Its specific title: MOLECULAR GENETIC ANALYSIS AND ECOLOGICAL EVIDENCE REVEALS MULTIPLE CRYPTIC SPECIES AMONG THYNNINE WASP POLLINATORS OF SEXUALLY DECEPTIVE ORCHIDS

1. MOLECULAR GENETIC ANALYSIS AND
ECOLOGICAL EVIDENCE REVEALS MULTIPLE
CRYPTIC SPECIES AMONG THYNNINE WASP
POLLINATORS OF SEXUALLY DECEPTIVE
ORCHIDS
PREPARED BY:
NURFARAH AIN LIMIN (P74703)

2. INTRODUCTION
Thynnine wasps
Neozeleboria cryptoides
Male: strong & active
flyers
Female: flightless & live
underground
Sex pheromone: 2-ethyl-5-
propylcyclohexan-1,3-dione
(chiloglottone 1)
Males showed
equal preference
for synthetic
compound
Mating:
Females emerge from ground &
emit volatile sex pheromone to
attract males.
Males respond in seconds, readily
locate hidden females-proves
chemical cues are vital
Specific pollinator
of sexually
deceptive orchids

3. Chiloglottis trapeziformis
Sexually deceptive
Orchids
 Produce same novel compound
to attract male N.cryptoides.
 Chiloglottone 1:
semiochemical involved in
intra-/interspecies
communication
Pollination occur during
premating routine/
attempted to mate with
flower
Pollination is highly specific
with average of 1.1
pollinator species per
orchid
Pollination by sexual deception
-Sexually lure male wasps by
mimicry of sex pheromone

4.  Bioassays with synthetic chiloglottones indicate 2
mechanisms to control orchid-pollinator specificity:
1. A single specific compound is required for pollinator
attraction. Eg: C. trapeziformis & chiloglottone 1,
with different compounds attract different specific
pollinators
2. Two/more compounds in a particular blend trigger
specific attraction. Eg: Allopatric Chiloglottis species
known to use same semiochemical but attract
different pollinator species

5.  Prediction: the pollinator pheromones will be either
identical to orchid semiochemical as in
C.trapeziformis/N.cryptoides or will share same major
components in similar proportions
 Neozeleboria wasps that appear to be prime candidate for
cryptic speciation associated with chemical rather than
morphological change
Aim:
 To investigate this prospect among those species of
Neozeleboria genus that are involved in orchid pollination

6. Materials & methods
 The wasp genus Neozeleboria are divided into:
Proxima MonticolaCryptoides
Impatiens
3 clades
Sub-clade

7. All wasp samples are associated with orchids in one three ways:
 Specific orchid pollinators whose behavior included attempted
copulation, pollen removal & pollen deposition
 Minor responders. They occasionally respond to orchids whose
behavior didn’t result in pollination (Eg: approach only)
 Responders to synthetic semiochemicals but not yet known to be
pollinators or minor responders

8.  Samples represent 18 sites across South-
Eastern Australia, from northern New
South Wales to Tasmania
 3 wasps chosen for sequencing from each
site. Sequencing is performed at:
- 1 mitochondrial locus: Cytochrome c
oxidase 1 (CO1)
-2 nuclear loci: rhodopsin & wingless

9. DNA extraction, PCR
& Sequencing
Sequencing
analysis
Samples and groups of
interest
Statistical parsimony
analysis
Population genetic
analysis
Morphological
identification
Consideration of candidate cryptic
taxa

10. DNA extraction, PCR & sequencing
PCR reaction (40µl) contained approximately:
 1-5ng of DNA template
 PCR buffer
 dNTPs
 Forward & reverse primers
 Taq Polymerase
Amplification:
 Performed with 3 min denaturation at 94°C
 30 cycles with annealing at 49°C followed by 10 min extension at 72°C
PCR products were purified with minor modification using ExoSAP-IT clean-up protocol

11. SEQUENCE ANALYSIS
 The 3 molecular data sets were analyzed both
separately and together. Maximum likelihood
analyses & bootstrap tests are conducted
 For comparison with maximum likelihood analyses,
Bayesian and parsimony tree-estimation was
performed

12. SAMPLES & GROUPS OF INTEREST
 24 groups of interest are identified
 Based on inspection of combined 3 loci
maximum likelihood best tree and associated
bootstrap values

13. Statistical parsimony analysis
 To identify groups of interest by following method of
Templeton et al.
 In brief,the method estimates genealogical relationships
among sequences & identifies sub-networks
 CO1 data sets are compiled and trimmed to remove missing
data before being analyzed separately for Proxima,
Cryptoids & Monticola

