Research Article Journal of Orthoptera Research 2023, 32(2): 153-170 Geographic variation in the calling songs and genetics of Bartram’s round- winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species TIMOTHY G. Forrest!, MICAELA ScCosle!, OLIVIA BRUECKNER!, BRITTANIA BINTz2, JOHN D. SPOONER? 1 Department of Biology, University of North Carolina at Asheville, Asheville, NC, USA. 2 Department of Chemistry and Physics, Western Carolina University, Cullowhee, NC, USA. 3 Department of Biology, University of South Carolina at Aiken, Aiken, SC, USA. Corresponding author: Timothy G. Forrest (tforrest@unca.edu) Academic editor: Ming Kai Tan | Received 13 October 2022 | Accepted 8 February 2023 | Published 21 September 2023 https://zoobank. org/E6535B4D-60EE-4FF6-82 D9-E2899E64FCE7 Citation: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram’s round- winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi. org/10.3897/jor.32.96295 Abstract Previous work on Bartram’s round-winged katydid, Amblycorypha bar- trami Walker, found inconsistencies in song variation across the species’ range. Individuals of purported populations of A. bartrami from sandhills across the southeastern US were collected, recorded, and their genes were sequenced to better understand their population structure and evolution. Significant differences in songs, morphology, and genetics were found among populations from Alabama (AL), Georgia (GA), North Carolina (NC), and South Carolina (SC), and they differed from those of individu- als collected from the type locality in Florida (FL). Males from all popula- tions produced songs composed of a series of similar syllables, but they differed in the rates at which syllables were produced as a function of tem- perature. At temperatures of 25°C, the calling songs of males from popula- tions in northern AL and GA were found to have the highest syllable rates, those from SC had the lowest rates, and those from NC were found to pro- duce songs with doublet syllables at rates that were intermediate between those of males from FL and those of AL and GA. These song differences formed the basis for cluster analyses and principal component analyses, which showed significant clustering and differences in song spectra and morphology among the song morphs. A Bayesian multi-locus, multi- species coalescent analysis found significant divergences from a panmictic population for the song morphs. Populations from GA and AL are closely related to those of A. bartrami in FL, whereas populations from NC and SC are closely related to each other and differ from the other three. Large river systems may have been important in isolating these populations of flightless katydids. Based on the results of our analyses of songs, morphol- ogy, and genetics, three new species of round-winged katydids from the southeastern coastal plain and piedmont are described. Keywords massively parallel sequencing, multi-locus multi-species coalescent model, new species Introduction The round-headed katydids of North America (Amblycorypha Stal, 1873) consist of three species groups—oblongifolia, rotundi- folia, and uhleri—that differ in morphology and size (Rehn and Hebard 1914, Walker 2004). Walker et al. (2003) reviewed the rotundifolia complex and described two species, Amblycorypha bar- trami Walker, 2003 and A. alexanderi Walker, 2003, based on dif- ferences in their calling songs and ecology. All three species in the complex from the eastern United States are cryptic, with calling song being the only useful character for distinguishing between A. rotundifolia, A. alexanderi, and A. bartrami. Bartram’s round- winged katydid, A. bartrami, occurs primarily in xeric longleaf pine and turkey oak habitats in the southeastern United States. During Walker et al’s (2003) research, it became apparent that popula- tions of supposed A. bartrami near Aiken, South Carolina differed significantly in calling songs from typical A. bartrami from Florida. The specimens were designated A. nr bartrami at the time. Other populations (e.g., in North Carolina) also exhibited song anoma- lies that indicated more thorough investigations were needed. Two of us (TGF and JDS) undertook a broader examination of A. bartrami across its range, including collecting DNA and using molecular data to understand the population structure and evolu- tion within this species. Because the song rates of A. nr bartrami in South Carolina are similar to those of A. parvipennis, whose popu- lations are all west of the Mississippi River, we also include data from populations of A. parvipennis in Arkansas and Missouri. In this paper, we describe the variation in calling song, mor- phology, and genetics of populations of purported A. bartrami. We present the first molecular phylogenetic data from widespread populations in the rotundifolia complex, which show significant Copyright Timothy G. Forrest et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) 154 divergence among them. Members of the rotundifolia group, includ- ing A. bartrami, are flightless, which probably influences gene flow among populations. Therefore, we discuss the phylogeography of A. bartrami and how our genetic results relate to isolation and spa- tial population structure, particularly concerning river drainages and fragmentation of the longleaf pine habitat. Clustering analy- ses across populations also detected previously unidentified popu- lation differences in song and morphology. The significant genetic, acoustical, and morphological variation we discovered reveal new species that were, at one time, considered Amblycorypha bartrami. Materials and methods Fieldwork.