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Deep cox1 divergence and hyperdiversity of Trigonopterusweevils in a New Guinea mountain range (Coleoptera,

Curculionidae)ALEXANDER RIEDEL, DAAWIA DAAWIA & MICHAEL BALKE

Submitted: 31 March 2009Accepted: 8 July 2009doi:10.1111/j.1463-6409.2009.00404.x

Riedel, A., Daawia, D. & Balke, M. (2010). Deep cox1 divergence and hyperdiversity

of Trigonopterus weevils in a New Guinea mountain range (Coleoptera, Curculionidae).—

Zoologica Scripta, 39, 63–74.

Trigonopterus is a little-known genus of flightless tropical weevils. A survey in one locality,

the Cyclops Mountains of West New Guinea, yielded 51 species, at least 48 of them unde-

scribed. In this study, we show that mtDNA sequencing, or DNA barcoding, is an effective

and useful tool for rapid discovery and identification of these species, most of them mor-

phologically very difficult to distinguish even for expert taxonomists. The genus is hyperdi-

verse in New Guinea and different species occur on foliage and in the litter layer.

Morphological characters for its diagnosis are provided. Despite their external similarity,

the genetic divergence between the species is high (smallest interspecific divergence 16%,

mean 20%). We show that Trigonopterus are locally hyperdiverse and genetically very

strongly structured. Their potential for rapid local biodiversity assessment surveys in Mela-

nesia is outlined (a-diversity); providing a regional perspective on Trigonopterus diversity

and biogeography is the next challenge (b-diversity).

Corresponding author: Alexander Riedel, Staatliches Museum fur Naturkunde, Erbprinzenstr

13, D-76133 Karlsruhe, Germany. E-mail: [email protected]

Daawia Daawia, Jurusan Biology, FMIPA-Universitas Cendrawasih, Kampus Waena-Jayapura,

Papua, Indonesia. E-mail: [email protected]

Michael Balke, Zoologische Staatssammlung, Munchhausenstr. 21, D-81247 Munchen, Germany.

E-mail: [email protected]

IntroductionBeetles form a major part of global biodiversity and with

62 000 described species and possibly more than 150 000

undescribed ones, the weevils (Curculionoidea) are the

dominating group among them (Oberprieler et al. 2007).

Weevils are especially diverse in tropical forests. Although

they constitute a high proportion of terrestrial biodiver-

sity, they are largely ignored during surveys and decision

making in conservation programmes. For example, during

a Rapid Biodiversity Assessment (RAP) of Conservation

International in the Cyclops mountains of West New Gui-

nea, only four entomologists (covering butterflies and

aquatic insects) out of 30 scientists participated, whereas

the remainder studied vertebrates or plants (Richards &

Suryadi 2002). One of the reasons behind this imbalance

is certainly the ‘taxonomic impediment’ (Hoagland 1996):

the lack of taxonomic expertise for many of the megadi-

verse taxa such as weevils (Wheeler et al. 2004). In fact,

many of the species are still undescribed and it is almost

impossible to identify the described ones without compari-

son with the historic type material. A commonly applied

remedy in all sorts of biodiversity studies is sorting to

morphospecies by parataxonomists. This approach has its

merits, but the results depend strongly on the experience

of the parataxonomist, regardless of the academic level or

cultural background. Not surprising, sorting to morpho-

species (and the authors include themselves here when it

comes to taxa they are not particularly familiar with) has

be shown to suffer often from high error rates (Krell

2004).

DNA sequencing has recently been promoted as a tool

to help overcome this impediment, DNA barcoding being

currently the most popular approach (Hebert et al. 2003;

Schindel & Miller 2005; Stoeckle & Hebert 2008). How-

ever, surprisingly few studies have to date sought to use

DNA sequence data, or barcode data, for rapid biodiversity

ª 2009 The Authors. Journal compilation ª 2009 The Norwegian Academy of Science and Letters d Zoologica Scripta, 39, 1, January 2010, pp 63–74 63

Zoologica Scripta

assessment. Initial studies by Smith et al. (2005) (Madaga-

scan ants) and Borisenko et al. (2008) (Surinamese small

mammals) found good congruence between morphological

identification and barcode clusters. Among the ants, some-

times more conservative morphospecies than molecular

‘species’ delineation (i.e. genetically diverse morphospecies

with up to 16% cox1 p-distance), and higher b-diversity

from molecular taxonomic units vs. morphospecies were

found. This means that different localities have very simi-

lar looking species which parataxonomists tend to lump

together into the same morphospecies, a well-documented

problem with morphospecies sorting, cf. Krell (2004).

