Molecular Phylogenetics and Evolution 57 (2010) 84–100 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Phylogeny of the tribe Athetini (Coleoptera: Staphylinidae) inferred from mitochondrial and nuclear sequence data Hallvard Elven *, Lutz Bachmann, Vladimir I. Gusarov Department of Research and Collections, National Centre of Biosystematics, Natural History Museum, University of Oslo, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway a r t i c l e i n f o Article history: Received 13 October 2009 Revised 18 April 2010 Accepted 25 May 2010 Available online 8 June 2010 Keywords: Athetini Lomechusini Ecitocharini Atheta Thamiaraea Halobrecta Meronera Thendelecrotona Bayesian analysis Maximum Parsimony analysis a b s t r a c t The Athetini are the largest and taxonomically most challenging tribe in the subfamily Aleocharinae. We present the ﬁrst molecular phylogeny of Athetini. Nucleotide sequences were obtained from three genome regions for 58 athetine and 23 non-athetine species. The sequenced genes are cytochrome oxidase subunits 1 and 2 (2030 bp), tRNA-Leucine 1 and 2 (154 bp), 16S (628 bp, partial sequence), NADH dehydrogenase subunit 1 (54 bp, partial sequence), and the nuclear 18S gene (999 bp, partial sequence). The Athetini were recovered as paraphyletic with respect to Lomechusini and Ecitocharini. Lomechusini were recovered as polyphyletic, with Myrmedonota grouping separately from Pella and Drusilla. The basal topology of Athetini remained largely unresolved but many apical clades were well supported, e.g. Geostiba + Earota, Pontomalota + Tarphiota, Mocyta + Atheta (Oxypodera) + Atheta (Mycetota), Liogluta + Atheta (Thinobaena) + Atheta (Oreostiba), and Lyprocorrhe + Atheta (Datomicra). The monophyly of Atheta was refuted, as several species of Atheta formed well supported clades with members of other genera. Additionally, the following groups were rejected: Strigotina (=Acrotonina) and Dimetrotina sensu Newton et al. (2000), Acrotona sensu Brundin (1952), Liogluta series (Yosii and Sawada, 1976), Atheta (Dimetrota) and Atheta (Alaobia) sensu Smetana (2004). New tribal placements are proposed for four genera: Halobrecta is removed from Athetini and provisionally placed in Oxypodini; Thendelecrotona is removed from Athetini and treated as Aleocharinae incertae sedis; Meronera and Thamiaraea are included in the Athetini. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Staphylinid beetles (Coleoptera: Staphylinidae) represent one of the major radiations within the phylum Arthropoda. The family is believed to have originated in the early Triassic (240 mya), and was already a diverse group by the mid-Cretaceous (120 mya) (Grimaldi and Engel, 2005). Today, the family comprises almost 50,000 described species in 31 subfamilies (Thayer, 2005). However, the number of undescribed species is believed to be several times larger (Grimaldi and Engel, 2005). Staphylinids are widespread on all continents except Antarctica and occupy virtually every terrestrial habitat. Some lineages within the Staphylinidae have proved particularly successful in terms of species number and ecological diversity. The tribe Athetini Casey, 1910 represents one of the family’s major radiations. Cases of adaptive radiation are well documented in other groups of organisms, and though the particular causes can be complex (see e.g. Davies et al., 2004 on the rise of the ﬂowering plants), they are often explained as a result of key adaptive innova* Corresponding author. Fax: +47 22851837. E-mail addresses: [email protected] (H. Elven), [email protected] uio.no (L. Bachmann), [email protected] (V.I. Gusarov). 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.05.023 tions, colonization of new territories, or global shifts in either climate or species communities (Gavrilets and Losos, 2009). Adaptive radiations may occur rapidly (see e.g. Kocher, 2005), and the short time span between cladogenetic events may complicate the task of inferring phylogenies. The Athetini are the largest tribe in the subfamily Aleocharinae Fleming, 1821, and comprises more than 170 genera and thousands of described species worldwide (Newton et al., 2000). The tribe nests within the so-called ‘‘higher” Aleocharinae (Ashe, 1994, 2005), a monophyletic group of at least 46 tribes (Ashe, 2007) supported by several morphological characters (Ashe and Newton, 1993; Ashe, 2005, 2007). Athetines exploit most terrestrial habitats, and are particularly abundant in humid microhabitats rich in decomposing organic matter, such as leaf litter, decaying wood, dung, carrion, mushrooms, mammal burrows, and riparian zones. Examples of more unusual habitats include ant nests and fermenting tree sap. Both adults and larvae are usually predators on micro-arthropods and possibly other microinvertebrates. While ecologically diverse, the members of Athetini generally show little morphological variation, and the tribe is considered the taxonomically most challenging group within the Aleocharinae (Newton et al., 2000). The classiﬁcation of Athetini has been H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 unstable, and several conﬂicting and competing classiﬁcations for the tribe exist (see Yosii and Sawada, 1976; Seevers, 1978; Muona, 1979; Lohse et al., 1990; Newton et al., 2000; Smetana, 2004). An important reason for this conﬂict is that all classiﬁcations of Athetini following the World catalogue of Bernhauer and Scheerpeltz (1926) and Scheerpeltz (1934) have been based on regional faunas, resulting in only partial overlap in the taxon coverage. Another reason is the high level of conﬂict in the morphological characters themselves. Even the tribe itself is not well characterized, and its monophyly has never been tested in a rigorous phylogenetic analysis. Seevers (1978) considered the so-called ‘‘athetine bridge” of the aedeagus (see e.g. Muona, 1987: Fig. 1) a possible diagnostic character of the tribe. However, the ‘‘athetine bridge” is present in several other tribes as well, e.g. Lomechusini Fleming, 1821, and may be a synapomorphy for a larger clade. Athetini were originally introduced as ‘‘group Athetae” (Casey, 1910), a subtribe of the tribe Myrmedoniini Thomson, 1867 (currently, the valid name for Myrmedoniini is Lomechusini). For some time the athetines were treated as a subtribe within Myrmedoniini (e.g. Fenyes, 1918; Bernhauer and Scheerpeltz, 1926; Scheerpeltz, 1934), which included additional subtribes now recognized as separate tribes or placed in tribes other than Athetini and Lomechusini. Fenyes (1921) appears to be the ﬁrst to refer to Athetini as a tribe, but he simply used the name as a replacement for Myrmedoniini on the grounds that the genus Atheta Thomson, 1858 was larger and more representative for the tribe than Myrmedonia Erichson, 1837 (=Drusilla Leach, 1819). Eventually, Athetini became accepted as a tribe separate from Lomechusini (Myrmedoniini) (Benick and Lohse, 1974; Seevers, 1978; Newton et al., 2000; Smetana, 2004). In most classiﬁcations, Atheta is by far the largest genus in Athetini, but the delimitation of Atheta varies substantially between authors. For example, Seevers (1978) recognized only two species of Atheta in his North American checklist, stating that the genus comprised ‘‘less than a dozen species” worldwide. In contrast, the Catalogue of Palaearctic Coleoptera (Smetana, 2004) lists 843 85 valid species of Atheta. When treated in the broader sense, Atheta is usually subdivided in multiple subgenera (the Palaearctic Catalogue lists 47). However, the systematic ranks of these genusgroup names tend to vary between authors or sometimes even between papers of the same author (see discussion in Muona, 1995). The overall trend is to raise rank, and many taxa listed as subgenera of Atheta in early catalogues are today treated as genera (cf. Bernhauer and Scheerpeltz, 1926 and Smetana, 2004). None of the ranking decisions have been backed by phylogenetic analyses, and while other athetine genera are usually supported by unique morphological characters (e.g. Lohse, 1971; Benick and Lohse, 1974), the genus Atheta appears to be deﬁned by a combination of plesiomorphic character states only. Few phylogenetic analyses involving Athetini have been published, and none of these addressed the phylogeny of Athetini in detail. Steidle and Dettner (1993) investigated the abdominal tergal gland of adult Aleocharinae, and included a phylogenetic analysis of 11 aleocharine tribes using ﬁve morphological and four chemical characters. Athetini were represented by eight species belonging to four genera, but phylogenetic relationships within the tribe were not addressed as all athetines were lumped in a single terminal taxon. Athetini formed a trichotomy with Myrmedoniini (=Lomechusini) and Aleocharini Fleming, 1821, with Oxypodini Thomson, 1859 forming a sister group to the three tribes. Ahn and Ashe (2004) used morphological characters of adult beetles to investigate the phylogeny of the tribe Myllaenini Ganglbauer, 1895 and included in their analysis two representatives of Athetini (Atheta and Pontomalota Casey, 1885). Athetini was not recovered as monophyletic: the clade containing the two athetine genera also included Lomechusini and Oxypodini (both represented by two genera). Ashe (2005) used larval and adult morphology to investigate the basal phylogeny of Aleocharinae and included three representatives of Athetini (Atheta, Geostiba Thomson, 1858, and Pontomalota). He found strong support for a monophyletic ‘‘higher” aleocharine clade, excluding four tribes of ‘‘basal” Aleocharinae: Gymnusini Heer, 1989, Deinopsini Sharp, 1883, Trichopseniini Fig. 1. Saturation plots for seven data partitions. (A–C) Mitochondrial protein coding regions (CO1, CO2, and partial NADH1); (A) 1st codon positions; (B) 2nd codon positions; (C) 3rd codon positions; (D–E) mitochondrial RNA coding regions (Leu1, Leu2, and partial 16S); (D) paired sites; (E) unpaired sites; (F–G) nuclear RNA coding region (partial 18S gene); (F) paired sites; and (G) unpaired sites. The choice of substitution models is based on the Akaike information criterion. 