(Orthoptera: Prophalangopsidae)?

applyparastyle “fig//caption/p[1]” parastyle “FigCapt”

Biological Journal of the Linnean Society, 2020, 131, 434–448. With 5 figures.

Does hunger lead to hybridization in a genus of sexually cannibalistic insects (Orthoptera: Prophalangopsidae)? Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020

JULIAN R. DUPUIS1,2*, , KEVIN A. JUDGE3,4*, BRYAN M. T. BRUNET2,5, SHAWNA OHLMANN CHAN3 and FELIX A. H. SPERLING2

1Department of Entomology, University of Kentucky, Lexington, KY 40546, USA 2Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada 3Department of Biological Sciences, MacEwan University, Edmonton, Alberta T5J 4S2, Canada 4Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada 5Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada

Received 17 March 2020; revised 27 May 2020; accepted for publication 28 May 2020

Allochronic isolation can be a strong mechanism for reproductive isolation and speciation. However, imperfect allochrony and the expression of phenological plasticity can erode temporal barriers to gene flow and result in hybridization between divergent lineages. Here, we combine behavioural ecology and genomics to investigate this scenario in two closely related species of grigs in the genus Cyphoderris. These species exhibit a unique mating system whereby females feed on the fleshy hind wings of the male during copulation, and copulation with conspecific males is more likely in food-restricted females than in well-fed females. In western Canada, Cyphoderris buckelli and Cyphoderris monstrosa are sympatric but largely allochronically separated, with C. buckelli breeding earlier. However, their breeding seasons can overlap, leading to potential for older C. buckelli females to mate with young C. monstrosa males to obtain resources via sexual cannibalism. We used behavioural assays to test whether female feeding status affects the propensity for interspecific mating between C. buckelli females and C. monstrosa males. We then tested for hybridization and gene exchange in wild populations of both species, using morphology, mitochondrial DNA and genome-wide nuclear markers. We found that interspecific courtship and mating can occur, but the relationship between food restriction and increased propensity for hybridization was not significant. Although we observed intraspecific population genetic structure in both species, we found no signatures of hybridization in the morphological or genetic datasets, which suggests that postmating reproductive barriers might be preventing successful hybridization in the wild.

ADDITIONAL KEYWORDS: Cyphoderris – genomics – genotyping by sequencing –grig – population genetics – Prophalangopsidae – sexual selection.

INTRODUCTION (e.g. host preference; Feder et al., 1994). Although assortative mating via temporal isolation can be an Allochrony, or divergence in breeding time, can automatic route to divergence (e.g. an ‘automatic’ be a potent mechanism for assortative mating, magic trait sensu Servedio et al., 2011), cases exist reproductive isolation and, ultimately, speciation in where allochrony is not perfect and hybridization sympatry (Hendry & Day, 2005; Taylor & Friesen, occurs between lineages that overlap phenologically 2017). This temporal isolation can act both as an (Yamamoto & Sota, 2012; Burban et al., 2016). These incipient speciation process (Simon et al., 2000; cases highlight the complex nature of gradual or Santos et al., 2007; Yamamoto & Sota, 2009) and in plastic temporal isolation, whereby allochrony is often concert with other traits to facilitate divergence interacting with other selective pressures to instigate or maintain genetic divergence (Pashley et al., 1992; *Corresponding authors. E-mail: [email protected]; Hendry & Day, 2005; Matsubayashi et al., 2010). Our [email protected] understanding of the dynamics and evolutionary effects

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 131, 434–448 434 HYBRIDIZING GRIGS IN WESTERN CANADA 435 of allochrony in these systems is greatly dependent on Furthermore, in both C. strepitans and Cyphoderris the identification and quantification of interbreeding buckelli Hebard, 1933, nutritionally deprived females between divergent lineages or populations, a process mount males sooner (Johnson et al., 1999) and are that is greatly expedited using genomic approaches more likely to mate, copulate and feed on male hind (Dupuis & Sperling, 2016; Taylor & Friesen, 2017). wings than non-nutritionally deprived females (Judge

In this study, we assess the potential for breakdown et al., 2011). Although male C. strepitans can mate Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 of reproductive isolation between closely related species successfully with either their fleshy hind wings or gin of hump-winged grigs (Cyphoderris spp., Orthoptera: traps disabled, if both are disabled they are unable to Prophalangopsidae) in western Canada (Fig. 1A). mate (Sakaluk et al., 1995). Virgin males (those with no From an evolutionary ecology perspective, this group hind wing damage) are also generally more successful is best known for its unique breeding system, whereby at mating (Morris et al., 1989). Taken together, these females feed on the fleshy hind wings of the males observations can be explained if females receive a during copulation and ingest both wing tissue and nutritional benefit from feeding on the hind wings, and haemolymph from the resulting wound, causing non- males exploit this by using their fleshy hind wings and fatal, but permanent, damage (Dodson et al., 1983). gin traps, respectively, to entice and coerce females to Simultaneously, males use two pairs of hooks on the mate (Sakaluk et al., 1995). dorsal side of their eighth and tenth abdominal tergites, Studies of sexual selection in Cyphoderris have, called the gin trap, to clasp the ventral surface of the to date, focused on intraspecific mating dynamics. female’s abdomen during spermatophore transfer However, in coniferous forests of southern British (Dodson et al., 1983). In Cyphoderris strepitans Morris Columbia and Alberta, the distributions and habitats & Gwynne, 1978, experimental removal of male hind of C. buckelli and Cyphoderris mostrosa Uhler, 1864 wings greatly decreases the probability of successful overlap (Buckell, 1924). Cyphoderris monstrosa spermatophore transfer (Eggert & Sakaluk, 1994), but has a relatively widespread geographical range in gin trap engagement in males lacking hind wings can mountainous regions in the Rocky Mountains from facilitate successful spermatophore transfer, suggesting central British Columbia and south-west Alberta into a coercive function of the gin trap (Sakaluk et al., 1995). Idaho and in the Cascade Mountains from British Columbia into Oregon; Cyphoderris buckelli has a much more restricted distribution and is limited to A) southern British Columbia and Northern Washington and Idaho (Morris & Gwynne, 1978). Morphologically, these species can be distinguished by the subgenital plate on the ventral tip of the abdomen, where C. monstrosa has a characteristic pair of spines shaped like the nail-pulling claw of a hammer that are lacking in C. buckelli (Fig. 1B, C). The mating periods of these species are predominately allochronic, with C. buckelli mating in late spring/early summer and C. monstrosa mating later in the summer; however, there can be overlap in their breeding periods (Supporting Information, Fig. S1). Interestingly, recent surveys have identified some intermediate individuals (Fig. 1D), albeit at a low frequency (~0.5% based on ~1000 scored individuals), from a sympatric location B) C) D) where the species ranges overlap (population NPL; K.A.J., unpublished observation). Given the presence of morphologically intermediate, putative hybrid individuals and the sexually cannibalistic mating dynamics of these species, we postulate the following hypothesis of interspecific interaction: if breeding periods of these species overlap during times when food Figure 1. Male grig morphology. A, habitus of male resources are scarce (e.g. during summer droughts), Cyphoderris monstrosa. B–D, lateral view of distinctive then older, nutritionally deprived C. buckelli females sternal process on the subgenital plate (arrow) in male might choose preferentially to mate with young, Cyphoderris buckelli (B, no hook), C. monstrosa (C, virgin C. monstrosa (i.e. with no hind wing damage) prominent hook) and putative hybrid (D, reduced, blunt in comparison to older C. buckelli with damaged hind hook), used for species identification. wings. Thus, plasticity in allochronic isolation might