14. Population genetic analysis
 Performed Analysis of Molecular Variance
(AMOVA) for mtDNA data set
 To investigate the partitioning of genetic
variation within and among various priori
groupings of samples

15. Morphological identification
 To assess whether groups of interest could be distinguished by
current set of 40 morphological characteristics used to identify
Neozeleboria wasp
 One individual from each group was examined closely by 3rd author
(Graham R.B.)
 Emphasis on structure of male genitalia. A difference was defined
as a minimum of three minor differences in the un-dissected male
genitalia
 Specimens were coded & submitted for identification without
collection of biological data. As blind test, thus identification was
based only on morphological

16. Consideration of candidate cryptic taxa
 Probable cryptic taxa were identified
by being morphologically similar but
genetically distinct
 With strong support from non-
molecular evidence such as different
orchids associations

17. RESULTS

18. Statistical parsimony
analysis of CO1
Population genetic
analysis of CO1
Congruence among
genetic methods
Morphological analysis
 Monticola clade:
Sample 12 representing
a single sequence of
pollinator C.triceratops
(Tasmania) was
retained in same
network as Group 13
representing pollinators
of C.turfosa on
mainland
 Cryptoides clade: Two
largest networks
matched Group 7 & 8
 Proxima clade: mtDNA
sequencing at CO1
failed for Group 3
• Monticola clade: Due to
tight congruence
between orchid
associations & group of
interest containing 3 or
more samples, only minor
adjustment required to
match groups of interest
(GOI)
• Cryptoides clade: Not
possible to partition the
data among orchids
associations for AMOVA
because all but 1 sample
was associated with
C.trapeziformis/C. valida
that both uses
chilogglottone 1
• Proxima clade: Due to a
lack of sufficient
replication, AMOVA was
not feasible
• The phylogenetic
analysis across 3
genes, statistical
parsimony &
population genetic
analyses at mtDNA
CO1 gene provide
strong support for the
genetic
distinctiveness of the
numbered samples &
GOI
• It was concluded
that 3 named
species were
represented:
• N.cryptoides (Group
7 & 8)
• N.proxima (2, 3, 4)
• N.tabulata (20)
• 3 samples were
morphologically
distinct & assumed as
new species (1, 17 &
18)
• All were classified
into major clades
(Proxima, Cryptoides
or Monticola)

19. DISCUSSIONS
 Consistent with prediction made, a relationship between defined wasp groups, their
orchid associations & semiochemicals was found
Monticola clade Crypticoides clade Proxima clade
 There is evidence for
pheromone sharing
among pollinators.
Eg: Wasp groups 12-
14 were associated
with orchid taxa that
use chiloglottone 4
 All wasps appear to
respond to
chiloglottone 1only
 Illustrate pheromone
sharing among
Crypticoides &
Monticola
 Wasp Group 4
associated with C .
trilabra that produces
chiloglottone1 & 3
 Wasp Group 2
associated with C.
seminuda
(chiloglottone 1)

20.  Majority of wasp groups were associated with 1 orchid
species
 Despite strong association with 1 orchid, there is
pheromone sharing among wasp groups
 The collective phylogenetic, statistical parsimony,
population genetic, ecological & geographic
evidences support the hypothesis that the wasp
samples & GOI (Group 1-24) are taxonomically distinct

21. Are there cryptic species?
 Group 7 within Cryptoides clade. It was morphologically
identified as N.cryptoides but was characterized by a deep
phylogenetic divergence from N.cryptoides (Group 8).
 All 4 groups within Impatiens complex (Group 21-24) were
morphologically indistinguishable
 Within Proxima clade, Groups 2-4 were identified to be
morphologically as N.proxima
 Overall, up to 16 groups out of 24 may represent genetically
distinct but cryptic species

22. IMPLICATIONS FOR EVOLUTION OF
NEOZELEBORIA WASP
 Genitalia are predicted to evolve rapidly & divergently
under sexual selection. It may be the 1st morphological
characters to define newly formed species
 In this study, multiple cryptic thynnine wasp taxa that
lacked diagnostic differences in male genitalia were
uncovered
 This is tally with the observation: chemical rather than
visual cues are most important for wasp mate attraction.

23. IMPLICATIONS FOR EVOLUTION OF THE EVOLUTION
AND DIVERSIFICATION OF ORCHIDS
 The exploitation of Neozeleboria wasps by Chiloglottis
orchids led to hypothesise that this strong association
might limit the available pool of pollinators and
therefore, orchid speciation
 But the findings of multiple cryptic species in
Neozeleboria suggest that the wasp genus maybe larger
than previously thought
 So rather than constraining evolution of orchid genus,
species diverstiy within the wasp may have actually
enabled diversification by providing diverse pool of
species pollinator