While sequence data can thus assist in the rapid delivery

of biodiversity assessment of relevant data, it has also been

shown that incomplete lineage sorting and organelle cap-

ture might lead to erroneous results (e.g. Monaghan et al.

2006). Consequently, taxa require individual evaluation. In

this study, we explore the potential of DNA sequencing or

barcoding for rapid surveys of a very diverse but neglected

group of phytophagous beetles in Melanesia.

The taxon

Trigonopterus Fauvel is a genus of flightless, apterous wee-

vils placed in the Cryptorhynchinae of Curculionidae

(Morimoto 1978; Alonso-Zarazaga & Lyal 1999), distrib-

uted from Eastern Sumatra to Samoa and from New Cale-

donia to the Philippines. The centre of its diversity

appears to be in New Guinea. Trigonopterus inhabit tropi-

cal forests, both on foliage and in the litter layer. In New

Guinea, the foliage-frequenting species are a prominent

element of the local arthropod fauna. Sometimes more

than 50% of the phytophagous beetles collected by beat-

ing forest undergrowth are Trigonopterus. They possess a

pectoral receptacle into which the rostrum and the anten-

nae can fit when the weevil is alarmed. Trigonopterus wee-

vils attain a typical defense position, thanatosis, by folding

the legs over the ventral side of the body. Also, the elytra

are fused with each other and with the thorax, thus form-

ing a solid capsule. In combination with an extraordinary

well-sclerotized cuticle, the body of these weevils can

withstand considerable pressure.

Trigonopterus weevils are excellent indicator organisms

for biodiversity monitoring in Melanesia, being present

and easily collected in the majority of natural habitats.

Their a-diversity is high and our unpublished data also

suggest very high b-diversity. Trigonopterus’s ability to dis-

perse over degraded habitat is presumably very limited

because of their flightlessness and small size. Yet, Trigon-

opterus remained poorly known. The majority of the 90

named species were described before 1930 (Pascoe 1885;

Faust 1898; Heller, 1916). The species are usually very

similar to each other externally, and genitalia need to be

examined for safe identification. However, not a single

illustration of these structures has ever been published.

Preparation of specimens, especially extraction of their

genitalia, is difficult and time-consuming. The fusion of

body sutures and the extensive defense adaptations make a

fast dissection, comparable with that of other beetles,

impossible even for an expert. Thus, the routine identifica-

tion of a large number of specimens on the basis of mor-

phological characters alone is impossible for Trigonopterus.

The area

The Cyclops Mountains are one of the most clearly

defined and isolated New Guinea mountain ranges, situ-

ated on the North coast of Indonesian Papua (formerly

Irian Jaya), close to the border with Papua New Guinea

(Fig. 1). They extend over 45 km from Tanahmerah Bay

in the West, to Jayapura, the capital of Papua, in the East.

To the South, they are bordered by Lake Sentani; to the

North, they drop to the Pacific Ocean. The highest peak,

Mt. Rafeni, is 1880 m high (van Royen 1965; Polhemus &

Richards 2002).

The Cyclops Mountains represent a former island that

was accreted to northern New Guinea in the Tertiary.

There are uncertainties about the precise timing of events,

but the formation of the island arc may have begun in the

Eocene, while the collision occurred between Miocene and

Pliocene (Monnier et al. 1999). Probably, the same island

arc also included all or some of the following terranes: Ar-

fak, Biak, Japen, Van Rees, Foja, Torricelli, Prince Alexan-

der, Adelbert, Finisterre and Saruwaged (Polhemus 2007).

The vegetation of the Cyclops Mountains was studied

in detail by van Royen (1965). Forest types change quite

markedly with elevation and range from tropical lowland

forest to montane forest dominated by Araucaria and Not-

hofagus in the upper elevations. Such montane habitats are

well-isolated from others in the region. The nearest area

would be the Bewani Mountains in the East (c. 100 km),

the Central Range in the South (c. 130 km) and the Foja

Mountains in the West (c. 150 km). These montane habi-

tats are likely to harbour a variety of endemic plant and

animal species. Sir David’s Long-beaked Echidna (Zaglos-

sus attenboroughi) is so far known to inhabit the upper ele-

vations of the Cyclops Mountains only. It was described as

recently as 1998 by Flannery and Groves. There are no

endemic bird or butterfly species recorded, which can be

accounted for by their high mobility. A look at other, less

mobile species would be rewarding to identify endemism

on species level.

