Phylogeny of the water strider genus Gerris Fabricius (Heteroptera: Gerridae) based on COI mtDNA,...

Phylogeny of the water strider genus Gerris Fabricius(Heteroptera: Gerridae) based on COI mtDNA, EF-1anuclear DNA and morphology

J A K O B D A M G A A R D and F E L I X A . H . S P E R L I N GZoological Museum and Zoological Institute, University of Copenhagen, Copenhagen, Denmark and Department of

Environmental Science, Policy and Management, University of California, Berkeley, California, U.S.A.

Abstract. Phylogenetic relationships between water striders (Heteroptera: Gerridae)

of genus Gerris Fabricius were examined using molecular and morphological

characters. The molecular dataset was 820 bp DNA from the 3¢ half of the

mitochondrial gene encoding cytochrome oxidase subunit I and 515 bp DNA from

the nuclear gene encoding elongation factor 1 alpha. The morphological dataset was

a slightly modi®ed version of a previously published dataset. Representatives from

all eight recognized species groups of Gerris, as well as six species from three

related genera, including Gigantometra gigas, Limnoporus esakii, L. rufoscutellatus,

Aquarius najas, A. conformis and A. paludum, were included. Unweighted parsimony

analyses of the COI sequences gave a topology with strong support for only those

nodes that were already recognized as closely related based on morphological

characters. Similar analyses of EF-1a gave a cladogram with a topology quite

different from that based on morphology and COI. Unweighted parsimony analyses

of the `total evidence' dataset largely supports the traditional view of Gerris

phylogeny. Finally, the implications of the reconstructed phylogeny in relation to

biogeography and ecological phylogenetics of Gerris is discussed.

Introduction

Water striders are familiar inhabitants of the surface ®lm of

different types of aquatic habitats all over the world. These

bugs are conspicuously adapted for life on the water surface,

especially with respect to locomotion, feeding, reproductive

behaviour and life history (Andersen, 1982; Spence, 1989;

Spence & Andersen, 1994). The pond skater genus Gerris

Fabricius includes forty-two species with a predominantly

Holarctic distribution. A few species extend into the

Afrotropical Region, and a single species, G. ®rmus Drake &

Hottes, is recorded from Mexico (Andersen, 1993a). Fossils in

Baltic amber (Eocene/Oligocene) have been assigned to

Gerris, giving a minimum age of 40±35 my for the genus

(Andersen, 1998). Gerris can be distinguished from the related

genera Limnoporus StaÊl and Aquarius Schellenberg by the

relatively short ®rst antennal segment, nonspinous connexival

corners and details in the male phallus (Andersen, 1993a).

Species of Gerris are found in lakes and ponds, often in

company with Limnoporus species. Unlike Aquarius, Gerris is

generally found to inhabit more temporary habitats, and no

Gerris species are exclusively con®ned to lotic waters.

Gerris is one of the best studied genera of water striders in

terms of wing polymorphism, habitat selection, mating

systems, etc. (Spence, 1989; Andersen, 1993b, 1997; Spence

& Andersen, 1994). The striking pattern of permanent and

seasonal wing polymorphism found in Gerris has intrigued

natural historians for decades, and in its integration with

systematics we are starting to understand the evolution of such

traits, even though the environmental and genetic mechanisms

behind them are still poorly understood.

Andersen (1993a) reviewed the character complexes in

genitalia and metasternal scent apparatus of particular

phylogenetic and taxonomic importance, and presented the

®rst attempt to classify Gerris and its subgenera into

monophyletic species-groups. He demonstrated that subgenus

Gerriselloides (Hungerford & Matsuda), as de®ned by

Andersen (1975) and Kanyukova (1982), was a paraphyletic

assemblage and restricted the subgenus to the Palearctic G.

lateralis group. He elevated the eastern Palearctic G.

gracilicornis group to a new subgenus, Macrogerris, and

grouped the remaining species in presumed monophyletic

Correspondence: Jakob Damgaard, Zoological Museum and

Zoological Institute, University of Copenhagen, Universitetsparken

15, DK-2100 Copenhagen, Denmark. E-mail: [email protected]

# 2001 Blackwell Science Ltd 241

Systematic Entomology (2001) 26, 241±254Systematic Entomology (2001) 26, 241±254

species groups in subgenus Gerris s. str. Finally, Andersen

(1993a) discussed the biogeographical and ecological implica-

tions of the reconstructed phylogeny of Gerris.

Water strider phylogenies based on morphological datasets

have repeatedly faced challenges from phylogenetic recon-

structions based on DNA sequences. Examples are the

Holarctic genus Limnoporus (Sperling et al., 1997), the

Pantropical marine Halobates Eschscholtz (Damgaard et al.,

2000a) and the cosmopolitan genus Aquarius (Damgaard et al.,

2000b). Although these studies have not greatly changed the

traditional view of water strider phylogenies, the inclusion of

new molecular data has provided a way to test the established

morphologically based phylogenies and have led to a better

understanding of phylogenetic relationships among water

striders. These new analyses have questioned the taxonomic

validity of several closely related species pairs, as well as

deeper relationships between more distantly related species,

genera and higher level taxa. Furthermore, the use of

molecular markers to evaluate well established morphological

phylogenies adds new insight into the limitations of different

kinds of data when analysed separately or in combination. The

focus of this study was to test the morphologically based

phylogeny of Gerris with new DNA sequence data, and to

review and re-evaluate the biogeographical implications and

evolution of ecological and behavioural traits in the context of

a well corroborated phylogeny.

