Distinctive but Functionally Convergent Song Phenotypes

JOURNAL OF NATURAL HISTORY, 2018 VOL. 52, NOS. 25–26, 1603–1635 https://doi.org/10.1080/00222933.2018.1478011

Distinctive but functionally convergent song phenotypes characterize two new allopatric species of the Chrysoperla carnea-group in Asia, Chrysoperla shahrudensis sp.nov.and Chrysoperla bolti sp.nov.(Neuroptera:Chrysopidae) Charles S. Henrya, Stephen J. Brooksb, James B. Johnsonc, Atsushi Mochizukid, Alinaghi Mirmoayedie and Peter Duellif aEcology and Evolutionary Biology, University of Connecticut, Storrs, CT, USA; bDepartment of Life Sciences, The Natural History Museum, London, UK; cDivision of Entomology, University of Idaho, Moscow, ID, USA; dEntomology Department, Institute for Agro-Environmental Sciences NARO, Tsukuba City, Japan; eDepartment of Plant Protection of Razi University, Kermanshah, Iran; fBiodiversity and Conservation Biology, WSL Swiss Federal Research Institute, Birmensdorf, Switzerland

Chrysoperla shahrudensis sp.nov.isdiscoveredinnorthernIran, ARTICLE HISTORY co-occurring with at least ﬁve other cryptic species of the Received 29 November 2017 Chrysoperla carnea-group. It is distinguished by the volley period Accepted 9 May 2018 and tonality of its courtship duetting song. Another Asian species KEYWORDS from alpine meadows of northern Kyrgyzstan, previously Convergent evolution; C. ‘adamsi-K’ but here named Chrysoperla bolti sp. nov., has a courtship song; duetting; song distinct from but convergent with both C. shahrudensis and Eurasia; parallel speciation; North American Chrysoperla adamsi. Coordinated duets can be sibling species; systematics; established in the laboratory between individuals of vibration C. shahrudensis and recorded songs of either C. bolti or C. adamsi. Such functional song equivalence in distinct allopatric species suggests that repeated episodes of parallel speciation can drive the origin of cryptic species diversity in lacewings. Morphology, life history, and ecology of larvae and adults of C. shahrudensis and C. bolti are then formally described. Adding C. shahrudensis to a large mitochondrial DNA data set for ≈ 21 species shows it to be similar to neither C. adamsi nor C. bolti, further supporting independent, convergent evolution of song rather than song similarity due to relationship. Although C. bolti and C. shahrudensis are both from Asia and share some basic temporal song features, the two taxa are distinct, allopatric bio- logical species. www.zoobank.org/urn:lsid:zoobank.org:pub:D9B7BDC9-6C09-468B-A6B-D378628EC557

Introduction The green lacewing species Chrysoperla carnea (Stephens) was once thought to be distributed across the entire North Temperate Zone (Tjeder 1960; Tauber and Tauber 1986). However, it is now understood that the original taxon comprises at least 21 distinct but morphologically cryptic sibling species, reproductively isolated from each

Published online 29 Jun 2018 1604 C.S. HENRY ET AL. other by complex species-specific, substrate-borne vibrational duetting songs (Henry et al. 2013). These species, collectively assigned to the ‘carnea’ species-group of Chrysoperla (Brooks 1994), generally exhibit large, often extensively overlapping geographic distributions. Throughout its range, each species retains its distinctive courtship signal, including in regions where it might be sympatric or even syntopic with other members of the carnea-group (e.g. compare the song of Chrysoperla agilis Henry et al. in Switzerland, the Azores, or Fairbanks, Alaska – see Henry et al. 2003, 2011). Wherever there is geographic heterogeneity, cryptic species diversity is high in the carnea-group. For example, at least five of the eight carnea-group species recognized in North America are largely syntopic in the far western mountains of that continent (Henry et al. 2013). In Eurasia, the known complement of sibling species is 12 (Henry et al. 2013), and at least five of these at a time can be found living together in close association in mountainous regions (Duelli et al. 2015). Asia is relatively under-collected and its carnea- group fauna is poorly understood (e.g. see Mirmoayedi et al. 2014), except in the south and Far East (Henry et al. 2010, 2015). Recent opportunities to collect in Iran and the Kyrgyz Republic therefore presented a chance to sample the carnea-group from western and central Asia, respectively. The recent trip to Iran included authors P. Duelli and A. Mirmoayedi and their colleague D. Bolt (MTM Consulting, Switzerland) and took place during August 2014. This trip, plus earlier collecting in Iran by Dr H. Heidari (Centre for Sustainable Development, Iran) in July 2002 (Henry et al. 2014), revealed the presence in that country of five described species: Chrysoperla carnea (Stephens), Chrysoperla pallida Henry et al., C. agilis Henry et al., Chrysoperla heidarii Henry et al., and Chrysoperla zastrowi sillemi (Esben-Petersen). Two additional widespread species, Chrysoperla mediterranea (Hölzel) and Chrysoperla lucasina (Lacroix), occur in nearby Armenia and Georgia (Henry et al. 2014; Duelli et al. 2015) and are probably also present in Iran, although song-confirmed specimens have not yet been found there (but see Mirmoayedi and Thierry 2002, who nevertheless included C. lucasina in the lacewing fauna of Iran based on morphology). Also collected during the 2014 trip were six gravid females of a new ‘song species’ of the carnea-group, from which more than 20 offspring were reared to adulthood. We describe here the external appearance of all life stages of this Iranian species, naming it Chrysoperla shahrudensis sp. nov. for the collecting site of the holotype (Shahrud). Its species-specific vibrational courtship songs and duets are also described for both sexes, including song features that distinguish the taxon from all other cryptic species in the carnea-group. Comparative ecological and song playback observations of C. shahrudensis are included, and its phylogenetic affinities within the carnea-group are inferred using p-distances and branching topology calculated from mitochondrial DNA sequences. Another recent collecting trip of P. Duelli and D. Bolt was to Kyrgyzstan in central Asia during June and July 2016. Three distinct but undescribed song species were collected there, all of which had been found by P. Duelli on a previous trip to that country in May and June, 1995 (Aspöck et al. 1996). One of these species, informally known as C. ‘adamsi KR’ or ‘adamsi-K’ (Wells and Henry 1998; Henry, Wells et al. 1999), exhibits some intriguing acoustic similarities to C. shahrudensis. Thus, parallel speciation (Schluter and Nagel 1995) due to convergent evolution of courtship songs in species having non-overlapping geographic ranges is once again shown to be an important theme JOURNAL OF NATURAL HISTORY 1605 underlying evolution and speciation in the carnea-group (Wells and Henry 1998; Henry et al. 2012, 2014). Given these apparent song similarities between C. ‘adamsi-K’ and C. shahrudensis, we present here a formal description and comparative diagnosis of this central Asian species as Chrysoperla bolti sp. nov., named in honour of our colleague and collector, Daniel Bolt. Included are comprehensive acoustic and morphological data pertaining to all its life stages, ecological and song playback observations, and phylogenetic inferences from mitochondrial DNA.