Apart from its rich biodiversity, its importance as a

water catchment area for nearby Sentani and the provincial

capital Jayapura is one of the reasons why the area is offi-

cially protected in the 22 500 ha Cyclops Mountains

Hyperdiversity of Trigonopterus weevils d A. Riedel et al.

64 Zoologica Scripta, 39, 1, January 2010, pp 63–74 d ª 2009 The Authors. Journal compilation ª 2009 The Norwegian Academy of Science and Letters

Reserve (Ratcliffe 1984). However, in recent years, popula-

tion pressure increased markedly, especially from the Sen-

tani area. Illegal gardening using slash and burn

agriculture is evident, in areas up to an elevation of about

700 m. In December 2006, following heavy rainfalls, land-

slides occurred on the Southern slopes that caused major

damage to environment and infrastructure. The area of

the Cyclops Mountains is severely threatened and effective

protection measures are urgently needed.

Materials and methodsField sampling

Field work was conducted on 6 days within 2 weeks in

November ⁄ December 2007. A transect was sampled

between 300 and 1520 m elevation (Fig. 1). Specimens

collected from foliage in the upper elevations (1200–

1520 m) may be somewhat under-represented as the area

is very hard to reach and was visited only once when

weather conditions were favourable.

Two collecting methods were applied, based on previ-

ous experience that different species of Trigonopterus occur

on the foliage and in the litter layer. The former were col-

lected by hand and by using a beating sheet, beating the

lower vegetation along the trail. Foliage higher than about

2.5 m was not covered in this survey. Specimens were col-

lected in a tube of absolute ethanol. The tube was changed

after an elevational interval of 200–300 m. Elevations were

read from the altimeter of a Suunto X-Lander.

Edaphic species were collected by sifting the litter layer.

A beetle sieve (after Reitter, as sold by H. Winkler, Vienna)

was used, which consists of 1 cm hardware cloth fixed in a

ripstop nylon sifter. Sampling consisted of vigorous sifting

of leaf litter and twigs down to, but not including, soil. Sift-

ing for each sample was performed for about 30–45 min

Fig. 1 Map of New Guinea with enlarged

selection of the Cyclops Mountains. The

sampled transect is indicated by dots.

A. Riedel et al. d Hyperdiversity of Trigonopterus weevils


until 5–10 L of sifted litter had accumulated. Precise sample

locations were selected based on suitable terrain and the

presence of good litter accumulations. It was attempted to

cover the full elevational range of the transect evenly with

litter samples. It was attempted to cover a variety of litter

types (near big trees ⁄ between undergrowth ⁄ on ridge ⁄ on

level ground) within each sample location within a distance

of about 25 m from the fixed sampling locality. Coordinates

were measured with Garmin GPSmap 60CSx. Samples were

carried back to the hotel in the evening. Altogether, 16 litter

samples were collected.

The litter samples were processed to maximize speci-

mens obtained within a short period. First of all, sample

bags were carefully opened and specimens found on top of

the litter ⁄ on the cloth of the bag were taken and preserved

in absolute ethanol. During the first night after sampling,

this method yielded a relatively large number of speci-

mens. On the following day, portions amounting 1–2

handsful of sifted litter were placed on a white rubber

cloth and placed in the sun. Specimens usually started

walking and became visible after 5 min. Subsequently, the

litter was placed into ecclectors (Winkler and Konzelman

ecclectors) for another 2–3 days. When no additional spec-

imens appeared, the dry litter was disposed. A total of

1265 specimens of Trigonopterus were collected.

Diagnostic characters of Trigonopterus and selection of

specimens

Although Trigonopterus is morphologically and phylogenet-

ically clearly defined, published information on how to

separate it from similar weevils is insufficient. A study to

identify autapomorphies of the genus is in progress (by A.

Riedel). Characters that were used in this study to diag-

nose Trigonopterus are outlined below, as a number of

other weevil genera occurring in the area could easily be

confounded with Trigonopterus.

Trigonopterus species are usually subglabrous, without or

with only few scales, whereas the majority of other Cryp-

torhynchinae have a dense vestiture of scales or setae.

Exceptions occur in both groups; therefore, this character

is only useful for presorting. Furthermore, only apterous

Cryptorhynchinae with a reduced or absent metanepister-

num were considered to be closely related to Trigonopterus.

