Phylogeny and classification of the tribe Hydaticini

Phylogeny and classification of the tribe Hydaticini
(Coleoptera: Dytiscidae): partition choice for Bayesian analysis
with multiple nuclear and mitochondrial protein-coding genes
Blackwell Publishing Ltd
KELLY B. MILLER, JOHANNES BERGSTEN & MICHAEL F. WHITING
Submitted: 28 August 2008
Accepted: 11 March 2009
doi:10.1111/j.1463-6409.2009.00393.x
Miller, K. B., Bergsten, J. & Whiting M. F. (2009). Phylogeny and classification of the tribe
Hydaticini (Coleoptera: Dytiscidae): partition choice for Bayesian analysis with multiple
nuclear and mitochondrial protein-coding genes. — Zoologica Scripta **, ****–****.
A phylogenetic analysis of the diving beetle tribe Hydaticini Sharp (Coleoptera: Dytiscidae:
Dytiscinae) is presented based on data from adult morphology, two nuclear (histone III and
wingless) and two mitochondrial (cytochrome c oxidase I and II) protein-coding genes. We
explore how to best partition a data set of multiple nuclear and mitochondrial protein-coding
genes by using Bayes factor and a penalized modification of Bayes Factor. Ten biologically
relevant partitioning strategies were identified ranging from all DNA analysed under a single
model to each codon position of each gene treated with a separate model. Model selection
criteria AIC, AICc, BIC and four ways of traversing parameter space in a hierarchical likelihood ratio test were applied to each partition. All unique partitioning and model combinations
were analysed with Bayesian methods. Results show that partitioning by codon position and
genome source (nuclear vs. mitochondrial) is strongly favoured over partitioning by gene. We
also find evidence that Bayes Factor can penalize overparameterization even when comparing
nested models. Species groups showing a strong geographical pattern were generally highly
supported, however, the sister group relationship of an isolated Madagascan and Australian
species were shown to be artefactual with a long-branch extraction test. The following
conclusions were supported in both the selected method of partitioning the Bayesian analysis
and combined parsimony analyses: (i) the tribe Hydaticini is monophyletic (ii) the genus
Hydaticus Leach is paraphyletic with respect to Prodaticus Sharp (iii) the subgenus Hydaticus
(Hydaticus) is monophyletic, and (iv) the subgenus H. (Guignotites) Brinck is paraphyletic with
respect to Prodaticus and the subgenera H. (Pleurodytes) Régimbart and H. (Hydaticinus)
Guignot. Based on these results, Hydaticus and Prodaticus are each recognized as valid genera
and Guignotites, Hydaticinus and Pleurodytes are each placed as junior synonyms of Prodaticus
(new synonymies).
Corresponding author: Kelly B. Miller, Department of Biology and Museum of Southwestern Biology,
University of New Mexico, Albuquerque, NM 87131, USA. E-mail: kbmiller@unm.edu
Johannes Bergsten, Department of Entomology, Swedish Museum of Natural History, Box 50007,
SE-104 05 Stockholm, Sweden. E-mail: johannes.bergsten@nrm.se
Michael F. Whiting, Department of Biology, Brigham Young University Provo, UT 84602, USA.
E-mail: michael_whiting@byu.edu
Introduction
Throughout much of the world, members of the tribe
Hydaticini Sharp are conspicuous and important components of the aquatic beetle fauna. The members of this group
are medium to large in size (8.5–20.5 mm) and are often
abundant and species rich in many regions. Most are tropical
or subtropical, but there are also numerous temperate
species. They are among the most attractively coloured of all
diving beetles with many species characterized by spots,
fasciae or stripes (e.g. Figs 1–6). Most species occur in ponds
with dense vegetation, but many occur also in areas with
mineral substrates, and some prefer slow lotic habitats.
Hydaticini are characterized by several synapomorphies
including; (i) the anterolateral margin of the metasternal
wing linear (Fig. 7) (convex in other diving beetles, e.g. Fig. 8)
(ii) males with an apparent stridulatory device comprised of a
series of modified setae (pegs) along the apicodorsal margin
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
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Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Figs 1–6 Hydaticini species, habitus. 1, H. modestus. 2–3, H. dineutoides; 2, dorsal habitus; 3, ventral surface of thorax and abdomen. 4,
H. bivittatus. 5, H. modestus. 6, H. speciosus. bar = 1.0 mm.
of the protibia and a field of pits on the dorsal surface of the
probasotarsus (Fig. 10) (apparatus absent in a few species),
and (iii) the anterior margin of the larval prementum bilobed.
The tribe is part of the subfamily Dytiscinae, and is closely
related to three other tribes, Aubehydrini, Eretini and
Aciliini, though relationships among these groups are not
firmly established (Miller 2000, 2001, 2003). One analysis by
Miller (2003) suggested that the tribe may not be mono2
phyletic, with Aciliini and Eretini nested within Hydaticini,
though this was not strongly supported. Another analysis
based only on 18S rRNA data by Ribera et al. (2002) found
Notaticus Zimmermann (Aubehydrini) and Hyderodes Hope
(Hyderodini) nested within Hydaticini.
The group as a whole has not been revised since Sharp’s
(1882) monograph, but there are regional revisions for
North America (Roughley & Pengelly 1981), South America
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
Figs 7–11 Aubehydrini and Hydaticini
species. 7–8, metasternum and metacoxae,
lines indicate anterolateral margins of
metasternal wing; 7, H. aruspex; 8, N. fasciatus.
9, H. aruspex, left metatrochanter, metafemur
and metatibia. 10, H. aruspex, profemur,
protibia, and protarsus, lines indicate reticulations and short spines associated with
stridulatory device. 11, H. dorsiger, protarsus,
line indicates accessory spinous setae on
dorsal surface of protarsomere I (reticulations
on protarsomere II not shown). Bar =1.0 mm.
(Trémouilles 1994), Australia (Watts 1978), Africa (Guignot
1961), India (Vazirani 1968), northern Europe (Nilsson
1981), and the main portion of Europe and Asia (Galewski
1985; Zaitzev 1953; Zimmermann & Gschwendtner 1937). A
few species groups have also been revised (Vazirani 1969;
Wewalka 1975, 1979). Considerable species-level work
remains to be done, and the group is in need of a comprehensive revision.
Hydaticini has a long history of consistent recognition
since Sharp (1882) erected it to include two genera, Hydaticus
Leach and Prodaticus Sharp. An exception is Balfour-Browne’s
(1950) placement of Hydaticus in the subtribe Hydaticina of
Dytiscini. Prodaticus includes two species, P. pictus Sharp from
the Middle East and India, and P. africanus Rocchi, from
northern Africa. They are characterized by equal-length
metatarsal claws and a large number of small adhesive discs
on the expanded male pro- and mesotarsomeres. The series
of bifid setae on the posterior surface of the metatibia is in an
irregular line parallel to the dorsal margin of the tibia. Hydaticus
Leach, a much larger genus with over 130 species (Nilsson
2001), has been divided into four subgenera (Nilsson 2001).
Hydaticus (Pleurodytes) Régimbart was originally described at
the genus rank, but was placed as a subgenus of Hydaticus by
Roughley & Pengelly (1981). This taxon includes two species,
H. (P) dineutoides Sharp from Malaysia and H. (P) epipleuricus
Régimbart from Burma. These two species are characterized
by wide elytral epipleurae (Fig. 3), flattened apicolateral
margins of the elytra, and uniformly black colouration dorsally
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
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Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Figs 12–19 Hydaticini species. 12–14, left
metatibia, posterior aspect; 12, H. aruspex; 13,
H. xanthomelas; 14, H. flavolineatus. 15–16,
left mesofemur, posterior aspect; 15, H.
aruspex; 16, H. flavolineatus. 17–18, right
mesotarsus, ventral aspect, line indicates
brush of setae on mesotarsomere I; 17, H.
aruspex; 18, H. luczonicus. 19, H. fabricii, apex
of male median lobe, right lateral aspect.
bar = 1.0 mm except Fig. 19 bar = 0.25 mm.
(Fig. 2). Hydaticus (Hydaticinus) Guignot is based on a single
Neotropical species, H. xanthomelas (Brullé), known from
lowland South America. This species lacks fine punctation on
the anterior surfaces of the metafemur and metatibiae and the
posterior metatibial bifid setae are in a long series parallel to
the dorsal margin of the metatibia (Fig. 13).
Hydaticus (Hydaticus) is Holarctic and includes seven species
(two in the Nearctic region, four in the Palaearctic and a single
Holarctic species). The subgenus is characterized by the
anterior surface of the metatibia with large punctures interspersed with many small punctures, the metafemora with the
anterior surface with dense punctation in two sizes (Fig. 9),
and the series of bifid setae on the posterior surface of the
metatibia in a series that is straight and parallel to the dorsal
margin (Fig. 12).
The subgenus H. (Guignotites) Brinck is the largest genusgroup in the tribe with nearly 130 species (Nilsson 2001). Its
4
members lack fine punctation on the anterior surfaces of the
metafemur and metatibiae and the posterior metatibial bifid
setae are in a series that curves ventrad basally and is not
parallel with the anterior margin (Fig. 14). This group is
largely circumtropical with a few species extending ranges
north into the Holarctic region.
A comprehensive phylogenetic analysis has not been previously proposed for this taxon. An analysis by Roughley &
Pengelly (1981) presented a number of characters of potential
use for elucidating the phylogeny, but the included taxa were
only a limited representation of the world fauna. The monophyly of the several genus groups in the tribe have not been
adequately tested. A phylogenetic analysis would not only
serve to improve the classification of the tribe, but it would
allow tests of the evolution of several character systems
such as those associated with sexual conflict (Miller 2003), an
apparent sexual acoustic signalling device present in males
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
(Fig. 10, Larson & Pritchard 1974), and a wide variety of
colour patterns (e.g. Larson 1996). This project seeks to
establish a phylogenetic hypothesis and an improved classification for future investigation of various aspects of the biology
of this tribe.
A second goal of this project is to explore issues related to
partitioning data in model based, total evidence approaches
to phylogenetic analysis. Model based phylogenetic inference
is increasingly used with molecular data, and recently it has
become possible to combine molecular and morphological
data in a Bayesian total evidence approach (Lewis 2001;
Nylander et al. 2004). Although methods are in development
to eliminate the model choice step (e.g. reversible jump
Markov Chain Monte Carlo (Huelsenbeck et al. 2004; Alfaro
& Huelsenbeck 2006), not yet available for nucleotide models
in MRBAYES), in practice, most model-based approaches still
require the a priori choice of an appropriate model, and for
this various criteria have been suggested (Sullivan & Joyce
2005; Kelchner & Thomas 2006). The most commonly used
are likelihood ratio tests of nested models such as those
implemented in MODELTEST (Posada & Crandall 1998).
Alternatives include the Akaike Information Criterion (AIC)
(Posada & Buckley 2004), the Bayesian Information Criterion
(BIC) (Posada & Buckley 2004), Bayes Factor (Nylander
et al. 2004) and Decision Theory approaches (Minin et al.
2003; Abdo et al. 2005). In addition to the model choice
criteria, the pool of candidate models from which to choose
varies. Whereas Modeltest chooses from a pool of 56 models,
only 24 of these can be implemented in (for example)
MRBAYES, but these still only constitute 8 of the 203 possible
time-reversible substitution rate models (Huelsenbeck et al.
2004). In other words, there is a plethora of nonsimple
considerations in justifying choice of a model a priori.
Problems are increasing as supermatrices with multiple
genes are becoming standard and another less explored consideration becomes necessary, the appropriate partitioning of
data. Many studies force a single substitution model onto
multiple genes, but the hazard of this is well-known since the
resulting ‘compromise’ model parameters may be inappropriate for particular genes or partitions and in a Bayesian
context can result in either inflated or deflated posterior
probability values (Brown & Lemmon 2007). A few studies
have begun to explore the effect of partition choice and the use
of Bayes Factor (BF) to select the most appropriate partitioning
(Castoe et al. 2004; Nylander et al. 2004; Brandley et al. 2005;
Castoe et al. 2005; Castoe & Parkinson 2006). These have
shown that, whereas in theory, Bayes Factor should be able to
penalize overparameterization, in practice nested model
comparisons always prefer the most parameter rich model
with ‘very strong support.’ This led Brandley et al. (2005) to
question the scale of interpretation of the Bayes Factor for
use in phylogenetics. Latrillot & Philippe (2006) gave a plau-
sible explanation for this and suggested the harmonic mean
estimator (HME) of the marginal likelihood as the source of
error. The HME as estimated by the Markov Chain Monte
Carlo (MCMC) sample grossly underestimates the dimensional penalty by its biased sampling towards high-likelihood
regions and therefore overestimates the marginal likelihood.
Furthermore, this bias is predicted to be more pronounced in
higher-dimensional models, which could explain why Bayes
Factor does not seem to penalize overparameterization
appropriately (Latrillot & Philippe 2006). McGuire et al.
(2007) therefore used the alternative Bayesian Information
Criterion (BIC) and decision-theoretic methodology (DT),
with stronger penalties for overparameterization, to select
partitioning strategy. However, Bayes Factor was recently
given new credibility for use in Bayesian phylogenetics and
data partitioning from a simulation study by Brown &
Lemmon (2007). They found that use of the subjective cut-off
value BF = 10 (Kass & Raftery 1995) resulted in a false positive
(type I) error rate close to 5%, that is, similar to the frequent
use of a α = 0.05. Accordingly, Brown & Lemmon (2007)
suggest that, contrary to rising beliefs, Bayes factor does not
select more partitions than what the data requires. In this
study we explore how to best partition multiple nuclear and
mitochondrial protein coding genes in a Bayesian context.
We evaluate both Bayes Factor and a penalized modification
as criteria for choosing a partitioning scheme that balances
between a realistic model and assigning more parameters
than limited data can possibly estimate.
