctional genera (14 species) of Acrocerinae were sampled, including protein with carbamoyl phosphate synthetase (CPSase), four species each from the species-rich and widely distrib- aspartate transcarbamoylase and dihydroorotase active uted genera Acrocera and Ogcodes. A single species also sites, catalysing the Wrst three steps of de novo pyrimidine was included from Tasmania of the enigmatic and cosmo- biosynthesis (Freund and Jarry, 1987). CAD is a large politan Pterodontia. gene complex (6.6+ kb) that exhibits both conservation Outgroups were selected from three of the main lower and signiWcant variability in coding regions, along with brachyceran infraorders: Dialysis Walker (Xylophagomor- relatively few unpredictable introns in Diptera, make it a pha: Xylophagidae), Tabanus L. (: Tabanidae) promising candidate for investigating phylogenetic rela- and two species of Stratiomyidae (Stratiomyomorpha), tionships of holometabolous insects at multiple taxo- Actina Meigen and Antissa Walker. Since the two Stratiomyi- nomic levels (Moulton and Wiegmann, 2004). Moulton dae species were sequenced without overlap for diVerent and Wiegmann (2004) used the CPSase region of CAD to genes, they were combined as a single chimeric ‘stratiomyid’ investigate eremoneuran Diptera relationships, recover- gene sequence. Acroceridae has long been closely associated ing a well resolved and well supported phylogeny concor- with Nemestrinidae in the superfamily Nemestrinoidea (sum- dant with previous estimates using morphology and other marized by Yeates and Wiegmann, 2005). Four species of frequently used genes. Nemestrinidae were selected: Trichophthalma Westwood Primer sequences used to amplify and sequence the four from Australia, Nemestrinus Latreille from Israel, and two sampled gene regions are presented in Table 2. The fully species of Hirmoneura Meigen from Chile. concatenated dataset comprised ca. 5.5 kb of unaligned gene sequence (CAD: 850 bp; COI: 1.5 kb; 16S: 500 bp; 28S: 2.2. DNA extraction, gene ampliWcation, and sequencing 2.5–2.8 kb). DNA ampliWcations using PCR were performed using the following cycling parameters. CAD Adult specimens were collected by hand netting or Mal- (fragment 1, sensu Moulton and Wiegmann, 2004) was aise traps or reared from Weld collected juvenile spiders. generated with a single primer pair using a touchdown Specimens were subsequently placed into 95–100% EtOH protocol: initial denaturation at 94 °C (4 min); Wve cycles of and stored at ¡80 °C. Genomic DNA was extracted from 94 °C (30 s), 54 °C (30 s.) and 72 °C (1 min 30 s); 37 cycles of whole (small) specimens or thoracic muscle tissue from 94 °C (30 s), 51 °C (30 s) and 72 °C (1 min 30 s); 72 °C (3 min) large specimens. Voucher specimens are deposited in the for Wnal annealing. A ca. 500 bp fragment of 16S rDNA (3Ј- North Carolina State University insect collection and Cali- end) was generated using a single primer pair with one fornia Academy of Sciences collection. Extractions were primer modiWed from Simon et al. (1994) with the following carried out using either the guanidinium isothiocyanate PCR protocol: initial denaturation 92 °C (3 min); Wve cycles S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 811 ) g continued on next page ( ngham; May 1997; B.M. Wiegmann B.M. 1997; May ngham; Y Irwin January, 2000; I. Jimenez July, 2000; K.C. Holston Schlinger E.I. Irwin, Schlinger E.I. Irwin, Irwin, E.I. Schlinger E.I. Irwin, Schlinger E.I. 1999; D. Defoe 1999; M.E. Irwin M.E. Cooloola Section; October, 1996; D.K. Yeates D.K. 1996; October, Section; Cooloola Wiegmann Wiegmann Wiegmann Schlinger E.I. Irwin, DQ631978 Spain: Almeria Prov.:Almerimar; 1999; April,M.E. Irwin AY144441 16S rDNA CPSase 28S rDNA COI NCSU#99-07-09-76 AY140854 AF539867 AY144385 — USA: California: SanDiego; June 2000; S.L. Winterton NCSU#95-05-11-41 AY140848 AF539871 AY144409 DQ631995 ISRAEL:Arava Valley:Shiraf NatureReserve; March, 1995;

Phillipi EIS#010944 AY140886 — AY144438 DQ631977 Chile: Curico Prov.:5 kmE Curico; March, 1998;Barriga J.E. Brimley EIS#009955 AY140853 AF539887 AY144397 DQ631979 USA: North Carolina: GreatSmoky Mountains N.P.; June, (Phillippi) EIS#010914 AY140856 AF539865 AY144391 DQ631969 Chile: NubleProv.: Las Trancas; January, 2000; D.K. Yeates Walker NCSU#99-07-21-54 AY140887 AF539895 AF238513, AF238537, AF238561 DQ631993 USA: Georgia: E Schlinger EIS#010916 AY140889 AF539881 AY144399 — Chile: Quillota Prov.: Palma de Ocoa, January, 2001; M.E. Irwin, (Say) NCSU#99-07-21-46 AY140888 AF539869 AF238505, AF238527, AF238551 DQ631989 USA: North Carolina: nearLinville; May, 1996; L. Yan Westwood EIS#009956 AY140851 AF539866 AY144400, AY144401 — New Zealand: Otira Valley;January, 1999; L.J. Boutin sp. NCSU#98-02-19-39 AY140849 AF539868 AY144383 DQ631994 AUSTRALIA: Queensland: Great Sandy National Park, vittatus (Say) NCSU#99-07-21-56 — — AY144382 — USA: North Carolina: GreatSmoky N.P.: May 1994; B.M. sp. 1 NCSU#98-02-11-36 AY140850 AF539899 AY144410 DQ631988 CHILE: Quillota Prov.: Palma de Ocoa; November, 1997; B.M. sp. 2 N/A DQ537936 DQ537938 DQ537937 DQ631999 CHILE:Quillota Prov.: Palmade Ocoa, November,1997; M.E. nr. sp. EIS#011191 AY140885 AF539882 AY144398 — Chile: QuillotaProv.: Palma de Ocoa, December, 1999; M.E. sp.1 NCSU#99-07-21-48 AY140852 AF539877 AY144396 — Forest; National Wenatachee Fork: South Washington; USA: sp. EIS#009964 AY140859 AF539901 AY144439, AY144440, sp.2 EIS#007416 — AF539904 — — Bolivia: Sana CruzProv.:3 km Brasilia; N March, 1999; M.E. sp.1 EIS#011194 AY140879 AF539898 AY144434, AY144435 DQ632000 Costa Rica: Guanacasta, Prov.: Palo VerdeFila Catalenia; sp. NCSU#99-07-21-55 AY140891 AF539870 — DQ631992 USA: Pennsylvania:Alleghaney State Forest:August B.M. 1994; sp.1 sp.2 sp.3 EIS#007222 NCSU#99-07-21-49 AY140855 AY140857 AF539890 EIS#010940 AF539879 AY144394, AY144395 AY144392, AY144393 AY140858 AF539863 — AY144390 — Chile: QuillotaProv.: Palma de Ocoa, November, 1997; M.E. Chile: QuillotaProv.: Palma de Ocoa, November, 1997; M.E. — Chile: Curico Prov.:15 km E Curico; March,1998;J.E. Barria Ocnaea Lasia Lasia carbonanicus Lasia Lasia Ocnaea Arrhynchus penai Arrhynchus Astomella Archipialea Apsona muscaria Eulonchus marialiciae Eulonchus smaragdinus Nemestrinus aegyptiacus Hirmoneura Antissa Hirmoneura Trichophthalma Tabanus rufofrater Actina viridis Dialysis elongata Gerstaecker PANOPINAE Wiedemann ACROCERIDAE NEMESTRINIDAE TABANIDAE STRATIOMYIDAE XYLOPHAGIDAE Table 1 Table study in used this Exemplars Exemplar EIS#/NCSU#No. Accession Genbank data Collection 812 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 ereville; January, V pe: 37Willowmore; NW km Cape: 37Willowmore; NW km ca: Eastern Cape: 43 NE Willowmore; km public South Africa: Eastern Ca May 1998;Azofeifa. A. Tp.Foso. Blakemore, L.J. Boutin Schlinger, G. Barriga H.M. Yan, D. Kavanaugh, C.E. Griswald, H.B. Liang, D. Ubick, D. Ubick, Liang, H.B. Griswald, C.E. Kavanaugh, D. Yan, H.M. D.Z. Dong Webb March, 2001;Azofeifa J.A. Republic South Africa: Eastern November, 1999; M. Hauser Irwin November, 1999; M.E. Irwin,E.I.Schlinger, F.D. Parker Schlinger E.I. Irwin, 2001; M.E. Irwin, E.I. Schlinger, Rin’ha, Mananjara; January, 2000; Rin’ha Boutin L.J. and November, 1999;Parker F. Skevington Blakemore and L.J. Boutin D. Ubick, Liang, H.B. Griswald, C.E. Kavanaugh, D. Yan, H.M. D.Z. Dong 31996 Republic South Afri DQ631973 R. 1999; January, Pass; Arthurs Island: South Zealand: New DQ631974 Chile:Quebrada de la Plata; November, 1997; M.E. Irwin,E.I. DQ631976 Australia: Tasmania: 2.4Gladstone; km S December, 1998; J.A. DQ631991 Australia: Queensland:Fernvale; October,S.L. 1999; Winterton AY144432, AY144433 AY144429 AY144419 AY144422 9873 AY144386, AY144387 DQ631981 Re 16S rDNA CPSase 28S rDNA COI EIS#009960 AY140874 AF539885 AY144427, AY144428, EIS#010917 AY140870 AF539876 AY144415 DQ631990 Madagascar: Ranamofana N.P.; December, 1999; E.I. Schlinger EIS#011170 AY140881 AF539888 AY144402 — New Caledonia: Mt. Nihgua; November, 2000; E.I. Schlinger EIS#010885 AY140868 AF539892 AY144388 DQ631985 China: Yunnan Prov.: Nujiang State Nature Reserve; July, 2000;