This excludes fully winged genera such as Imathia (Curcu-

lioninae: Storeini) or Semiathe (Cryptorhynchinae), which

possesses an elongate metanepisternum. Trigonopterus was

diagnosed based on the complete loss of the metanepister-

num and by an apomorphic structure of the tarsus with

minute claws and a deeply incavated articulation of tarso-

mere 4. One fully winged species of Semiathe (code

ARC689 on the tree) was included as outgroup, as well as

one wingless species of Telaugia (ARC602) and an unde-

scribed genus of wingless Cryptorhynchinae (ARC600).

The latter two species possess a reduced subtriangular

metanepisternum and tarsi of the plesiomorphic type with

larger divaricate claws.

Previous experience had shown that many Trigonopterus

species differ by more or less subtle external morphologi-

cal characters, such as size, sculpture, dorsal outline and

lustre. Based on these characters, a pre-selection was

made. It was attempted to include at least three specimens

of each ‘morphospecies’ if series were available. These

were selected from a maximum range, i.e., different speci-

mens were chosen from samples of the lowest ⁄ the highest

elevation. Genital characters could not be consulted at this

stage because the genitalia are held inside the abdomen by

extensive muscle tissue. It is only after maceration that the

genitalia can be safely extracted from the specimen. Thus,

these preliminary ‘morphospecies’-hypotheses needed revi-

sion in a second step after genital characters had been

studied and specimens were properly mounted. Altogether,

222 Trigonopterus were selected, plus 3 outgroups.

Preparation of specimens

DNA was extracted nondestructively to allow subsequent

examination of genital morphology and photographic doc-

umentation, followed by museum storage of voucher spec-

imens. Standard procedures of beetle dissection had to be

modified: the beetles were placed individually in an

embryo dish containing absolute ethanol. The prothorax

was carefully pulled from the mesothorax, rupturing the

thin connecting membrane between the two and thus

splitting the body into an anterior and a posterior part.

Both parts were used for DNA extraction. After the

extraction, the digested specimens were stored in ethanol

and softened in 10% acetic acid for 1 day before further

preparation. After the extraction of the genitalia, the ante-

rior and posterior body parts were put together again.

This step works surprisingly well in Trigonopterus, because

the mesothorax forms a distinct anterior neck which fits

into the prothorax, forming a natural plug connection.

Winged beetles of comparable size would usually fall

apart after such a preparation as the elytra get detached

from the thorax easily. Instead, in such groups, the abdo-

men is removed while slightly opening up the elytra. In

Trigonopterus, this standard procedure cannot be used

because the elytra are fused with each other and to the

base of the abdomen. Thus, removal of the abdomen

would inflict irreparable damage to the specimen.

However, because of the fusion of elytra and thorax in

Trigonopterus, it is safe to split these specimens between

meso- and prothorax. This is also useful for the subse-

quent preparation of the genitalia. The abdominal tip is

often deeply retracted dorsally into the elytra. It can be



pushed ventrad and opened up with a pin that is inserted

anteriorly through the thoracic opening. The genitalia are

then removed from the abdominal apex. They can be

pulled out in better condition from the extracted specimen

than possible in fresh ones: extensive muscle tissue sur-

rounding the long ductus ejaculatorius respectively ductus

spermathecae is cleared by enzyme digestion. In specimens

with intact and somewhat hardened tissue, these parts

often tear off and remain in the specimen when the genita-

lia are removed.

Thus, with the preparation outlined above, the DNA

extracted specimens remain without visible damage and

can be used as type material without any problem. The

genitalia of all the extracted specimens were macerated

with 10% KOH, rinsed in diluted acetic acid, stained with

an alcoholic Chlorazol Black solution and stored in glyc-

erol in microvials attached to the pin of the specimens.

Photographs were taken with an automated Leica Z6 APO

(Leica Microsystems, Wetzlar, Germany), a JVC KY70

camera (JVC Professional Products) and Automontageªsoftware (Syncroscopy, Cambridge, UK).