Materials and methods
Taxon sampling
Forty-five species of Hydaticini were included in the analysis
(Table 1). All five currently recognized genus groups (Prodaticus, Hydaticus (Hydaticus), H. (Guignotites), H. (Hydaticinus),
and H. (Pleurodytes)), were newly sampled for DNA sequencing.
Within the large H. (Guignotites) an attempt was made to
include as many of the informal species groups historically
used by such workers as Guignot (1961). Specimens were
identified by the first two authors. Outgroup taxa were
included from several other tribes in the Dytiscinae (Table 1).
Trees were rooted using Dytiscus verticalis, a member of the
tribe Dytiscini, the sister group to the remaining tribes in
Dytiscinae (Miller 2000, 2001, 2003).
DNA sequences
Methods for DNA extraction, amplification and sequencing
closely followed Miller et al. (2007). DNAs were extracted
with Qiagen DNEasy kit (Valencia, California, USA) using
the animal tissue protocol. In most cases, thoracic muscle
tissue was removed and extracted. In some cases a single
metathoracic leg was extracted. As with Cybistrini, many
Hydaticini are large and preserve poorly in ethanol (Miller
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
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Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Table 1 Taxa used in analysis including locality data, KBM voucher numbers, and GenBank accession numbers for DNA sequences.
Tribe
Species
Collection data
Voucher #, GenBank ## (COI/COII/H3/Wnt)
Aciliini
Graphoderus liberus (Say, 1825)
KBMC-Grli69, DQ813693/DQ813795/DQ813759/AF392016
Aubehydrini
Dytiscini
Notaticus fasciatus
Zimmermann, 1928
Dytiscus verticalis Say, 1823
USA: New York: Tompkins Co., Ringwood Pres.,
19 September 2000, KB Miller
BOLIVIA: Dpto Beni, Prov. Cercado, 9.5 km N Trinidad,
14°46′34″S 64°58′00″W 17 June 1999, KB Miller
NEW YORK: Tompkins Co., Ithaca, 26 May 2000, KB Miller
Hyderodini
Hyderodes shuckardi Hope, 1838
Eretini
Hydaticini
Hydaticini
Eretes australis
(Erichson, 1842)
Hydaticus bihamatus
(Aubé, 1838)
H. bimarginatus (Say, 1830)
H. bivittatus (Laporte, 1835)
Hydaticini
H. bowringii (Clark, 1864)
Hydaticini
H. caffer (Boheman, 1848)
Hydaticini
H. capicola (Aubé, 1838)
Hydaticini
H. consanguineus (Aubé, 1838)
Hydaticini
H. dineutoides (Sharp, 1882)
Hydaticini
H. dorsiger (Aubé, 1838)
Hydaticini
H. exclamationis (Aubé, 1838)
Hydaticini
Hydaticini
H. fabricii (MacLeay, 1825)
H. flavolineatus (Boheman, 1848)
Hydaticini
H. galla (Guérin-Méneville, 1849)
Hydaticini
H. grammicus (Germar, 1830)
Hydaticini
H. humeralis (Régimbart, 1895)
Hydaticini
H. lativittis (Régimbart, 1895)
Hydaticini
H. leander (Rossi, 1790)
Hydaticini
H. litigiosus (Régimbart, 1880)
Hydaticini
H. luczonicus (Aubé, 1838)
Hydaticini
Hydaticini
H. major (Régimbart, 1899)
H. matruelis (Clark, 1864)
Hydaticini
Hydaticini
H. nigrotaeniatus
(Régimbart, 1895)
H. orissaensis (Nilsson, 1999)
Hydaticini
H. parallelus (Clark, 1864)
Hydaticini
Hydaticini
H. philippensis (Wehncke, 1876)
H. quadrivittatus
(Blanchard, 1853)
Hydaticini
6
AUSTRALIA: Victoria, roadside pool ~20 km W Cowwarr,
38°00′52″S 146°32′03″E, 7 November 2000, KB Miller
AUSTRALIA: S Australia, shallow roadside pool ~15 km
N Kingston, 13 November 2000, KB Miller
NEW CALEDONIA: South Prov. Dumbea, near road to Mt Koghis,
NCI, 3 November 2001, Balke and Wewalka
USA: Florida: Naples, 2 January 1999, K Binder
NAMIBIA: Waterberg Park, Onjoka Spring, 20°24.65′S
17°21.221′E, 25 May 2004, KB Miller
JAPAN: Honshu, Kawasaki, Kameyama City: Mie Pref.,
1 August 2000, Y Utsunomiya
SOUTH AFRICA: Eastern Cape Province, 2 km N Sterkstroom
31°31.063′S 26°31.687′E, 1398m, 21 January 2005, J Bergsten
SOUTH AFRICA: Eastern Cape Province, 31°19.613′S
26° 41.828′E, 1706 m, 21 January 2005 J Bergsten
NEW CALEDONIA: South Province, Dumbea, swamp at road to
upper Dumbea Riv., 4 November 2001, Balke and Wewalka
INDONESIA: Borneo: Central Kalimantan, Schwaner Range,
upper Kahayan basin, River Ogé, 24 July 2004, P Mazzoldi
GHANA: Volta Region, Rd btwn Nkwanta and Odumase,
08°15′32.2″N 000°26′33.7″E, 15–17 June 2005, KB Miller
SOUTH AFRICA: Eastern Cape Province, 2 km N Sterkstroom
31°30.233′S 26°32.160′E, 1414 m, 20 January 2005, J Bergsten
PHILIPPINES: Boracay Central, 26 September 2000, J Bergsten
GHANA: Greater Accra Region, Shai Hills Resource Res.,
05°53.426′N 000°02.623′E, 1 June 2005, KB Miller
SOUTH AFRICA: Eastern Cape Province, Dwesa NR 32°17.621′S
28°48.885′E, 109 m, 24 January 2005, J Bergsten
CHINA: Yunnan: 2 km N of Shizong, 10 September 2000,
J Bergsten
GHANA: Volta Reg., Rd btwn Nkwanta and Odumase,
08°15′32.2″N 000°26′33.7″E, 15–17 June 2005, KB Miller
GHANA: Volta Region, Rd btwn Nkwanta and Odumase,
08°15′32.2″N 000°26′33.7″E, 15–17 June 2005, KB Miller
PORTUGAL: Odemira: Vila Nova de Milfontes, Planalto do
Malhao, 6 June 2000, J Bergsten
INDIA: Andamen Islands, Havelock, Village 5, 6 April 2004,
P Bohman
VIETNAM: Mui Ne: Lotus Lake, 10 km N Mui Ne, 6 January 2004,
F Johansson
CHINA: Yunnan: 4 km S Shizong, 13 September 2000, J Bergsten
SOUTH AFRICA: Eastern Cape Province, Near Dwesa NR
32°17.027′S 28 °47.506′E, 188 m, 23 January 2005, J Bergsten
MADAGASCAR: Montagne d’Ambre, 1 January 2005, J Bergsten
INDIA: Karnataka, Jog Falls, 14°16.480′N 74°44.436′E,
6 October 2004, KB Miller
AUSTRALIA: Victoria, roadside pool ~20 km W Cowwarr,
38°00′52″S 146°32′03″E, 7 November 2000, KB Miller
PHILLIPINES: Boracay, 26 September 2000, J Bergsten
AUSTRALIA: Queensland, Eubenange Swamp, 17°24′53″S
145°58′91″E, 4 August 2003, CHS Watts
KBMC-Nofa52, FJ796625/–/FJ796545/AF392036
KBMC-Dyve24, DQ813692/DQ813794/DQ813758/
AF392012
KBMC-Hdsh104, DQ813694/DQ813796/DQ813760/
AF392018
KBMC-Erau103, FJ796579/–/FJ796506/FJ796547
KBMC-Hybh205, FJ796581/FJ796628/FJ796508/FJ796548
KBMC-Hybi204, FJ796582/FJ796629/–/FJ796549
KBMC-Hybv465, FJ796584/FJ796631/FJ796510/–
KBMC-Hybo121, FJ796583/FJ796630/FJ796509/AF392020
KBMC-Hycf403, FJ796585/FJ796632/FJ796511/FJ796550
KBMC-Hycp405, FJ796589/FJ796636/FJ796514/FJ796552
KBMC-Hycn212, FJ796587/FJ796634/–/FJ796551
KBMC-Hydi321, FJ796590/FJ796637/FJ796515/–
KBMC-Hydo423, FJ796591/FJ796638/FJ796516/FJ796553
KBMC-Hyex400, FJ796592/FJ796639/FJ796517/FJ796554
KBMC-Hyfa119, FJ796593/FJ796640/FJ796518/AF392022
KBMC-Hyfl422, FJ796594/FJ796641/FJ796519/FJ796555
KBMC-Hyga398, FJ796595/FJ796642/FJ796520/FJ796556
KBMC-Hygr117, FJ796596/FJ796643/FJ796521/AF392023
KBMC-Hyhu426, FJ796597/FJ796644/FJ796522/FJ796557
KBMC-Hylv417, FJ796601/FJ796648/FJ796526/FJ796561
KBMC-Hyle195, FJ796598/FJ796645/FJ796523/FJ796558
KBMC-Hyli310, FJ796599/FJ796646/FJ796524/FJ796559
KBMC-Hylu270, FJ796600/FJ796647/FJ796525/FJ796560
KBMC-Hyma120, FJ796602/FJ796649/FJ796527/AF392024
KBMC-Hymt402, FJ796603/FJ796650/FJ796528/FJ796562
KBMC-Hyni399, FJ796604/FJ796651/–/–
KBMC-Hyor332, FJ796605/FJ796652/FJ796529/FJ796563
KBMC-Hypr106, FJ796607/FJ796654/FJ796530/AF392025
KBMC-Hyph203, FJ796606/FJ796653/–/FJ796564
KBMC-Hyqv241, FJ796609/–/–/FJ796566
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
Table 1 Continued.
Tribe
Species
Collection data
Voucher #, GenBank ## (COI/COII/H3/Wnt)
Hydaticini
Hydaticini
H. rhantoides (Sharp, 1882)
H. ricinus (Wewalka, 1979)
KBMC-Hyrh206, FJ796610/FJ796656/FJ796531/FJ796567
KBMC-Hyri271, FJ796611/FJ796657/FJ796532/FJ796568
Hydaticini
H. rimosus (Aubé, 1838)
Hydaticini
Hydaticini
Hydaticini
H. rivanolis (Wewalka, 1979)
H. satoi (Wewalka, 1975)
H. servillianus (Aubé, 1838)
Hydaticini
H. speciosus (Régimbart, 1895)
Hydaticini
H. subfasciatus (Laporte, 1835)
Hydaticini
H. ugandaensis (Guignot, 1936)
Hydaticini
H. ussherii (Clark, 1864)
Hydaticini
H. vittatus (Fabricius, 1775)
Hydaticini
H. wattsi (Daussin, 1980)
Hydaticini
H. xanthomelas (Brullé, 1838)
Hydaticini
H. aruspex Clark, 1864
Hydaticini
H. cinctipennis Aubé, 1838
Hydaticini
H. continentalis
J. Balfour-Browne, 1944
H. seminiger (DeGeer, 1774)
H. transversalis
(Pontoppidan, 1763)
Prodaticus pictus Sharp, 1882
CHINA: Yunnan, 4 km S Shizong, 13 September 2000, J Bergsten
VIETNAM: Mui Ne: Lotus Lake, 10 km N Mui Ne, 6 January 2004,
F Johansson
COSTA RICA: Guanacaste Province, Barra Honda Natl. Park,
pools in Quebrada La Palma, 12 January 2004, Short and Lebbin
PAPUA NEW GUINEA: Alotau, 14 July 2001
CHINA: Yunnan, 2 km S Shizong, 15 September 2000, J Bergsten
SOUTH AFRICA: Eastern Cape Province, 2 km N of Sterkstroom
31°31.063′S 26°31.687′E, 1398 m, 21 January 2005 J Bergsten
GHANA: Volta Reg., rd btwn Nkwanta and Odumase,
08°15′32.2″N 000°26′33.7″E, 15–17 June 2005, KB Miller
BOLIVIA: Dpto Sta Cruz, Province. Chiquitos 2.7 km S San Jose,
17°52′20″S 60°44′28″W, 27 June 1999, KB Miller
GHANA: Volta Region, rd btwn Nkwanta and Odumase,
08°15′32.2″N 000°26′33.7″E, 15–17 June 2005, KB Miller
GHANA: Western Region, Ankasa Resource Reserve,
05°16.566′N 002°38.342′W, 7–8 June 2005, KB Miller
INDIA: Maharashtra, 16°34.992′N 73°35.221′E,
1 October 2004, KB Miller
AUSTRALIA: Queensland, Eubenange Swamp, 17°24′53″S
145°58′91″E, 4 August 2003, CHS Watts
BOLIVIA: Departemento Sta Cruz, Province, Velasco, 1.5 km
SE San Ignacio, 24 June 1999, KB Miller
USA: New York: Schuyler Co., Texas Hollow, 6 September 2000,
KB Miller
USA: New York: St. Lawrence Co., Macomb Twp., Fish Cr. marsh,
23 May 2000, 44°28′20″N 75°33′48″W, KB Miller
RUSSIA: Volgograd Oblast, 4 km E Khmelevskoy Lake S River
Don, 2 May 2002, J Bergsten
Sweden: Östergötland, E Lundqvist
RUSSIA: Volgograd Oblast, Krasnoslobodsk, 15 May 2001,
J Bergsten
United Arab Emirates (U.A.E.): Wadi Bih Darn, Lat 25.48°,
Lon 56.04° 22 February–1 March 2007 A van Harten
Hydaticini
Hydaticini
Hydaticini
et al. 2007). To avoid this problem, thoracic muscle from
many specimens was dissected in the field soon after collection. This tissue was immediately placed in ethanol and later
extracted for use with PCR. Vouchers and other identified
material are deposited in the Museum of South-western
Biology Arthropod Collection (MSBA).
