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Molecular Ecology. 2019;00:1–13.
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wileyonlinelibrary.com/journal/mec
1 | INTRODUCTION
Sexual reproduction is a common strategy among multicellular
organisms, despite its two‐fold cost relative to asexual reproduc‐
tion (Maynard Smith, 1978). This apparent paradox has stimulated
research exploring the potential benefits of sexual reproduction
that would explain its prevalence (Barton & Charlesworth, 1998;
Burke & Bonduriansk y, 2017; Otto, 2009). Where evolutionary lin‐
eages that reproduce in different ways are in competition, valuable
evidence about the selective advantages of different strategies can
be gleaned (van der Kooi & Schwander, 2014; Neiman, Meirmans,
Schwander, & Meirmans, 2018). For example, the gradual upstream
range expansion of sexual Potamopyrgus antipodarum snails recorded
over 20 years ( Wallace, 1992), suggests short‐term local competitive
advantage of sex in this species that is supported by evidence from
parasite infection rates (Gibson, Xu, & Lively, 2016; Jokela, Dybdahl,
& Lively, 2009). Unfortunately, such systems are ephemeral and thus
their availability can be a limiting factor in the study of why sex is
Received:21June2018
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Revised:17Ju ne2019
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Accepted:30July2019
DOI : 10.1111 /mec.152 03
ORIGINAL ARTICLE
Loss and gain of sexual reproduction in the same stick insect
MaryMorgan‐Richards1 | Shelley S. Langton‐Myers2 |StevenA.Trewick1
Thisisanop enaccessarticleundert hetermsoftheCreat iveCommonsAttributio n‐NonCommercialLicense,whichpermit suse,dis tributionandreproduction
in any medium, provided the original work is prop erly cited and is not used for commercia l purpos es.
©2019TheAuthors.Molecular Ecology published by Jo hn Wiley & Sons Ltd
1Wildlife & Ecology, Massey University,
Palmerston North, New Zealand
2EcoQuest Education Foundation,
Whakatiwai, New Zealand
Correspondence
Mary Morgan‐Richard s, Wildlife & Ecolog y,
Massey University, Palmerston North, New
Zealand.
Email: m.morgan‐richards@massey.ac.nz
Funding information
MasseyUniversit y,Grant/AwardNumber:
MU RF‐201 6
Abstract
The outcome of competition between different reproductive strategies within a sin‐
gle species can be used to infer selective advantage of the winning strategy. Where
multiple populations have independently lost or gained sexual reproduction it is pos‐
sible to investigate whether the advantage is contingent on local conditions. In the
New Zealand stick insect Clitarchus hookeri, three populations are distinguished by
recent change in reproductive strategy and we determine their likely origins. One
parthenogenetic population has established in the United Kingdom and we provide
evidence that sexual reproduction has been lost in this population. We identify the
sexual population from which the parthenogenetic population was derived, but show
that the UK females have a post‐mating barrier to fertilisation. We also demonstrate
that two sexual populations have recently arisen in New Zealand within the natural
range of the mtDN A lineage that other wise characte rizes parthenoge nesis in this
species. We infer independent origins of males at these two locations using microsat‐
ellite genotypes. In one population, a mixture of local and nonlocal alleles suggested
males were the result of invasion. Males in another population were most probably
the result of loss of an X chromosome that produced a male phenotype in situ. Two
successful switches in reproductive strategy suggest local competitive advantage for
outcrossing over parthenogenetic reproduction. Clitarchus hookeri provides remark‐
able evidence of repeated and rapid changes in reproductive strategy, with competi‐
tive outcomes dependent on local conditions.
KEYWORDS
Clitarchus hookeri, evolution of sex, fertilization barrier, Phasmids, sexual reproduction
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MORGAN‐RICH ARDS et Al .
so common (Barton & Charlesworth, 1998; Maynard Smith, 1978;
Mirzag haderi & Hör andl, 2016; Otto, 2 009). Altho ugh it might be
expected that a single reproductive strategy within a species would
prevail in all circumstances, local factors can determine local adap‐
tive advantage (Tabata, Ichiki, Tanaka, & Kageyama, 2016). Our
study with temporal sampling provides evidence that local effects
are impor tant to the outcome of competition between reproductive
strategies within a single stick insect species.
The maintenance of sexual reproduction in nature has been
addressed by contrasting the traits of sexual and related asexual
lineages, and ideal study systems have natural replication, with inde‐
pendently derived populations displaying contrasting reproductive
strategies (see reviews; Neiman et al., 2018; Neiman & Schwander,
2011). Examples of this circumstance include the snail Potamopyrgus
antipodarum whose different clonal lineages are derived from the
same sexual species (Gibson et al., 2016; Jokela et al., 2009), and
Timema stick insects that contain multiple species pairs of sexual/
asexual lineages (Bast et al., 2018; Schwander & Crespi, 20 09;
Schwander,Henry,&Crespi,2011).Anoptimalstudysystemwould
provide replication but lack the confounding effects of ploidy vari‐
ation and hybridisation that exist in many study systems (Kearney,
2005). The stick insect species Clitarchus hookeri meets these opti‐
mal criteria; replicates of sexual and asexual populations exist, and
all individuals assessed so far are diploid (Myers, Trewick, & Morgan‐
Richards, 2013). However, the range of mechanisms employed by
asexually reproducing organisms can influence our abilit y to isolate
the benefits of sex (Neiman & Schwander, 2011). In stick insects,
parthenogenetic reproduct ion can be apomictic (without recombina‐
tion; Schwander & Crespi, 2009), or automictic (with recombination;
Marescalchi & Scali, 2003) and thus reduction of heterozygosity in
subsequent generations. The outcome of automixis is similar to self‐
ing in plants and can cause inbreeding depression (Barrett, 2002).
It is not yet known whether parthenogenetic C. hookeri reproduces
with recombination and resulting homozygosity or without.
Stick insects (Phasmids) have provided many examples of re‐
cently derived asexual lineages (Scali, 2009; Schwander & Crespi,
2009), with the switch from sexual reproduction to parthenogenesis
having occurred independently numerous times within this family
(Ghiselli, Milani, Scali, & Passamonti, 2007; Schwander et al., 2011).
Some lineages contain facult ative par thenogens (Mantovani & Scali,
1992) that provide the oppor tunit y to investigate competition mod‐
els of the two reproductive strategies. Facultative parthenogenetic
populations have the potential to switch from asexual to sexual
reproduction if males are available, and we can distinguish three
scenarios in which asexual lineages revert to sexual reproduc tion:
swamping, introgression, and male genesis. Swamping could follow
colonization by sexual males and females, that might involve long‐
distance dispersal into an empty habitat patch. The genetic signature
of such a population would be distinct from adjacent parthenoge‐
neticpopulationsbuthavethesameallelesandmtDNAhaplotypes
as found in sexual populations further afield. Introgression involves
establishment of males from outside the area. These males would
successfully fertilize eggs of local females and so restore sexual re‐
production. This scenario would mix local alleles and alleles distinc‐
tive to sexual populations, resulting in a population with high allelic
diversity relative to surrounding all‐female populations, but retain
mtDNAfrom thelocalparthenogeneticlineage. Alternatively,suc‐
cessful natural male genesis could occur via in situ nondisjunction of
the sex chromosome. Loss of X chromosomes during egg production
in some parthenogenetic insects can produce locally derived males
(e.g., Brock, Lee, Morgan‐Richards, & Trewick, 2018; Pijnacker &
Ferwerda, 1980; Scali, 2009). If these males are fer tile and restore
sexual reproduction to the population, it will contain only alleles and
mtDNA haplotypesfrom the locallineage,butobserved heterozy‐
gosity will rise from levels when males were rare (if parthenogenetic
reproduction is automictic).
In New Zealand, most northern populations of the common dip‐
loid stick insect C. hookeri have an even sex ratio and all reproduc‐
tion is assumed to be sexual (Langton‐Myers, Holwell, & Buckley,
2019; Morgan‐Richards, Trewick, & Stringer, 2010; Myers, Buckley,
& Holwell, 2015; Myers, Holwell, & Buckley, 2017), but males are
absent from many southern locations (Figure 1). A widespread
FIGURE 1 Three questions arise
from the distribution of male and
female Clitarchus hookeri stick insects.
Parthenogenetic populations surround
sexual populations at Otaki and Wilton,
so from where did the males come?
Human‐mediated dispersal has resulted in
a parthenogenetic population in the UK,
but from where in New Zealand did this
all‐female lineage originate? Blue (sexual)
and pink (asexual) shading indicates the
natural extent of sexual and asexual
populations in New Zealand
Origin of UK females?
Origin of Otaki males?
Origin of Wilton males?
