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Abstract

The outcome of competition between different reproductive strategies within a single species can be used to infer selective advantage of the winning strategy. Where multiple populations have independently lost or gained sexual reproduction it is possible 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 mtDNA lineage that otherwise characterizes parthenogenesis in this species. We infer independent origins of males at these two locations using microsatellite genotypes. In one population, a mixture of local and non‐local alleles suggested males were the result of invasion. Males in another population were most likely 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 remarkable evidence of repeated and rapid changes in reproductive strategy, with competitive outcomes dependent on local conditions. This article is protected by copyright. All rights reserved.
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:21June2018 
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  Revised:17Ju ne2019 
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  Accepted:30July2019
DOI : 10.1111 /mec.152 03
ORIGINAL ARTICLE
Loss and gain of sexual reproduction in the same stick insect
MaryMorgan‐Richards1| Shelley S. Langton‐Myers2|StevenA.Trewick1
Thisisanop enaccessarticleundert hetermsoftheCreat iveCommonsAttributio n‐NonCommercialLicense,whichpermit suse,dis tributionandreproduction
in any medium, provided the original work is prop erly cited and is not used for commercia l purpos es.
©2019TheAuthors.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
MasseyUniversit y,Grant/AwardNumber:
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).Anoptimalstudysystemwould
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‐
neticpopulationsbuthavethesameallelesandmtDNAhaplotypes
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
mtDNAfrom thelocalparthenogeneticlineage. 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 haplotypesfrom the locallineage,butobserved 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‐
pansionbyasinglemtDNAlineage(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 | MATERIALSANDMETHODS
2.1 |Clitarchus hookeri
The commonNew Zealand mānukastick insect (the“smooth stick
insect ” in the UK ) is frequently found on the Myrtaceae species
Leptospermum scoparium (mānuka) and Kunzeasp.(kānuka),col
lectivelyknown as tea‐tree. Adult female mānukastick 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
theTrescoAbbeyGardensin1940,wherenumerousplantspecies
FIGURE 2 Similarit yofmtDNA
sequences indicates the probable
origins of Clitachus hookeri stick insect
populations. (a) Sample locations in
New Zealand, coloured to highlight two
intraspecificmtDNAlineagesasshown
in b (pink par thenogenetic; blue from
Taranaki). (b) Phylogenetic relationships
offullmtDNAdiversitysampledinNew
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 | Breedingexperiments
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
Hemisphereautumn2013(August,September,October).Onimpor 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).Aswithgeneration‐2,we obser vedcopulation (Februaryand
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 | Naturalpopulations
We collected stick insects from six locations within the New Zealand
rangeof the parthenogenetic mtDNA lineage(Morgan‐Richardset
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,wecollectedadultinsectsinautumn(April2003)
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)withintheOtari‐Wilton'sBushnativeforestremnant
(=Wilton). The recently planted mānukaand kānukaon 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 | DNAextraction,amplificationandsequencing
We dissected leg muscle from fresh, frozen, or alcohol‐preserved
C. hookeri,and extractedgenomic DNA using asalting‐outmethod
(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-
neiousv9(Kearseetal.,2012),andexcludedthetRNA‐Leugenebe
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‐
terizednuclearDNAvariationat10independentmicrosatelliteloci
(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
GenescanLiz‐500(AppliedBiosystems)asaninternalsizestandard.
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 | Populationgeneticanalyses
2.5.1 | MitochondrialDNA
WeexaminedmtDNACOIvariationfor125individuals(46newse
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,
implementingaGTR+I+GmodelofDNAevolution(Tavaré,1986).
Weusedsubsets of the mtDNAhaplotypedata (1,307 bp) to infer
median‐joining networks (Bandelt, Forster, & Rohl, 1999) using
POPART(Leigh&Bryant,2015).
2.5.2 | Lineageageestimation
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 threeratesbasedon observedin
traspecificnucleotide mutations(Gratton,Konopiński,&Sbordoni,
2008; Haag‐Liaut ard et al., 2008; Ney, Frederick, & Schul, 2018; see
Table1). A relaxed,uncorrelated, lognormal molecular clock model
(Drummond, Ho, Phillips, & Rambaut, 2006) was applied, in order to
6 
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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).Analternativemodel,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 | Microsatelliteloci
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.