Molecular data
Four genes were sequenced for analysis, cytochrome c oxidase I
(COI) and II (COII), histone III (H3) and wingless (wnt). Primers
used for amplification and sequencing were derived from
several sources (see online Supporting Information Table S1).
For some taxa, COI and COII were amplified and sequenced
together using several combinations of primers and the intervening approximately 60 bp leucine-coding tRNA sequence
(which was length variable in many taxa) was excised. In
most cases, COI and COII were amplified and sequenced
separately without the intervening tRNA sequence. For
KBMC-Hyrm335, FJ796612/FJ796658/FJ796533/FJ796569
KBMC-Hyrv389, FJ796613/–/FJ796534/FJ796570
KBMC-Hysa209, FJ796614/FJ796659/FJ796535/FJ796571
KBMC-Hysr401, FJ796617/FJ796661/FJ796538/FJ796573
KBMC-Hyso421, FJ796616/FJ796660/FJ796537/FJ796572
KBMC-Hysu23, FJ796618/FJ796662/FJ796539/FJ796574
KBMC-Hyug424, FJ796620/FJ796664/–/–
KBMC-Hyus425, FJ796621/FJ796665/FJ796541/FJ796576
KBMC-Hyvi331, FJ796622/FJ796666/FJ796542/FJ796577
KBMC-Hywa242, FJ796623/FJ796667/FJ796543/FJ796578
KBMC-Hyxa67, FJ796624/FJ796668/FJ796544/AF392028
KBMC-Hyar68, FJ796580/FJ796627/FJ796507/AF392019
KBMC-Hyci20, FJ796586/FJ796633/FJ796512/AF392021
KBMC-Hyco211, FJ796588/FJ796635/FJ796513/–
KBMC-Hyse125, FJ796615/–/FJ796536/AF392026
KBMC-Hytr192, FJ796619/FJ796663/FJ796540/FJ796575
BMNH 833204, FJ796626/FJ796669/FJ796546/–
certain taxa, markers or portions of markers could not be
amplified or sequenced. GenBank numbers are indicated in
Table 1.
DNA fragments were PCR amplified using AmpliTaq Gold
(Applied Biosystems), Platinum Taq (Invitrogen) or HotMaster
Taq (Eppendorf) on a DNA Engine DYAD Peltier Thermal
Cycler. Amplification conditions are presented in online
Supporting Information Table S2. Contamination was mediated using negative controls, and fragments produced from
PCR were examined using gel electrophoresis. Products were
purified using Montage PCR96 Cleanup Kit (Millipore) and
cycle sequenced using ABI Prism Big Dye (version 3) using
the same primers used to amplify. Sequencing reaction
products were purified using Sephadex G-50 Medium and
sequenced using an ABI 3730xl DNA analyser (DNA
Sequencing Center, BYU). Gene regions were sequenced in
both directions. Resulting sequence data were examined and
edited using the program Sequencher (Genecodes 1999).
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
7
Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Morphology
The morphological characters analysed in this analysis are
described in Appendix I. Many of these characters have been
used previously in dytiscid classification and they are described
in general treatments including Sharp (1882), Balfour-Browne
(1950), Guignot (1961) and Larson et al. (2000). There are
several characters included, however, that are specific to
Hydaticini. Some of these are new and are discussed in
greater detail. Others were first introduced in the literature
including papers by Galewski (1985), Hernando & Fresneda
(1994), Nilsson (1981), Roughley & Pengelly (1981), Satô
(1961), Trémouilles (1994) and Wewalka (1975, 1979). Some
of these characters have been described more thoroughly and
tested in other phylogenetic projects (Miller 2000, 2001,
2003; Miller et al. 2007) (online Supporting Information
Table S3). Coded states are presented in online Supporting
Information Table S4.
Analysis
Sequence alignment was done manually using Sequencher
(Genecodes 1999) since the sequences are, in most taxa, not
length variable and alignment is unambiguous. Notaticus
fasciatus has a three base pair indel in wingless which was
aligned based on conservation of reading frame and conversion
to amino acids for alignment (Miller 2003) (online Supporting
Information Table S5). Similarly, one species of Hydaticini,
H. bihamatus, exhibits several three, or multiples of three,
base pair indels in COI which were aligned in the same way
(online Supporting Information Table S5). Alternative alignments were examined and analysed, but did not affect the
results of the analysis.
Data were analysed using parsimony and the program
NONA (Goloboff 1995) implemented using WinClada
(Nixon 2002) with the ‘heuristics’ search option and the commands set to hold 5000 trees total (‘h 5000’), 60 replications
(‘mu*60’), 40 trees held per replication (‘h/40’), and multiple
TBR + TBR (‘max*’). Character 34 was treated as additive.
Trees were examined and analysed under different optimizations and character distributions on the resulting topologies
were examined using WinClada. Support for branches was
measured using bootstrap values. These were calculated in
NONA as implemented by WinClada using 1000 replications,
10 search reps, 1 starting tree per rep, ‘don’t do max*(TBR)’,
and save the consensus of each replication. We also ran a
maximum likelihood analysis using GARLI (Zwickl 2006) on
the DNA data alone, specifying a single GTR + I + Γ model
across all genes and leaving other settings as default in the
configuration file (GARLI version 0.96b8). Clade support
values were estimated with 200 bootstrap replications.
To explore the effect of partition choice with multiple
protein-coding genes, we identified nine different logical
partitioning schemes ranging from forcing a single model to
8
Table 2 Partitioning multiple nuclear and mitochondrial protein
coding genes. Partitions include genome (nuclear/mitochondrial),
genes, third codon positions, all codon positions, and combinations
of these. Morphological characters were treated with a separate Mk
or Mk + Γ model for all analyses (Lewis 2001).
First
Number of
partitions
Morphology/
DNA
1
2
3a
3b
4
5a
5b
7
9
13
(only DNA)
X
X
X
X
X
X
X
X
X
Nuclear/
Mitochondrial
Genes
Second
Third
Codon position
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
all genes (with a separate Mk model for morphology) to treating
all codon positions from all genes as separate partitions
(Table 2). All 35 different components of the 9 partitioning
schemes were analysed with MRMODELTEST (Nylander
2004b) and MrAIC (Nylander 2004a). MRMODELTEST is a
version of MODELTEST (Posada & Crandall 1998) that limits
the hLRT to the 24 models supported in MRBAYES (Ronquist
& Huelsenbeck 2003) and includes the four different directions of traversing parameter space explored by Posada &
Crandall (2001). Pol (2004) found that varying the sequence
for which parameters were tested with hLRT often led to
different model choices which in some cases led to conflicting
phylogenetic hypotheses. MrAIC differs from the AIC and
BIC calculated in MODELTEST (and MRMODELTEST) in that
the likelihood score is, as appropriate, optimized under each
model using searches with PHYML (Guindon & Gascuel
2003). We recorded the model choice under the four hLRT
directions, AIC, AICc (corrected AIC for small sample size)
and BIC (online Supporting Information Table S6). All nonidentical model choice × partitioning scheme combinations
(a total of 57) including a MAX (GTR + Γ + I) and MIN model
(JC) for all partitions were analysed with the parallel version
of MRBAYES vs. 3.1.2 (Altekar et al. 2004) on a 408 processor
Opteron (1.8 GHz, 2 Gb memory) Beowulf cluster at Imperial
College, London.
We acquired DNA of Prodaticus pictus at a late stage of revision of the submitted manuscript and were able to sequence
all but a portion of COI and wnt. Given the circumstances,
we wanted to include this taxon for taxonomic purposes, but
we were not able to include it in these time-consuming
analyses. Data from P. pictus was included in the presented
favoured Bayesian analysis (7BIC, see below) as well as in the
parsimony and likelihood analysis. In all runs we used one
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
cold and three incrementally heated chains (Temperature = 0.2)
and sampled every 1000 steps for 10 million generations.
This was repeated twice for all combinations, and samples
from the two runs were pooled, discarding the first 5 million
generations as burn-in, unless otherwise stated. Prior and
proposal settings were left as default. Among partition rate
variation was allowed with a ratemultiplier using the ‘prset
ratepr = variable’ command in MRBAYES (see Marshall et al.
2006). The morphological characters were given an Mk
model (Lewis 2001) in all analyses after rejecting as an
informative model component (the inclusion did not increase
the log of the harmonic mean of the likelihood). We treated
character 34 as additive and accounted for the fact that only
parsimony informative characters were included. Standard
convergence diagnostics for the independent runs, as implemented in MRBAYES, were checked to ensure that sampling
was long enough and had reached the same stationarity. The
topology with highest posterior probability and the majorityrule consensus with posterior probability clade support values
were recorded and compared across the runs of all combinations. We used the log of the harmonic mean of the likelihood
at the stationary phase (after discarding the burn-in) to calculate
Bayes Factor and a modification (see below) in the increase of
LnL for accepting additional parameters as recommended by
Pagel & Meade (2004). Branch lengths are not included when
mentioning the number of free parameters for different models
for practical purposes since this number is constant across all
tested models.
Results
Model choice for the 35 partitions varied depending on the
criteria used (online Supporting Information Table S6). For
example depending on the direction of traversing parameter
space the partition with first and second codon positions of
H3 resulted in a model choice of SYM + Γ with direction 1,
K80 + I with direction 2, K80 + Γ with direction 3 and SYM + I
with direction 4. Evidently, when a proportion of invariable
sites (I) is tested in the presence of the Γ parameter (Γ) (direction
1 and 3) I is rejected, whereas when the Γ -parameter is tested
in the presence of I (direction 2 and 4), Γ is rejected. Likewise
the model chosen by AIC, AICc and BIC varied especially
with small partitions. For example, model choice for the first
codon partition of H3 resulted in GTR + Γ with AIC, JC
with AICc and K80 + I with BIC (and SYM + Γ or SYM + I
with hLRT).
In a comparison of the partitioning schemes, in general,
partitioning by codon position resulted in greater likelihood
than partitioning by gene (online Supporting Information
Table S7). The log of the HME of the marginal likelihood is
about 570 log-likelihood units (LnLU) higher with scheme 3b
(first + second and third codon positions) than 3a (nuclear vs.
mitochondrial genes), although there are the same number
of parameters in each. This is in addition to an original
improvement of 600 LnLU over the unpartitioned molecular
data (2). Additional partitioning of 1st and 2nd codon positions
(4) increases HME by an another 100 LnLU. Partitioning by
gene (5a) is 500–600 LnLU worse than codon partitioning
(3b, 4) although 5a has > 20 additional free parameters. The
alternative five-partition scheme (5b, separating nuclear and
mitochondrial genes and also partitioning third codon
positions) is overwhelmingly favoured with the HME 1400
LnLU higher than results from partitioning by gene (5a) and
800 LnLU higher than partitioning by all three codon positions but not nuclear and mitochondrial genes separately (4).
Adding two more partitions (7, similar to 5b but with an extra
partitioning of first and second codon positions for both the
nuclear and mitochondrial group) further improves the HME
by about 100 LnLU. Alternatively, partitioning each gene
and separating third codon position for each gene adds two
partitions and around 10–20 free parameters, but without any
improvement in LnLU. Finally, the maximum partitioning
(13 partitions of separate codon positions for each gene)
improves the HME by a modest 10–100 LnLU compared with
results from seven partitions while adding another 10–60 free
parameters.
According to the suggested use of Bayes Factor (Kass &
Raftery 1995a 2*Ln Bayes factor of > 10 is interpreted as
model 1 being strongly favoured over model 0. Using this
criterion, the 13 partitioning scheme with the maximum
GTR + Γ + I model is favoured. This model resulted in an
estimated marginal likelihood of –25 660, 40 LnLU more
than the second best choice (13hLRT2 and 4) which resulted
in a 2*Ln Bayes Factor of 80. Thus, this model is strongly
favoured despite adding 50 free parameters for a total of 132.
Since concerns have been raised about the scale of interpretation of Bayes Factor in phylogenetics and that the HME
may overestimate the real marginal likelihood, more severely
so for high-dimensional models, we implemented a recommendation by Pagel & Meade (2004). The Bayes Factor test
penalizes overparametrization through the specified priors.
Pagel & Meade (2004) in the context of mixture models note
that given their priors, each GTR rate matrix added to their
mixture model requires an improvement of about 30 loglikelihood units to return a Bayes Factor of 0. 0 is equivalent
to no preference for either the simpler or the more complicated model. More than 30 units is therefore required for
preferring the more complicated model, and they suggested
that for an extra GTR model to be accepted, thereby adding
another seven free parameters, it should improve the likelihood
with 70–80 log-likelihood units. This would conveniently
equal a requirement of a 10 log-likelihood unit’s improvement
per parameter, and corresponded with their priors to a log
Bayes factor of about 40, that is, strongly supported. Thus,
we calculated the ‘PM-factor’, PM = ΔLnL/Δp (p = number
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
9
Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Figs 20–22 Hydaticini species, female
genitalia, ventral aspect. 20, H. aruspex. 21,
H. flavolineatus. 22, H. dorsiger.
of free parameters) and we accepted additional partitions
only when this value was 10 or greater (online Supporting
Information Table S8). When multiplied by 2 the PM-factor
is identical to what Castoe et al. (2005) called the ‘relative
Bayes Factor’ and the threshold we employed would consequently be the same as a ‘relative Bayes Factor’ of > 20. Application of this criterion clearly implies preference of 3b over
2 (PM = 109–120) and 5b over 4 (PM = 62.6–265.4). It
marginally implies preferences of 4 over 3b (PM = 9.4–14.2)
and 7 over 5b (PM = 7.7–12.3, for model choice favoured for
each partitioning, 7-BIC vs. 5b-hLRT1, PM = 11.5) both of
which refers to splitting the lumped first and secnd codon
position partition into two separate partitions. It implies
rejection of the addition of another 6 partitions in the maximum
13 scheme (PM = 0–2.6), and 9 partitions is rejected since
this scheme increased the number of parameters but not
LnL. Among the model choice criteria within the seven
partitions, where Bayes Factor would choose the maximum
GTR + Γ + I for all partitions (2*Ln Bayes factor = 32), the
PM factor selects the simplest model combination chosen by
BIC with only one of the seven partitions given the max
GTR + Γ + I model (42 parameters, LnL = –25 802, alterna10
tive hLRT1, 45 parameters –25 784, PM = 6, alternative
MAX, 66 parameters, LnL = –25 763, PM = 1.6). The topology inferred from the seven partitions-BIC selected models
is shown in Fig. 23.
