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Wallaceaphytis: an unusual new genus
of parasitoid wasp (Hymenoptera:
Aphelinidae) from Borneo
Andrew Polaszeka, Thomas Ayshforda, Bakhtiar Effendi Yahyab &
Lucian Fusuc
a Department of Life Sciences, Natural History Museum, London,
UK
b Institute for Tropical Biology and Conservation, Universiti
Malaysia Sabah, Kota Kinabalu, Malaysia
c Faculty of Biology, Al. I. Cuza University, Iasi, Romania
Published online: 07 Nov 2013.
To cite this article: Andrew Polaszek, Thomas Ayshford, Bakhtiar Effendi Yahya & Lucian Fusu
(2014) Wallaceaphytis: an unusual new genus of parasitoid wasp (Hymenoptera: Aphelinidae) from
Borneo, Journal of Natural History, 48:19-20, 1111-1123, DOI: 10.1080/00222933.2013.852264
To link to this article: http://dx.doi.org/10.1080/00222933.2013.852264
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Wallaceaphytis: an unusual new genus of parasitoid wasp (Hymenoptera:
Aphelinidae) from Borneo
Andrew Polaszek
a
*, Thomas Ayshford
a
, Bakhtiar Effendi Yahya
b
and Lucian Fusu
c
a
Department of Life Sciences, Natural History Museum, London, UK;
b
Institute for Tropical
Biology and Conservation, Universiti Malaysia Sabah, Kota Kinabalu, Malaysia;
c
Faculty of
Biology, Al. I. Cuza University, Iasi, Romania
(Received 2 October 2013; accepted 2 October 2013; first published online 7 November 2013)
Wallaceaphytis Polaszek and Fusu gen. nov. (type species Wallaceaphytis kikiae
Ayshford and Polaszek sp. nov.) is described from Danum Valley, Sabah, in
Malaysian Borneo. Although known from just a single female individual, the
genus is extremely unusual morphologically, being the only member of the large
subfamily Aphelininae with four-segmented tarsi. The form of the fore wings and
head are also unique in the subfamily, and its status as a new genus is confirmed
by analysis of nuclear ribosomal DNA. DNA sequence analysis was undertaken
by comparison with more than 60 aphelinid sequences from GenBank. The
sequence for the standard DNA barcode region (cytochrome oxidase c subunit
I; COI) is provided. The new genus is named in honour of Alfred Russel Wallace,
co-discoverer of the theory of evolution by natural selection. The new genus and
species are published on the exact date of the centenary of his death.
http://www.zoobank.org/urn:lsid:zoobank.org:pub:700D2B2A-1586-4100-85D2-
24844EFE3F90
Keywords: Aphelininae; Chalcidoidea; chalcids; phylogeny; Sabah; Malaysia;
Alfred Russel Wallace; DNA barcode; non-destructive DNA extraction
Introduction
Within the large and megadiverse parasitoid superfamily Chalcidoidea (“chalcid
wasps”), Aphelinidae is one of the smaller families, containing 1300 species belonging
to 36 genera (Noyes 2013). Species of Aphelinidae are mostly primary parasitoids or
hyperparasitoids of Hemiptera, mainly Sternorrhyncha (Aleyrodidae, Aphididae,
Coccidae, Diaspididae & Pseudococcidae, among others), although several genera
are known to include species that are parasitoids of insect eggs (Polaszek 1991).
The large subfamily Aphelininae was recently the subject of a major phylogenetic
analysis based on morphological characters, which resulted in the description of four
new genera (Kim & Heraty 2012). This analysis includes a key to all of the currently
recognized 16 genera within the subfamily. The new genus described below differs
radically in several characters from any of the currently valid genera of Aphelininae,
and these differences are discussed in detail below.
*Corresponding author. Email: ap@nhm.ac.uk
Journal of Natural History, 2014
Vol. 48, Nos. 19–20, 1111–1123, http://dx.doi.org/10.1080/00222933.2013.852264
© 2013 Crown Copyright published by Taylor and Francis
This is an Open Access article. Non-commercial re-use, distribution, and reproduction in any medium, provided the
original work is properly attributed, cited, and is not altered, transformed, or built upon in any way, is permitted. The
moral rights of the named author(s) have been asserted.