Schlinger EIS#011171 AY140873 AF539903 AY144407, AY144408 — Madagascar: Diego-Suarez Prov.: 7 km N. Jo Schlinger EIS#010897 AY140890 AF539889 AY144416 DQ631975 USA:Kansas: Montgomery Co.; July, 1997; H. Guarisco pictus Schlinger EIS#010962 AY140865 AF539891 — — New Zealand: South Island: ArthursPass, January,R. 1999; (Fabricius) EIS#010945 AY140863 AF539893 AY144414 DQ631983 Spain: CuidadProv.: SierraSanta de Maria; April, 1999; M.E. (Hudson) EIS#009959 AY140869 AF539884 AY144430, AY144431, mbriatus Brunetti EIS#010918 AY140872 AF539872 AY144406 DQ631984 Madagascar: Bellevue, December, 1999; M.E.Irwin Westwood EIS#010952 AY140860 AF53 W (Walker) NCSU#99-07-21-50 AY140867 AF539886 AY144420, AY144421, Westwood NCSU#96-12-02-25 — — AY144411 — USA: California: Santa Ynez; E.I. Schlinger ) (Fabricius) EIS#010947 AY140871 AF539897 AY144423, AY144424 DQ631997 nis Y sp. (nr. sp. NCSU#99-07-21-52 AY140875 AF539906 AY144425, AY144426 — Guatemala: EsquintlaPalin; nr. May 1997; S.L. Winterton, D. sp.1 EIS#010884 AY140862 AF539905 AY144413 DQ631982 China: Yunnan Prov.: Nujiang State Nature Reserve; July, 2000; sp.2 EIS#010948 AY140864 AF539894 — DQ6 sp. NCSU#99-07-21-51 AY140866 AF539900 AY144417, AY144418, sp.3 EIS#011195 AY140878 AF539864 — DQ631972 Costa Rica: Prov. Puntarenas, Est. Agujas. Send. Zamia. 300m. 3 sp. EIS#011197 AY140880 AF539878 AY144389 DQ631980 Costa Rica: PuntarenasProv.: 6 km Cerro S Rincon; February– sp. EIS#007220 AY140861 AF539902 AY144412 DQ631971 Chile:Quillota Prov.: Palmade Ocoa, November1997; M.E. sp.1 EIS#011172 AY140882 AF539896 AY144405 — Madagascar: FianarantsoaProv.: 30 km SE Fandriana continued longirostris Megalybus Ocnaea Phillippi) Parahelle stuckenburgi Acrocera orbicula Acrocera Philopota Terphis Thyllis crassa Thyllis philopotoides Acrocera Acrocera bulla Holops Ogcodes Thyllis Thyllis splendens (New 1 Genus Undescribed Caledonia) (Hildebrandt) Ogcodes leptisoma Ogcodes basalis Psilodera a Ogcodes canadensis albo Paracyrtus PHILOPOTINAE Schlinger ACROCERINAE Table 1 ( Exemplar EIS#/NCSU# No. Accession Genbank data Collection S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 813

of 92 °C (15 s), 48 °C (45 s), 62 °C (2 min 30 s); 29 cycles of 92 °C (15 s), 52 °C (45 s), 62 °C (2 min 30 s); Wnal annealing at 62 °C for 7 min. A ca. 2.5 kb section near the 3Ј-end of 28S rDNA was generated using primers from Hamby et al. (1988) modiWed according to the published sequence of Drosophila melanogaster Meigen (Hancock et al., 1988; Yang et al., 2000). Three initial PCR fragments were gener- ated: rc28C to 28E (fragment1), rc28D to 28K (fragment 2), and rc28Q to 28Z (fragment 3). Internal primers were used in cycle sequencing reactions for fragment 1 (rc28P and 28P) and fragment 2 (28H and rc28H). The ampliWcation protocol for 28S was a standard three step PCR (without a touchdown step): initial denaturation 95 °C (1 min); 30 cycles of 95 °C (1 min), 45 °C (1 min), 72 °C (2 min); Wnal annealing at 72 °C for 7 min. The entire length of COI DNA (ca. 1.5 kb) was ampliWed using two primer pairs from Simon et al. (1994): initial denaturation 95 °C (5 min); 33 cycles of 93 °C (20 s), 50 °C (40 s), 72 °C (2 min); Wnal December, 1998; D.K.Yeates Schlinger E.I. Irwin, Schlinger March–April, 2000; F.Umana annealing at 72 °C for 5 min. The primer TY-J-1460 begins

mens for ease of locating these specimens in NCSU and California and NCSU in specimens these locating of ease for mens at tRNA-tyrosine and ampliWes into COI while TL2-N- 3014 begins in tRNA-Leucine in the complementary direction. All PCR products were run on a 1% agar electro- phoresis gel and speciWc bands excised prior to puriWcation using QIAquick gel extraction kits (Qiagen Sciences, Mary- land, USA). Sequences were obtained using ABI d-Rhodamine Ter- minator Cycle Sequencing Ready Reaction with Amp- litaq® DNA Polymerase FS (PE Applied Biosystems, Foster City, CA, USA). To all CAD sequencing reactions 2 l of DMSO (dimethyl sulphoxide) was added to improve the sequencing product (Moulton and Wiegmann, 2004). Sequences were gel fractionated and based identiWed on an ABI Prism™ 377 DNA sequencer (PE Applied Biosys- tems). Sequencing electropherograms were edited and contigs assembled and proofed using Sequencher™ 4.1 (GeneCodes Corp., Michigan, USA). While the number of taxa sequenced for all genes in this study is extensive, there were a few instances where taxa could not be successfully ampliWed and sequenced for at least one gene (Table 1).

2.3. Alignment and sequence characteristics

A fundamental part of the use of sequence data in sys- tematics is the statement of homology explicitly hypothe- sized in any alignment (Buckley et al., 2000). Since both ribosomal and protein-encoding sequences were obtained, EIS#010925EIS#003117 AY140877 AF539875 AY144403, AY144404 AY140884 AF539874 AY144384 DQ631987 Chile:Quillota Prov.: Palmade Ocoa, February, 2000; M.E. DQ631998 USA:California: Santa BarbaraCo.;October, 1997; E.I. alignment of sequences was idiosyncratic, capitalizing on the relative strengths and weaknesses of manual and auto-

mated alignment protocols. We used both automated and (Osten manual alignment procedures to obtain estimates of ribo- somal and protein-encoding gene alignment to improve our

sp. EIS#011196 AY140883 AF539880 —ability DQ631986 Costa Rica: LimonProv.: Res. Biol Hitoy CerereSend. Esparel; to more accurately recover homologous phyloge- sp. EIS#009957 AY140876 AF539883 AY144436, AY144437 DQ631970 Australia: Tasmania: Chauncyvale Wildlife Sanctuary; netic signal.