The inferred topologies for the different model choice ×
partitioning scheme combinations differ only minimally
from each other with the exception of the results from assignment of a JC model to all partitions. This topology differed
most but is clearly rejected based on both HME-based factors.
Among the other topologies (47) areas of incongruence
were restricted to three alternatives. First, Hyderodes is in
an alternative placement in 2 of the 47 analyses as sister to
the clade Notaticus + Eretes + Graphoderus. Second, the clade
H. luczonicus + H. orissaensis + H. bihamatus is resolved as
H. bihamatus + H. orissaensis as sisters in 35 of the analyses
and as H. luczonicus + H. orissanensis as sisters in 12. However,
posterior probability of the resolution in this clade was low
throughout the topologies for both alternatives (pp = 0.50–0.82).
The third incongruency involved the species H. nigrotaeniatus
and H. parallelus. As a clade H. nigrotaeniatus + H. parallelus was
resolved as sister to the remaining Guignotites (eight analyses)
or as sister to the H. vittatus + H. speciosus groups (38 analyses,
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
Fig. 23 Topology with highest posterior probability (P = 0.043) from the preferred 7BIC Bayesian analysis of morphology and four genes.
Numbers on branches are posterior probability support values and branch lengths, jointly estimated from all seven partitions, are averaged
over the last 5 million generations.
as in Fig. 23). The first alternative occurred in the 3b, 4 and
13BIC analyses but was weakly supported in the last (pp = 0.58).
The 13AICc analysis resulted in a third alternative with
H. parallelus sister to the H. vittatus + H. speciosus groups and
H. nigrotaeniatus sister to the remaining H. (Guignotites).
These positions are the two alternative placements of the
clade H. nigrotaeniatus + H. parallelus in the remaining 46
analyses. Whereas this third alternative was found in the
topology with highest posterior probability, resolution of the
entire topology between the two terminals was collapsed in
the consensus. A sister relationship between these two taxa
has never been proposed based on morphology and they are
optimized as having two of the three longest terminal
branches in the ingroup (Fig. 23). This led us to investigate
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
11
Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Fig. 24 Topology from 7BIC-exclusion runs with H. nigrotaeniatus and H. parallelus placed as indicated by the long branch extraction test.
Double numbers on internodes are posterior probability support values from (A, B) —A. when H. parallelus was excluded and —B. when
H. nigrotaeniatus was excluded (only one number indicates these values were identical).
the possibility of an effect from long-branch attraction.
Using the long-branch extraction test (Bergsten 2005; Siddall
& Whiting 1998) we successively excluded H. nigrotaeniatus
and H. parallelus and reran all 57 model choice × partitioning
analyses, recording changes in the placement of these taxa
12
and others. In all analyses with 5b partitioning or higher and
H. parallelus excluded, H. nigrotaeniatus moves from its original
placement (Fig. 23) to being sister to the remaining H.
(Guignotites) (Fig. 24). In contrast, for the same partition
schemes with H. nigrotaeniatus excluded, H. parallelus remains
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
in its original phylogenetic resolution (Fig. 24). In the 13BIC
analysis, the only one with H. nigrotaeniatus + H. parallelus
resolved as sister to the remaining H. (Guignotites), the result
is the opposite. Hydaticus nigrotaeniatus remains in this position
with H. parallelus excluded but H. parallelus is resolved as in
Fig. 24 with H. nigrotaeniatus excluded. Finally, in the 13AICc
analysis which originally had the two species resolved separately, neither taxon changed in phylogenetic position with
the other excluded (Fig. 24). Consequently, the response to
the taxon removal test fulfills the expected prediction of an
LBA artefact (Bergsten 2005; Siddall & Whiting 1998). Some
of the results from the 5a partitions and below responded
slightly differently to the long-branch extraction treatment.
However, since these partitioning schemes were overwhelmingly rejected as inferior to partition 5b and above, we will
not discuss these in detail. Full information is available as
online Supporting Information.
The combined parsimony analysis resulted in two equally
parsimonious cladograms (L = 5503, CI = 29, RI = 48), one
of which is shown in Fig. 25. The other tree differed only in
the resolution of the three closely related species H. fabricii,
H. rivanolis and H. ricinus. Although there are a few disagreements between this analysis and the Bayesian results, especially
regarding some ‘deeper’ branches, only in the H. vittatusgroup are disagreements supported by bootstrap values of
> 0.5 (Fig. 25). We tested if the two potentially problematic
long-branched taxa H. nigrotaeniatus and H. parallelus are the
cause for lack of support of the four deeper internodes in
H. (Guignotites) (below the H. nigrotaeniatus + H. parallelus
clade) by rerunning the bootstrap analysis with H. nigrotaeniatus
and H. parallelus excluded. This resulted in two of the four
internodes supported with bootstrap values of 70 (H. (Guignotites)) and 63 (H. (Guignotites) excluding the H. bihamatus
group). Also the support for Hydaticus increased from 70 to
88, indicating that these long branched taxa were probably
attracted to the outgroup terminals in several of the bootstrap replicates, a well-known phenomenon (Bergsten
2005).
The maximum likelihood analysis on the DNA data alone
using GARLI (not shown) resulted in an identical topology to
the favoured partitioned Bayesian analysis (Fig. 23) except
for the clade H. orissaensis + H. bihamatus (bt = 0.68) and the
H. vittatus species group which was identical to the parsimony
result (Fig. 25). Both parsimony and unpartitioned likelihood
places the African H. bivitattus basal in the otherwise oriental
H. vittatus clade (no bootstrap support) and H. quadrivittatus +
H. philippensis as sister species (likelihood bt = 0.64, parsimony
bt = 0.62) whereas partitioned Bayesian analysis places H.
bivittatus as sister to H. satoi (pp = 0.87), and H. quadrivittatus
as basal to the clade (pp = 1.0) with strong support (Fig. 23).
In all analyses the tribe Hydaticini is monophyletic but the
genus Hydaticus is paraphyletic due to the nesting within it of
the other recognized genus, Prodaticus (bootstrap = 70, pp = 1.0;
Figs 23 and 25). Of the subgenera, Hydaticus sensu stricto is
monophyletic (bt = 100, pp = 1.0) whereas Hydaticus (Guignotites) is paraphyletic (pp = 1.0) due to the nesting within it of
Prodaticus and the subgenera H. (Hydaticinus) and H. (Pleurodytes)
(Figs 23 and 25). Within H. (Guignotites), clades correspond
roughly to various previously recognized species groups and
show a strong biogeographical pattern. The H. bihamatus
(bt = 81, pp = 1.0) group is sister to the remaining H.
(Guignotites) (and Prodaticus and other subgenera) (pp = 0.63)
and has an Oriental distribution with some species also
reaching the Palearctic and Australian regions. The next
clade (the H. subfasciatus group) includes members which are
entirely Neotropical (bt = 84, pp = 1.0), with one species, H.
bimarginatus extending into the southern and eastern Nearctic.
The monotypic subgenus H. (Hydaticinus) is strongly associated with Neotropical relatives (bt = 96, pp = 1.0). The next
clade is Afrotropical (bt = 64, pp = 1.0) and consists of species
from the H. sobrinus species group, with H. dorsiger sister
(bt = 100, pp = 1.0) to the other three species. The H. leander
group (bt = 100, pp = 1.0) is sister to the H. fabricii group
(bt = 97, pp = 1.0), with both consisting of rather unicolorous
species. The H. leander group is Afrotropical with some
reaching the Mediterranean and Middle East. The H. fabricii
group is Oriental-Australian and Palearctic. Finally, the
H. speciosus and H. vittatus groups are together monophyletic
(bt = 63, pp = 0.84) with an entirely Afrotropical clade (the
H. speciosus group, bt = 70, pp = 0.98) and an Oriental-AustralianPalaearctic clade (the H. vittatus group, bt = 98, pp = 1.0),
with this second group having a single Afrotropical species,
H. bivittatus. The subgenus H. (Pleurodytes) is convincingly
nested within the Oriental members of the H. vittatus group
(bt = 92, pp = 1.0), and the Afrotropical clade includes species
of the speciosus group sensu Guignot (1961). The species P. pictus
is strongly supported as sister group to the H. vittatus +
H. speciosus groups (bt = 93, pp = 1.0). The positions of the
Australian H. parallelus and the Madagascar endemic, H.
nigrotaeniatus (in the H. sobrinus group), as rigorously
investigated (see above), are uncertain, but their sister group
relationship (bt = 66, pp = 1.0) is most probably artificial.
Hydaticus parallelus may have an intermediate position between
the clade H. leander group + H. fabricii group and the H.
vittatus + H. speciosus groups + Prodaticus whereas H. nigrotaeniatus seems to have a basal position within H. (Guignotites).
Discussion
Partitioning strategies
Our combined results imply guidelines for selecting among
alternative partitioning strategies for a data set of multiple
nuclear and mitochondrial protein-coding genes for Bayesian
analyses. Earlier workers have investigated the same question
but for data sets that combine protein-coding genes with
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
13
Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Fig. 25 One of two most parsimonious cladograms (L = 5503, CI = 29, RI = 48) from combined parsimony analysis with morphological
characters mapped using ‘fast’ optimization in WinClada. Second tree differed only in the resolution of the P. rivanolis, P. ricinus, P. fabricii cade.
Black hash marks indicate unambiguous changes, white hash marks indicate homoplasious changes or reversals. Numbers above hash marks
are character numbers, those below hash marks are state numbers. Numbers on internodes are support values from 1000 bootstrap replicates.
Dendrogram in lower left depicts branch lengths of the same tree based on number of changes (all changes in combined parsimony analysis
including morphology and DNA sequence data) mapped using ‘fast’ optimization in WinClada.
14
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
ribosomal DNA (Castoe et al. 2004; Nylander et al. 2004;
Brandley et al. 2005; Strugnell et al. 2005; Castoe & Parkinson
2006), or introns and tRNAs (McGuire et al. 2007), which
either differ in classes of biologically relevant partitions or do
not have multiple genes of both sources of coding DNA
(Castoe et al. 2005; Li et al. 2008). We found genome source
(nuclear vs. mitochondrial) and codon position to be the most
important partitions, far more important than partitioning by
individual genes. Apart from having a higher evolutionary
rate (> 8 times higher in our data), mitochondrial genes have
a significantly higher AT content than nuclear genes (88% vs.
47% in our data set), both certainly responsible for the
increase in 600 LnL units when partitioned. Not accommodating this kind of bias in model-based optimality approaches
is known to be a hazard that can lead to incorrect phylogenetic inferences (Lockhardt et al. 1994). The compromise
estimation of base frequencies (A: 0.303–0.334, T: 0.366–
0.396: 95% credible intervals) from the unpartitioned
DNA data set is a serious misrepresentation of both DNA
sources (nuclear, A: 0.237–0.292, T: 0.181–0.226; mitochondrial, A: 0.397–0.431, T: 0.449–0.482) from the 3a partition
scheme.
As a general rule, the same codon position in different
genes evolves more similar than do different codon positions
in the same gene. For example treating third codon positions
from all genes as a separate partition from first and second
(3b), results in a 500LnL units higher likelihood compared to
separating all four genes, wingless, histone 3, COI and COII
(5 A) despite many more parameters. This conclusion is
broadly in agreement with earlier work. Brandley et al. 2005)
found that partitioning the codon positions of the ND1 gene
had the largest effect on the mean LnL, and was favoured
even over alternatives with more total partitions. Partitioning
third codon positions in two mitochondrial protein-coding
genes resulted in a much higher harmonic mean likelihood
than dividing the two genes in Castoe et al. (2005). McGuire
et al. (2007) concluded that a minimum of four partitions
corresponding to one nuclear (introns) and three mitochondrial partitions after codon positions stabilized the topology,
branch lengths and posterior probability estimates. Li et al.
(2008) not only showed that three partitions representing the
codon positions across 10 nuclear loci resulted in the greatest
improvement in AIC and BIC values, but also that 10 genespecific partitions performed as badly as having no partitions
at all. Nylander et al. (2004) found that allowing rate variation
with a Γ parameter within a DNA partition consisting of three
protein-coding and one ribosomal gene had a much larger
effect than allowing rate variation between four gene partitions. The Γ shape parameter in this case will mostly model
rate variation across codon positions. However, whereas the Γ
shape parameter models as good as possible the systematic
rate differences between codon positions if not partitioned,
our results clearly show that this alternative is inferior to codon
partitioning. In our 5a analysis four separate Γ parameters
model codon rate variation within each gene, whereas in the
simpler codon partitioning (4) three Γ parameters model rate
variation within each codon class. Both allow rate variation
between partitions, genes in the former and codons in the
latter. Despite 11 more free parameters 5a is inferior by more
than 600 LnLU. Thus, we make the general recommendation
to partition protein-coding multigene Bayesian analyses by
codons and by genome source, but not only by genes as is
commonly done (see also Li et al. 2008).