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Material and methods
Collecting
In September and October 2012 a major multidisciplinary joint expedition from the
Natural History Museum, London, UK, and Universiti Malaysia Sabah, Kota
Kinabalu, Malaysia, undertook extensive sampling and surveying of arthropods
and other invertebrates in the Danum Valley and Maliau Basin Conservation
Areas. A wide range of collecting techniques was employed, including the use of
Winkler bags for soil samples, Malaise traps and yellow pan traps for day-flying
insects, and a specially modified sweep net for very small insects found in under-
growth and foliage. The “Noyes-net”(Noyes 1982) is a heavy-duty, long-handled
sweep net with 4-mm wire mesh screening the collecting bag, ensuring that only
specimens with a maximum length of 4 mm or thereabouts are collected. Sweeping
the undergrowth and foliage for several minutesresultsinanaccumulation of usually
several hundred microarthropods in the collecting bag, as well as associated debris,
seeds etc. These can either be collected directly into a container of 80–100% ethanol
for sorting under a microscope later, or the emergent insects can be aspirated indivi-
dually. In the latter case, less sorting is needed subsequently, but a large proportion of
the catch can be lost or overlooked. The latter technique was used in the present case,
with the sample sorted back in London several weeks later. Immediate recognition by
the second author (TA) that a particular specimen was clearly something extremely
unusual, prompted a special study of this individual, which was then subjected to the
non-destructive DNA extraction protocol described below.
DNA extraction and slide-mounting
Genomic DNA was extracted using the DNeasy
®
blood and tissue kit (Qiagen,
Hilden, Germany) from the whole specimen, using a non-destructive method slightly
modified from the manufacturer’sprotocol(Noyes2010). The specimen was removed
from ethanol, dried briefly on absorbent paper to remove any visible traces of liquid,
and immersed in ATL lysis buffer containing proteinase K in a 1.5 ml microtube
(“Eppendorf
®
”). Specimen lysis was achieved by overnight incubation at 55°C (or for
at least 8 hours) after which all internal tissues have been digested, while the exoske-
leton remains intact in the enzyme–buffer mix (throughout this time vortexing should
be avoided to reduce the risk of damaging the specimen). The lysis buffer containing
DNA was transferred by pipetting to a new 1.5 ml microtube, and processed as
described in the kit, except that the final DNA elution was into 100 µl. The extracted
specimen was immediately washed by pipetting 1 ml distilled water into the micro-
tube, changing the water after 30 min, and finally transferring the specimen into 80%
ethanol. If the specimen is not thoroughly washed, crystals can form upon its surface
on contact with ethanol. In the unlikely case of crystal formation, these can be
dissolved by placing the specimen for a few minutes in warm distilled water.
Specimens extracted with this method can be directly mounted without a previous
alkaline treatment (e.g. maceration in 10% KOH), or critical-point dried or chemi-
cally dried using hexamethyldisilazane, but air drying is likely to result in the specimen
collapsing. For slide-mounting, the specimen can be dehydrated through graded
alcohols up to 100% and permanently slide-mounted in Canada balsam after clearing
with clove oil. In the present case, the specimen was dissected and mounted following
the protocol described by Noyes (1982), from the 100% ethanol/clove oil stage
1112 A. Polaszek et al.
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onward, with the modification that dissection takes place in Canada balsam to reduce
specimen movement and the consequent risk of losing the dissected parts.
Polymerase chain reaction (PCR) and sequencing
The standard barcode region (Hebert et al. 2003) and the 28S rDNA D2 and D3
expansion regions were amplified by PCR using the LCO1490/HCO2198 primer pair of
Folmer et al. (1994)andD23F(5′-GAG AGT TCA AGA GTA CGT G-3′;Park&
Foighil 2000)/28Sb (5′-TCGGAAGGAACCAGCTACTA-3′, aca D3B; Nunn et al.
1996), respectively. We performed standard 25-μl PCRs containing 2.5 μl of 10× PCR
buffer, 0.75 μlof50m
MMgCl
2
,0.2μl dNTPs solution (25 mMeach), 1.25 μlofeach
primer (10 μM), 0.3 μlTaq polymerase (5u/μl Biotaq, Bioline), 6 μl DNA extract, and PCR
grade water to final volume. PCR conditions for cytochrome oxidase c subunit I (COI)
were 94°C for 2 min, followed by 40 repeated cycles of 94°C for 30 s, 42°C for 50 s and 72°
C for 35 s, a final extension at 72°C for 10 min and incubation at 10°C. The same
conditions were used for the amplification of 28S rDNA except annealing for 30 s at 55°
C. The PCR products were visualized on a 1% agarose gel.
Both DNA strands were sequenced at the Natural History Museum Life Sciences
DNA Sequencing Facility using the same primers used for the PCR. Sequences were
edited using Pregap4 v1.5 and Gap v4.10 in Staden Package (Bonfield et al. 1995) and
sequence verification was conducted by comparing forward and reverse sequences.
All sequences are deposited on GenBank (Accession numbers BankIt1665361 Seq1
KF718961 [28S ribosomal]; BankIt1665361 Seq1 KF718962 [CO1]).