—Fieldwork occurred mostly at night, and katydids were collected by listening for and finding males as they called or by searching vegetation for males and females using headlights. In some cases, males and females were collected during the day using sweep nets in areas and from vegetation likely to harbor katydids. Katydids were housed in 10 x 10 x 10 cm cages (either clear plas- tic or screened) with ad libitum water and food (apple, lettuce, oats, or a dry high-protein artificial diet; Gwynne 1988). For some individuals, we removed a hind leg that was stored at -80°C for DNA extraction and sequencing (see below). Collection sites were typical A. bartrami habitats of longleaf pine, turkey oak sandhills distributed throughout the southeastern US, including Alabama (AL) (30: 12, Cleburne Co.), Florida (FL) (4: 19, Liberty Co.), Georgia (GA) (434: 49, Gordon Co.), North Carolina (NC) (92: 19, Richmond Co.), and South Carolina (SC) (4¢: 39, Aiken Co. [SCA]; 63: 89, Edgefield Co. [SCE]; 64: 42, Georgetown Co. [SCG]). Collection sites for A. parvipennis include Arkansas (AR) (5: 39, Faulkner Co.) and Missouri (MO) (34: 02, Shannon Co.). Because the songs of GA and AL specimens were found to have similar features and females from each population duetted with males from each population, their data were combined in many analyses and were designated GAL. Acoustic recordings and analyses.—Calling songs of free-ranging males in the field or caged males in the laboratory were recorded with Sennheiser ME66 shotgun microphones and either a Tascam DAP-1 DAT recorder or a Marantz PMD-670 solid-state recorder. The sampling rate for the digital recordings was either 44 or 48 kHz. Time and frequency characteristics of the calling songs were deter- mined with Audacity 2.3 or using the seewave package in R (Sueur et al. 2008, Sueur 2018, R Core Team 2020, RStudio team 2020). To reduce noise and echoes, laboratory recordings were made with mi- crophones 0.5 m from the caged males with the substrate between the male and microphone covered with Sonex acoustic foam. The songs of A. bartrami are relatively uniform, and a com- plete cycle of wing movement (syllable = phonatome, Baker, and Chesmore 2020) is indicated by repeated patterns in the time waveforms of the songs (Walker et al. 2003). Syllable rates of katy- dids vary with temperature, and these relationships differ among species, making syllable rate a distinguishing character (Walker 1975). For consistency, one of us (TGF) measured syllable rates during the sustained portion of calling songs (see also Walker et al. 2003). When possible, the rates were based on 10 syllables. However, in some recordings, the sustained series had fewer than 10 syllables at consistent rates. In those cases, the rates were de- termined based on 4-8 (typically 5) syllables. We also included recordings from previous work in our analyses (Shaw et al. 1990, Walker et al. 2003). T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER Spectral variation among populations was also examined at two different temporal levels: 30 s of calling song and for indi- vidual syllables. Because spectra can be influenced by the record- ing environment, we used songs with high signal-to-noise ratios (x+SE=43+1.4 dB). Before spectral analyses, we removed low-fre- quency noise from the recordings using a finite impulse response bandpass filter (5 kHz-22 kHz, hanning window length = 512). The average spectra of each recording were normalized to prob- ability mass functions during each discrete Fourier transform (DFT: window length = 2048 with 0% overlap), and Kolmogorov- Smirnov (K-S) distances (Gasc et al. 2013) or relative frequency dissimilarities (Deecke and Janik 2006) were computed between each pair of recordings. The K-S distances are the maximum differ- ence between the cumulative probability mass functions of each spectrum in the pair. Relative frequency dissimilarity is a percent- age based on the sum of the ratio of minimums and maximums across all frequencies in the two spectra. Only single recordings from each male were used (30 s recordings—GAL: 4, FL: 3d’, NC: 44, SC: 63, A. parvipennis: 53; single syllables—GAL: 4¢, FL: 36, NC: 5d), SC: 86, A. parvipennis: 53). A hierarchical cluster analysis (hclust function in R) was performed on the distance/dissimilar- ity matrices to produce a dendrogram showing the relationships among the individuals’ songs or syllables (Sueur 2018). To test for differences among populations, we used distance-based redun- dancy analysis (db-RDA, ade4 package in R) and a principal co- ordinate analysis (PCoA, ade4 package in R) on the distance/dis- similarity matrices with population as a factor. We then ran Monte Carlo simulations (N =10000) to test for significant clustering by population under the H, of the db-RDA output (Sueur 2018). Morphological measurements.