86 H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 LeConte and Horn, 1883, and Mesoporini Cameron, 1959. The three members of Athetini nested among the ‘‘higher” Aleocharinae, but resolution within this clade was low and Athetini was not retrieved as monophyletic. The only published phylogenetic study of the Aleocharinae based on molecular markers (Thomas, 2009) included two mitochondrial genes (12S and 16S) and eight aleocharine tribes. Athetini were represented by three taxa (Atheta sp., Leptonia sp., and Sableta infulata Casey, 1910). The very limited dataset proved insufﬁcient for testing the monophyly of Athetini or for resolving relationships between Athetini and other tribes. Despite the lack of a formal phylogeny addressing the tribe Athetini, there are several relatively recent classiﬁcations that can be treated as preliminary phylogenetic hypotheses based on an evaluation of morphological characters (Yosii and Sawada, 1976; Seevers, 1978; Muona, 1979; Lohse et al., 1990; Newton et al., 2000; Smetana, 2004). Some of these works explicitly list and/or discuss morphological characters supporting particular suprageneric groups (Yosii and Sawada, 1976; Seevers, 1978; Newton et al., 2000), while others simply present classiﬁcation as a key, catalogue, or checklist (Muona, 1979; Lohse et al., 1990; Smetana, 2004), presumably based on a synthesis of available information (see e.g. Muona, 1987). Table 1 lists all previously proposed suprageneric groups within the Athetini. From the position of the Code (Newton and Thayer, 1992; ICZN, 1999) some of the proposed names are not available. Some were proposed as informal names (e.g. as ‘‘series” (Yosii and Sawada, 1976) or ‘‘groups” (Seevers, 1978)), others have failed to meet certain requirements of the Code (e.g. some names introduced by Seevers (1978) and Muona (1979)). However, regardless of name availability, they all represent morphology-based phylogenetic hypotheses amenable to testing. The goal of this study is to infer a phylogeny for the tribe Athetini by means of molecular markers. We aim to investigate the relationship between genera and suprageneric groups within the Athetini, and to test the monophyly of some of these groups. An important subgoal of this study is to test the monophyly of the large genus Atheta and some of its many proposed subgenera. 2. Material and methods 2.1. Taxon selection and sampling A total of 81 species representing 11 aleocharine and 1 tachyporine tribe were included in this study (Table 2). The tribe Athetini was represented by 58 species in 27 genera, 20 of which were represented by single species while 6 were represented by 2–3 species each. The genus Atheta was represented by 25 species in 11 subgenera, 6 of which were represented by 2–4 species. Atheta s. str. (in the sense of Benick and Lohse, 1974) was represented by 2 species including the type of the genus, A. graminicola (Gravenhorst, 1806). The main objective of this study was to investigate relationships between suprageneric groups within the Athetini, and to this aim we seeked to maximize the number of suprageneric groups represented in our study. Our dataset included representatives of 16 suprageneric groups, of which 14 were represented with their type genus and 7 also with the type species of the type genus. Table 1 lists all proposed suprageneric groups within Athetini and their representation in this study. In addition to the Athetini, 9 additional tribes of ‘‘higher” Aleocharinae were represented. The tribes Lomechusini and Homalotini Heer, 1839 were each represented by 4 species in 3 genera. Other tribes were represented by just single species. The ‘‘basal” Aleocharinae were represented by Gymnusa variegata Kiesenwetter, 1845 from the tribe Gymnusini. Tachinus proximus Kraatz, 1855 from the subfamily Tachyporinae MacLeay, 1825 was included as a non-aleocharine outgroup taxon. Also included were 4 aleocharine genera whose current tribal placements are unresolved or doubtful: Halobrecta Thomson, 1858, Meronera Sharp, 1887, Thamiaraea Thomson, 1858, and Thendelecrotona Paśnik, 2007. All specimens were collected directly into 96–100% ethanol and stored at 20 °C prior to sorting and extraction. Label information for the included specimens is provided in Supplementary Table 1. The initial systematic assignment of the included species (Table 2) is based on a compromise between several non-fully compatible views on the systematics of Athetini (Brundin, 1952; Benick and Lohse, 1974; Seevers, 1978; Muona, 1979; Newton et al., 2000; Smetana, 2004). 2.2. DNA extraction, PCR ampliﬁcation and sequencing DNA was extracted from a leg, the head, or head plus prothorax using the Qiagen DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer’s protocol for animal tissue. DNA extraction was performed on vacuum dried samples without prior crushing, and exoskeletons were retrieved afterwards for dry mounting with the rest of the voucher. DNA yield was low due to the small amount of tissue, and most extractions were therefore optimized for increased yield and concentration by using extended lysis time (24 h) and reduced amount of elution buffer (2 100 ll). Three target regions were ampliﬁed by PCR using the primer combinations listed in Table 3. The ﬁrst region covered most of the mitochondrial cytochrome oxidase subunit 1 and 2 (CO1 and CO2) genes and the tRNA-Leucine 2 (Leu2) gene, and was ampliﬁed in three overlapping fragments. The second region covered the 30 end of the mitochondrial 16S ribosomal RNA (16S) gene, the tRNALeucine 1 (Leu1) gene, and a small part of the NADH dehydrogenase subunit 1 (NADH1) gene. The third region covered an internal part of the nuclear 18S ribosomal RNA (18S) gene. Most PCR ampliﬁcations were set up in 25 ll reaction volume containing 3 ll template DNA extract, 2.5 mM MgCl2 (Applied Biosystems), 1 ABI GeneAmp PCR buffer (Applied Biosystems), 0.8 mM GeneAmp dNTPs (Applied Biosystems), 0.5 lM of each primer (MWG-Biotech AG), and 1 U ABI AmpliTaq DNA Polymerase (Applied Biosystems). The following modiﬁcations were done when amplifying the 16S-Leu1-NADH1 fragment: MgCl2: 2 mM, PCR buffer: 0.96, dNTPs: 0.64 mM, primers: 0.4 lM of each. Mitochondrial targets were ampliﬁed using an initial denaturation step of 94 °C for 3000 , followed by 30 cycles of 94 °C for 10 , annealing temperature Ta for 3000 , and 72 °C for 20 , and a ﬁnal extension step at 72 °C for 100 . For the nuclear target region the initial denaturation step was extended to 20 and the number of cycles was increased to 35. Annealing temperatures are provided in Table 3. PCR products were puriﬁed using ExoSAP-IT (Stratagene). If secondary products were obtained, the product with the expected length was cut out from 1% agarose gel and puriﬁed using the MN NucleoSpin Extract II gel extraction kit (Macherey–Nagel). Puriﬁed PCR products were sequenced using the ABI BigDye Terminator Cycle Sequencing Kits v1.1 and v3.1 (Applied Biosystems) following basically the manufacturer’s instructions. PCR products were sequenced in both directions using the original PCR primers and some internal sequencing primers (Table 3). Cycle sequencing products were cleaned using Sephadex (GE Healthcare) and subsequently analyzed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Voucher specimens are deposited at the Natural History Museum, University of Oslo (ZMUN). 