♀♀
♀♀
♀♀
♀♀
♀♀
♂
♀
♂
♀
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MORGAN ‐RICHA RDS et Al.
parthenogenetic lineage appears to have resulted from range ex‐
pansionbyasinglemtDNAlineage(Buckley,Marske,&Att anayake,
2010; Morgan‐Richards et al., 2010). Throughout the southern
and eastern North Island and eastern South Island of New Zealand
(Figure 2), reproduction is inferred to be entirely parthenogenetic
(Wu, Twort, Crowhurst, Newcomb, & Buckley, 2017); males have
been recorded only sporadically and at very low density (Buckley et
al., 2010; Morgan‐Richards et al., 2010). However, two sexual pop‐
ulations of C. hookeri that are surrounded by all‐female populations
are described in the present study. Both of these sexual populations
are within the geographic range of the parthenogenetic mtDNA
lineage (Figure 1), but feed on plants that had been transported
and planted by humans. Surprisingly, C. hookeri is also found in the
United Kingdom (UK), where an all‐female population was founded
by accidental human‐mediated dispersal (Figure 1).
Captive breeding experiments show that, if they have mated,
C. hookeri females from sexual populations produce all offspring
via sex. In contrast , even when provided with males, females from
the parthenogenetic lineage experience a barrier to fertilisation.
Despite retaining sexual signals (Nakano, Morgan‐Richards, Godfrey,
& Clavijo McCormick, 2019) and mating, only about 5% of of fspring
from these females resulted from sexual reproduction (Morgan‐
Richards et al., 2010). This resistance to fertilisation might prevent a
successful transition to sexual reproduction via males colonizing the
range of the parthenogenetic lineage unless there exists an indirect
sons' effect (Kawatsu, 2015; Morgan‐Richards et al., 2010). Here,
we use both temporal and spatial sampling of wild populations of the
New Zealand stick insect C. hookeri to study three putative transi‐
tions in reproductive strategy and attempt to determine whether the
outcome of competition between different reproductive strategies
is contingent on local conditions. We provide evidence that C . hook‐
eri populations have undergone multiple sexual‐asexual transitions.
We utilize mating experiments to test the permanency of asexual
populations. We compare genetic variation (mtDNA haplotypes
and nuclear microsatellite loci) to determine which New Zealand
C. hookeri population is the closest relative of the asexual popula‐
tion established in the UK, and use this relationship to infer the UK
population's reproductive history. Genetic variation is used to deter‐
mine the likely origin of the two sexual C. hookeri populations in New
Zealand that are surrounded by all female populations. We distin‐
guish between three possible scenarios to explain the origin of these
two sexual populations: swamping, introgression, or male genesis.
2 | MATERIALSANDMETHODS
2.1 | Clitarchus hookeri
The commonNew Zealand mānukastick insect (the“smooth stick
insect ” in the UK ) is frequently found on the Myrtaceae species
Leptospermum scoparium (mānuka) and Kunzeasp.(kānuka),col‐
lectivelyknown as tea‐tree. Adult female mānukastick insects are
about 9 cm long; the males are shorter (~7 cm) and thinner (Figure 1).
We collected adult and juvenile Clitarchus hookeri from natural sites
around New Zealand to obtain a representative sample of their
sexual and geographic diversity (Figure 2, Table S1). In the UK, pop‐
ulations of stick insects have established from human‐mediated dis‐
persal (Jewell & Brock, 2002). Clitarchus hookeri was first recorded in
theTrescoAbbeyGardensin1940,wherenumerousplantspecies
FIGURE 2 Similarit yofmtDNA
sequences indicates the probable
origins of Clitachus hookeri stick insect
populations. (a) Sample locations in
New Zealand, coloured to highlight two
intraspecificmtDNAlineagesasshown
in b (pink par thenogenetic; blue from
Taranaki). (b) Phylogenetic relationships
offullmtDNAdiversitysampledinNew
Zealand inferred with Maximum likelihood
(COI 1,389 bp), colours as in networks
and map, scale represents nucleotide site
distances. (c) Parthenogenetic lineage
haplotype network (medium joining, COI
1,307 bp) in pink, with the haplot ypes of
male individuals indicated. (d) Haplotypes
from Taranaki lineage (blue) includes UK
specimens (median joining, COI 1,307 bp).
Circle sizes in c & d are proportional to
sample size, and number of nucleotide
substitutions that differentiate haplotypes
are indicated on branches
(a) (b)
(c) (d)
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MORGAN‐RICH ARDS et Al .
from New Zealand were planted in 1911 and in the 1930s (Brock,
1987; Godley, 1997). Nine C. hookeri individuals were collected by
Paul Brock from the Isles of Scilly UK (five Tresco; four St Mary's;
one Bryher Island) and held in captivity in the UK to obtain eggs.
2.2 | Breedingexperiments
To assess the ability of the all‐female UK lineage to return to sexual
reproduction in captivity we imported UK C. hookeri eggs to New
Zealand to raise and mate with New Zealand male C. hookeri. To
achieve this Paul Brock collected 922 eggs from nine C. hookeri (re‐
ferred to here as generation‐1) from the Isles of Scilly UK in Northern
Hemisphereautumn2013(August,September,October).Onimpor t
to New Zealand, these were held in a physical cont ainment facility
(PC level 2) for 12 months. We monitored the eggs as they hatched
between May 2014 and February 2015 (Southern Hemisphere win‐
ter, spring, summer; generation‐2) and raised some of the nymphs,
which were housed and fed individually, to maturity. Moving from
the Northern Hemisphere to the Southern Hemisphere resulted
in many Scilly Isle individuals maturing before adult males were
available in New Zealand; this constrained the origin and number
of potential mates we provided. Ten of the adult generation‐2 fe‐
males were each provided with a single male C. hookeri collected
from either Karapiro or Kerikeri in New Zealand (January 2015).
We observed whether copulation took place and collected eggs
that were laid (Table S2). Generation‐3 hatched from these eggs be‐
tween September 2015 and January 2016 (Table S2). We sexed all
nymphs using external morphological differences (Morgan‐Richards
et al., 2010; Stringer, 1970), and raised a portion of (all‐female) gen‐
eration‐3 individuals to maturity. This generation, which was more
closely synchronised with wild New Zealand populations, matured
later in the season and allowed us to pair more than one adult male
per female. We provided 13 adult generation‐3 females with males
from the Wilton and Taranaki populations (2–4 males with each fe‐
male).Aswithgeneration‐2,we obser vedcopulation (Februaryand
March 2016), collected eggs, and sexed all nymphs that hatched
(generation‐4 hatched between September and December 2016).
We used the frequency of males that hatched from the eggs to esti‐
mate the proportion of offspring resulting from sexual reproduction,
assuming that half of all of fspring resulting from fusion of sperm and
egg (sex) would be male. We genotyped a subset of nymphs to con‐
firm that males were the result of sex rather than nondisjunction of
FIGURE 3 Genotypes of Clitachus hookeri stick insec ts suggest that the UK population was derived from a sexual New Zealand
population, that Otaki males result from population mixing, and Wilton males result from in situ male genesis (nondisjunction of X
chromosome). Bayesian assignments of individual stick insect genotypes (10 microsatellite loci) to three genetic clusters (optimal model
K = 3) is shown by three colours. Collecting locations indicated on the map are coloured to indicate dominant genotype assignment cluster
Tresco
Isles of Scilly
New zealandUnited kingdom
Urenui
Gordon
Rotorangi
Tarata
Tresco
Opanuku
Karapiro
Stony Bay
East Cape
2003
2013
2003
2016
Otaki
Wilton
Gisborne
Turitea
Peel Forest
Manaroa
Opanuku
Gisborne
Karapiro
Urenui
Stony Bay
Peel Forest
Turitea
Wilton
Otaki
East Cape
Gordon
Manaroa
Rotorangi
Tarata
♀♀ ♂
♀
♂
♀
♂
♀
♀♀
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MORGAN ‐RICHA RDS et Al.
one of their X chromosomes and to test our assumption that half the
offspring resulting from sexual reproduction were female (Table S2).
Nondisjunction during egg formation is rare but has been recorded
among unmated C. hookeri females in captivity, which produce fewer
than one son for every 300 daughters (Morgan‐Richards et al., 2010;
Salmo n, 1955) .