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 131, 434–448 436 J. R. DUPUIS ET AL. combine with the sexually cannibalistic nature of ambient temperature, humidity and light during the these species to break down reproductive barriers. collecting trip, and upon arrival at the University of In this study, we investigate the prediction Lethbridge campus they were housed in a controlled- of breakdown of allochronic separation via environment chamber at 20 °C, 70% relative humidity, hybridization between old C. buckelli females and with a 12 h–12 h light–dark cycle.

young C. monstrosa males. Using methods similar Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 to a previous study investigating the dynamics of sexual cannibalism in C. buckelli (Judge et al., Experimental design 2011), we conduct a behavioural assay to evaluate Our behavioural assay took place in two phases. the possibility of such interspecific mating. We Phase 1 was an initial intraspecific pairing, in which all then survey populations from across the western females were predicted to reject male mating attempts Canadian range of these species for morphological, because they were well fed (Judge et al., 2011). mitochondrial DNA (mtDNA) and genome-wide This was followed by a period when half the female single nucleotide polymorphism (SNP) variation to C. buckelli were starved to induce mating interest. identify putative hybrid individuals or signatures Then phase 2 occurred, in which all C. buckelli females of introgression. This is the first investigation of were paired with a virgin male C. monstrosa. We interspecific interactions in Cyphoderris and adds predicted that: (1) fed females would continue to reject to the growing literature of this evolutionarily male mating attempts; and (2) starved females would charismatic group of insects. be more likely to mate with a heterospecific male. This experimental design was chosen because: (1) it made maximal use of females, which are difficult to collect in large numbers and so far impossible to rear MATERIAL AND METHODS in the laboratory; and (2) it provided a limited range of Behavioural assay experimental conditions (i.e. starved vs. fed) in which to assay the possibility of interspecific copulation. Study animals and husbandry Both phases of the experiment took place in a dark To test the behavioural potential for hybridization room at ~20 °C during the dark phase of the 12 h–12 h between species, we collected 18 female C. buckelli light–dark cycle in which the animals were housed in from several populations in British Columbia, the laboratory. Illumination was provided by both a Canada (Supporting Information, Table S1) in 2011. single 40 W-equivalent red LED bulb mounted ~50 cm Male C. buckelli and C. monstrosa (N = 42 and 22, above the trial arenas and a red LED headlamp worn respectively) were also collected in 2011, but from by the observer. single, separate populations: C. buckelli from Dick Hart Recreation Area, British Columbia and C. monstrosa near Paul Lake, British Columbia. Males used in the Phase 1: intraspecific pairings behavioural assays were random subsets (N = 18) Female C. buckelli were each paired with a randomly of non-virgin C. buckelli and virgin C. monstrosa, as assigned male C. buckelli. Before initiating each evidenced by their damaged and undamaged hind pairing, males and females were held under separate wings, respectively. Although this difference in male overturned plastic cups (4.5 cm in diameter) on a paper mating status is confounded with the difference in towel that was used as substrate during the experiment. species, it represents the natural situation faced by After a 2 min acclimation period, the cups were raised female C. buckelli in a sympatric location at the end gently, and an overturned plastic deli container (9 cm of their breeding season and the beginning of the in diameter, 8 cm high), which then served as the arena, C. monstrosa breeding season, when most, if not all, was placed over the male–female pair. The 18 pairings male C. buckelli and few, if any, male C. monstrosa were initiated in a randomly determined order, have mated (K.A.J., unpublished observation). Species staggered by 5 min intervals and lasted 3 h. During identification was based on morphology, following the experiment, the observer watched each active Morris & Gwynne (1978). pair in sequence (maximum N = 18) for ~1 s. Thus, All animals were housed individually in plastic deli each trial was observed as often as once every 18 s, containers (9 cm in diameter, 8 cm high) with a water- although in practice this interval was longer (1–2 min) filled, cotton-stoppered microfuge tube for moisture, given that behavioural note-taking took a few seconds. a piece of cardboard egg carton for shelter and one Resulting behavioural observations can be considered piece of cat chow (Iams Original with Chicken) and accurate to within 1 min, and that is the precision with a minimum of five grains of bee pollen for food. This which we noted the timing of the following events: (1) diet corresponds to the high-quality diet described by first contact; (2) initiation of courtship singing by the Judge et al. (2011). Animals were kept in conditions of male; (3) initiation of mounting of males by females;