Astheseparthenogeneticstickinsects couldpotentiallyreproduce
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,wescoredthesex‐linkedlocus
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 Theageofthemostrecentcommonancestor(MRCA)oftheparthenogeneticlineageofClitarchus hookeriwasestimatedusingfiveinsectmitochondrialDNAmutationrates
derived from different taxa and calibrations
Ratetype Tax a mtDNAgenes
Age of calibra‐
tion (years)
Substitutions per site
per million years Reference
Ageofpar thenogenMRCA
Constantpopsize
(million years) SE of mean
Bayesianskyline
(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: Thestickinsectdatasethad101haplotypesand1,350bpofCOI‐COIImtDNAsequence.MolecularclockanalysesusedastrictclockandeitheraconstantpopulationsizeoraBayesianskyline
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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 7
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)
mtDNAparthenoge
netic clade
Microsatellites
Inferredreproductive
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 DeltaK (Evanno, Regnaut, & Goudet, 20 05).
3 | RESULTS
3.1 | Transitiontoasexualreproduction
3.1.1| Breedingexperiments
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
hookeriontheIslesofScilly.Allnymphsthathatched(generation‐2)
were female (n = 677). Ten adult females of generation‐2 laid eggs
after mating with conspecific New Zealand males (n = 201). These
eggshatchedinto82daughtersbutnosons(generation‐3).Atotalof
13 adult females of this third generation laid 492 eggs after mating,
whichhatched44 0daughtersandsevensons.Althoughwehaddata
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 | Mitochondrialdiversity
Mitochondrial DNA (cytochrome oxidase I & II) was sequenced
and aligned with existing data (n= 125; DNA alignmentsavailable
from Dryad: https ://doi.org/10.5061/dryad.b7t80m5), represent
ing 101 distinct C. hookerihaplotypesinthe1,389bpalignment.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
ZealandhadhaplotypesthatarepartoftheparthenogeneticmtDNA
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
themostrecentcommonancestorofthemtDNAlineageassociated
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
mtDNAlineagewasaliveabout38,000–28,000yearsagoarebased
onintraspecificratesforthesamemtDNAgeneregion,derivedfrom
a lineage sister to the Phasmids (Orthoptera) with similar generation
times (Table 1).
3.1.3 | Populationgeneticstructure
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
    
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 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
notbeendetectedandmtDNAhaplotypesare sister totheparthe
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 11i 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
ancestorofthewidespreadmtDNAlineageassociatedwithparthe
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).TheparthenogeneticmtDNAline
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 | OriginofIslesofScilly(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
TrescoAbbeyGardens,Isles ofScilly,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
andoursamplecontainedneithermtDNAnornucleargeneticvari
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 | Rapidacquisitionofresistancetofertilisation
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 | Transitionfromparthenogenetictosexual
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)ofsexualreproductio n.WeusedmtDN Aandnucl earmark
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 sexualindividuals (scenario 1: swamping), mtDNAand
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 oflocal maternal mtDNAandadditionof alleles
from a sexual population. This introgression scenario is what we
10 
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   MORGAN‐RICH ARDS et Al .
observedinOtaki; malesandfemaleshadmtDNAhaplotypes 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 weobservedjustthispattern;theparthenogeneticmtDNA
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 | Localadvantagesofsexualreproduction
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
asexuallineages(Neiman&Schwander,2011).Alocaladvantagefor
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 exactUK mtDNAhaplotype in New
Zealand, however, the UK haplotype does nest within the Taranaki
mtDNAdiversity,suggestingeitherarecentswitchtoparthenogen
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(ratherthanapomictic)reproduction. Aswitchtosexual
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.
AtOtaki,thesuccessofsexforC. 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
    
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 11
MORGAN ‐RICHA RDS et Al.
facilities was provided by Shaun Nielsen, Paul Barrett, Susan Wells,
YurakYeongandJamesConnell.SimonFKHillsprovidedexpertise
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.
AUTHORCONTRIBUTIONS
M.M.R.,andS.A.T.conceivedtheproject,collectedtheinsectsand
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.analyseddata.Allauthorscontributedto design andwrit
ing the manuscript.
DATAAVA I L AB I LI T YS 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_Toolkit.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
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https ://doi.org/10.1186/s12864‐017‐4245‐x
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SS,TrewickSA.Lossandgainofsexualreproduc tioninthe
same stick insect. Mol Ecol. 2019;00:1–13. ht t ps : //doi.
org /10.1111/mec.1520 3
... In some species, rare spontaneous males can also be produced from unfertilized eggs (e.g. Carson, 1967;Morgan-Richards et al., 2019;Pijnacker, 1969). Intriguingly, allfemale populations often persist over many generations (Burns et al., 2018;Morgan-Richards, 2023;Tsurusaki, 1986), even in close proximity to mixed-sex populations (Miller et al., 2024a). ...