2.3. Sequence alignment Alignment of the CO1, CO2, and NADH1 genes was straightforward. For the 16S and 18S rRNA genes, secondary structure was Table 1 Representation of suprageneric groups of Athetini in the present study and in recent classiﬁcations of the tribe. Taxon name d, Represented taxa; –, not represented taxa. u Unavailable name. i Informal name. 1 Liogluta. 2 Pontomalota and Tarphiota. 3 Trichiusa. 4 As Acrotona series. 5 As Atheta series. 6 As Acrotonae. 7 As Athetae. 8 As Dimetrotae. 9 As Geostibae. 10 As Thamiaraeae or Thamiaraea group. Recent classiﬁcations The type genus The type species of the type genus Yosii and Sawada (1976) Seevers (1978) Muona (1979) Lohse et al. (1990) Newton et al. (2000) Smetana (2004) d d d d d – d d – d – d d – – d – d d – – d d – d d d d – – – d d – d – d d1 – – d – d2 d – – d d3 – d – d d – – – d – – d – – – – – – – – d – – d – – d d4 – d5 – d – – – – – – – d – – – – – – – – – – – – d6 – d7 – – – – d8 d d9 d – – – – – – d – – – d10 d – d – d d d – – d – – – – d – – – – – – – – – d – d – – – d d – – – – – – – d – – – – – – – – – – – – – d – d – – – – d – d – – – – – – – – – – – – – – – – – d – – – – – – – – – – – – – d – – d – d – – – H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 Acrotonina (Seevers, 1978) Amischina Muona, 1979u Athetina Casey, 1910 Callicerina Jacobson, 1908 Coprothassa series (Yosii and Sawada, 1976)i Coptotermoeciina Kistner and Pasteels, 1970 Dadobiina Muona, 1979u Dimetrotina (Seevers, 1978) Doliponta group (Seevers, 1978)i Geostibina (Seevers, 1978) Goniusa group (Seevers, 1978)i Hydrosmectina Muona, 1979u Liogluta series (Yosii and Sawada, 1976)i Microceroxenina Kistner, 1970 Nasutiphilina Kistner, 1970 Plagiarthrina Cameron, 1926 Schistogeniina Fenyes, 1918 Sea-shore group (Seevers, 1978)i Strigotina Casey, 1910 Taxicerina Lohse, 1989 Termitotelina Kistner, 1970 Thamiaraeina Fenyes, 1921 Trichiusa group (Seevers, 1978)i Trichomicrina Muona, 1979u Xenotae (Seevers, 1978)u Representation in our study At least one species 87 Species name Tribe/subtribe ZMUN barcode label GenBank Accession numbers Country CO1-Leu2-CO2 16S-Leu1-NADH1 18S Tachyporini 10002542 GQ980859 GQ980968 GQ981067 Norway ‘‘Basal” Aleocharinae Gymnusa variegata Kiesenwetter, 1845 Gymnusini 10002641 GQ980860 GQ980969 GQ981068 Romania Cordalia obscura (Gravenhorst, 1802) Aleocharini Aleocharini Ecitocharini Falagriini 10002579 10002570 10002592 10002651 GQ980861 GQ980862 GQ980863 GQ980864 GQ980970 GQ980971 GQ980972 GQ980973 GQ981069 GQ981070 – GQ981071 Norway Norway Peru Greece Bolitochara pulchra (Gravenhorst, 1806) Homalotini 10002591 GQ980865 – GQ981072 Norway Bolitochara pulchra (Gravenhorst, 1806) Gyrophaena congrua Erichson, 1837 Gyrophaena fasciata (Marsham, 1802) Gyrophaena fasciata (Marsham, 1802) Homalotini 10002596 GQ980866 GQ980974 GQ981073 Norway Silusida marginella (Casey, 1893) Homalotini Homalotini Homalotini Homalotini 10002584 10002585 10002572 10002625 GQ980867 GQ980868 GQ980869 GQ980870 GQ980975 GQ980976 GQ980977 GQ980978 GQ981074 GQ981075 GQ981076 GQ981077 Norway Norway Norway USA Silusida marginella (Casey, 1893) Homalotini 10002624 GQ980871 GQ980979 GQ981078 USA Hoplandria lateralis (Melsheimer, 1846) Hoplandriini 10002550 GQ980872 GQ980980 GQ981079 USA Drusilla canaliculata (Fabricius, 1787) Lomechusini 10002604 GQ980873 GQ980981 GQ981080 Norway Drusilla canaliculata (Fabricius, 1787) Myrmedonota sp. Myrmedonota sp. Pella caliginosa (Casey, 1893) Pella caliginosa (Casey, 1893) Pella humeralis (Gravenhorst, 1802) Myllaena audax Casey, 1911* Oxypoda praecox Erichson, 1839 Placusa sp. prope tachyporoides (Waltl, 1838) Lomechusini 10002601 GQ980874 GQ980982 GQ981081 Norway Lomechusini Lomechusini Lomechusini Lomechusini Lomechusini Myllaenini Oxypodini Placusini 10002615 10002614 10002617 10002616 10002569 10002598 10002637 10002541 GQ980876 GQ980877 GQ980878 GQ980879 GQ980880 GQ980881 GQ980882 GQ980883 GQ980984 GQ980985 GQ980986 GQ980987 GQ980988 – GQ980989 GQ980990 GQ981083 GQ981084 GQ981085 GQ981086 GQ981087 GQ981088 GQ981089 GQ981090 USA USA USA USA Norway USA Germany USA Strigotina Strigotina Strigotina Strigotina Strigotina 10002544 10002543 10002547 10002632 10002588 GQ980884 GQ980885 GQ980886 GQ980887 GQ980888 GQ980991 GQ980992 GQ980993 GQ980994 GQ980995 GQ981091 GQ981092 GQ981093 GQ981094 GQ981095 USA USA USA Russia Germany Tribe Athetini Acrotona sp. prope Acrotona sp. prope Acrotona sp. prope Lypoglossa lateralis assecla (Casey, 1910) austiniana (Casey, 1910) austiniana (Casey, 1910) (Mannerheim, 1830) Mocyta fungi (Gravenhorst, 1806) Strigotina 10002589 GQ980889 GQ980996 GQ981096 Germany Nehemitropia lividipennis (Mannerheim, 1830) Strigotina Strigotina Strigotina 10002540 10002559 10002648 GQ980890 GQ980891 GQ980892 GQ980997 GQ980998 GQ980999 GQ981097 GQ981098 GQ981099 USA USA Greece Strigota ambigua (Erichson, 1839) Strigotina 10002571 GQ980893 GQ981000 GQ981100 USA Strigota ambigua (Erichson, 1839) Strigotina 10002575 GQ980894 GQ981001 GQ981101 USA Amischa analis (Gravenhorst, 1802)* Amischa nigrofusca (Stephens, 1832)* Alpinia sp. prope alpicola (Miller, 1859)* Amischina 10002623 GQ980895 – GQ981102 Norway Amischina Athetina Athetina 10002622 10002644 10002646 GQ980896 GQ980897 GQ980898 – – GQ981002 GQ981103 GQ981104 GQ981105 Norway Romania Norway Athetina Athetina Athetina Athetina Athetina 10002619 10002618 10002578 10002580 10002653 GQ980899 GQ980900 GQ980901 GQ980902 GQ980903 GQ981003 GQ981004 GQ981005 GQ981006 GQ981007 GQ981106 GQ981107 GQ981108 GQ981109 – USA USA Norway Norway France Mocyta fungi (Gravenhorst, 1806) Mocyta scopula (Casey, 1893) Mocyta scopula (Casey, 1893) Amidobia talpa (Heer, 1841) Atheta (?) klagesi Bernhauer, 1909 Atheta (?) klagesi Bernhauer, 1909 Atheta (Alaobia) gagatina (Baudi di Selve, 1848) Atheta (Alaobia) gagatina (Baudi di Selve, 1848) Atheta (Alaobia) membranata G. Benick, 1974* H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 Subfamily Tachyporinae Tachinus proximus Kraatz, 1855 ‘‘Higher” Aleocharinae excluding Athetini Aleochara moerens Gyllenhal, 1827 Aleochara moerens Gyllenhal, 1827 Ecitophya gracillima Mann, 1925* 88 Table 2 List of specimens used in this study. Sub- and suprageneric placements are based on a synthesis of available literature. Included type species of the respective genera and Atheta subgenera are underlined. Species with incomplete sequence data are marked with an asterisk (*). Full label information is listed in Supplementary Table 1. Atheta Atheta Atheta Atheta Atheta Atheta (Alaobia) pallidicornis (Thomson, 1856) (Alaobia) pallidicornis (Thomson, 1856) (Ceritaxa) pervagata G. Benick, 1974 (crassicornis-gr.) crassicornis (Fabricius, 1793) (crassicornis-gr.) modesta (Melsheimer, 1844) (crassicornis-gr.) modesta (Melsheimer, 1844) 10002627 10002626 10002652 10002640 10002621 10002620 10002560 GQ980904 GQ980905 GQ980906 GQ980907 GQ980908 GQ980909 GQ980910 GQ981008 GQ981009 GQ981010 GQ981011 GQ981012 GQ981013 GQ981014 GQ981110 GQ981111 GQ981112 GQ981113 GQ981114 GQ981115 GQ981116 Czech Republic Czech Republic Hungary Hungary USA USA Norway Athetina 10002556 GQ980911 GQ981015 GQ981117 Norway Athetina Athetina Athetina Athetina Athetina Athetina Athetina Athetina Athetina 10002554 10002583 10002582 10002545 10002558 10002639 10002564 10002566 10002606 GQ980912 GQ980913 GQ980914 GQ980915 GQ980916 GQ980917 GQ980918 GQ980919 GQ980920 GQ981016 GQ981017 GQ981018 GQ981019 GQ981020 GQ981021 GQ981022 GQ981023 GQ981024 GQ981118 GQ981119 GQ981120 GQ981121 GQ981122 GQ981123 GQ981124 GQ981125 GQ981126 USA Norway Norway USA USA Romania USA Norway Norway Atheta (s. str.) graminicola (Gravenhorst, 1806) Athetina Athetina Athetina Athetina Athetina Athetina Athetina 10002642 10002638 10002586 10002548 10002557 10002635 10002561 GQ980921 GQ980922 GQ980923 GQ980924 GQ980925 GQ980926 GQ980927 GQ981025 GQ981026 GQ981027 GQ981028 GQ981029 GQ981030 GQ981031 GQ981127 GQ981128 GQ981129 GQ981130 GQ981131 GQ981132 GQ981133 France Romania Kenya Norway Norway Romania Norway Atheta (s. str.) graminicola (Gravenhorst, 1806) Athetina 10002562 GQ980928 GQ981032 GQ981134 Norway Atheta (Thinobaena) vestita (Gravenhorst, 1806) Atheta (vaga-group) vaga (Heer, 1839)* Athetina 10002613 GQ980929 GQ981033 GQ981135 Norway Athetina Athetina 10002655 10002590 GQ980930 GQ980931 GQ981034 GQ981035 – GQ981136 France Norway Athetina 10002573 GQ980932 GQ981036 GQ981137 Norway Athetina Athetina Athetina 10002634 10002633 10002643 GQ980933 GQ980934 – GQ981037 GQ981038 GQ981039 GQ981138 GQ981139 GQ981140 Russia Russia France Athetina 10002546 GQ980935 GQ981040 GQ981141 Czech Republic Athetina Athetina Athetina Athetina 10002602 10002600 10002636 10002649 GQ980936 GQ980937 GQ980938 GQ980939 – GQ981041 – GQ981042 GQ981142 GQ981143 GQ981144 GQ981145 Czech Republic Czech Republic Russia Norway Athetina 10002565 GQ980940 GQ981043 GQ981146 USA Athetina Athetina Athetina Athetina Athetina Athetina Athetina Athetina 10002607 10002608 10002610 10002609 10002595 10002594 10002605 10002567 GQ980941 GQ980942 GQ980943 