—To test for differences in morpho- logical characters among populations, we positioned preserved, pinned museum specimens so that digital images (11Mpix) could be taken of their dorsal and lateral aspects. In each photo, a scale in the same focal plane as the structures to be measured al- lowed calibrated measurements to be made with ImageJ software (Schneider and Rasband 2012). Measures (to the nearest 0.1 mm) included pronotal length along the midline (PrnL), maximal pro- notal width (PrnW), tegminal length (TegL) and width (TegW), hindwing exposure (HwEx), femur and tibia lengths of the hindleg (FemL and TibL, respectively), and for females, ovipositor length (OviL). See also Walker et al. (2003). Measurements for each char- acter were analyzed using ANOVA to test for differences among populations. We also used principal component analysis (PCA, ade4 package in R) on the matrix of morphological measures with population as a factor and conducted Monte Carlo simulations (N = 10000) to test for significant clustering by population under the H, of the PCA output. DNA extraction and sequences. —Genomic DNA was extracted from the proximal portion of the frozen femur of individuals from field populations of purported A. bartrami (AL (24: 19, Cleburne Co.), FL (16: 19, Liberty Co.), GA (10: 29, Gordon Co.), NC (4¢: 09, Richmond Co.), A. nr bartrami SC (06: 19, Aiken Co.; 13: 09, Edgefield Co.; 24: 22, Georgetown Co.) and for A. parvipennis in AR ( 24: 19, Faulkner Co.) and MO (2: 09, Shannon Co.). We used the standard protocol for the Qiagen DNeasy tissue kit (Qia- gen, Valencia, CA) and stored the gDNA extracts at either -20°C or -80°C until they were used for PCR amplification and sequencing. Because reliance on a single barcoding gene might cause prob- lems in phylogenetic analyses (Moulton et al. 2010), massively JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) 155 T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER parallel sequencing was performed to simultaneously interrogate regions of mitochondrial, and nuclear DNA for analysis. In particu- lar, we sequenced the cytochrome oxidase subunit I (COI, 658 bp) mitochondrial gene and a large region of nuclear ribosomal DNA (rDNA) that included portions of 28S and 18S rDNA as well as the entire region of 5.8S rDNA and two internal-transcribed spac- ers ITS1 and ITS2 (~3700 bp). The COI gene is a barcoding gene that has short divergence times and has been used extensively in molecular systematics (Hebert et al. 2003). Ribosomal genes (28S, 5.8S, and 18S) are relatively conserved with little change over long periods of time, whereas the internal transcribed spacers are more labile and thus have been used successfully to distinguish cryp- tic species in some taxa (Li and Wilkerson 2005, Li et al. 2010). Additionally, we sequenced three nuclear genes, histone 3 (HIS), tubulin-alpha I (TUB), and wingless genes (WNG), that have been used in tettigoniid phylogenies (Mugleston et al. 2013). PCR amplification.—Published primer pairs were used to amplify the regions of interest (Table 1). The Roche FastStart High Fidel- ity PCR System (Millipore Sigma, St. Louis, MO) was used for all amplifications. PCR amplification for the ~3700 bp rDNA region used conserved primers LR7 and NS19b with an initial denatura- tion at 95°C for 2 min followed by 35 cycles of 60 sec at 95°C, 60 sec at 50°C, and 5 min at 68°C plus an additional 20 sec- onds each successive cycle. The final PCR extension was 7 min at 72°C. PCR reactions (50 pL total volume) contained final reagent concentrations of 2.5 U of Roche FastStart High Fidelity enzyme blend, 1.8 mM MgCl, 0.4 1M each forward and reverse primer, 4% DMSO, and 0.2 mM each dNTP. Reverse touchdown ampli- fication of COI used LCO1490 and HCO2198 primers and had thermal cycling parameters including an initial denaturation of 95°C for 2 min followed by 6 cycles of 30 sec at 94°C, 90 sec at 45°C, and 60 sec at 72°C and an additional 34 cycles of 30 sec at 94°C, 90 sec at 49°C and 60 sec at 72°C with a final extension of 7 min at 72°C. PCR reactions (25 pL total volume) contained final reagent concentrations of 5 U of Roche FastStart High Fidelity enzyme blend, 1.8 mM MgCl, 0.6 11M each forward and reverse primer, 6.25% DMSO, and 0.2 mM each dNTP. Tubulin-alpha I genes were amplified with 294F1 and 294R1 primers, histone 3 genes