(4) initiation and termination of hind wing feeding; feeding rate, male hind wing damage, and female (5) initiation and termination of gin trap engagement; mating rate. Unlike parametric statistical tests, and (6) initiation and termination of spermatophore permutation tests do not assume underlying transfer. From these observed times, the following distributions and are therefore useful for datasets variables were calculated: (1) latency to contact (time where parametric assumptions are likely to be violated

of first contact minus time of pairing initiation); (2) (e.g. small sample sizes, as in our data). Briefly, Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 latency to court (time of first courtship minus time of we randomly shuffled (without replacement) the first contact); (3) total number of mountings; (4) total treatment values (starved or fed) among the observed number and average duration of hind wing feedings; values for each response variable and then calculated (5) total number and average duration of gin trap a difference between the permuted treatment means. engagements; and (6) duration of spermatophore We replicated this process 10 000 times and calculated transfer. If the male was completely unresponsive for the proportion of times the permuted difference the first 5 min of the assay, he was replaced with a new exceeded the observed difference, which is equivalent male. Finally, if any hind wing feeding was observed, to a P-value in a two-tailed test. We also calculated 95% we photographed the hind wings of the male 24 h after confidence intervals from 10 000 bootstrap replicates the trial to assess the amount of hind wing damage for starved and fed females for all response variables. inflicted, using the same methods as in the study by These calculations were conducted using the PopTools Judge et al. (2011). add-in (Hood, 2009) in Microsoft Excel.

Dietary manipulation Preparation of morphological and molecular Immediately after pairing with a C. buckelli male, specimens each female was assigned randomly to one of two We collected specimens of Cyphoderris during the dietary treatments: (1) a high-quality diet, which was summer of 2013 from several populations across British a continuation of the diet all females received before Columbia and western Alberta, Canada (Table 1; phase 1; or (2) a starvation treatment, in which females Supporting Information, Table S2). Individuals were received no food but ad libitum water as before. Dietary hand-captured and either euthanized by freezing at manipulation continued for 8 days. We measured the −20 °C and preserved in 100% ethanol or kept alive mass of all females before dietary manipulation and for behavioural experiments. After these behavioural on days 5 and 7 after the dietary manipulation. The experiments were finished, individuals either died and change in mass was assessed after each measurement were preserved in 70% ethanol or were euthanized by of mass, and the dietary manipulation was validated freezing at −20 °C and preserved in 100% ethanol. by body mass loss of ~15%. Phase 2 of the behavioural For morphological measurements, we dissected assay took place 8 days after phase 1. individuals and removed pertinent body parts that were then photographed in standardized orientation submerged in 100% ethanol. For molecular datasets, Phase 2: interspecific pairings DNA was extracted from a single leg for live- Phase 2 of the mating experiment proceeded exactly as frozen specimens using DNeasy Blood and Tissue phase 1 except that females were paired with a virgin kits (Qiagen), with the optional RNase A (Sigma- male C. monstrosa, assigned randomly. The same Aldrich) treatment, following the manufacturer’s observations were recorded as in phase 1, with one recommendations. We ethanol precipitated the addition. In phase 1 we noticed that females tended resulting DNA extracts, eluted them in 50 µL Millipore to walk around the margins of the container almost water, and quantified and qualified theDNA using continuously. To test whether the dietary manipulation a Qubit 1.0 dsDNA BR assay kit (Invitrogen) and a had an effect on female activity, we recorded the Nanodrop ND-1000 (Thermo Scientific), respectively. amount of time spent walking during a 2 min time span at ~80 min into each trial. All individuals were housed individually after the experiment until they Morphology died and were then preserved in 70% ethanol for Owing to small sample sizes of females and juveniles subsequent measurement of morphology. and high levels of morphological sexual dimorphism in these species (K.A.J., unpublished observation), we measured morphological characters for adult Statistical analysis for behavioural assays males only. In preparing specimens for morphological We used permutation tests (Legendre & Legendre, measurement, we removed the head and hind legs and 1998) as described by Judge et al. (2011) to test for then took photographs of three different body parts/ differences between dietary treatment and female regions from standardized orientations (the posterior

Table 1. Numbers of Cyphoderris samples collected at each population during the summer 2013 field season for genotyping by sequencing dataset

Species Pop AM JM AF Tot

Cyphoderris buckelli DH 4 0 0 4 Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 FK 3 0 2 5 FPL 5 0 0 5 JC 3 0 0 3 KC 3 0 0 3 NPL 5 1 0 6 OSM 3 1 1 5 RC 1 0 0 1 UO1 4 0 1 5 Total 31 2 4 37 Cyphoderris monstrosa BR 3 0 2 5 CW 5 0 0 5 ERD 2 0 0 2 FT 2 0 0 2 GDUR 5 0 0 5 HW97 3 1 1 5 ML 4 0 1 5 NPL 4 1 4 9 SVR 4 0 0 4 WR 1 1 2 4 Total 33 3 10 46

Abbreviations: AF, adult female; AM, adult male; JM, juvenile male; Pop, population; Tot, total. head, the lateral surface of both hind legs, and the species. This analysis was carried out using IBM SPSS posterior dorsum) using a digital camera (Lumenera Statistics v.25 (IBM Corporation, 2017). INFINITY 1-3C) mounted on a stereomicroscope (Wild M5). Images were captured using INFINITY CAPTURE v.5.0.2 (Teledyne Lumenera), saved as jpegs Genotyping by sequencing and analysed using software from SB Morphometrics Genotyping by sequencing (GBS) libraries were prepared (Rohlf, 2016). Specifically, image files were bundled (with DNA normalized to 20 ng/µL) following Poland together using tpsUtil v.1.70, and landmarks were et al. (2012), using the restriction enzymes PstI and placed in the resulting TPS file with tpsDig2 v.2.26. MspI. Duplex-specific nuclease (Zhulidov et al., 2004) We measured five linear dimensions from the collected and complexity reduction following Sonah et al. (2013) photographs: (1) head width (HW; maximal distance (a C added to the reverse primer) were implemented in perpendicular to the frontal plane); (2) maxillae span the library preparation, and 96 individual libraries were (MS; distance between the cardo-stipes articulations pooled into a single lane of 100 bp single-end sequencing of the left and right maxillae); (3) hind femur length on a HiSeq 2000 (Illumina). Some individuals yielded (FL); (4) hind femur depth (FD; maximal depth poor-quality DNA in the first round of extractions, and perpendicular to femur length); and (5) gin trap span for these we generated and sequenced GBS libraries (GTS; distance between the left and right hooks on from DNA extracted from a second leg. the eighth abdominal tergite). These dimensions are We used Stacks v.2.1 (Catchen et al., 2011, 2013) to all highly repeatable (data not shown), represent process the raw GBS data and call SNPs using default characteristics spread across all three body tagmata, parameters and settings unless otherwise noted. First, and are all predicted to be related directly to male process_radtags was used to demultiplex and filter mating success (e.g. Sakaluk et al., 1995; Judge & raw data files (removing reads with low quality scores/ Bonanno, 2008). uncalled bases and rescuing reads with barcode/cutsite We used principal components analysis (PCA) to errors). We then used denovo_map.pl to execute the reduce the morphological dataset to a few uncorrelated Stacks pipeline, allowing three mismatches between composite dimensions that could be used to visualize stacks within and between individuals (-M and -n, the morphological similarity between populations and respectively). We called the final SNP datasets using