Materials and methods Collecting, rearing and identification Chrysoperla shahrudensis sp. nov.: Six gravid adult females of this species were collected in the Islamic Republic of Iran between 1200 and 2300 m elevation during August 2014 by P. Duelli, A. Mirmoayedi and D. Bolt. Collecting data are shown in Table 1. Each individual was isolated in a 250-ml clear-plastic arena, placed under long-day photo- period (17 : 7 h light : dark), and provided with water and a Wheast-based diet (Hagen and Tassan 1970). Because field-mated females will not sing (Henry and Busher 1987), it was necessary to rear progeny of mated individuals to determine species identity. Eggs were therefore collected and the larvae were raised individually in 28-ml clear-plastic cups at 20–26°C on sterile Ephestia kuehniella (Zeller) moth eggs plus locally available species of Aphidae. Newly spun cocoons were shipped (in compliance with USDA APHIS- PPQ permit P526P-07–06006) to Storrs, CT, USA, where songs were recorded from quarantined virgin adults 3–5 days after eclosion (see below). We were able to obtain living adult offspring from all six females. Song-identified individuals were preserved either as dried specimens or in 96% ethanol for molecular systematic studies (Price et al. 2015). Several first-, second-, and third-instar larvae were taken from rearing stocks and preserved in alcohol for morphological study. Voucher specimens were deposited in (i) the University of Connecticut Invertebrate Collection, Storrs, CT, USA (UCMS); (ii) the Natural History Museum, London (BMNH); (iii) the collection of Peter Duelli, Zürich,

Table 1. Collecting sites for living Chrysoperla shahrudensis sp. nov. in Iran (western Asia) and Chrysoperla bolti sp. nov. in Kyrgyzstan (central Asia). Locality Elevation GPS coordinates Date collected Specimens tested for song Pass Qazvin to Alamut Valley, Iran 2267 36.38°N, 50.21°E 5 August 2014 4 ♀,1♂ progeny of 1 field (Populus, Alnus) C. shahrudensis Shahrud, Iran (agricultural: 1296 36.37°N, 54.99°E 7 August 2014 7 ♀,6♂ progeny of 3 field vegetable crops; pistachio) C. shahrudensis Shahrud, Iran (alfalfa) 1286 36.60°N, 54.98°E 7 August 2014 1 ♀,3♂ progeny of 2 field C. shahrudensis S Kara Balta, Kyrgyzstan (high- 1700 42.60°N, 73.85°E 27 May 1995 1 ♀,1♂ progeny of 2 field grass meadows) C. bolti Chichkan Valley, Kyrgyzstan (high- 1750 42.12°N, 72.80°E 1 June 1995 7 ♀,5♂ progeny of 2 field grass meadows) C. bolti N Kyzyl-Unkyur, Kyrgyzstan (high- 1350 41.48°N, 73.05°E 11 June 1995 2 ♀,2♂ progeny of 4 field grass meadows) C. bolti Alamüdün District, Kyrgyz. (high- 1950–2056 42.58°N, 74.48°E 25 June 2016; 12 8 ♀,15♂ progeny of 5 field grass meadows) July 2016 C. bolti Elevation is in metres, geographic coordinates are in decimal format, and each specimen tested for its song (i.e. recorded to disk) is the offspring of a gravid female collected at the specified locality. 1606 C.S. HENRY ET AL.

Switzerland (WSL); (iv) the Laboratory of Entomology, Faculty of Agriculture, Tottori University, Tottori, Japan (LETU); and (v) the W.F. Barr Museum, University of Idaho, Moscow, ID, USA (WFBM). Chrysoperla bolti sp. nov.: A total of 24 gravid females of this species were collected by one of us (P. Duelli) in the northern part of the Kyrgyz Republic between 1350 and 2060 m elevation during May and June 1995 and, accompanied by D. Bolt, on 25 June and 12 July 2016. We were able to obtain living adult oﬀspring from eight females in 1995 and ﬁve females in 2016. Collecting data are shown in Table 1. Methods for rearing, shipping, preserving and vouchering adults and larvae were the same as described above for C. shahrudensis.

