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: hallvard.elven@nhm.uio.no (H. Elven), lutz.bachmann@nhm.
uio.no (L. Bachmann), vladimir.gusarov@nhm.uio.no (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
–
–
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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
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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
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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. Scand. 23, 347–359.
Ahn, K.-J., Ashe, J.S., 2004. Phylogeny of the Myllaenini and related taxa (Coleoptera:
Staphylinidae: Aleocharinae). Cladistics 20, 123–138.
Ashe, J.S., 1985. Fecundity, development and natural history of Meronera venustula
(Erichson) (Coleoptera: Staphylinidae: Aleocharinae). Psyche 92, 181–204.
Ashe, J.S., 1994. Evolution of aedeagal parameres of aleocharine staphylinids
(Coleoptera: Staphylinidae: Aleocharinae). Can. Entomol. 126, 475–491.
Ashe, J.S., 2005. Phylogeny of the tachyporine group of subfamilies and ‘basal’
lineages of the Aleocharinae (Coleoptera: Staphylinidae) based on larval and
adult characteristics. Syst. Entomol. 30, 3–37.
Ashe, J.S., 2007. Aleocharinae. Version 25 April 2007. In: The Tree of Life Project.
Available from: <http://tolweb.org/>, <http://tolweb.org/Aleocharinae/9777/
2007.04.25> (verified 10.12.2008).
Ashe, J.S., Newton, A.F., 1993. Larvae of Trichophya and phylogeny of the
tachyporine group of subfamilies (Coleoptera: Staphylinidae). Syst. Entomol.
18, 267–286.
Benick, G., 1973. Die von Prof. Dr. Z. Kaszab in der Mongolei gesammelten Arten der
Subfamilie Athetae (Col. Staphyl.). Nouvelle Rev. d’Entomol 3, 211–217.
Benick, G., Lohse, G.A., 1974. 14. Tribus: Callicerini (Athetae). In: Freude, H., Harde,
K.W., Lohse, G.A. (Eds.), Die Käfer Mitteleuropas. Band 5. Staphylinidae II
(Hypocyphtinae und Aleocharinae). Pselaphidae. Goecke & Evers, Krefeld, pp.
72–220.
Bernhauer, M., Scheerpeltz, O., 1926. Staphylinidae VI. Coleopterorum Catalogus 82,
499–988.
Brundin, L., 1940. Zwei neue nordische Atheta-Arten aus der islandica-Gruppe (Col.
Staphylinidae) Vorläufige Mitteilung. Entom. Tidskr. 61, 131–133.
Brundin, L., 1952. Acrotona-Studien (Gattung Atheta, Col., Staphylinidae). Entom.
Tidskr. 73, 93–145.
Carapelli, A., Vannini, L., Nardi, F., Boore, J.L., Beani, L., Dallai, R., Frati, F., 2006. The
mitochondrial genome of the entomophagous endoparasite Xenos vesparum
(Insecta: Strepsiptera). Gene 376, 248–259.
Casey, T.L., 1910. New species of the staphylinid tribe Myrmedoniini. Memoirs on
the Coleoptera, vol. 1. New Era, Lancaster, Pennsylvania, pp. 1–183.
Clary, D.O., Wolstenholme, D.R., 1985. The mitochondrial DNA molecule of
Drosophila yakuba: nucleotide sequence, gene organization, and genetic code.
J. Mol. Evol. 22, 252–271.
Cunningham, C.W., 1997. Can three incongruence tests predict when data should be
combined? Mol. Biol. Evol. 14, 733–740.
Davies, J.T., Barraclough, T.G., Chase, M.W., Soltis, P.S., Soltis, D.E., Savolainen, V.,
2004. Darwin’s abominable mystery: Insights from a supertree of the
angiosperms. Proc. Natl. Acad. Sci. USA 101, 1904–1909.
Farris, J.S., Källersjö, M., Kluge, A.G., Bult, C., 1995. Testing significance of
incongruence. Cladistics 10, 315–319.
Fenyes, A., 1918. Coleoptera. Fam. Staphylinidae. Subfam. Aleocharinae. In:
Wytsman, P. (Ed.), Genera Insectorum 173A. Louis Desmet-Verteneuil,
Bruxelles, pp. 1–110.
Fenyes, A., 1921. New genera and species of Aleocharinae with a polytomic synopsis
of the tribes. Bull. Mus. Comp. Zool. 65, 17–36.
Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for
amplification of mitochondrial cytochrome c oxidase subunit I from diverse
metazoan invertebrates. Mol. Marine Biol. Biotechnol. 3, 294–299.
Ganglbauer, L., 1895. Die Käfer von Mitteleuropa. Die Käfer der österreichischungarischen Monarchie, Deutschlands, der Schweitz, sowie des französischen
und italienischen Alpengebietes. Zweiter Band. Familienreihe Staphylinoidea. 1.