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 131, 434–448 HYBRIDIZING GRIGS IN WESTERN CANADA 439 populations with a population map assigning all TCATAAAGATATTGG-3′) and LepR1 (5′-TAAACTT individuals to one population and used alternative CTGGATGTCCAAAAAATCA-3′); polymerase chain filtering strategies to create two datasets, one tailored reaction conditions, clean-up and sequencing followed to population genetic analyses and one to phylogenetic Dupuis & Sperling (2015). We combined forward and analyses. For the population genetic dataset, we reverse reads with Chromaseq v.1.31 (Maddison &

required loci to be present in one population and 1% of Maddison, 2018a) in Mesquite v.3.51 (Maddison & Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 individuals to be processed in populations and generated Maddison, 2018b), checked for erroneous base calls by a vcf output with a single SNP per catalogue locus. We eye, aligned sequences using MAFFT v.7.305b (Katoh then identified poor-quality individuals from this vcf file & Standley, 2013), and trimmed 5′ and 3′ ends of the using the individual missingness output in VCFtools alignment to remove variably missing data between v.0.1.15 (Danecek et al., 2011) (those with > 70% individuals. We conducted ML tree searches on the missing data after removal of loci with > 50% missing trimmed mtDNA alignment using IQ-Tree as above, genotypes). We used VCFtools to remove poor-quality except that we did not use a model incorporating individuals and duplicate individuals (resulting from an ascertainment bias as we did for the SNP-based multiple DNA extractions; see above) and removed loci analysis. All trees were visualized with FigTree with > 30% missing data and a minor allele frequency v.1.4.4 (Rambaut & Drummond, 2010) and compared < 5% to generate the final population genetic dataset. with the ape v.5.3 (Paradis et al., 2004) and phytools For the phylogenetic dataset, we removed the same v.0.6.99 (Revell, 2012) packages in R v.3.4.4 (R Core individuals as for the population genetic dataset and Team, 2018). used populations to remove loci with > 50% missing data and a minor allele frequency < 5% and generate a phylip- formatted output with a single SNP per catalogue locus. RESULTS We used individual-based Bayesian clustering in Behavioural assays STRUCTURE v.2.3.4 (Pritchard et al., 2000) to assess genetic structure (i.e. the number of unique genetic In phase 1 of the behavioural assay (intraspecific clusters, K) in the population genetic dataset. We pairings), all female C. buckelli experienced vigorous assessed K = 1–20, each with 20 replicates of 250 000 courtship singing by conspecific males, but no mating sampled generations (after 50 000 burn-in generations), (hind wing feeding or spermatophore transfer) correlated allele frequencies (Falush et al., 2003) and resulted from these interactions. However, at least two the admixture model. To determine the optimal value males were observed using their gin traps successfully of K, we calculated LnPr(X|K) natural logarithm of the to clasp their paired female as she circled the probability of the data (Pritchard et al., 2000) and ΔK experimental arena (Sakaluk et al., 1995) (Supporting (Evanno et al., 2005) with Clumpak v.1.1 (Kopelman Information, Video S1). Both females pulled away et al., 2015), and MedMed/Mean K and MaxMed/Mean K from the male after being gin trapped (Supporting (median of medians, median of means, maximum Information, Video S1), and one of these females died a of medians, maximum of means of K, respectively) few days after phase 1 ended (Supporting Information, (Puechmaille, 2016) with StructureSelector (Li & Table S1). Liu, 2018) (using a threshold of 0.5, we refer to these In phase 2 (interspecific pairings), all male as the Puechmaille statistics). To assess fine-scale C. monstrosa courted female C. buckelli vigorously substructure, we conducted hierarchical analysis, (Supporting Information, Video S2), and either gin following Pritchard & Wen (2004) and Vähä et al. (2007). trap contact, hind wing feeding or spermatophore For the phylogenetic dataset, we conducted maximum transfer was observed in 12 of 17 pairings (Supporting likelihood (ML)-based tree searches in IQ-Tree v.1.6.7 Information, Table S1). In all of the instances where (Nguyen et al., 2015) using the best-fitting model of gin trap contact was observed, it seemed to be initiated evolution predicted by ModelFinder (Kalyaanamoorthy by the male as the female walked past or over his et al., 2017), including an ascertainment bias appropriate abdomen (Sakaluk et al., 1995). When this occurred, for SNP-based datasets with no invariable sites (Lewis, females usually spent some time feeding on the hind 2001). We assessed node support using 10 000 replicates wings of the male, but also appeared to struggle to free of both ultra-fast bootstrap (ufBS; Hoang et al., 2018) themselves, sometimes by biting the hind legs and and the Shimodaira–Hasegawa approximate likelihood dorsal abdomen of the male (Supporting Information, ratio test (SH-aLRT; Guindon et al., 2010). Video S3). In two cases, females did not successfully extricate themselves, and a spermatorphore was transferred (Supporting Information, Video S4). Two Mitochondrial DNA pairings were stopped early: one because the female We amplified ~680 bp of cytochrome c oxidase subunit pulled away part of her abdominal cuticle during a I (COI) using the primers LepF1 (5′-ATTCAACCAA struggle to free herself from the male’s gin trap, and