Song recording, analysis and comparison To understand which song features were measured, first in C. shahrudensis and then in C. bolti, it is necessary to provide background information on the known differences among the basic types of lacewing songs. By doing this, song methods can be specified more accurately and clearly. Unfortunately, some conflation of Results with Methods then becomes unavoidable. Substrate-borne lacewing signals were recorded to computer disk at 25 ± 1°C, using methods, software and equipment described in recent publications (e.g. see Henry et al. 2012). In brief, an infrared optical microphone (OM1 by Schwenk, Willimantic, CT, USA) detected vibrational signals from the reflective plastic-wrap surface of a small 110-ml arena housing the insect. The signals were digitized at 500 Hz, low-pass filtered above 200 Hz, and analysed on a computer running WINDOWS with specialized hardware and software. Chrysoperla shahrudensis sp. nov.: The songs of 19 individuals (7 males, 12 females; progeny of five of the six field-inseminated females) were archived and analysed. In C. shahrudensis, the song consisted of a single volley of abdominal vibration (‘volley’ defined in Henry 1979), usually followed by a low-frequency, atonal rumble (Figure 1(a), ‘rumble’ defined in Henry, Wells et al. 1999). This single-volley song type is shared within the carnea-group by Chrysoperla adamsi Henry et al. and, typically without the rumble, by Chrysoperla plorabunda (Fitch), C. agilis, C. heidarii, C. zastrowi, and (documented in the present study, below) C. bolti (Figure 1(b)). In this type of song, the volley unit may be repeated many times at a fixed period by a singing individual (Figure 1(a)), but when a duet is established, the partners exchange single volley units alternately in synchrony (see figs 2a and 3 in Henry et al. 2013). Thus in species like C. plorabunda, C. shahrudensis, C. adamsi and C. bolti the volley (plus optional rumble) is also the ‘shortest repeated unit’ or SRU (the SRU, also known as the ‘song,’ is defined in Henry 1985), whereas in other species, e.g. C. carnea (Stephens) or C. pallida, a cluster of multiple volleys functions as the SRU(Henryetal.2002). Also, the rate of abdominal vibration or oscillation – the dominant frequency – can and usually does change over the course of each volley. Consequently, there are up to six significant song features that must be measured in ‘single-volley’ songs: volley duration, rumble duration (if present), volley period (i.e. SRU period) during an iterative solo or duet, and the dominant frequency at the start, middle and end of the volley (Figure 1(a)). For C. shahrudensis, such measurements were taken from at least 10 songs (= SRUs) per individual. Therefore, all tabulated means are grand means. Two-tailed JOURNAL OF NATURAL HISTORY 1607

a. C. shahrudensis solo song (4 SRUs) dominant frequency: 140 middle end 120 start 100 80 rumble 60 40

Frequency (Hz) 20 0 volley/SRU period duration

rumble

0 seconds 246 8 10 12

b.C. bolti solo song (4 SRUs) dominant frequency: 140 start middle 120 end 100 80 60 40 20 Frequency (Hz) volley/SRU period 0 duration

(no rumble)

0 seconds 2 4 6 8 10 12 Time (s)

Figure 1. Oscillographs (lower of each pair of traces) and sonographs (upper traces) of (a) a solo vibrational song of Chrysoperla shahrudensis sp. nov. from northern Iran and (b) a solo vibrational song of Chrysoperla bolti sp. nov. from Kyrgyzstan, Asia, illustrating convergent song phenotypes in these two Asian species. Each song was recorded at 25 ± 1°C and is drawn to the same time scale of 12 s. Start, middle, end, and rumble sections of an individual volley are marked by the arrows, while duration and period of the primary volley are bracketed. 1608 C.S. HENRY ET AL. t-tests for independent samples (Snedecor and Cochran 1980) were used to assess the magnitude of differences between the sexes. Chrysoperla adamsi and C. bolti sp. nov.: The same six song features were also measured at 25 ± 1°C in at least 10 SRUs per individual extracted from the solo (spontaneous) songs of the two carnea-group species whose courtship songs most closely resembled those of C. shahrudensis,i.e.C. adamsi from western North America and C. bolti sp. nov. from northern Kyrgyzstan, Asia. For C. adamsi, solo songs were recorded and measured from 40 individuals (24 males, 16 females) collected in Idaho, Oregon and California between 1996 and 2015 (e.g. see Henry and Wells 2010). For C. bolti, songs were recorded and measured from 41 individuals (23 males, 18 females) reared from a total of 13 gravid females collected in 1995 (Henry, Wells et al. 1999) and 2016. Using grand means, song differences between the sexes in C. bolti were evaluated, and the song phenotype of C. bolti was compared to that of C. shahrudensis to determine whether these two Asian species were better interpreted as conspecifics or as an example of convergent evolution between species (sensu Losos 2011). A similar feature-by-feature song comparison was made between C. bolti and North American C. adamsi.Two-tailedt-tests for independent samples were used throughout; all statistical tests and analyses were performed using STATISTICA v.12 (Statistica (data analysis software system), version 12 [2015]).

Song playback tests To assess the acceptability of adamsi and bolti songs to duetting individuals of C. shahrudensis, we conducted several simple song playback trials using previously recorded songs of the three species. The ability of three males and three females of C. shahrudensis to duet with conspecific versus heterospecific signals was then noted and their responses were digitally recorded (e.g. Figure 2(c)). To do this, the SPIKE2waveform program (Spike2 for Windows software, version 5 [2003]) was configured for simultaneous input to and output from separate channels of a CED1401plus Intelligent Laboratory Interface (Cambridge Electronic Design Ltd, Cambridge, UK). We played back stimulus signals (CED analogue output) to each lacewing in a 110-ml arena by placing the latter on an electronic shaker (model 132–203; Labworks Inc., Costa Mesa, CA, USA) powered by a high-quality audio amplifier. Each stimulus signal consisted of nine repeating SRUs (Figure 2(c)). All vibrational responses were detected and recorded (CED digital input) from the reflective plastic-wrap cover of the arena using the Schwenk optical microphone. Ambient temperature was maintained at 25 ± 0.5°C. The low-frequency stimulus signal plus all responses to it were low-pass filtered at 200 Hz and digitized at 500 Hz. More detailed descriptions of similar methods can be found elsewhere (Henry et al. 2012, fig. 1 in Henry et al. 2013). Using the same playback system, we also conducted informal tests of the responsive- ness of males and females of C. shahrudensis and C. bolti to the songs of carnea-group species with which each might co-occur in nature. For C. shahrudensis,thoseincluded C. carnea, C. mediterranea, C. pallida, C. heidarii, C. lucasina, C. agilis and C. zastrowi sillemi. For C. bolti, sympatric species included C. ‘carnea-K’ and C. ‘downesi-K,’ which are apparently the only other members of the carnea-group living in Kyrgyzstan. JOURNAL OF NATURAL HISTORY 1609 a. C. shahrudensis heterosexual duet (4 SRUs exchanged)

female’s male’s rumble rumble

0 seconds 246810 12

b.C. bolti heterosexual duet (3 SRUs exchanged)