DNA sequencing and sequence analysis

Genomic DNA was extracted from individual beetles using

the DNeasy tissue kit (Qiagen, Hilden, Germany). Using

standard PCR protocols and double-stranded sequencing

with PCR primers, we sequenced the 5¢ end of cyto-

chrome c oxidase 1 (cox1). At first, standard primers were

used: LCO1490: 5¢-GGTCAACAAATCATAAAGA-

TATTGG-3¢, HCO2198: 5¢-TAAACTTCAGGGTGAC-

CAAAAAATCA-3¢ (Folmer et al. 1994). For about 30

samples in which PCR amplification with these primers

failed, primers adjusted for Cryptorhynchinae were

used: LCO1490-JJ CHACWAAYCATAAAGATATYGG,

HCO2198-JJ AWACTTCVGGRTGVCCAAARAATCA

(Astrin & Stuben 2008). Sequences were edited using the

Sequencher 4.2 software package (GeneCodes Corp., Ann

Arbor, MI, USA) and submitted to GenBank [accession

numbers FN429126 - FN429350 (will be added in proof)].

Sequence data analysis

A data matrix was generated in Sequencher and exported

to MacClade (Maddison & Maddison 2000) to inspect the

amino acid sequence to confirm the nucleotide alignment

and for quality control. As no stop codons or other suspi-

cious features were detected, we assembled a 658-bp data

set in PAUP* 4.0b10 (Swofford 2002), trimming the

sequence ends to some degree to avoid too many missing

data. The data were then exported, and maximum-likeli-

hood analysis ran in GARLI (Zwickl 2006). Data structure

was also visualized by compiling a neighbor-joining tree in

PAUP*, using uncorrected p-distances. Taxon DNA soft-

ware (written by Gaurav Vaidya, Singapore. http://code.

google.com/p/taxondna/; http://taxondna.sourceforge.net/)

was used to study genetic divergences in our data set, to

cluster sequences at different set thresholds (Meier et al.

2006) and to calculate the smallest interspecific distance

(Meier et al. 2008).

According to Hebert et al. (2003), cox1 divergence even

between closely related hexapod species is roughly

between 6% and 15%, depending on the order. We thus

evaluated clustering at such thresholds as a potentially fast

tool to estimate diversity with our data set. We have

shown elsewhere that divergence between recent sister

species might be much smaller (Monaghan et al. 2006;

Balke et al. 2007), but understand that the magnitude of

genetic divergence between prospective species has to be

evaluated strictly on a case-by-case basis. We also use

nested cladistic phylogenetic analysis as implemented in

the program TCS 1.13 (Clement et al. 2000). The obtained

statistical networks subdivide the variation on the basis of

the level of homoplasy within the data themselves, i.e. dis-

tinguish between long (homoplastic) and short (nonhomo-

plastic) branches. This provides a relative measure of

divergence within a given data set, rather than a predeter-

mined phenetic cut-off value (Monaghan et al. 2006).

ResultsInitial morphospecies count

On the basis of the initial study of the external morphol-

ogy, we recognized 40 morphospecies.

Second morphospecies count

After examination of genital morphology of all specimens,

we recognized a total of 51 Trigonopterus species.

Sequence data

The sequences had no indels and were thus easily aligned

on the nucleotide level. Total cox1 uncorrected p-distance

in the 222 Trigonopterus sequences was 0.0–27.5%. Both

neighbor-joining and maximum-likelihood analysis pro-

duced strongly structured trees, with deeply structured

monophyletic clusters. These visually obvious clusters

were congruent with the 51 morphospecies based on exter-

nal plus genital morphology. The smallest interspecific

morphospecies distance was 16.5% (mean 20.5%) in Trig-

onopterus. Intraspecific distances ranged from 0.0% to

8.8% (mean 1.2%).

Species recognition based on DNA sequence data

Clustering the data at thresholds of 8–12% revealed 51

clusters that corresponded to the 51 recognized morpho-

species (Fig. 2). Clustering at 13% revealed 50 clusters,

but only 49 clusters contained only one morphospecies,



one cluster contained members of species 7 and 8; cluster-

ing at 14%: 48 clusters, 45 of them corresponding to rec-

ognized morphospecies; 15%: 46 ⁄ 42; 16%: 31 ⁄ 30 (i.e.,

there was one cluster lumping 21 morphospecies), etc.

Lowering the threshold to 7% has the opposite effect,

retrieving 52 clusters, 50 of which correspond to the given

morphology based taxonomy, members of species 12 were

split into two clusters; 6%: 52 ⁄ 50 (species 12 split); 5%:

53 ⁄ 49 (species 11 and 12 both split); 4%: 56 ⁄ 47 (species

30, 39 and 11 split in two each, species 12 split into three

species); 3%: 56 ⁄ 47 (species 30, 39 and 11 split in two

each, species 12 split into three species); 2%: 59 ⁄ 45 (spe-

cies 30, 39 and 11 split in two each, species 12 and 51

split into three species each).