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 131, 434–448 440 J. R. DUPUIS ET AL. the second because the male attacked the female. As individuals have previously been collected (Fig. 3 inset, predicted by our hypothesis, starved females were population NPL). Sixty-four adult males were included more likely to feed on male hind wings, did more in the final morphological dataset (Table 2; Supporting damage to male hind wings when feeding and were Information, Table S3). more likely to mate than fed females. However, none Principal components analysis of this adult male

of these relationships was statistically significant (all dataset using five morphological traits (FD, FL, HW, MS Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 P > 0.191; Fig. 2). and GTS) resulted in two principal components (PCs) that explained 92.82% of the variation in the original traits (Supporting Information, Table S4). GTS loaded Morphology negatively on PC1 and in strongly positive manner on Eighty-three individuals (including juveniles and PC2, whereas all four other traits loaded in a strongly adult females used for GBS but not morphology) were positive manner on PC1 and a weakly positive manner collected for morphological and genetic analysis, with on PC2 (Supporting Information, Table S4). Principal the collection localities for these populations centred component 1 separated the two species, with only two around an area where both species are present in close individuals having relatively intermediate placement proximity, including the locality where intermediate (Fig. 4), neither of which came from populations at or near the location where individuals with intermediate 1.0 genitalia have been found (NPL), nor did they show genetic intermediacy (see below, genotyping by 0.8 p = 0.329 sequencing). The second principal component showed g some spread of individuals, but finer-scale groupings 0.6 did not correspond to any geographical populations

eedin (Fig. 4). 0.4 op. F

Pr Genotyping by sequencing 0.2 The GBS sequencing resulted in 145.2 million reads, 0.0 of which 110.4 million reads (per individual maximum, minimum and average: 3.2 million, 48.5 thousand 8 and 1.2 million, respectively) were retained after p = 0.191 initial data filtering. The SNP calling resulted in 244 229 SNPs, and after we removed low-quality and Damage 6 duplicated individuals (resulting from multiple DNA 4 extracts; see Material and Methods) and conducted Wing final filtering, our population genetic and phylogenetic 2 datasets consisted of 78 individuals and 3042 and Hind 16 794 SNPs, respectively. 0 In STRUCTURE analysis of the population genetic dataset, we found mostly consistent support for the 0.8 best value of K (for all STRUCTURE results and best K support, see Supporting Information, Fig. S2): 0.6 LnPr(X|K) plateaued at K = 3, ΔK supported K = 2 and K = 3, and the Puechmaille statistics generally 0.4 supported K = 4. Clustering at K = 2 separated

op. Mam g p = 0.214 C. buckelli from C. monstrosa (Fig. 3A), and at K = 3 Pr 0.2 a unique cluster of C. monstrosa from populations FT and GDUR was observed. Owing to sample size 0.0 constraints, we analysed the two clusters from K = 2 StarvedHigh Food hierarchically, and both had support for K = 2, 3 and Diet Treatment 4 as the best value of K. For the C. buckelli cluster, LnPr(X|K) supported K = 3, ΔK supported K = 2 and Figure 2. The effect of dietary treatment on interactions K = 3, and the Puechmaille statistics supported K = 4 between starved or high-food female Cyphoderris buckelli and K = 5. For the C. monstrosa cluster, LnPr(X|K) and male Cyphoderris monstrosa. Error bars represent 95% supported K = 3, ΔK supported K = 4, and the confidence intervals generated by bootstrapping; P-values Puechmaille statistics supported K = 3. Given the are the result of permutation tests. sample sizes of these clusters and their biological

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 131, 434–448 HYBRIDIZING GRIGS IN WESTERN CANADA 441 Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020

Figure 3. Results of STRUCTURE analyses of 3042 single nucleotide polymorphisms. A, all individuals at K = 2 (K: the number of unique genetic clusters). B, hierarchical analyses of two clusters corresponding to Cyphoderris monstrosa and Cyphoderris buckelli, each at K = 3. C, population averages from hierarchical analyses (barplots in B) displayed as pie charts, with diameter indicating sample size per locality. Dot shape in C denotes species at each collection locality, with a grey star indicating that both species were collected (locality NPL; also with two pie charts). plausibility, we considered K = 3 as the best value of (Fig. 5; Supporting Information, Fig. S3). Many of K for each species, which resulted in geographically the same intraspecific clusters from STRUCTURE associated clustering within each species (Fig. 5). Most analyses were also recovered, although if admixed of the population samples showed very little admixture individuals were observed in STRUCTURE, those between each of the K = 3 intraspecific genetic clusters, clades were generally not well supported in the ML except for populations in the south-west portion of our analysis. sampling range. For direct comparison between the GBS and mtDNA datasets, mid-rooted ML consensus trees in Figure 5 Mitochondrial DNA have had individuals removed for which we failed to The final mtDNA dataset consisted of 641 bp (77 obtain either GBS or mtDNA data (four individuals parsimony informative sites) for 79 individuals (34 not shown for GBS and five for mtDNA; full trees C. buckelli and 45 C. monstrosa). As with the GBS data, are provided in Supporting Information, Figs S2, S3). we observed reciprocal monophyly between C. buckelli The GBS ML consensus resulted in well-supported and C. monstrosa (Fig. 5; Supporting Information, reciprocal monophyly of C. buckelli and C. monstrosa Fig. S4), and some of the most divergent of the