GQ980944 GQ980945 GQ980946 GQ980947 GQ980948 GQ981044 GQ981045 GQ981046 GQ981047 GQ981048 GQ981049 GQ981050 GQ981051 GQ981147 GQ981148 GQ981149 GQ981150 GQ981151 GQ981152 GQ981153 GQ981154 Norway Norway Czech Republic Czech Republic USA USA Norway USA Athetina 10002568 GQ980949 GQ981052 GQ981155 USA Athetina Athetina Athetina Dadobiina 10002599 10002628 10002597 10002630 GQ980950 GQ980951 GQ980952 GQ980953 GQ981053 GQ981054 – GQ981055 GQ981156 GQ981157 GQ981158 GQ981159 USA USA USA Norway Geostibina 10002587 GQ980954 GQ981056 GQ981160 Norway Pontomalota opaca (LeConte, 1863) Hydrosmectina ‘‘Sea-shore genera” 10002650 10002577 GQ980955 GQ980956 GQ981057 GQ981058 GQ981161 GQ981162 USA USA Pontomalota opaca (LeConte, 1863) ‘‘Sea-shore genera” 10002574 GQ980957 GQ981059 GQ981163 USA Atheta (Datomicra) celata (Erichson, 1837) Atheta Atheta Atheta Atheta Atheta Atheta Atheta Atheta Atheta (Datomicra) celata (Erichson, 1837) (Datomicra) dadopora (Thomson, 1867) (Dimetrota) aeneipennis (Thomson, 1856) (Dimetrota) cinnamoptera (Thomson, 1856) (Dimetrota) hampshirensis Bernhauer, 1909 (Dimetrota) hampshirensis Bernhauer, 1909 (Dimetrota) setigera (Sharp, 1869) sp. ex gr. lippa (Microdota) subtilis (W. Scriba, 1866) Atheta Atheta Atheta Atheta Atheta Atheta Atheta (Mycetota) laticollis (Stephens, 1832) (Mycetota) pasadenae Bernhauer, 1906 (Oreostiba) bosnica Ganglbauer, 1895 (Oxypodera) kenyamontis Pace, 1986 (ravilla-group) ravilla (Erichson, 1839) (ravilla-group) ravilla (Erichson, 1839) (s. str.) contristata (Kraatz, 1856) Atheta (Xenota) myrmecobia (Kraatz, 1856) Atheta (Xenota) myrmecobia (Kraatz, 1856) Boreophilia hyperborea (Brundin, 1940) Boreostiba sp. Dalotia coriaria (Kraatz, 1856)* Dinaraea aequata (Erichson, 1837) Liogluta microptera Thomson, 1867 Liogluta microptera Thomson, 1867 Liogluta nigropolita (Bernhauer, 1907)* Lyprocorrhe anceps (Erichson, 1837) Micratheta caudex (Casey, 1910) Philhygra debilis (Erichson, 1837) Philhygra debilis (Erichson, 1837) Philhygra fallaciosa (Sharp, 1869) Philhygra fallaciosa (Sharp, 1869) Philhygra iterans (Casey, 1910) Philhygra iterans (Casey, 1910) Schistoglossa gemina (Erichson, 1837) Stethusa dichroa (Gravenhorst, 1802) Stethusa dichroa (Gravenhorst, 1802) Stethusa spuriella (Casey, 1910) Stethusa spuriella (Casey, 1910) Tomoglossa ornatella (Casey, 1910)* Dadobia immersa (Erichson, 1837) Geostiba circellaris (Gravenhorst, 1806) Hydrosmecta sp. 89 (continued on next page) H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 Athetina Athetina Athetina Athetina Athetina Athetina Athetina South Africa GQ981173 GQ981066 GQ980967 USA USA USA USA France GQ981165 GQ981166 GQ981167 GQ981168 GQ981169 10002612 Tribe unknown Thamiaraea cinnamomea (Gravenhorst, 1802)* Thendelecrotona sp. 10002553 10002552 10002551 10002563 10002629 Athetini/Thamiaraeini Athetini/Thamiaraeini Athetini/Thamiaraeini Athetini/Thamiaraeini Athetini/Thamiaraeini GQ980959 GQ980960 GQ980961 GQ980962 GQ980963 GQ981061 GQ981062 – – – Greece USA GQ981172 GQ981082 10002647 10002576 Athetini/Oxypodini Lomechusini (?) Meronera venustula (Erichson, 1839) Thamiaraea americana Bernhauer, 1907 Thamiaraea americana Bernhauer, 1907 Thamiaraea brittoni (Casey, 1911)* Thamiaraea brittoni (Casey, 1911)* Taxa with uncertain tribal assignment Halobrecta sp. cf. halensis Mulsant and Rey, 1873 Trichiusina Athetini incertae sedis GQ980966 GQ980875 GQ981065 GQ980983 USA GQ981164 GQ981170 GQ981171 GQ981060 GQ981063 GQ981064 GQ980958 10002593 10002611 10002539 ‘‘Sea-shore genera” Tarphiota fucicola (Mäklin in Mannerheim, 1852) Trichiusa ursina Notman, 1920 Earota dentata (Bernhauer, 1906) GQ980964 GQ980965 18S 16S-Leu1-NADH1 ZMUN barcode label GenBank Accession numbers The level of sequence saturation was assessed by plotting p-distances against corrected genetic distances for all pairs of sequences. Separate plots were made for paired and unpaired regions of the pooled mitochondrial RNA genes, paired and unpaired regions of the 18S gene, and 1st, 2nd and 3rd codon positions of the pooled protein coding genes. P-distances were calculated in PAUP* 4.0b10 (Swofford, 2002), while corrected genetic distances were calculated in distphase (PHASE package: Jow et al., 2002) using best-ﬁtting substitution models determined with Modeltest 3.7 (Posada and Crandall, 1998) under the Akaike information criterion. Only taxa with sequence information for all three target regions were included in this analysis. Tribe/subtribe CO1-Leu2-CO2 inferred through comparison with published secondary structures of Apis mellifera (Gillespie et al., 2006), and used as a guide for manual sequence alignment in Mega 3.1 (Kumar et al., 2004). Published secondary structures of Xenos vesparum (Carapelli et al., 2006) were likewise used for aligning the Leu1 and Leu2 tRNA genes. Conspeciﬁc samples generally yielded identical or highly similar sequences, and in these cases a consensus sequence was inferred for the species for use in the downstream analyses. The morphology-based assessments of conspeciﬁcity were ﬁrst veriﬁed through Neighbor-Joining analyses of each of the three target regions, and by assessing the level of within- and between-species sequence variation in the CO1/CO2 region. With a few exceptions, within-species sequence variation was in the range of 0–1.1%, while between-species sequence divergence exceeded 6.2%. Conspeciﬁc sequences which differed by more than 1.1% in the CO1/ CO2 region, or did not group as monophyletic in the initial Neighbor-Joining analysis, were kept separate in downstream analyses. 2.4. Sequence saturation Species name Table 2 (continued) USA USA H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 Country 90 2.5. Partition homogeneity The dataset was tested for incongruence between partitions using the incongruence length difference (ILD) test of Farris et al. (1995) implemented as Hompart option in PAUP* 4.0b10. The three target regions were tested against each other, and each gene was tested against each of the other six. Constant sites were excluded prior to comparison as recommended by Cunningham (1997). Only taxa with sequence information for all three target regions were included in this analysis. 2.6. Phylogenetic analyses Maximum Parsimony analyses were performed in TNT 1.1 (Goloboff et al., 2003) using ratchet, fusing, and sectorial searches with 2000 initial addition sequences. Gaps were treated as missing data. Bootstrap support values were calculated using 1000 pseudoreplicates, each with 50 initial addition sequences. The main parsimony analysis was performed using implied weighting (Goloboff, 1993) with the constant of concavity set to 3 (moderate downweighting of homoplastic characters). Only taxa with sequence information for all three target regions were included in this analysis. Five additional analyses were performed exploring alternative weighting schemes or datasets: (1) equal character weights, (2) all taxa included, (3) third codon positions excluded, (4) mitochondrial markers only, and (5) nuclear marker only. Bayesian analyses were performed in PHASE 2.0 (Jow et al., 2002) and in MrBayes 3.1 (MPI version) (Ronquist and Huelsenbeck, 2003). Seven data partitions were deﬁned and modeled independently: paired and unpaired regions of the nuclear 18S gene, paired and unpaired regions of the mitochondrial RNA genes (pooling Leu1, Leu2, and 16S together), and 1st, 2nd and 3rd codon positions of the mitochondrial protein coding genes (pooling CO1, CO2, 91 H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 Table 3 Primers used for PCR ampliﬁcation and DNA sequencing. Positiona Primer site Sequence (50 –30 ) Reference TY-J-1460 1460 LCO1490 1514 C1-J-1718 1718 HCO2198 2173 C1-J-2183 2183 a2411b 2411 C1-2416ra 2416 C1-2441 fa 2441 C1-2798fb 2798 C1-2807fb 2807 TL2-N-3014 3014 b C2-3045 fa 3045 C2-3368rcb 3368 C2-N-3389b 3389 C2-N-3661 3661 C2-3772 ra 3772 N1-J-12585 12585 LR-N-13398 13398 18s ai 446 18s bi 1393 PCR primer combinations Forward primer Reverse primer Tyr CO1 CO1 CO1 CO1 CO1 CO1 CO1 CO1 CO1 Leu2 Leu2 CO2 CO2 CO2 CO2 NADH1 16S 18S 18S TAC AAT TTA TCG CCT AAA CTT CAG CC GGT CAA CAA ATC ATA AAG ATA TTG G GGA GGA TTT GGA AAT TGA TTA GTT CC TAA ACT TCA GGG TGA CCA AAA AAT CA CAA CAT TTA TTT TGA TTT TTT GG GCT AAT CAT CTA AAA ACT TTA ATT CCW GTW G GCT AAT CAT CTA AAA ATT TTA ATT CC CCT ACA GGA ATT AAA ATT TTT AGT TGA TAA GC CCT CGA CGT TAT TCA GAT TAC CC TAT TCT GAT TAT CCA GAT GCT TA TCC AAT GCA CTA ATC TGC CAT ATT A CAG ATT AGT GCA ATG GAT TTA AGC CAT TGA TGT CCA ATA GTT TTA ATA GT TCA TAA GTT CAR TAT CAT TG CCA CAA ATT TCT GAA CAT TGA CCA GAG ACC ATT ACT TAC TTT CAG CCA TCT GGT CCC TTA CGA ATT TGA ATA TAT CCT CGC CTG TTT AAC AAA AAC AT CCT GAG AAA CGG CTA CCA CAT C GAG TCT CGT TCG TTA TCG GA Simon et al. (1994) Folmer et al. (1994) Simon et al. (1994) Folmer et al. (1994) Simon et al. (1994) Normark