Research Article Journal of Orthoptera Research 2018, 27(2): 97-106 Viability and fertility of hybrid New Zealand tree weta Hemideina spp. (Orthoptera: Anostostomatidae) NATASHA E. MckEAN!, STEVEN A. TREwWICK!, MELISSA J. GrRiFFIN', Eopy J. DowLe!, MArY MorGAN-RICHARDS'! 1 Ecology Group, Institute of Agriculture and Environment, Massey University, Palmerston North 4442, New Zealand. Corresponding author: Natasha E. Mckean (tarsha9990 @gmail.com) Academic editor: Corinna S. Bazelet | Received 10 June 2017 | Accepted 21 January 2018 | Published 29 June 2018 http://zoobank.org/D3780808-6977-4DF8-9920-89096FBBBCF1 Citation: Mckean NE, Trewick SA, Griffin MJ, Dowle EJ, Morgan-Richards M (2018) Hybrid viability and fertility of New Zealand tree wéta Hemideina spp. (Orthoptera: Anostostomatidae). Journal of Orthoptera Research 27(2): 97-106. https://doi.org/10.3897/jor.27.14963 Abstract Natural hybridization between species provides an opportunity to study the mechanisms that maintain independent lineages and may help us understand the process of speciation. The New Zealand tree wéta spe- cies Hemideina thoracica produces F, hybrids where it lives in sympatry with two closely related species: Hemideina crassidens and Hemideina trewicki. This study looked at the viability and fertility of F, hybrid weta between H. thoracica and H. crassidens that were collected from the wild and kept in captivity. The hybrids appeared to have normal viability from the late juvenile stage, with all male wéta maturing at a late instar. Male F, hybrids displayed normal mating behavior and one male produced offspring in captivity. In contrast to Haldane’s rule, female F, hybrids appeared to be infertile; they refused to mate and did not produce eggs. No evidence of Wolbachia infection was identified in any of the three North Island Hemid- eina species. Key words Haldane’s rule, Hybridization, introgression, sexual exclusion, Wolbachia Introduction Natural hybridization between species provides an opportu- nity to study the mechanisms that maintain independent lineages and may help us understand the process of speciation (Butlin 1987, Barton and Gale 1993). Hybrid animals often have lower fitness than individuals of their parent species (Harrison 1993 and references therein) as a result of incompatible genetic combina- tions, such as Dobzhansky-Muller incompatibilities (Dobzhansky 1937, Muller 1942, Bolnick and Near 2005), mismatched chro- mosomes (Shaw and Wilkinson 1980, Searle 1993), incompatible cellular organelles (e.g. mitochondria: Ellison and Burton 2008) and symbiotic bacteria (e.g. intracellular Wolbachia infections: Bor- denstein et al. 2001; gut bacteria: Brucker and Bordenstein 2013). However, low hybrid fitness also arises through natural and sexual selection when an intermediate phenotype is a disadvantage (Sve- din et al. 2008). If hybrids are viable they might nevertheless have limited fer- tility. If fertility of F, hybrids is very low, fertility levels usually improve in subsequent generations of backcross hybrids (Mallet et al. 1998, Mallet 2005, Descimon and Mallet 2009), and even limited fertility provides the possibility for introgression that can have important consequences for species interactions. Adaptive alleles that arise in one population/species can be transferred to another through hybridization, known as adaptive introgression, as seen in wing coloration in Heliconius butterflies (Pardo-Diaz et al. 2012). In some cases, when hybridization reduces fertility a unimodal hybrid (or tension) zone forms. Unimodal hybrid zones are geographically constrained; with most individuals in the zone having mixed ancestry, and the width of the hybrid zone depending on hybrid disadvantage and dispersal of the species (Jiggins and Mallet 2000). If hybridization is more limited, a bi- modal hybrid zone may result, where parental forms overlap and predominate with a few individuals of mixed ancestry amongst them. Bimodal hybrid zones are typically associated with assorta- tive mating so, in tree weta, reproductive character displacement is a likely outcome that increases assortative mating (Dieckmann and Doebeli 1999, Jiggins and Mallet 2000). Where two species compete for the same resources, hybridization may prevent one from successfully out-competing the other. Alternatively sexual exclusion, where one species (often the males of that species) out-competes the other for mates, can limit fitness through re- productive interference (Gréning and Hochkirch 2008), as ob- served between the tetrigids: Tetrix ceperoi and Tetrix subulata (Hochkirch et al. 2007). Tree weta (Orthoptera: Anostostomatidae: Hemideina) are a ge- nus of seven nocturnal arboreal insects, with high morphological and ecological similarity (Field and Bigelow 2001, Dewhurst 2012, Bulgarella et al. 2014). They are hypothesized to have speciated in allopatry during the Pliocene or earlier (Trewick and Morgan- Richards 2005), and they now have broadly parapatric distribu- tions (Trewick and Morgan-Richards 1995, Bulgarella et al. 2014). Hybridisation in the wild between the New Zealand tree wéta Hemideina thoracica (White) and both Hemideina crassidens (Blan- chard) and Hemideina trewicki Morgan-Richards has recently been confirmed with genetic data, but so far only F, hybrids have been confirmed (Mckean 2014, Mckean et al. 2016). These three Hemid- eina species each have distinct karyotypes (i.e. different numbers of JOURNAL OF ORTHOPTERA RESEARCH 2018, 27(2) 98 chromosomes with some differing in size and shape; Morgan-Rich- ards 1995, 1997, 2000, Mckean et al. 2015). Karyotype differences are generally seen as presenting barriers to gene flow by disrupting meiosis and rendering F, hybrids infertile. However, some tree wéta species naturally comprise multiple chromosome races that are ca- pable of interbreeding in the wild (Morgan-Richards 1997, 2000, Morgan-Richards et al. 2000, Morgan-Richards and Wallis 2003). The apparent tolerance of chromosome rearrangements displayed in this orthopteran lineage might influence fertility of interspecies hybrids. Karyotype, mtDNA haplotypes, and alleles at four nuclear DNA loci were found to differentiate parent populations of H. tho- racica and H. trewicki in a large area of sympatry in Hawke’s Bay. These markers (except mtDNA, which is maternally inherited) were heterozygous in individuals who were phenotypically intermediate in abdominal coloration (orange rather than yellow or brown), ab- dominal bands (faint rather than striking or non-existent), abdomi- nal stripe (a series of spots rather than a stripe or the absence of a stripe) and the number of spines on the prolateral hind tibia (typi- cally between the three spines seen in H. thoracica and the four in H. crassidens/H. trewicki, with a half-sized medial spine on each leg being common, or three spines on one leg and four on the other). A similar situation was seen in the Manawatu area of sympatry be- tween H. thoracica and H. crassidens, where karyotype, mtDNA and three nuclear DNA markers were found to differentiate the two spe- cies (with some introgression detected relative to allopatric popu- lations). All individuals, which had an intermediate phenotype (the same phenotype as for H. thoracica x H. trewicki hybrids), were heterozygous for these markers (Mckean et al. 2016). Whether hy- bridization occurs between H. crassidens and H. trewicki is currently unknown due to the morphological similarities of these two spe- cies, and unknown distribution boundaries due in part to clearance of native forest where the two are hypothesized to have historically met (Trewick and Morgan-Richards 1995). A lack of gene flow sug- gests that H. thoracica x H. trewicki hybrids, which are found at a frequency of 1% of wéta in sympatry, are infertile, but genetic and morphological data suggest a low, but potentially significant, level of introgression between H. thoracica and H. crassidens, where hy- brid frequency is ~3 in every 100 wéta (Mckean et al. 2016). Introgression is the signal of past hybridization, and an abil- ity to successfully hybridize might be of fundamental importance to the future of a species, while climates and environments con- tinue to change (Grant and Grant 1993, Allendorf et al. 2001, Becker et al. 2013, Taylor et al. 2015, Sivyer et al. 2018). There is evidence that H. crassidens formerly occupied much of central North Island that is now the range of H. thoracica (Bulgarella et al. 2014). Isolated populations of H. crassidens remain in regions of high elevation, which suggests they are adapted to colder en- vironments. With global warming, H. crassidens might continue to be displaced, but this depends on the ecological and sexual interaction between species. Adult tree wéta often form harems in tree cavities during the summer and autumn (Wehi et al. 2013), and mixed species harems in areas of overlap suggest that spe- cies recognition is not complete (Trewick and Morgan-Richards 1995, Wehi et al. 2017). Of F, hybrids collected in the wild, the majority had an H. thoracica father and H. crassidens mother (Mc- kean et al. 2016), which suggests sexual exclusion by H. thoracica males; as in Hemideina spp. the females do not appear to actively choose their mates. Although females will sometimes resist mat- ing, resistance times are similar whether mating occurs or not (Field and Jarman 2001). This gives rise to the possibility that hybridization may be an important factor in the coexistence/ex- clusion of these species. N.E. MCKEAN, S.A. TREWICK, M.J. GRIFFIN, E.J. DOWLE AND M. MORGAN-RICHARDS Haldane (1922) observed that where one sex is absent, rare, or infertile in F, hybrids, it is usually the heterogametic sex. In tree weta this is the male, as tree weta, like most Orthoptera, have an XO sex determination system where females have two copies of the sex chromosome and males one (White 1940, Morgan-Richards 1997, Morgan-Richards and Wallis 2003). Based on this, if there is a difference between the sexes, we would expect male F, hybrids to have lower viability and/or fertility than female F, hybrids. Given their apparent tolerance of karyotype variation, the high degree of infertility in wéta might have another source. Wolbachia is an endosymbiotic intracellular bacteria that infects a large propor- tion of the arthropod and nematode phyla (Werren et