2.3 | Naturalpopulations
We collected stick insects from six locations within the New Zealand
rangeof the parthenogenetic mtDNA lineage(Morgan‐Richardset
al., 2010): four locations where males have not been recorded and
two locations where males were locally abundant (Figure 3; Table
S1). These collections included one sexual population on private
property where C. hookeri are seasonally common and feed on vari‐
ous non‐native host plants including plum (Prunus domestica; Lupin
Road,Otaki).Here,wecollectedadultinsectsinautumn(April2003)
and nymphs in spring (October 2013). At Wilton (Otari‐Wilton's
Bush in Wellington), we located a C. hookeri population where
males were uncommon in the 1960s (Salmon, 1991) and 200 0s
(Morgan‐Richards et al., 2010) but were at similar frequencies to
females in 2016. In 2003 stick insects were collected from native
plants(kānuka)withintheOtari‐Wilton'sBushnativeforestremnant
(=Wilton). The recently planted mānukaand kānukaon which the
2016 sample were feeding had been germinated from local seed and
raised at a plant nursery in Taupo (30 0 km north of the site; personal
communication Eleanor Burton, Wellington City Council).
2.4 | DNAextraction,amplificationandsequencing
We dissected leg muscle from fresh, frozen, or alcohol‐preserved
C. hookeri,and extractedgenomic DNA using asalting‐outmethod
(Sunnuck & Hales, 1996) or the Qiagen DNeasy tissue kit . We ampli‐
fied and sequenced a mitochondrial genome fragment comprising
the 3′ end of c ytochrome ox idase I (COI), tRNA‐Leu cine, and cy‐
tochrome oxidase II (COII), using a combination of primers C1‐J‐2195
and TK‐N‐3785, L2‐N‐3014 and TL2‐J‐3034 (Simon et al., 1994),
and standard conditions described elsewhere (Morgan‐Richards &
Trewick, 2005). We aligned and visually checked sequences using ge-
neiousv9(Kearseetal.,2012),andexcludedthetRNA‐Leugenebe‐
tween COI and COII as this was missing from some specimens due to
its use in PCR priming. We translated coding sequences to check for
stop codons, frame shifts, and amino‐acid substitutions that might
indicate nuclear copies. We included C. hookeri sequences from pre‐
vious studies (Genbank: GU299870.1‐GU299966.1, AY940431.1,
EU492994.1‐EU492973.1; Buckley et al., 2010; Morgan‐Richards &
Trewick, 2005; Morgan‐Richards et al., 2010).
For 111 individuals across 15 population samples, we charac‐
terizednuclearDNAvariationat10independentmicrosatelliteloci
(Myers et al., 2017). Multiplex PCR used thermal cycling conditions
of 95°C for 5 min, followed by 28 cycles of 95°C for 30 s, anneal‐
ing temperature of 60°C for 1 min 3 0 s, and 72°C for 30 s and one
additional cycle of 60°C for 30 min. Reac tions were resolved on an
ABI Prism 3100 Genetic Analyzer (PE Applied Biosystems), with
allele size determined using the Microsat plugin in Geneious with
GenescanLiz‐500(AppliedBiosystems)asaninternalsizestandard.
To determine error rate we re‐genotyped six individuals at random
and scored alleles blind.
This insect species has two large metacentric X chromosomes
in females, and one X chromosome in males (Morgan‐Richards &
Trewick, 2005). We inferred that one locus (Ch29‐14; Myers et al.,
2017) was sex‐linked, as males always had a single allele. We con‐
firmed that males inherited their mother's allele but not their father's
by genotyping sons.
2.5 | Populationgeneticanalyses
2.5.1 | MitochondrialDNA
WeexaminedmtDNACOIvariationfor125individuals(46newse‐
quences) across 41 population samples, and uploaded new unique
haplotype sequences to GenBank (accession numbers MK532396,
MK606153‐MK606171). The aligned data set is available on Dryad
(https ://doi.org/10.5061/dryad.b7t80m5) and at www.evolv
es.massey.ac.nz. For the full data set (1,389 bp), we inferred relation‐
ships amo ng haplotype s using Maximum L ikelihood with PH YML,
implementingaGTR+I+GmodelofDNAevolution(Tavaré,1986).
Weusedsubsets of the mtDNAhaplotypedata (1,307 bp) to infer
median‐joining networks (Bandelt, Forster, & Rohl, 1999) using
POPART(Leigh&Bryant,2015).
2.5.2 | Lineageageestimation
One mitochondrial lineage is associated with the all‐female popu‐
lations that are geographically restrained to eastern and southern
locations of New Zealand (Morgan‐Richards et al., 2010). The distri‐
bution of this lineage, may be associated with range and population
expansion (Buckley et al., 2010). To infer the approximate time of
the most recent common ancestor (MRCA) of this lineage, we im‐
plemented a molecular clock analysis in be ast v2.5.0 (Bouckaert et
al., 2014), using a 1,350 bp alignment of mtDNA sequence of COI
and COII from 101 C. hookeri individuals. We evaluated nucleotide
substitution models using jm odel test v 0.1.1 (Posada, 2008) and
found GTR + G to be a suitable model. No species specific rates of
molecular evolution exists for C. hookeri, nor are there fossils for
calibration, and using inferred timing of range expansion associated
with climate cycling for calibration would introduce circularity to
our analy sis. Instead we use d five differe nt insect DNA sub stitu‐
tion rates to calibrate our analyses. From the literature we obtained
two rates based on insect mitochondrial interspecific divergence
dates (Clarke, Levin, Kavanaugh, & Reimchen, 2001; Papadopoulou,
Anastasiou,&Vogler,2010),and threeratesbasedon observedin‐
traspecificnucleotide mutations(Gratton,Konopiński,&Sbordoni,
2008; Haag‐Liaut ard et al., 2008; Ney, Frederick, & Schul, 2018; see
Table1). A relaxed,uncorrelated, lognormal molecular clock model
(Drummond, Ho, Phillips, & Rambaut, 2006) was applied, in order to
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MORGAN‐RICH ARDS et Al .
evaluate the clock‐like behaviour of the dat a. The posterior probabil‐
ity of nonzero rate variance was close to zero (mean of rate variance
~0.0 014), and therefore a simpler strict‐clock model w as used in sub‐
sequent analyses. Because our data are from a subdivided species
with a signature of population grow th for some lineages (Buckley et
al., 2010), we used two models for the tree prior; Coalescent con‐
stant population size, and Coalescent Bayesian Skyline (Drummond,
Rambaut,Shapiro,&Pybus,2005).Analternativemodel,Coalescent
Extended Bayesian Skyline, assumes a single population but allows
for any number of changes in population size; however, with this
more parameter rich model, MCMC simulations failed to converge
on a stati onary distrib ution of posterior probabilities (e.g., ESS poste‐
rior = 166). Unless stated otherwise, default parameters were used.
Markov Chain Monte Carlo (MCMC) simulations had chain lengths of
10 million, sampling ever y 1,000 generations. We assessed conver‐
gence through visual inspection of posterior statistics in tracer v1.5
(Rambaut & Drummond, 2007).
2.5.3 | Microsatelliteloci
To estimate observed and expected heterozygosity per population
sample (HO, HE) and average number of alleles per locus (Na) we used
arlequin v3.5 (Excoffier & Lischer, 2010). We scored the sex‐linked
locus as either homoz ygous for all males or as a single allele with
missing data for all males. The results of these two coding methods
did not alter population inferences from downstream analyses, so
we present only the results from coding locus Ch29‐14 as sex‐linked.
Astheseparthenogeneticstickinsects couldpotentiallyreproduce
via automixis or apomixis, we tested our population samples for evi‐
dence of deviations from Hardy‐Weinberg expectations (exact tests;
autosomal loci only). For those samples that showed significant
departures from Hardy‐Weinberg expectations, we tested for het‐
erozygote deficienc y using global Hardy‐Weinberg exact tests for
multiple samples (U test), which are suitable for small samples and
large numbers of alleles (Rousset & Raymond, 1995). We estimated
pairwise FST for each population sample and used an exact prob‐
ability test of population dif ferentiation (Raymond & Rousset, 1995)
with gene pop v4.2 (Rousset, 20 08). To estimate population structure
and assign individuals to clusters based on their genotypes at 10
nuclear loci, we used Bayesian model‐based analyses implemented
in struct ure v2.3.4 (Falush, Stephens, & Pritchard, 2007; Pritchard,
Stephens,&Donnelly,20 00).Again,wescoredthesex‐linkedlocus
as either homozygous for all males or as a single allele with missing
data. A s these yiel ded the sam e optimal K we present the results
from coding locus Ch29‐14 as sex‐linked. For each struc ture run
we conducted analyses with K ranging from one to seven, using an
admixture model with correlated allele frequencies (Pritchard et al.,
2000). Each run used 500,00 0 MCMC iterations following a 50,000
iteration burnin, based on recommendations provided by Gilbert et
al. (2012). For each K set, we conducted ten replicate analyses. To
identif y the number of population clusters (K) with the best fit to
our data, we used stru cture ha rverster (Earl & VonHoldt, 2012)to
TABLE 1 Theageofthemostrecentcommonancestor(MRCA)oftheparthenogeneticlineageofClitarchus hookeriwasestimatedusingfiveinsectmitochondrialDNAmutationrates
derived from different taxa and calibrations
Ratetype Tax a mtDNAgenes
Age of calibra‐
tion (years)
Substitutions per site
per million years Reference
Ageofpar thenogenMRCA
Constantpopsize
(million years) SE of mean
Bayesianskyline
(million years) SE of mean
Interspecific Darkling beetles COI 10.5 million 0.0177 Papadopoulou et al.