0 seconds 24681012

c. Duetting response of a C. shahrudensis female to other species

stimulus response stimulus response stimulus response

to C. shahrudensis to C. bolti to C. adamsi

0 s05101520 25 30 35 40 455 55 60 65 70 Time (s)

Figure 2. Oscillographs of duetting interactions. (a) 12 s of a typical duet between a male and a female of Chrysoperla shahrudensis sp. nov., showing the partners politely exchanging single volley- plus-rumble units or shortest repeated units (SRUs). (b) 12 s of a typical duet between a male and a female of Chrysoperla bolti sp. nov., showing the partners politely exchanging single SRUs. (c) 71 s oscillograph showing a female of C. shahrudensis duetting with a conspeciﬁc for the ﬁrst 24 s, with C. bolti for the next 25 s, and with Chrysoperla adamsi (North America) for the last 22 s. In each of those three duets, the female being tested is capable of synchronizing precisely with the pre- recorded playback signal. As discussed in the text, C. shahrudensis shows no such duetting response to playbacks of songs of any carnea-group species other than C. bolti and C. adamsi. All duets were recorded at 25 ± 1°C. 1610 C.S. HENRY ET AL.

Morphology of adults and larvae Adults were inspected for the states of 22 morphological characters historically used for species delineation in the carnea-group (see Henry et al. 2012 for details). Twenty adult specimens (8 males, 12 females) identified acoustically as C. shahrudensis were examined. Localities in Iran (Table 1) were Pass Qazvin to Alamut Valley (3 ♀♀) and Shahrud (8 ♂♂,9♀♀). In Kyrgyzstan, 14–16 adult specimens (8 males, 6–8 females) from the Alamüdün District identified acoustically as C. bolti were similarly studied. Larvae identified to species by the songs of their parents were used for anatomical study. Specimens were placed directly into 70% ethanol or boiled in distilled water with a drop of liquid detergent, allowed to cool, and transferred to 70% ethanol with 5% glycerine. They were then viewed at magnifications up to 50 × to note patterns and details of head and body markings. Larval markings in C. shahrudensis and C. bolti were described and compared with each other and with the patterns seen in other species of the carnea-group documented in the published literature. For C. shahrudensis, 24 larvae were examined. These had been reared from field-collected females taken at two sites in Iran (Pass Qazvin to Alamut Valley and Shahrud; see Table 1). The sample included 6 first-, 6 second-, and 12 third-instars. For C. bolti,30first-, 8 second-, and 18 third-instar larvae were reared from two adjacent sites in the Alamüdün District, Kyrgyzstan (see Table 1) and viewed. Representative third-instar larvae of C. shahrudensis and C. bolti were then illustrated, emphasizing head markings.

Life history and ecology As in past studies of carnea-group lacewing species, habitat data and plant associations were recorded for each specimen of C. shahrudensis and C. bolti at the time of collection (Table 1). Using song analysis of progeny grown from all field-collected gravid females, we also determined which other species of the carnea-group were sympatric with C. shahrudensis in northern Iran and with C. bolti in northern Kyrgyzstan. To these records were added data from Chrysoperla specimens collected by H. Heidari in the vicinity of Shahrud (Iran) in July 2002. A small number of the offspring of field-collected C. shahrudensis and C. bolti were reared from the third larval stadium until adult eclosion under short-day, cool conditions (≈ 8 : 16 h light : dark; 22°C : 16°C warm : cool) to induce winter diapause and concomitant colour changes (Duelli 1992; Duelli, Johnson, et al. 2014).

Evolutionary relationships Using techniques described previously (Takano et al. 2011; Henry et al. 2012), DNA was extracted and 4630 base-pairs (bp) of the mitochondrial genome were amplified, sequenced and aligned from multiple individuals of 22 species and subspecies of the carnea group, plus three outgroup species from the Chrysoperla pudica-group. Specimens included two individuals (from two different mothers) of C. shahrudensis from Shahrud, three of C. bolti from different sites in northern Kyrgyzstan, and four of C. adamsi from one site in Oregon and three sites in central and southern California. Outgroup taxa comprised one individual of C. pudica (Navás) from Wolfdrift, Republic of South Africa; four specimens of Chrysoperla comanche (Banks) from southern California, USA; and three specimens of JOURNAL OF NATURAL HISTORY 1611

Chrysoperla ruﬁlabris (Burmeister) from southeastern Arizona, USA. In total, 89 specimens (operational taxonomic units) were sequenced and analysed. The data set comprised four protein-coding mitochondrial genes: 1000 bp of NADH dehydrogenase subunit-2 (ND2), 1226 bp of cytochrome c oxidase subunit I (COI), 685 bp of cytochrome c oxidase subunit II (COII), and 1719 bp of NADH dehydrogenase subunit- 5(ND5). Primers and polymerase chain reaction conditions were the same as speciﬁed in Table 4 in Henry et al. (2012). Mitochondrial gene sequences for two specimens of C. shahrudensis have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers LC101829–LC101836. Accession numbers for the other 87 operational taxonomic units, including three specimens of C. bolti (as C. ‘adamsi-K’), were published previously (Henry et al. 2012; Price et al. 2015). Evolutionary divergences among selected taxa and groups of taxa were estimated as the number of base substitutions per site averaged over all sequence pairs between selected specimens, species, or groups of species. Methods of analysis included uncorrected p-distances and distances calculated using the Kimura two-parameter model, gamma-distributed, as implemented in MEGA v.7.0 (Kumar et al. 2016). A range of phylogenetic analyses were also performed on the full mitochondrial data set (4630 bp). We selected an evolutionary tree that was inferred from the Maximum Likelihood method under the General Time Reversible model, using a discrete gamma distribution to model evolutionary rate variation while allowing some sites to remain invariant (GTR+G+I, as implemented in MEGA v.7.0; Kumar et al. 2016). Statistical support for branches on the tree was determined by running 150 bootstrap replicates.