al. 2008). In arthropods, Wolbachia is estimated to infect about 65% of spe- cies (Hilgenboecker et al. 2008) including many grasshoppers and crickets (Werren and Windsor 2000, Mandel et al. 2001, Bella et al. 2010). Wolbachia is known to manipulate the reproductive biology of many of its hosts to its own advantage (Werren et al. 2008). Some of the currently known host-reproductive manipula- tions include male killing, induction of parthenogenesis, femini- zation of genetic males, forced production of haploid individuals in haplodiploid systems, and cytoplasmic incompatibility (Wer- ren 1997). Wolbachia is hypothesized to have a role in arthropod speciation via induction of cytoplasmic incompatibility (Werren 1998 and references therein). Wolbachia infections appear to be re- sponsible for maintaining hybrid zones between the well-studied grasshopper subspecies Chorthippus parallelus parallelus and Chort- hippus parallelus erythropus via two different forms of cytoplasmic incompatibility (Bella et al. 2010). Although recently detected in New Zealand insects (Bridgeman et al. 2018), it is not known whether the tree wéta lineage (with many hybrid zones; Morgan- Richards and Wallis 2003) contains this intracellular parasite. Here, we describe the viability and fertility of hybrids between Hemideina thoracica and H. crassidens, using F, hybrids collected in the wild and held in captivity. We sought evidence of Wolbachia infections to assess whether this common intracellular parasite has potential to limit reproductive compatibility among these weta species. Methods Sampling and captive conditions.—Eleven F, hybrid tree weéta were captured from native forest in Turitea Valley (S40.47184, E175.60943) and Kahutawera Valley (S40.431725, E175.674595), Manawatu, New Zealand (Fig. 1, Table 1). Hybrid identity was tested and confirmed using genetic markers for eight of the 11 in- dividuals which died or were euthanized during the course of this study, and were preserved as specimens in alcohol (Table 1; F, hy- brids; Morgan-Richards 1995, Mckean et al. 2016). The other three putative hybrid individuals were assumed to be F, hybrids as their phenotypes were completely consistent with the F, hybrids that had been genetically identified. No cryptic hybrids were identi- fied in previous studies of tree wéta from this population (Trewick and Morgan-Richards 1995, Morgan-Richards and Gibbs 2001, Bulgarella et al. 2014, Mckean et al. 2016). Live wéta were held in individual containers at a constant temperature of 14°C. They were given a suitable daytime roost cavity made from harakeke (Phormium tenax) flower stalk, and were fed palatable leaves from at least three native plant species each week and 80% soy protein pellets (Griffin et al. 2011). Body size of F, hybrids.—No significant difference in body size be- tween adult females of the two parent species has been found in JOURNAL OF ORTHOPTERA RESEARCH 2018, 27(2) N.E. MCKEAN, S.A. TREWICK, M.J. GRIFFIN, E.J. DOWLE AND M. MORGAN-RICHARDS z= North Island ® Hemideina thoracica crassidens X thoracica @ Hemideina crassidens South Island i | @ Hemideina trewicki 99 Fig. 1. Distribution of the three North Island New Zealand species of tree wéeta (Hemideina) and an H. thoracica x H. crassidens F,, hybrid. The distributions of the species were taken from Morgan-Richards and Wallis (2003) and Morgan-Richards (2000). Table 1. Sampling information, size and results for mating behavior in both sexes and egg production in hybrid females. weta Hybrid 1 Hybrid 2 Hybrid 3 Hybrid 4 Hybrid 5 Hybrid 6 Hybrid 7 Hybrid 8 Hybrid 9 Hybrid 10 Hybrid 11 Legend: *See Table 3, + See section ‘Mating Behavior’ in methods. : Genetically Tibia Length Instar at . Age since Maturity Sample Location ; Sex Age . Behavior ; Confirmed Hybrid (mm) Maturity (Final Molt) Live Kahutawera valley Yes M Adult 23.63 10 Normal; mated* NA Live Turitea valley Yes M Adult 24.01 10 Normal; mated* NA Live Turitea valley Yes M Adult 23.62 10 Normal; mated* NA Live Kahutawera valley Yes F Adult 22°92 10 Resisted Mating+ 6 months Live Kahutawera valley No F Adult 21.26 10 Resisted Mating+ 4 months Live Kahutawera valley No F Adult 23.76 10 Partial Resistance+ 3 months Preserved Kahutawera valley Yes F Adult 2126 10 NA 6 months Preserved Kahutawera valley No F Adult 2S 10 NA 3 months Preserved Kahutawera valley Yes M Juvenile 16 10 NA NA Live Kahutawera valley Yes M Sub-adult 18.42 10 NA NA Preserved Kahutawera valley Yes M Adult 2d 10 NA NA JOURNAL OF ORTHOPTERA RESEARCH 2018, 27(2) Z Z Z Ze 2 2, Le ZL, Z Z, *Z, > > > 00000 FS > DB ts P 100 this zone of sympatry (Mckean et al. 2016). To detect signs of im- paired growth (hybrid inviability) the hind tibia length of both dead and living adult hybrids was measured with electronic cali- pers and compared via ANOVA to wild adult females of both par- ent species measured in a previous study (15 H. thoracica and 19 H. crassidens; Mckean et al. 2016), and to a separate sample of adult males from both species (25 H. thoracica and 22 H. crassidens), that were sampled from the same locations as the hybrids. Hind tibia length is a reliable proxy for body size in tree wéta (Minards et al. 2014, Bulgarella et al. 2014.) The sex of hybrids and instar at maturity for male wéta were both recorded. Maturity is deter- mined in tree wéta by the shape and size of the cerci or ovipositor. Tibia length data for each sex were compared via ANOVA. Mating behavior.