Material and methods

DNA sequences and protocols

DNA was sequenced from twenty-two species of Gerris

selected to represent all subgenera and species-groups

recognized by Andersen (1993a). In addition, DNA was

sequenced from species belonging to closely related genera,

including Aquarius conformis Drake & Hottes, A. najas (De

Geer), A. paludum (Fabricius), Limnoporus esakii (Miyamoto),

L. rufoscutellatus (Latreille) and Gigantometra gigas (China).

The last species has been proposed as the closest relative of the

three above-mentioned genera (Andersen, 1993a, 1995). The

target sequences were 830 bp from the 3¢ half of the

mitochondrial gene encoding cytochrome oxidase subunit I

(COI), corresponding to position 2184±3013 in Drosophila

yakuba (Genbank accession no. NP006902) and 523 bp of the

nuclear gene encoding elongation factor-1 alpha (EF-1a),

corresponding to position 2413±2935 in D. melanogaster

(Genbank accession no. X06870). Some water strider DNA

sequences were available from studies by Sperling et al. (1997)

(Genbank accession nos U83337±U83345) and Damgaard

et al. (2000b) (Genbank accession nos AF200255±AF200737).

See Table 1 for collection data and accession numbers.

For most species, DNA was extracted from alcohol-

preserved adults, but for a few species live specimens were

Table 1. Species, geographic localities and GenBank accession numbers.

Subgenus/species-group Species Locality Collector COI EF-1a

Macrogerris Gerris gracilicornis (Horvath) Japan, Kochi T. Harada AF251100 AF251080

Gerriselloides G. lateralis Schummel Denmark, Grib Skov J. Damgaard AF251102 AF251082

G. asper (Fieber) Switzerland, Berne J.R. Spence AF251101 AF251081

G. gillettei-group G. sphagnetorum Gaunitz Sweden, BjaÈdjesjoÈ J. Damgaard AF251103 AF251083

G. incognitus Drake & Hottes USA, New Mexico G. Arnqvist AF251104 AF251084

G. pingreensis Drake & Hottes Canada, Alberta J.R. Spence U83345 AF200275

G. gillettei Lethierry & Severin USA, New Mexico G. Arnqvist AF251105 AF251085

G. argenticollis-group G. argenticollis Parshley USA, Virginia J.T. Polhemus AF251111 AF251092

G. nepalensis-group G. nepalensis Distant Japan, Kochi T. Harada AF251106 AF251086

G. odontogaster-group G. argentatus Schummel Denmark, Gl. Holte J.R. Spence AF251107 AF251087

G. buenoi Kirkaldy Canada, Ottawa F.A.H. Sperling U83343 AF251089

G. babai Miyamoto China, Tianjin J.R. Spence AF251108 AF251090

G. odontogaster (Zetterstedt) Denmark, Gl. Holte J.R. Spence AF251109 AF251088

`G. thoracicus-group' G. thoracicus Schummel Denmark, TaÊstrup J. Damgaard AF251114 AF251093

G. costae poissoni Wagner & Zimmermann Spain, Pyrenees V. Michelsen AF251113 AF251094

G. swakopensis-group G. swakopensis StaÊl Kenya, Masai Mara J.R. Spence AF251112 AF251097

G. lacustris-group G. lacustris L. Denmark, Gl. Holte J.R. Spence AF200735 AF200278

G. maculatus Tamanini Tunesia, Tamara ZMUC expedition AF251116 AF251099

G. gibbifer Schummel Denmark, Bornholm J. Damgaard AF251117 AF251098

`G. marginatus-group' G. latiabdominis Miyamoto Japan, Sakurai T. Harada AF251110 AF251091

G. comatus Drake & Hottes Canada, Ottawa F.A.H. Sperling U83344 AF251096

G. alacris Hussey U.S.A., Pennsylvania J.R. Spence AF251115 AF251095

Outgroup taxa Gigantometra gigas (China) Viet-Nam Lab culture D. Currie AF200245 AF200280

Limnoporus esakii (Miyamoto) Japan, Honshu J.R. Spence U83341 AF200263

L. rufoscutellatus (Latreille) Finland, Hanko J.R. Spence U83337 AF200268

Aquarius najas (De Geer) Denmark, Lellinge AÊ N.M. Andersen AF200736 AF200262

A. conformis (Uhler) U.S.A., Kentucky J. Krupa AF200255 AF200272

A. paludum (Fabricius) Denmark, Agersù F.A.H. Sperling AF200737 AF200266

# 2001 Blackwell Science Ltd, Systematic Entomology, 26, 241±254

242 J. Damgaard and F. A. H. Sperling242 J. Damgaard and F. A. H. Sperling

killed in a freezer at ± 70 °C and DNA was extracted later.

Heads, abdomens and legs were stored in 70% alcohol as

voucher specimens, and are deposited in the Zoological

Museum, University of Copenhagen.

A phenol/chloroform extraction method was used for Gerris

alacris, G. argentatus, G. asper, G. babai, G. gibbifer, G.

odontogaster and G. swakopensis. Later, the CTAB extraction

protocol (Doyle & Doyle, 1987) was used for Gerris lateralis

and G. sphagnetorum. Because this method only gave 30 mL

DNA in solution, template DNA for G. sphagnetorum was

exhausted and DNA had to be extracted from another

specimen, although from the same collection. For all

remaining species, the QiaAmp tissue kit protocol (QIAGEN

Inc., Santa Clara, California) was used, which included at least

2 h digestion of tissue with Proteinase K and gave a volume of

300 mL DNA in solution.