Previous studies on the behaviour of Bayes Factor for
selection of models or partition schemes in phylogenetics
have all resulted in the most parameter-rich model being
chosen, and in all comparisons of nested models the more
parameter rich model has been favoured (2*Ln Bayes
Factor > 10 (Nylander et al. 2004; Brandley et al. 2005; Castoe
et al. 2005; Castoe & Parkinson 2006)). We also found that
Bayes Factor selects the most parameter rich model with
partitioning of all genes and all three codon positions and
each of the 12 molecular partitions assigned a GTR + Γ + I
model (13MAX). However, in contrast to previous studies,
we found examples of nested models where Bayes Factor
selects a simpler model. For example in the two partition
scheme with assignment of either an Mk or a Mk + Γ model
to the morphological matrix and GTR + Γ + I to the DNA
partition there is no support for adding the G parameter (2*ln
BF = 0.02). Likewise, in a comparison of 5b-hLRT3 and
5b-Max which differ in 3rd codon position partitions for
COI + COII and H3 + WNT, Bayes Factor selects GTR + Γ
over GTR + Γ + I with ‘strong’ support (LnL = –25873.29
vs. LnL = –25876.68, 2*ln BF = 6.78) according to the interpretation scale of Kass & Raftery (1995). This is empirical
evidence that Bayes Factor actually can penalize overparameterization, even for nested comparisons, as is theoretically expected (Nylander et al. 2004). Nevertheless, it seems
clear that Bayes Factor does not penalize to a satisfying degree
(but see Brown & Lemmon 2007). Latrillot & Philippe 2006)
convincingly argue that this is due to the MCMC HME
grossly overestimating the marginal likelihood especially for
more parameter rich models. The Metropolis Coupled
MCMC (MC3) is too efficient in only sampling the high
lkelihood areas of parameter space. To accommodate this
concern, we used the implicit recommendation by Pagel &
Meade (2004) requiring at least a 10LnL increase in the
HME per additional free parameter before accepting a more
complicated model. Whereas > 10LnLU is an artificial value
(just as > 10 represents ‘very strong support’ for the 2*Ln
Bayes Factor), it might do a rough job in correcting the
harmonic mean bias demonstrated by Latrillot & Philippe
(2006). In our case such a threshold resulted in a scheme with
seven partitions, with partitioning by each codon position
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
15
Hydaticini phylogeny & data partition choice • K. B. Miller et al.
and by genome source (nuclear and mitochondrial). A
maximum of 150 LnL units can be gained using the 13partition alternative, but at the price of a considerable 90
additional free parameters estimated from the data. While
Brown & Lemmon (2007) found the Bayes Factor to behave
appropriately, they do note a possible increase in the variance
of 2LnBF towards smaller data set sizes. Intuitively, only
estimation error can be gained by assigning a GTR + Γ + I
model to a partition of 109 nucleotides such as second codon
positions in H3. In our data set this partition has only four
variable sites and two of the six substitution types of the GTR
model are not even present. In comparison, this threshold
level would also imply selection of a simpler model than the
most complex models chosen in the studies by Castoe et al.
(2005), Castoe & Parkinson (2006), Brandley et al. (2005) and
Nylander et al. (2004). That being said, it is clear that
Bayesian analyses are more sensitive to oversimplified model
violations than to overspecified models of evolution (Huelsenbeck & Rannala 2004). More studies investigating data partitioning, small partition size, estimation error of parameters,
and the effect of different model priors (especially branch
length priors, see Yang & Rannala 2005) on the Bayes Factor
and its penalizing ability are warranted.
Variation in clade support values and topological inference
across the model choice criteria and 10 partitioning schemes
exist but are not extensive. As in previous studies clade
support values show both increasing and decreasing trends,
between < 0.7 and > 0.95 for different clades, when partitions
increased (Nylander et al. 2004; Brandley et al. 2005; Castoe
et al. 2005). For example, support for H. lativittis + H. speciosus
increased from 0.64 (3aBIC) to 0.99 (13AICC), whereas the
clade H. seminiger + H. aruspex + H. continentalis decreased
from 0.99 (2) to 0.62 (13hLRT3). The posterior probability
of the best topology decreased from the overly simplified
MIN-models (0.29–0.82) and two partition models (0.31–0.33)
to less than 0.16 for all analyses more complex than 3a. This
is similar to the finding by Nylander et al. (2004) that the
number of topologies in a 95% credible set of trees increased
with more complex models. One topological difference was
resolution of the clade H. bihamatus + H. orissaensis + H.
luczonicus in which no resolution ever had higher support than
0.82, apart from the MIN models ( JC) which (in all partition
schemes) resolved the relationship H. bihamatus + H. luczonicus
with up to 0.97 support. This clade clearly needs additional
data to be unambiguously resolved. The other topological
difference in the ingroup between analyses resolves around
the Madagascar endemic H. nigrotaeniatus and the Australian
endemic H. parallelus. The long branch extraction test indicates that their sister group relationship is artificial, but more
interesting is that their placement varied depending on the
partitioning. Partitioning by all three codon positions or by
only the third without partitioning by nuclear and mitochon16
drial genome (3b and 4), resolved this group as sister group
to the remaining H. (Guignotites). And a single model choice ×
partition scheme combination (13AICc) resolved the two
terminals in phylogenetic positions indicated in the extraction
test in the topology with highest posterior probability but
with support value of < 0.5. This particular combination
combines the highest number of partitions separating all
codon position for all genes, with the Corrected Akaike
Information Criteria as the model choice criteria which most
severely penalizes over-parametrization when sample size is
small (Posada & Buckley 2004). Especially in the nuclear
gene fragments, sample size (number of nucleotide sites) for
a single codon position were very small (wingless, 155; H3,
109) and for these AICc selected simple JC, JC + I and K80 + Γ
whereas, for example, the uncorrected AIC selected GTR + Γ
for 4 of 6 partitions. Actually the two independent runs of
13AICC differed in their inference of the clade, but HME of
the marginal likelihood differed by only 1 LnL unit (and the
samples were consequently pooled). Does this suggest that a
scheme of highly partitioned data sets with simple models for
all (small) partitions is most likely to escape artifacts and may
be the way forward for analyses of large multigene matrices?
Whereas our single example of analysis of this data set is
insufficient to definitively establish this, it does suggest a
future direction for more comprehensive investigation.
Classification
In a previous analysis (Miller 2003), Hydaticini was found to
be paraphyletic with respect to a group including Eretini and
Aciliini. An analysis by Ribera et al. (2002) placed Hyderodes
(Hyderodini) and Notaticus (Aubehydrini) within Hydaticini.
The position of Hyderodes was regarded as weakly supported
by the authors, but the placement of Notaticus within the
group was regarded as well-founded. Our analysis, which
includes the most extensive character and taxon sampling
within Hydaticini to date, supports monophyly of Hydaticini
and a sister group relationship between Hydaticini and a clade
containing Aubehydrini, Aciliini and Eretini with Hyderodini
and Dytiscini outside this clade (Figs 23–25). Hyderodes is
most certainly not a member of Hydaticini since it lacks all
the structural apomorphies of the tribe and has several plesiomorphies that suggest a much closer relationship with Dytiscus,
a conclusion supported by this analysis (Figs 23–25) and
several previous analyses (Miller 2000, 2001, 2003). The
position of Notaticus is, however, more unstable through
several analyses of this subfamily. In addition to its position
nested within Hydaticini, proposed by Ribera et al. (2002), it
has also been found as sister to the clade Hydaticini +
Eretini + Aciliini by Miller (2000) and as sister to Aciliini
(Miller 2003). This analysis, which includes better character
sampling, but not extensive taxon sampling outside Hydaticini,
places it as sister to Aciliini + Eretini with relatively strong
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
branch support (Figs 23–25). The larva, recently described
(Miller et al. 2007), possesses characteristics similar to
hydaticines, but many of these appear to be plesiomorphies
(Fig. 25). Even so, there seems to be mounting evidence that
Notaticus, which, like Hyderodes, lacks the structural synapomorphies shared by hydaticines, is not a member of Hydaticini.
Exactly where it fits in the phylogeny remains ambiguous.
The monophyletic Hydaticini is divided into two prominent
clades (Figs 23–25). One corresponds with the subgenus
Hydaticus sensu stricto as historically recognized. The other
includes H. (Guignotites) with Prodaticus, H. (Hydaticinus) and
H. (Pleurodytes) nested well within it (Figs 23–25). The character
combination used historically to diagnose H. (Hydaticinus) is
the absence of fine punctures on the anterior surfaces of the
metafemur and metatibia combined with a linear series of
bifid setae on the posterior surface of the metatibia (Fig. 13).
This combination makes it similar to both Hydaticus s. str. and
H. (Guignotites). Evidence from this analysis, however, strongly
indicates that the species is nested within H. (Guignotites), is
closely related to several other Neotropical hydaticines
(Figs 23–25), and is sister to the species H. (G) subfasciatus.
The linear series of metatibial setae is a reversal (Fig. 25).
Hydaticus (Pleurodytes), with two Oriental species, is characterized by a very broad elytral epipleuron (Fig. 3), a unique
habitus (Fig. 2), and uniformly black dorsal colouration (Fig. 2),
a unique feature within Hydaticini. Evidence presented here
indicates that it is nested within H. (Guignotites) and is closely
related to several other Oriental hydaticines (Figs 23–25).
The unique features exhibited by these two species are
apomorphic within H. (Guignotites).
The main features characterizing Prodaticus are the subequal
metatarsal claws and a substantially larger number of small,
protarsal adhesive discs in the male than other species of
Hydaticini. In most respects, Prodaticus specimens are similar
to H. (Guignotites) specimens. They lack fine punctation on
the anterior surfaces of the metafemur and metatibia, there is
a small, linear brush of setae on the ventral surface of mesotarsomere I in the male, and the posterior line of setose
punctures on the metafemur are dense and in a well defined
line. Our analyses is conclusive in that it is nested well within
H. (Guignotites) with high support values. The linear series of
setae on the posterior surface of the metatibia, however, is in
a line parallel to the dorsal margin as in Hydaticus sensu stricto,
necessesarily interpreted as independently evolved. These
results suggest the basis for an improved classification for the
tribe Hydaticini. Because of the clear evidence for two large,
well defined clades (Figs 23–25), we propose recognition of
two genera within Hydaticini, Hydaticus and Prodaticus. The
groups Prodaticus, H. (Pleurodytes) and H. (Hydaticinus) are clearly
nested within a larger clade corresponding to the group H.
(Guignotites) (Figs 23–25). Of the genus-group names Prodaticus,
Hydaticinus, Pleurodytes and Guignotites, Prodaticus is the oldest
name and has priority. Therefore, the following new genus
group synonymies are established: Guignotites Brinck 1943,
Pleurodytes Régimbart (1899) and Hydaticinus Guignot 1936 =
Prodaticus Sharp (1882) new synonyms.
Genus Hydaticus Leach 1817
Hydaticus Leach 1817, type species: Dytiscus transversalis
Pontoppidan 1763, by subsequent designation of Curtis
1825.
Icmaleus Gistel 1856, type species: Dytiscus transversalis
Pontoppidan 1763, by subsequent designation of Nilsson &
Roughley 1997.
Diagnosis. Hydaticus includes species of Hydaticini with the
following character combination: (1) anterior surfaces of
metafemur and metatibia with fine punctation (Fig. 9) (2)
series of bifid setae on posterior surface of metatibia in a
linear series, approximately parallel to dorsal margin (Fig. 12),
(3) basal brush of setae on male mesotarsomere I large, forming
a broad brush (Fig. 15), and (4) gonocoxae of female apically
sharply acute, knifelike (Fig. 20).
Taxon content. This genus includes seven species, two in the
eastern Nearctic region (H. cinctipennis and H. piceus), four
in the Palearctic region (H. transversalis, H. continentalis,
H. seminiger and H. schelkovnikovi Zaitzev) and one Holarctic
species (H. aruspex).
Discussion. This small, but distinctive, genus includes nearly
the entire Holarctic element in the Hydaticini. In contrast to
the largely tropical Prodaticus, the seven species in this group
are north temperate occurring in cold pools including bogs
and fens. Several of the species are well-known in North
America and Europe and there are good revisions including
most of the species such as those by Roughley & Pengelly
(1981), Nilsson (1981, 1996), Nilsson & Holmen (1995) and
Zimmermann & Gschwendtner (1937).
Genus Prodaticus Sharp, 1882
Prodaticus Sharp (1882); type species: Prodaticus pictus Sharp
(1882), by monotypy.
Pleurodytes Régimbart (1899); type species: Hydaticus dineutoides
Sharp (1882), by monotypy, new synonym.
Isonotus Guignot 1936, type species: Dytiscus vittatus Fabricius
1775 by original designation, preoccupied, replaced by
Guignotites Brinck 1943.
Guignotities Brinck 1943, type species: Dytiscus vittatus
Fabricius 1775 according to Article 67.8 of the Code, new
synonym.
Hydaticinus Guignot 1950, type species: Hydaticus rectus
Sharp (1882) (= Dytiscus xanthomelas Brullé 1838 by original
designation, new synonym.
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
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Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Diagnosis. This taxon is diagnosible within Hydaticini based
on the combination of: (i) anterior surfaces of metafemur and
metatibia without fine punctation (plesiomorphic within
Hydaticini) (ii) series of bifid setae on posterior surface of
metatibia curved ventrad proximally (Fig. 14), except in the
Neotropical P. xanthomelas (Fig. 13) and the Palaearctic P. pictus
and P. africanus Rocchi which have this series in a nearly
straight line (iii) basal brush of setae on male mesotarsomere
I small, linear (Fig. 16), (iv) posterior surface of metafemur
with well defined, curved line of dense, short setae (Fig. 16),
and (v) gonocoxae of female apically relatively broad (not
strongly knifelike) (Figs 21 and 22). Additional characters
used historically are difficult to homologize such as the
degree of angle of the posterolateral angles of the pronotum
(Roughley & Pengelly 1981). Although Hydaticus specimens
have this angle approximating 90° and many Prodaticus have
this angle much more acute and directed posteriorly, Prodaticus
species also exhibit a broad range of angles, some approaching
an angle similar to Hydaticus. This character was not coded in
this analysis.
