Association of insect life stages using DNA sequences: the larvae of Philodytes umbrinus (Motschulsky) (Coleoptera: Dytiscidae)
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Systematic Entomology (2005), 30, 499–509
Association of insect life stages using DNA sequences:
the larvae of Philodytes umbrinus (Motschulsky)
(Coleoptera: Dytiscidae)
K E L L Y B . M I L L E R 1 , Y V E S A L A R I E 2 , G . W I L L I A M W O L F E 3 and
MICHAEL F. WHITING1
1
Department of Integrative Biology, Brigham Young University Provo, Utah, U.S.A.,
2
Department of Biology, Laurentian University, Sudbury, Ontario, Canada and
3
Department of Biological and Environmental Sciences, Georgia College and State University, Milledgeville, Georgia, U.S.A.
Abstract. Insect life stages are known imperfectly in many cases, and classifica-
tions are based often on only one or a few semaphoronts of a species. This is
unfortunate as information in alternative life stages often is useful for scientific
study. Although recent examples of DNA in taxonomy have emphasized the
identification of indistinguishable species, such sequence data facilitate the associ-
ation of life history stages and hold considerable promise in phylogenetic analysis,
evolutionary studies, diagnostics, etc. These concepts are discussed here and an
example is provided from diving beetles (Dytiscidae: Coleoptera). Three unknown
larval specimens of an apparent species of Laccophilinae collected in Namibia were
associated with the species Philodytes umbrinus (Motschulsky) using DNA sequence
data. An 806-bp portion of the gene cytochrome oxidase I was sequenced from the
unknown larvae. Several identified adult specimens of species of Laccophilinae
from Namibia were also sequenced, including two P. umbrinus specimens and
specimens from four Laccophilus Leach species. Additional species of Laccophilus
from other areas of the world also were sequenced, as were specimens of Agabetes
acuductus (Harris), Australphilus saltus Watts, Neptosternus boukali Hendrich &
Balke and a species of Laccodytes Régimbart. Parsimony analysis resulted in two
most parsimonious trees with the unknown larva unambiguously resolved in a
group with both adult specimens of P. umbrinus (bootstrap value ¼ 100%). The
average pairwise p-distance between the unknown larva and adult P. umbrinus
specimens averaged 0.09% (0–0.14%), compared with an average divergence
between other conspecifics in the analysis of 0.24% (0–0.82%) and an overall
average divergence between species of 13.49% (1.90–19.86%). Based on this, the
unknown larvae were assigned to P. umbrinus. The larvae are diagnosed and
described and their relationship with other Laccophilinae is discussed.
Introduction stage. An incomplete knowledge of the life stages of a
species renders unavailable a potential wealth of character
For many groups of insects, classifications are based largely and natural history information that may be of particular
or entirely on a single life stage or even a single sex of a life interest for ecological and evolutionary studies, phyloge-
netic analysis and diagnostics. The rate of acquisition of
this knowledge is hindered by the challenges involved in
making associations between life stages. For example, dif-
Correspondence: Kelly B. Miller, Department of Integrative ferent sexes of a particular species may be rarely collected
Biology, Brigham Young University Provo, Utah 84602, U.S.A. together, or larvae of a certain species may be difficult to
E-mail: kelly.miller@byu.edu rear for various reasons.
# 2005 The Royal Entomological Society 499500 K. B. Miller et al.
DNA taxonomy (the use of a particular sequence of possible and providing the larval and pupal exuviae for
DNA to diagnose or delimit species) is a current contro- taxonomic use. Larvae can be reared from eggs oviposited
versy (e.g. Hebert et al., 2002; Lipscomb et al., 2003; Tautz in the laboratory, giving advantages of positive identifica-
et al., 2003; Moritz & Cicero, 2004; Will & Rubinoff, 2004). tion of species through association with the ovipositing
Regardless of differing theoretical or practical views adult, preservation of intact specimens rather than just
regarding the larger ‘barcoding’ programme, a portion of exuviae, and acquisition of multiple larval instars.
variable DNA sequence can aid diagnosis minimally. However, rearing of larvae is labour intensive in these
Circumstances might include the identification of a limited beetles (Alarie et al., 1989) and difficult to attain in the
set of well-known taxa (such as groups of economically or remote field.