PCR ampli®cations for both gene regions were carried out in

a thermal cycler in 51 mL of a cocktail containing 2 mL template,

5 mL of each primer (5 mM), 14 mL ddH2O, 20 mL dNTP (GATC

0.5 mM each) and 5 mL 103 Promega PCR-reaction buffer

(15 mM MgCl2). After a `hot start' with 2 min of denaturation at

94 °C, the reaction was paused at 72 °C and 0.2 mL Taq-

polymerase (5 U/mL) was added. Ampli®cation parameters for

each of the following 35 cycles were as follows: 94 °C for 1 min

(denaturation), 45 °C for 1 min (annealing) and 72 °C for

1.5 min (extension). For EF-1a the annealing temperature was

raised to 50±55 °C in cases where there were multiple bands.

The target segment of COI was delimited by the primers C1-

J-2183 to TL2-N-3014 (reproduced from Simon et al., 1994).

Because the segments were too long to be ampli®ed and sequen-

ced in one step, two internal primers were used for ampli®cation

with the end-primers, namely C1-N-2609 (from Damgaard

et al., 2000a) to work with C1-J-2183, and a new primer, C1-J-

2456 (5¢TTAGCAAATTCTTC AATTGA 3¢), to work with

Tl2-N-3014. These two sequences had an overlap of 153 bp.

To amplify and sequence EF-1a, the primers M2412 and

rcM52.6 from Damgaard et al. (2000b) were used. The PCR

product was electrophoresed on a 2% NuSieve gel, stained with

ethidium bromide and sized against a fX174/HaeIII

(Boehringer Mannheim, Mannheim, Germany) DNA ladder

under UV light. PCR products were cleaned with a QIAquick

PCR Puri®cation Kit (QIAGEN Inc.). Cycle sequencing was

carried out using a Perkin Elmer/ABI Dye Terminator Cycle

Sequencing Kit and run on a thermocycler using the pro®les

recommended by the manufacturers. Cycle sequencing products

were cleaned using Centrisep columns or ethanol precipitation

and sequenced using a Perkin Elmer ABI377 Automated

Sequencer (Applied Biosystems Inc., Foster City, California).

DNA sequence for each species was con®rmed with both sense

and anti-sense strands. Because both gene segments are protein

coding and relatively conserved in amino acid sequences,

alignment was unproblematically performed in the program

Sequencher (Gene Codes Corporation, Ann Arbor, Michigan).

Morphological characters

Characters used in the morphological analyses were those

used by Andersen (1993a) for twenty-two species of Gerris,

three species of Aquarius, Limnoporus rufoscutellatus and

Gigantometra gigas. The data matrix is shown in

Table 2. Andersen (1993a) should be consulted for de®nitions

of the characters and their states. Andersen used Limnoporus

canaliculatus (Say) among the outgroup taxa because this

species could be scored for characters only found in wingless

morphs. Because L. esakii is found to be the sister species to all

other species of Limnoporus, this species was used as outgroup

instead of L. canaliculatus. The two species are scored

identically, except for character 29, which is scored as

character 19 in Andersen & Spence (1992), and characters

related to the wingless morph of L. canaliculatus. Some

characters (20, 25, 30, 32, 42, 48 and 63) are conditionally

de®ned and can only be scored if certain structures are present.

These characters are scored as inapplicable (denoted by a

question mark). All multistate characters were treated as non-

additive (states unordered).

Phylogenetic analyses

Phylogenetic reconstructions were obtained by both max-

imum parsimony and maximum likelihood. Unweighted

parsimony analyses of various datasets were performed using

PAUP* 4.0 b2 (Swofford, 1998) in combination with MacClade

3.05 (Maddison & Maddison, 1992). As the number of taxa

and the size of the data matrix often precluded more thorough

searches, heuristic searches were carried out with twenty

random-taxon-addition replicates. In case of multiple equally

parsimonious cladograms, a successive weighting procedure

(Farris, 1969) was applied to reduce the number of cladograms.

The resulting cladograms were only considered if they were a

subset of the most parsimonious cladograms.

Maximum likelihood analyses were conducted in PAUP*.

The 50% parsimony bootstrap search was used as the starting

point for NNI branch swapping under likelihood. The

Hasegawa±Kishino±Yano (Hasegawa et al., 1985) model of

sequence evolution was implemented using observed nucleo-

tide frequencies, two substitution types: TI/TV (transition/

transversion) ratio initially estimated by MacClade from the

50% bootstrap cladogram, and estimation of the rate hetero-

geneity, a, according to a G distribution. When a was

estimated, this value was used for estimation of a new TI/TV

ratio. At the completion of this search, the estimated a and TI/

TV values were used for a heuristic search using NNI branch

swapping to ®nd the cladogram with the highest ln-likelihood

(Swofford et al., 1996).

To determine whether signi®cant incongruence exists

between the nucleotide datasets and the morphological dataset,

Incongruence Length Difference (ILD) tests (Farris et al.,

1995) were conducted by excluding all invariant sites and

using the partition homogeneity test in PAUP* with 100

iterations.