—Six hybrid wéta (three males, three females) were provided with one potential mate of each parent species, on different nights, in a Perspex tank (60 cm x 60 cm x 60 cm) (Table 1). Mating trials were observed for 30 min in the evening when tree wéta are most active (Kelly 2006a). For male wéta, success- ful transfer of spermatophores was recorded as well as attempts to mate, defined as curling the abdomen to position for mating. Other mating behavior prior to this, such as following the female or rapid twitching of the palps that indicated the male had scent- ed the female, and running the palps over the female’s abdomen, were recorded (Field and Jarman 2001 and references therein). As male mating behavior has been well described elsewhere, the male F, hybrids’ behavior was compared to what is known from pre- vious work which details the parental species’ behavior. Female tree wéeta do not appear to actively choose or approach male wéta (Field and Jarman 2001 and references therein), so their accept- ance or active resistance to mating was recorded. Resistance was defined as any behavior that appeared to obstruct mating attempts by the male including moving away, stridulating (a defensive/ag- gressive gesture in tree weta; Field 2001, Field and Glasgow 2001), and biting and kicking the male to dislodge him. Acceptance was defined as the female staying still and allowing copulation to be initiated and completed, as evidenced by the successful transfer of a spermatophore. Egg production.—Females of both parent species begin producing eggs as soon as they reach maturity (N.E.M. personal observation, >50 females 2012-2013). Eggs inside the ovarioles of mature fe- males typically vary in developmental stage and range from very small undeveloped yellow eggs through to large black mature eggs with a thick outer casing (Griffin 2011). After laying, the embryo case expands and turns from black to brown and eventually yellow (Stringer 2001). Four F, hybrid adult females and 18 H. crassidens females were given soil slightly deeper than the length of the ovi- positor to lay eggs in (Table 2). Conditions were otherwise the same as detailed in captive conditions above. After approximately 100 days (StDev = 35.9) the eggs laid were removed and counted. Each wéta was euthanized, dissected and the number of unlaid mature eggs counted under a dissecting microscope. Additional data were obtained from a preserved hybrid female euthanased before she laid eggs (n=5 in Table 2). Male fertility —Two adult F, hybrid males, which were adults at the time of the study, were each provided with virgin females of both parent species, as above (Table 3). They were observed until a mating occurred and then left together in the tank overnight. Female wéta were removed the next morning and placed in a con- tainer with a layer of soil slightly deeper than the length of the N.E. MCKEAN, S.A. TREWICK, M.J. GRIFFIN, E.J. DOWLE AND M. MORGAN-RICHARDS Table 2. Average number of eggs +/- standard deviation for H. crassidens females vs. F, hybrid females. Sample Age since Eggs Eggs Eggs size maturity (days) (unlaid) (laid) (total) H. crassidens 18 201 +/- 70.7 26+/-30.9 65+/-32 91+/- 26.5 F, Hybrids 5 139 +/- 47.8 0 0 0 Table 3. Results of captive breeding experiments with F, hybrid H. thoracica x H. crassidens fathers and mothers of both parent spe- cies. Growth of eggs was both physical expansion and changing color from black to brown or yellow. Male Female No. Eggs Laid Growth Hatched H. crassidens 50 Yes 0 Hybrid 1x — H. crassidens 35 Yes 0 H. thoracica 111 Yes 0 ) H. thoracica oF Yes 4 Hybrid 2 x ; H. crassidens ovipositor. After a period of oviposition the female was removed, the eggs counted and placed back into the soil. As little is known about triggers for embryo growth and hatching in wéta, the eggs were stored outside, exposed to the ambient winter temperature fluctuations experienced by the wild population from which they were derived. Expansion and hatching were recorded the follow- ing summer (approximately 9 months after laying). Wolbachia detection.—Two methods were used to obtain evidence of infection by the bacteria Wolbachia: amplification of DNA se- quences using Wolbachia specific Polymerase Chain Reactions (PCR) primers, and whole genome sequencing and alignment to a reference Wolbachia genome. For amplification of specific Wol- bachia DNA sequences, DNA was extracted from three tree wéta specimens representing each of the three North Island species (H. thoracica, H. crassidens and H. trewicki). Tissue was taken from the hind femur and testes or ovariole of each tree wéta specimen and DNA isolated using a salting out method (Trewick and Morgan- Richards 2005). Wolbachia-specific primers (Appendix 1) were used in PCR with wéta DNA, and DNA from an introduced gregarious parasitoid wasp (Nasonia vitripennis) known to be infected with Wolbachia as a positive