(2010)
0.1701 1.57E‐ 03 0.1537 1.55E‐03
Interspecific Ground beetles ND2, COI,
COII , Cyt‐ B
150,000 0.0285 Clarke et al. (20 01) 0.1055 1.15E‐ 03 0.0964 1. 01E‐0 3
Intraspecific Fruit‐flies ~60% of
mtDNA
200 generations 0.062aHaag‐Liautard et al.
(2008)
0.0493 4.44E‐04 0.044 4.89E‐ 04
Intraspecific Katydids COI 1 0, 740 0.0792 Ney et al. (2018) 0.0381 3.85E‐04 0.0355 4.37E‐0 4
Intraspecific Swallowtail Butterflies COI 10–11,000 0.0960 Gratton et al.
(2008)
0.0315 3.66E‐ 04 0.0281 3.72E‐0 4
Note: Thestickinsectdatasethad101haplotypesand1,350bpofCOI‐COIImtDNAsequence.MolecularclockanalysesusedastrictclockandeitheraconstantpopulationsizeoraBayesianskyline
model.
a6.2 × 10–8 per site per fly generation (as study insects have ~1 generation per year we converted to 0.062 substitutions per site per million years).
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MORGAN ‐RICHA RDS et Al.
TABLE 2 Higher genetic variation detected in sexual Clitarchus hookeri stick insect population samples compared to parthenogenetic population samples (number of alleles [Na], observed
heterozygosity [HO], and expected heterozygosity [HE]) using 10 microsatellite loci, * denotes where genotype propor tions deviated from expectations based on the Hardy‐Weinberg
assumptions (using all nine autosomal loci with Fisher's exact test; p < .05)
Location Region Males common mtDNA(n)
mtDNAparthenoge‐
netic clade
Microsatellites
Inferredreproductive
strategynCluster(K = 3) NaHOHE
Opanuku Auckland Yes 4No 3 1 2.3 0.56 0.60 Sexual
Stony Bay Coromandel Yes 7No 8 1 4.4 0.71 0.68 Sexual
Karapiro Waikato Yes 8No 13 12.8 0.38 0.49 Sexual
East Cape East C ape No 5No 3 1 1.7 0.07 0.60 Parthenogenetic
Urenui, Tarat a,
Rotorangi, Gordon
Taranaki Yes 20 No 18 22.0 NA NA Sexual
Tresco, Isles of Scilly UK No 9No 10 21.0 00Parthenogenetic
Gisborne Gisborne No 5Yes 6 3 1.3 0.06 0.38 Parthenogenetic
Turitea Manawatu No 3Yes 8 3 1.2 0* 0.40 Automictic
parthenogenetic
Otaki 2003 Wellington Yes 3Ye s 5 2, 3 2.3 0.64 0.54 Sexual
Otaki 2013 Wellington Ye s 2Ye s 6 2, 3 2.8 0. 52 0.54 Sexual
Otaki combined 11 3.1 0.54 0.53
Wilton 20 03 Wellington No 6Yes 4 3 1.3 0.08 0.56 Parthenogenetic
Wilt on 2016 Wellington Yes 7Yes 16 31.4 0.25 0.35 Sexual
Wilton combined 20 1.5 0.25 0.36
Manaroa Marlborough
Sounds
No 2Yes 4 3 1.0 00Parthenogenetic
Peel Forest Canterbur y No 2Yes 6 3 1.1 00.30 Parthenogenetic
Note: The stick insects were sampled from 14 New Zealand and one UK loc ation.
8
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MORGAN‐RICH ARDS et Al .
identif y optimal K based on the posterior probability of the data for
a given K, and the Delta‐K (Evanno, Regnaut, & Goudet, 20 05).
3 | RESULTS
3.1 | Transitiontoasexualreproduction
3.1.1 | Breedingexperiments
Stick insect eggs were impor ted into New Zealand from the UK
(n = 922). These eggs were the product of nine females (genera‐
tion‐1) collected from the parthenogenetic population of Clitarchus
hookeriontheIslesofScilly.Allnymphsthathatched(generation‐2)
were female (n = 677). Ten adult females of generation‐2 laid eggs
after mating with conspecific New Zealand males (n = 201). These
eggshatchedinto82daughtersbutnosons(generation‐3).Atotalof
13 adult females of this third generation laid 492 eggs after mating,
whichhatched44 0daughtersandsevensons.Althoughwehaddata
from two generations, the females were assumed to be genetically
identical and were pooled. From all offspring (laid after mating) over
both generations, we estimated that approximately 2.6% of nymphs
were the product of sexual reproduction (number of sons × 2; to ac‐
count for daughters also produced by sex; Table S2).
We genotyped 30 offspring from three generation‐3 mothers,
selecting male and female nymphs who had hatched within a week
of each other because sexual and parthenogenetic embryos de‐
velop at different rates (Morgan‐Richards et al., 2010). Of the 30
offspring, 21 were females with genotypes identical to their mother.
Three daughters were not identical to their mother, suggesting they
resulted from fusion of gametes (egg and sperm). These daughters
were heterozygous at five or six loci (depending on their father's
genotype) where their parents differed. All six genotyped sons
were heteroz ygous at these same loci, with the exception of locus
Ch29‐14 (Myers et al., 2017), where all males genotyped had only
the maternal allele observed in the Isles of Scilly, UK population
sample. Examination of our genotyping data for 142 individuals in‐
dicates that locus Ch29‐14 is sex‐linked. The genotype information
confirmed that the males were produced via the fusion of sperm and
egg (i.e., sex), rather than nondisjunction of one of their X chromo‐
somes. Within our nonrandom sample of 30 nymphs hatched in the
same week from three mothers, 21 (70%) were the result of asexual
reproduction (Table S2).
3.1.2 | Mitochondrialdiversity
Mitochondrial DNA (cytochrome oxidase I & II) was sequenced
and aligned with existing data (n= 125; DNA alignmentsavailable
from Dryad: https ://doi.org/10.5061/dryad.b7t80m5), represent‐
ing 101 distinct C. hookerihaplotypesinthe1,389bpalignment.All
specimens of C. hookeri collected from the UK had the same haplo‐
type. Haplotype genealogy (Figure 2) revealed that the UK haplo‐
type joined a cluster of 17 haplotypes from specimens collected in
Taranaki, New Zealand. Despite intensified sampling in this region,
no New Zealand C. hookeri sampled had the UK haplotype that dif‐
fered by three substitutions from the most similar Taranaki haplo‐
type (0.23%; Figure 2d). Male specimens were collected from three
locations contributing to the Taranaki haplotype cluster, where they
were as common as females (n = 28 males, 23 females). Two Taranaki
samples contained only females (Tarata n = 3; Rotorangi n = 6).
All stick insects collected from southern North Island, New
ZealandhadhaplotypesthatarepartoftheparthenogeneticmtDNA
lineage previously identified (pink in Figure 2). This included all spec‐
imens sequenced from the sexual populations at Otaki (n = 5) and
Wilton (n = 14), as expected from their location, but not expected
from their reproductive mode (Table 2). We estimated the age of
themostrecentcommonancestorofthemtDNAlineageassociated
with parthenogenetic reproduction by using rates of molecular evo‐
lution inferred for insects (Table 1). Our oldest estimate of about
170,000 years ago is based on an interspecific rate of nucleotide
substitution (Papadopoulou et al., 2010) and is almost certainly too
old due to the time dependency of the molecular clock (Ho, Phillips,
Cooper, & Drummond, 20 05; Molak & Ho, 2015). Estimates that
suggest the most recent common ancestor of the parthenogenetic
mtDNAlineagewasaliveabout38,000–28,000yearsagoarebased
onintraspecificratesforthesamemtDNAgeneregion,derivedfrom
a lineage sister to the Phasmids (Orthoptera) with similar generation
times (Table 1).
3.1.3 | Populationgeneticstructure
Ten polymorphic nuclear microsatellite loci were amplified and
scored for 111 individuals collected from 15 locations (data available
on Dryad: https ://doi.org/10.5061/dryad.b7t80m5). Repeat amplifi‐
cation and scoring of individuals resolved the same genotypes, sug‐
gesting a low error rate (<0.016). Numbers of alleles per locus ranged
from two to 11. Expected and observed heterozygosity levels were
highest in sexual population samples (Stony Bay) and zero in some
asexual population samples (e.g., Isles of Scilly UK; Manaroa NZ).