et al. (1999) Modiﬁcation of C1-J-2441; Simon et al. (1994) Modiﬁcation of C1-J-2441; Simon et al. (1994) Modiﬁcation of C1-J-2797; Simon et al. (1994) Modiﬁcation of C1-J-2797; Simon et al. (1994) Simon et al. (1994) Modiﬁcation of TL2-J-3037; Simon et al. (1994) Modiﬁcation of C2-N-3389; Simon et al. (1994) Simon et al. (1994) Simon et al. (1994) Modiﬁcation of TK-N-3785; Simon et al. (1994) Simon et al. (1994) Simon et al. (1994) Whiting et al. (1997) Whiting et al. (1997) Target gene(s) Fragment length Annealing temp. (°C) LCO1490 C1-J-2183 C1-2441 fa N1-J-12585 18s ai LCO1490 TY-J-1460 TY-J-1460 C1-J-1718 C1-2441 fa C1-2807 fb CO1 CO1 + Leu2 CO1 + Leu2 + CO2 16S + Leu1 + NADH1 18S CO1 CO1 CO1 + Leu2 CO1 + Leu2 CO1 + Leu2 + CO2 CO1 + Leu2 + CO2 902 831 1331 813 947 659 956 1554 1296 1220 854 45 56 53 49–53 70–74 45 45 45 45 52–58 45 Primer name C1-2416ra TL2-N-3014 C2-3772 ra LR-N-13398 18s bi HCO2198 C1-2416ra TL2-N-3014 TL2-N-3014 C2-N-3661 C2-N-3661 a Positions of mitochondrial primers relate to the Drosophila yacuba mitochondrial genome (Clary and Wolstenholme, 1985). Positions of 18S primers relate to the Apis mellifera 18S gene (Gillespie et al., 2006). b Internal sequencing primer. and NADH1 together). Separate analyses were run with taxa lacking information for one of the three target regions either included or excluded. Analyses in PHASE were performed using the model RNA7D+dG6+I for paired sites. The model was chosen as a biologically plausible model of paired-site evolution. Unpaired regions and codon positions were modeled using the most general time reversible model (REV+dG6+I). Paired sites lacking information for one of the paired bases were treated as unpaired. Each analysis was performed with four independent runs using different starting seeds. The analyses were allowed a burn-in of 10 million generations followed by 20 million generations of sampling every 200 generations. Posterior trees were summarized in a majority rule consensus tree. Branch lengths were estimated with optimizer (PHASE package), and averaged over ten independent replicates. Analyses in MrBayes were performed using the model HKY+I+C for 3rd codon positions and GTR+I+C for the six other partitions. Models were selected using MrModelTest 2.2 (Nylander, 2004) under the Akaike information criterion. Model parameters were allowed to evolve during the run, starting with ﬂat priors. Each analysis was performed using the default settings of two independent runs, each with three heated and one cold chain. The analyses were allowed a burn-in of 25 million generations followed by 75 million generations of sampling every 1000 generations. Branch lengths were inferred during the run, and posterior trees were summarized in a majority rule consensus tree. All Bayesian analyses were performed at the Bioportal computer facility (http://www.bioportal.uio.no) at the University of Oslo, Norway. 3. Results 3.1. Alignment The concatenated sequence alignment contained 85 sequences and 3865 positions after trimming (Table 4). A total of 237 positions were excluded from all downstream analyses due to ambiguous sequence alignment. Of the remaining characters, 2190 were constant, 258 were uninformative, and 1180 were parsimony informative. Complete sequences were obtained for 72 terminal taxa, while 13 terminal taxa lacked data for one of the three target regions (Table 2). The sequence alignment is provided as Online supplementary material. 3.2. Sequence saturation and partition homogeneity The saturation plots for seven partitions are shown in Fig. 1. Only 3rd codon positions showed high levels of saturation in our dataset. Moderate levels were indicated for 1st codon positions, while all other partitions showed fairly low levels of saturation. Paired RNA coding sites were less saturated than unpaired sites, and the nuclear 18S gene was generally less saturated than the mitochondrial genes. The ILD tests (Table 5) revealed statistically signiﬁcant incongruence between 18S and several of the mitochondrial genes (16S, Leu1, Leu2, and NADH1), and also between 16S and the NADH1 and CO2 genes. However, several of the included genes contained very few parsimony informative characters (i.e. Leu1, Leu2, and NADH1, Table 4), and there was no signiﬁcant conﬂict when the target regions were tested against each other in their 92 H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 Table 4 Details on the concatenated alignment of the three target regions used in this study. Gene Partition Excluded positions Constant Uninformative Parsimony informative 16S Paired sites Unpaired sites – 123a 163 150 39 12 77 64 279 349 Leu1 Paired sites Unpaired sites – 12a 24 16 6 2 8 5 38 35 NADH1 1st codon positions 2nd codon positions 3rd codon positions – – – 1 8 1 3 3 1 14 7 16 18 18 18 18S Paired sites Unpaired sites 52b 34b 449 323 27 24 33 57 561 438 CO1 1st codon positions 2nd codon positions 3rd codon positions 1c 1c 1c 328 435 10 32 30 17 131 26 464 492 492 492 b c Total CO1-Leu2 boundary Noncoding sites 2 – – – 2 Leu2 Paired sites Unpaired sites – 11a 24 14 8 3 8 13 40 41 CO2 1st codon positions 2nd codon positions 3rd codon positions – – – 98 142 4 25 15 11 61 27 169 184 184 184 237 2190 258 1180 3865 Total a Included positions Positions excluded due to ambiguous alignment. One hairpin excluded due to length polymorphism. One codon at the 50 end of CO1 excluded due to gene length polymorphism. Table 5 ILD test p-values. Pairwise comparisons between three target regions and between seven genes. Comparisons indicating signiﬁcant incongruence (p < 0.05) are marked with an asterisk (*). Fragment 2 Fragment 1 16S-Leu1-NADH1 Fragment–fragment comparisons 16S-Leu1-NADH1 – 18S 0.495 CO1-Leu2-CO2 0.365 Gene 2 CO1-Leu2-CO2 – 0.995 – Gene 1 16S Gene–gene 16S Leu1 NADH1 18S CO1 Leu2 CO2 18S Leu1 comparisons – 0.960 – 0.040* 1.000 0.025* 0.030* 0.610 1.000 0.530 0.365 0.010* 1.000 NADH1 18S CO1 Leu2 CO2 – 0.005* 0.890 0.785 0.435 – 1.000 0.005* 0.380 – 1.000 0.125 – 1.000 – entirety. Comparisons between longer stretches of DNA are probably less sensitive to local substitution biases and may therefore more reliably reﬂect the level of conﬂict between partitions. 3.3. Phylogenetic analyses Maximum Parsimony using implied weighting and excluding taxa with incomplete sequences yielded a single most parsimonious tree with score 755.97 (bootstrap consensus tree shown in Fig. 2). A majority rule consensus tree for the Bayesian analysis in MrBayes with all taxa included is shown in Fig. 3. Trees from the other analyses can be found in Supplementary Figs. 3–11. Table 6 lists all clades retrieved with posterior probability (PP) P 0.75 in one or more of the four Bayesian analyses, or bootstrap support P50% in one or more of the six parsimony analyses. For both analyses in MrBayes, convergence was assessed by tracing the average standard deviation of split frequencies between two independent runs (Supplementary Fig. 1). The values stabilized below 0.03 after 25 million generations, indicating that stationarity had been reached. In PHASE, log likelihood values and tree priors were examined in order to ensure that independent runs returned the same values, and that they had stabilized before sampling was initiated. Log likelihoods stabilized after less than 300,000 generations and priors on trees after about 600,000 generations (Supplementary Fig. 2). The resulting tree topologies were largely congruent when poorly supported nodes were ignored. In general, only few deep nodes were recovered with bootstrap support P50% in the parsimony analyses, whereas several were well supported in the Bayesian analyses (PP = 0.99–1.00). The basal topology of the main Athetini clade (Fig. 3, clade 4) was essentially unresolved, but many apical clades were recovered with good statistical support both in the parsimony analyses and in the Bayesian analyses. When using equal character weights in Maximum Parsimony, many apical clades had higher bootstrap support than under implied weighting (Table 6, Fig. 2 and Supplementary Fig. 4). However, the support for many basal nodes was lower with equal weighting. The exclusion of 3rd codon positions from the alignment decreased the statistical support for most apical clades (Table 6 and Supplementary Fig. 6). This was expected, since quickly evolving sites are needed to resolve recent nodes. The exclusion of 3rd positions did not improve the resolution of the basal nodes in the tree. The inclusion of the 13 taxa with missing sequence data in parsimony analysis reduced the support for most clades in the tree, whereas the topology and clade supports in the Bayesian analyses were fairly unperturbed by the inclusion of these taxa (Table 6). There was no signiﬁcant conﬂict between trees inferred from mitochondrial and nuclear markers analyzed separately (Supplementary Figs. 7 and 8), and combining all markers in a single analysis signiﬁcantly improved the support for a large number of nodes in the tree (Table 6). H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 93 Fig. 2. Bootstrap consensus tree inferred in TNT using Maximum Parsimony with implied weighting (K = 3). Only taxa with complete sequence data are included. Clade numbers are shown in circles, bootstrap support values P50% are shown beneath branches. 