2.3.1. Ribosomal genes Pterodontia Sphaerops appendiculata Turbopsebius diligens Sacken) Turbopsebius Philippi Alignment of 16S rDNA sequences was performed using EIS (E.I. Schlinger Collection)and NCSU (North Carolina State University collection) numbers identify individual voucher speci Academy of Science insectcollections (future depository EIS of collection). ClustalX (ver. 1.8) (Thompson et al., 1997). Rather than 814 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832

Table 2 Primer sequences used to amplify genes examined in this study Gene Primer code Sequence (5Ј end) Reference CAD 54F GTN GTN TTY CAR ACN GGN ATG GT Moulton and Wiegmann (2004) 405R GCN GTR TGY TCN GGR TGR AAY TG COI TY-J-1460 TAC AAT TTA TCG CCT AAA CTT CAG CC Simon et al. (1994) C1-N-2191 CCC GGT AAA ATT AAA ATA TAA ACT TC C1-J-2183 CAA CAT TTA TTT TGA TTT TTT GG TL2-N-3014 TCC ATT GCA CTA ATC TGC CAT ATT A 16S LR-J-12887 CCG GTT TGA ACT CAG ATC ATG T Simon et al. (1994) SR-N-13398b CRC YTG TTT AWC AAA AAC AT 28S rc28C CCG AAG TTT CCC TCA GGA TAG C Hancock et al. (1988) 28P GGC TTA CGC CAA ACA CTT CTA CGC rc28P TGG TAT GCG TAG AAG TGT TTG GC 28E CCT TAT CCC GAA GTT ACG rc28D CCG CAG CTG GTC TCC AAG 28H GGT TTC GCT GGA TAG TAG rc28H CTA CTA TCC AGC GAA ACC 28K GAA GAG CCG ACA TCG AAG rc28Q GGA CAT TGC CAG GTA GGG AGT T 28Z GCA AAG GAT AAG CTT CGA TGG using the default values for gap insertion and gap extension gap extension parameters can have a signiWcant, and poten- penalties, a two-stage alignment procedure was used for tially biased, inXuence on alignment (Morrison and Ellis, 16S using secondary structure and an elision matrix. Use of 1997; Hickson et al., 2000). To address this issue experimen- secondary structure to guide ribosomal alignment has been tally within our data, we used an elision matrix (sensu shown to improve the accuracy of both manual and auto- Wheeler et al., 1995) as an a posteriori weighting scheme for mated sequence alignment (Kjer, 1995, 2004; Titus and multiple alignments generated by varying the gap opening/ Frost, 1996; Hickson et al., 1996, 2000; Mugridge et al., gap extension penalties in ClustalX. Multiple alignments 2000; Xia et al., 2003). A secondary structure model for were generated using the ‘slow/accurate’ option in ClustalX the 3Ј-end (Domain V) of the 16S rDNA molecule was to calculate distances between sequences and then arbi- constructed for Acrocera sp. 1 (GenBank Accession: trarily varying the gap opening/gap extension penalties as AY140864; EIS#10948) (Fig. 2) based comparatively on follows: 15/10, 20/4, 15/8, 20/10, 24/4, 15/6, 15/12, and 12/4. published models of Delphinia picta (Fabricius) () These parameters generated alignments that ranged from by Buckley et al. (2000) and Drosophila melanogaster relatively open (many gaps inserted) (e.g., 12/4) to tightly () on the Gutell website (Cannone et al., compressed (relatively few gaps inserted) (e.g., 20/10). Simi- 2002). While there were many nucleotide diVerences (with lar results were found using this technique by Hedin and corresponding compensatory changes in stem regions) Maddison (2001) for jumping spider (Salticidae) 16S rDNA between the three Xy sequences, the secondary structure sequences. Because this empirical approach speciWes no a model for Domain V is relatively conserved between these priori criterion for choosing among alternative alignments, published models and that of Acrocera. Structural conser- each alignment is concatenated into a single combined vation of Domain V is concordant with previous studies on alignment, or elision matrix (Wheeler et al., 1995; Hedin secondary structure in other groups (e.g., spi- and Maddison, 2001). This matrix was analysed using ders: Hedin and Maddison, 2001; Smith and Bond, 2003; PAUP¤ 4.0b10 in a parsimony analysis (see details below) Lepidoptera, Cicadellidae, , Coleoptera, Hyme- and the resultant strict consensus cladogram compared noptera: Buckley et al., 2000; Odonata: Misof and Fleck, with strict consensus cladograms from analyses (with iden- 2003). One structural element, helix H2077, has been shown tical search parameters) of each of the individual align- by Misof and Fleck (2003) to be highly variable in structure ments. The replication of identically aligned sites within the and phylogenetically informative between Odonata fami- elision matrix serves as a de facto weighting function for lies. This is not the case in these dipteran families. The sec- sites that do not change their positional homology under ondary structure model for Acrocera sp. was used to diVering alignment parameters. Topological congruence construct a gap penalty mask for input into ClustalX dur- between elision and individual analyses was assessed by ing proWle alignment. The mask superimposes higher gap comparing the number of common nodes between consen- penalties in stem regions so that gaps are preferentially sus cladograms of the individual alignments with that of inserted in less conserved loop regions (Thompson et al., the elision matrix consensus cladogram. Nodes were scored 1997). as either “present” or “absent”. “Present” nodes were those Even with the imposition of secondary structure-based found in both consensus cladograms, while “absent” nodes gap penalties, application of global default gap insert and represented alternative topologies found for that clade in S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 815

Fig. 2. Acrocera sp. (Genbank Accession Number: AY140864): secondary structure model of the 3Ј end of mitochondrial large subunit (16S). Conserved stem regions indicated by blocks. 816 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 the individual alignments. Topological resolution and con- determined with a reasonable level of conWdence were iden- gruence under diVering parameters thus were used as crite- tiWed and the eVects of their inclusion or exclusion on sub- ria for choice among alternative alignments (Wheeler et al., sequent phylogenetic analyses of the combined data were 1995). examined. For 16S data, three alignments (i.e., 20/4, 15/6, and 15/ 12) had the lowest ratio of absent to present nodes (Table 2.4. Phylogenetic analyses and nodal support 3), although the alignment using the gap: extension penal- ties 20/4 had the highest cladogram resolution (i.e., fewest Parsimony and Bayesian likelihood analyses were con- polytomies) with 35 nodes compared with 25 and 29 nodes ducted using PAUP¤4.0b10 (SwoVord, 1999) and Mr Bayes for the other two alignments, respectively. This alignment 3.1 (Huelsenbeck and Ronquist, 2001; Ronquist and Huel- was chosen as the preferred alignment for the subsequent senbeck, 2003), respectively. The number of taxa dictated a phylogenetic analysis. Two other alignments (15/10 and 12/ heuristic tree searching protocol, using 20 replicate random 4) had greater cladogram resolution than 20/4, with 35 and addition sequences with tree bisection and reconnection 41 nodes, respectively, but both had a much greater number (TBR). Bootstrap support values (Felsenstein, 1985) for the of conXicting nodes with the elision matrix consensus clad- parsimony analyses were calculated from 10,000 heuristic ogram and were less suitable. search (TBR) pseudoreplicates of re-sampled data sets, The extensive hypervariability within 28S rDNA expan- each with 10 random additions. Bremer (or branch) sup- sion regions makes their alignment even more problematic. port values were determined using TreeRot ver. 2.0 (Soren- We conducted an initial automated alignment in ClustalX son, 1999). Modeltest 3.07 (Posada and Crandall, 1998) was using the default gap insertion: gap extension penalties and employed using the Bayesian Information Criterion (BIC) then these were manually adjusted in MacClade (ver. 4.06) to determine the best-Wt model of sequence evolution for (Maddison and Maddison, 2000). Stem and loop regions each individual gene. The GTR+I+G model was found to were identiWed using a 28S secondary structure model for be most applicable for 16S, 28S, and COI genes while the D. melanogaster by Hancock et al. (1988). Boundaries TVM+I+G model was most appropriate for CAD. Bayes- of variable length regions were delimited by identifying ian analyses where preformed using Mr Bayes 3.1 (Ron- relatively conserved stem and adjacent loop regions and quist and Huelsenbeck, 2003) employing a Monte Carlo determining the last position whose alignment preserves Markov Chain (MCMC) method with constant characters unambiguous adjacent positional homology. excluded and four MCMC chains run simultaneously for 5,000,000 generations. Trees were sampled every 100th gen- 2.3.2. Protein-encoding genes eration resulting in 50,000 trees. Two runs were run simul- Alignment of protein-encoding genes was done manu- taneously with identical trees generated from each analysis. ally with reference to the translated amino acid sequence. The MCMC chains achieved stationarity after 190,000 gen- Alignment of cytochrome oxidase I (COI), a mitochondrial erations, so the Wrst 1900 trees were discarded as the burn- transmembrane protein, was undertaken with reference to in phase for each analysis. Posterior probabilities were two-dimensional structural models previously published determined from majority-rule consensus trees generated for hexapods (Lunt et al., 1996; Hedin and Maddison, from the remaining saved trees. 2001) and using the Drosophila mitochondrial reading An incongruence test was used in the parsimony analy- frame in MacClade. DiVerent structural domains were ses to estimate the level of congruence between the four W identi ed, including conserved hydrophobic helix regions gene partitions. The partition-homogeneity test (HTF) and relatively less conserved hydrophilic amino acid strings (sensu Johnson and Soltis, 1998), which applies a signiW- where gaps may be preferentially inserted. Alignment of cance level to the ILD test (Farris et al., 1995) was imple- CAD was done manually using MacClade based on amino mented using PAUP¤4.0b10 with a minimum of 5000 acid translations (standard genetic code). replicate. Invariant characters were always excluded before For both ribosomal and protein-encoding genes, ambig- running the HTF test as the proportion of invariant charac- uously aligned regions where positional homology is not ters is greatly diVerent between the data partitions