medically important taxa), or groups in which traditional The recent discovery of adults and putative larvae of
diagnostic techniques are exceptionally difficult. Current Philodytes umbrinus (Motschulsky) in the Skeleton Coast
examples have focused primarily on the identification of National Park, Namibia, provided the impetus for this
species when specimens are especially similar or indistin- study. Philodytes Balfour-Browne is a diving beetle genus
guishable using traditional types of data. For example, in the tribe Laccophilini (Laccophilinae) comprising a single
DeSalle & Birstein (1996) used polymerase chain reaction species, P. umbrinus. The species is relatively widespread in
(PCR) assays to distinguish between otherwise indistinguish- Africa and portions of the Middle East, but is not especially
able eggs of several species of sturgeon (Acipenseridae). well known. Therefore, photographs of the habitus and
Paquin & Hedin (2004) used DNA sequence data to distin- aedeagus of a male specimen from the Oasis Spring locality
guish between previously unidentifiable immature stages of (see Table 1) are provided (Fig. 1). The adult is pale greenish
species of Cicuria Menge (Araneae: Dictynidae). Miller et al. when alive, but fades to a translucent tan colour when dried
(1997, 1998, 1999) developed strategies for the identification and pinned. This species superficially resembles a large
of larvae of canegrubs (Coleoptera: Scarabaeidae) using tra- Laccophilus Leach, and the genus was described originally
ditional techniques for those species that were amenable to as a subgenus of Laccophilus before being elevated by
these data, and DNA sequence data for those that were Balfour-Browne (1939). Adult Philodytes share with other
indistinguishable. Other examples of the diagnostic uses of Laccophilini genera: (1) a concealed scutellum; (2) the pre-
DNA sequence data include the identification of indistin- sence of a single metatarsal claw; (3) metatarsomeres with
guishable immature forms of trematodes (Digenea; Jousson posterolateral lobes; and (4) female gonocoxae fused medially
et al., 1998, 1999), tapeworms (Cestoda; Dezfuli et al., 2002) into a knifelike structure and the rami together fused and
and redhorse suckers (Myxostoma, Catostomidae; Wirgin serrate ventrally. Diagnostic generic adult characters include:
et al., 2004). (1) two metatibial spurs which are apically simple; (2) the
In some cases, however, DNA sequence data may have prosternal process apically simple, narrow apically and
utility beyond the simple identification of difficult speci- weakly carinate; (3) the base of the pronotum distinctly
mens. The association of life stages could make available angulate; and (4) the pro- and mesofemora and tibia not
valuable character and natural history information that densely punctate. Many of these characters are probably
would be lacking otherwise. For example, sequence data plesiomorphies.
may allow the confident association of males and females, The larvae of four genera of Laccophilini, Africophilus
castes in social species, or larva and adults. Variable DNA Guignot, Australphilus Watts, Laccophilus Leach and
sequence characters are potentially useful as they overlap Neptosternus Sharp, have been described and their charac-
between semaphoronts, whereas other characters (such as ters examined in a cladistic analysis (Alarie et al., 2000). In
morphology and behaviour) may not. Although sometimes addition, the larva of Agabetes acuductus (Harris), a mem-
indirectly suggested as a potential strength of the use of ber of Agabetini (Laccophilinae or Agabetinae, depending
DNA sequence data in taxonomy (Barrett & Hebert, 2005; on the classification used), has been described and its rela-
Kress et al., 2005), in entomology, such associations have tionship as sister to Laccophilini established based on larval
been few. characters (Alarie et al., 2002), supporting similar conclu-
Diving beetle (Dytiscidae) larvae provide a rich assort- sions made earlier based on adult characters (Burmeister,
ment of character information useful for phylogenetics, 1990; Miller, 2001). Here, we describe for the first time the
evolutionary studies and diagnostics. They are encountered larvae of the genus Philodytes. As the larvae collected could
frequently and are important predators in fresh water. not be separated safely from those of adults of local
Most species of dytiscids, however, are unknown as larvae, Laccophilus species, a molecular association is reported.
with even the well-studied Nearctic fauna known for only
about 20% of the species (Larson et al., 2000). The associa-
tion of diving beetle larvae with adults is challenging as Materials and methods
larvae typically are short lived and seasonal, making them
more rarely collected than the longer lived adults. Several Specimens
similar species often co-occur, making association through
common occurrence unreliable. Larvae can be reared Adults of P. umbrinus were collected from two localities
through pupae to adults in the laboratory, making larval in the Skeleton Coast National Park; putative larvae of
identification and confident association with adults P. umbrinus were collected from one of these locations
# 2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509Matching insect live stages via DNA barcoding 501
Table 1. Species and specimens used in the analysis, collection locality information and GenBank accession numbers for cytochrome oxidase
I (COI) sequences.
Specimen Collection locality GenBank accession
Agabetes acuductus U.S.A.: New York: St. Lawrence Co., Macomb TwP. Fish Cr. DQ112634
marsh, 44 280 2000 N 075 330 4800 W, 23.v. 2000 (Miller)
Australphilus saltus Watts Australia: Victoria, Brodribb R. at Sardine Cr. N Orbost, DQ112635
37 300 5100 S 148 320 3700 E, 22 November 2000, KB Miller, leg.