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 131, 434–448 442 J. R. DUPUIS ET AL. 0.97 (0.14) 0.90 0.82 (0.10) 0.98 (0.17) 0.94 (0.07) 0.83 (0.06) 1.04 (0.07) 1.17 (0.01) 1.15 (0.11) 0.94 (0.12) 1.07 (0.15) 1.21 (0.11) 1.23 (0.17) 1.29 1.16 (0.05) 1.26 (0.09) 1.33 (0.02) 1.27 (0.10) 1.13 (0.08) 1.24 (0.11) 1.10 (0.11) GTS (mm) Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 5.96 (0.26) 5.42 5.79 (0.17) 6.26 (0.25) 6.04 (0.25) 5.88 (0.19) 6.08 (0.26) 6.25 (0.17) 5.92 (0.10) 5.81 (0.12) 5.87 (0.27) 5.13 (0.22) 5.31 (0.09) 5.54 5.35 (0.26) 5.06 (0.22) 4.96 (0.18) 5.15 (0.14) 5.06 (0.09) 4.84 (0.14) 5.17 (0.22) MS (mm) 6.13 (0.23) 5.87 5.93 (0.24) 6.21 (0.24) 5.99 (0.23) 6.17 (0.10) 6.20 (0.15) 6.60 (0.13) 6.08 (0.12) 6.10 (0.15) 6.21 (0.25) 5.44 (0.19) 5.56 (0.20) 5.81 5.61 (0.05) 5.53 (0.02) 5.15 (0.15) 5.46 (0.10) 5.43 (0.09) 5.17 (0.15) 5.44 (0.16) HW (mm)

9.45 (0.37) 9.31 (0.43) 9.78 (0.04) 9.61 (0.37) 9.70 (0.42) 9.19 (0.17) 9.31 (0.27) 9.14 (0.18) 9.31 (0.19) 11.68 (0.46) 10.49 11.34 (0.29) 11.83 (0.18) 11.82 (0.25) 11.38 (0.12) 12.05 (0.33) 12.56 (0.21) 11.27 (0.03) 11.53 (0.35) 11.81 (0.37) 10.31 FL (mm) 2.77 (0.14) 2.49 2.61 (0.13) 2.78 (0.0) 2.80 (0.09) 2.76 (0.06) 2.93 (0.09) 3.00 (0.04) 2.76 (0.02) 2.73 (0.06) 2.68 (0.13) 2.40 (0.12) 2.39 (0.16) 2.75 2.43 (0.07) 2.37 (0.10) 2.49 (0.05) 2.40 (0.09) 2.35 (0.05) 2.48 (0.10) 2.30 (0.13) FD (mm) 1 4 4 4 3 5 2 2 5 3 4 1 3 5 3 3 5 3 4 33 31 N Average WR SVR NPL* ML HW97 GDUR FT ERD CW BR Average UO1 RC OSM NPL* KC JC FPL FK DH Population Mean adult male trait values for morphological traits measured in populations of Cyphoderris

Cyphoderris monstrosa

Cyphoderris buckelli Table 2. Table Species Numbers in parentheses are standard deviations. sample size. N , maxillae span; MS, head width; HW, gin trap span; GTS, femur length; FL, femur depth; FD, Abbreviations: been observed. putative hybrid individuals have where both species are found and intermediate, *Population

) this, we found no morphological or genetic signatures BR CW of hybridization with any of the population-level 2 a ERD datasets. Taken together, we were able to find support FT for only part of the prediction laid out in our original GDUR 1 HWY97 hypothesis. Our results suggest that hybrid matings

ML between C. buckelli and C. monstrosa can occur in Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 . monstros NPL the laboratory, but that if they do in the wild, some 0 SVR factor might be preventing the persistence of hybrids. WR It is unclear whether this is the result of pre- (e.g. -1 DH unsuccessful fertilization after hybrid mating) or post- FK iC FPL zygotic (e.g. hybridization producing infertile offspring) barriers, or whether the intermediate, putative hybrid -2 JC KC individuals are simply cases of anomalous morphology. NPL Both C. buckelli and C. monstrosa tend to mate

C. buckell OSM

PC2 (+0.09FL,+0.26FD,+0.13MS,+0.13HW, +0.77GTS -3 RC on vegetation, but during dusk and the first few -1 1 -2 0 2 UO1 hours after dark during the breeding season, males PC1 (+0.96FL,+0.91FD,+0.96MS,+0.94HW,-0.63GTS) can both be found singing on the ground and low on shrubs and trees. As the night progresses, male Figure 4. Scatterplot of principal component (PC) 1 and C. monstrosa move higher up trees, predominantly PC2 from principal components analysis of morphological lodgepole pine, and aggressively defend territories data, including only males. Species and populations are indicated by colour and symbol shape, respectively. Factor (Mason, 1996), whereas male C. buckelli stay in the loadings are given in parentheses on each axis, and trait understorey and undergrowth, moving often and abbreviations are as follows: FD, femur depth; FL, femur without the same degree of territoriality (Morris length; GTS, gin trap span; HW, head width; MS, maxillae et al., 2002). Thus, in locations where breeding of span. both species overlaps (e.g. locality NPL, Fig. 3) there is opportunity for young male C. monstrosa and older intraspecific clusters/clades identified with the GBS C. buckelli females to encounter each other when data were recovered as well-supported clades with the both are actively seeking a mate. Using behavioural mtDNA data (e.g. the C. monstrosa cluster of GDUR assays, we were able to demonstrate that interspecific and FT populations). However, in general there was courtship and mating between female C. buckelli and much less intraspecific resolution with this dataset male C. monstrosa is possible, and that, with respect and short intraspecific branch lengths (see scales in to male C. monstrosa, there is no behavioural isolation Fig. 5); divergence between most of the closely related between these species. Although our data trended haplotype pairs/groups were the result of one or two toward a relationship between food deprivation and base differences. This lack of resolution explains most increased interspecific mating, this relationship was of the apparent fine-scale conflict when compared with not statistically significant, unlike previous studies the GBS-based phylogeny (Fig. 5). examining the effects of diet on intraspecific mating in C. strepitans (Johnson et al., 1999) and C. buckelli (Judge et al., 2011). If there is some biological basis for the trend, the lack of significance in our present DISCUSSION study could be the effect of: (1) the small sample size Our study used behavioural assays to investigate constrained by the difficulty of collecting adult females; the potential for interspecific mating between older, (2) insufficiently severe manipulation of nutritional food-starved, female C. buckelli and young, male status; or (3) other factors besides female nutritional C. monstrosa, and surveyed populations for hybrid status driving interspecific pairings. Density, which is individuals using both morphological and genetic predicted to affect encounter rates between the species, analyses. We found that courtship and mating can is one such factor that has received relatively little occur between these species in the aforementioned attention in studies of mating dynamics, despite its conditions (older, food-starved, female C. buckelli and theoretical importance (Kokko & Rankin, 2006). More young, male C. monstrosa). Although it appears that comprehensive evaluation of the activity patterns, food restriction might increase the propensity for this local densities and proportions of mated and virgin hybridization, none of these trends were statistically individuals of both species would be invaluable for significant. Mitochondrial diversity was low in understanding the potential for interspecific mating both species, but intraspecific population structure, interactions in Cyphoderris. particularly in the genome-wide SNP dataset, was The results of our behavioural experiment both apparent in both C. buckelli and C. monstrosa. Despite highlight the extent of conflict between the sexes