Nested cladistic phylogenetic analysis with TCS software

set to 95% connection limit identified 62 statistical net-

works, in which nine of the 51 morphospecies were split

into two or three networks: 2, 8, 11, 12, 30, 38, 39, 48

and 51. Number of ‘perfect clusters’ was thus 42.

Taxonomy ⁄ new species

The 51 Cyclops species were compared with our complete

image database of type specimens for the Trigonopterus

described from the Papuan region. Three of them could

be tentatively assigned to an available species name as they

agreed relatively well with characters of the type speci-

mens. However, no final identification could be given as

the type series of Trigonopterus obnixus (Faust) possibly

contain more than one species and as the type localities of

Trigonopterus oblongus (Pascoe) and Trigonopterus illex

(Faust) are quite distant from the site of this study and

minor differences do exist. Thus, our survey uncovered 48

new species.

Habitat specialization

Of the 51 species, 20 are edaphic, while 31 are foliage-

frequenting species (Figs S1–S51). Both habitats appear

clearly separated in Trigonopterus: none of the edaphic spe-

cies was collected while beating foliage, whereas only two

specimens of foliage-frequenting species were found in lit-

ter samples. Such a clear separation is remarkable in Cryp-

torhynchinae that are usually xylophagous and may be

found occasionally as ‘tourists’ on foliage.

DiscussionSpecies count

The first morphospecies hypotheses which were merely

based on external morphology showed two different types

of conflict with molecular data. First, externally similar spe-

cies had been overlooked. In two cases, three species had

been lumped (species 19 ⁄ 20 ⁄ 21; sp. 26 ⁄ 27 ⁄ 30); in seven

cases each, two species were lumped (sp. 7 ⁄ 8; sp. 9 ⁄ 10; sp.

16 ⁄ 17; sp. 18 ⁄ 22; sp. 23 ⁄ 25; sp. 33 ⁄ 34; sp. 46 ⁄ 47).

Second, a small number of errors occurred when assign-

ing specimens to the initial 40 ‘morphospecies’. Eleven

specimens were assigned to the wrong ‘morphospecies’.

This can be clearly attributed to the difficult working con-

dition, sorting unmounted specimens in alcohol, which

does not allow viewing all details and also impedes com-

paring a number of specimens side by side.

However, after studying the genital morphology and

revising our morphospecies delineations, we found congru-

ence between morphological and molecular data. This

means that all members of any given morphospecies also

form a monophyletic group on the cox1 tree (Fig. 3). In

this study, the morphospecies were supported by long

Fig. 2 Two Trigonopterus species from the Cyclops Mountains.

Habitus of male (left) and its aedeagus (right). Note that species

externally very similar usually can be safely separated by

morphology of male genitalia.



branches and were intuitively obvious from the tree topol-

ogy alone (but see below for the ‘automatization’ issue).

The smallest interspecific distance was 16.5% (mean

20.5%), values remarkably high for Coleoptera (Meier

et al. 2008); intraspecific distances were smaller, ranging

from 0 to 8.8% (average 1.2%). In six species, the intra-

specific distance was higher than 4%: sp. 8 (4.0–5.7%), sp.

12 (4.4–8.0%), sp. 39 (4.5%), sp. 48 (4.7–8.8%), sp. 30

(4.9%), sp. 11 (5.1–5.9%). No morphological differences

could be detected within these genetically more divergent

species, and such cases can only be addressed properly

based on more sophisticated sampling and an iterative

approach using different sources of information and more

material, i.e. integrative taxonomy.

Species should not be characterized solely based on

DNA sequence data (Tautz et al. 2002), and our data set

shows that morphologically identical specimens from the

same locality can diverge as much as 8.8% cox1 p-distance

(e.g. also Balke et al. 2008). Whether these are cryptic spe-

cies or not cannot and should not be decided with the

information available now.

In a case where morphology and mtDNA data reveal

monophyletic, congruent clusters, seeking to automatize

species-richness estimation to some degree is tempting.

Tools have to be fast and easy to use if they are to be use-

ful in routine studies. Clustering at preset thresholds is as

such a very fast and easy to use method. Though threshold

setting is arbitrary, we suggest that in our local approach

it would have been an efficient diversity approximator.