Theil: Staphylinidae, Pselaphidae. Druck und Verlag von Carl Gerold’s Sohn,
Wien, vi + 880pp.
Gavrilets, S., Losos, J.B., 2009. Adaptive radiation: contrasting theory with data.
Nature 323, 732–737.
Gillespie, J.J., Johnston, J.S., Cannone, J.J., Gutell, R.R., 2006. Characteristics of the
nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of
Apis mellifera (Insecta: Hymenoptera): structure, organization, and
retrotransposable elements. Insect. Mol. Biol 15, 657–686.
Goloboff, P.A., 1993. Estimating character weights during tree search. Cladistics 9,
83–91.
Goloboff, P.A., Farris, J.S., Nixon, K., 2003. T.N.T.: Tree Analysis Using New
technology. Program and Documentation. Available from: <http://www.zmuc.
dk/public/phylogeny/TNT>.
Grimaldi, D., Engel, M.S., 2005. Evolution of the Insects. Cambridge University press,
New York.
Gusarov, V.I., 2002. A revision of Nearctic species of the genus Geostiba Thomson,
1858 (Coleoptera: Staphylinidae: Aleocharinae). Zootaxa 81, 1–88.
Gusarov, V.I., 2003a. A catalogue of the athetine species of America north of Mexico
(Coleoptera: Staphylinidae: Aleocharinae: Athetini). Available from: <http://
nhm.ku.edu/ksem/peet/catalogs/cataweb.htm> (verified 7. 10.2009).
Gusarov, V.I., 2003b. A Revision of the Nearctic Species of the Genus Stethusa Casey,
1910 (Coleoptera: Staphylinidae: Aleocharinae). Zootaxa 239, 1–43.
Gusarov, V.I., 2003c. Revision of some types of North American aleocharines
(Coleoptera: Staphylinidae: Aleocharinae), with synonymic notes. Zootaxa 353,
1–134.
5. Conclusions
Obtaining a robust classification for Athetini has historically
proved very difficult. We found the molecular markers used in this
study a powerful tool for investigating athetine phylogeny and for
testing previous morphology-based hypotheses. Several previously
proposed groups were retrieved as monophyletic while many were
firmly rejected. Interestingly, we also uncovered a number of
unexpected relationships that deserve a closer morphological
investigation. Furthermore, our study demonstrated unreliability
of certain morphological characters as indicators of phylogenetic
relationship.
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. The
project was supported by the National Center for Insect Biodiversity (Natural History Museum, University of Oslo), and in part
funded by the Norwegian Research Council Grant 180681/D15
to V. Gusarov.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2010.05.023.
100
H. Elven et al. / Molecular Phylogenetics and Evolution 57 (2010) 84–100
Gusarov, V.I., 2004. A revision of the Nearctic species of the genus Halobrecta
Thomson, 1858 (Coleoptera: Staphylinidae: Aleocharinae) with notes on some
Palaearctic species of the genus. Zootaxa 746, 1–25.
ICZN, 1999. International Code of Zoological Nomenclature. Fourth Edition. The
International Trust for Zoological Nomenclature, London, xxix + 306pp.
Jow, H., Hudelot, C., Rattray, M., Higgs, P.G., 2002. Bayesian phylogenetics using an
RNA substitution model applied to early mammalian evolution. Mol. Biol. Evol.
19, 1591–1601.
Kincaid, T., 1961. The staphylinid genera Pontomalota and Thinusa. Privately
published. The Calliostoma Company, Seattle, Washington, 10p.
Kocher, T.D., 2005. Ghost of speciation past. Nature 435, 29–30.
Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular
evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5,
150–163.
Lohse, G.A., 1971. Über gattungsfremde Arten und Artenkreise innerhalb der
‘‘Großgattung” Atheta Thomson. Verh. Ver. Naturw. Heimatforsch. Hamburg 38,
67–83.
Lohse, G.A., 1989. Ergänzungen und Berichtigungen zu Freude-Harde-Lohse ‘‘Die
Käfer Mitteleuropas” Band 5 (1974). In: Lohse, G.A., Lucht, W.H. (Eds.), Die Käfer
Mitteleuropas. 1. Supplementband mit Katalogteil. Goecke & Evers, Krefeld, pp.
185–240.
Lohse, G.A., Klimaszewski, J., Smetana, A., 1990. Revision of Arctic
Aleocharinae of North America (Coleoptera: Staphylinidae). Coleopts Bull.
44, 121–202.
Lohse, G.A., Smetana, A., 1988. Four new species of Geostiba Thomson from the
Appalachian Mountains of North Carolina, with a key to North American species
and synonymic notes (Coleoptera: Staphylinidae: Aleocharinae). Coleopts Bull.
42, 269–278.
Moore, I., 1956. A revision of the Pacific coast Phytosi with review of the foreign
genera (Coleoptera: Staphylinidae). Trans. San Diego Soc. Nat. Hist. 12, 103–
152.