Taxon content. Prodaticus includes nearly 130 diverse species
from throughout the tropical portions of the world with a few
occurring in the Holarctic region. Species previously placed
in H. (Guignotites), H. (Pleurodytes) and H. (Hydaticinus) are
members of this group. There are several distinct clades
within Prodaticus, but division into subgenera would be difficult
at this time with the available character and taxon sampling.
However, informal species groups are useful and described
below.
Discussion. Prodaticus pictus and P. africanus were previously
placed in their own genus. The most distinctive characters
present in the two species are apomorphic such as the larger
number of small adhesive setae on pro- and mesotarsomeres
I–III, unique colouration, and subequal metatarsal claws.
The species lack the fine punctation on the anterior surfaces
of the metatibia and metafemur and the series of bifid setae
on the posterior surface of the metatibia is linear, not curved
as in most other species of Prodaticus, though at least some of
these, such as P. parallelus and P. rectus, have this series of setae
linear, as well. Our results strongly indicate that P. pictus is
nested well within species historically placed in H. (Guignotites)
and these names are synonymized here. As Prodaticus has
priority, it is the valid name of this taxon.
Species of Prodaticus previously in H. (Guignotites) have
been divided into several informal, and, in some cases, overlapping species groups (Guignot 1961; Satô 1961; Wewalka
1975, 1979). In many cases, these groups appear to circumscribe clades, but in many cases they do not (Fig. 26). Because
of our limited sampling of these groups and obvious problems
with their status as monophyletic entities, we have elected not
18
to assign all our species to groups. However, several groups
are clearly monophyletic, and we believe that it is useful to
recognize and discuss them. Of the informal species groups
delimited by Guignot (1961), only the P. sexguttatus group is
not included in this study, and it is not clear to which of the
included taxa members of this group may be most closely
related.
The P. vittatus group (Fig. 26) is one of the most recognizable groups within Prodaticus. Most of its members are characterized by the pronotum yellow and black and the elytron
black with two lateral yellow stripes that meet posteriorly
(Fig. 4; Char. 6). The group occurs from southern Africa
through India and the Oriental Region to Australia. The species
are, in some cases, very similar and numerous subspecies have
been described. Despite having had focused investigation
(Satô 1961; Wewalka 1975), the taxonomy of the group
requires additional work. Old records are not reliable because
of misidentification of similar species and nomenclatural
problems. The P. vittatus group as now defined is an OrientalAustralian-Palearctic clade, with only P. bivittatus reaching
the Afrotropical region. Significantly, the species P. dineutoides
Sharp is resolved nested within the Oriental clade (Figs 23–25).
This species, along with P. epipleuricus Régimbart, has historically been placed in the subgenus H. (Pleurodytes). Although
unique in the group by being entirely black (Fig. 2), having
very broad epipleurae (Fig. 3), and a distinctive habitus (Figs 2
and 3). Prodaticus dineutoides is very convincingly nested
within Prodaticus and the P. vittatus group (Figs 23–25).
The P. speciosus group is similar in many respects to the
P. vittatus group and is resolved as its sister (Fig. 26). This
group includes some of the largest hydaticine species. Most
members of this group are longitudinally vittate on the elytra
or have a single lateral stripe along the elytra.
The P. fabricii group (Fig. 26) was revised by Wewalka
(1979). Most of the species in this group are uniformly
irrorate on the elytra and yellow on the pronotum, though
some have more extensive black regions. The main character
uniting its members is the median lobe of the aedeagus which
has a dense brush of setae at its apex (Fig. 19; Char. 27). The
P. fabricii group occurs in the southern Palaearctic, Oriental
and northern Australian regions with numerous closely
related species occurring throughout the Pacific Islands. In
the analysis, this group is resolved as sister to P. wattsi Daussin
and near relative of P. grammicus (Germar), which are not
particularly similar in general characteristics including male
genitalia. They lack the defining morphological features of
the P. fabricii group (notably the setal brush at the apex of the
male median lobe (Fig. 19)). Prodaticus concolor Sharp (not
included in this analysis) was regarded as closely related to
the P. fabricii group by Wewalka (1979) despite lacking
the setal brush. This suggests that these taxa collectively
may represent a natural group despite the lack of known
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
Fig. 26 Hydaticini clade from consensus of two most parsimonious trees showing correspondence with previous classifications (Wewalka 1975,
1979; Guignot 1961; Nilsson 2001) and a new classification system based on this analysis.
morphological synapomorphies. Support for this clade is
relatively high (Figs 23–25). Sister to the P. fabricii group is a
clade including the likewise rather uniformly irrorate species
in the P. leander group from African and Palaearctic regions.
This group corresponds with the P. leander/leander group of
Guignot (1961).
The P. subfasciatus group (Fig. 26) is united by only DNA
sequence characters, but it unites all included New World
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
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Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Prodaticus. There are numerous additional species of Prodaticus
in the Neotropical region that were not included, and future
additional phylogenetic tests may prove interesting by determining whether all New World Prodaticus are together
monophyletic. The only truly Nearctic species in Prodaticus,
P. bimarginatus (Say), is a member of this group. This group
includes the species P. xanthomelas (Brullé) which was previously placed in the subgenus H. (Hydaticinus). Support for its
placement with other Neotropical Prodaticus and as sister to
P. subfasciatus Laporte is strong (Figs 23–25).
Several other groups appear to be strongly supported
monophyletic groups based on DNA sequence characters
and morphological features which are not discrete (see Fig. 26
for classification of groups). The P. luczonicus group includes
large dark Oriental and northern Australian species with a
general elytral colour pattern of a lateral yellow border, a subbasal and postmedian bands and a subapical spot (Régimbart
1899). The P. caffer group corresponds with the P. leander/
sobrinus group of Guignot (1961) and includes numerous
irrorate species from Africa. This group also includes the
widespread African and Arabian P. dorsiger Aubé from the
P. leander/leander species group of Guignot (1961).
The Australian P. parallelus Clark and Madagascar endemic
P. nigrotaeniatus Régimbart do not seem to have any close
relatives among the sampled species (see above).
Character evolution
One of the most distinctive and consistent synapomorphies
for the tribe Hydaticini is the straight anterolateral margin of
the metasternum (the anterior margin of the metasternal
wing) (Fig. 7; Char. 24). This feature is not found in similar
form elsewhere within Dytiscidae (where it is convex to
varying degrees, e.g. Fig. 8), and appears to be present in all
Hydaticini.
A series of bifid setae on the posterior surface of the metatibia
is an important character system (Char. 19) for members of
Dytiscinae and related taxa (Miller 2000, 2001, 2003; and
references therein). In Hydaticini, members of Hydaticus
have this series parallel to the dorsal margin of the metatibia
(Fig. 12), a plesiomorphic state. One species, H. cinctipennis
Aubé, has the series slightly curved ventrad basally, but not to
as great a degree as in Prodaticus. Members of Prodaticus have
the series distinctly curved ventrad basally (Fig. 14), except
P. xanthomelas, P. parallelus, P. pictus and P. africanus, which have
the series similar to Hydaticus (Fig. 13). This was (in part) the
basis for placement of P. xanthomelas in its own subgenus and
P. pictus and P. africanus in their own genus, but the state is a
reversal in these taxa (Fig. 25). Prodaticus specimens also have
a dense, irregular line of setae that is also parallel to the dorsal
margin of the metatibia. There seems to be a general relationship of curvature of this line of setae to a shortening of the
metafemur (Figs 12–14). Members of Aciliini and Eretini,
20
which have very short metafemora, have the line of bifid setae
short and oblique to the dorsal margin of the metatibia
(Miller 2000, 2001, 2003).
Guignot (1961) and Roughley & Pengelly (1981) emphasized a structure in the male they called an ‘epipenite’ in the
classification of genera within Hydaticini. This sclerite is
closely associated with the preputial membrane which extends
between the ventral margins of the lateral lobes. They proposed that a ventral position of the epipenite (on the ventral
surface of the preputial membrane) is a synapomorphy of
Hydaticus whereas a dorsal position of the epipenite (on the
dorsal surface of the preputial membrane) is a synapomorphy
of Prodaticus. As suggested by Roughley & Pengelly (1981),
and as demonstrated by Miller (2000, 2001) the epipenite is
homologous with the ventral sclerite of the male median lobe
in other dytiscines. This sclerite forms the ventral surface of
a tube through which the spermatophore is passed. The
ventral sclerite is reduced in Aciliini, Aubehydrini, Eretini
and Hydaticini when compared with Cybistrini, Dytiscini
and Hyderodini and is incorporated more thoroughly into a
membrane, the preputial membrane, extending between the
lateral lobes (Miller 2000, 2001, 2003). Based on examination
of many species of Hydaticini, it appears that the shape of the
apex of the ventral sclerite varies between species, but its
position on the dorsal or ventral surface of the preputial
membrane is not a reliable character. Rather than being
orientated dorsally or ventrally, instead it appears that the
ventral sclerite is variously incorporated into the membrane,
but in a continuous manner across Hydaticini. This character
was not coded for analysis.
Colour patterns have been important for classifying species
in Hydaticini, but most are difficult to homologize across
taxa. Only two characters were coded here, the presence or
absence of two lateral, longitudinal yellow stripes uniting
posteriorly (Fig. 4; Char. 6), which characterizes the P. vittatus
group (except P. dineutoides) (Fig. 25), and the presence or
absence of a transverse yellow basal band (Figs 1 and 6;
Char. 5). This last feature is homoplasious within Hydaticini
with at least five origins and three subsequent losses (Fig. 26),
and many taxa are dimorphic for this feature. Other than this,
members of Hydaticini range from entirely black (Fig. 2) to
irrorate to varying degrees (e.g. Fig. 5) to dramatically
marked with maculae or stripes (e.g. Figs 1, 4 and 6). Colour
patterns may exhibit some evolutionary response to habitat,
such as living in rock pools or on mineral substrates (Young
1960; Larson 1996). Colour patterns are often species specific
and useful for diagnoses, and some larger groups may be
defined by general colour patterns, such as the P. vittatus
group or the P. fabricii group which has members which are
nearly uniformly irrorate. However, many groups exhibit
considerable variety in colour patterns, and species often vary
across their ranges.
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
Members of this tribe have characters suggesting that a
form of sexual conflict has influenced the evolution of their
behaviour and morphology (Miller 2003). Males have suckershaped pro- and mesotarsal adhesive setae used to better
adhere to female dorsal surfaces, whereas females have the
pronotum and humeral angles of the elytron modified with
deep cavities and irregular grooves which presumably inhibit
the ability of the male sucker-setae to adhere (Miller 2003).
The female modifications are often continuously variable
between individuals in a given population of a species from
nearly unmodified to strongly modified (Roughley & Pengelly
1981). Presumably, this set of characters represents, in part,
an intersexual ‘arms race’ as each sex seeks to gain greater
control over the decision to mate (Alexander et al. 1997).
However, an additional sexual dimorphism exhibited in this
group is an apparent stridulatory device on male prolegs
comprised of a series of pegs on the dorsomedial margin of
the tibia and a field of reticulate pits on the dorsal surface of
tarsomere I (Fig. 10; Char. 7). The stridulatory device appears
to have been lost in some species of Prodaticus including
P. dregei (Aubé) (Larson 1996), and P. ugandaensis (Guignot)
(Fig. 25). The presence of a stridulatory device in most
Hydaticini, which is presumably a means for males to signal
to potential mates, would seem to be discordant with the
normal view of sexual conflict. This is because females who
respond to a signalling male have presumably already made
the decision to mate and would be unlikely to resist male
mating attempts. Although a phylogenetic analysis is unlikely
to fully clarify some of the seemingly conflicting evolutionary
issues surrounding sexual conflict in Hydaticini, having a
well-founded phylogenetic hypothesis could provide a better
understanding of the historical constraints and order of
evolution of features associated with the scenario.
Revised classification of Hydaticini
For full details regarding taxon names, refer to Nilsson (2001).
Hydaticini Sharp, 1882
Hydaticus Leach, 1817
Icmaleus Gistel, 1856
Prodaticus Sharp, 1882
Guignotites Brinck, 1943, new synonym
Isonotus Guignot, 1936 (pre-occupied)
Hydaticinus Guignot, 1950, new synonym
Pleurodytes Régimbart, 1899, new synonym
Key to genera of Hydaticini
1 Metafemur with dense, fine punctation on anterior surface, metatibia with numerous large, setose punctures and also dense,
fine punctation on anterior surface (Fig. 9); series of bifid setae on posterior surface of metatibia in linear series, parallel to dorsal margin (Fig. 12), in one known species, ??, series perceptively curved; basal brush of setae on mesotarsomere I in form of
large brush (Fig. 17); posterior surface of metafemur with irregular series of punctures (Fig. 15); female gonocoxae together
sharply acute, knifelike (Fig. 20) .............................................................................................................................Hydaticus Leach
1′′ Metafemur without fine punctation on anterior surface, metatibia with only large, setose punctures and no fine puncation
on anterior surface; series of bifid setae on posterior surface of metatibia parallel to dorsal margin (Fig. 13) or curved ventrad
basally (Fig. 14); basal brush of setae on mesotarsomere I in small, linear series (Fig. 18); posterior surface of metafemur with
regular, dense series of setose punctures in curved line (Fig. 16); female gonocoxae together apically broader, not strongly knifelike (Figs 21 and 22). .............................................................................................................................................. Prodaticus Sharp
Acknowledgements
Authors thank the following individuals for help with specimens, field work and other aspects of support for various
projects: M. Balke, O. Brekhov, K. Binder, S.L. Cameron, J.R.