Laccodytes sp. Peru: Rio Tambopata, Explorers Inn, jnct Rio Tower, 12 50.2080 S DQ112651
069 17.6030 W, 10.xii. 2003 (Miller)
Laccophilus adspersus Boheman Namibia: Skeleton Coast NP, spring, mouth of Khumib R, DQ112649
18 52.6600 S 012 25.5390 E, 13.v. 2004 (Miller & Wolfe)
L. adspersus [2] Namibia: Etosha NP, Devilwater Spr., 18 59.2660 S 015 15.5930 E, DQ112636
18.v. 2004 (Miller & Wolfe)
L. adspersus [3] Same as above DQ112637
L. congener Omer-Cooper Namibia: Skeleton Coast NP, spring, mouth of Khumib R, DQ112640
18 52.6600 S 012 25.5390 E, 13.v. 2004 (Miller & Wolfe)
L. congener [2] Namibia: Etosha NP, Salvida Spring, 19 02.0930 S 016 16.1550 E, DQ112638
23.v. 2004 (Miller & Wolfe)
L. congener[3] Same as above DQ112639
L. continentalis Gschwendtner Namibia: Etosha NP, Devilwater Spr., 18 59.2660 S 015 15.5930 E, DQ112641
18.v. 2004 (Miller & Wolfe)
L. continentalis [2] Same as above. DQ112642
L. horni van den Branden U.S.A.: Arizona: Cochise Co., Bear Cr., Huachuca Mts, DQ112643
31 22.7960 N 110 21.8140 W, 9.v. 2003 (Miller)
L. horni [2] Same as above DQ112644
L. lineatus Aubé Namibia: Skeleton Coast NP, Oasis Spring, DQ112648
19 26.7460 S 012 49.3010 E, 14.v. 2004 (Miller & Wolfe)
L. lineatus [2] Same as above DQ112650
L. maculosus Say U.S.A., New York: Oswego Co. Boylstown Township, DQ112645
marsh nr Boylstown Center, 18.viii. 2000 (Miller)
L. maculosus [2] Same as above DQ112646
L. maculosus [3] U.S.A.: New York: Tompkins Co., Ithaca, 23.x. 2000 (Miller) DQ112647
Neptosternus boukali India: Karnataka, Agumbe Ghats, 13 29.8520 N 075 04.2210 E, DQ112652
Hendrich & Balke 09.x. 2004 (Miller)
Philodytes umbrinus (Motschulsky) Namibia: Skeleton Coast NP, spring, mouth of Khumib R, DQ112653
18 52.6600 S 012 25.5390 E, 13.v. 2004 (Miller & Wolfe)
P. umbrinus [2] Namibia: Skeleton Coast NP, Oasis Spring, DQ112654
19 26.7460 S 012 49.3010 E, 14.v. 2004 (Miller & Wolfe)
P. umbrinus (larva) Same as above DQ112655
Laccophilus poecilis GenBank AY334246
(see Table 1). These larvae appeared typical of (Table 1) to root the resulting cladogram. Two or more speci-
Laccophilinae, but were larger than most Laccophilus lar- mens of several species were sequenced in order to examine
vae (including Laccophilus larvae collected at the same intraspecific variation in the DNA.
locality), just as adult Philodytes are larger than most
adult Laccophilus in this region. Only two genera of
Laccophilinae are expected to occur here. DNA sequences
To associate larvae and adults of P. umbrinus confidently,
several species of Laccophilus collected in Namibia were DNAs were extracted using a Qiagen DNEasy Kit
sequenced for the same portion of the mitochondrial gene (Valencia, California, U.S.A.) and the protocol for animal
cytochrome oxidase I (COI) (Table 1). Species from other tissue. Adult males which could be identified confidently
Laccophilus groups were included from elsewhere in the were extracted in two ways. A slit was made along the side
world (Table 1), including a single Laccophilus sequence from of the thorax of large specimens allowing removal of thor-
GenBank (L. poecilis Klug, Table 1). Although other laccophi- acic muscle tissue into extraction buffer with the remaining
line genera are unlikely to occur in this area of Africa, speci- portions of specimens retained as vouchers. For small
mens of Australphilus saltus Watts from Victoria, Australia, specimens, the abdomen was removed, and all remaining
Laccodytes sp. from Madre de Dios, Peru and Neptosternus portions were placed in extraction buffer. After incubation,
boukali Hendrich & Balke from Karnataka, India were also the material was retrieved from the buffer and vouchered in
sequenced (Table 1). Agabetes acuductus was included the collection of KBM and the Brigham Young University
# 2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509502 K. B. Miller et al.
(A) (B) (C) based on conservation of the codon reading frame and
amino acid sequence. The length difference was regarded
as unproblematic as the length-variable region was unin-
formative. Sequences were aligned and trimmed to length
using Sequencher, resulting in 812 characters. Data were
analysed using parsimony in the program NONA (Goloboff,
1995), as implemented using the ‘heuristics’ menu option in
the program WinClada (Nixon, 1999–2002) and the follow-
ing settings: hold 5000 trees total (‘h 5000’), forty replica-
tions (‘mu*40’), forty trees held per replication (‘h/40’) and
(D) multiple TBR þ TBR (‘max*’) swapping. Trees were exam-
ined and analysed under different optimizations using
WinClada. Bootstrap values were calculated using
WinClada and the options ‘1000 replicates’, ‘10 search
reps’, ‘1 starting tree per rep’ and ‘Don’t do max*.’
Characters were weighted equally and gaps were treated as
missing data. Pairwise numbers of nucleotide differences
were calculated with the program MEGA 2.1 (Kumar et al.,
2001) using the ‘Calculate distances’ option and ‘Nucleotide:
p-distance’ model option for distances. The p-distance, the
Fig. 1. Philodytes umbrinus. A, Dorsal habitus; B–D, aedeagus,
proportion of sites in which two sequences differ, was calcu-
left lateral aspect (B), ventral aspect (C), right oblique aspect (D). lated by dividing the number of nucleotide differences by the
total number of nucleotides (Kumar et al., 2001).
(BYU) Frozen Insect Tissue Collection (MFW). A sus-
pected Philodytes larva was slit along the side of the thorax,
placed in entirety in buffer and retrieved after incubation
Larval analysis
for vouchering. This extracted larva was cleared, slide
mounted and vouchered in KBM.
Instar III specimens were disarticulated and mounted on
An 806-base pair portion of COI was amplified and
standard glass slides with Hoyer’s medium ringed with clear
sequenced (Ribera et al., 2001, 2003a, b, 2004; Balke
nail polish. Examination at magnifications of 80–800 was
et al., 2004). The primer pair C1-J-2183 (‘Jerry’) and
performed using an Olympus BX50 compound microscope
TL2-N-3014 (‘Pat’) (Simon et al., 1994) was used to amplify
(Olympus, Melville, NY, U.S.A.) equipped with Nomarsky
this region, employing PCR on a DNA Engine DYAD
differential interference optics.