Results Song phenotype Chrysoperla shahrudensis sp. nov.: Adult males and females produced a single type of song (= SRU), which was used both in solitary calling and duetting (Figures 1(a) and 2(a); Tables 2 and 3). The SRU was iterative and more often disyllabic than monosyllabic, consisting of a single ≈ 735-ms primary volley that was usually (355/421 SRUs = 84% of the time) followed by an atonal rumble ≈ 570 ms long. This SRU repeated on average every 2.38 s when the song was being delivered in a solo context (Figure 3, Table 3). Songs of males and females differed slightly but significantly: males exhibited longer volleys and rumbles, repeated more slowly, than females (Table 2). During the first third (≈ 245 ms) of the volley proper, the dominant frequency decreased very sharply from a mean maximum of 76 Hz to a minimum of ≈ 35 Hz, before rising again more gradually to ≈ 60 Hz during the remaining portion of the volley (25°C). The rumble, when present, was of low but variable frequency. Heterosexual duetting in C. shahrudensis consisted of each individual repeatedly answering single SRUs (volley-plus-rumble units) of its partner with the same kind of SRU. The result of that interaction was a long series of repeated SRUs, wherein the responses of the partners alternated precisely with one another (Figure 2(a)). As in other lacewings of the carnea- group (Henry 1979), the duet ended with the male repeatedly tapping the genital area of the female with the tip of his abdomen just before copulation. Chrysoperla bolti sp. nov.: This species also produced a single type of song for solos and duets (Figures 1(b) and 2(b); Tables 3 and 4). The SRU was iterative, typically monosyllabic and consisted of a single ≈ 930-ms volley rarely followed (118/592 1612 ..HNYE AL. ET HENRY C.S.

Table 2. Values at 25 ± 1°C of the principal features of solo songs for males versus females of Chrysoperla shahrudensis sp. nov. from Iran (western Asia). Frequency measures of main volley, Hz Start Middle End Main volley duration, ms Rumble duration, ms Volley period, sec C. shahrudensis A.78.15 ± 4.19 B.34.95 ± 2.37 C.60.02 ± 3.26 E.798.20 ± 54.27 G.770.72 ± 123.31 I.2.643 ± 0.156 Males (n =7) C. shahrudensis Females A.74.56 ± 5.72 B.36.35 ± 2.16 D.64.07 ± 2.84 F.696.61 ± 49.73 H.467.28 ± 80.74 J.2.231 ± 0.129 (n = 12) t-statistic 1.45 −1.32 −2.84 4.16 6.32 6.22 df 17 17 17 17 16 17 p 0.167 0.205 0.011 0.00066 0.00001 0.000009 Each value is the mean of the means of n individuals (≥ 10 ‘shortest repeated units’/individual) in the population subsample, ±SD (standard deviation). Pairwise comparisons were tested for statistical significance using two-tailed t-tests for independent samples. For all variables marked with the same letter, the difference between the means is not statistically significant (p > 0.05). See Song recording, analysis and comparison in the Methods section for details. Table 3. Values at 25 ± 1°C of the principal features of solo songs for Chrysoperla shahrudensis sp. nov. from Iran (western Asia), Chrysoperla bolti sp. nov. from Kyrgyzstan (central Asia), and Chrysoperla adamsi from western North America. Frequency measures of main volley, Hz Start Middle End Main volley duration, ms Rumble duration, ms Volley period, sec C. shahrudensis A.75.88 ± 5.39 D.35.83 ± 2.29 E.62.58 ± 3.53 H.734.04 ± 70.91 K.568.43 ± 174.23 L.2.383 ± 0.245 (Iran) (n = 19) C. bolti B.81.57 ± 3.71 E.61.65 ± 2.71 G.41.39 ± 3.65 I.930.41 ± 105.11 (infrequent) M.3.824 ± 0.307 (Kyrgyzstan) (n = 41) C. F. D. J. K. N. C. adamsi (N. America) (n = 40) 73.35 ± 3.09 55.62 ± 1.69 37.55 ± 3.53 801.10 ± 77.42 628.71 ± 219.93 3.221 ± 0.380 HISTORY NATURAL OF JOURNAL Data from both males and females are pooled in this table. Each value is the mean of the means of n individuals (≥ 10 ‘shortest repeated units’/individual) in the population subsample, ±SD (standard deviation). Pairwise comparisons were tested for statistical significance using two-tailed t-tests for independent samples. For all variables marked with the same letter, the difference between the means is not statistically significant (p > 0.05). See Song recording, analysis, and comparison in the Methods section for details. 1613 1614 C.S. HENRY ET AL.

C. shahrudensis (N=19) 10 C. adamsi (N=40) C. bolti (N=41)

Number of individuals per bin 2

0 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 Volley period, s

Figure 3. Frequency distributions of volley period measured in solo vibrational songs recorded at 25 ± 1° C from individuals of Chrysoperla shahrudensis sp. nov. (red/grey bars), Chrysoperla bolti sp. nov. (blue/ dark grey bars), and Chrysoperla adamsi (green/light grey bars). Measurements were calculated as individual averages; N = number of individuals from which individual averages were calculated.