Cryan, C. Deschodt, I. & J. Hansen, T. Kondo, T.L. McCabe,
M. Michat, G. Morse, A.N. Nilsson, M. Satô, C. Scholtz,
G.J. Svenson, J.M. Urban, Y. Utsenomyia, C.H.S. Watts,
Q.D. Wheeler, and G.W. Wolfe. We thank E. Marais of the
National Museum of Namibia for advice and permit arrangements, and J. Patterson of the Skeleton Coast National Park
in Namibia. Authors thank the staves at Explorers Inn and
Posada Amazonas, Tambopata Reserve, Peru. Thanks to J.
Ledezma, Museo de Historia Natural ‘Noel Kempff Mercado’,
Santa Cruz, Bolivia. Thanks also to A. Southwood, Department of Economic Affairs, Environment and Tourism,
Greenacres, E. Cape Prov., S. Africa, for permit arrangements. Financial research support for KBM and MFW came
in part from National Science Foundation grants #DEB0073088, #DEB-9983195, #DEB-0329115 and #DEB-0515924.
Financial support to JB came from Helge Ax:son Johnsons
Stiftelse and Stiftelsen J.C. Kempes Minnes Stipendiefond.
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Appendix I
Morphological characters analysed in the cladistic analysis
of Hydaticini
Head
1. Frontoclypeal suture: (0) incomplete; (1) complete.
2. Mandible, mesal line of setae: (0) discontinuous, not
extending along apicoventral surface, with an isolated patch
of setae medially on ventral surface; (1) with continuous line of
setae from mesal margin in curve along apicoventral surface.
Pronotum
3. Female cuticular modification: (0) not modified as following;
(1) Pronotum and humeral angles of elytron with deep,
irregular grooves and pits. Since the unique modifications to
the pronotum and elytron present in Dytiscus marginalis,
Hyderodes shuckardi, and Thermonectus succinctus are
apomorphic we have not coded them.
Elytron
4. Apicoventral setal patch on elytron: (0) absent; (1) present.
5. Transverse basal macula on elytron: (0) absent; (1) present
(Figs 1 and 6).
6. Two longitudinal yellow lines on black elytron: (0) absent;
(1) present (Fig. 4).
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
23
Hydaticini phylogeny & data partition choice • K. B. Miller et al.
Prolegs
7. Protarsal stridulatory device: (0) absent; (1) present. Males of
many Hydaticini have a stridulatory device on the front legs
in the form of a reticulate pattern of pits on the dorsal surface
of protarsomere II opposing a series of short, stout pegs on
the apicodorsal surface of the protibia (Fig. 10).
8. Accessory medial spinous setae on dorsal surface of basal
tarsomere of male: (0) absent (Fig. 10), (1) present (Fig. 11).
Males of many Hydaticini species have a series of spines
across the dorsal surface of protarsomere I (Fig. 11). These
are minute or absent in other species.
9. Male anteroapical protarsal spur: (0) absent; (1) present.
10. Male posteroapical protarsal spur: (0) absent; (1) present.
Mesolegs
11. Basal brush of setae ventrally on male mesotarsomere I:
(0) absent; (1) present (Figs 17 and 18).
12. Basal brush of setae ventrally on male mesotarsomere I:
(0) small (Fig. 18), (1) large (Fig. 17). This is coded as
inapplicable (‘–’) in taxa lacking the basal brush of setae.
13. Posteroapical marginal setae on mesotibia: (0) absent
medially; (1) present in a continuous line.
Metalegs
14. Metacoxal process and medial portion of metacoxa: (0) not
deeply concave laterally, metacoxa entirely visible near base
of metafemur (Fig. 7); (1) deeply concave laterally such that
a portion of metacoxa not visible when viewed in ventral
aspect (Fig. 8).
15. Anterior surface of metafemur: (0) without punctures or
with few, large punctures; (1) with many small punctures
interspersed among few large punctures (Fig. 9).
16. Posterior surface of metafemur: (0) with series of punctures
sparse and in poorly defined line (Fig. 15), (1) with series of
punctures distinctly setose, dense and in well defined curved
line (Fig. 16).
17. Metatibial spurs: (0) simple; (1) bifid.
18. Anterior surface of metatibia: (0) with only large, setaebearing punctures or without punctures; (1) with many fine
punctures (Fig. 9).
19. Posterodorsal series of setae on metatibia: (0) a linear series,
nearly parallel to dorsal margin of metatibia (Figs 12 and 13),
(1) a linear series, curved ventrad basally, not parallel to
dorsal margin of metatibia (Fig. 14), (2) a closely spaced
series oblique to long axis of metatibi.
20. Posteroapical setae on metatibia: (0) simple; (1) bifid
(Figs 12–14).
21. Adpressed apical series of setae on anterior and posterior
margins of metatarsomeres I–IV: (0) absent; (1) present.
22. Natatory setae on posteroventral margin of metatibia and
metatarsomeres of female: (0) absent; (1) present.
23. Metatarsal claws: (0) different lengths, anterior claw
shorter than posterior; (1) same length.
24
Metathorax
24. Anterior margin of metasternal wings: (0) curved (Fig. 8);
(1) straight (Fig. 7).
Abdomen
25. Series of transverse carinae dorsally on abdominal
segment II: (0) absent; (1) present.
Male genitalia
26. Setae along apicoventral margin of male median lobe: (0)
absent; (1) present.
27. Apical setal clump on median lobe: (0) absent; (1) present
(Fig. 19).
Female genitalia
28. Gonocoxae: (0) not knifelike (Figs 21 and 22), (1) knifelike
(Fig. 20).
29. Subapical setal pencil on gonocoxa: (0) absent; (1) present
(Figs 20–22).
Larva habitus
30. Body shape: (0) not jackknifed; (1) jackknifed, abruptly
bent medially.
Larva head
31. Occipital foramen: (0) not deeply excised; (1) deeply excised
on dorsal and ventral margins.
32. Stemmata: (0) not different in size; (1) different in size,
two ocelli very large.
33. Cardo of maxilla: (0) narrow, without row of long setae;
(1) very broad, with medial and lateral margins bearing long
setae.
34. Number of maxillary palpomeres (of subdivided ones, and not
including the palpiger): (0) 4; (1) 5; (2) 6; (3) 7; (4) 8.
35. Apicomedial margin of labial prementum: (0) unmodified; (1)
bilobed, with two projections arising from margin of prementum; (2) with a single, elongate, generally spinous projection.
36. Serrations on mandible: (0) absent; (1) present.
Supporting information
Additional Supporting Information may be found in the
online version of this article:
Table S1 Primers used for amplification and sequencing.
Table S2 Amplification conditions used in PCR reactions.
Table S3 Character correspondence between this analysis
and previous published analyses incorporating them. Numbers
refer to character numbers in the analyses, those marked with
“–” were not included in the prior analysis.
Table S4 Data matrix of assigned states of 36 morphological
characters for 54 species of Dytiscidae. Characters marked
with “+” are additive. Characters coded as “−” are inapplicable.
Characters coded with “?” are unobserved. Characters coded
with “*” are polymorphic and equal states 0 and 1.
Zoologica Scripta, 2009 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters
K. B. Miller et al. • Hydaticini phylogeny & data partition choice
Table S5 Aligned portions of wingless and COI showing
positions of indels.
Table S6 Model for each of 35 partitions used in 9 partitioning
schemes as chosen based on different criteria. MrModeltest
(Nylander, 2004a, = MrM in table) uses four ways of traversing parameter space (Posada & Crandall, 2001) to select one
of 24 models implemented in MrBayes. MrAIC (Nylander,
2004b) selects a model based on AIC, AICc or BIC but likelihood scores are estimated with PHYML searches under each
model. The maximum is GTR+I+G and minimum is JC for
all tests.
Table S7 The log of the harmonic mean of sampled likelihood values (HME) and number of free parameters (P) in
selected models for partitions × model choice combinations.
MrM = MrModeltest. Note that branch length is not
included in number of free parameters. For the calculation of
number of free parameters each GRT+I+G model gives 10
free parameters (5 for the substitution matrix, 3 for the base
frequencies, and one each for the alpha and invariable sites
parameters) + the number of partitions – 1 for the among partition
rate multipliers. Entries marked with * were calculated from
one run that had reached a better space than the second run.
Table S8 Tests based on log of estimated harmonic mean of
likelihoods (see Table 9) for partitions × model choice combinations showing 2*Ln Bayes Factor and the increase in
Ln L as a ratio with the increase in number of free parameters
(ΔLn L/ΔP) for several comparisons. BF < 0 indicates less
partitioned model has highest HME; ΔLn L/ΔP = ∞ indicates
second model partition in comparison with less parameters
than first; NA = # free parameters equal.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by
the authors. Any queries (other than missing material) should
be directed to the corresponding author for the article.
© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 2009
25
Table S1. Primers used for amplification and sequencing.
Gene
Primer
Direction
Sequence (5’-3’)
COI
C1-J-1718 (“Mtd6”)
1
For
GGA GGA TTT GGA AAT TGA TTA GTT CC
COI
1
Rev
CAA CAT TTA TTT TGA TTT TTT GG
1
Rev
TCC AAT GCA CTA ATC TGC CAT ATT A
For
GGA TCA CCT GAT ATA GCA TT CCC
Rev
CCC GGT AAA ATT AAA ATA TAA ACT TC
C1-J-2183 (“Jerry”)
COI
TL2-N-3014 (“Pat”)
COI
1
C1-J-1751 (“Ron”)
1
COI
C1-N-2191(“Nancy”)
COI
NabCOI-R
Rev
GCT ACT ACA TAA TAT GTA TC
COII
F-lue
2
For
TCT AAT ATG GCA GAT TAG TGC
COII
2
Rev
GTA CTT GCT TTC AGT CAT CTW ATG
Rev
GAG ACC AGT ACT TGC TTT CAG TCA TC
For
ATG GCT CGT ACC AAG CAG ACG GC
Rev
ATA TCC TTG GGC ATG ATG GTG AC
For
GAR TGY AAR TGY CAY GGY ATG TCT GG
9b
COII
R-lys
H3
Haf3
H3
3
Har
2
4
Wnt
LepWg1
Wnt
LepWg2a4
Rev
ACT ICG CAR CAC CAR TGG AAT GTR CA
Wnt
5
For
CGY CTT CCW TCW TTC CGW GTY ATC
5
Rev
CCG TGG ATR CTG TTV GCH AGA TG
Wnt
WgDytF1
WgDytR1
1
4
2
5
Simon et al. (1994)
Whiting (2002)
3
Colgan et al. (1998)
Brower and Egan (1997)
Miller (2003)
Table S2. Amplification conditions used in PCR reactions.
H3
Hot start
Denature
95º (12min)
94º (0.5min) 48-50º (1min) 70º (1.5min)
COI, COII 95º (12min)
Wnt
94º (1min)
95º (12min) Taq Gold / HotMaster 94º (1min)
95º (2min) Taq Platinum
Anneal
Extension
Cycles
40
50-52º (1min) 60-68º (1.5min) 40
46-54º (1min) 70º (1.5min)
40
Table S3. Character correspondence between this analysis and previous published analyses incorporating them.
Numbers refer to character numbers in the analyses, those marked with “−” were not included in the prior analysis.
Miller et al.,
Character
Miller, 2000
Miller, 2001
Miller, 2003
1
−
40
4
2
1
34
3
1
3
−
−
−
8
4
8
71
8
6
5
−
−
−
−
6
−
−
−
−
7
−
−
19
−
8
−
−
20
−
2007
−
9
11
75
16
−
10
12
76
17
11
11
−
−
27
−
12
−
−
28
−
13
15
−
22
13
14
6
89
32
19
15
−
−
38
−
16
−
−
37
−
17
30
93
39
−
18
−
−
42
−
19
29
82
44
23
20
32
83
46
−
21
34
97
50
−
22
36
98
51
24
23
35
101
52
25
24
5
−
14
−
25
7
59
15
5
26
−
−
56
30
27
−
−
−
−
28
42
−
63
−
29
44
−
65
37
30
−
−
69
−
31
−
−
72
−
32
−
−
73
−
33
−
−
76
−
34
−
−
79
41
35
−
−
80
42
36
−
−
90
−
Table S4. Data matrix of assigned states of 36 morphological characters for 54 species of Dytiscidae. Characters
marked with “+” are additive. Characters coded as “-” are inapplicable. Characters coded with “?” are unobserved.
Characters coded with “*” are polymorphic and equal states 0 and 1.
0000000001 1111111112 2222222223 333333
1234567890 1234567890 1234567890 123456
+
Dytiscus verticalis
1101000001 0-01000000 001011010? ??????
Hyderodes shuckardi
0100000000 1101000000 0010100110 000200
Notaticus fasciatus
0100000000 0-11000001 1100000100 000-0-
Graphoderus liberus
0100000000 0-10001021 1100000011 111021
Eretes australis
0100000000 0-00000021 0100000011 111021
Hydaticus aruspex
0110*01000 1110100101 1101000110 000110
H. cinctipennis
0110101000 1110100101 110100011? ??????
H. continentalis
0110101000 1110100101 110100011? ??????
H. seminiger
0110001000 1110100101 1101000110 000110
H. transversalis
0110101000 1110100101 110100011? ??????
H. bihamatus
0110101100 1010010011 110100001? ??????
H. bimarginatus
0110001000 1010010011 110100001? ??????
H. bivittatus
0110011000 1010010011 110100001? ??????
H. bowringii
0110011000 1010010011 110100001? ??????
H. caffer
0110001100 1010010011 110100001? ??????
H. capicola
0110001100 1010010011 110100001? ??????
H. consanguineus
0110001100 1010010011 110100101? ??????
H. dineutoides
0110001000 1010010011 110100001? ??????
H. dorsiger
0110001100 1010010011 110100001? ??????
H. exclamationis
0110001000 1010010011 110100001? ??????
H. fabricii
0110001100 1010010011 110100101? ??????