Peltier Thermal Cycler (MJ Research, Bio-Rad Labs.,
Inc., Hurcules, CA, U.S.A.). Amplification conditions
Morphometric analysis. The part to be measured was
were 95 C (12 min) for one cycle, 94 C (1 min),
adjusted so that it was, as nearly as possible, parallel to
48–52 C (1 min), 72 C (1 min) for forty cycles, and 72 C
the plane of the objectives. The characters and terms used
(7 min) for one cycle. Contamination was mediated using
in the morphometric analysis are mainly those employed in
negative controls. Fragments produced from PCR were
recent studies of larval Laccophilinae (Alarie et al., 2000,
examined using gel electrophoresis. Products were purified
2002). This system recognizes individual setae and primary
using a Montage PCR96 Cleanup Kit (Millipore, Billerica,
and secondary setae, which is a distinct advantage over
MA, U.S.A.) and cycle sequenced using ABI Prism Big Dye
previous systems of setal nomenclature, such as that of
(version 3, Applied Biosystems, Foster City, CA, U.S.A.),
Wolfe & Roughley (1985). However, to ensure correct
using the same primers as employed for amplification.
interpretations of some terms, the following notes of
Sequencing reaction products were purified using
explanation are provided.
Sephadex G-50 Medium (Sigma-Aldrich, St. Louis, MO,
Head length (HL): total head length including the fronto-
U.S.A.) and sequenced using an ABI 3730xl DNA analyser
clypeus measured medially along the epicranial stem.
(DNA Sequencing Center, BYU). The gene region was
Head width (HW): maximum width measured posterior to
sequenced in both directions and data examined and edited
the stemmata.
using Sequencher (Genecodes, 1999).
Length of frontoclypeus (FCL): measured from apex of the
nasal to the back of the ecdysial suture.
Occipital foramen width (OcW): maximum width measured
DNA sequence analysis along the dorsal margin of the occipital foramen.
Length of antenna: derived by adding the length of each indi-
The alignment of sequences was relatively unambiguous vidual antennomere; antennomeres are indicated by the capital
as most taxa were length invariable. However, one taxon letter A and a number corresponding to the antennomere con-
(Laccodytes sp.) had a six-base-pair indel which was aligned sidered; A30 is the lateral elongation of antennomere III.
# 2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509Matching insect live stages via DNA barcoding 503
Length of maxillary and labial palpus: derived by adding The number of secondary setae present on the anteroven-
the length of each individual palpomere. tral (AV) margin of the femur includes additional setae which
Length of legs: derived by adding the length of each individ- cannot be numbered because of a lack of instar I specimens.
ual segment including the longest claw; the length of each
segment was taken at the longest point, except for the tro-
chanter, which includes only the proximal portion (the Results
length of the distal portion is included in the femoral length).
Dorsal length of last abdominal segment (LLAS): includes Two most parsimonious trees of length 738 (CI ¼ 53,
the whole sclerite measured from the anterior margin of the RI ¼ 73) were obtained, one of which is shown in Fig. 2.
prescutum to the apex of the siphon. Siphon refers to the The only differences concerned the group containing
dorsal prolongation of the abdominal segment VIII (¼ last the three specimens of L. maculosus, which collapsed to a
abdominal segment); the length of the siphon was deter- tritomy in the consensus.
mined by calculating the difference between the dorsal and The unknown larva grouped unambiguously with the
ventral lengths of the segment. adult specimens of P. umbrinus. The average distance
Length of urogomphus: derived by adding the length of between these three specimens (p-distance) was 0.09%
each individual urogomphomere; urogomphomeres are (range, 0–0.14%) (Table 2). Divergences between conspeci-
indicated by Uro (e.g. Uro1 for urogomphomere I). The fics in this analysis were small, averaging 0.24% (range,
length of urogomphomere II is not included in the descrip- 0–0.82%) (Table 2). Divergences between all species aver-
tion because it was broken in all examined specimens. aged 13.49% (range, 1.90–19.86%) (Table 2), with those
Length of maxillary galea: maximum length measured from between species of Laccophilus averaging 12.0% (range,
the apex of the galea to the margin of the maxillary stipes. 1.9–15.5%).
Length of palpifer: maximum length measured along lateral
margin.
Pectens: refers to comblike spinula [¼ ‘e´cailles pectine´es’ of Description of larvae of Philodytes Balfour-Browne
Bertrand (1928) ] observed on the posterodorsal margin of (Figs 4, 5)
coxa and anteroventral posteroventral margin of femur and
tibiae, respectively. Diagnostic combination. Instar III of Philodytes can be
The individual measurements defined above were used to distinguished from those known larvae of other genera of
calculate several ratios that help to characterize the body Laccophilinae by the following combination of features:
shape, as described elsewhere (Alarie et al., 2000, 2002). head capsule distinctly constricted posteriorly, with
temporal spines (Fig. 3); lamellae clypeales spatulate
Chaetotaxic analysis. Primary and secondary setae and (Fig. 3); spinulose epipharyngeal band present; antenna
pores were identified on the cephalic capsule, head > 0.60 HW; antennomere I elongate, subequal in length
appendages, legs, last abdominal segment and urogomphi to antennomere II; lateral elongation of antennomere III
according to the systems proposed for the Laccophilinae (A30 ) 0.70 length of antennomere IV; antennomere I
(Alarie et al., 2000). without secondary setae; primary pore ANf present;
Ag. acuductus
100 P. umbrinus (larva)
P. umbrinus
P. umbrinus [2]
Au. saltus
Neptosternus sp.