The departure of genotype proportions from Hardy‐Weinberg ex‐
pectations was significant (p < .05) in one population sample with a
deficit of heterozygotes, as expected of an automictic parthenogen
(Turitea; Tables 2 and S3). Obser ved heterozygosity was significantly
higher in the six population samples with males (mean = 0.432) than
the six all‐female populations (mean = 0.022; t test p = .0008). The
sexual population sample from Wilton (2016) had fewer alleles and
lower heterozygosity compared to sexual population samples from
further north (Figure 3; only four polymorphic loci, no deviations
from Hardy‐Weinberg expectations; chi‐squared 11.49; probabil‐
ity 0.17; Tables 2 and S3). The Otaki population samples (2003 and
2013) had eight polymorphic loci and genotype proportions met
Hardy‐Weinberg expectations, as did all northern sexual population
samples (Tables 2 and S3).
Our population samples were genetically differentiated from one
another except for just seven (of 91) pairwise comparisons (exac t G
test; p < .05). The highest pairwise FST estimate was between the
UK and Manaroa samples (Table S4). Model‐based assignment of
|
9
MORGAN ‐RICHA RDS et Al.
genotypes to clusters met an optimal fit to the genetic data with
three population clusters (K = 3). Under this model, individuals col‐
lected from the same location had high probabilities of being as‐
signed to the same cluster (Figure 3). Samples from nor thern New
Zealand sexual populations grouped together (Opanuku, Stony Bay,
Karapiro; Figure 3). This cluster of northern sexual populations also
included the three individuals from East Cape, where males have
notbeendetectedandmtDNAhaplotypesare sister totheparthe‐
nogenetic lineage. The UK Isles of Scilly specimens grouped with
high assignment probability with the Taranaki samples, and the
south‐eastern New Zealand populations formed the third group
(Figure 3). A ll 11i ndividuals ( 2003 + 2013) from t he sexual Ot aki
population had low assignment probabilities (0.56–0.69), due to the
presence of alleles t ypical of the south‐eastern asexual lineage and
Taranaki (except for t wo individuals who had alleles typic al of the
northern lineage; Figure 3). By contrast, all the individuals sampled
from Wilton in 2016 clustered with the south‐eastern asexual lin‐
eage, with high assignment probability (0.71–0.99). The small sample
(n = 4) from Wilton 2003 contained some allelic variation (three loci
with two alleles each) but only a single individual (at a single locus)
was heterozygous.
4 | DISCUSSION
The capacity of Phasmids to reap the benefits of asexual reproduc‐
tion are evident in the transition from sexual to parthenogenetic
reproduction inferred for multiple independent lineages around the
world (Scali, 2009; Schwander & Crespi, 2009). For the New Zealand
species Clitarchus hookeri we estimated the most recent common
ancestorofthewidespreadmtDNAlineageassociatedwithparthe‐
nogenetic reproduction to have existed about 38,000–28,000 years
ago. This coincides with the last glacial maximum (Rother et al., 2014)
and is consistent with southward range expansion of this lineage
when land connection allowed passage between North and South
Islands(Trewick&Bland,2012).TheparthenogeneticmtDNAline‐
age is sister to another in East Cape, North Island (Morgan‐Richards
et al., 2010), where a coastal refugia for this species during the LGM
has been inferred (Buckley et al., 2010). The pattern of geographic
parthenogenesis seen in C. hookeri (Morgan‐Richards et al., 2010) is
the expected result of asexual reproduction being under positive se‐
lection when populations are growing during range expansion (Law
& Crespi, 2002).
4.1 | OriginofIslesofScilly(UK)population
To infer the reproductive mode of the ancestral population from
which the UK C. hookeri were derived, we examined genetic varia‐
tion within natural populations in New Zealand. Evidence from both
mitochondrial haplotypes and nuclear genotypes revealed the UK
specimens were genetically most similar to Taranaki stick insects, a
result that is consistent with plant collecting in New Zealand for the
TrescoAbbeyGardens,Isles ofScilly,UK(Godley,1997),providing
an accidental source of stick insect eggs. Three of the five popula‐
tions of C. hookeri sampled in Taranaki had an even sex ratio, and
high heterozygosity, evidence that sexual reproduction is the pre‐
dominant mode of reproduction in that region. In contrast, all
C. hookeri specimens sampled in the Isles of Scilly (UK) were female
andoursamplecontainedneithermtDNAnornucleargeneticvari‐
ation, as expected of an obligate parthenogenetic population. It is
possible that this population resulted from a single female or single
egg transferred with the soil of a plant seedling from New Zealand,
as parthenogenetic reproduction would have provided reproductive
assurance in colonization (Baker's Rule: Baker, 1955).
4.2 | Rapidacquisitionofresistancetofertilisation
by a parthenogen
In sexual populations of C. hookeri, mated females produce only sex‐
ual offspring, although each female is capable of parthenogenetic
reproduction if males are absent (Morgan‐Richards et al., 2010;
Salmon, 1955). Our C. hookeri data suggest that the Isles of Scilly
(UK) population was derived from a sexually reproducing popula‐
tion (Taranaki, NZ), therefore, individuals from the UK population
could be expected to return to 100% sexual reproduc tion if mated.
However, we showed that captive females derived from the UK pop‐
ulation had a barrier to fertilisation although no resistance to mat‐
ing; their eggs and the offspring that hatched were mostly the result
of parthenogenetic reproduction (~97%). Their barrier to fertilisa‐
tion was similar to the ~5% sexually‐produced offspring previously
documented for mated females of the geographic parthenogenetic
lineage in N ew Zealand (Morgan‐ Richards et al., 2010). Although
apparently derived recently (~100 generations) from a Taranaki
population, the UK lineage has developed resistance to fertilisation
without apparent behavioural change. If there are costs associated
with sperm storage or sexual receptivity we would expect selection
to drive decay of sexual traits (Schwander, Crespi, Gries, & Gries,
2013). Therefore, the UK C. hookeri may have lost the propensity
for sexual reproduction through selection involving sexual conflict.
4.3 | Transitionfromparthenogenetictosexual
reproduction
Populations that naturally experience competition between individ‐
uals reproducing in different ways offer opportunities to determine
what factors contribute to the local selective advantage (or disad‐
vantage)ofsexualreproductio n.WeusedmtDN Aandnucl earmark‐
ers to determine the likely origin of two sexual C. hookeri populations
within the range of the NZ parthenogenetic lineage. We expected
that if these sexual populations were derived from long‐distance
dispersal of sexualindividuals (scenario 1: swamping), mtDNAand
nuclear markers would show concordance in the placement of the
samples within the genetic diversity of sexual C. hookeri. In contrast,
the introduction of allopatric males (scenario 2: introgression), would
result in retention oflocal maternal mtDNAandadditionof alleles
from a sexual population. This introgression scenario is what we
10
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MORGAN‐RICH ARDS et Al .
observedinOtaki; malesandfemaleshadmtDNAhaplotypes that
were part of the typical par thenogenetic lineage in this region, but
their nuclear genomes contained alleles from two different geno‐
typic clusters (sexual Taranaki and local parthenogenetic), resulting
in a signal of mixture expressed as low assignment probabilities to
clusters (Figure 3). Population samples separated by 10 years, (20 03
and 2013) indicated introgression and establishment of sexual re‐
production occurred prior to 2003, but high heterozygosity was re‐
tained in 2013.
If males had arisen via spontaneous loss of an X chromosome
in situ (scenario 3: male genesis) only local nuclear alleles and local
mtDNA haplotypes would be expected in their descendants. At
Wilton weobservedjustthispattern;theparthenogeneticmtDNA
haplotype and all nuclear alleles were those expected from the re‐
gion. While Wilton samples from 2003 when males were rare and
2016 when males were common shared the same alleles at all loci,
the 2016 sample had higher heterozygosity (0.08 compared to
0.025). These data suggest the most likely origin of males at Wilton
was through in situ loss of an X chromosome, although we can‐
not exclude introgression and coincidental loss of all novel alleles.
Given our small 2003 sample it is possible that we would miss alleles
from outside the region if invasion involved just a few males. Over
13 years the sex ratio at Wilton changed from highly female skewed
to an equal number of males and females, suggesting a rapid increase
in successful sexual reproduction. Together, these observations sug‐
gest that the sexual populations at Wilton and Otaki constitute two
independent and recent transitions to sexual reproduction from
asexual reproduc tion in C. hookeri.