16,794 SNPs 641 bp mtDNA GDUR005 GDUR005 GDUR004 GDUR004 GDUR002 GDUR002 GDUR003 GDUR003 FT007 FT007 FT003 FT003 PBM002 PBM002

ERD002 HW97004 Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 WR018 WR004 WR005 ERD002 WR008 HW97006 WR004 CW002 HW97006 CW005 HW97004 NPL016 HW97002 NPL004 HW97003 HW97003 HW97001 WR008 CW005 NPL001 CW002 NPL020 CW007 WR005 CW001 CW007 SVR005 CW001 SVR004 BR006 SVR002 SVR005 SVR001 BR005 BR005 HW97002 BR003 HW97001 BR002 WR018 BR006 BR003 BR001 ML001 NPL007 SVR004 NPL016 ML004 NPL012 NPL007 NPL020 ML007 NPL009 NPL012 NPL003 SVR001 NPL001 SVR002 ML001 BR002 NPL021 NPL003 ML002 ML005 0.07 avg. NPL004 NPL021 0.02 avg. changes/site ML004 NPL009 changes/site ML007 ML002 ML005 BR001

C. buckelli C. monstrosa

FK015 KC006 KC006 KC005 KC005 KC002 KC002 FK015 FK013 FK013 FK007 FK007 FK004 FK004 RC001 NPL006 JC003 OSM005 JC002 FPL003 UO1008 OSM004 UO1005 OSM001 UO1002 OSM003 UO1001 NPL011 OSM004 DH004 OSM003 RC001 OSM005 JC002 OSM001 JC003 DH008 UO1002 NPL005 UO1001 NPL006 UO1008 FPL004 UO1005 DH006 DH006 DH004 DH008 NPL011 NPL005 FPL002 FPL002 FPL008 FPL008 FPL003 FPL004 FPL001 FPL001 DH003 DH003

Figure 5. Comparison of maximum likelihood consensus trees from the genotyping by sequencing (GBS; left) and mitochondrial DNA (mtDNA; right) datasets. Only individuals present in both datasets are included. in the genus Cyphoderris and shed new light on C. buckelli (Judge et al., 2011). Female C. buckelli, all a previous experiment that supported a material well fed, never mounted a conspecific male in phase 1 benefits explanation for hind wing feeding in of our behavioural experiment. However, two of these

18 females were gin trapped by conspecific males required to substantiate this hypothesis (Bull et al., and escaped (e.g. Supporting information, Video S1). 2011). In contrast to the GBS dataset, few populations Likewise, in phase 2, nine females were gin trapped formed clusters or monophyletic groups with the COI by C. monstrosa males (four of nine well fed; five of dataset (Fig. 5). This seemed mostly attributable to a eight starved), some up to three times. All but two lack of information in this dataset, which is apparent

of the gin-trapped females freed themselves without from the short intraspecific branch lengths in the main Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 mating, both of which were in the starved treatment. clades of both species. Again, the eastern populations Although noted for C. strepitans (Sakaluk et al., showed the most phylogenetic distinction in the entire 1995), previous work on C. buckelli failed to observe dataset, supporting a hypothesis of strong isolating gin trapping as a coercive male strategy (Judge et al., pressures of the Rocky Mountain range. The higher 2011). This is likely to be attributable to the fact that information content of the GBS dataset is unsurprising Judge et al. (2011) made focal observations of their given the proven utility of this style of genomic dataset mating pairs of C. buckelli only once every 20 min, for population genetics and phylogenetics (e.g. Andrews whereas we observed mating pairs continuously. et al., 2016). Our sampling centred around an area If male C. buckelli use gin trapping regularly as where both species are in close proximity and where a coercive mating strategy then females in poor intermediate individuals have been observed (Fig. 3 nutritional condition in their experiment might simply inset, population NPL); therefore, more geographically have been unable to extricate themselves from a male’s comprehensive sampling across the ranges of these gin trap compared with females in good condition. species should provide a finer-scale assessment of Furthermore, if, as our data suggest, C. monstrosa population structure in both species. males use gin trapping regularly, then their overall These results serve as a foundation for future larger size (Fig. 4), and therefore strength, might research in this system, with two aspects of the greatly increase the likelihood of: (1) gin trapping any present study being important to address in future encountered female C. buckelli; and (2) clasping that efforts. First, although we sampled populations where female until after successful spermatophore transfer, intermediate individuals have been observed, we were thus facilitating hybrid mating. Several females in unable to generate genetic data from any intermediate our experiment sustained damage to their abdominal individuals. Increased sampling of these populations cuticles as a result of gin trapping (see Supporting or adoption of genomic techniques more amenable Information, Fig. S5), suggesting both high costs to to museum specimens (e.g. whole-genome shotgun interspecific pairings and that it might be possible to sequencing; Sproul & Maddison, 2017) will be needed identify female C. buckelli that experience attempted to characterize the genetics of these putative hybrids. interspecific mating in the field. In the presence Second, although we have demonstrated interspecific of high costs to females of resisting male mating copulation between C. buckelli and C. monstrosa, attempts, females are predicted to increase their this does not translate directly to the viability and mating rate beyond their preferred optimum. Such fecundity of hybrid individuals. Rearing hybrid broods mating systems (e.g. water striders, family Gerridae) to both evaluate hybrid viability and confirm that are characterized by convenience polyandry (Thornhill intermediate morphology results from hybrid mating & Alcock, 1983; Rowe, 1992; Anrnqvist & Rowe, 2005). would provide strong evidence to substantiate these We speculate that in conditions of nutritional stress, hypotheses. Given that Cyphoderris has never been C. buckelli females might engage in convenience cultured successfully in the laboratory, development of interspecific mating. rearing methods would greatly increase our ability to Although we found no signatures of hybrid individuals investigate the viability of hybridization in this system with either the mtDNA or GBS datasets, we did observe and address many potentially confounding variables genetic population structure in both species. This in the study of sexual conflict between these species structure was most pronounced in the GBS dataset, (e.g. individual age, body condition, mating status). where we found K = 3 to be well supported in both species (Fig. 3B). Populations in the eastern part of the sampled distribution were more highly differentiated ACKNOWLEDGEMENTS in STRUCTURE analysis (Supporting Information, Fig. S2) and sister to the other clusters in phylogenetic J.R.D. and K.A.J. contributed equally to this work. analyses (Fig. 5), suggesting that the Rocky Mountains Genotyping by sequencing libraries were prepared might be a stronger isolating factor than geographical at Institut de Biologie Intégrative et des Systèmes at (Euclidean) distance (Fig. 3C). The distribution of Université Laval and sequenced at McGill University’s admixture between the other two clusters in each Génome Québec Innovation Centre. Sanger sequencing species supports this inference, although quantitative was conducted at the Molecular Biology Service Unit evaluation of the effect of landscape features would be at the University of Alberta. We thank Terrence Chang