SRUs = 20% of the time) by an atonal rumble of highly variable length, frequency (37– 70 Hz, n = 348), and intensity. The SRU repeated on average every 3.82 s when the song was being delivered in a solo context (Figure 3, Table 3). Songs of males and females were very similar, differing only in the consistently higher (by 2 Hz) volley frequencies of females (Table 4). During the course of each volley, the dominant frequency decreased linearly from a mean maximum of 82 Hz to a minimum of ≈ 41 Hz (25°C). Heterosexual duetting in C. bolti consisted of each individual repeatedly answering single SRUs of its partner with the same kind of SRU. The result of that interaction was a long series of repeated SRUs, wherein the responses of the partners alternated precisely with one another (Figure 2(b)). Duets ended in copulation when the male repeatedly tapped the genital area of the female with the tip of his abdomen. The songs of Iranian C. shahrudensis, Central Asian C. bolti, and North American C. adamsi were similar to one another in their basic tempo (= volley period; see Figure 1(a,b); Table 3) and pattern of single-volley alternation during duets (Figure 2 (a–c)). For example (see Figure 3, Table 3), the mean SRU period for solo songs of C. shahrudensis (2.38 s) was only about 26% shorter than that of C. adamsi (3.22 s) and 38% shorter than that of C. bolti (3.82 s). Furthermore, duetting conspecific partners in all three species alternately exchanged single SRUs, over the course of a long series of repeated SRUs (Figure 2(a,b)). However, it can be seen from Figure 3 that the frequency Table 4. Values at 25 ± 1°C of the principal features of solo songs for males versus females of Chrysoperla bolti sp. nov. from Kyrgyzstan (central Asia). Frequency measures of main volley, Hz Start Middle End Main volley duration, ms Rumble duration, ms Volley period, sec C. bolti Males A.80.48 ± 2.95 C.60.79 ± 2.32 E.40.66 ± 3.14 F.944.39 ± 112.14 (infrequent) G.3.892 ± 0.320 (n = 23) C. bolti Females B.82.96 ± 4.18 D.62.75 ± 2.84 E.42.33 ± 4.10 F.912.54 ± 95.50 (infrequent) G.3.736 ± 0.273 (n = 18) t-statistic −2.22 −2.44 −1.49 0.96 – 1.65 df 39 39 39 39 – 39 P 0.032 0.019 0.145 0.342 – 0.106 ORA FNTRLHISTORY NATURAL OF JOURNAL Each value is the mean of the means of n individuals (≥ 10 ‘shortest repeated units’/individual) in the population subsample, ±SD (standard deviation). Pairwise comparisons were tested for statistical significance using two-tailed t-tests for independent samples. For all variables marked with the same letter, the difference between the means is not statistically significant (p > 0.05). See Song recording, analysis, and comparison in the Methods section for details. 1615 1616 C.S. HENRY ET AL. distribution of SRU periods for solo songs of C. shahrudensis overlapped that of C. adamsi but not that of C. bolti. Hence, the songs of the two Asian species were less similar to each other than C. shahrudensis was to allopatric C. adamsi. Also present in Asia were two undescribed species from Kyrgyzstan, informally named C. ‘carnea-K’ and C. ‘downesi-K’ in reference to the carnea-group species most closely similar to each in ecology, morphology and song (Wells and Henry 1998). These two species produced their own unique multi-volley SRUs when duetting (data not shown), in sharp contrast to the single-volley SRUs characteristic of C. shahrudensis, C. bolti or C. adamsi.

Song playback tests Living individuals of C. shahrudensis were capable of establishing normal duets with recorded signals of either C. adamsi or C. bolti (Figure 2(c)). Of the three males tested, two duetted with their own and both heterospecific signals, whereas the third responded only to its own song. However, none of the males answered all nine SRUs of either heterospecific stimulus signal. In contrast, all three females of C. shahrudensis duetted accurately with their own and both heterospecific signals, responding correctly to all nine SRUs of each type of playback stimulus (e.g., Figure 2(c)). It was also observed (data not shown) that individuals of C. shahrudensis did not respond at all to the recorded songs of any other Eurasian member of the carnea-group with which it could conceivably co-occur in nature, i.e. not to C. carnea, C. mediterranea, C. pallida, C. heidarii, C. lucasina, C. agilis, nor C. zastrowi sillemi. Similarly, C. bolti showed no response to playbacks of recorded songs of C. ‘carnea-K’ or C. ‘downesi-K,’ with which it lives in sympatry in northern Kyrgyzstan.

Adult and larval morphology The adult and larval morphologies of C. shahrudensis sp. nov. and C. bolti sp. nov. are formally described later in the Taxonomy section. Figure 4 includes photographs of adult females of both new species. Photographs of typical third-instar larvae and drawings of their head capsules are shown in Figures 5 and 6. For additional methodological details, refer to fig. 4 in Henry et al. (2002) explaining claw basal dilation, fig. 4(a) in Henry et al. (2006) depicting the orientation of the Rs-M crossvein, fig. 5 in Henry et al. (2002) explaining the genital lip/chin ratio, figs 3 and 4 in Henry et al. (1993) illustrating the tignum, and fig. 4 in Henry et al. (2014) showing a larval head capsule that bears a fronto-medial spot.

Life history and ecology Chrysoperla shahrudensis sp. nov.: Specimens of this species were found occurring naturally in alfalfa fields or in low bushes and trees at the edges of crop fields in northern Iran, at elevations around 1200 m. A single female was collected from Populus sp. on a windy mountain pass at 2300 m (Table 1). Other carnea-group species abundantly collected from this same region in 2002 and 2014 included C. carnea (2014), C. heidarii (2014) and C. agilis (2002, 2014). The presence of C. carneas. str. in Iran represented a significant range extension for this species, well beyond its previously documented eastern limit in the Caucasus of Armenia and Georgia (Duelli et al. 2015). JOURNAL OF NATURAL HISTORY 1617

Figure 4. Adult of (a) Chrysoperla. shahrudensis sp. nov., non-diapausing green form; (b) C. shahrudensis, diapausing sandy-brown form, and (c) Chrysoperla bolti sp. nov., all-season green form.

More rarely found in Iran was C. pallida (2014: Beth Oili, 422 m), which also constituted a significant range extension beyond the Caucasus. Another species occasionally found in Iran was C. zastrowi sillemi (2002: Gorgan, 88 m; 2014: Alamut Lake, 1825 m), currently known to be widespread from western Africa to India (Henry et al. 2010) and north to the Caucasus of Armenia (Henry et al. 2014). The only non-carnea-group Chrysoperla species collected in northern Iran was Chrysoperla mutata (McLachlan), previously known to occur there (Brooks 1994; p. 163; Thierry et al. 2004) and present as expected in both 2002 and 2014 at elevations from 1200 to 2000 m. Chrysoperla bolti sp. nov.: This species was present in Kyrgyzstan at elevations between 1350 and 2056 m, in the tall grasses and herbaceous plants that characterize alpine meadows. It was rarely found in taller shrubbery or trees at the edges of fields, and it showed no significant association with evergreen vegetation. In contrast, the undescribed Kyrgyz species C. ‘carnea-K’ and C. ‘downesi-K’ occupied other niches. The former was very common at lower elevations, associated with croplands or the shrubs and young trees at the edges of fields, whereas the latter was typically collected from conifers growing at various elevations. Although generally not found in syntopy, there were a few sites where all three Kyrgyz species were collected, i.e. South Kara Balta in 1995 and the Alamüdün District in 2016. Adults of C. shahrudensis reared since pupation under cool, short-day conditions entered reproductive diapause, characterized by an absence of reproductive activity and by a waxy sheen to the cuticle (described in MacLeod 1967, p. 1346). These diapausing adults also changed colour, but to varying degrees. Whereas some individuals simply became lighter green, others changed completely to a light sandy brown (Figure 4(b)). When switched back to long-day conditions, adults would gradually become bright green again. In contrast, neither C. bolti nor C. adamsi exhibited a colour 1618 C.S. HENRY ET AL.