Agreement between morphological species count and

mtDNA clusters was perfect at 8–12% (Fig. 2). At 2%, we

found 59 clusters, which is a 15% overestimation of diver-

sity, with 88% perfect clusters (or 12% error). At 16%, we

found only 31 clusters, which is a 39% underestimation of

diversity (or error), with only 58% perfect clusters, i.e.

only 30 clusters are congruent with some of the total of

51 recognized morphospecies.

Computation of statistical networks breaking down vari-

ation on the basis of the information contained in the data

set itself revealed 62 networks; assuming these were

species means 21% diversity overestimation, and 42

networks perfectly corresponding to morphospecies, that

is, an error of 18%.

Implications for taxonomy and diversity assessment

At present, only 46 species of Trigonopterus were formally

described from the entire Papuan region, including the

Moluccas, the Bismarck archipelago and the Solomon

Fig. 3 Trigonopterus species from the

Cyclops Mountains. Problems in sorting

morphospecies based on external

characters, caused by sexual dimorphism

and intraspecific variation. Upper row:

Difference in habitus is bigger between

female (left) and male (middle) of species

10 than between males of species 10

(middle) and species 9 (right). Lower

row: Species 18; female (left); smooth

male (middle); striate male (right).



islands (Gressitt 1982), including 40 species from New

Guinea. Only four species were recorded from mainland

West New Guinea (Setliff 2007). Thus, the number of

species recorded within a 2-week survey in parts of only

one small mountain range in the region (51 species)

exceeds the number of species known from the entire

region. A comparison with historic type material revealed

that at most three out of our 51 species belong to

described species, thus at least 94% or 48 of the species

found are undescribed. A simple (and obviously oversim-

plified) arithmetic extrapolation would lead to an expected

number of 1500 species worldwide and to 767 species

from the Papuan region. Based on personal field observa-

tions, these numbers appear quite conservative. Thus,

although the number of described Trigonopterus species

does not appear very impressive, the genus is in fact

hyperdiverse, that is, very species rich as well as abundant.

New technologies to speed up the process of making this

plethora of mostly similar species identifiable would be

highly welcome.

In recent years, there has been an intense debate about

the benefits of DNA taxonomy or DNA barcoding (e.g.

Hebert & Gregory 2005; Will et al. 2005 exemplifying

opposing views). What is the practical experience from

our study?

1 Technically, the method applied works well for Trigon-

opterus. The strongly sclerotized specimens endure the

extraction process well and the morphological charac-

ters can be studied even more easily after an extraction

than before.

2 cox1 data helped to sort very similar specimens rapidly

into well-defined entities or clusters which we, based

on additional morphological evidence, suggest to rep-

resent species. Our starting hypothesis based on exter-

nal morphological characters suggested the presence

of 40 species, which means that despite expert taxo-

nomic sorting, 11 species or 22% had been over-

looked. There was an especially severe

underestimation of diversity among the numerous

similar foliage-frequenting species (29%). It can be

stated with confidence that the percentage of over-

looked species would have been much higher if the

process had been performed by a nonexpert in weevil

taxonomy (see Krell 2004). This error rate is higher

than relying merely on cox1 in conjunction with com-

putation of statistical networks; it is also higher than

the results of clustering with a preset threshold

between 2% and 15%.

3 The preparation of genitalia is critical for a reliable

species assignment based on morphology, but it is a

time-consuming task. If this is carried out with a qual-

ity as that required for subsequent taxonomic work, c.

15 specimens can be dissected and processed per day.

The speed of DNA barcoding depends much on the

equipment and the workflow, but under average condi-

tions, the latter should be about two to three times fas-

ter. Moreover, it is easier to automatize. However, it

must be noted that once the fauna of a given area is

well-known and documented, it may again be possible

to identify a great proportion of specimens faster using

traditional techniques, for example, by combining mor-

phological data with ecological data (e.g., the elevation

of a given sample ⁄ food-plants, etc.). DNA barcoding

could then be restricted to the ‘doubtful’ specimens

that are hard or impossible to identify by morphologi-

cal means. This has proved to be an effective strategy

before, especially under sympatric conditions (Petersen

et al. 2007).

4 Five species (sp. 17, 20, 26, 30, 34; Figs S1–S51) were

represented by females only. Morphological differences

between males and females are frequently bigger than

that between specimens of the same sex of closely

related species (Fig. 4, species 10), and the morpholog-

ical characters critical for species identification are

found only in males (Fig. 5). Therefore, it is difficult

to decide about the species-status of these specimens

based on morphological data alone.