... These processes could all contribute to the maintenance of reproductive mosaics in facultative systems, but their roles remain unknown. While previous studies have reported barriers to fertilization in all-female populations of facultative parthenogens (Larose et al., 2023;Morgan-Richards et al., 2019), no previous study has investigated the contribution of both evolutionary (i.e., genotypic) changes in all-female populations and existing maternal effects in mixed-sex populations to geographical parthenogenesis. ...
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Facultatively parthenogenetic animals could help reveal the role of sexual conflict in the evolution of sex. Although each female can reproduce both sexually (producing sons and daughters from fertilized eggs) and asexually (typically producing only daughters from unfertilized eggs), these animals often form distinct sexual and asexual populations. We hypothesized that asexual populations are maintained through female resistance as well as the decay of male traits. We tested this via experimental crosses between individuals descended from multiple natural sexual and asexual populations of the facultatively parthenogenic stick-insect Megacrania batesii. We found that male-paired females descended from asexual populations produced strongly female-biased offspring sex-ratios resulting from reduced fertilization rates. This effect was not driven by incompatibility between diverged genotypes but, rather, by both genotypic and maternal effects on fertilization rate. Furthermore, when females from asexual populations mated and produced sons, those sons had poor fertilization success when paired with resistant females, consistent with male trait decay. Our results suggest that resistance to fertilization resulting from both maternal and genotypic effects, along with male sexual trait decay, can hinder the invasion of asexual populations by males. Sexual conflict could thus play a role in the establishment and maintenance of asexual populations.
... This reduction in heterozygosity is expected to expose deleterious recessive alleles as well as to deplete allelic diversity, thereby resulting in reduced performance in the short term and reduced ability to adapt to environmental change on longer timescales. However, while the genomic consequences of automixis have been investigated in the laboratory (reviewed in Jaron et al. 2021) or modeled mathematically (Engelstädter 2017), much less is known about how such changes are manifested in wild populations, and even less is known about their phenotypic and fitness consequences in natural environments (but see Morgan-Richards et al. 2019;Jaron et al. 2022). A number of studies have compared the performance of sexual and asexual animals in the laboratory (Browne et al. 1988;Kenny 1996;Cullum 1997;Mee et al. 2011;Sukumaran and Grant 2013), but given the strong environment dependence of life history and fitness (Saether and Engen 2015), there is a need for research on wild populations in fully natural environments. ...
... Spontaneous male genesis is possible in diploid species with XX XO sexual karyotypes, where meiotic/developmental error results in the loss of one X chromosome in parthenogenically produced XX offspring (Pijnacker and Ferwerda 1980;Scali 2009). The viability and reproductive functionality of spontaneous males have not been studied in any phasmid (see Morgan-Richards et al. 2019). However, in the obligate asexual snail Potamopyrgus antipodarum, low rates of male production and reduced functionality in asexually produced males were found to make male invasion in asexual populations unlikely (Neiman et al. 2012;Jalinsky et al. 2020). ...
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Transitions from sexual to asexual reproduction have occurred in numerous lineages, but it remains unclear why asexual populations rarely persist. In facultatively parthenogenetic animals, all-female populations can arise when males are absent or become extinct, and such populations could help to understand the genetic and phenotypic changes that occur in the initial stages of transitions to asexuality. We investigated a naturally occurring spatial mosaic of mixed-sex and all-female populations of the facultatively parthenogenetic Australian phasmid Megacrania batesii. Analysis of single-nucleotide polymorphisms indicated multiple independent transitions between reproductive modes. All-female populations had much lower heterozygosity and allelic diversity than mixed-sex populations, but we found few consistent differences in fitness-related traits between population types. All-female populations exhibited more frequent and severe deformities in their (flight-incapable) wings but did not show higher rates of appendage loss. All-female populations also harbored more ectoparasites in swamp (but not beach) habitats. Reproductive mode explained little variation in female body size, fecundity, or egg hatch rate. Our results suggest that transitions to parthenogenetic reproduction can lead to dramatic genetic changes with little immediate effect on performance. All-female M. batesii populations appear to consist of high-fitness genotypes that might be able to thrive for many generations in relatively constant and benign environments but could be vulnerable to environmental challenges, such as increased parasite abundance.