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 131, 434–448 446 J. R. DUPUIS ET AL. for assistance in specimen collection, Taylor Becker In: Gwynne DT, Morris GK, eds. Orthopteran mating systems: for assistance with wet laboratory procedures, and sexual competition in a diverse group of insects. Boulder: Bill Cade, Paul De Luca, Andrew Mason and Glenn Westview Press. Morris for helpful discussions. Thank you to Thor Veen Dupuis JR, Sperling FA. 2015. Repeated reticulate evolution and two anonymous reviewers for helpful comments in North American Papilio machaon group swallowtail butterflies. PLoS One 10: e0141882. on the manuscript. This research was supported by Downloaded from https://academic.oup.com/biolinnean/article/131/2/434/5893746 by MacEwan University Libraries user on 07 December 2020 Natural Sciences and Engineering Research Council Dupuis JR, Sperling FA. 2016. Hybrid dynamics in a species of Canada Discovery Grants to F.A.H.S. 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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Results of behavioural assays. Table S2. Detailed specimen information. Table S3. Raw morphological data. Table S4. Eigenvalues and variance of principal components analysis. Figure S1. Phenogram of historical field collections (K.A.J., unpublished information) of Cyphoderris buckelli (blue) and Cyphoderris monstrosa (red) from the populations assessed in the present study. The number of males captured is displayed on a logarithmic scale owing to disproportionately large sample sizes in 2009. Figure S2. All STRUCTURE results, including LnPr(X|K), ΔK, and Puechmaille statistics and barplots for all values of K for analyses of all individuals and the hierarchical analysis of the Cyphoderris buckelli and Cyphoderris monstrosa clusters. Figure S3. Maximum likelihood (ML) consensus tree for single nucleotide polymorphism (SNP) dataset for all individuals. Branch support is Shimodaira–Hasegawa approximate likelihood ratio test (SH-aLRT)/ultra-fast bootstrap (ufBS), with good support indicated by > 0.8/0.95. Figure S4. Maximum likelihood (ML) consensus tree for mitochondrial DNA dataset for all individuals. Branch support is Shimodaira–Hasegawa approximate likelihood ratio test (SH-aLRT)/ultra-fast bootstrap (ufBS), with good support indicated by > 0.8/0.95. This tree is rooted with a sequence from Pseudorhynchus acuminatus (Orthoptera: Tettigoniidae) retrieved from GenBank (accession NC_033992.1). Figure S5. Photograph of the ventral abdomen of one of two female Cyphoderris buckelli that received a spermatophore from a male Cyphoderris monstrosa during phase 2 of the behavioural experiment. Arrows indicate melanization, an insect immune response, presumably as a result of puncture wounds caused by the male’s gin trap. Video S1. An intraspecific interaction from phase 1 of the behavioural experiment, in which a male Cyphoderris buckelli uses his gin trap to clasp a female C. buckelli by her ventral abdomen as she walks over his dorsum. Note that before the female is gin trapped, the male appears at several points to be attempting use his gin trap to clasp the edge of the plastic arena. The damaged hind wings of the male can be seen whenever he faces away from the camera. Video was recorded using a hand-held Canon PowerShot SD790 IS with a red LED headlamp for illumination. Video S2. An interspecific interaction from phase 2 of the behavioural experiment, in which a male Cyphoderris monstrosa vigorously courts a female Cyphoderris buckelli. Courtship consists of stridulation (rubbing together of his fore wings) and periodically turning to present his posterior abdomen to the female. Note that whenever the female passes his posterior, there is a short motion of the distal abdomen of the male, where his gin trap is located. The undamaged hind wings of the male can be seen whenever he faces away from the camera. Video was recorded using a hand-held Canon PowerShot SD790 IS with a red LED headlamp for illumination. Video S3. An interspecific interaction from phase 2 of the behavioural experiment, in which a female Cyphoderris buckelli is mounted on the dorsum of a male Cyphoderris monstrosa, apparently clasped by his gin trap. The female periodically feeds on the hind wings of the male and also appears to struggle to free herself by occasionally biting his dorsal abdomen and hind legs. The male appears to be trying actively to engage his genitalia. Video was recorded using a hand-held Canon PowerShot SD790 IS with a red LED headlamp for illumination. Video S4. An interspecific interaction from phase 2 of the behavioural experiment, in which a female Cyphoderris buckelli receives a spermatophore from a male Cyphoderris monstrosa and is then released by his gin trap. Video was recorded using a hand-held Canon PowerShot SD790 IS with a red LED headlamp for illumination. File S1. Population genetic dataset [3042 single nucleotide polymorphisms (SNPs)]. Genotypes are in STRUCTURE format. File S2. Phylogenetic dataset [16 794 single nucleotide polymorphisms (SNPs)]. DNA alignment is in fasta format. File S3. Mitochondrial DNA dataset (641 bp). DNA alignment is in fasta format.