Figure 5. Dorsal view of third-instar larval head capsule (left half) of Chrysoperla shahrudensis sp. nov. The drawing represents the typical condition seen in 24 individuals from two populations in Iran. The colour insert is a typical mature third-instar larva of the species. change to brown during diapause, although in C. bolti enhanced red pigmentation typically appeared on the genae and laterally on the frons (P.D., unpublished data).

Evolutionary relationships Summarized in Table 5 are evolutionary divergences among song-convergent C. shahrudensis, C. bolti and C. adamsi. Shown also are divergences between and among those three species and six other species with very diﬀerent songs that are widespread in Europe and western Asia: C. carnea, C. pallida, C. agilis, C. lucasina, C. mediterranea and C. heidarii. Uncorrected p-distances are shown; other divergence models produced relative distance values very similar to the uncorrected ones. The p- distances were nearly twice as large between species on diﬀerent continents (e.g. JOURNAL OF NATURAL HISTORY 1619

Figure 6. Dorsal view of third-instar larval head capsule (left half) of Chrysoperla bolti sp. nov. The drawing represents the typical condition seen in 18 individuals from two populations in northern Kyrgyzstan. The colour insert is a typical mature third-instar larva of the species.

C. shahrudensis versus C. adamsi) as they were between sympatric species. That distinction applied just as strongly to pairs of allopatric species with similar (convergent) songs as it did to pairs of allopatric species with dissimilar songs. In summary, divergence indices did not support a close relationship among the song-convergent species, nor were those three species particularly similar to any other species in the carnea-group. These results also indicated that C. bolti should not be considered a sub-population of the species C. shahrudensis. Similarly, the topology of the mitochondrial tree (Figure 7) clearly showed that C. shahrudensis, C. bolti and C. adamsi occupied separate regions of phylogenetic space, conﬁrming that they were not each other’s closest relatives. This supplementary ﬁgure also includes geographic site information for all specimens used in 1620 C.S. HENRY ET AL.

Table 5. Evolutionary divergences among selected taxa and groups of taxa of the Chrysoperla carnea-group green lacewings, estimated as the number of base substitutions per site averaged over all sequence pairs between the taxa or groups. Comparison p-distance carnea-group/pudica-group (outgroup) 0.061 Eurasian species/North American species 0.022 C. shahrudensis/C. adamsi (N. America) 0.024 C. shahrudensis/C. carnea 0.016 C. shahrudensis/C. bolti 0.014 C. shahrudensis/C. mediterranea 0.009 C. bolti/C. adamsi (N. America) 0.020 C. bolti/C. pallida 0.015 C. bolti/C. carnea 0.007 Uncorrected p-distances are shown. Comparisons among the three song-convergent species are highlighted in bold font. The ingroup/outgroup and Eurasian/North American comparisons are taken from an earlier study that used similar methods (Table 4 in Henry et al. 2014). the phylogenetic analysis and illustrates typical song phenotypes for 13 species mentioned frequently in the manuscript.

Discussion Both C. shahrudensis and C. bolti can be somewhat bluish-green as living adults (Figure 4(a, c)), are found in west/central Asia, and share the same basic style of single-volley alternation during bouts of duetting (Figure 1(a,b)). From this, one could infer that they are mere populations of a single species. However, specimens of C. shahrudensis are consistently paler green than those of bolti, although our sample sizes are too small to say whether a subtle diﬀerence in body colour is a reliable morphological character state. Furthermore, we lack evidence that the geographical ranges of these two taxa overlap, and their songs do not appear to be similar enough to constitute clinal variation of a single song type. Although nothing can be truly resolved without more ﬁeld work in the vast region between Kyrgyzstan and Iran, we can nevertheless help shed light on the matter by closer examina- tion of song phenotype, adult and larval morphology and ecophysiology.

Song phenotype and duetting behaviour Although volley duration is the song feature that is most similar in C. shahrudensis (Iran) and C. bolti (Kyrgyzstan), it nevertheless diﬀers by 21% (Table 3). More importantly, volley/SRU period, which is known to have the greatest impact on species recognition in carnea-group species (Wells and Henry 1992a, 1992b), diﬀers by 38% (Table 3) and shows no overlapping variation (Figure 3). Furthermore, the three principal frequency (tonal) song traits of C. shahrudensis and C. bolti are not similar (Figure 1(a,b)), and the characteristic post-volley rumble usually found in C. shahrudensis is absent in 80% of SRUs recorded from the C. bolti population. By these criteria alone, C. bolti should be considered a distinct species. Clinal variation in measurable song parameters has been documented in several lacewing species. In the best-known examples, i.e. Old World C. zastrowi sillemi and JOURNAL OF NATURAL HISTORY 1621