Clade stability was estimated using two different para-

meters: bootstrap and branch support (a.k.a. Bremer support or

decay index; Bremer, 1994). Bootstrap values were generated

in PAUP* from 500 replicates, each with ten random-addition

heuristic searches. Branch support values were obtained in


Phylogeny of Gerris 243Phylogeny of Gerris 243

PAUP* by using the `converse constraints' approach to obtain

branch support for the most stable clades (Bremer, 1994). In

order to assess the degree of support provided by each dataset

when analysed together, the partitioned branch support (a.k.a.

partitioned Bremer support; Baker & DeSalle, 1997; Baker

et al., 1998) was calculated for all datasets with reference to

the combined cladogram.

Results

Comparison of the two genes

A 820 bp segment of COI for twenty-two species of Gerris

and Limnoporus and a 780 bp segment for Gigantometra gigas

and species of Aquarius were sequenced. A total of 263 bp

(32.07%) varied between all taxa and 210 bp (25.61%) were

phylogenetically informative, with twenty-®ve (11.9%) in ®rst,

four (1.90%) in second and 181 (86.19%) in third codon

positions. The ratio of transitions to transversions was 1.72 for

all sites (9.13 in ®rst + second codon positions and 1.48 in third

codon positions). The predominance of transitions has been

documented widely for insect mtDNA (Simon et al., 1994),

including gerrine water striders of genera Limnoporus (Sperling

et al., 1997) and Aquarius (Damgaard et al., 2000b). The

average base composition was 35.0% A, 14.6% C, 13.2% G

and 37.1% T. For the different positions, the average base

composition for ®rst codon position was 35.9% A, 12.7% C,

22.9% G and 28.4% T; second codon position was 20.5% A,

23.7% C, 14.9% G and 40.8% T; and third codon composition

was 48.7% A, 7.4% C, 1.9% G and 42.1% T. The sequence is

therefore A/T rich with an overall average of 64.3% A + T. The

third codon position was extremely A/T rich with 90.8% A + T.

A 515 bp segment of EF-1a was sequenced for twenty-eight

species of gerrine water striders. EF-1a sites that consistently

displayed double peaks on electropherograms after multiple

sequencing runs were assumed to be heterozygous (for Papilio

see Reed & Sperling, 1999; for Aquarius see Damgaard et al.,

2000b). In the EF-1a matrix, twenty-eight sites were scored as

dimorphic, and thirteen individuals were scored as hetero-

zygous. Most of the dimorphisms were synonymous third

position variations. Of these, fourteen were C/T, ®ve were A/

G, two were A/T and one was G/T. Two dimorphisms were

non-synonymous in second position with one A/G and one G/T

variation. Finally, one A/G and one A/T non-synonymous

variation occurred in ®rst positions.

Of the 515 bp sequenced, 128 (24.85%) varied between all

taxa and seventy-nine (15.34%) were phylogenetically in-

Table 2. Data matrix of sixty-three morphological characters scored for twenty-two species of Gerris, three species of Aquarius, two species of

Limnoporus and Gigantometra gigas. De®nitions of characters and character states taken from Andersen (1993a). A questionmark (?) denotes

inapplicable or missing character scores.

1 1111111112 2222222223 3333333334 4444444445 5555555556 6661234567890 1234567890 1234567890 1234567890 1234567890 1234567890 123

G. gigas 1111111111 112121112? 1111131221 1111111111 1?11111?11 1111111112 111

L. esakii 1112221112 1122122112 111112222? 1111111211 1?11111?11 1112121112 21?L. rufoscutellatus 1112221112 1122122112 112112222? 1111111211 1?12111?12 1112111112 21?

A. najas 2121212312 2122211111 1222?21211 1111111111 1?11111?12 1111121112 111

A. paludum 2121212312 1122211111 1222?22211 1111111211 1?12122221 1121111112 113

A. conformis 2121212312 1122211211 1222?3122? 1?11111211 1?11112231 1121111112 11?G. lateralis 2211222112 122211112? 1221111221 1121111111 2121122111 1211111122 112

G. asper 2211222112 122211112? 1221111221 1121111111 2121122111 1211111122 112

G. gracilicornis 2211222112 112221112? 2221121222 1121212111 2222122111 1111212222 113G. sphagnetorum 2211212212 1122111111 2221111221 1121121111 2121122111 1111212222 122

G. incognitus 2211212222 1122111111 2221111221 1122121111 2121122111 1211212222 122

G. pingreensis 2211212222 1122111111 2221111221 1122121111 2121122111 1211212222 122

G. gillettei 2211212222 1122111111 2221111221 1122121111 2121122111 1211212222 122G. argenticollis 2211212212 112221312? 2221221222 2221221111 2121222312 2211312222 113

G. nepalensis 2211212312 112211112? 2222?21221 1222121111 2121122112 2111312222 122

G. latioabdominis 2211212222 112221312? 2221121222 2221221111 2121222312 1111312222 123

G. argentatus 2211212212 112221312? 2221221222 2221321111 2221222312 3211312222 123G. babai 2211212212 112221312? 2221221222 2221321111 2221222312 3211312222 123

G. buenoi 2211212212 112221312? 2221221222 2221321111 2221222312 3211312222 123

G. thoracicus 2211222212 112221212? 2221111222 2221221111 2121222312 1111312222 123

G. odontogaster 2211212212 112221312? 2221221222 2221321111 2221222312 3211312222 123G. alacris 2211212312 112231312? 2221221222 2221221112 2121222312 1111312222 123

G. comatus 2211212322 112221312? 2221121222 2221221112 2121222312 1111312222 123

G. swakopensis 2211212212 112221312? 2221121212 2222322123 2121222312 3211312222 123G. costae poissoni 2211222212 112231212? 222111122? 2221221111 2121222312 1111312222 12?