... Analogous situations were studied in parthenogenetic stick insects. Post-mating barrier to fertilisation were found in some lineages 103 , while parallel decay of female pheromones and contact signals used in communication with males, sperm storage organs, and lost the ability to fertilise eggs was found in other lineages 104 . Nevertheless, the easiest strategy seems to be an allotopy with respect to related sexual forms, for example, by expanding their range beyond the boundaries of parental species distribution. ...
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Obligatory parthenogenesis in vertebrates is restricted to squamate reptiles and evolved through hybridisation. Parthenogens can hybridise with sexual species, resulting in individuals with increased ploidy levels. We describe two successive hybridisations of the parthenogenetic butterfly lizards (genus Leiolepis) in Vietnam with a parental sexual species. Contrary to previous proposals, we document that parthenogenetic L. guentherpetersi has mitochondrial DNA and two haploid sets from L. guttata and one from L. reevesii, suggesting that it is the result of a backcross of a parthenogenetic L. guttata × L. reevesii hybrid with a L. guttata male increasing ploidy from 2n to 3n. Within the range of L. guentherpetersi, we found an adult tetraploid male with three L. guttata and one L. reevesii haploid genomes. It probably originated from fertilisation of an unreduced triploid L. guentherpetersi egg by a L. guttata sperm. Although its external morphology resembles that of the maternal species, it possessed exceptionally large erythrocytes and was likely sterile. As increased ploidy level above triploidy or tetraploidy appears to be harmful for amniotes, all-female asexual lineages should evolve a strategy to prevent incorporation of other haploid genomes from a sexual species by avoiding fertilisation by sexual males.
... However, these dispersal methods are unlikely in R. mikado since their eggs do not possess either a sponge-like structure or the ability to stick to the branches. While anthropogenic translocation is another possible explanation [65,72,73], it is highly improbable that ancient anthropogenic translocation occurred tens of thousands or even thousands of years ago, given the limited association between R. mikado and humans. Thus, the somewhat plausible human-mediated dispersal of R. mikado is accidental long-distance transportation with plant seedlings in the past few centuries, coinciding with the advent of steam engines. ...
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Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect Ramulus mikado across Japan, based on a multifaceted molecular approach [cytochrome oxidase subunit I (COI) haplotypes, nuclear simple sequence repeat markers and genome-wide single nucleotide polymorphisms]. Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.
... However, such a re-expression of the sexual pathway would require the molecular bases underlying the sexual functions of both sexes to be maintained. Re-expression of sex is therefore possible at least shortly after the transition to parthenogenesis, which may also have been the case in the stick insects Clitarchus hookeri (Morgan-Richards et al., 2019) and Bacillus rossius (de Vichet, 1944). Occasional male production and re-expression of the sexual pathway in otherwise obligate parthenogens paves the way for cryptic sex, which could contribute to explaining their long-term persistence (Freitas et al., 2023). ...
... /2022 chromosome can be lost in situ in parthenogenically produced offspring, resulting in a genetic male (Pijnacker & Ferwerda, 1980;Scali, 2009). The viability and reproductive success of spontaneous males has not been studied in any phasmid species, so the ability for spontaneous males to successfully turn an established asexual population to sexual is currently unknown (see Morgan-Richards, Langton-Myers & Trewick, 2019). However, in an obligate asexual marine snail Potamopyrgus antipodarum, low rates of male production and reduced functionality in asexually-produced males were found to make male invasion in asexual populations unlikely (Neiman et. ...
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Transitions from sexual to asexual reproduction have occurred in numerous lineages across the tree of life but many questions remain about how such transitions occur and why asexual populations rarely persist. In facultatively parthenogenetic animals, all-female populations can arise when males are absent or become extinct and, if such populations can persist asexually for many generations, they could ultimately give rise to obligately asexual species. However, the initial stages of this process remain poorly understood. The facultatively parthenogenetic Australian phasmid Megacrania batesii exhibits a spatial mosaic of mixed-sex populations that reproduce predominantly sexually and all-female sites that reproduce exclusively via parthenogenesis. We used this system to compare genetic and phenotypic parameters among multiple natural populations that vary in reproductive mode and geographic location. Analysis of single nucleotide polymorphisms (SNPs) collected from reduced representation whole genome data showed populations grouping by geographic location rather than reproductive mode, with little gene-flow between them. Mixed-sex populations had drastically higher heterozygosities than all-female populations. Phenotypic analysis revealed considerable inter-population variation in rates of deformities in (non-functional) wings, with higher mean frequency and severity of deformities in all-female sites. All-female sites also harboured more parasites, but only in swamp habitats. However, reproductive mode did not explain variation in mean female fecundity, egg hatching success, or missing appendages. These results indicate that local transitions to parthenogenetic reproduction can lead to increased rates of developmental abnormalities and increased vulnerability to disease but can also occur without substantial fitness consequences despite dramatic reductions in genetic diversity and heterozygosity.