Muona, J., 1979. Staphylinidae. In: Silfverberg, H. (Ed.), Enumeratio Coleopterorum
Fennoscandiae et Daniae. Helsingin Hyönteisvaihtoyhdistys, Helsinki, pp. 14–
28.
Muona, J., 1987. Some aspects of Aleocharinae systematics—a response to Dr. G.A.
Lohse. Entomol. Blätter 83, 19–24.
Muona, J., 1995. Taxonomic notes on the genus Philhygra Mulsant & Rey
(Coleoptera, Staphylinidae). Entomol. Medd. 63, 11–16.
Newton, A.F., Thayer, M.K., 1992. Current classification and family-group names in
Staphyliniformia (Coleoptera). Fieldiana Zool., N. S. 67, iv + 92pp.
Newton, A.F., Thayer, M.K., Ashe, J.S., Chandler, D.S., 2000. 22. Staphylinidae
Latreille, 1802. In: Arnett, R.H., Thomas, M.C. (Eds.), American Beetles, vol. 1,
Archostemata, Myxophaga, Adephaga, Polyphaga: Staphyliniformia. CRC Press,
LLC, Boca Raton, FL, pp. 272–418.
Normark, B.B., Jordal, B.H., Farrell, B.D., 1999. Origin of a haplodiploid beetle lineage.
Proc. R. Soc. Lond. B 266, 2253–2259.
Nylander, J.A.A., 2004. MrModeltest v2. Program distributed by the author.
Evolutionary Biology Centre, Uppsala University.
Pace, R., 2007. Description of Amischa paolettii sp. Nov. and Thamiaraea tsitsilasi sp.
nov. from the Australian region (Coleoptera, Staphylinidae). Mem. Mus. Victoria
64, 7–11.
Pace, R., 2008. New records of Aleocharinae from Ecuador and Peru, with the
description of new species, new subgenera and new genera (Coleoptera,
Staphylinidae). In: Memoirs on Biodiversity Volume I. Biodiversity of South
America I. World Biodiversity Association Onlus, pp. 225–398.
Paśnik, G., 2007. A new genus and species of the aleocharine tribe Thamiaraeini
(Coleoptera, Staphylinidae) from South Africa. Zootaxa 1527, 31–38.
Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution.
Bioinformatics 14, 817–818.
Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19, 1572–1574.
Scheerpeltz, O., 1934. Staphylinidae VIII. Coleopterorum Catalogus 130, 1501–1881.
Seevers, C.H., 1965. The Systematics, Evolution and Zoogeography of Staphylinid
Beetles Associated with Army Ants (Coleoptera, Staphylinidae). Fieldiana Zool.
47, 137–351.
Seevers, C.H., 1978. A generic and tribal revision of the North American
Aleocharinae (Coleoptera: Staphylinidae). Fieldiana Zool. 71, 1–289.
Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution,
weighting, and phylogenetic utility of mitochondrial gene sequences and a
compilation of conserved polymerase chain reaction primers. Ann. Entomol.
Soc. Am. 87, 651–701.
Smetana, A., 2004. Subfamily Aleocharinae Fleming, 1821. In: Löbl, I., Smetana, A.
(Eds.), Catalogue of Palaearctic Coleoptera, vol. 2. Hydrophyloidea–Histeroidea–
Staphylinoidea. Apollo Books, Stenstrup, pp. 353–494.
Steidle, J.L.M., Dettner, K., 1993. Chemistry and morphology of the tergal gland of
freeliving adult Aleocharinae (Coleoptera: Staphylinidae) and its phylogenetic
significance. Syst. Entomol. 18, 149–168.
Swofford, D.L., 2002. PAUP*. Phylogenetic Analysis using Parsimony (* and other
Methods). Version 4.0b10. Sinauer Associates, Sunderland, MA.
Thayer, M.K., 2005. Staphylinidae Latreille, 1802. In: Beutel, R.G., Leschen, R.A.B.
(Eds.), Coleoptera, Beetles Volume 1: Morphology and Systematics
(Archostemata, Adephaga, Myxophaga, Polyphaga partim). Handbook of
Zoology, Vol. IV. Part 38. Walter de Gruyter, Berlin, pp. 296–344.
Thomas, J.C., 2009. A Preliminary Molecular Investigation of Aleocharine Phylogeny
(Coleoptera: Staphylinidae). Ann. Entomol. Soc. Am. 102, 189–195.
Whiting, M.F., Carpenter, J.C., Wheeler, Q.D., Wheeler, W.C., 1997. The Strepsiptera
Problem: phylogeny of the holometabolous insect orders inferred from 18S and
28S ribosomal DNA sequences and morphology. Syst. Biol. 46, 1–68.
Yosii, R., Sawada, K., 1976. Studies on the genus Atheta Thomson and its allies
(Coleoptera, Staphylinidae). II. Diagnostic characters of genera and subgenera
with descriptions of representative species. Contrib. Biol. Lab. Kyoto Univ. 25,
11–140.