Table 3 Nodal agreement comparison between the elision matrix and individual alignments for 16S rDNA sequence data Gap cost/extension cost Nodes present Nodes absent Total number nodes Ratio (present/absent) Overall sequence length (bp) 15/10 30 8 38 3.75 598 20/4¤ 32 3 35 10.67 612 15/8 22 3 25 7.33 607 20/10 29 10 39 2.90 599 24/4 27 12 39 2.25 602 15/6 24 1 25 24.00 604 15/12 27 2 29 13.50 603 12/4 27 14 41 1.93 614 Bold and asterisk row identiWes gap and extension penalties used in the selected 16S alignment. S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 817

(Cunningham, 1997a,b). The distribution of the HTF test est Diptera fossils (middle Triassic), and oldest dates for the statistic (P-value) is estimated by calculating the most par- group estimated by Wiegmann et al. (2003). The minimum simonious tree length for the original partition and then for age constraint for the root was set at 155 MYA based on replicates of randomized partitions of the same size as each ages of the oldest deWnitive fossils of Nemestrinidae (e.g., original partition. Removal of invariant sites is important Archinemestriinae) (Ansorge and Mostovski, 2000). Neme- because each randomized partition is composed of a ran- strinoidea were relatively diverse during the late dom mixture of characters from each original partition, and early Cretaceous, although most fossils are represented and thus it is possible for multiple replicate of the smaller by Nemestrinidae with only two Acroceridae fossils known partitions to be composed of large numbers of invariant from these periods (Mostovki and Martinez-Delclòs, 2000). characters, potentially resulting in substantially biased test results. To investigate the relative contribution of the data 3. Results partitions to the arrangement of individual nodes on the combined tree(s), Partitioned Bremer Support (PBS; Baker 3.1. Sequence details, nucleotide composition, and and DeSalle, 1997) values were determined using TreeRot substitution pattern ver. 2.0 and PAUP¤4.0b10 using 20 random addition sequences for each constraint tree, with tree bisection and The total aligned sequence length for this analysis was reconnection (TBR). 6099 base pairs (bp) (16S D 612 bp; 28S D 3093 bp; CAD D 893 bp; COI D 1498 bp), although this was reduced 2.5. Estimating dates of divergence to 5453 bp once ambiguously aligned sites were removed across all data partitions. Ribosomal gene fragments (16S Comparisons of gene sequence variation over time and 28S) have large proportions of hypervariable indels enable estimation of historical dates of divergence (clado- corresponding to loop regions in the secondary structure genesis) with or without the aid of dates provided from the model for each. There were almost no indels in the protein- fossil record. Traditional estimations of substitution rate encoding genes (CAD and COI) although there is a dele- based on a molecular clock (Zuckerkandl and Pauling, tion of two amino acids in CAD in Acrocera and Sphaerops 1965) have been shown to vary considerably between genes Philippi at nucleotide position 327. There were also amino (e.g., Lin and Danforth, 2004) and between taxa (e.g., Wu acid insertions at position 633 in Terphis (Leu) and Philo- and Li, 1985; Britten, 1986). Recent approaches for estimat- pota (Glu) (Philopotinae), and a neighboring variable ing divergence times use stochastic methods that do not amino acid insertion at position 636 for many acrocerine assume a constant rate of evolution and therefore account and philopotine acrocerids. for this rate heterogeneity between lineages (Sanderson, Typical of insect genomes in general, there is a distinct 1997, 2002; Thorne et al., 1998; Kishino et al., 2001; Thorne A/T bias in nucleotide proportions (A D 32.6%, C D 15.1%, and Kishino, 2002). The basic concept of this approach is G D 18.8%, and T D 33.4%) in the four gene fragments sam- that substitution rates tend to be more similar between pled here. Base frequencies for the entire dataset were heter- nearby branches on a tree than between more distant ogeneous across the 47 included taxa (2 D 495.965, branches (Wiegmann et al., 2003). We used a parametric, d.f. D 138, P D 0.000). Average base frequencies for CAD Bayesian approach for estimating divergence times among were A D 34.3%, C D 15.1%, G D 19.4%, and T D 31.1%, lineages using the programs estbranches and multidivtime although the A/T bias was considerably greater in third (Thorne and Kishino, 2002). An assumed topology, onto codon positions (79.8%) than in either Wrst (55.3%) or sec- which sequence divergence rates are estimated, was sourced ond (60.5%) codon positions (Fig. 3A). A similar pattern of from a tree generated in this study (see Fig. 5) as the best nucleotide proportions was found in COI (A D 30.9%, hypothesis of acrocerid evolution based on quantitative C D 16.9%, G D 14.3%, and T D 37.8%), with even greater A/ data. Divergence time estimates were bounded extraneously T bias in third codon positions (88.8%). While both 16S and using dates from the fossil record. The oldest deWnitive fos- 28S genes show strong A/T bias across all sites (Fig. 3B), sil known for an acrocerid or nemestrinid lineage was used this bias was more pronounced in loop regions (91.8% and to generate minimum age constraints for particular nodes. 82.3%, respectively) as they are free of constraints of com- Four minimum age constraints were used, representing ages pensatory base pair changes characteristic of stem regions. of the oldest known fossils for that group, including The loop regions of 16S had only 0.37% C present. 130 MYA for Hirmoneura (Nemestrinidae) (Hirmoneura Transversions are more common than transitions in all richterae Mostovski and Martinez-Delclòs (Lower Barre- genes, as is common in groups with strong A/T bias in their mian)) (Mostovki and Martinez-Delclòs, 2000), 45 MYA gene sequence. Plots of uncorrected pairwise sequence for the “Terphis-Megalybus” clade (Philopotinae) (Prophil- divergence against number of nucleotide substitutions (not opota succinea Hennig) (Baltic Amber) (Hennig, 1966), and shown) displayed an expected linear relationship for all 45 MYA for Acrocerinae partim (i.e., exclusive of Acrocera genes and did not show any sign of saturation at greater and Sphaerops) (Villalites electrica Hennig) (Baltic Amber) divergences. As expected, sequence divergence was lowest (Hennig, 1966). The maximum age constraint for Brachy- between closely related taxa (between members of the same cera was set to 240 MYA corresponding to ages of the old- genus) and greatest between outgroup and ingroup taxa, 818 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832

Fig. 3. Average nucleotide frequencies: (A) COI and CAD, divided by codon position; (B) 16S and 28S, divided by stem and loop regions. although for genes such as CAD and 16S, Nemestrinidae ella), with an average of 18.2%. Average sequence diver- sometimes showed greater divergence from its putative sis- gence was lower for 28S (9.1%), and ranging from identical ter-group, Acroceridae, than some of the far outgroup taxa. sequences for closely related species of Eulonchus to 23.9% Uncorrected sequence divergence for 16S ranged from 0.2% (Psilodera vs. Acrocera). CAD displays the greatest (between Eulonchus spp.) to 31.5% (Hirmoneura vs. Astom- sequence divergence for all the genes, ranging from 0.4% S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 819