... Numerous animal lineages have undergone evolutionary transitions from sexual to asexual reproduction, but many questions remain about how such transitions occur. In facultatively parthenogenetic animals, female individuals are capable of both sexual and asexual reproduction: females that mate produce both sons and daughters from fertilised eggs (although mated females can also produce some offspring parthenogenetically, as occurs in Timema stick insects; Arbuthnott et al., 2015;Schwander et al., 2010), while females that avoid mating usually produce only daughters that develop parthenogenetically from unfertilised eggs (although it is possible for virgin females to occasionally produce sons parthenogenetically through the rare loss of an X chromosome, as occurs in a number of stick insect species; Brock et al., 2012;Morgan-Richards et al., 2019;Pijnacker, 1969). In facultatively parthenogenetic organisms, allfemale populations can be established if females do not mate. ...
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In facultatively parthenogenetic populations, the prevalence of sexual reproduction depends on whether females mate and therefore produce sons and daughters or avoid mating and produce daughters only. The relative advantage of mating in such species may depend on a female's own reproductive origin (i.e. development from a fertilised or unfertilised egg) if parthenogenesis reduces heterozygosity similar to sexual inbreeding, or if it inhibits mating, sperm storage or fertilisation. But effects of reproductive origin on development and performance are poorly understood. Using the facultatively parthenogenetic stick insect, Extatosoma tiaratum, we quantified morphology, mating probability, and reproductive success in mated versus unmated females of sexual versus automictic (parthenogenetic) origin. We found strong evidence that increased homozygosity negatively impacted some traits in parthenogenetically produced females: compared to sexually produced females, parthenogenetically produced females were smaller and more prone to deformities in vestigial wings, but not more prone to fluctuating asymmetry in their legs. Parthenogenetically produced females received fewer mating attempts and avoided mating more often than sexually produced females. Yet, contrary to the expectation that sex should rescue parthenogenetic lineages from the detrimental effects of increased homozygosity, parthenogenetically produced females gained no net reproductive benefit from mating, suggesting that physiological constraints limit fitness returns of sexual reproduction for these females. Our findings indicate that advantages of mating in this species depend on female reproductive origin. These results could help to explain spatial distributions of sex in facultatively parthenogenetic animals and evolutionary transitions to obligate asexuality. Read the free Plain Language Summary for this article on the Journal blog.
... Despite many New Zealand taxa apparently surviving in numerous 'microrefugia' during the glacial periods of the Pleistocene (Wallis and Trewick 2009;Wood et al. 2017), so far, few concordant hybrid zones that date to LGM divergence have been detected. Some forest species may have survived the LGM only in northern and coastal New Zealand and expanded south and inland during interglacials (Buckley et al. , 2010Marshall et al. 2009;Morgan-Richards et al. 2019). Either there was no opportunity for populations from alternative refugia to meet or hybrid zones have not yet to be been identified where these lineages made contact. ...
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Hybridisation is commonly observed in geographical zones of contact between distinct lineages. These contact zones have long been of interest for biogeographers because they provide insight into the evolutionary and ecological processes that influence the distribution of species as well as the process of speciation. Here we review research on hybrid zones and zones of past introgression, both terrestrial and marine, in Aotearoa New Zealand. Many of New Zealand’s hybrid zones occur between lineages or species that diverged prior to the Last Glacial Maximum (LGM), with numerous divergences dating to the early Pleistocene or Pliocene. Few secondary contact zones have been detected in terrestrial plants and in marine taxa. This may reflect a lack of the intensive sampling required to detect hybrid zones in these groups but for plants may also indicate widespread Pleistocene survival across the country. Lastly, we suggest avenues for research into New Zealand hybrid zones that are likely to be fruitful.