control. Standard PCR conditions for these primers were followed (Braig et al. 1998, Heddi et al. 1999, Baldo et al. 2006) (Appendix 1). PCRs were repeated to rule out prob- lems with reaction conditions. One PCR product longer than the expected Wolbachia fragment from the CoxA primer pair was am- plified. This long DNA fragment was sequenced at the Massey Ge- nome Service with a capillary AB13730 Genetic Analyzer (Applied Biosystems Inc.), and then visualized and trimmed in Geneious 6.1.7 (Biomatters LTD; Kearse et al. 2012) software. The resulting 269 bp sequence was compared to public databases using the Ba- sic Local Alignment Search Tool (BLAST) algorithm on the NCBI website. Total genomic DNA from two tree wéta specimens (an H. thoracica male collected from the Kahutawera Valley and an H. crassidens male collected from a South Island population) were separately processed through parallel, high-throughput sequencing (Illumina HiSeq 2500) for a separate phylogenetic study (Dowle 2013). Briefly, DNA was extracted from a single male individual (testes tissue), fragmented, prepared using the ThruPLEX DNA-seq Kit (Rubicon Genomics) and used to generate 100 bp paired-end JOURNAL OF ORTHOPTERA RESEARCH 2018, 27(2) N.E. MCKEAN, S.A. TREWICK, M.J. GRIFFIN, E.J. DOWLE AND M. MORGAN-RICHARDS sequence on a Hi-Seq 2000 (BGI). This resulted in 5,191,884 100 bp paired-end sequences 200 bp apart for the H. thoracica speci- men and 17,434,429 100 bp paired-end sequences for the H. crassidens specimen. An annotated reference Wolbachia genome was obtained from New England Biolabs (http://tools.neb.com/ wolbachia, originating from infection of Brugia malayi; Foster et al. 2005). Reads were trimmed to remove index sequences using sol- exaQA (Cox et al. 2010) before mapping to the Wolbachia genome using the default settings with Bowtie 2 (Langmead and Salzberg 2012). Results were visualised with Tablet v1.7.0_35 (Milne et al. 2010). Sequences that matched parts of the Wolbachia genome were compared with published data using the NCBI (National Library of Medicine) GenBank BLAST search algorithm to determine their similarity to Wolbachia DNA sequences from other hosts. This ena- bled us to determine whether the sequences came from the Wol- bachia genome or another related bacterial species, which could be determined by sequence similarity. Results Phenotype F, hybrids.—Hybrids were identified by genetic markers and intermediate phenotypes, and no morphologically cryptic hybrids were identified (Mckean et al. 2016). The sex ratio of F, hybrids in our small sample was even (five females, six males). All but two hybrid wéta examined were adults (or reached adulthood in captivity - two wéta) providing no evidence of reduced hybrid viability. There was no significant size difference between adult F, hybrid females and adult females of the two parent species from the same location with ANOVA; F = 2.575, P = 0.09 (Fig. 2A), how- ever male F, hybrids were significantly larger than males of either parent species (ANOVA; F = 8.969, P = 0.00049; Fig. 2B). The five adult male hybrids matured at the tenth instar as determined by comparing their hind tibia lengths to data of wéta trimorphism in Hemideina crassidens (Kelly and Adams 2010, Bulgarella et al. 2015). Although one male did not reach maturity (Hybrid 10; Table 1), as a ninth instar sub-adult he would have been an adult at the tenth instar, as determined by growth/size charts from previous studies (Spencer 1995, Kelly and Adams 2010). A 2 24 i La Tibia Length (mm) Ro Ro ho = 20 19 H. thoracica H. crassidens F1 Hybrids 101 Mating behavior.—All three F, hybrid males mated with females of both species (Table 1). Each male exhibited normal and similar mating behavior to females of both species he was housed with (Field and Jarman 2001 and references therein), and was accepted by females of both species. In contrast, two of three hybrid females actively resisted mating. The third allowed the H. thoracica male to begin copulation several times, but then dislodged him and pro- ceeded to bite him and display other resistance behaviors. She al- lowed mating to occur once with the H. crassidens male, and then resisted all subsequent mating attempts, and was the only hybrid female wéta that was observed to accept a spermatophore. Fertility. —None of the five female F, hybrids contained eggs in any stage of development when killed and dissected as adults. This contrasts with 18 H. crassidens females that each laid and/or con- tained an average of 91 eggs (Table 2). Females that were mated to the hybrid males laid 35-111 eggs (except one H. crassidens female that died soon after mating with Hybrid 2). Some eggs from every female showed signs of expansion after 6 — 8 months, with many eggs increasing in size and changing color from black to light brown or yellow (Table 3). Four eggs by male Hybrid 2 and his H. thoracica female mate expanded and then hatched to produce off- spring. The nymphs were inferred to be phenotypically normal, as no obvious morphological differences were seen under a dissect- ing microscope. The color of nymphs is uniformly grey (dorsal) and yellowish white (ventral) at this stage regardless of species, so no inferences could be drawn about eventual color phenotype (whether the F, generation look the same as F,, or resemble the weta of the parent species). No other eggs hatched during the study, including the eggs produced by the control wéta (Table 3). Wolbachia.