G. lacustris 2211212213 112221312? 2221121222 2221121111 2121222312 1111312222 113

G. gibbifer 2211212313 112231312? 2221121222 2221121111 2121222312 1111312222 113

G. maculatus 2211212313 112231312? 2221121222 2221121111 2121222312 1111312222 113



formative with three (3.8%) in ®rst and seventy-six (96.2%) in

third positions. The ratio of transitions to transversions was

2.96 for all sites (0.78 for ®rst + second positions and 3.49 for

third positions). The TI/TV ratio is lower than the 4.25 found

in heliothine moths (Cho et al., 1995) and the 3.81 found in

Aquarius (Damgaard et al., 2000b), but higher than the 2.8

found in Ips bark beetles (Cognato, 1998). The average base

composition was 26.0% A, 23.3% C, 25.7% G and 25.0% T.

For the different codon positions, the average base composi-

tion for ®rst codon position was 31.4% A, 17.9% C, 38.4% G

and 12.4% T; second codon position was 30.1% A, 22.7% C,

18.7% G and 28.5% T; and third codon composition was

16.3% A, 29.5% C, 19.9% G and 34.4% T. The average

composition of each base is therefore roughly equal overall as

well as between the different positions.

Phylogenetic analyses

COI. An unweighted parsimony analysis of the COI dataset

gave a single MPC (most parsimonious cladogram) of 950

steps (Table 3 summarizes cladogram statistics). This clado-

gram is shown with branch lengths, bootstrap and branch

support values in Fig. 1. The cladogram includes clade

numbers 1, 4, 6, 10, 12, 14 and 15 (Table 4). Surprisingly,

G. argenticollis was placed as the sister species to all other

Gerris. To rule out the possibility of contamination, COI from

another specimen of G. argenticollis from Ontario, Canada,

was sequenced, giving an identical DNA sequence. To test for

saturation of substitutions in the gene, the number of inferred

steps from the MPT was plotted against pairwise comparisons

of the uncorrected genetic differences (Philippe et al., 1994).

The result shown in Fig. 2 was an almost straight line, only

indicating some degree of saturation of COI among the most

distantly related taxa. An unweighted parsimony analysis of

only transversions gave ®ve MPCs, each of 381 steps

(CI = 0.3255, RI = 0.5105). A successive weighting procedure

did not reduce the number of MPCs. A strict consensus

contains clade numbers 1, 2, 4, 7, 10 and 12 (Table 4). Finally,

the COI sequence data was used in generating a cladogram

based on maximum likelihood. A TI/TV ratio of 2.4 and an

among site variation of 0.13 gave the ln-likelihood ±

5302.1399. The cladogram is sixteen steps longer than the

MPC, and contains clade numbers 2, 4, 7, 9±12, 14 and 15

(Table 4).

EF-1a. Sixty-four MPCs (each of 287 steps) were obtained

from an unweighted parsimony analysis of the EF-1anucleotide dataset, which by successive weighting were

reduced to eight cladograms (see Table 3 for cladogram

statistics). Figure 3 shows the strict consensus cladogram with

bootstrap and branch support values, and includes clade

numbers 1, 4, 5, 10, 12 and 13 (Table 4). The EF-1a dataset

was tested for saturation, and a relatively straight line was

obtained that indicated virtually no saturation (Fig. 4). An

unweighted parsimony analysis of only transversions gave 515

MPCs, each of sixty-®ve steps (CI = 0.7385; RI = 0.7733),

which by successive weighting were reduced to twenty-eight

cladograms. A strict consensus of these includes clade numbers

1, 4, 9±12, 14 and 15 (Table 4). Finally, the EF-1a sequence

data were used in generating a cladogram based on maximum

likelihood. A TI/TV ratio of 3.65 and an among site variation

of 0.134 gave the highest likelihood (2177.8308). This is seven

steps longer than the MPC and includes clade numbers 1, 4, 5,

9, 10, 12 and 15 (Table 4).

Morphology

An unweighted parsimony analysis of the morphological

dataset gave twelve MPCs, each of 119 steps, which by

successive weighting were reduced to four cladograms (see

Table 3 for cladogram statistics). Figure 5 shows the strict

consensus of these cladograms with bootstrap and branch

support, and includes clade numbers 1±4, 7±12, 14 and 15

(Table 4). The cladogram is congruent with the established

morphologically based phylogeny (Andersen, 1993a: Fig. 23).

Combined analyses

Unweighted parsimony analyses were performed on the

morphology and nucleotide sequence datasets in different

combinations, as well as in `total evidence' analyses. An

unweighted parsimony analysis of the `total nucleotide' dataset

gave eight MPCs, each of 1252 steps, which by successive

weighting were reduced to a single cladogram that includes

Table 3. Summary of parsimony analyses of molecular, morphological and variously combined datasets.

PIC = phylogenetically informative characters.