4.4 | Localadvantagesofsexualreproduction
The best opportunities to understand the advantages of sexual re‐
production over asexual reproduction come from studying natural
replicates of the competition that plays out between sexual and
asexuallineages(Neiman&Schwander,2011).Alocaladvantagefor
a particular reproductive strategy can be inferred when one strat‐
egy switches to another. The all‐female population in the UK was
apparently derived from a population in the Taranaki region of New
Zealand and was therefore likely to have been sexual (or recently
derived f rom a sexual popul ation). Accordingly, we infer a r ecent
change from sexual to asexual reproduction. Intriguingly, our experi‐
mental work suggests this recent switch to asexual reproduction has
been accompanied by a rapid loss of sexual propensity in the form
of a barrie r to fertilisati on. An alternati ve scenario to conside r is
that the C. hookeri population was historically asexual at the time of
transfer to the UK. In this instance the UK population would be de‐
rived from an asexual Taranaki population. This is plausible consider‐
ing we have not sampled the exactUK mtDNAhaplotype in New
Zealand, however, the UK haplotype does nest within the Taranaki
mtDNAdiversity,suggestingeitherarecentswitchtoparthenogen‐
esis in the UK lineage, or a switch to sexual reproduction in some
Taranaki populations. Given our evidence from Otaki and Wilton
males, it is indeed possible that Taranaki populations have switched
from asexual to sexual reproduction, but the nuclear data give no
indication of recent invasion. Despite the reproduc tive history of
the original Taranaki population, finding the UK C. hookeri produced
>90% of their of fspring asexually after mating with conspecifics is
significant considering the promiscuous nature of Clitarchus species
(Langton‐Myers et al., 2019). Conflic t between the sexes over egg
fertilisation might influence the outcome of competition bet ween
reproductive strategies (Burke & Bonduriansk y, 2017; Gerber &
Kokko, 2016; Kawatsu, 2013). Models suggest that par thenogenetic
populations are unlikely to establish in the presence of males if sex‐
ual conflict results in males coercing facultative parthenogens into
sexual reproduction (Kawatsu, 2013, 2015).
Two separate New Zealand populations of C. hookeri provide
evidence that sexual reproduction can replace parthenogenesis de‐
spite the numerical reproductive advantage provided by the latter.
Heterozygote deficit within our parthenogenetic samples suggests
automictic(ratherthanapomictic)reproduction. Aswitchtosexual
reproduction from automictic parthenogenesis provides the benefits
associated with outcrossing; increased heterozygosity and increased
allelic diversity. Evidence for the switch in our study suggests these
benefits can locally outcompete the numerical advantage of par the‐
nogenetic reproduction.
AtOtaki,thesuccessofsexforC. hookeri might be linked to in‐
trogression from allopatric males that resulted in novel genotypes,
increased allelic variation, and higher heterozygosity. By contrast, at
Wilton, putative‐local males with local alleles reveal that rapid and
successful switching to sexual reproduction cannot be attributed
solely to increased allelic diversity provided by outsourced males. In
this study system, we have spatial and temporal sampling to infer the
outcome of competition among reproductive strategies. Continued
observation will reveal whether switches from asexual to sexual
reproduction are stable (Innes & Ginn, 2014) and whether males
continue to expand their range. The value of temporal sampling for
documenting the rapid spread of sex and identifying short‐term local
effects in wild populations are clear. Our study suggests the short‐
term advantages of outcrossing can enable sex to successfully out‐
compete automictic parthenogenetic reproduction. Females with
lower resistance to fertilisation might gain indirect reproductive
success through their sons inheriting an ability to overcome fer til‐
isation barriers (Kawatsu, 2015). This new study system with multi‐
ple independent transitions in reproductive strategy presents a rare
opportunity to focus on location‐specific forces that provide sexual
reproduction with a competitive advantage over parthenogenetic
reproduction.
ACKNOWLEDGEMENTS
Collection and research permits were provided by the Welling ton
City Council, with additional assist ance from Eleanor Burton and
Rewi Elliot . Assista nce with colle cting sp ecimens was p rovided by
Jenny Gillam, Ted and Bee Trewick, Esta Chappell, Mary Robinson,
Ralph Powlesland, Lorraine Peoples, Paul Brock, Malcolm Lee, Mike
Nelhams. Help with insect husbandr y and maintenance of PC2
|
11
MORGAN ‐RICHA RDS et Al.
facilities was provided by Shaun Nielsen, Paul Barrett, Susan Wells,
YurakYeongandJamesConnell.SimonFKHillsprovidedexpertise
with molecular clock analyses. Massey Universit y provided research
funding (MURF –Women's award 2016 to MMR). This manuscript
was improved by constructive criticism from Maurine Neiman and
two anonymous reviewers.
AUTHORCONTRIBUTIONS
M.M.R.,andS.A.T.conceivedtheproject,collectedtheinsectsand
raised their offspring in captivit y. M.M.R . received funding and gen‐
erated genotypes and haplotype sequences. M.M.R., S. A.T., and
S.S.L.‐M.analyseddata.Allauthorscontributedto design andwrit‐
ing the manuscript.
DATAAVA I L AB I LI T YS TATE MEN T
Genbank accession numbers MK532396, MK606153‐MK606171.
Aligned m tDNA sequences an d microsatallite genotyp es are pro‐
vided via Dryad (https ://doi.org/10.5061/dryad.b7t80m5). During
the review process all data can be downloaded from: http://evolv
es.massey.ac.nz/DNA_Toolkit.htm.
ORCID
Mary Morgan‐Richards https://orcid.org/0000‐0002‐3913‐9814
Shelley S. Langton‐Myers https://orcid.org/0000‐0003‐2904‐0981
Steven A. Trewick https://orcid.org/0000‐0002‐4680‐8457
REFERENCES
Baker, H. G. (1955). Self‐compatibilit y and establishment after ‘long dis‐
tance’ dispersal. Evolution, 9, 347–348.
Bandelt, H. J.,Forster,P., & Rohl,A. (1999). Median‐joiningnetworks
for inferring intraspecific phylogenies. Molecular Biology and
Evolution, 16, 37–48. https ://doi.org/10.1093/oxfor djour nals.mol‐
bev.a026036
Barret t, S. C. H. (2002). The evolution of plant sexual diversity. Nature
Reviews Genetics, 3, 274–284. https ://doi.org/10.1038/nrg776
Barton, N. H., & Charlesworth, B. (1998). Why sex and recombina‐
tio n? Science, 281, 1986–1990. https ://doi.org/10.1126/scien
ce. 281 .5 385 .1986
Bast, J.,Parker,D.J.,Jalvingh, K., Dumas, Z., Van Tran, P.,Jaron, K.,…
Schwander, T. (2018). Consequences of asexuality in natural popula‐
tions: Insights from stick insect s. Molecular Biology and Evolution, 35,
1668–1677. https ://doi.org/10.1093/molbe v/msy058
Bouckaert, R.,Heled, J., Kühner t, D.,Vaughan, T.,Wu,C.‐H.,Xie, D.,…
Drummond,A.J.(2014).BEAST2:AsoftwareplatformforBayesian
evolutionary analysis. PLOS Computational Biology, 10, 1–6. https ://
doi.org/10.1371/journ al.pcbi.1003537
Brock, P. D. (1987). A third New Zealand stick insect (Phasmatodea)
established in t he British Isles, with notes on the other species, in‐
cludingacorrection.InM.Mazzini&V.Scali(Eds.),Stick insects phy‐
logency and reproduction: 1st international symposium on stick insect s
(pp. 125–132). Siena, Italy: University of Siena.
Brock, P. D., Lee, M., Morgan‐Richards, M., & Trewick, S. A. (2018).
Missing stickman found: The first male of the parthenogenetic New
Zealand Phasmid Genus Acanthoxyla Uvarov, 1944 discovered in the
United Kingdom. Atropos, 60, 16 –23 .
Buckley, T. R., Marske, K., & Attanayake, D. (2010). Phylogeogr aphy
and ecological niche modelling of the New Zealand stick in‐
sect Clitarchus hookeri (White) support survival in multiple
coastal refugia, Journal of Biogeography, 37, 682–695. https ://doi.
or g /10.1111/ j.13 65‐2 69 9. 20 0 9. 022 39. x
Burke, N. W., & Bonduriansky, R. (2017). Sexual conflict, facultative asex‐
uality, and the true paradox of sex. Trends in Ecology & Evolution, 32,
646–652. https ://doi.org/10.1016/j.tree.2017.06.002
Clarke, T. E., Levin, D. B., Kavanaugh, D. H., & Reimchen, T. E. (20 01).