(Archipialea vs. Arrhynchus) to 41.8% (Tabanus vs. Astom- ters included in the alignment, 231 (23%) were phylogeneti- ella), with an average of 25.1%. Sequence divergence for cally informative and 716 were constant. With so many COI with thirds positions included ranged from 1.0% minimum-length trees obtained, there was almost no reso- (Arrhynchus vs. Trichophthalma) to 26.8% (Psilodera vs. lution in the strict consensus tree except for the grouping of Eulonchus), with an average of 20.1%. When third positions Acrocera and Ocnaea with Sphaerops, and a small clade of were excluded, sequence divergences ranged from 2.0% closely related philopotine genera. In both analyses boot- (Arrhynchus vs. Trichophthalma) to 16.1% (Terphis vs. Turb- strap support for almost all nodes was weak with very few opsebius), with an average of 10.2%. values over 50%. Due to their high variability and signiW- cant conXict with previously proposed relationships (e.g., 3.2. Individual gene analyses Acroceridae and Philopotinae monophyly), third positions of COI were removed from all subsequent analyses of the 3.2.1. 16S rDNA sequence data COI partition. Parsimony analysis of the 45 taxa sequenced for 16S resulted in 22 equally parsimonious trees with a length of 3.2.4. CAD (CPSase region) DNA sequence data 1434, consistency index (CI) D 0.32 and retention index Forty-Wve taxa were sequenced for CAD with 834 char- (RI) D 0.57 (Fig. 4). Of the 506 included characters, 242 acters included in the alignment. Parsimony analysis (48%) were phylogenetically informative and 211 were con- resulted in a single tree of length 3077, CI D 0.35 and stant. Many nodes have relatively high bootstrap values but RI D 0.61 (Fig. 4). More than half of the aligned characters there is a large polytomy of unresolved relationships for (494 or 59%) were phylogenetically informative and 262 much of the Acroceridae. Stratiomyidae and Tabanidae are characters were constant. Nemestrinidae were not found to placed with relatively weak support with Nemestrinidae. be the sister-group to Acroceridae in this data set, as indi- Acrocera and Sphaerops are clearly placed as the sister cated by the large sequence divergences between the two clade to the rest of the Acroceridae, although Sphaerops groups. Acroceridae are monophyletic, and internal rela- renders Acrocera paraphyletic. Ocnaea is placed with Archi- tionships completely resolved throughout this tree with pialea and Arrhynchus in a well-supported clade next to most nodes supported by relatively high bootstrap values Acrocera and Sphaerops. Relationships for the rest of the except for relationships between acrocerine and panopine family are not well resolved or if resolved are not well sup- genera. Again, Acrocera and Sphaerops were recovered as ported. the sister-group to the rest of Acroceridae. Philopotinae was monophyletic although diVering slightly in internal 3.2.2. 28S rDNA sequence data topology to 28S. Panopinae were monophyletic in the Forty-one taxa were sequenced for 28S and parsimony CAD-based tree, except for placement of the panopine analysis resulted in 21 trees with a length of 2005, CI D 0.59 genus Astomella as sister to Pterodontia. and RI D 0.62 (Fig. 4). Of the 2628 characters included in the alignment, 494 (19%) were phylogenetically informative 3.3. Combined dataset phylogeny and 1745 were constant. Similar to 16S data, numerous ter- minal and some basal nodes are relatively well supported We combined the four data partitions despite indications W with high bootstrap values, but many of the intermediate from the HTF test statistic that they hold signi cantly incon- nodes corresponding to acrocerid subfamilial relationships gruent individual phylogenetic signals. The HTF is consid- are not resolved at all. Nemestrinidae are recovered as a sis- ered conservative in its estimation of congruence in ter-group to a monophyletic Acroceridae. Acrocera and phylogenetic signal between data sets (Cunningham, 1997a), Sphaerops are again relatively well supported as the basal but empirical evidence has shown that statistical congruence clade of Acroceridae. While many internal nodes are not tests such as the HTF may give misleading indications of resolved in this data set, all genera are monophyletic and incongruence depending on speciWc attributes of character well supported. The only subfamily recovered as monophy- conXict within data sets, especially whether the conXict is letic for this dataset are Philopotinae. randomly distributed or limited to speciWc taxa or clades (Dolphin et al., 2000; Yoder et al., 2001; Winterton et al., 3.2.3. COI DNA sequence data 2001). We calculated HTF as a heuristic measure of congru- Parsimony analysis of the 32 taxa sequenced for COI ence among data partitions to serve as descriptor of signal resulted in 245 trees with a length of 3270, CI D 0.36 and contribution and conXict among the four gene partitions, but RI D 0.47 (Fig. 4). From the aligned sequence length of not as an absolute arbiter of combinability. Initial inspection 1485 characters, 636 (43%) characters were phylogeneti- of the individual trees produced from each gene showed cally informative and 755 constant. A strict consensus pro- some distinct similarities and diVerences. While COI (all duced a completely nonsensical phylogeny where none of positions) shows highly discordant phylogenetic signal when the families, subfamilies, and most genera are recovered as compared to both of the ribosomal genes and CAD, the monophyletic. Excluding third positions produced 11,092 latter three genes individually support many of the same trees and a largely unresolved phylogeny (not shown) higher-level relationships, thereby indicating some degree of (length of 939, CI D 0.41 and RI D 0.61). Of the 990 charac- phylogenetic concordance between partitions. 820 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832

Fig. 4. Strict consensus trees resulting from parsimony analyses for individual 16S, 28S, COI, and CAD data sets. Bootstrap values (>50%) are given below branches. Abbreviations: AC, Acrocerinae; PA, Panopinae; PH, Philopotinae. S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 821

Fig. 5. Single most parsimonious phylogram with branch lengths generated from combined 16S, 28S, CAD, and COI data for 47 taxa in both maximum parsimony and Bayesian analyses (length D 7741; CI D 0.4; RI D 0.57). Third positions for COI were excluded. Numbers on nodes represent bootstrap (parsimony)/Bremer support (parsimony)/posterior probabilities (Bayesian). Thickened branches represent bootstrap and posterior probabilities 785%. 822 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832

The combined dataset comprised 47 taxa with a total chus. Excluding third positions only for the CAD data gave aligned sequence length of 4958 characters, of which 1449 an identical topology to the aforementioned Wnal combined (29%) were phylogenetically informative and 2964 were tree with COI third positions excluded. Excluding third constant. Analyses using parsimony methods returned a positions for both COI and CAD resulted in lack of nodal single tree with a length of 7741 steps (CI D 0.40, RI D 0.57) resolution in basal relationships between panopine and ter- (Fig. 5). Bayesian analysis returned an almost identical tree, minal acrocerine genera. The eVect of COI on tree topology diVering only in estimates of relationship among outgroups. was examined by excluding the COI data from the analysis. The combined molecular data set yields a well supported A single tree (length D 6628 steps, CI D 0.41, RI D 0.59) was and highly resolved phylogeny with a large number of recovered of similar topology to the combined tree except nodes for which bootstrap proportions and posterior prob- for Ogcodes being placed between Acrocera + Sphaerops abilities were high. Seventy percent of the resolved nodes and the rest of Acroceridae. show support above 85%, and many are supported at 100%. Not all taxa were sequenced for all genes, so PBS values Bremer support values are also high for these same nodes were calculated on a phylogeny generated for only a subset and are well correlated until rapidly reaching maximum of 28 taxa for which complete sequence data were available. possible bootstrap values (100%). Estimated branch lengths While not ideal, these values are still indicative of the rela- by parsimony and Bayesian likelihood were relatively uni- tive contributions of the various data partitions to the over- form throughout the tree, with few long branches. As found all phylogeny using the larger taxon sample. Parsimony in separate analyses of 28S, 16S, and CAD, the combined analysis of the pruned taxa set resulted in a single most par- data set had Acrocera + Sphaerops placed as sister to all simonious tree of length 6402 steps (CI D 0.445; RI D 0.509) remaining Acroceridae with relatively strong support (Fig. 6). This pruned tree is congruent in topology to that (bootstrap (BS) D 100/Bremer support D 15/posterior prob- found in analyses of the full taxon set. As with bootstrap ability (PP) D 100). Philopotinae are strongly supported and Bremer support values in the full taxa set phylogeny, (98/20/100) and intergeneric relationships are also reliably bootstrap, and PBS values are relatively high across most recovered with strong support. Only the internal relation- of the tree and are well correlated until reaching maximum ships of the sample species of Thyllis are not well resolved. possible bootstrap values. Like in the full analysis, mono- Whilst relationships among closely related terminal taxa phyly of Nemestrinioidea is only weakly supported (e.g., congeners) and basal clades within the family are well (BS D 63; summed Bremer support D 4) and only by small supported, some intermediate nodes are not. In particular, contributions from the ribosomal data partitions. Individ- Acrocerinae are polyphyletic in these analyses, with three ual monophyly of both Nemestrinidae and Acroceridae are clades recovered. Acrocerine genera such as Acrocera well supported by all genes. Acrocera + Sphaerops are also + Sphaerops (i.e., Acrocerinae sensu stricto) are sister to the strongly supported by all genes as the sister clade to the rest of the family, while Turbopsebius + Paracyrtus form remaining Acroceridae. As found in the individual analysis, another clade sister to the remaining members of the sub- 16S does not support a monophyletic Philopotinae (16S family (the latter two clades referred herein as Acrocerinae PBS D¡7) although all other data does (summed Bremer partim). In no analysis of separate or combined data sets do support D 16). Acrocerinae partim (i.e., exclusive of Acrocerinae form a monophyletic clade and most internal Acrocera + Sphaerops) remain paraphyletic. PBS values of relationships of the group are only weakly supported. The all basal nodes of Acrocerinae partim are weakly supported most consistently obtained relationships for acrocerine taxa by 28S and COI and weakly contradicted by 16S and CAD. are the grouping and placement of Acrocera + Sphaerops The grouping of Acrocerinae partim with Panopinae is and Turbopsebius + Paracyrtus, respectively. Panopinae are strongly supported by the COI data but contradicted by the monophyletic with high posterior probability (100%), but other genes resulting in a weak summed Bremer support (6) with relatively weak bootstrap (56%) and Bremer (7) for that node. Monophyly of Panopinae is again not sup- supports. Overall, the monophyly of individual panopine ported by 16S data (PBS D¡6) but is supported by CAD, genera is well supported, but intergeneric relationships are 28S, and COI (summed Bremer support D 3). largely unresolved. Third positions in protein-encoding genes may have a 3.4. Historical divergence time estimates signiWcant misleading eVect on phylogenetic reconstruction (Foster and Hickey, 1999; Chang and Campbell, 2000). The topology resulting from the simultaneous analysis When third positions for COI were included in the com- of the pruned taxa set (28 taxa) (Fig. 6) was used as the bined data set, parsimony analysis recovered Wve equally input tree for the divergence time analyses. Fig. 7 shows the parsimonious trees of length 10,252 steps (CI D 0.379; divergence time estimates plotted on a phylogram where RI D 0.53) (not shown) with a similar consensus tree to that branching nodes correspond to means of the probability generated from the Bayesian analysis. Topology of this tree distributions for the node age on a geological time-scale. is similar in some respects to the tree recovered from the Red bars represent the range of dates around a node within analysis with COI third positions excluded, but it is also 95% of the probability of that node age. Age estimates very diVerent in many areas, most notably in lack of mono- ranged from 72 MYA (divergence of Acrocera and phyly for any subfamily and even genera such as Arrhyn- Sphaerops; node 6) to 18 MYA (Ogcodes spp.; node 21) on S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 823