No. of No. of Cladogram No. of

Dataset characters PICs length cladograms CI RI

COI 820 210 950 1 0.3811 0.4463

EF-1a 515 79 287 64 0.5296 0.5846

Morphology 63 54 119 12 0.6050 0.8374

COI + EF-1a 1335 289 1252 8 0.4105 0.4679

COI + morphology 883 264 1088 2 0.3989 0.5159

EF-1a + morphology 578 133 418 5 0.5359 0.6840

`Total evidence' 1398 343 1388 3 0.4222 0.5215



clade numbers 1, 2, 4, 6, 7 and 9±15 (Table 4). An unweighted

parsimony analysis for only transversions gave ®ve MPCs,

each of 456 steps (CI = 0.3772; RI = 0.5267), which by

successive weighting were reduced to a single cladogram that

includes clade numbers 1±4, 7, 10 and 12 (Table 4). The ILD

test gave P = 0.27, which indicates that the two molecular

partitions are not signi®cantly incongruent. When the pairwise

uncorrected genetic distances from the two datasets were

plotted against each other (Fig. 6), the uncorrected genetic

distances from EF-1a were found to be generally smaller than

for COI, and the majority of variable characters for closely

related taxa arose from COI, whereas for the more distantly

related species the dominance of COI levelled off. When the

two genes are corrected for differences in length, COI contains

about 25% more variable sites and 40% more phylogenetically

informative characters. An unweighted parsimony analysis of

the COI and morphology datasets gave two MPTs, each of

1088 steps, which by successive weighting were reduced to a

single cladogram containing clade numbers 1, 2, 4, 7±12, 14

and 15 (Table 4). The ILD test gave P = 0.85, which indicates

no signi®cant incongruence between the two partitions. An

unweighted parsimony analysis of EF-1a + morphology gave

®ve MPCs, each of 418 steps, which by successive weighting

were reduced to two cladograms that include clade numbers 1±

4, 6±12 and 14. The ILD test gave P = 0.03, which indicates

signi®cant incongruence between EF-1a and morphology.

Finally, an unweighted `total evidence' analysis of the three

datasets gave three MPCs, each of 1388 steps. A successive

Fig. 1. Single most parsimonious cladogram resulting from an unweighted parsimony analysis of 820 bp of the 3¢ half of COI conducted in

PAUP* using a heuristic search with twenty random addition replicates. Numbers above branches indicate branch length. The ®rst number below

each branch indicates bootstrap support from 500 replicates with ten random-addition heuristic searches per replicate. The second number below

each branch is branch support.



weighting procedure was not successful in reducing the

number of cladograms. Figure 7 shows the strict consensus

cladogram with bootstrap and branch support values, and

includes clade numbers 1, 2, 4 and 6±15 (Table 4). The ILD

test for the data partitions gave P = 0.27, which indicates that

the three datasets are not signi®cantly incongruent. Table 5

shows the partitioned branch support for the consensus

cladogram (Fig. 7).

Table 4. Summary of phylogenetic analyses of the three datasets (morphology, EF-1a and COI). Tot. nuc. = COI + EF-1a data, Tot.

ev. = COI + EF-1a + morphology, MP = maximum parsimony, TV = transversions only, ML = maximum likelihood, Mor. = morphology.

Tot. nuc.

COI COI COI EF1a EF1a EF1a COI EF1a Tot.

Clade no. MP TV ML MP TV ML Mor. MP TV + Mor. + Mor. Ev.

(1) G. gibbifer + G. maculatus X X ± X X X X X X X X X

(2) G.lacustris-group ± X X ± ? ± X X X X X X

(3) G. costae + G. thoracicus ± ? ± ? ± ± X ± X ± X ±

(4) G. alacris + G. comatus X X X X X X X X X X X X

(5) 2 + 3 + 4 ± ± ± X ± X ± ± ± ± ± ±

(6) 5 + G. swakopensis X ± ± ± ± ± ± X ± X X X

(7) G. odontogaster group ± X X ± ± ± X X X X X X

(8) `derived Gerris' ± ± ± ± ± ± X ± ± X X X

(9) 8 + G. nepalensis ± ± X ± X X X X ± X X X

(10) G. gillettei-group X X X X X X X X X X X X

(11) Gerris s. str. ± ± X ± X ± X X ± X X X

(12) Gerriselloides X X X X X X X X X X X X

(13) 11 + 12 ± ± ± X ? ± ± X ± ± ± X

(14) Gerris s.l. X ± X ± X ± X X ± X X X

(15) Gerris + Aquarius X ± X ± X X X X ± X ± X

Fig. 2. Number of inferred steps based on Fig. 1 against uncorrected (p) distances for pairwise comparisons between 820 bp nucleotide sequences

of COI in PAUP*.



Discussion

Combination of datasets

The data partitions yielded three different cladograms of

widely varying topology, and produced a consensus cladogram

from these partitions that failed to resolve numerous relation-

ships. Based on the ILD tests, it is evident that the

phylogenetic signals from COI and morphology show no

signi®cant incongruence. With the inclusion of EF-1a, the

incongruence increases, and the combination of EF-1a and

morphology is signi®cantly incongruent. Damgaard et al.

Fig. 3. Strict consensus of eight equally parsimonious cladograms obtained by successive weighting of sixty-four equally parsimonious

cladograms resulting from parsimony analysis of 515 bp of EF-1a conducted in PAUP* using a heuristic search with twenty random-addition

replicates. Format for numbers as in Fig. 1.



(2000b) Aquarius found no signi®cant incongruence between

these two datasets in their study.

The different signal in EF-1a is poorly supported and

largely dissolves when combined with the other partitions.