Laccodytes sp.
100 L. horni
L. horni [2]
100 L. maculosus [2]
L. maculosus
L. maculosus [3]
100 L. adspersus [2]
Fig. 2. One of two most parsimonious cla-
L. adspersus [3]
dograms derived from parsimony analysis of L. adspersus
cytochrome oxidase I (COI) sequences from L. poecilis
Laccophilinae specimens. Branch lengths are 100 L. lineatus
proportional to the number of nucleotide L. lineatus [2]
changes mapped using ‘fast’ optimization in 95 L. continentalis
WinClada. Numbers above the branches are 100 L. continentalis [2]
bootstrap values (percentage). Branches 98 L. congener
without numbers have bootstrap values of L. congener [2]
less than 50%. L. congener [3]
# 2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509504 K. B. Miller et al.
Table 2. Pairwise distances between taxa in analysis expressed as a percentage of nucleotide differences (p-distances). Values of 0–1% indicated in bold.
A B C D E F G H I J K L M N O P Q R S T U V
Au. saltus 12.93
L. adspersus 11.84 11.97
L. adspersus [2] 11.97 11.84 0.41
L. adspersus [3] 11.84 11.97 0.14 0.54
L. congener 12.93 13.61 10.88 10.88 10.88
L. congener [2] 12.93 13.61 10.88 10.88 10.88 0.00
L. congener [3] 12.93 13.61 10.88 10.88 10.88 0.00 0.00
L. continentalis 13.06 13.61 11.97 11.97 11.97 1.90 1.90 1.90
L. continentalis [2] 13.61 13.47 11.43 11.70 11.43 1.90 1.90 1.90 0.54
#
L. horni 14.83 11.70 14.29 14.69 14.29 14.97 14.97 14.97 15.51 15.24
L. horni [2] 14.69 11.56 14.42 14.83 14.42 14.83 14.83 14.83 15.37 15.10 0.14
L. lineatus 16.05 14.42 12.93 12.79 12.93 12.11 12.11 12.11 11.84 11.56 14.56 14.69
L. lineatus [2] 16.05 14.42 12.93 12.79 12.93 12.11 12.11 12.11 11.84 11.56 14.56 14.69 0.00
L. maculosus 12.93 11.16 12.24 12.11 12.24 12.52 12.52 12.52 12.79 12.79 12.24 12.38 14.01 14.01
L. maculosus [2] 12.93 11.29 12.24 12.11 12.24 12.65 12.65 12.65 12.93 12.93 12.38 12.52 14.15 14.15 0.82
L. maculosus [3] 12.79 11.29 12.11 11.97 12.11 12.52 12.52 12.52 12.79 12.79 12.11 12.24 13.88 13.88 0.41 0.41
L. poecilis 18.78 18.37 18.23 18.64 18.23 19.18 19.18 19.18 19.46 19.18 17.82 17.96 19.86 19.86 16.73 17.01 16.73
Laccodytes sp. 12.93 13.74 9.12 9.12 9.12 9.39 9.39 9.39 10.20 9.93 14.29 14.42 12.11 12.11 11.84 11.97 11.84 17.69
Ne. boukali 16.33 13.74 14.56 14.69 14.56 14.29 14.29 14.29 14.97 14.69 15.37 15.24 16.60 16.60 14.29 14.69 14.29 13.47 19.05
P. umbrinus 14.42 13.61 12.52 12.93 12.52 14.69 14.69 14.69 14.69 14.83 15.24 15.10 14.15 14.15 13.88 14.56 14.15 12.38 19.46 16.05
P. umbrinus [2] 14.42 13.74 12.52 12.93 12.52 14.69 14.69 14.69 14.69 14.83 15.37 15.24 14.29 14.29 14.01 14.69 14.29 12.38 19.59 16.19 0.14
P. umbrinus (larva) 14.42 13.61 12.52 12.93 12.52 14.69 14.69 14.69 14.69 14.83 15.24 15.10 14.15 14.15 13.88 14.56 14.15 12.38 19.46 16.05 0.00 0.14
A, Ag. acuductus; B, Au. saltus; C, L. adspersus; D, L. adspersus [2]; E, L. adspersus [3]; F, L. congener; G, L. congener [2]; H, L. congener [3]; I, L. continentalis; J, L. continentalis [2]; K,
L. horni; L, L. horni [2]; M, L. lineatus; N, L. lineatus [2]; O, L. maculosus; P, L. maculosus [2]; Q, L. maculosus [3]; R, L. poecilis; S, Laccodytes sp.; T, Neptosternus sp.; U, P. umbrinus; V,
P. umbrinus [2].
2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509Matching insect live stages via DNA barcoding 505
Fig. 4. Philodytes umbrinus. Abdominal segment VIII and proxi-
mal portion of urogomphus, dorsal aspect. Scale ¼ 1 mm.
Fig. 3. Philodytes umbrinus. Instar III, head capsule, dorsal aspect.