—The fbpA and Wol16S primers failed to amplify a DNA fragment when used with tree wéta DNA, but produced a DNA fragment with the positive control (a wasp know to be in- fected with Wolbachia). The Wsp and CoxA primers gave a series of weakly amplified DNA fragments longer than that expected from the Wolbachia genome. A consistent DNA fragment amplified with the CoxA primers was 200 bp longer than the positive control. 24> 22> 20° 18 16 H. thoracica H. crassidens F1 Hybrids Fig. 2. A. Tibia length of adult female F, hybrids compared with adult females from the two parent species, showing no significant difference; B. Tibia length of F, hybrid males compared with males of the two parent species, showing a significant difference: p-value = 0.0001. JOURNAL OF ORTHOPTERA RESEARCH 2018, 27(2) 102 No close sequence match was found when compared to DNA se- quences on the database Genbank, including Wolbachia sequences. None of the > 17 million H. crassidens next-generation short read DNA sequences mapped to the Wolbachia genome. Howev- er, eight 100 bp DNA sequences from genomic H. thoracica DNA shared similarity with Wolbachia. Six identical DNA sequence reads mapped to one location, all with the same ten mismatches. The other two reads mapped to a different location on the Wol- bachia genome, differing at nine sites (mismatches). However, the paired-end for all eight of these sequence reads (100-300 bp downstream from the putative-bacteria DNA sequence) did not map to the Wolbachia genome sequence. Comparing the pu- tative Wolbachia sequences to the Genbank database identified these sequences as: 1) 93% similarity with the 16S rRNA gene from various members of the Chlamydiae phylum, with six of these matches belonging to the Rhabdochlamydia genus, and 2) 93% match for three 28S gene fragments from Simkania negeven- sis, which also belongs to the Chlamydiae phylum. As similarity with Wolbachia sequences was lower (90-91%), it is likely that the H. thoracica wéta was infected with a bacteria species from the chlamydia family, not closely related to Wolbachia. Both the 16S and 28S rRNA genes are highly conserved among bacteria, and of the > 22 million DNA short-sequences from the wéta none mapped to Wolbachia-specific regions of the Wolbachia ge- nome. A separate study of other Orthoptera confirmed that this level of data was sufficient for detection of Wolbachia infections (Bridgeman et al. 2018). Discussion The size of H. thoracica x H. crassidens hybrids fell within the normal range expected for the parent species (with males at the larger end), and many hybrids were found as adults in the wild, therefore we have no positive evidence of hybrid inviability or abnormal development. There could be some inviability early in development, during the pre-hatching or early instar phases, but it appears that at least by the time F, hybrids have reached the larger instars (5" to 7"), they are as successful as a typical wéta of either parent species. Female tree wéta all mature at the tenth instar but males can mature at the eighth, ninth or tenth instar (Spencer 1995, Kelly and Adams 2010), resulting in a wide size range of adult males. All F, hybrid males in this study matured at the tenth instar, which may be important in understanding their reproductive success (if any). Male tree wéta compete for females via competition for tree cavities (resources) that females use as refuges during the day (Spencer 1995, Field 2001). Tenth instar males have much larger mandibles than eighth instar adult males, which are used during male-male competition, but which also limit their mobility, leading to the hypothesis that smaller males actively search for and mate with females which are away from their tree cavities foraging at night (Field 2001, Kelly 2004, Kelly 2006a, Kelly and Adams 2010). If male F, hybrids all mature at a later instar, it is unclear whether this is an advantage or disadvan- tage for reproductive success. As the ratios of other male wéta ma- turing at different instars may play a part in determining success in controlling harems, more research into reproductive success among males in this location is needed to determine the outcome of these 10“ instar hybrid males. Our observations of mating were limited to experimental pairs (rather than harems, which are common in the wild; Wehi et al. 2013) and this might have influenced the behaviors exhib- ited and observed. Female wéta were not given a choice of mate, N.E. MCKEAN, S.A. TREWICK, M.J. GRIFFIN, E.J. DOWLE AND M. MORGAN-RICHARDS which could have also influenced mating behavior. However, mating behavior appeared to be normal for our limited sample of F, hybrid males when paired with adult H. thoracica and H. crassidens females. One of the hybrid males had been found in the wild with a harem consisting of two adult H. crassidens fe- males. This male produced offspring in