Additional sequences for the phylogenetic analysis obtained mostly by J.-W. Kim
and J. Heraty (unpublished data) were retrieved from GenBank. Sequences were first
aligned using MEGA 5.05 (Tamura et al. 2011) and the ClustalW algorithm for the
ML analysis, or were manually aligned using the secondary structure models follow-
ing Gillespie et al. (2005) and the alignment of Munro et al. (2011) for the Bayesian
analysis. Phylogenies were estimated using maximum likelihood in MEGA 5.05 and
Bayesian methods in MrBayes version 3.2 (Ronquist & Huelsenbeck 2003). Analyses
were run using a GTR + G + I model of nucleotide substitution as this was
determined as the most appropriate model with MEGA 5.05. In MrBayes the analysis
was run for 10,000,000 Markov Chain Monte Carlo generations, with trees and lnLs
sampled every 100 generations. Likelihood stationarity occurred after 15,000 genera-
tions that were discarded as “burn in”.
Results
Sequence analysis
The aligned sequences of the 28S rDNA D2 and D3 expansion region produced a
sequence matrix 1121 base pairs long when automatically aligned using the ClustalW
algorithm or 1074 base pairs long in the case of the secondary structure alignment.
Maximum likelihood analysis resulted in one best-scoring tree with Wallaceaphytis
placed as sister group to a clade formed by all Centrodora species used in the analysis
(Figure 1), but this relation is only weakly supported (54% bootstrap support). In the
bootstrap consensus tree Wallaceaphytis,Centrodora,Aphytis, and Aphelinus are all
part of an unresolved polytomy (tree not shown). In the Bayesian analysis, the exact
position of Wallaceaphytis was not resolved either, as it was placed in a polytomy
with Marietta,Aphytis and Aphelinus (Figure 2).
Journal of Natural History 1113
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Figure 1. Maximum likelihood tree based on 28S-D2 and D3 sequences. Bootstrap values
based on 1000 replications are shown for nodes with more than 50% bootstrap support.
1114 A. Polaszek et al.
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Figure 2. Majority rule consensus Bayesian tree based on 28S-D2 and D3 sequences. Posterior
probability values are indicated in bold above nodes.
Journal of Natural History 1115
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Taxonomy
Wallaceaphytis Polaszek and Fusu gen. nov.
Type species Wallaceaphytis kikiae Ayshford and Polaszek sp. nov.
(Figures 3–11)
Description/generic diagnosis
Morphology. Antenna with three segments (Figure 5), scape, pedicel and a single
flagellar segment. Anellus present, narrower on its internal side. Scape narrow,
length 5× maximum width. Maximum width of pedicel 1.6× maximum width of
scape. Flagellum length 2.9× maximum width, and 1.4× scape. Head strongly trans-
verse (Figures 4 and 8) 2.9× as wide as long in dorsal view (unmounted specimen
Figure 8); 1.3× as wide as maximum width of mesosoma in dorsal view (Figure 8).
Mandible very small, with two teeth and a small truncation; mandibular glands
Figure 3. Wallaceaphytis kikiae holotype female; mesosoma and metasoma.
1116 A. Polaszek et al.
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elongate, parallel-sided. Maxillary palp two-segmented, labial palp one-segmented.
Lateral ocellus separated from eye margin by slightly more than the maximum width
of ocellus. Pronotum centrally membranous, each side with a robust seta at the
lateral edge, a fine seta adjacent to it, and two fine setae further towards the
centre. Mid-lobe of mesoscutum with two setae laterally. Each side lobe of mesoscu-
tum with two setae; tegula with a robust seta; axilla without setae; scutellum trans-
verse, with two pairs of setae. Propodeum elongate centrally, projecting posteriorly,
with a central process, and without crenulae (Figure 3). Propodeal spiracle
without anterior groove. Mesofurca of typical Aphytini form (Figure 7; see Heraty
et al. 1997). All tarsi four-segmented. Fore wing 3.8× as long as maximum width of
disc (excluding marginal fringe); submarginal vein with a single seta; stigmal vein
well-developed (Figure 6). Fore wing without setae below marginal vein, remainder
of wing very sparsely setose. Anterior gastral sterna without projections (see Woolley
1988, p.469). T1–T6 of gaster each with a pair of setae, T7 (syntergum) with two pairs
of setae, and in the form of a single sclerite, undivided and without epiproct.
Figure 4. Wallaceaphytis kikiae holotype female; head: dorsal (above), ventral (below).
Journal of Natural History 1117
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Figure 5. Wallaceaphytis kikiae holotype female; antennae.
Figure 6. Wallaceaphytis kikiae holotype female; fore wing.
1118 A. Polaszek et al.
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Comments
Wallaceaphytis presents a combination of characters that is so far unique among the
family Aphelinidae. Within the family, Wallaceaphytis is clearly a member of the sub-
family Aphelininae, as shown above based on DNA sequence data, and as follows based
on morphological data. The reduced number of antennal segments (three in the present
case), elongate and parallel-sided mandibular glands, medially membranous pronotum,
propodeal spiracles without anterior grooves, sterna without anterior apodemes,
Aphytis-like mesofurca and presence of a syntergum, exclude all other subfamilies.