3.4. Phylogenetic relationships The following presentation of phylogenetic relationships is based on the results from the Bayesian analyses, and on the Maximum Parsimony analysis using implied weighting and excluding incomplete sequences. Clade numbers refer to Table 6 and to Figs. 2 and 3. The ‘‘higher” Aleocharinae formed a well supported clade (clade 1). Within this clade, the tribes Athetini, Lomechusini and Ecitocharini Seevers, 1965 formed a monophyletic group (clade 3), with 94 H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 Fig. 3. Majority rule consensus tree inferred in MrBayes using all taxa. Ln likelihood = 62,115.8. Clade numbers are shown in circles, posterior probabilities are shown beneath branches. Dashed branches denote taxa with incomplete sequence data. Athetini being paraphyletic with respect to Lomechusini and Ecitocharini. The lomechusine genera Pella Stephens, 1835 and Drusilla formed a sister clade (15) to the athetine genera Geostiba and Earota Mulsant and Rey, 1874 (clade 14), and together the four genera formed a sister clade (13) to the remaining Athetini, Lomechusini and Ecitocharini (clade 4). These relationships all had posterior probabilities of 1.00 in the Bayesian analyses, but only clades 14 and 15 had bootstrap supports P95% in the parsimony analysis. The lomechusine genus Myrmedonota Cameron, 1920 nested within the main Athetini clade, forming a moderately well supported H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 clade with Meronera (clade 56: PP = 0.97–0.99). Ecitophya gracillima Mann, 1925, representing the tribe Ecitocharini, formed a well supported sister group to Stethusa Casey, 1910 within the main Athetini clade (clade 53: PP = 1.00). Sequence data for the 18S gene was missing for Ecitophya Wasmann, 1900, and it was therefore not included in all analyses. The basal topology of the main Athetini clade was largely unresolved, but many apical clades were recovered with high statistical support. Most genera represented with multiple specimens were recovered as monophyletic. Atheta was retrieved as non-monophyletic, as many species of Atheta formed separate, well supported clades with members of other genera (i.e. clades 19, 28, 31, 37, 40, 45, and 47). Two of the four genera with unresolved tribal assignment nested within the main Athetini clade (clade 4). For Meronera, a sister group relationship with Myrmedonota was indicated (see above), while the phylogenetic position of Thamiaraea within clade 4 could not be established. The two other genera, Halobrecta and Thendelecrotona, both nested outside the Athetini–Lomechusini– Ecitocharini clade. 4. Discussion 4.1. The Athetini–Lomechusini–Ecitocharini clade Our most important result was to ﬁnd the tribes Lomechusini and Ecitocharini nested within the Athetini (clade 3). The bootstrap support for clade 3 was low (58%), but it was recovered with a posterior probability of 1.00 in all Bayesian analyses. The tribe Lomechusini in the currently accepted sense (Newton et al., 2000; Smetana, 2004) is separated from Athetini by the mesoventral process of the mesothorax being short and broad, and the galea being long to very long. However, there is certain variation in both characters, making the assignment of some genera to Lomechusini ambiguous. The three genera of Lomechusini included in our analyses were not recovered as a monophyletic group, but were split into two well supported but very distant clades. The genera Pella and Drusilla (clade 15) grouped with the athetine genera Geostiba and Earota (clade 14) to form a well supported sister group (clade 13) to the remaining Athetini (clade 4). The lomechusine genus Myrmedonota nested inside the main Athetini clade forming a moderately well supported clade with Meronera (clade 56: PP = 0.97–0.99). The tribal assignment of Meronera has been debated, and it has variously been placed in Oxypodini (Seevers, 1978), Tachyusini Thomson, 1859 (Ashe, 1985), or Falagriini Mulsant and Rey, 1873 (Pace, 2008). However, most recent treatments consider it a member of the Lomechusini (e.g. Newton et al., 2000). The type genus of Lomechusini was not included in the current study, but based on the shape of the mesoventral process and male genitalia, it seems more closely related to Drusilla and Pella than to Meronera and Myrmedonota. Ecitocharini is one of several tribes of highly derived myrmecophiles associated with Old and New World army ants. It was described by Seevers (1965) and originally included four genera associated with New World ants of the genus Eciton Latreille, 1804. Seevers himself considered Ecitocharini closely related to Athetini, and his main purpose for establishing the tribe was ‘‘to emphasize their evolutionary and ecological divergence”. Our study included Ecitophya gracillima as a representative of Ecitocharini. It nested within the main Athetini clade (clade 4), forming a well supported clade with the New World genus Stethusa (clade 53: PP = 1.00). Stethusa is a fairly generalized non-myrmecophile athetine (Gusarov, 2003b), and its close relationship with Ecitophya was therefore surprising. Finding all included Lomechusini and Ecitocharini species nested within Athetini calls for a revision of the classiﬁcation of 95 the three tribes. It is clear that despite the highly derived morphology, Ecitocharini does not deserve tribal rank and should be included in Athetini. Furthermore, the genus Myrmedonota should be included in the Athetini. However, several key taxa were missing in our analysis, including the type genera of both Lomechusini and Ecitocharini. We do not wish to propose formal changes in classiﬁcation until a broader taxon sampling has been conducted. 4.2. Suprageneric groups within Athetini All members of Athetini with the exception of Geostiba and Earota nested within the main Athetini clade (clade 4). The basal relationships within clade 4 were poorly resolved, and it was not possible to reliably identify larger subclades within this clade. Nevertheless, many apical clades were well supported, providing new insights about many of the previously proposed suprageneric groups within Athetini. 4.2.1. Subtribe Geostibina Seevers, 1978 Of the seven genera originally included in Geostibina, only Geostiba, Gaenima Casey, 1911, and Crephalia Casey, 1910 are still considered valid genera within Athetini (Gusarov, 2003a). The two latter were not available for our analyses, but based on morphology, they do not appear closely related to Geostiba (Lohse and Smetana, 1988; Gusarov, unpublished). In our analyses, Geostiba grouped with Earota to form the well supported clade 14 outside the main Athetini clade (clade 4). This was surprising, as a close relationship between Geostiba and Earota has never been suggested before. Earota has variously been placed in Thamiaraeina Fenyes, 1921 (Seevers, 1978) or Athetina Fleming, 1821 (Smetana, 2004), or been listed without subtribal assignment (Newton et al., 2000). There is however at least one morphological character supporting the Geostiba + Earota clade: sensillum a of the epipharynx reduced (e.g. Gusarov, 2002: Figs. 2 and 4), as opposed to long, fully developed in the remaining Athetini and Lomechusini (e.g. Gusarov, 2003b: Fig. 6). In the future, the name Geostibina might be used for clade 14, but the close association between this clade and Lomechusini means that formal changes should await a closer investigation of the Athetini–Lomechusini relationship. 