Figure 4 shows that the poor phylogenetic signal of EF-1acannot be explained as a result of saturation, and is more likely

to be caused by the restricted number of phylogenetically

informative characters related to the slow mutation rate in this

gene. Therefore, its signal is probably swamped by the greater

amount of information yielded by COI with its faster mutation

rate. Phylogenetic studies of > 1000 bp of EF-1a have shown

the marker to be excellent for solving intrageneric relation-

ships in Lepidoptera (Cho et al., 1995; Reed & Sperling, 1999)

and a longer strand would probably have increased its utility in

the present study. The `total nucleotide analysis' resolved

many of the clades found in the `total evidence analysis', that

were probably caused by the combination of COI with a

relatively high mutation rate and EF-1a with a slower rate. As

shown in Fig. 5, the phylogenetic signal for closely related taxa

mostly arises from COI, whereas EF-1a becomes increasingly

supportive, probably because the information yieded by EF-1abecomes more signi®cant with increasing saturation of

information yielded by COI at higher taxonomic levels.

In the `total evidence' cladogram (Fig. 7), COI supplies

37.5%, EF-1a 35.4% and morphology 27.2% of the total

branch support (Table 5). Therefore, we conclude that even

though analyses of EF-1a gave a topology different than the

other partitions, this topology is poorly supported in other

datasets, and all three partitions contribute equally to the

clades found in the `total evidence' cladogram.

The species groups of Gerris

All of the datasets strongly support the sister-species

relationship between G. asper and G. lateralis, and thereby

subgenus Gerriselloides. Andersen (1993a) omitted the G.

gracilicornis-group from Gerriselloides and elevated it to

subgenus Macrogerris, a belief supported by all present

analyses. Strong support was also found for the Nearctic sister

species G. gillettei and G. pingreensis, and their inclusion in a

monophyletic group together with the Nearctic G. incognitus

and the Palearctic G. sphagnetorum. Andersen (1993a)

recognized convincing, morphological similarities between

the species and named the assemblage the G. gillettei-group.

The data strongly support the sister-group relationship

between the Palearctic G. babai and G. odontogaster and the

Nearctic G. buenoi. Andersen (1993a) grouped these three

species in the G. odontogaster-group along with the Palearctic

G. argentatus, and this topology is recognized in all of the

combined analyses, even though the support is relatively low.

All of the analyses show a relationship between G. alacris and

G. comatus. Andersen (1993a) included these and three

additional species in the Nearctic G. marginatus-group, and

assigned the members of the group to two different clades

based on differences in the male genitalia. Even though G.

Fig. 4. Number of inferred steps based on Fig. 2 against uncorrected (p) distances for pairwise comparisons between 515 bp nucleotide sequences

of EF-1a in PAUP*.



alacris and G. comatus were assigned to different clades in the

G. marginatus-group, strong support was found here for their

monophyly, and hence the monophyly of the G. marginatus-

group. Andersen (1993a) further included the Palearctic G.

latiabdominis in the G. marginatus-group based on the

presence of lateral stripes of silvery pubescence on the

pronotum, but none of the analyses here supports this, and

this similarity to members of the group must be due to

convergence.

Strong support was found for the relationship between G.

gibbifer and G. maculatus. Andersen (1993a) included these

along with G. lacustris in the G. lacustris-group, but the group

is poorly supported in the analyses of morphological data, and

was not found in analysis of molecular data. On the other hand,

it was found in analyses of all combinations of data, and was

well supported in the `total evidence' analysis; therefore, we

accept the G. lacustris-group. Finally, Andersen (1993a)

suggested a relationship between G. thoracicus and G. costae

on the basis of two supposed synapomorphies (rufous or

yellowish brown pronotal lobe and relatively short, stout legs),

but interpreted them both as reversals. Based on the analysis of

molecular data, when analysed alone or in combination with

Fig. 5. Strict consensus of twelve equally parsimonious cladograms resulting from an unweighted parsimony analysis of sixty-three

morphological characters conducted in PAUP* using a heuristic search with twenty random-addition replicates. Format for numbers as in Fig. 1.



morphological data, there is no support for a close relationship

between these species, and we therefore consider the `G.

thoracicus-group' to be a polyphyletic assemblage.

Phylogeny of Gerris

Analysis of both molecular datasets indicates a relationship

between the G. lacustris-group, the G. marginatus-group, G.

thoracicus and G. costae. The analyses of COI further link G.

swakopensis to this group as the sister species to G. costae,

although this condition is contradicted by analyses of both EF-

1a and the morphological data. Andersen (1993a) placed these

species along with G. argenticollis, G. latiabdominis and the

G. odontogaster-group, in a `derived Gerris' clade. This clade

is not supported by the molecular data, but is present, though

weakly supported, in the combined analyses of morphological

and molecular data. Hypotheses about the relationships could

well be tested against sequencing data in order to validate or

contradict them. Andersen (1993a) suggested that G. nepa-

lensis is the sister taxon to the `derived Gerris'. This

relationship is not supported in the molecular analyses, but it

is recognized and relatively well supported in the `total

evidence' analysis. The analyses of COI and the combined data

indicate a sister-group relationship between the G. gillettei-

group and the `derived Gerris + G. nepalensis' clade, except

for the exclusion of G. argenticollis. Andersen (1993a)

recognized this basal position of the G. gillettei-group and

elevated the entire assemblage to subgeneric rank, Gerris s. str.