with H3 AF and H3 AR primers, and wingless genes with WG550F and WGABRZ primers, respectively. Tubulin-alpha I, his- tone 3, and wingless genes were amplified in independent PCR reactions with thermal cycling parameters having an initial dena- turation at 95°C for 2 min followed by 35 cycles of 30 sec at 94°C, 30 sec at 50°C, and 50 sec at 72°C with a final 7 min extension at 72°C. PCR reactions (25 pL total volume) contained final reagent concentrations of 5 U of Roche FastStart High Fidelity enzyme blend, 1.8 mM MgCl, 0.6 1M each forward and reverse primer, 6.25% DMSO, and 0.2 mM each dNTP. Amplicon products were quantified using an Agilent 2100 Bioanalyzer and DNA 1000 kit (Agilent Technologies, Inc., Santa Clara, CA). Library preparation and massively parallel sequencing (MPS).—PCR products were diluted to a concentration of 0.2 ng/L and enzy- matically fragmented and tagged with MPS sequencing adapters using the Illumina Nextera XT Library Prep kit (Illumina, Inc., San Diego, CA). Limited-cycle PCR was used to add flow cell adapt- ers and multiplexing barcodes to fragmented libraries. Flow cell adapters enable library fragments to anchor to the surface of the solid support where sequencing occurs. Barcodes allow for post- sequencing parsing of sample-dependent data, which permits a high degree of multiplexing per sequencing run. Solid-phase reversible immobilization (SPRI) beads were used to purify the prepared libraries via the removal of unincorporated primers and dNTPs that could affect sequencing downstream. Libraries were then normalized to ensure equal representation of each sample, and equal volumes were pooled to create a master library for se- quencing. Sequencing was performed on an Illumina MiSeq using a v3 2 x 300 cycle kit (Illumina, Inc., San Diego, CA). Assembly, validation, and alignment.—Sequence analyses were car- ried out using Geneious Prime 2020.1.2. NextGen Fastq sequenc- es were first set as paired reads and trimmed using BBDuk with a minimum quality Q30 and a minimum length of 20. These reads were then assembled to GenBank (Clark et al. 2016) refer- ence sequences (COI: HQ968170 and ITS/5.8s ribosomal genes: AM888963) of Scudderia furcata, another phaneropterine katydid, and two other sequences from members of Amblycorypha (tubulin- alpha I: KF571404 and wingless: KU550854.1). Major vote con- sensus sequences were extracted from these assemblies, inspected for quality, and searched for within NCBI using BLAST (Altschul et al. 1990). All alignments were made using Clustal Omega 1.2.2 with fast clustering, a cluster size of 100, and 3 refinement itera- tions (Sievers et al. 2011). Table 1. Primer pairs and annealing temperatures for PCR and expected size for sequences. Primer Sequence 5’3’ Anneal (°C) %GC Amplicon Size (bp) Ref COI Primers F LCO1490 GGTCAACAAATCATAAAGATATTGG 59.7 32.0 Folmer et al. 1994 RHCO2198 TAAACTTCAGGGTGACCAAAAAATCA 64.5 34.6 658 Folmer et al. 1994 28S and 18S rDNA Primers F LR7 TACTACCACCAAGATCT 53.6 41.2 Vigalys and Hester 1990 RNS19b CCGGAGAGGGAGCCTGAGAAC 68.9 66.7 ~3700 Bruns Lab, UC Berkeley Histone 3 Primers F H3 AF ATGGCTCGTACCAAGCAGACV 50.0 55.6 Colgan et al. 1998 RH3 AR ATATCCTTRGGCATRATRGTG 50.0 40.5 0375 Colgan et al. 1998 wingless (wg) Primers F WG550F ATGCGTCAGGARTGYAARTGY 50.0 47.6 Mugleston et al. 2013 R WGABRZ CACTTNACYTCRCARCACCAR 50.0 50.0 ~450 Mugleston et al. 2013 tubulin-alpha I Primers F 294F1 GAAACCRGTKGGRCACCAGTC 50.0 eS HS) Buckman et al. 2012 R 294R1 GARCCCTACAAYTCYATTCT 50.0 42.5 ~350 Buckman et al. 2012 JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) 156 Phylogenetic analysis. —Phylogenetic relationships were inferred us- ing Bayesian analysis in *BEAST2, which uses the Markov chain Monte Carlo (MCMC) process to explore tree space based on pos- terior probabilities (Bouckaert et al. 2019). We used BEAUTi2 to generate the analysis parameters. We set each gene sequence as a separate partition in the multi-locus, multi-species coalescent analysis and allowed the program to integrate analytical popula- tion size. We set the site substitution model for all genes to HKY with frequencies empirically estimated. The analysis was run un- der a strict clock for each gene partition with priors, using the birth-death model to estimate birth and death rates during the analysis. The number of MCMC iterations was 1.2E8, which was sufficient for the model to reach stationarity after a 20% burn- in. The output of each *BEAST2 run was inspected using Tracer vl1.7.2., and the trees were visualized and annotated using Densi- Tree v2.2.7 and TreeAnnotator v2.6.6, respectively. TreeAnnotator produced trees with maximum clade credibility for each gene tree and for the species tree that resulted from the coalescent analysis. We used different random seeds to conduct 5 *BEAST2 analyses to ensure that the random process adequately covered tree space and that the output trees generated were robust. We ran the analy- ses with all