Scale ¼ 1 mm.
(A)
maxillary palpus > 2.30 length of labial palpus; palpifer
< 0.30 length of maxillary palpomere I; galea about CS
0.30 length of maxillary palpomere I; maxillary
A
palpomere I without secondary setae; labial palpomere II CS AD
slightly shorter than palpomere I; length of mandible V
> 2.60 width, slightly dentate along medial margin; AV
pronotum without neck constriction; legs elongate,
metathoracic legs about 3.20 HW; natatory setae
present on dorsal margin of tibia and tarsus (Fig. 5B);
metatarsus elongate, subequal in length to metacoxa and
metafemur; pectens present (Fig. 5); abdominal segments PR NS
IV–V membranous ventrally; last abdominal segment (B)
slightly constricted at point of insertion of urogomphi
(Fig. 4); siphon broad with a crescentic setal pattern
NS
comprising numerous secondary spines (Fig. 5); urogomphus
elongate, about 2 LLAS, with secondary setae (Fig. 5);
urogomphomere I with subbasal suture (Fig. 5). D
CS
Description of Philodytes umbrinus (Motschulsky), instar
III (n ¼ 2)
Head. (Fig. 3). HL ¼ 1.66–1.77 mm (mean ¼1.71 mm); Fig. 5. Philodytes umbrinus. A–B, prothoracic leg, instar III, ante-
HW ¼ 1.38–1.45 mm (mean ¼ 1.41 mm); FCL ¼ 0.79– rior surface (A), posterior surface (B). Sensillar series:
0.82 mm (mean ¼ 0.81 mm). Cephalic capsule (Fig. 3) A ¼ anterior; AV ¼ anteroventral; AD ¼ anterodorsal;
subquadrate, longer than broad (HL/HW ¼ 1.16–1.29), CS ¼ comblike spinulae or pectens; D ¼ dorsal; NS ¼ natatory
distinctly constricted at level of occipital region, HW/ setae; PR ¼ proximal; V ¼ ventral. Scale ¼ 0.5 mm.
# 2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509506 K. B. Miller et al.
OcW ¼ 1.53–1.80; ecdysial suture well developed; occipital Abdomen (Fig. 4). LLAS ¼ 1.29–1.32 mm (mean ¼
suture absent; frontoclypeus slightly convex mesally, 0.46– 1.30 mm). Eight-segmented, dorsally sclerotized; segments
0.47 HL, extending to about level of lateral lobes I–VI membranous ventrally, segments VII–VIII completely
(¼ adnasalia); apical margin of frontoclypeus with about 40 sclerotized; terga I–VII with an anterodorsal transverse
spatulate setae [‘lamellae clypeales’ of Bertrand (1972)]; carina; segments I–VII each with a pair of spiracular
spinulose epipharyngeal band [‘area o banda spinulosa del openings; segment VIII shorter than HW, LLAS/
palato’ of de Marzo (1979)] present; gular suture not visible; HW ¼ 0.73–0.79, slightly constricted posterior to
ocularium present, stemmata visible ventrally and subdivided insertion of urogomphi. Siphon. Short, 0.32–0.33 LLAS,
into two vertical series; tentorial pits visible ventrally on each broad apically. Chaetotaxy and porotaxy. Secondary tergal
side of middle at about midlength; occipital foramen indented setation present; segment VIII with a dorso-apical tuft of
ventrally. Antenna. Four-segmented, shorter than HW (length secondary spines.
of antenna/HW ¼ 0.72–0.75); A4 < A1 ¼ A2 ¼ A3, A2/
A3 ¼ 0.94–0.97; lateral elongation of antennomere III long, Urogomphus (Fig. 4). Two-segmented (urogomphomere
A30 /A4 ¼ 0.66–0.70; antennomere III without a II broken). Length of urogomphomere I ¼ 2.54 mm
ventroapical spinula. Mandible. Falciform, length 2.89– (n ¼ 1), 1.95 LLAS; 1.80 HW. Chaetotaxy and
2.91 width, 0.43–0.46 HL; mandibular channel present, porotaxy. Urogomphomere I with several spinelike or
medial margin slightly dentate dorsally, pubescent hairlike secondary setae along lateral and medial margins,
ventroapically. Maxilla. Stipes trapezoidal; cardo and galea respectively.
present, lacinia absent; galea short, 0.33 length of
palpomere I; palpifer similar to palpomeres, 0.26–0.29 Distribution and biology. Widespread in Africa and
length of palpomere I; palpus 3-segmented, slightly shorter portions of the Middle East. In the Skeleton Coast of
than antenna (length of antenna/length of maxillary Namibia, P. umbrinus were collected from two sites.
palpus ¼ 1.18–1.21); palpomere I ¼ II > III; length of Larvae and adults were collected at Oasis Spring, an
palpomere III/length of palpomere II ¼ 0.77–0.81. Labium. extremely large spring habitat on the edge of a large dune
Prementum subrectangular, broader than long, without field. The specimens were collected in a small side pond that
spinulae; palpus 2-segmented, much shorter than maxillary was shallow with a largely mineral (sand) substrate and
palpus (length of maxillary palpus/length of labial minimal vegetation. Of fourteen specimens collected (by
palpus ¼ 2.64–2.75), palpomere II 0.83–0.89 length of GWW), seven were teneral. Furthermore, one of us
palpomere I. Chaetotaxy and porotaxy. Head capsule with (GWW) collected seven specimens from Botswana
several secondary setae; parietale with 7–8 elongate lateral (Okavanga, Delta) on 20.v. 2001, one of which was
spines; head appendages without secondary setae except for teneral. Adults were collected also at a spring at the
one lateroproximal seta on mandible. mouth of the Khumib River. This is a large, deep pool
with extensive vegetation at its upper end. This pool is
Thorax. Pronotum elliptical to subtrapezoidal dorsally; separated from the ocean by a large dune and may
length of pronotum about 2 length of mesonotum; occasionally be inundated by sea water.