4.2.2. Subtribes Strigotina Casey, 1910 and Acrotonina Seevers, 1978 Casey (1910) introduced the subtribe Strigotina for the genus Strigota Casey, 1910. Seevers (1978) later proposed the subtribe Acrotonina for Acrotona Thomson, 1859 and Strigota, apparently overlooking that an available name already existed. In most recent works, neither subtribe is recognized as valid (Muona, 1979; Lohse et al., 1990; Smetana, 2004). The rank and content of the genus Acrotona varies signiﬁcantly between authors. In its broadest sense, Acrotona includes virtually all athetines with inﬂexed hypomera invisible in lateral view (e.g. Brundin, 1952). In its narrowest sense (Benick and Lohse, 1974), many Acrotona-like species groups are placed in separate genera or subgenera. In our analyses, Acrotona s. str. grouped with Strigota to form the fairly well supported clade 58 (PP = 1.00). Other members of Acrotona s. lat. included in this study did not group within this clade: Lypoglossa Fenyes, 1918 grouped with Atheta (Alaobia) gagatina (Baudi di Selve, 1848) (clade 37), Mocyta Mulsant and Rey, 1874 formed a well supported clade with the Atheta subgenera Mycetota Ádám, 1987 and Oxypodera Bernhauer, 1915 (clade 19), while the position of Nehemitropia Lohse, 1971 remained unresolved. Thus, our study refutes Acrotona in the broad sense of e.g. Brundin (1952), and Strigotina (=Acrotonina) in the broad sense of e.g. Newton et al. (2000). Our results conﬁrm the opinion of Muona (1987) that inﬂexed hypomera may be of limited value for classiﬁcation of Athetini. However, the close relationship be- 96 Table 6 Clades retrieved with bootstrap support P50% (parsimony analyses) or posterior probability P0.75 (Bayesian analyses) in one or more analysis. Clade supports below these values are not shown. Taxa with incomplete sequence data are listed in parentheses. These taxa were excluded from many analyses. Groups containing one or more taxa with incomplete sequence data are marked with (*). Clade Included taxa Bayesian analysis MrBayes 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 ‘‘Higher” Aleocharinae* ‘‘Higher” Aleocharinae* excluding Hoplandriini, Homalotini and Aleocharini Athetini*, Lomechusini, (Ecitocharini) Main Athetini clade* Hoplandriini, Homalotini, Aleocharini Hoplandriini, Homalotini Homalotini spp. Bolitochara, Silusida Gyrophaena spp. Falagriini, (Myllaenini), Oxypodini, Placusini, Halobrecta, Thendelecrotona Oxypodini, Placusini (Myllaenini), Thendelecrotona Geostiba, Earota, Pella, Drusilla Geostiba, Earota Pella, Drusilla Pella spp. Pella caliginosa 1 + 2 Atheta kenyamontis, A. klagesi, A. laticollis, A. pallidicornis, A. pasadenae, Mocyta spp., Nehemitropia, Thamiaraea* Atheta kenyamontis, A. laticollis, A. pasadenae, Mocyta spp. Atheta kenyamontis, A. laticollis Atheta kenyamontis, Mocyta spp. Mocyta spp. Atheta laticollis, A. pasadenae Atheta graminicola, A. contristata, A. setigera, A. crassicornis, A. modesta, Boreostiba Atheta crassicornis, A. modesta 1 + 2 Atheta crassicornis, A. modesta 1 Atheta modesta 1 + 2 Atheta graminicola, A. contristata, A. setigera, Boreostiba Atheta graminicola, A. contristata, A. setigera Atheta graminicola, A. contristata Atheta aeneipennis, A. cinnamoptera, Boreophilia Atheta aeneipennis, A. cinnamoptera Atheta bosnica, A. vestita, A. gagatina, (A. membranata), A. subtilis, (Alpinia), (Dalotia), Liogluta spp.*, Lypoglossa (Atheta membranata), (Dalotia) Atheta bosnica, A. vestita, A. gagatina, (Alpinia), Liogluta spp.*, Lypoglossa Atheta gagatina, A. hampshirensis, A. subtilis, Lypoglossa Atheta gagatina, Lypoglossa Atheta hampshirensis, A. subtilis Atheta bosnica, A. vestita, (Alpinia), Liogluta spp.* Atheta bosnica, A. vestita, Liogluta spp.* Atheta bosnica, A. vestita Liogluta spp.* (Liogluta microptera 1), (L. nigropolita) Atheta myrmecobia, Trichiusa Atheta celata, A. dadopora, Lyprocorrhe TNT All taxa Complete sequences All taxa Complete sequences Default parametersa Equal weights All taxa included 3rd pos. excluded Mitochondrial markers Nuclear marker 1.00 1.00 1.00 1.00 0.81 0.91 1.00 1.00 1.00 0.91 0.80 1.00 1.00 1.00 1.00 1.00 1.00 – 1.00 1.00 1.00 1.00 0.87 0.85 1.00 1.00 1.00 – – n/a 1.00 1.00 1.00 1.00 1.00 – 1.00 1.00 1.00 1.00 – 0.83 1.00 1.00 1.00 0.93 – 0.99 1.00 1.00 1.00 1.00 1.00 – 1.00 0.99 1.00 1.00 – 0.76 1.00 1.00 1.00 – – n/a 1.00 1.00 1.00 1.00 1.00 – 99 – 58 – – – 71 80 100 – – n/a 73 95 99 99 100 – 96 – – – – – – – 100 – – n/a – – 72 100 100 – 100 – – – – – 66 82 100 – – – 63 87 98 99 100 – 99 – 54 – – – 66 95 100 – – n/a 75 97 99 98 100 – 98 – – – – – – 76 100 – 53 n/a – 75 – 89 100 – 50 – – – – – 66 – – – – n/a – – 93 74 60 56 1.00 – 0.90 1.00 – – 1.00 – 0.91 1.00 – – 1.00 – 0.97 1.00 0.78 0.92 1.00 – 0.95 1.00 0.75 0.97 56 – – 55 – – 77 83 – 92 – – – – – – – – – – – – – – – – – – – – – – – – – – 1.00 – 0.93 0.95 0.99 1.00 0.98 0.99 0.77 1.00 – 0.92 1.00 0.99 1.00 1.00 0.96 – 1.00 – 0.90 0.99 0.97 1.00 1.00 1.00 – 1.00 – 0.88 0.99 0.96 1.00 1.00 0.99 – 94 – 90 – – 91 – 86 – 100 – 80 – 61 91 83 92 – 93 – 89 – – 94 – 88 – 90 81 – – – 76 – 84 – 78 – 76 – – 73 – 77 – – – – – – – – – – 0.86 0.91 – 0.97 – 1.00 1.00 1.00 1.00 1.00 – 1.00 n/a – – 1.00 0.81 n/a 1.00 1.00 n/a n/a – 1.00 – – – 0.92 0.85 1.00 1.00 0.99 1.00 1.00 – 1.00 n/a – 0.85 1.00 0.88 n/a 1.00 0.96 n/a n/a 0.76 1.00 n/a – – – – n/a 99 95 n/a n/a – 54 n/a – – 58 – n/a 100 92 n/a n/a – – – – – – – – 99 98 98 91 – – n/a – – – – n/a 99 84 n/a n/a – 52 n/a – – – – n/a 96 93 n/a n/a – – n/a – – – – n/a 74 – n/a n/a – – H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Maximum parsimony PHASE a Default parameters for the parsimony analyses were: implied weighting with K = 3; all markers and all sites included; thirteen taxa with incomplete sequences excluded. The ﬁve additional analyses each altered one of the default parameters. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 Atheta dadopora, Lyprocorrhe Atheta ravilla, (A. vaga), Dadobia, Pontomalota, Tarphiota Pontomalota, Tarphiota Atheta ravilla, (A. vaga), Dadobia Atheta ravilla, (A. vaga) (Ecitocharini), Micratheta, Nehemitropia, Schistoglossa, Stethusa spp. Micratheta, Nehemitropia, Schistoglossa (Ecitocharini), Stethusa spp. Stethusa spp. Dinaraea, Meronera, Myrmedonota Meronera, Myrmedonota Acrotona spp., Strigota, Meronera, Myrmedonota, Micratheta Acrotona spp., Strigota Acrotona sp. prope austiniana, Strigota Acrotona sp. prope assecla, Meronera Philhygra spp. Philhygra debilis 1 + 2, P. iterans Philhygra fallaciosa, P. iterans Philhygra debilis 1 + 2 Amidobia, Atheta sp. ex gr. lippa, (Amischa spp.) Amidobia, Atheta sp. ex gr. lippa (Amischa spp.) Atheta klagesi, A. pallidicornis, Thamiaraea spp.* Thamiaraea spp.* Thamiaraea americana, (Thamiaraea brittoni) Atheta klagesi, A. pallidicornis – 1.00 1.00 0.93 – 0.78 0.84 1.00 0.98 0.78 0.97 – 1.00 – – 1.00 0.88 – 1.00 0.99 – 1.00 0.97 1.00 1.00 1.00 – 1.00 1.00 0.75 n/a – – n/a 1.00 0.81 0.98 – 1.00 – – 1.00 0.91 – 1.00 n/a 1.00 n/a – n/a n/a 1.00 – 1.00 1.00 0.97 – – – 1.00 0.91 – 0.99 – 1.00 – – 1.00 0.96 – 1.00 – 0.85 1.00 – 1.00 1.00 1.00 – 1.00 1.00 0.94 n/a – – n/a 1.00 – 0.99 0.81 1.00 – – 1.00 0.96 – 1.00 n/a 1.00 n/a – n/a n/a 1.00 – 72 99 75 n/a – – n/a 91 – 51 – – 64 – – 80 – 100 n/a – n/a – n/a n/a 99 50 – 100 – n/a – – n/a 74 – – – – 80 – 56 – 53 100 n/a – n/a – n/a n/a 100 – 61 99 57 53 – – 57 – – – – – 55 – – 82 – 100 – – 100 – 70 95 99 – 74 100 72 n/a – – n/a 87 – – – – 52 – 57 94 – 99 n/a – n/a – n/a n/a 99 – – 95 – n/a – – n/a – – – – – – – – 74 – 99 n/a – n/a – n/a n/a 99 – – 90 57 n/a – – n/a – – – – – – 53 – – – – n/a – n/a – n/a n/a – H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 97 tween Acrotona s. str. and Strigota hypothesized by Seevers (1978) was conﬁrmed. 4.2.3. Seevers’ (1978) ‘‘sea-shore genera” and the subtribe Dadobiina Muona, 1979 The Nearctic genera Tarphiota Casey, 1893 and Pontomalota were informally grouped together as ‘‘sea-shore genera” by Seevers (1978) due to their strictly intertidal habitat. He emphasized that the genera were not necessarily related, but others have hypothesized a close relationship between these and Thinusa Casey, 1893, another Nearctic sea-shore genus (Moore, 1956; Kincaid, 1961; Ahn and Ashe, 1992; Ahn, 1997). However, no subtribal name has yet been proposed for the group. In our study, Pontomalota and Tarphiota formed a strongly supported clade in all analyses (clade 48: PP = 1.00, bootstrap = 99%). They further grouped with Atheta ravilla (Erichson, 1839), A. vaga (Heer, 1839), and Dadobia immersa (Erichson, 1837) to form the well supported clade 47 (PP = 1.00, bootstrap = 72%). The genus Dadobia Thomson, 1858 includes a single Palaearctic species remarkable in its strongly depressed body, an adaptation to subcortical habitat. The subtribal name Dadobiina was proposed for Dadobia by Muona (1979), but the name was never made available. Clade 47 constitutes yet another example of a well supported clade including both fairly generalized (Atheta spp.) and morphologically rather derived taxa (see also 4.1: Ecitophya + Stethusa). 4.2.4. Subtribe Dimetrotina Seevers, 1978 Seevers (1978) proposed the subtribe Dimetrotina for 10 genera with pronotum slightly less convex and hypomera slightly more exposed than in Acrotonina. He himself questioned the phylogenetic validity of the group. Our study included four of the genera originally included in Dimetrotina: Dimetrota Mulsant and Rey, 1873 and Datomicra Mulsant and Rey, 1874 (as subgenera of Atheta), Fusalia Casey, 1911 (=Thamiaraea), and Amischa Thomson, 1858. Our results refute the hypothesis of a monophyletic Dimetrotina (or even a monophyletic Dimetrota: see Section 4.3.1). The included members of Dimetrotina nested in different parts of the tree, and several formed well supported clades with members of other genera. 