populations separated and with putative ‘species’ that A FL M04 2004_02 HHH HH B FL Mo? 2007_10 \ | \ 1 iil Mil | Hi! | | WTA t IH) it l | | i y ii | EE il Cc FLMO3 2007_06 | il) did) qt | | E prose D FLMO3 2007_065 . Frequency (kH T BA : Amplitude 3 Fig. 1. A-C. Oscillograms (30s) of calling songs of 3 male A. bartra- mi from Liberty Co., Florida. Songs consist of a long duration, sus- tained main series of (~100) syllables preceded by several (15-20) short-duration series of 1-7 syllables; D. Oscillogram showing 16 syllables within yellow highlighted portion of the main series of C; E. Oscillogram and spectrogram showing the fine temporal struc- ture and frequency content of 3 syllables highlighted in D. T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER were suspected based on differences in syllable rate functions with temperature (see below). Deposition of specimens, recordings, and sequences.—Unless otherwise indicated, the specimens are currently housed at the University of North Carolina at Asheville (UNCA). The collection, along with types, will be transferred to the Florida State Collection of Arthro- pods (FSCA), Gainesville, FL. Recordings will be made available through the Macaulay Library of Natural Sounds at the Cornell Lab of Ornithology and Singing Insects of North America (SINA) website. Sequencing data have been uploaded to the National Center for Biotechnology Information (NCBI) under BioProject PRJNA906584. Results Song variation.—Figs 1-6 show the temporal structure of call- ing songs among populations. For all populations, the calling songs are a series of easily quantified, repeated syllables repre- senting a single cycle of wing movement. The calling songs of A. bartrami and A. parvipennis do not exhibit the extreme song complexity of the virtuoso Amblycorypha, which have 4 syllables A S¢ MOt 2003_05 se B SC MO1 2019_1005 | | EE) Cc SC M02 2003_01 Hl | Ss E 20 s | toa t. Zo Ff 4 z = D SC M02 2003_01s = a § wo L= “ g = a L Time (s) Fig. 2. A-C. Oscillograms (30s) of calling songs of 3 male A. nr bartrami from Aiken Co., South Carolina. Songs consist of a long duration, sustained main series of syllables preceded by several shortduration series of 1-5 syllables; D. Oscillogram of 15 syl- lables highlighted in C; E. Oscillogram and spectrogram showing the fine temporal structure and frequency content of 3 syllables highlighted in D. JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER that may be produced with varying syntaxes (Walker and Dew 1972, Walker 2004). In most cases, the songs of A. bartrami consist of a longer duration, sustained (main) series of syllables preceded by 1 to >20 shorter series that typically increase in amplitude. Calling songs vary significantly among the popula- tions in several ways. Temporal variation.—Songs of males from the Florida panhan- dle (N=3<: 9 series) have sustained portions with significant- ly more syllables (x+SE=107+22) than all other populations (GAL: 841, N=40: 53 series; NC: 1741, N=8<: 30 series; SC: 2542, N=11¢: 112 series; A. parvipennis: 24+3 N=6: 39 series; Fig. 7). Males from NC nearly always produced syllables in dou- blets during the sustained main portion of their calling song (Fig. 5E). Of the 517 syllables produced in 30 main series from 8 males, 482 were doublets, 8 (2%) were singlets, and 9 (5%) were triplet syllables. Series that precede the main series of calling songs have, on average, 5-6 syllables for males from FL, whereas those in songs of males from other populations have fewer (GAL: 3-8; NC: 2-3; SC: 2-4; A. parvipennis: 1-2; Fig. 7). Males of A. parvipennis rarely (13%, 5 of the 39 series from 6 males) produce syllables preceding the main series of their calling songs (Fig. 7). See Suppl. material 3. A GAMO1) 2007_12 B GA Mot) 2007_14 Cc GA Mot) 2007_15 Frequency (kHz) 0.00 Time (5) Fig. 3. A-C. Oscillograms (30s) of calling songs of 1 male A. bar- trami from Gordon Co., Georgia. The long-duration sustained, main series of syllables are rarely preceded by shorter series as found in the songs from other supposed populations of A. bar- trami; D. The yellow highlighted portion of C; E. Oscillogram and spectrogram of 3 syllables highlighted in D. 157 Syllable rate variation.—Based on the relationships of syllable rates with temperature (Fig. 8, Suppl. material 2), there are at least 4 different song types across the populations we sampled. Males from northern GA and northern AL (GAL) have functions with the greatest slopes (m=0.81, Fig. 8: green) and rates of ~13.1s"! at 25°C. SC males (A. nr bartrami) have the slowest syllable rates at ~5.8s! at 25°C (m=0.29, Fig. 8: orange), which is very similar to that of A. parvipennis ~5.0s! at 25°C (m=0.16, Fig. 8: black). Males from northern FL and the FL panhandle produce syllables at inter- mediate rates of 10.0s! at 25°C (Fig. 8: red). Males from western AL had songs with syllable rates similar to those of FL males (Fig. 8: pink). Because NC males produced songs with syllable dou- blets, two rates were calculated. The faster syllable rate, within a doublet (m=0.58, 11.6s" at 25°C), falls between rates for songs of FL males and those of GA and AL males whereas the slower rate, between doublets (m=0.17, ~4.0s' at 25°C), was slower than the rates of SC A. nr bartrami and A. parvipennis males. Syllable variation.