Rapid evolution in the Nebria gregaria group (Coleoptera: Carabidae)
and the paleogeography of the Queen Charlotte Islands. Evolution,
55, 1408–1418. https ://doi.org/10.1111/j.0014‐3820. 2001.tb006
62.x
Drumm ond, A. J., Ho, S . Y. W., Phillips, M . J., & Rambaut, A . (2006).
Relaxed phylogenetics and dating with confidence. PLOS Biology, 4,
699710. https ://doi.org/10.1371/journ al.pbio.0040088
Drummond, A. J., Rambaut, A., Shapiro, B., & Pybus, O. G. (2005).
Bayesian coalescent inference of past population dynamic s from mo‐
lecular sequences. Molecular Biology and Evol ution, 22, 1185–1192.
htt ps ://doi.org /10.1093/mol be v/msi103
Earl,D.A.,&VonHoldt,B.M.(2012).STRUCTUREHARVESTER:Aweb‐
site and program for visualizing STRUC TURE output and implement‐
ing the Evanno method. Conservation Genetics Resources, 4, 359–361.
htt ps ://doi.org /10.10 07/s12686‐011‐954 8‐7
Evanno, G., Regnaut , S., & Goudet, J. (2005). Detecting the num‐
ber of clusters of individuals using the software STRUCTURE: A
simulation study. Molecular Ecology, 14, 2611–2620. https ://doi.
org/10.1111/j.1365‐294X.2005.02553.x
Excoff ier, L., & Lische r,H. E . L. (2010). Arle quin suite ver 3. 5: A new
series of programs to perform population genetics analyses under
Linux and Windows . Molecular Ecology Resources, 10, 564–567.
https ://doi.org/10.1111/j.1755‐0998.2010.02847.x
Falush, D., Stephens, M ., & Pritchard, J. K. (2007). Inference of popu‐
lation structure using multilocus genotype data: Dominant markers
and null alleles. Molecular Ecology Notes, 7, 574–578. https ://doi.
org /10.1111/j.1471‐8286. 20 07.0175 8. x
Gerber, N., & Kokko, H. (2016). Sexual conflict and the evolution of asex‐
uality at low population densities. Proceedings of the Royal Society B:
Biological Sciences, 283(1841), 20161280. https ://doi.org/10.1098/
rspb.2016.1280
Ghisel li, F.,M ilani, L., S cali, V., & Passam onti, M. (20 07). The Leptynia
hispanica species complex (Insecta Phasmida): Polyploidy, partheno‐
genesis, hybridization and more. Molecular Ecology, 16 , 4256–4268.
https ://doi. org/10.1111/j .1365‐294X. 20 07.03 471. x
Gibson,A.K.,Xu,J.Y.,&Lively,C .M.(2016).Within‐populationcovari‐
ation bet ween sexual reproduction and susceptibility to local para‐
sites. Evolution, 70, 20 49–2060. ht tps ://doi.o rg/10.1111/evo.130 01
Gilber t,K.J.,Andrew,R. L., Bock,D.A .N.G., Franklin,M .T.,Moore,
B., Kane, N. C.,… Vines, T.H. (2012). Recommendations for uti‐
lizing and reporting population genetic analyses: The repro‐
ducibility of genetic clustering using the program STRUCTURE.
Molecular Ecology, 21, 4925–49 30. ht tps ://doi. org/10.1111/
j.1365‐294X.2012.05754.x
Godley,E.J.(1997).The1907expeditiontotheAucklandandCampbell
Islands, and an unpublished repor t by B.C. A ston. Tu atara, 33,
13 3 –1 57.
Gratton, P.,Konopiński,M. K., & Sbordoni,V. (2008). Pleis tocene evo‐
lutionary history of the Clouded Apollo (Parnassius mnemosyne):
Genetic signatures of climate cycles and a ‘time‐dependent’ mi‐
tochondrial substitution rate. Molecular Ecology, 17, 4248–4262.
https ://doi.org/10.1111/j.1365‐294X.2008.03901.x
Haag‐Liautard, C ., Coffey, N., Houle, D., Lynch, M., Charles worth, B., &
Keightley,P.D.(2008).Direct estimation ofthemitochondrialDNA
12
|
MORGAN‐RICH ARDS et Al .
mutati on rate in Drosophila melanogaster. PLOS Biology, 6, 1706–1714.
https ://doi.org/10.1371/journ al.pbio.0060204
Ho,S.Y.W.,Phillips,M.J.,Cooper,A.,&Drummond,A .J.(2005).Time
dependency of molecular rate estimates and systematic overestima‐
tion of recent divergence times. Molecular Biology and Evolution, 22,
1561–1568. ht tps ://doi.org/10.1093/molbe v/msi145
Innes, D. J ., & Ginn, M. (2014). A pop ulation of sexua l Daphnia pulex
resist s invasion by asexual clones. Proceedin gs of the Royal Society
B: Biological Sciences, 281, 20140564. ht tps ://doi.org /10.1098/
rspb.2014.0564
Jewell, T.,&Brock, P.D.(2002). A reviewof the New Zealandstick in‐
sects: New genera and synonymy, keys, and a catalogue. Journal of
Orthoptera Research, 11, 189–197. https ://doi.org/10.1665/1082‐
6467(2002)011[0189:AROTNZ]2.0.CO;2
Jokela, J., Dybdahl, M. F., & Lively, C. M . (2009). The maintenance of sex ,
clonal dynamics, and host‐parasite coevolution in a mixed population
of sexual and asexual snails. The American Naturalist, 174(S1), S43–
S53. ht tps ://doi.or g/10.1086/59908 0
Kawatsu, K. (2013). Sexual conflict over the maintenance of sex: Effects
of sexually antagonistic coevolution for reproductive isolation of
parthenogenesis. PLoS ONE, 8, 1–11. https ://doi.org/10.1371/journ
al.pone.0058141
Kawatsu, K. (2015). Breaking the parthenogenesis fertilization barrier:
Direct and indirect selection pressures promote male fertilization off
parthenogenetic females. Evolutionary Ecology, 29, 49–61. https ://
doi.org /10.10 07/s10682‐0 14‐9749‐0
Kearney, M. (2005). Hybridization, glaciation and geographical parthe‐
nogenesis. Trends in Ecology a nd Evolution, 20, 495–502. https ://doi.
org/10.1016/j.tree.2005.06.005
Kearse,M.,Moir,R.,Wilson,A.,Stones‐Havas,S.,Cheung,M.,Sturrock,
S., … Drummond, A . (2012). Geneious basic: An integrated andex‐
tendable desktop software platform for the organization and anal‐
ysis of sequence data. Bioinformatics, 28, 1647–1649. https ://doi.
org/10.1093/bioin forma tics/bts199
Lang ton‐Myers, S. S., Holwell, G. I., & Bu ckley, T. R. (2019). Weak prem at‐
ing isolation between Clitarchus stick insect species despite diver‐
gent male and female genital morphology. Journal of Evolutiona ry
Biology, 32, 398–411. https ://doi .org/10.1111/ jeb.13 424
Law, J. H., & Crespi, B. J. (2002). The evolution of geographic par theno‐
genesis in Timema walking‐sticks. Molecular Ecology, 11, 1471–1489.
https ://doi.org/10.1046/j.1365‐294X.2002.01547.x
Leigh,J.W.,&Bryant ,D.(2015).P OPART:Full‐featuresoftwa reforhap‐
lotype network construction. Methods in Ecology and Evolution, 6,
1110–1116. https : //doi.o rg /10.1111 /20 41‐210X .12410
Mantovani,B.,&Scali,V.(1992).Hybridogenesisandandrogenesisinthe
stick‐insect bacillus rossius‐grandii benazzii (Insecta, Phasmatodea).
Evolution, 46, 783–796.
Maresc alchi, O., & Sc ali, V. (2003). Au tomictic pa rtheno genesis in the
diploid‐triploid stick insect Bacillus atticus and its flexibility lead‐
ing to heterospecific diploid hybrids. Invertebrate Reproduction
& Development, 43, 163–172. https ://doi.org/10.1080/07924
259.20 03.9652535
Maynard Smith, J. (1978). The evol ution of sex. Cambridge, UK: Cambridge
University Press.
Mirzaghaderi, G., & Hör andl, E. (2016). The evolution of meiotic sex and
its alternatives. Proceedings of the Royal Society B: Biological Scie nces,
283(1838), 20161221. https ://doi.org/10.1098/rspb.2016.1221
Molak, M.,&Ho,S.Y.W.(2015).Prolongeddecayofmolecularr atees‐
timatesformetazoanmitochondrialDNA.Pe erJ , 3, e821. https ://doi.
org /10.7717/pee rj.821
Morgan ‐Richards, M ., & Trewick, S. A . (2005). Hybr id origin of a par‐
thenogenetic genus? Molecular Ecology, 14, 2133–2142. https ://doi.
org/10.1111/j.1365‐294X.2005.02575.x
Morgan‐Richards, M., Trewick , S. A., & Stringer, I. A. N. (2010).