Whether subgenera Gerriselloides or Macrogerris is the sister

group to Gerris s. str. is ambiguous in all analyses. Although

the analyses of the morphological data gave relatively strongly

support for Andersen's (1993a) belief that Gerriselloides is the

most basal clade in Gerris, this is contradicted in analyses of

both COI and the combined data. We therefore suggest that

Macrogerris is the basal subgenus in Gerris.

Biogeographical implications

The distributions of Gerriselloides and the G. lacustris-

group cover most of the Palearctic Region, and several species

have broadly overlapping ranges. Closely related species,

however, are usually allopatric, showing both north±south and

east±west vicariance, e.g. the sister species G. lateralis + G.

asper and G. gibbifer + G. maculatus. Andersen (1993a)

included other pairs of sister species, but these ideas need to be

veri®ed on the basis of molecular data. Subgenus Macrogerris

and the monotypic G. nepalensis-group are endemic to eastern

Asia, and the most widespread species of Macrogerris, G.

gracilicornis, is sympatric with G. nepalensis (Andersen,

1993a). Andersen (1993a) included the Mexican G. ®rmus and

the eastern Palearctic G. latiabdominis in the otherwise

Nearctic G. marginatus-group. The analysis here shows that

the af®nity with G. latiabdominis probably is due to

convergence. The inclusion of G. ®rmus still needs to be

tested with DNA sequence data, but until then, the G.

marginatus-group and the monotypic G. argenticollis-group

are the only strictly Nearctic species-groups in Gerris. The G.

swakopensis-group occurs in all sub-Saharan Africa, including

Fig. 6. Uncorrected (p) distances for pairwise comparison between 820 bp nucelotide sequences for COI against 515 bp nucleotide sequences of

EF-1 a.



Madagascar (Andersen, 1993a), and is therefore endemic to the

Afrotropical Region. Finally, we can con®rm Andersen's

(1993a) hypothesis of `trans Beringian' relationships in the

G. gillettei-group and the G. odontogaster-group.

Ecological phylogenetics

Andersen (1993a,b) used his reconstructed phylogeny of

Gerris based on morphological characters to interpret the

evolution of morphological, developmental and ecological

patterns. Andersen (1993b) showed that the ancestral state for

Gerris is wing dimorphism, even for non-diapausing adults.

The replacement of Gerriselloides with Macrogerris as the

most basal subgenus in Gerris does not require one to postulate

any additional changes in character states. Andersen (1993a)

stated that species of Macrogerris, including Chinese G.

gracilicornis are wing-dimorphic, whereas Andersen (1993b:

Fig. 2) stated that Japanese G. gracilicornis are monomorphic

long-winged. Therefore, additional work should be performed

on this subgenus to resolve whether the monomorphic or

dimorphic long-winged condition is ancestral. Changes toward

the monomorphic long-winged condition in the diapausing

generation were found to have arisen twice, once in G.

gracilicornis and once in all `derived Gerris', except for

reversals to wing dimorphism in the G. lacustris-group.

Changes toward the monomorphic long-winged condition in

the non-diapausing generation were found to have arisen twice,

once in the `G. thoracicus-group', and once in G. latiabdo-

minis (Andersen, 1993b). Because G. costae and G. thoracicus

no longer are considered to be sister taxa, the changes toward

the monomorphic long-winged condition in the non-diapausing

generation must have arisen three times. The evolution of wing

dimorphism in Gerris is correlated with the habitat preferences

of the species, in which the basal, permanently wing dimorphic

species Gerriselloides and in the G. gillettei- and G.

nepalensis-groups are restricted to more stable habitats,

whereas the `derived Gerris' are either permanently or

Fig. 7. Strict consensus of three equally parsimonious cladograms resulting from unweighted parsimony analyses of the combined molecular and

morphological data conducted in PAUP* using a heuristic search with twenty random-addition replicates. Format for numbers as in Fig. 1.



seasonally wing-dimorphic or monomorphic long-winged and

occupy more unstable habitats.

Acknowledgements

This study was carried out in the Department of Entomology,

Zoological Museum (ZMUC) and the Department of

Evolutionary Biology, Zoological Institute (ZIUC), both in

the University of Copenhagen, and the Department of

Environmental Sciences, Policy and Management, University

of California, Berkeley. We are indebted to Anthony Cognato,

Mike Caterino and May Kuo, University of California,

Berkeley, and Pia Friis, Peter Gravlund and Sheila Tang (all

at ZIUC) for technical advice and support; and Nils Mùller

Andersen (ZMUC), Henrik Glenner and Bo Vest Pedersen

(both at ZIUC) for valuable comments on the manuscript. We

are also thankful to the following persons for collecting

specimens for this study: GoÈran Arnquist (Department of

Animal Ecology, University of UmeaÊ, Sweden), Doug Currie

(Toronto, Canada), Tetsuo Harada (Department of Biology,

Osaka City University, Japan), James J. Krupa (University of

Kentucky, Lexington, U.S.A.), Verner Michelsen

(Entomological Department, Zoological Museum, University

of Copenhagen, Denmark), John T. Polhemus (Colorado

Entomological Museum, Englewood, Colorado, U.S.A.), B.S.

Smith (Ithaca College, Ithaca, New York, U.S.A.) and John R.

Spence (University of Alberta, Edmonton, Canada). This work

was supported by grants from the Danish Natural Science

Research Council (grant no. 9502155 to J.D.) and the

California Agriculture Experiment Station (to F.A.H.S).

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Accepted 5 April 2000



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