Wallaceaphytis superficially resembles Ablerus, especially the unusual fore wing, but
can be easily excluded from that genus and from the subfamily Azotinae (family
Azotidae) by the above combination of characters. Eretmocerus has been included in
Aphelininae by some authors, and also has four-segmented tarsi, while the antennae are
five-segmented in females and three-segmented in males. However, in virtually all other
respects the two genera are very distinct, and Eretmocerus appears to be only distantly
related to Aphelininae. Marlatiella is the only known genus that also has three-segmen-
ted female antennae, but has five-segmented tarsi and very different wing characters.
Molecular DNA analysis supports the status of Wallaceaphytis as a distinct genus
with unresolved affiliations or allied more closely with Centrodora, but this relation-
ship is weakly supported. Despite the Bayesian analysis placing Wallaceaphytis in a
polytomy with Marietta,Aphytis and Aphelinus, morphology, and the maximum
likelihood analysis, suggest a closer relationship with Centrodora. In the key to genera
of Aphelininae by Kim and Heraty (2012)Wallaceaphytis keys immediately with
Eretmocerus because of the four-segmented tarsi, but is easily separated from this
genus by the three-segmented female antenna, and long marginal vein. Although
Wallaceaphytis is superficially similar to Ablerus, the molecular analysis (as with the
comparison of morphology discussed above) clearly shows that this genus belongs to
Aphelininae and not to Azotinae.
Figure 7. Wallaceaphytis kikiae holotype female; mesofurca.
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Figure 9. Wallaceaphytis kikiae holotype female; habitus.
Figure 8. Wallaceaphytis kikiae holotype female; habitus.
1120 A. Polaszek et al.
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Wallaceaphytis kikiae Ayshford and Polaszek sp. nov.
Description
In addition to the genus-level characters above, the following characters are likely to
be of species-level significance if additional species of Wallaceaphytis are discovered:
Colour. Head dorsally orange-yellow, brown on lower half of occiput (Figure 8).
Scape dark brown, pedicel and flagellum orange-yellow, flagellum darker basally.
Mesosoma and metasoma brown-black (Figures 9 and 10), a pale intersegmental area
behind propodeum laterally. Fore wing infuscate from base to slightly beyond stigma
vein (Figures 6 and 9). Legs brown, femora and tibiae paler at their bases; distal tarsal
segments darker than proximal segments (Figure 3).
Sculpture. Frons and face with transverse/reticulate sculpture. Mesoscutum
with reticulate sculpture in the form of large irregular cells, Fore wing
marginal vein with four robust setae and a smaller one at the junction with the
submarginal vein.
Additional characters
Ovipositor projecting beyond metasoma; 2.3× mid tibia. Second valvifers 3.3 × third
valvulae.
Figure 10. Wallaceaphytis kikiae holotype female; habitus.
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Holotype female. MALAYSIA: Borneo, Sabah, Danum Valley Field Study Centre,
Beach. 5°01' N, 117°48.75' E. 14 September 2012 screen-sweep (A. Polaszek).
Holotype dissected and slide-mounted in Canada balsam, deposited permanently in
Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Kota
Kinabalu, MALAYSIA.
Etymology
The genus name Wallaceaphytis is derived from the family name of Alfred Russel
Wallace, and the generic name Aphytis, to which it is related. Other genera within
the subfamily Aphelininae, to which Wallaceaphytis belongs, include Neophytis,
Paraphytis and Punkaphytis. The genus is described in honour of Wallace,
co-discoverer with Charles Darwin of the theory of evolution by natural selection.
Wallace’s collections and observations on the fauna and flora during his extensive
travels in South East Asia, including the islandofBorneo,ledtohisformulation
of the theory. The genus and species are described on the exact date of the
centenary of his death.
The specific epithet kikiae is based on, and in honour of, “Kiki”, the second
author’s mother, Mrs Christian Duke.
Acknowledgements
We thank Mohammad Hayat, John Noyes and Jim Woolley for improving earlier versions of
this paper with their comments and suggestions. AP and BEY thank Dr Abdul Fatah Amir,
Figure 11. Wallaceaphytis kikiae holotype female; habitus.
1122 A. Polaszek et al.
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Sabah Biodiversity Council, for the granting of Access Licence JKM/MBS.1000-2/2(77) under
which the field work in Sabah was carried out, and Glen Reynolds (Manager, Danum Valley
Field Studies Centre) for logistical support, and help with many other aspects of our work in
Sabah. LF’s research is currently supported by a grant from the Romanian National Authority
for Scientific Research, CNCS –UEFISCDI, project number PN-II-RU-TE-2012-3-0057. We
thank John Heraty, Jung-Wook Kim and Jason Mottern for making their aphelinid sequence
data available through GenBank.
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