Fig. 6. Pruned tree of 28 taxa sequenced for 16S, CAD, 28S, and COI genes. Single tree resulting from maximum parsimony analysis (length D 6402 steps; CI D 0.56; RI D 0.44). Third positions for COI were excluded. Partition Bremer Supports (PBS) are presented on each node for each gene (16S, CAD, 28S, and COI). Bootstrap values (>50%) are presented above PBS values. Thickened branches represent bootstrap values 785%. 824 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832

Fig. 7. Estimated divergence times for Nemestrinoidea and major clades of Acroceridae. Phylogram represents pruned taxon set of 28 taxa sequenced for all genes in Fig. 6. Time scale units are represented in millions of years before present day. Branching nodes correspond to means of the probability distri- butions for node ages while red bars represent the time interval for 95% probability of actual node age. Numbers on nodes correspond to those presented in Table 4. Vertical black bars represent minimum age constraints for particular node times based on dates of earliest deWnitive fossils known. For the root node, maximum and minimum age constraints are indicated based on maximum ages of Brachycera (240 MYA) and of the oldest deWnitive Nemestrinoi- dea fossil (155 MYA), respectively. S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 825

Table 4 certain instances, diminishing their value as an arbiter of Estimated dates of divergence for Nemestrinoidea and major lineages of data set combinability (Cunningham, 1997a,b; Winterton Acroceridae based on combined 16S, 18S, CAD, and COI DNA et al., 2001). Our position follows that of Wiens (1998), sequences and using the tree topology generated from phylogenetic analy- sis of the pruned taxa set (see Fig. 6) who argued that combined data should be considered the best average estimate of phylogeny and strongly discor- Node Estimated age (min., max.) (MYA) dant data sets viewed as questionable until additional 1 233 (217, 239) evidence to support them is found. We use monophyly of 2 190 (172, 209) W 3 184 (167, 203) well-de ned taxonomic groups based on concordance 4 134 (130, 146) with other data types (e.g., morphology) as a better judge 5 198 (173, 221) of data set congruence as suggested by Yoder et al. 6 84 (51, 123) (2001). Here the greatest recovery of well- established, 7 43 (21, 74) monophyletic groups was by combined data set and not 8 153 (127, 177) 9 128 (102, 153) by any individual data set. 10 116 (90, 143) Comparison of results from the combined data set 11 84 (60, 109) with individual partitions using concordance as a crite- 12 72 (48, 98) rion of gene performance (sensu Yoder et al., 2001) indi- 13 87 (62, 113) cates that the CAD data set strongly outperforms the 14 134 (108, 159) 15 82 (58, 107) mitochondrial and ribosomal data sets. Philopotinae are 16 126 (101, 152) a well-established group of Acroceridae based on several 17 123 (98, 148) autapomorphic morphological characters and recovery 18 116 (91, 142) of the monophyly of this group is a good measure of data 19 107 (82, 134) set performance through concordance. Both 28S and 20 23 (14, 36) 21 17 (9, 27) CAD recovered a monophyletic Philopotinae, although 22 113 (89, 138) tree resolution was lower in the 28S data (21 trees) than 23 102 (78, 127) in the CAD data, which produced a single, completely 24 79 (55, 104) resolved tree. Neither mitochondrial gene recovered Philopotinae as monophyletic. The COI data performed worst at recovering Acroceridae relationships, with the unconstrained nodes (average: 44.95 MYA; Std. Dev.: subfamilies and even genera scattered throughout the 12.95 MYA); the age estimate for divergence of Hirmoneura tree, and part of the ingroup nested deep within the out- spp. was 16 MYA, but it was constrained by a minimum group. age for the genus (node 4) (see Table 4). The estimated divergence of Acroceridae and Nemestrinidae is 233 MYA 4.1. Nemestrinoidea phylogeny (range: 217–239 MYA) while the Acroceridae diverges basally into the Acrocera + Sphaerops clade and the rest of The monophyly of families Acroceridae and Nemes- Acroceridae at 198 MYA (173–221 MYA). The major lin- trinidae as separate distinct lineages is strongly supported eages of Acroceridae were present by the late Jurassic and by molecular data and both families have been previously early Cretaceous. The greatest amount of cladogenesis well characterized based on various morphological char- occurred during the Cretaceous with all genera present by acters. By contrast, Nemestrinoidea is not well supported the end of that period with the only acrocerid divergences according to current evidence, although this was not the occurring during the subsequent periods being intrageneric main objective of the study and is diYcult to test without speciation events. broader taxon sampling. Schlinger (1960a) and later Nag- atomi (1992) and GriYths (1994), suggest that Acroceri- 4. Discussion dae are more closely related to Tabanidae than to Nemestrinidae, a theory not widely supported (see Yeates The debate on combining disparate data in simulta- and Wiegmann, 2005). Although a close relationship neous analyses is vigorous and ongoing (Huelsenbeck between Acroceridae and Nemestrinidae is favored by and Bull, 1996; Lutzoni, 1997; Wenzel and Siddall, 1999; most studies, a monophyletic Nemestrinoidea has not Cunningham, 1997a; Yoder et al., 2001). Advocates of been unequivocally proven in any quantitative analysis ‘total evidence’ analyses point to numerous examples of (Yeates, 1994, 2002). Moreover, Tabanidae have been robust, well-resolved combined phylogenies generated conclusively shown to be members of Tabanomorpha from disparate data sets despite homogeneity tests indi- without any close relationship with Acroceridae or cating incongruence between one or more of those indi- Nemestrinidae (Woodley, 1989; Wiegmann et al., 2003; vidual data sets (e.g., Winterton et al., 2001; Yoder et al., Yeates, 2002). Nemestrinidae and Acroceridae share a 2001; Cryan et al., 2004; Michel-Salzat and WhitWeld, number of apomorphic characters with and 2004; Downie and Gullan, 2004). Combinability tests Eremoneura, including reduction in number of cerci such as HTF have been shown to be inconsistent under segments in the female from two to one, lack of tibia spurs 826 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 and larval spiracles present anterodorsally on the penulti- opinae. Philopotinae are well supported as a monophy- mate or ultimate abdominal segment (Woodley, 1989; letic group in most analyses. Yeates, 1994). Moreover, Yeates et al. (2002) found that Nemestrinoidea have greater similarity in ventral nerve 4.3. Placement of Acrocera and Sphaerops and the polyphyly chord arrangement to Muscomorphan groups Asiloidea of Acrocerinae and than to other lower Brachyceran infraor- ders. Hennig (1973) placed Bombyliidae with Nemestrini- Our molecular data analyses show Acrocerinae, as previ- dae and Acroceridae in Nemestrinoidea based on the ously delimited, to be polyphyletic. In the combined analy- larval parasitic lifestyle of the three families. Bombyliidae sis and in three individual analyses, Acrocera and have more recently been found to be sister group to the Sphaerops are placed as sister to the rest of Acroceridae rest of Asiloidea (Woodley, 1989; Yeates, 2002). Clearly, with relatively high levels of statistical support. Acrocerinae the parasitic lifestyle of the larvae of these three families partim are paraphyletic with respect to Panopinae, has arisen independently at least twice. Yeates and Irwin although this is the only region of the combined phylogeny (1992) suggested that larvae of the bombyliid, Heterotr- where statistical support for several nodes is weak. Two opus Loew, are likely soil predators representing the plesi- clades are recovered among the paraphyletic Acrocerinae omorphic condition in Bombyliidae and that the partim, one comprising Paracyrtus and Turbopsebius, the ectoparasitic lifestyle is derived in the family. All Nemes- other comprising Holops, Psilodera, Pterodontia, and trinidae and Acroceridae are endoparasitic except for the Ogcodes. Chilean genus Sphaerops, which is ectoparasitic (Schlin- Morphologically, adult Acrocera and Sphaerops are sim- ger, 1987). Our data support a similar scenario in which ilar to other Acrocerinae by having relatively small heads, the ectoparasitic habit is best considered a unique feature bulbous bodies, and reduced wing venation. Larval mor- of Sphaerops independently derived from endoparasitic phology and behaviour of both genera are however, quite nemestrinoid ancestors. diVerent from the rest of the family. All acrocerids for which the Wrst instar planidial larvae are known have well 4.2. Placement of subfamilies sclerotized body segments with numerous long setae or scales and move actively about with looping as well as leap- The classiWcation and implied phylogenetic relation- ing or Xicking movements (King, 1916; Schlinger, 1960b, ships of Acroceridae has been established as comprising 2003). Acrocera Wrst instar larvae lack both sclerotization three subfamilies based on morphology and host use (Sch- and long setae and move around