—Fig. 9 shows the variation in the fine tempo- ral structure of syllables produced by males from each popula- tion. The impulses in each syllable are probably the result of the scraper engaging and releasing a single tooth on the file. Males from FL have a single pulse train followed by a longer A AL MO4j 2007_07 | vil | rr een | B AL Mos) 2007_10 AL Mo6y 2007_23 AL M06) 2007_235 Frequency (kHz) Amplitude 9.00 0.05 O10 0.15 0.20 025 0.20 Time (s) Fig. 4. A-C. Oscillograms (30s) of calling songs of 3 male A. bar- trami from Cleburne Co., Alabama. Syllable rates of sustained main series are similar to those of males from north Georgia (Figs 3, 7); D. Oscillogram of 17 syllables highlighted in C; E. Yel- low highlighted portion of D showing detailed temporal structure and spectral composition of 3 syllables. JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) NC M04 2006 NC M06 2006 NC M02 2004_23 3 20 zi fé 18 b sis to + ah i 0 a> 4t2 : Angitute ae fo8) sS +3 naif = ‘ #6 20h at ek r & 4 45 20 25 2 NC M02 2004_23s. iw] Frequency (kHz) n Amplitude Time (s} Fig. 5. A-C. Time waveforms (30s) of calling songs for 3 NC A. bar- trami males. Note the variation in the number of syllables that pre- cede the main sustained train of syllables; D. Highlighted portion in C; E. Oscillogram and spectrogram of highlighted portion of D with 6 syllables in 3 doublets. Doublet syllable rates were faster than those of FL A. bartrami and slower than those of GA-AL A. bar- trami. FL, GA, AL, and SC males rarely produced doublet syllables. terminal pulse train in the syllable. In all other populations, males exhibited two pulse trains made up of 1-5 pulses before the terminal pulse train. Spectral variation.—The signals produced by males are broadband, with most of the energy between 10-15 kHz (Panel E of Figs 1-6). Hi- erarchical cluster analyses using K-S distance and relative frequency dissimilarity metrics indicate that significant spectral variation exists between populations of A. bartrami, A. nr bartrami, and A. parvipen- nis at the level of calling song and syllables (Figs 10, 11, respectively, Suppl. material 4). Although there is much overlap among popula- tions in the dendrograms (Figs 10A, B, 11A, B), principal coordinate analyses calculated on the distance/dissimilarities show significant clustering within populations and differences among populations. Average spectra computed over the entire calling song (30s) differed significantly from random when using population as a factor for both distance metrics (K-S distance, Monte-Carlo test simulation, N=10000, p=0.032, first two components explain 77% of total vari- ance; relative frequency dissimilarity, Monte-Carlo test simulation, N=10000, p=0.018, first two components explain 43% of total vari- ance; Figs 10C, D). The same was found when the average spectra were calculated on smaller time scales associated with single sylla- bles (K-S distance, Monte-Carlo test simulation, N= 10000, p<0.008, T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER A par M01 2007_14 A par M02 2007_25 A par MOT 2007_09 D A par MO7 2007_095 Frequency (kHz) TT Bie Cy “ihe Sb is Amplitude _| te h 0 = | 5 e i | “10 ¢ i 2 | “15 | -20 | 2 | -30 Amplitude Fig. 6. A-C. Oscillograms (30s) of calling songs of 3 male A. parvi- pennis; D. Oscillogram of 10 syllables highlighted in C; E. Fine tem- poral structure and spectrogram of 3 syllables highlighted in D. first two components explain 74% of total variance; relative frequen- cy dissimilarity, Monte-Carlo test simulation, N=10000, p<0.002, first two components explain 32% of total variance; Figs 11C, D). Morphological variation.—Morphological characters differed among some of the populations (Table 2, Suppl. material 1). The only significant differences among females’ character measurements were pronotal length (PrnL), where GAL females have significantly shorter PrnL (5.99+0.23 mm) than SC females (6.60+0.07 mm), and respective A. parvipennis females (6.96+0.19 mm). The lack of significant differences among any other female morphological measurements may, in part, be due to the small sample sizes as- sociated with many of the populations. There were many differences in male size among some of the populations. Similar to our findings for female PrnL, GAL males also had significantly shorter PrnL (5.08+0.14 mm) than SC males (6.03+0.09 mm) and A. parvipennis males (6.15+0.13 mm). For nearly every morphological measurement (TegL, TegW, FemL, TibL), GAL and A. parvipennis were shorter than the other popula- tions (Table 2). GAL males differed significantly from NC