4.2.5. Liogluta series (Yosii and Sawada, 1976) The Liogluta series was proposed by Yosii and Sawada (1976) for Liogluta Thomson, 1858 and ﬁve additional genera (Schistoglossa Kraatz, 1856, Aloconota Thomson, 1858, Tomoglossa Kraatz, 1856, Callicerus Gravenhorst, 1802, and Geostiba), based on some characters of ligula and prementum. Their concept of Liogluta was broader than traditional views and included the type species of the Atheta subgenera Thinobaena Thomson, 1859 and Oreostiba Ganglbauer, 1895. The Liogluta series was not retrieved as monophyletic in our study, but we found strong support for a close relationship between Liogluta, Thinobaena and Oreostiba (clade 40: PP = 1.00, bootstrap = 99%), as hypothesized by Yosii and Sawada. 4.3. Genus Atheta Thomson, 1858 The systematics of the genus Atheta has been the subject of much controversy. The modern concept of Atheta goes back to Ganglbauer (1895) who treated it as a large genus subdivided in multiple subgenera. Most subsequent publications by European authors are essentially variations of the Ganglbauer classiﬁcation (e.g. Benick and Lohse, 1974; Smetana, 2004), but the rank and scope of the different Atheta subgenera vary considerably between authors. Two alternative and poorly compatible classiﬁcations of Atheta were developed by North American authors (Seevers, 1978; adopted by Newton et al., 2000) and in Japan (Yosii and Sawada, 1976). Muona (1979, 1987) suggested a synthesis of the three 98 H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 alternative classiﬁcations, but his proposal was not readily accepted. In its narrowest possible sense (i.e. Atheta s. str. of Benick and Lohse (1974), the genus is diagnosed by having very distinctive and complex spermatheca (see Benick and Lohse, 1974; pp. 193, 196). In its broadest sense (e.g. Smetana, 2004) Atheta is diagnosable only by the lack of derived characters. Our study rejects the monophyly of Atheta in the broad sense. Members of Atheta were distributed throughout the main Athetini clade (clade 4), and many species of Atheta formed separate, well supported clades with members of other genera. Our ﬁndings support the view on Atheta as a paraphyletic species assemblage deﬁned by plesiomorphies only. Our results suggest several alternative narrower concepts of Atheta. The most strongly supported alternative is an equivalent of Atheta s. str. sensu Benick and Lohse, 1974, represented in our analyses by two species (clade 30: PP = 1.00, bootstrap = 91%). In this concept, Atheta is diagnosable by at least one unique morphological character, the very distinct shape of the spermatheca. 4.3.1. Subgenera of Atheta Fifteen subgenera or species groups of Atheta were represented in our study, six of them with more than one species. Fig. 4 shows the placement of Atheta subgenera within clade 3. The subgenus Datomicra, represented by two species, formed a well supported clade with the myrmecophilous genus Lyprocorrhe Thomson, 1859 (clade 45). This relationship was unexpected, but there are morphological characters in support of it: the two groups share the same pronotal setation pattern (Type I) and the distinct shape of the spermatheca. The subgenus Dimetrota was not recovered as monophyletic, but two of the included species, Atheta (D.) aeneipennis (Thomson, 1856) and A. (D.) cinnamoptera (Thomson, 1856), formed a well supported clade with the genus Boreophilia Benick, 1973 (clade 31). Our study thus refutes Benick’s (1973) hypothesis that Boreophilia is unrelated to Dimetrota. We reject a broad concept of subgenus Alaobia Thomson, 1858 (as adopted in Smetana, 2004) but found strong support for a close relationship between the Palaearctic Atheta (Alaobia) pallidicornis (Thomson, 1856) and the Nearctic A. klagesi Bernhauer, 1909, a species currently without subgeneric assignment (see Gusarov, 2003c) (clade 71: PP = 1.00, bootstrap = 99%). The members of Mycetota and Oxypodera grouped with the genus Mocyta to form a clade with high support in Bayesian analyses (clade 19). Strong support was found for a relationship between the Atheta subgenera Oreostiba and Thinobaena, and the genus Liogluta (clade 40), though only Thinobaena was represented with its type species (see also Section 4.2.5). 4.4. Some aleocharine genera with uncertain tribal placement The genus Halobrecta is traditionally placed in the tribe Athetini (e.g. Benick and Lohse, 1974; Smetana, 2004), based mainly on the 4-5-5 tarsal formula. However, Gusarov (2004) demonstrated that the genus lacks the ‘‘athetine bridge” of the aedeagus while sharing some characters with the tribe Oxypodini. In our study, Halobrecta consistently nested outside the Athetini–Lomechusini–Ecitocharini clade, supporting its exclusion from Athetini. The genus Meronera is usually placed in Lomechusini (see Section 4.1), but our study places it in Athetini. The genus Thamiaraea is variously treated as a member of tribe Thamiaraeini Fenyes, 1921 (e.g. Lohse, 1989; Paśnik, 2007; Pace, 2007), or as a subtribe Thamiaraeina within the Athetini (Seevers, 1978; Newton et al., 2000; Smetana, 2004). Thendelecrotona was recently described as another member of Thamiaraeini, which according to Paśnik (2007) contains 29 genera. Our study included three species of Thamiaraea and one representative of Thendelecrotona. The members of Thamiaraea formed a well supported monophyletic clade (69) within the main Athetini clade, conﬁrming that this genus belongs in the Athetini. Thendelecrotona nested outside the Athetini–Lomechusini–Ecitocharini clade, and its relation to Thamiaraea was thus not supported. These ﬁndings are supported by Thamiaraea possessing the ‘‘athetine bridge” of the aedeagus, and Thendelecrotona lacking it. Our study thus rejects the hypothesis of Thamiaraeini as a tribe separate from Athetini. Fig. 4. Detailed view on clade 3 of the Bayesian tree in Fig. 3 highlighting members of the genus Atheta and their subgeneric placement within the Athetini–Lomechusini– Ecitocharini clade. Dashed branches denote taxa with incomplete sequence data. H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100 99 4.5. Formal changes in classiﬁcation References Based on our results, we propose the following formal changes to classiﬁcation. Halobrecta is moved from Athetini and provisionally included in Oxypodini. Thendelecrotona is moved from Athetini and treated as Aleocharinae insertae sedis. Meronera and Thamiaraea are ﬁrmly placed in Athetini. Ahn, K.-J., 1997. Revision and systematic position of the intertidal genus Thinusa casey (Coleoptera: Staphylinidae: Aleocharinae). Entomol. Scand. 28, 75–81. Ahn, K.-J., Ashe, J.S., 1992. Revision of the intertidal aleocharine genus Pontomalota Casey (Coleoptera: Staphylinidae) with a discussion of its phylogenetic relationships. Entomol. 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The most important novel result was the paraphyly of Athetini with regard to Lomechusini and Ecitocharini. This discovery calls for a revision of all three tribes, but formal changes in classiﬁcation must wait until a broader taxon sampling of the tribes has been conducted. In particular, the phylogenetic position of Lomechusa, the type genus of Lomechusini, needs to be clariﬁed. Within Athetini, we discovered several clades with morphologically highly derived and generalized taxa related as sister groups. In two cases (Ecitophya and Lyprocorrhe) the derived taxa were myrmecophiles with pronounced morphological modiﬁcations related to their association with ants. The subfamily Aleocharinae includes many tribes and subtribes composed exclusively of highly derived myrmecophiles or termitophiles, and molecular markers may prove highly useful for re-evaluation of their relationships to non-myrmecophile ancestors. While many apical relationships were recovered with good support, our molecular markers were not able to resolve some of the most basal relationships in Athetini or in the genus Atheta. The estimated branch lengths in this part of the tree indicate that the tribe may have experienced an initial period of rapid radiation which made phylogentic inference at basal nodes difﬁcult. More markers and increased taxon sampling will undoubtedly help resolving some of these relationships. Acknowledgments We thank Sinan Anlasß, Igor Belousov, Alexander Derunkov, Martin Fikáček, Ilya Kabak, Derek Lott, György Makranczy, Alexander Ryabukhin, Alexey Shavrin, Alexey Tishechkin, Marc Tronquet, and Larry Watrous for providing specimens for DNA analysis. Galina Gussarova, Bjarte Jordal and Eirik Rindal provided valuable help with molecular methods and data analysis. Karsten Sund contributed excellent photos of beetles. We also thank three anonymous reviewers for valuable feedback on the paper. 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