by crawling only. Further- linger, 1987, 2003). Molecular data presented herein do more, mature acrocerid larvae typically emerge through the not corroborate this assessment. Our data suggest instead epigastric furrow (i.e., centre of Wrst sternite) of their spider that the existing classiWcation developed for Acroceridae host, but Acrocera always emerge through the dorsum of subfamilies only partially reXects natural groupings. the spider abdomen (Schlinger, 1987). Although the Wrst While Philopotinae and Panopinae are recovered as instar planidial larva of Sphaerops is unknown, it is the only monophyletic groups, Acrocerinae is polyphyletic. Pan- known ectoparasitic acrocerid. Schlinger (1987) presumed opinae are typically large, robust Xies with an extensive that these genera were highly derived. Instead, whilst adult covering of pile and regular wing venation similar to Acrocera and Sphaerops are morphologically similar to other families of Lower Brachycera. Panopine acrocerids other Acrocerinae, their diVerent larval morphology and exclusively parasitize mygalomorph spiders. While the behaviour actually lends additional support to placement subfamily is monophyletic (although weakly supported) as sister to the rest of the family by the molecular data. in the combined analysis of all sampled genes, support for The placement of Psilodera has been problematic and the group is not found in any of the individual trees; Pan- was suggested as the stem group for the Cyrtus–Opsebius opinae are a derived group placed as sister to Acrocerinae lineage of Acrocerinae (Schlinger, 1972). Our results con- partim in the combined tree. Acrocerinae sensu lato are Wrm that the two sampled taxa representing the Cyrtus– considered a derived or advanced subfamily based on Opsebius lineage form a close sister group relationship (i.e., morphological characters such as highly reduced wing with Paracyrtus and Turbopsebius), but they are not venation and relatively smooth shiny bodies (Schlinger, “derived directly” from Psilodera. Schlinger (1960a) indi- 1987, 2003). Both Acrocerinae s.l. and Philopotinae exclu- cated that Psilodera was likely the most “primitive” mem- sively parasitize araneomorph spiders. Many genera from ber of the subfamily with close relationships to both these two subfamilies have broad host ranges utilizing a Aspona (Panopinae) and Thyllis (Philopotinae). In the com- wide variety of genera and families; this is especially true bined molecular analysis, the acrocerine clade containing for widely distributed acrocerines Ogcodes and Acrocera. Holops, Psilodera, Pterodontia, and Ogcodes is weakly sup- Acrocerinae s.l. is not recovered as monophyletic in either ported, but Holops is sister to the rest of the clade with the combined or individual gene data analyses. Psilodera as an intermediate taxon. Acrocera + Sphaerops (Acrocerinae sensu stricto) are The phylogenetic position of Pterodontia is unclear, strongly supported as sister to the rest of the family while although some authors have placed it in Acrocerinae (e.g., Acrocerinae partim is paraphyletic with respect to Pan- Cole, 1919; Nartshuk, 1988; Barraclough, 1991) others have S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 827 placed it in Panopinae (e.g., Schlinger, 1960a). Pterodontia chus are closely related although they do not form a has a head and antennal shape, and wing venation similar monophyletic group as suggested by Schlinger (1971). to other acrocerine genera, but also has tibial spurs (Cole, 1919), characteristic of Panopinae (Schlinger, 1960a). Sch- 4.6. Acroceridae phylogeny and patterns in spider parasitism linger (1960a) suggested Pterodontia may be more closely related to Astomella than any Acrocerinae genera. In this Host rearing records are sparse and far from exhaustive, analysis, Pterodontia is sister to Ogcodes in Acrocerinae so generalizations regarding patterns in host selection and partim, indicating that tibial spurs are homoplasious in speciWcity in acrocerids can only be considered preliminary. Acroceridae as suggested by Barraclough (1991). Host rearing records by family are plotted on the full taxa set phylogeny recovered in this analysis (Fig. 8). Schlinger 4.4. Philopotinae (1987) presented an extensive list of spider taxa parasitized by acrocerids, demonstrating that Panopinae are host- Monophyly of Philopotinae has never been contested. restricted to Mygalomorphae spiders while Acrocerinae Several morphological features deWne this bizarre group of and Philopotinae are host-restricted to Aranaeomorphae Xies, including enlarged postpronotal lobes forming a collar spiders. Only Acrocera and Sphaerops have been reared around the head and a distinct arched body shape (Schlin- from spiders in Haplogynae; Acrocera from Plectreuridae ger, 1987). Our molecular data also strongly support the and Sphaerops from Segestriidae (Schlinger, 1987). All monophyly of the subfamily and its internal arrangement. other Acrocerinae and Philoptinae have been reared only Philopotine relationships broadly follow biogeographical from Entelegynae. While some genera are only known from regions with Afrotropical genera sister to New World gen- sporadic rearing records, Turbopsebius is known from mul- era and Oceanian genera sister to the rest. Schlinger (1961, tiple rearing records, the hosts of which all belonging to 1971, 2003) stated that Megalybus, Helle, and Parahelle, Agelenidae. The three cosmopolitan Acrocerinae genera form a monophyletic clade based on morphology and simi- (Acrocera, Pterodontia, and Ogcodes) are represented by lar trends in host–spider relationships, despite their highly numerous rearing records from multiple spider families. disjunct distribution. Our molecular data do not support Acrocera has been reared from seven families (Plectreuri- this grouping, instead placing Helle (endemic to New Zea- dae, Lycosidae, Agelenidae, Amaurobiidae, Clubionidae, land) as sister to an undescribed genus from New Caledo- Gnaphosidae, and Salticidae), while Pterodontia has been nia in a clade sister to all other Philopotinae. Moreover, reared from three families (Araneidae, Lycosidae, and Sal- Megalybus (Central America) is placed close to other New ticidae) (Schlinger, 1987). The greatest number of rearing World genera Terphis and Philopota, while Parahelle records are for the commonly encountered Ogcodes, which (endemic to Madagascar) is strongly supported as sister to is recorded parasitizing at least 15 families of Entelegynae, another Malagasy genus, Thyllis. This supports the conclu- including Amaurobiidae, Miturgidae, and multiple families sion by Hennig (1966) that Parahelle, Thyllis (and Dima- in Araneoidea, Lycosoidea, and Dionycha (Schlinger, 1987; crocolus Schlinger) form a monophyletic clade and that Barraclough and Croucamp, 1997). Helle is not closely related to Thyllis. Schlinger (1961, 2003) Of the 15 recognized Mygalomorphae families, Pano- suggested that Thyllis and Philopota comprise the “most pine acrocerids have been reared from spider hosts in Wve: primitive known stock of Philopotinae”. These two genera Ctenizidae, Theraphosidae, Dipluridae, Antrodiaetidae, are not closely related in this analysis and neither are and Migidae (Schlinger, 1987; Barraclough, 1991). There is placed basally. Instead, Philopota forms a clade with Ter- considerable overlap in rearing records, with many pano- phis and Megalybus (a relationship partly suggested by pine genera attacking two or rarely three families; no pano- both Schlinger (1960a) and Hennig (1966)), while Thyllis is pine genus attacks all Wve families. Astomella is recorded strongly supported as the most derived Philopotinae clade. from Ctenizidae, Migidae, and Theraphosidae (Barrac- lough, 1984; Schlinger, 1987). 4.5. Panopinae 4.7. Acroceridae historical divergences and the fossil record Schlinger (1987, 2003) hypothesized that Panopinae are the most “primitive” subfamily of Acroceridae, presumably The estimated date of divergence of the due to the presence of regular type wing venation similar to Acrocera + Sphaerops clade from the rest of Acroceridae is that in other lower Brachycera. By contrast, our data relatively old (early Jurassic) based on divergence times strongly indicate that Panopinae are a derived group within estimated from the molecular data presented in this study. the family. The “Ocnaea branch” of Panopinae as outlined The oldest deWnitive Acroceridae fossils are Archocyrtus by Schlinger (1968, 1973) with seven genera, is represented Ussatchov and Juracyrtus Nartshuk, both described from in this analysis by Ocnaea, Arrhynchus, and Archipialea, late Jurassic aged compression fossils from the Karatau and is recovered with high support. beds, Kazakhstan (Ussatchov, 1968; Nartshuk, 1996). Both Schlinger (1960a) implied that Apsona (endemic to New genera are ‘Acrocera’-like in appearance, with short anten- Zealand) is the most “primitive” panopine genus; a sugges- nae, short mouthparts, and wing venation similar to some tion borne out in our analyses. Apsona, Lasia, and Eulon- extant species of Acrocera. The elongate mouthparts of 828 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832