males in all measures except HwEx (PrnW: 3.64+0.08 vs 4.32+0.06 mm, respectively; TegL: 25.4+40.70 vs 28.2+0.21 mm, respectively; TegW: 7.83+0.21 vs 9.32+0.18 mm, respectively; FemL: 23.3+0.11 vs 27.340.38 mm, respectively; TibL: 25.140.20 vs 28.7+0.44 mm, respectively). GAL males had significantly shorter hind femurs (FemL) and hind tibiae (TibL) than that of all other purported A. bartrami populations (Table 2). JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER = fo] o SOEs ule Ul Se|qel|AS # UeEY\ (3) JG) { 80 3) Ta) [sy la To Jo Mean # Syllables Series Re Main Fig. 7. Mean (+SE) number of syllables as a function of the temporal relationship between series in male calling song from populations of supposed Amblycorypha bartrami (GA & AL: green, NC: blue, FL: red, SC: orange) and A. parvipennis (black). Triangles (X=0) represent the mean number of syllables in the sustained main series of songs aver- aged over the number of males shown in parentheses. Circles are the means for each series preceding the main series with the number of males contributing to the average indicated in parentheses. 15 10 Syllable Rate (s‘) 18 20 22 24 26 28 Temperature (°C) Fig. 8. Syllable rate as a function of temperature for populations of Amblycorypha bartrami (GA & AL: green, NC: blue, FL: red, AL: pink, SC: orange) and A. parvipennis (black). Solid symbols are recordings from our research and open symbols are recordings from other published work (Walker et al. 2003 for A. bartrami and A. parvipennis, Shaw et al. 1990 for A. parvipennis). Because the functions of syllable rate and temperature be- tween SC A. nr bartrami and A. parvipennis are so similar, it is im- portant to compare the morphological traits among them. Male SC nr bartrami had significantly longer tegmina (TegL: 26.2+0.44 vs 23.8+0.47 mm, respectively) and significantly longer hindwing exposure (HwEx: 2.74+0.17 vs 0.89+40.34 mm, respectively) than A. parvipennis males. Hindwing exposure is one of the key charac- teristics distinguishing A. parvipennis from all eastern members of the rotundifolia complex (Rehn and Hebard 1914). Principal component analysis indicates morphological differ- ences among the supposed populations of A. bartrami (Fig. 12). In 159 Fig. 9. Syllable variation among populations of A. bartrami. A. Florida N=3¢; B. Alabama N=36; C. Georgia N=1¢; D. South Carolina N=5d; E. North Carolina N=5<. Syllables consist of brief decaying impulses that are likely the result of the scraper engaging and releasing a single tooth on the file. Males from FL (A.) typi- cally have a single pulse train of 1-2 pulses preceding the longer terminal pulse train in the syllable. Males from all other popula- tions have two pulse trains (1-5 pulses) preceding the terminal pulse train. Scale bars: 100ms. particular, the slope of the ordination in the PCA for GAL males is negative, whereas it is positive for all other populations whose relationships are all parallel (Fig. 12). FL, NC, and SC males tended to be more similar in the averages of their morphologi- cal characters. Characters that contributed most to PC1 were TibL (20%), FemL (19%), PrnW (16%), TegL (16%), and TegW (16%), and those that contributed most to PC2 were HwEx (40%), PrnL (28%), and TegL (14%). Genetic variation. —Once sequences were processed and aligned, the lengths of consensus sequences were COI: 658 bp, H3: 333 bp, ITS1, ITS2, and 5.8S ribosomal genes = ITS3k: 3262 bp, TUB: 341 bp, and WNG: 371 bp. BLAST searches of each gene sequence, except for TUB, invariably matched those of Amblycorypha or other members of the Phaneropterinae (COI: all individuals >90% match to COI of Amblycorypha floridana [HQ983647.1, HQ983648.1, HQ983649.1], Amblycorypha oblongifolia [HQ983655.1, JN294610.1, KM532357.1, KM536809.1, KR144595.1] or Amblycorypha sp. [MG466233.1]; H3: all individuals 100% match to histone 3 gene of Amblycorypha sp. [KF571154.1]; ITS3k: all individuals >98% match to 28S Mi- crocentrum rhombifolium or Scudderia furcata; WNG: all individuals JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) 160 T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER Table 2. Mean (SE, N) of morphological measures (mm) from populations of supposed Amblycorypha bartrami and populations of A. parvipennis.* Sex Population PrnL PrnW TegL TegwW FemL TibL OviL Females FL 652% 4.33° 28.6 9.11 29.6 30.6 9.83 (NA, 1) (NA, 1) (NA, 1) (NA, 1) (NA, 1) (NA, 1) (NA, 1) GAL 599? 3.86? 26.0 8.07 P Bo ea 26.6 10.7 (0.23, 5) (0.13, 5) (0.67, 5) (0.34, 5) (0.49, 5) (0.59, 5) (0.38, 5) NC 20527 4.58* 28.0 9.63 29:9 31.0 11.0 (NA, 1) (NA, 1) (NA, 1) (NA, 1) (NA, 1) (NA, 1) (NA, 1) SC 6.60° 4.12? 26.6 Sal2 27.6 PR Lar 9.93 (0.07, 17) (0.08, 17) (0.47, 13) (0.25, 15) (0.40, 15) (0.40, 14) (0.25, 17) A. par 6.96° 4.52° 25.8 7.81 PA SA 28.3 9.9 (0.19, 3) (0.07, 3) (0.78, 3) (0.27, 3) (0.54, 3) (0.68, 3) (0.54, 2) Males FL 5.88 4.01% 29.7? 