captivity, hence it is likely that this male, along with at least some others, are behaving in the wild in a manner typical of males from the parent species. One significant limitation of this study is the lack of control mat- ing crosses and mating behavior comparisons for parental spe- cies from the same populations, as previous mating studies were conducted with Hemideina crassidens from southern populations (Field and Jarman 2001, Kelly 2006b, c). There have been few studies of H. thoracica mating behavior (Wehi et al. 2013) and there is a general lack of understanding of mating outcomes in this lineage (Field and Jarman 2001). Unfortunately, the trig- gers for embryo development and hatching are unknown for tree weta, making laboratory crosses difficult and prone to fail- ure, as evidenced by the control wéta embryos failing to hatch. Therefore no inferences can be drawn about the success of these crosses relative to parental crosses, but given that laboratory crosses are sometimes successful when virgin females are mated to single males, it does not appear that sperm competition has to take place in these species to induce fertility in females (Morgan- Richards 2000, Stringer 2001, present study), and can probably be ruled out as an explanation for lack of fertility. In contrast to the males, the female F, hybrids did not show typical mating behavior, but this may be irrelevant to fertility if they cannot produce eggs. The lack of egg production in all five F, female hybrids is probably biologically important, despite the small sample, because it contrasts with that observed in adult H. crassidens females kept in the same conditions (Table 2). A lack of eggs was also never observed in more than 50 mature par- ent females of both species that were dissected (N.E.M. personal observation, 2012-2013). The absence of eggs suggests that F, females may typically be infertile, whereas at least some F, male hybrids are fertile, as was evident from the offspring produced in captivity. Male F, hybrids being partially fertile while females are in- fertile contrasts with the usual variation between the sexes in re- duced fertility (Haldane’s rule) and may be of interest for future research. Haldane’s rule applies across many animal taxa, includ- ing others with a XO sex determination system (Haldane 1922, and one analysis found that it applied in 99% of 223 cases of sex-specific hybrid sterility and 90% of 115 cases of sex-specific hybrid inviability (Laurie 1997). Infertility as opposed to invi- ability appears to be the most normal sex-skewed outcome, as heterogametic infertility is known to outnumber heterogametic inviability about 10:1 in Drosophila and mammals (Wu and Da- vis 1993). The mechanism behind Haldane’s rule is still unclear although two main hypotheses are X chromosome to autosome imbalance, and incompatibilities between the sex chromosomes. It is also possible that there are multiple causes underlying this phenomenon, but with so few sex determination systems for comparison a conclusive inference is elusive (Coyne 1985, Wu and Davis 1993, Turelli 1998). There is also evidence that the ge- netic basis of inviability in heterogametic hybrids differs from the genetic basis for infertility (Coyne 1985). Exceptions such as this may eventually shed light on why this rule applies so well to the majority of species. There are some contradictions to Haldane’s rule in other XO systems, such as the field crickets Teleogryllus oce- anicus and Teleogryllus commodus (Moran et al. 2017), although JOURNAL OF ORTHOPTERA RESEARCH 2018, 27(2) N.E. MCKEAN, S.A. TREWICK, M.J. GRIFFIN, E.J. DOWLE AND M. MORGAN-RICHARDS whether contradictions to Haldane’s rule are more common in XO systems is unknown. One question remaining unanswered in the present study is where the barriers to reproduction are. As bimodal hybrid zones are typically associated with pre-mating rather than post-mating barriers (Jiggins and Mallet 2000 and references therein), the situ- ation here is somewhat unusual. As there does not appear to be assortative mating between these species pairs (Field and Jarman 2001, Morgan-Richards et al. 2001, Wehi et al. 2017), it suggests that barriers are more likely to be the result of genetic constraints. It is not known at what stage the production of F, hybrids is lim- ited, but as intermediate forms are far less common than expected if the species were freely interbreeding (Mckean et al. 2016) some reproductive constraint must operate. Barriers are hypothesised to be at the post-mating pre-zygotic stage or early in development, and it is possible that the wéta use unknown behavioral mecha- nisms to limit interbreeding. A bimodal hybrid zone in two spe- cies of chrysomelid beetles (Chyrsochus cobaltinus and C. auratus) also involves stronger post-zygotic barriers than pre-zygotic barri- ers (Peterson et al. 2005), so the association of assortative mating and bimodal hybrid zones has exceptions. A later study of these same beetles also showed a significant sex-bias in the production of offspring (most had mtDNA haplotypes and hence mothers from one species), despite mating occurring in both directions in the wild, and