metanotum length subequal to mesonotum length, both
slightly broader than width of posterior margin of Identification. The suspected larvae of P. umbrinus
pronotum; all terga with posterotransverse carina; grouped unambiguously with adult P. umbrinus using
mesopleural region with spiracular opening on each side; parsimony analysis of a portion of COI sequence (Fig. 2).
secondary setation present on each tergum; thoracic venter Nucleotide divergence between these specimens was less
membranous. than 0.14%, compared with an average of 0.24% for
other conspecifics and 13.49% between species. These
Legs (Fig. 5). Five-segmented; metathoracic legs longest, divergence patterns are similar to values obtained for
about 1.40 length of prothoracic legs, and 3.50 HW; meta- other insect taxa (e.g. Hebert, 2004) and for
[coxa ¼ femur ¼ tarsus > tibia > trochanter]; tarsus with mitochondrial gene divergences between animals in
two claws, posterior claw slightly shorter than anterior claw general (Avise, 2000). The distances between known adult
on meso- and metathoracic legs, subequal to slightly longer on specimens of P. umbrinus and the suspected larvae are well
metathoracic leg; anterior metathoracic claw 0.22–0.25 within the range of a number of nucleotide differences
metatarsus length; ventral marginal spinulae strongly between specimens of the same species in the analysis. In
developed on tibia and tarsus; posterodorsal, anteroventral addition, morphology shows that these are laccophilines
and posteroventral pectens present on coxa, femur and and the size of specimens is larger than expected for
tibia, respectively. Chaetotaxy and porotaxy. All primary Laccophilus occurring in this area of Africa. Combined,
setae and pores of generalized colymbetine present; seta the evidence indicates that the suspected larvae are, in
CO7 inserted proximally on all legs; seta TI4 inserted fact, P. umbrinus.
ventrally and proximally on tibiae; position and number
of secondary setae as indicated in the supplementary Taxonomic notes. The subfamily Laccophilinae
material; natatory setae present on dorsal margin of tibia comprises thirteen genera worldwide (Nilsson, 2001), six
and tarsus. of which are now known as larvae (Alarie et al., 2000,
# 2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509Matching insect live stages via DNA barcoding 507
2002; this paper). Based on the larval morphology of the considerably between taxa in structure and ecology.
Laccophilinae, Philodytes clearly is related to Neptosternus, However, it is uncommon to find a female laying eggs
Australphilus and Laccophilus, as these genera share the and it is difficult to rear the eggs through several nymphal
following apparent apomorphies: (1) the presence of instars to adults for identification and positive association
natatory setae on the legs (Fig. 5B); (2) the presence of of egg cases with particular species (Breland & Dobson,
elongated metatibiae and metatarsi (about twice as long 1947). DNA sequence data may aid in facilitating these and
as metafemora); (3) the proximal articulation of the similar associations.
primary seta CO7 present on all coxae; and (4) the We recognize some limitations of our procedure in that
presence of elongated urogomphi. we did not ascertain the entire extent of variation in COI in
Larvae of P. umbrinus morphologically resemble those of a given species, nor the extent of variation in this gene
Laccophilus, sharing the following unique character states between closely related species in a given group. Instead,
within the Laccophilinae: (1) the presence of an epipharyn- we focused only on likely candidate species from a small
geal band (homoplasious in Africophilus); (2) the absence of geographical region, and thus possibly failed to sequence
swimming hairs on the femur (Fig. 5B); (3) the presence of adults of the actual species to which our larvae belong. For
pectens on the legs (Fig. 5); (4) the presence of a tuft of example, it may belong to a species of Laccophilus not
secondary spines on the dorsal surface of the siphon included in our analysis, or perhaps to an undescribed
(Fig. 4); (5) the presence of secondary setae on urogompho- species of Philodytes of which we are unaware. However,
mere I; and (6) a proximal (¼ subbasal) suture on urogom- we believe that this is unlikely based on the small amount of
phomere I (Fig. 4). A close morphological similarity variation in COI between the specimens. Ours was a limited
between the larvae of these two genera is also indicated problem with few alternative solutions. We were unable to
by the number and position of secondary setae on the rear suspected larvae to associate with adults, and it is
legs (see supplementary material). Compared with other unlikely that anyone will ever carry out this procedure.
known larvae of Laccophilus, instar III of P. umbrinus DNA sequences promised additional information for
can be distinguished by: (1) a distinct neck constriction resolving our specific problem.