931° 28.0° 28.8° (0.05, 3) (0.14, 3) (0.72, 3) (0.17, 3) (0.38, 3) (0.59, 3) GAL 5.08° 3.64 25a 7283" 253° 25.15 (0.14, 7) (0.08, 7) (0.70, 7) (0.21, 7) (0.11, 7) (0.20, 7) NC 5.84 4.32? DS 22 9:32" DP ae 28.7% (0.04, 9) (0.06, 9) (0.21, 9) (0.18, 9) (0.38, 6) (0.44, 6) SC 6.03? 4.00% 262 8.21 27312 Pca (0.09, 17) (0.09, 17) (0.44, 17) (G.17;.17) (0.42, 15) (0.34, 15) A. par 6.15* Baye 23.88 rae parma 26,6"° (0.13, 8) (0.08, 8) (0.47, 8) (0.25, 8) (0.48, 8) (0.41, 8) * Comparisons of means within each sex were made for each morphological trait. Means within a column followed by different letters are significantly different (ANOVA, Tukey honest significant difference posthoc test, P<0.05). >99% match to WNG Amblycorypha longinicta |KU550854.1] or Am- blycorypha sp. [KF571288.1]. There was no genetic variation in H3 among all samples; therefore, we did not include H3 sequences in any further analyses. For all individuals sequenced, TUB sequences matched tubulin-alpha I sequences of members in the Tettigonii- dae, with 11 individuals matching (85-97% identical) sequences in the Phanertopterinae (Syntechna and Trigonocorypha), 11 individuals matching (79-80% identical) Lipotactes maculatus (Lipotactinae), and one individual matching 82% of the tubulin-alpha I sequence of Kuzicus megaterminatus (Meconematinae). Because these match- es for TUB were so varied, we ran the *BEAST2 analyses with and without TUB sequences included. Multiple runs of our multispecies coalescent analyses pro- duced identical phylogenetic topologies with only small differenc- es in the posterior probabilities at the nodes. Effective sample sizes (ESS) for every parameter of the models were always over 1500. Gene trees.—Gene trees based on COI, ITS3k, and WNG sequences were similar to the species trees generated in our analysis (Figs 13, 14). Gene trees using TUB sequences differed substantially from the species trees. The estimated mutation rate of mitochondrial gene COI was about 45X that estimated for ITS3k, about 10X that for TUB, and almost 15X that for WNG. Our gene tree for COI using the Bayesian coalescent approach showed high support for most populations (AL, GA, NC, SCG, AR A. parvipennis, MO A. parvipennis with all posterior probabili- ties >0.97, Fig. 13A). The greatest uncertainty involved the two South Carolina populations (SCA and SCE) where we had data from only single individuals. There was high support for grouping the South Carolina population (Georgetown Co., SCG) with the North Carolina population (posterior probability = 1.0). Support values for our ribosomal gene trees were variable. The tree supported monophyly of A. parvipennis (posterior = 1.0), grouped the two individuals from South Carolina together (SCA and SCE, posterior probability = 0.98), grouped two of the SCG individuals with all North Carolina individuals (posterior prob- ability = 1.0), grouped all GA individuals with most of the AL indi- viduals, and grouped the two FL individuals (posterior probability = 0.79) (Fig. 13B). The gene trees for our nuclear sequences (TUB and WNG) dif- fered more from the population/species trees than the COI and ITS3k gene trees. Interestingly, the A. parvipennis populations clus- tered in the middle of both gene trees (Fig. 13C, D). Species/population trees.—The output of our coalescent analyses (trees with maximum clade credibility) indicated genetic diver- gences among all populations that we sampled (Fig. 14A). Our phylogenetic analyses showed that AR and MO populations of A. parvipennis differed genetically and are more closely related to each other than they are to all supposed A. bartrami populations we sampled (posterior probability = 1.0). The population tree in- dicates (Fig. 14A) that A. bartrami populations from AL and FL split more recently and that there was an earlier divergence from populations in GA. Populations of supposed A. bartrami in the west (AL, FL, and GA) differ from those in the east (NC and SC). Interestingly, populations from within SC differ from each oth- er genetically although they have identical syllable rates in their songs. South Carolina A. nr. bartrami from Georgetown Co., SC were found to be more closely related to NC ‘bartrami’ than to populations in Edgefield Co. or Aiken Co., SC A. nr. bartrami. Note that the NC and Georgetown Co. SC populations are also closer geographically (see Phylogeography below). When the analysis was done with individuals grouped by call- ing song information (Fig. 14B), song morphs from GAL (GA and AL) diverged from those in FL and differed (posterior probability = 1.0) from the two eastern song morphs NC and SC (posterior JOURNAL OF ORTHOPTERA RESEARCH 2023, 32(2) T. G. FORREST, M. SCOBIE, O. BRUECKNER, B. BINTZ AND J. D. SPOONER A wT (o>) oO (om) N oO = ao = o oOo x= °o oO ooo oooooclucwcmcmcUW CGO ee eer Oo fo a nS SS SS SS SS Senonnrtr Pet an ppt ace S "o-.07 OOF Ooo: Heo. goto. Bs Sra sO ENE eee) Siar a, Amal Pele Sean tO OOO One 9 Fal oe Z ZO Zz°*2z 0 oO oO oO oO ” ” n ” 7) 7) Recording B Oo i