5 Based on the molecular data, intraspecific morpho-

logical variation in the sexes could be clearly sepa-

rated from specific differences, e.g. in species 18

(Fig. 4).

6 Species were clearly delineated in this study. Molecular

and morphological data were perfectly congruent. This

means that the same (or a very similar) estimation of

species diversity based on inspection of tree topology

and divergence between clusters would have been made

based on molecular data alone. This may change to

some degree when material from allopatric populations

is included. However, having sampled some variety of

habitats over multiple sampling points and significant

altitudinal gradient, we do not find incongruence of

clusters because of introgression and ⁄ or lineage sorting

phenomena, as seen in other, similarly recent Melane-

sian radiations (Monaghan et al. 2006). Paraphyly of

one species appeared in a revision of Malagasy ants

based on both morphological and molecular cox1 data

(Fisher & Smith 2008).

From this study, DNA sequencing clearly emerges as an

efficient tool for the rapid and sustainable surveying of an

individual area. With this technique, the number of spe-

cies in samples from a given study site could be easily

determined, even by a nonexpert. Most importantly, data

thus gained can readily be compared with data from any

other area in the world because DNA sequence data are



Fig. 4 Maximum likelihood tree of 216 specimens of the genus Trigonopterus plus 3 outgroup representatives from the Cyclops

Mountains. Clusters correlate with the assignment of 51 morphospecies given to the right.



universal, unlike morphospecies descriptions which leave

room between different workers (Krell 2004).

DNA barcoding showed its potential to speed up taxo-

nomic work of a taxonomically demanding group. Based

on our previous experience with weevil taxonomy (Riedel

2001, 2002, 2006), we suggest that the final species count

could have also been achieved with morphological data

alone, but it would have been more time-consuming. This

resorts especially to the classification of unique specimens.

Additional field work would have been necessary to obtain

enough specimens for a decision of their status. Often this

is impossible, especially in the tropics. The same applies

to intraspecific variation. If long series are available, pref-

erably in combination with data on the ecology of the spe-

cies, then a decision upon the status of questionable

specimens can be made equally well with traditional meth-

ods. But the molecular approach proves to be an elegant

shortcut.

In future studies, we will include the Trigonopterus of

neighbouring localities, such as the Bewani, Torricelli or

Foja Mountains. This could lead to the discovery of closely

related sister species. Despite their superficial similarity, it

is possible that the species of the Cyclops Mountains are

phylogenetically rather distant – a suspicion nourished by

their long branches. Possibly, more closely related sister

species might occur allopatrically and they would break

down the long species branches. Determining this will be

the acid test for a general usage of DNA barcoding as a

general identification tool, even among allopatric popula-

tions. In the light of this study, one should be prepared for

a large number of species involved in this region.

More fieldwork in the Cyclops Mountains would be

rewarding, too. So far, the examined material was obtained

only from one transect on the Southern slopes. It would

be interesting to check if a transect on the Northern

slopes shows a different species composition. Relatively lit-

tle time was spent in the higher elevations above 1400 m.

The area between 1520 m and the peak was not sampled

at all. However, a high number of species were exclusively

found in the montane forests above 1000 m, such as species

4, 5, 6, 16, 17, 20, 24, 29, 30, 38, 39, 40, 44. If information

on endemic species is an argument for conserving habitats

of a given area, then Trigonopterus should be a taxon that

gives strong support for protection of the Cyclops.

AcknowledgementsWe thank J. Astrin (Bonn) for providing us Cryptorhyn-

chinae-specific primer sequences. Rene Tanzler (Munchen)

helped with some of the laboratory work.

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60

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02 3 4 5 6 7 8 9 10

(%)11 12 13 14 15 16

Fig. 5 Clustering the cox1 data at different

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Supporting InformationAdditional Supporting Information may be found in the

online version of this article:

Figs S1–S27 Habitus of different foliage-frequenting

Trigonopterus species from the Cyclops Mountains in

defense position. Illustration number equals number of

species as used throughout the text. Scale line 1 mm.

Figs S28–S51 Habitus of different Trigonopterus species

from the Cyclops Mountains in defense position. Illustra-

tion number equals number of species as used throughout

the text. (28–31) Foliage-frequenting species; (32–51)

edaphic species. Scale line 1 mm.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.



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