H. flavolineatus
0110101000 1010010011 110100001? ??????
H. galla
0110001100 1010010011 110100001? ??????
H. grammicus
0110001100 1010010011 1101000010 000110
H. humeralis
0110001000 1010010011 110100001? ??????
H. lativittis
0110001000 1010010011 110100001? ??????
H. leander
0110001100 1010010011 110100001? ??????
H. litigiosus
0110001100 1010010011 110100001? ??????
H. luczonicus
0110101100 1010010011 110100001? ??????
H. major
0110011000 1010010011 110100001? ??????
H. maturelis
0110001100 1010010011 110100001? ??????
H. nigrotaeniatus
0110001000 1010010011 110100001? ??????
H. orissaensis
0110101100 1010010011 110100001? ??????
H. parallelus
0110001000 1010010011 1101000010 000110
H. philippensis
0110011000 1010010011 110100001? ??????
H. quadrivittatus
0110011000 1010010011 110100001? ??????
H. rhantoides
0110001100 1010010011 110100101? ??????
H. ricinus
0110001100 1010010011 110100101? ??????
H. rimosus
0110*01000 1010010011 110100001? ??????
H. rivanolis
0110001100 1010010011 110100101? ??????
H. satoi
0110011000 1010010011 110100001? ??????
H. servillianus
0110001100 1010010011 110100001? ??????
H. speciosus
0110101000 1010010011 110100001? ??????
H. subfasciatus
0110101000 1010010011 110100001? ??????
H. ugandaensis
0110000000 1010010011 110100001? ??????
H. ussherii
0110101000 1010010011 110100001? ??????
H. wattsi
0110101100 1010010011 110100001? ??????
H. vittatus
0110011000 1010010011 110100001? ??????
H. xanthomelas
0110101000 1010110001 110100001? ??????
Prodaticus pictus
0110001000 1010010001 111100001? ??????
Table S5. Aligned portions of wingless and COI showing positions of indels.
Taxon
Position 106-168 of aligned wingless
Dytiscus marginalis
CGTTGGCAGTCAACGAGGCGGAAACAGCGCGCACGCTAATACGGCCAATTCAAACTCACATCT
Notaticus fasciatus
TGCCGGCAGCCAACGCGGGGGAAACAACGCACATGCAAATTC––-AAATGCAAACTCACATCT
Eretes australis
CGCAGGAAGTCAAAGAAA---------TGCGCACACAAACACAGCCAACGCCAACTCACACTT
Hydaticus aruspex
TGCTGGCAGTCAGCGAGGCGGAAACAGCGCGCACGCTAACAACGCAAATTCCAACTCACATCT
Hydaticus bimarginatus
TGCTGGCAGCCAGCGAGGCGGAAACAGCGCGCACGCTAACAACGCAAATTCCAACTCACATCT
Position 304-342 and 379-408 of aligned COI
Dytiscus marginalis
TTGATCAGTAGGAATTACAGCTCTTTTACTATTATTATC, AACTGATCGAAATTTAAATACTTCATTCTT
Thermonectus succinctus
ATGATCGGTCGGAATTACTGCTTTATTATTATTATTATC, AACAGACCGAAATTTAAATACTTCATTTTT
Hydaticus aruspex
CTGATCAGTAGGGATTACAGCTCTTTTATTACTCTTATC, AACTGATCGAAATTTAAATACATCATTTTT
Hydaticus bihamatus
TTGATCAGCTTT---------ATTATTATTATTATTAAC, AACTGATCGACATTTAAATAC---ATTATT
Hydaticus grammicus
TTGATCAGTAGGAATTACAGCTTTATTATTATTATTATC, AACTGATCGAAATTTAAATACGTCATTTTT
Table S6. Model for each of 35 partitions used in 9 partitioning schemes as chosen based on different criteria. MrModeltest (Nylander, 2004a, =MrM in table)
uses four ways of traversing parameter space (Posada & Crandall, 2001) to select one of 24 models implemented in MrBayes. MrAIC (Nylander, 2004b) selects
a model based on AIC, AICc or BIC but likelihood scores are estimated with PHYML searches under each model. The maximum is GTR+I+G and minimum is
JC for all tests.
4genes
coi-coii
h3-wnt
1pos.
2pos.
3pos.
12pos
coi
coii
h3
wnt
MrM hLRT1
GTR+I+G
GTR+I+G
SYM+I+G
GTR+I+G
GTR+I+G
GTR+G
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
SYM+I+G
MrM hLRT2
GTR+I+G
GTR+I+G
SYM+I+G
GTR+I+G
HKY+I
GTR+G
GTR+I+G
GTR+I+G
GTR+I+G
HKY+I+G
SYM+I+G
MrM hLRT3
GTR+I+G
GTR+I+G
SYM+I+G
GTR+I+G
HKY+I+G
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
SYM+I+G
MrM hLRT4
GTR+I+G
GTR+I+G
SYM+I+G
GTR+I+G
HKY+I+G
GTR+G
GTR+I+G
GTR+I+G
GTR+I+G
HKY+I+G
HKY+I+G
MrAIC AIC
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
GTR+G
GTR+I+G
GTR+I+G
HKY+I+G
HKY+I+G
GTR+I+G
MrAIC AICc
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
GTR+G
GTR+I+G
GTR+I+G
HKY+I+G
HKY+I+G
HKY+I+G
MrAIC BIC
GTR+I+G
GTR+I+G
K80+I+G
GTR+I+G
HKY+I+G
GTR+G
GTR+I+G
GTR+I+G
HKY+I+G
HKY+I+G
HKY+I+G
coi-ii1
coi-ii2
coi-ii3
h3-wnt1
h3-wnt2
h3-wnt3
coi-ii12
h3-wnt12
coi-12
coii-12
h3-12
MrM hLRT1
GTR+I+G
HKY+I+G
GTR+G
SYM+I+G
JC+I+G
HKY+G
GTR+I+G
SYM+I+G
GTR+I+G
HKY+I+G
SYM+G
JC+I+G
MrM hLRT2
GTR+I+G
HKY+I+G
GTR+G
GTR+I
JC+I
HKY+G
GTR+I+G
GTR+I+G
GTR+I+G
HKY+I+G
K80+I
GTR+I
MrM hLRT3
GTR+I+G
HKY+I+G
GTR+G
GTR+I+G
JC+I+G
GTR+G
GTR+I+G
GTR+I+G
GTR+I+G
HKY+I+G
K80+G
JC+I+G
MrM hLRT4
GTR+I+G
HKY+I+G
GTR+G
GTR+I
JC+I
HKY+G
GTR+I+G
GTR+I+G
GTR+I+G
HKY+I+G
SYM+I
GTR+I
MrAIC AIC
GTR+I+G
GTR+I+G
HKY+I+G
GTR+I+G
K80+I+G
GTR+G
GTR+I+G
GTR+I+G
GTR+I+G
HKY+I+G
GTR+G
GTR+I+G
MrAIC AICc
GTR+I+G
HKY+I+G
HKY+I+G
GTR+G
JC+G
HKY+G
GTR+I+G
GTR+I+G
GTR+I+G
HKY+I+G
K80+G
SYM+G
MrAIC BIC
GTR+I+G
HKY+I+G
HKY+G
GTR+G
JC+G
HKY+G
GTR+I+G
GTR+I+G
HKY+I+G
HKY+I+G
K80+G
JC+G
coi-1
coi-2
coi-3
coii-1
coii-2
coii-3
h3-1
h3-2
h3-3
wnt-1
wnt-2
wnt-3
wnt-12
MrM hLRT1
GTR+I+G
F81+I+G
GTR+G
GTR+G
HKY+I+G
HKY+G
SYM+G
JC
HKY+G
JC+G
JC+I*
HKY+G
MrM hLRT2
GTR+I+G
F81+I
GTR+G
GTR+G
HKY+I
GTR+I+G
SYM+I
JC
HKY+G
SYM+I
JC+I
HKY+G
MrM hLRT3
GTR+I+G
HKY+I+G
GTR+G
GTR+I+G
HKY+I+G
GTR+G
SYM+G
JC
GTR+G
JC+G
JC+I*
HKY+G
MrM hLRT4
GTR+I+G
F81+I
GTR+G
GTR+G
HKY+I
GTR+I+G
SYM+I
JC
HKY+G
SYM+I
JC+I
HKY+G
MrAIC AIC
GTR+G
HKY+I
HKY+G
GTR+I+G
HKY+I+G
GTR+G
GTR+G
JC
GTR+G
GTR+G
JC+G
GTR+G
MrAIC AICc
GTR+G
HKY+I
HKY+G
GTR+G
HKY+I
HKY+G
JC
JC
JC
JC+I
JC
K80+G
MrAIC BIC
GTR+G
HKY+I
HKY+G
GTR+G
HKY+I
HKY+G
K80+I
JC
HKY+G
JC+I
JC+G
HKY+G
*JC+I+G originally chosen but G removed since estimated shape equals infinity, which implies equal rates among sites.
Table S7. The log of the harmonic mean of sampled likelihood values (HME) and number of free parameters (P) in selected models for partitions × model choice
combinations. MrM = MrModeltest. Note that branch length is not included in number of free parameters. For the calculation of number of free parameters each
GRT+I+G model gives 10 free parameters (5 for the substitution matrix, 3 for the base frequencies, and one each for the alpha and invariable sites parameters) +
the number of partitions – 1 for the among partition rate multipliers. Entries marked with * were calculated from one run that had reached a better space than the
second run.
1
2
3a
3b
4
5a
5b
7
9
13
HME
P
HME
P
HME
P
HME
P
HME
P
HME
P
HME
P
HME
P
HME
P
HME P
MrM hLRT1
-27749
10
-27985
11
-27371
19
-26783
21
-26679
32
-27295
41
-25883
35
-25784
45
-25811
56
-25734
74
MrM hLRT2
-27749
10
-27985
11
-27371
19
-26783
21
-26698
27
-27298
36
-25879
38
-25780
46
-25784
64
-25700
82
MrM hLRT3
-27749
10
-27985
11
-27371
19
-26785
22
-26687
29
-27295
41
-25873
42
-25779
52
-25821
60
-25706
80
MrM hLRT4
-27749
10
-27985
11
-27371
19
-26783
21
-26693
28
-27288
36
-25879
38
-25780
46
-25776
68
-25700
82
MrAIC AIC
-27749
10
-27985
11
-27358
22
-26783
21
-26679
32
-27297
36
-25898
39
-25784
54
-25784
75 -25708*
93
MrAIC AICc
-27749
10
-27985
11
-27358
22
-26783
21
-26679
32
-27306
32
-25898
35
-25812
43
-25980
44
-25970
53
MrAIC BIC
-27749
10
-27985
11
-27374
15
-26783
21
-26693
28
-27306
32
-25895
34
-25802
42
-25861
43
-25759
64
MAX
-27749
10
-27985
11
-27358
22
-26785
22 -26681* 33
-27287
44
-25877
44
-25763
66
-25765
88
-25660
132
MIN
-33681
0
-33921
1
-33839
2
-30524
2
-33829
4
-30426
4
-30208
6
-30417
8
-30192
12
-30311
3
Table S8. Tests based on log of estimated harmonic mean of likelihoods (see Table 9) for partitions × model choice combinations showing 2*Ln Bayes Factor
and the increase in LnL as a ratio with the increase in number of free parameters (ΔLnL/ΔP) for several comparisons. BF < 0 indicates less partitioned model has
highest HME; ΔLnL/ΔP = ∞ indicates second model partition in comparison with less parameters than first; NA = # free parameters equal.
2 vs 3a
2 vs 3b
3a vs 3b
3a vs 4
3b vs 4
4 vs 5a
2*Ln ΔLnL/ 2*Ln ΔLnL/ 2*Ln ΔLnL/ 2*Ln ΔLnL/ 2*Ln ΔLnL/ 2*Ln ΔLnL/
4 vs 5b
5b vs 7
7 vs 9
7 vs 13
5b vs 13
2*Ln ΔLnL/ 2*Ln ΔLnL/ 2*Ln ΔLnL/ 2*Ln ΔLnL/ 2*Ln ΔLnL/
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
BF
ΔP
MrM hLRT1
1228
76.8
2404
120.2
1176
294.0
1382
53.2
206
9.4
<0
<0
1592
265.4
199
9.9
<0
<0
101
1.7
300
3.8
MrM hLRT2
1228
76.8
2404
120.2
1176
294.0
1346
84.1
170
14.2
<0
<0
1638
74.4
197
12.3
<0
<0
160
2.2
357
4.1
MrM hLRT3
1228
76.8
2399
109.0
1170
195.1
1367
68.4
197
14.1
<0
<0
1627
62.6
188
9.4
<0
<0
147
2.6
335
4.4
MrM hLRT4
1228
76.8
2404
120.2
1176
294.0
1356
75.3
180
12.9
<0
<0
1628
81.4
197
12.3
9
0.2
160
2.2
357
4.1
MrAIC AIC
1253
56.9
2404
120.2
1152
∞
1358
67.9
206
9.4
<0
<0
1562
111.6
230
7.7
<0
<0
151
1.9
381
3.5
MrAIC AICc
1253
56.9
2404
120.2
1152
∞
1358
67.9
206
9.4
<0
NA
1563
260.5
171
10.7
<0
<0
<0
<0
<0
<0
MrAIC BIC
1222
152.7
2404
120.2
1182
98.5
1362
52.4
180
12.9
<0
<0
1595
133.0
185
11.6
<0
<0
86
2.0
271
4.5
MAX
1253
56.9
2399
109.0
1146
NA
1355
61.6
209
9.5
<0
<0
1608
73.1
228
5.2
<0
<0
206
1.6
434
2.5
MIN
164
82.0
6793 3396.6 6629
NA
7055 3527.3 425
212.7
<0
<0
<0
<0
435
108.9
<0
<0
34
2.8
469
29.3