Fig. 8. Phylogeny of Acroceridae relationships with host spider families plotted against each genus. Host records summarized from Schlinger (1987). S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 829

Juracyrtus apparently incorrectly described by Nartshuk opinae may be expanded to accommodate these dislocated (1996) are more likely extraneous vegetative matter (Grim- genera, thus retaining three subfamilies. Additional data aldi and Engel, 2005). will be required to fully resolve acrocerid relationships. For Whilst Acrocerinae s.s. diverged from the rest of the example, male and female genitalic characters have not family relatively early (early Jurassic), the rest of Acroceri- been thoroughly explored and may provide important new dae appear to have radiated more recently, with divergence information. Moreover, given the phylogenetic utility of of Philopotinae from the rest of Acrocerinae molecular data for understanding acrocerid relationships, partim + Panopinae (based on taxon sampling in this study) additional sampling of key acrocerine genera such as Sab- occurring 45 million years later (late Jurassic). Philopotinae roskya Schlinger, Meruia Sabrosky, and Villalus Cole are strongly supported as monophyletic based on both should contribute to a better resolved and stable proposal morphological and molecular data. The chronology of for a revised classiWcation of the family. Our sample of pan- Philopotinae evolution based on divergence time estimates opine taxa is strongly biased toward New World genera. closely tracks continental plate movements during the Cre- We believe that additional taxa representing genera from taceous with separation of Laurasia from Gondwana. The other regions, such as Rhysogaster Aldrich (Oriental), Cor- New Zealand genus Helle and an undescribed genus from ononcodes Speiser (South Africa) and the diverse Austra- New Caledonia represent the sister lineage to the rest of the lian panopine fauna such as Lamarck and subfamily, diverging in the early Cretaceous as Gondwanan Mesophysa Macquart, would greatly improve estimates of continents began to fragment approximately 130 MYA. relationships. Subsequently, New World genera (Megalybus, Terphis, and Using concordance with other data and recovery of Philopota) diverged from Afrotropical genera (Parahelle monophyly of well-established groups like Philopotinae as and Thyllis) approximately 120–110 MYA, towards the criteria of data set performance, the combined data per- middle Cretaceous. Divergence patterns for Acrocerinae formed better than any single gene. Using the same criteria, and Panopinae are more diYcult to interpret based on CAD performed better than the ribosomal and mitochon- these results because of weak support for many of the basal drial genes for resolving acrocerid phylogeny, as has been branching patterns in both groups and because of the taxon found in other dipteran groups (e.g., Moulton and Wieg- sampling bias in Panopinae for New World taxa. mann, 2004). The greatest amount of cladogenesis in Acroceridae Schlinger (1981, 1987, 2003) suggested that there has been occurred during the Cretaceous (ca. 140–65 MYA), with all a long history of co-evolution between small-headed Xy para- major lineages and genera originating. Unfortunately, no sitoids and their spider hosts. While the age, diversity, and fossil acrocerids are known from this period, with the next specialization of their interactions are evidence of a long evo- oldest fossils known from Eocene and Oligocene amber lutionary association between acrocerids and spiders, there is deposits, between 55 and 20 million years of age (Hennig, little phylogenetic evidence for a pattern of reciprocal clado- 1966, 1968; Grimaldi, 1995; Hauser and Winterton, in genesis or host-tracking (Mitter and Brooks, 1983; Laban- press). This chronological gap suggests that fossil-based deira, 2002). Only 23 spider families are parasitized by corroborative support for a rapid Cretaceous acrocerid acrocerids and mapping fragmentary host records on the diversiWcation is lacking; such information would be useful phylogeny reveals few clear patterns except the distinction for ascertaining the validity of weakly supported nodes at between Mygalomorphae and Aranaemorphae spiders by the base of Panopinae and Acrocerinae (partim). some Acroceridae subfamilies. Overall, there is a distinct temporal separation in the evolution of both spiders and 5. Conclusions acrocerids, thus discounting any broad coevolution of host and parasitoid. Mygalomorphae spider fossils date back to The results from these analyses of nucleotide data from the (ca. 235 MYA) and major radiations of Araneo- four independent genes strongly support many aspects of morphae occurred during the late Palaeozoic or early Meso- the current morphology-based classiWcation of Acroceri- zoic (ca. 200–250MYA) (Coddington and Levi, 1991), before dae, e.g., Panopinae and Philopotinae are natural groups. major Acroceridae lineages appeared and diversiWed in the Acrocerinae s.l., however, are never recovered suggesting mid to late Mesozoic (200–60MYA). We may speculate that that a re-examination of characters that deWne the group the acrocerid phylogeny shows coevolutionary relationships may be warranted. Acrocerinae have highly reduced wing with their spider hosts at lower taxonomic levels, such as venation, rendering this often useful source of phylogenetic within subfamilies or genus groups, but this relationship is information of limited value for deWning the group. Acro- unlikely to be reXected at higher levels. One example is para- cerinae s.s., comprising Acrocera and Sphaerops, are sitism of Halpogynae spiders by Sphaerops and some Acro- strongly placed as the sister clade to the rest of Acroceridae, cera, while all more derived Aranaeomorphae parasitoids with some supporting evidence from larval morphology attack Entelegynae spiders. Spiders were already greatly and behavior. The rest of Acrocerinae will require further diversiWed before exposure to acrocerid parasitoids with examination and re-evaluation, with likely ultimate erec- some extant families of Araneomorphae already appearing in tion of new higher-level groupings (e.g., subfamilies) to the fossil record in the late Cretaceous (Coddington and accommodate these taxa. Alternatively, the concept of Pan- Levi, 1991). Our data suggest that Panopinae are not an early 830 S.L. Winterton et al. / Molecular Phylogenetics and Evolution 43 (2007) 808–832 acrocerid lineage that pioneered a parasitic foray into an the insect mitochondrial large subunit rRNA gene. Insect Mol. Biol. 9, ancient spider lineage. 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