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Why and how sexual reproduction is maintained in natural populations, the so‐called “queen of problems”, is a key unanswered question in evolutionary biology. Recent efforts to solve the problem of sex have often emphasized results generated from laboratory settings. Here, we use a survey of representative “sex in the wild” literature to review and synthesize the outcomes of empirical studies focused on natural populations. Especially notable results included relatively strong support for mechanisms involving niche differentiation and a near absence of attention to adaptive evolution. Support for a major role of parasites is largely confined to a single study system, and only three systems contribute most of the support for mutation accumulation hypotheses. This evidence for taxon specificity suggests that outcomes of particular studies should not be more broadly extrapolated without extreme caution. We conclude by suggesting steps forward, highlighting tests of niche differentiation mechanisms in both lab and nature and empirical evaluation of adaptive evolution‐focused hypotheses in the wild. We also emphasize the value of leveraging the growing body of genomic resources for non‐model taxa to address whether the clearance of harmful mutations and spread of beneficial variants in natural populations proceeds as expected under various hypotheses for sex. This article is protected by copyright. All rights reserved
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Background Stick insects (Phasmatodea) have a high incidence of parthenogenesis and other alternative reproductive strategies, yet the genetic basis of reproduction is poorly understood. Phasmatodea includes nearly 3000 species, yet only the genome of Timema cristinae has been published to date. Clitarchus hookeri is a geographical parthenogenetic stick insect distributed across New Zealand. Sexual reproduction dominates in northern habitats but is replaced by parthenogenesis in the south. Here, we present a de novo genome assembly of a female C. hookeri and use it to detect candidate genes associated with gamete production and development in females and males. We also explore the factors underlying large genome size in stick insects. Results The C. hookeri genome assembly was 4.2 Gb, similar to the flow cytometry estimate, making it the second largest insect genome sequenced and assembled to date. Like the large genome of Locusta migratoria, the genome of C. hookeri is also highly repetitive and the predicted gene models are much longer than those from most other sequenced insect genomes, largely due to longer introns. Miniature inverted repeat transposable elements (MITEs), absent in the much smaller T. cristinae genome, is the most abundant repeat type in the C. hookeri genome assembly. Mapping RNA-Seq reads from female and male gonadal transcriptomes onto the genome assembly resulted in the identification of 39,940 gene loci, 15.8% and 37.6% of which showed female-biased and male-biased expression, respectively. The genes that were over-expressed in females were mostly associated with molecular transportation, developmental process, oocyte growth and reproductive process; whereas, the male-biased genes were enriched in rhythmic process, molecular transducer activity and synapse. Several genes involved in the juvenile hormone synthesis pathway were also identified. Conclusions The evolution of large insect genomes such as L. migratoria and C. hookeri genomes is most likely due to the accumulation of repetitive regions and intron elongation. MITEs contributed significantly to the growth of C. hookeri genome size yet are surprisingly absent from the T. cristinae genome. Sex-biased genes identified from gonadal tissues, including genes involved in juvenile hormone synthesis, provide interesting candidates for the further study of flexible reproduction in stick insects. Electronic supplementary material The online version of this article (10.1186/s12864-017-4245-x) contains supplementary material, which is available to authorized users.
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Documenting natural hybrid systems builds our understanding of mate choice, reproductive isolation, and speciation. The stick insect species Clitarchus hookeri and C. tepaki differ in their genital morphology and hybridize along a narrow peninsula in northern New Zealand. We utilize three lines of evidence to understand the role of premating isolation and species boundaries: (1) genetic differentiation using microsatellites and mitochondrial DNA; (2) variation in 3D surface topology of male claspers and 2D morphometrics of female opercular organs; and (3) behavioral reproductive isolation among parental and hybrid populations through mating crosses. The genetic data show introgression between the parental species and formation of a genetically variable hybrid swarm. Similarly, the male and female morphometric data show genital divergence between the parental species as well as increased variation within the hybrid populations. This genital divergence has not resulted in reproductive isolation between species, instead weak perimating isolation has enabled the formation of a hybrid swarm. Behavioral analysis demonstrate that the entire mating process influences the degree of reproductive isolation between species undergoing secondary contact. Mechanical isolation may appear strong, while perimating isolation is weak. This article is protected by copyright. All rights reserved.
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Theory suggests that occasional or conditional sex involving facultative switching between sexual and asexual reproduction is the optimal reproductive strategy. Therefore, the true ‘paradox of sex’ is the prevalence of obligate sex. This points to the existence of powerful, general impediments to the invasion of obligately sexual populations by facultative mutants, and recent studies raise the intriguing possibility that a key impediment could be sexual conflict. Using Bateman gradients we show that facultative asexuality can amplify sexual conflict over mating, generating strong selection for both female resistance and male coercion. We hypothesize that invasions are most likely to succeed when mutants have negative Bateman gradients, can avoid mating, and achieve high fecundity through asexual reproduction – a combination unlikely to occur in natural populations.