(Fig. 3); and (2) the relatively shorter labial palpomere II In contrast, the proposed broader programme of
(< 0.90 length of palpomere I compared to > 1.00 in ‘DNA barcoding’ of all species on Earth (e.g. Hebert
Laccophilus). et al., 2002, 2003) seeks to characterize all extant species
Philodytes was made synonymous with Laccophilus by (or at least animals) using a single, short fragment of
Balke et al. (1997), but this synonymy was not recognized DNA. Our goal was not to provide diagnostic features
by Nilsson (2001) without explanation. The evidence does included in COI for this species, but, rather, to use
seem to suggest a close relationship between Philodytes and sequence data to work backwards to find diagnostic
Laccophilus but, without a more detailed examination of features in the morphology. Notwithstanding interesting
other genera in the subfamily, we hesitate to place the questions about molecular evolution, morphological
morphologically unique Philodytes back into synonymy characters are the ultimate fodder for studying evolution.
with the diverse genus Laccophilus at this time [following Simple identification of these larvae as P. umbrinus using
Nilsson (2001)]. DNA sequences contains relatively little of biological
interest, but, by adding to the identification a description
of larval structure, we have provided both the basis for
Discussion future identifications of unknown larval specimens using
morphology and the opportunity to explore the unique
The ability to use DNA sequence data to associate the character combinations present in P. umbrinus larvae
morphologically extremely different larvae and adults of within a phylogenetic and evolutionary context. We
Dytiscidae has the potential to dramatically alter our rate echo previous critics of ‘DNA taxonomy’ or ‘DNA bar-
of acquisition of knowledge of this character-rich life stage. coding’ (e.g. Lipscomb et al., 2003; Sperling, 2003; Will
Given the successful association of these specimens, similar & Rubinoff, 2004) by saying that a DNA-based pro-
success may be expected in associating adults and larvae gramme by itself severely reduces the intellectual content
of other beetles and other holometabolans whose rearing of taxonomy, would likely fail for practical and theore-
is difficult or impossible. Such taxa include mayflies tical reasons, and should not replace systematics based
(Ephemeroptera) for which larvae are well known taxono- on data from whole organisms. Were such a programme
mically, but adults much less so (Edmunds & Waltz, 1996). to be implemented, it should, at a minimum, incorporate
Where species-level classification is based almost entirely a comprehensive assessment of variation in sequences
on male morphology, females often are unassociated amongst individuals and populations, attune carefully
with males and DNA sequence data will aid in making to taxonomic hypotheses of species based on whole
associations. In others, such as Formicidae, males are rare organisms, and emphasize diagnostics and not species
and are often not confidently associated with females delimitations based on sequence data (Moritz & Cicero,
(worker, soldiers and queen castes) on which most taxon- 2004). If implemented properly, such a diagnostic tool
omy is based (Bolton, 1994). Egg cases (such as oothecae will greatly facilitate associations of the type presented in
in Mantodea) often are found in the field and vary this paper.
# 2005 The Royal Entomological Society, Systematic Entomology, 30, 499–509508 K. B. Miller et al.
Supplementary material Bolton, B. (1994) Identification Guide to the Ant Genera of the
World. Harvard University Press, Cambridge, Massachusetts.
Supplementary data is available in the full text version of Breland, O.P. & Dobson, J.W. (1947) Specificity of mantid oothe-
this article from http://www.blackwell-synergy.com. cae (Orthoptera: Mantidae). Annals of the Entomological Society
of America, 40, 557–575.
Burmeister, E.G. (1990) The systematic position of the genus
Agabetes Crotch within Dytiscidae (Coleoptera: Adephaga).
Acknowledgements
Quaestiones Entomologicae, 26, 221–238.
DeSalle, R. & Birstein, V. (1996) PCR analysis of black caviar.
Special thanks to E. Marais of the National Museum of Nature, 381, 197–198.
Namibia for advice and permit arrangements. J. Patterson Dezfuli, B.S., Capuano, S. & Congiu, L. (2002) Identification of
and other staff at the Skeleton Coast National Park in life cycle stages of Cyathocephalus truncatus (Cestoda:
Namibia provided extensive logistical support, took us to Spathebothriidea) using molecular techniques. Journal of
incredibly remote springs, and pulled our vehicles from Parasitology, 88, 632–634.
sand dunes on several occasions. We thank L. Davis, Edmunds, G.F. & Waltz, R.D. (1996) Ephemeroptera. An
T. Garnto, Z. Parmley and J. Wolfe for assistance in col- Introduction to the Aquatic Insects of North America, edition3rd
lecting specimens during 3.5 weeks in Namibia, often under edn. (ed. by R. W. Merritt & K. W. Cummins), pp. 126–163.
Kendell/Hunt Publishers, Dubuque, Iowa.
extreme circumstances. We thank S. Cameron for advice
Genecodes (1999) Sequencher, Version 3.1.1. Genecodes, Ann
and comments on the manuscript. Financial support for Arbor, Michigan.
portions of this project performed by KBM and MFW Goloboff, P. (1995) nona, Version 2.0. Fundación E. Instituto
was provided in part by National Science Foundation Miguel Lillo, Tucumán, Argentina.
grants #DEB-9983195 and #DEB-0329115. Financial sup- Hebert, P.D.N. (2004) Ten species in one: DNA barcoding reveals
port to YA was provided by the Natural Sciences and cryptic species in the neotropical skipper butterfly Astraptes
Engineering Research Council of Canada in the form of fulgerator. Proceedings of the National Academy of Science,
an operating research grant. 101, 14 812–14 817.
Hebert, P.D.N., Cywinska, A., Ball, S.L. & deWaard, J.R. (2002)
Biological identifications through DNA barcodes. Proceedings
of the Royal Society (B), 270, 313–321.
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