Geographic parthenogenesis and the common tea‐tree stick insect
of New Zealand. Molecular Ecology, 19, 1227–1238. https ://doi.
org /10.1111/j.1365 ‐294X .2010 .0 4542.x
Myers, S. S., Buck ley, T. R., & Holwell, G. I. (2015). Mate detection an d
seasonal variation in stick insect mating behaviour (Phamatodea:
Clitarchus hookeri). Behaviour, 152, 1325–1348. https ://doi.
org/10.1163/15685 39X‐00003281
Myers, S. S., Holwell, G. I., & Buckley, T. R. (2017). Genetic and morpho‐
metric data demonstrate alternative consequences of secondary
contac t in Clitarchus stick insects. Journal of Biogeography, 44, 2069–
2081. https ://doi.org/10.1111/jbi.13004
Myers,S.S.,Trewick,S.A.,&Morgan‐Richards,M.(2013).Multiplelines
of evidence suggest mosaic polyploidy in the hybrid par thenogenetic
stick insect lineage Acanthoxyla. Insect Conservation and Diversity, 6,
537–54 8. htt ps : //doi.org /10.1111/icad.12 00 8
Nakano,M.,Morgan‐Richards,M.,Godfrey,A.J.R.,&ClavijoMcCormick,
A. (2019). Par thenogenetic females of the stick insect Clitarchus
hookeri maintain sexual traits. Insects, 10(7), 202.
Neiman, M., Meirmans, P. G., Schwander, T., & Meirmans, S. (2018). Sex
in the wild: How and why field‐based studies contribute to solving
the problem of sex. Evolution, 72, 1–28. htt ps ://doi.o rg /10.1111/
evo.13 485
Neiman, M., & Schwander, T. (2011). Using parthenogenetic lineages to
identif y advantages of sex. Evolution ary Biology, 38, 115–123. https
://doi. org/10.1007/s11692‐011‐9113‐z
Ney,G ., Frederick, K.,&Schul, J. (2018).Apost‐pleistocene calibrated
mutation rate from insect museum specimens. PLoS Currents, 10.
https ://doi.org/10.1371/curre nts.tol.aba55 7de56 be881 79326 1f7e1
565cf35
Otto, S. P. (2009). The evolutionar y enigma of sex. The American
Naturalist, 174(S1), S1–S14. https ://doi.org /10.108 6/59 90 84
Papado poulou, A ., Anas tasiou, I ., & Vogler, A. P. (2010). Revisit ing the
insectmitochondrialmolecularclock:TheMid‐Aegeantrench cali‐
bration. M olecular Biology and Evolution, 27, 1659–1672. https ://doi.
org /10.1093/mol be v/m sq 051
Pijnacker,L.P.,&Ferwerda,M.A.(1980).Sexchromosomesandoriginof
males and sex mosaics of the parthenogenetic stick insect Carausius
morosus Br. Chromosoma, 114, 10 5–114. htt ps ://doi.or g/10.10 07/
BF 003 28 476
Posada, D. (2008). jModelTest: Phylogenetic model averaging. Molecular
Biology and Evolution, 25, 1253–1256. https ://doi.org/10.1093/
molbe v/msn083
Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference of pop‐
ulation structure using multilocus genotype data. Genetics, 155,
945–9 59.
Rambaut,A.,&Drummond,A.J.(2007).Tracer v1.5. Retrieved from ht tp.
beast.bio.ed.ac.uk/Tracer
Raymond, M., & Rousset, F. (1995). An exact test for popula‐
tion differentiation. Evolution, 49, 1280–1283. https ://doi.
org /10.1111/j.1558 ‐56 46 .1995.tb 04 4 56.x
Rother, H., Fi nk, D., Shulmei ster, J., Mifsud, C ., Evans, M., & Pu gh, J. (2014).
The early rise and late demise of New Zealan d's last glacial maximum.
Proceedi ngs of the National Academy of Sciences of the United States
of America, 111, 11630–11635 . ht tp s ://doi. org/10 .1073/p nas.14015
47111
Rousset, F. (2008). GENEPOP' 007: A complete re‐implementation of
the GENEPOP soft ware for W indows and Linux. Molecular Ecology
Resources, 8, 103–106. https ://doi.o rg /10.1111 /j.1471‐828 6. 20 07.
01931 .x
Rousset, F., & Raymond, M. (1995). Testing heteroz ygote excess and de‐
ficiency. Genetics, 140 , 1413–1419.
Salmon, J. T. (1955). Parthenogenesis in New Zealand stick insec ts.
Transactions of the Royal Society of New Zealand Zoolog y, 82,
1189–1192.
Salmon, J. T. (1991). The stick insect s of New Zealand. Auckl and, New
Zealand: Reed.
|
13
MORGAN ‐RICHA RDS et Al.
Scali,V.(2009).Met asexualstickinsects:Modelpathwaystolosingsex
andbringingitback.InI .Schön, K. Mar tens &P.V.Dijk (Eds.), Lost
sex (pp. 317–345). Dordrecht , the Netherlands: Springer. https ://doi.
or g/10.10 07/97 8‐90 ‐481‐277 0‐2
Schwander, T., & Crespi, B. J. (2009). Multiple direct transi‐
tions from sexual reproduction to apomictic parthenogenet‐
sis in Timema stick insects. Evolution, 63, 84–103. https ://doi.
org /10.1111/j.1558 ‐56 46 .200 8. 00 524. x
Schwander, T., Crespi, B. J., G ries, R ., & Gries, G. (2013). Neutral and
selection‐driven decay of sexual traits in asexual s tick insects.
Proceedi ngs of the Royal Society B: Biologica l Science s, 280 (1764) ,
20130823. https ://doi.org/10.1098/rspb.2013.0823
Schwander, T., Henry, L., & Crespi, B. J. (2011). Molecular evidence for
ancient asexuality in Timema stick insects. Current Biology, 21, 1129–
1134. https ://doi.org/10.1016/j.cub.2011.05.026
Simon,C.,Frati,F.,Beckenbach,A.,Crespi,B.,Liu,H.,&Flook,P.(1994).
Evolution, weighting, and phylogenetic utilit y of mitochondrial gene
sequences and a compilation of conser ved polymerase chain reac‐
tion primers. Annals of the Entomologica l Society of America, 87, 651–
701. https ://doi.org/10.1093/aesa/87.6.651
Stringer,I. A.N.(1970). The nymphal and imaginal stagesofthebisex‐
ual stick insect Clitarchus hookeri (Phasmidae: Phasminae). New
Zealand Entomologist, 4, 85–95. https ://doi.org/10.1080/00779
962.1970.9722927
Sunnuck, P., & Hales, D. F. (1996). Numerous transposed sequences of
mitochondrial cytochrome oxidase I‐II in aphids of the genus Sitobion
(Hemiptera:Aphididae).Molecular Bi ology and Evolution, 13, 510–524.
https ://doi.org/10.1093/oxfor djour nals.molbev.a025612
Tabata, J., Ichiki, R. T., Tanaka, H., & Kageyama, D. (2016). Sexual versus
asexual reproduction: Distinct outcomes in relative abundance of
parthenogenetic mealybugs following recent colonization. PLoS ONE,
1, e0156587. https ://doi.org/10.1371/journ al.pone.0156587
Tavaré,S.(1986).Someprobabilisticandstatisticalproblemsintheanal‐
ysisofDNAsequences.Lectures on Mathematics in Life Sciences, 17,
57– 86.
Trewick,S.A.,&Bland, K.J.(2012).Fire andslice:Palaeogeographyfor
biogeogr aphy at New Zealand's North Island/South Island juncture.
Journal of the Royal Society of New Zealand, 6758 , 153–183. https ://
doi.org /10.1080/03036 758. 2010.549493
van der Kooi, C. J., & Schwander, T. (2014). Evolution of sexuality via
different mechanisms in grass thrips (Thysanoptera: Aptinothrips).
Evolution, 68, 1883–1893.
Wallace, C. (1992). Parthenogenesis, sex and chromosomes in
Potamopyrgus. Journal of Molluscan Studies, 58, 9 3 –10 7.
Wu, C., Twort, V.G., Crowhurst, R. N., Newcomb,R. D., & Buckley, T.
R. (2017). A ssembling lar ge genomes: Ana lysis of the stick i nsect
(Clitarchus hookeri) genome reveals a high repeat content and sex‐bi‐
ased genes associated with reproduc tion. BMC Genomics, 18, 1–15.
https ://doi.org/10.1186/s12864‐017‐4245‐x
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