offspring in both sex-pairings being produced in equal numbers and with equal viability in laboratory crosses at the first instar (Monsen et al. 2007). The proposed explana- tion was asymmetric post-mating pre-zygotic barriers, or possi- ble asymmetric inviability later in development. As H. thoracica and H. crassidens appear to exhibit both a bimodal hybrid zone in the apparent absence of pre-mating barriers, and also a sex- biased production of F, offspring, there may be some similarities in the mechanisms causing reproductive isolation in these dispa- rate species pairs. Examples such as these may give insights into how bimodal hybrid zones are typically formed and maintained. Female infertility would prevent mtDNA passing the species boundary (introgressing), and this may explain why no evidence of mtDNA introgression has been seen in previous studies (Bulgar- ella et al. 2014, Mckean et al. 2016), despite evidence of a low level nuclear DNA and possible phenotypic introgression (Mckean et al. 2016). The low number of F, hybrids seen in the wild suggests that these wéta are forming a bimodal hybrid zone (Mckean et al. 2016), and with reduction in fertility of at least a 50% (due to female in- fertility), production of hybrids is probably costly. The most likely outcome in this scenario would appear to be reproductive character displacement or niche divergence limiting hybridization and loss of reproductive potential, as loss of reproductive compatibility allow- ing merging of the species would be unlikely with such a significant reduction in fertility (Dieckmann and Doebeli 1999, Jiggins and Mallet 2000). However, if one species uses sexual exclusion to mo- nopolize mates of the other, it could enable for the range expansion of this species. Given that most F, hybrids (8/9) were shown to have a H. thoracica father, this may in part explain how H. thoracica has been able to displace H. crassidens from much of its former range as climate has warmed since the last glacial maximum (Bulgarella et al. 2014). Introgression of adaptive alleles (e.g. cold tolerance) could enable continued expansion of H. thoracica, otherwise the hybrid zone is likely to settle where environmental selection and mate competition are at equilibrium. Further work to determine if male F, hybrids are more likely to successfully reproduce with H. thoracica or H. crassidens females would be valuable, because a bias at this point could influence introgressive asymmetry. 103 Neither of the two methods employed here provided evidence of Wolbachia infection in Hemideina. The primer pairs that amplify DNA from the common Wolbachia supergroups that infect arthro- pods (Simoes et al. 2011), and the mapping software used with NGS were sensitive enough to detect infection by another bacterial parasite, so it is highly likely that these wéta did not contain Wol- bachia although other New Zealand Orthoptera do (Bridgeman et al. 2018). A Chlamydia-like infection was detected in one of the weta in this study however, and as this bacteria also functions as an intracellular parasite (Wyrick 2000), it may be of interest. Wéta make good candidates for sexually transmitted diseases, as they generally have some level of promiscuity and have overlapping adult generations (Knell and Webberley 2004). Our sample of hybrid individuals was small, due to the low frequency of hybrids in the wild (Mckean et al. 2016), however, the results are biologically significant (i.e. F, males having at least some fertility, the infertility of female F, hybrids, thus being an exception to Haldane’s Rule), and raise questions about future in- teraction and survival of these species. In summary, both male and female F, hybrids are capable of reaching maturity, and although Wolbachia is not involved in limiting hybridization, there is at least a 50% (probably higher) reduction in F, hybrid fertility due to female infertility, which might have a strong limiting effect on in- trogression in the wild. There appears to be a contrast between complete failure by female F, hybrids to produce eggs and partial fertility of some male hybrids, suggesting this system provides an- other exception to Haldane’s rule that in interspecific hybrids the heterogametic sex (in this case males; XO) will have lower fertility than the homogametic sex (female Hemideina XX). Tree weéta are an interesting group for evolutionary studies, in part because they appear to have a high tolerance for chromo- some rearrangement that leads to many intraspecific hybrid zones. Much remains unknown about wéta biology, particularly with re- gard to species coexistence and production of hybrids where these weta meet in sympatry lends an extra layer of complexity to the situation. 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Forward / Reverse Locus Source ; Primers Wolbachia surface protein (wsp) Braig et al. 1998 Wsp81F/ Wsp691R Fructose-bisphosphate aldolase (fbpA) Baldo etal. 2006 fbpAF1/ fbpAR1 Cytochrome c oxidase, subunit I (coxA) Baldo et al. 2006 CoxAF1 / CoxAR1 Wolbachia specific portion of i Heddiet al. 1999 WollG6SF / Wol16SR 16S ribosomal RNA gene (wol16S) JOURNAL OF ORTHOPTERA RESEARCH 2018, 27(2)