95 zastrowi E153 RSA 67 zastrowi E154 RSA zastrowi E23 RSA 90 zastrowi E24 RSA C. z. sillemi sillemi E93 C India sillemi E193 S CA 92 sillemi E91 C India heidarii E234 Armenia heidarii E233 Armenia C. heidarii 100 lucasina E63 S Switzerland 70 lucasina E64 S Switzerland nipponensis A2 Aki1 Japan 98 nipponensis A2 Kyu9 Japan 100 agilis E130 Alaska agilis E132 N Italy lucasina E70 N Italy C. lucasina lucasina E42 Mediterranean 66 lucasina E41 Mediterranean 80 lucasina E69 N Italy 100 mediterranea E4 Greece mediterranea E6 Greece C. agilis 88 renoni E25 Hungary 100 renoni E28 Hungary 56 60 renoni E26 Hungary renoni E27 Hungary pallida E44 N Switzerland C. pallida 100 pallida E15 Greece pallida E16 Greece 58 100 pallida E152 W France pallida E146WSwitzerland 51 pallida E45 N Switzerland heidarii E232 Armenia 100 mediterranea E157 Pfyn Switz C. shahrudensis mediterranea E158 Pfyn Switz 98 100 agilis E133S Switzerland agilis E140 N Italy 96 61 shahrud E239 Shahrud N1 EURASIA 100 shahrud E241 Shahrud O1 100 agilis E122 Alaska C. bolti agilis E131 S Switzerland 70 bol ti E17 Kyrgyzstan 99 bol ti E18 Kyrgyzstan 99 67 bolt i E19Kyrgyzstan carnea E118 W France 69 carnea E120 Finland carnea E117 W France C. carnea 100 carnea E34 S Switzerland carnea E119 Finland carnea E49 N Switzerland OLD WORLD 100 nippon A1 Kyu6 Japan CHRYSOPERLA CARNEA-GROUP 100 nippon A1 Tsu4 Japan 100 nigrocapitata B32 Japan 100 nig rocapitata Ehi1 Japan 74 calocedrii E215 S CA 100 calocedrii E218 S CA C. calocedrii calocedrii E216 S CA 88 calocedrii E217 S CA ‘downesi 2’ E89 WA downesi E102 Alaska 100 NORTH 70 downesi E103 Alaska 100 downesi E101 Alaska C. downesi AMERICA ‘downesi 2’ E90 ID downesi E223 S CA 100 plorabunda E181 ID plorabunda E182 ID 100 52 johnsoni E84 WA johnsoni E85 ID C. johnsoni johnsoni E177 S CA 94 ‘downesi-mohave’ E207 S CA 100 adamsi E33 OR adamsi E168 C CA ‘downesi-mohave’ E206 S CA C. plorabunda 78 ‘downesi-mohave’ E205 S CA 99 ‘‘downesi-mohave’ E208 C CA plorabunda E179 C CA 100 adamsi E171 S CA ‘johnsoni-mohave’ E236 Inv CA ‘johnsoni-mohave’ E237 Inv CA C. adamsi johnsoni E37 SE AZ adamsi E164 C CA plorabunda E100 SW CA pudica D13 RSA CHRYSOPERLA PUDICA-GROUP rufilabris E211 SW AZ 100 rufilabris E209 SE AZ 100 rufilabris E210 SE AZ 69 comanche E187 S CA comanche E188 S CA 59 comanche E185 S CA 0.02 substitutions/site 93 90 comanche E186 S CA

Figure 7. Maximum likelihood phylogram of the cryptic species of the Chrysoperla carnea- group, based on 4630 bp of mitochondrial DNA sequence from the protein-coding genes ND2, COI, COII and ND5. The tree was inferred under the General Time Reversible model, using a discrete gamma distribution to model evolutionary rate variation while allowing some sites to remain invariant (GTR+G + I as implemented in MEGA v.7.0, Kumar et al. 2016). Numbers at the branch points are support values from 150 bootstrap replicates; branch lengths are proportional to the number of substitutions per site. Specimen numbers and geographical locations are shown for each operational taxonomic unit (twig) on the tree. The three song- convergent species discussed in the text are highlighted in colour: Chrysoperla shahrudensis in red, Chrysoperla bolti in blue and Chrysoperla adamsi in green. Thumbnail oscillographs of 12 s of song phenotype for 13 common species are placed on the ﬁgure at their approximate vertical positions on the phylogram. 1622 C.S. HENRY ET AL.

American Chrysoperla johnsoni Henry et al., the species ranges are vast and song features change gradually and continuously across each range (Henry 1993; Henry et al. 2006). In those examples, the song at one end of the cline is clearly recognizable as a variant of the song at the other end. In contrast, some of the differences between the songs of C. shahrudensis and C. bolti – e.g. frequency patterning within volleys, as seen in Figure 1 (a,b) – are discrete and invariant, as is typical of species-level differences seen in other carnea-group lacewings. This pattern of variation is not consistent with a hypothesis of clinal variation within a single widespread species. Therefore, these considerations also support separate species status for C. bolti. Nevertheless, the two types of songs are sufficiently compatible to allow interspecific duetting and mating, based on results of our staged song playback tests to living C. shahrudensis (Figure 2(c), middle section of oscillograph). Reproductive compatibility mediated by songs alone has been firmly documented in another pair of acoustically convergent but allopatric carnea-group species, Eurasian C. pallida and North American Chrysoperla calocedrii Henry et al. In that study, heterospecific individuals placed together immediately established duets and copulated with one another, producing viable hybrid offspring (Henry et al. 2012). It follows that populations of cryptic species such as C. shahrudensis and C. bolti (or C. pallida and C. calocedrii) can possess similar song phenotypes but still be considered valid species, if they are geographically isolated from each other and therefore cannot interact, duet and hybridize in nature (Henry and Wells 2010). Multiple independent instances of parallel speciation associated with convergent song evolution are therefore quite likely to occur in allopatry, and this has been substan- tiated by recent studies of functionally identical songs in carnea-group species-pairs occu- pying different continents. Documented examples include (i) C. bolti (Asia) and C. adamsi (North America), (ii) C. pallida (Eurasia) and C. calocedrii (North America), and (iii) C. heidarii (Eurasia) and C. plorabunda (North America) (Henry, Wells et al. 1999, 2012, 2014). The present study introduces a third participant, C. shahrudensis, to the example of parallel speciation found in the allopatric species pair C. bolti and C. adamsi. Interestingly, volley period range measured in North American C. adamsi overlaps that in both Asian C. shahrudensis and Asian C. bolti (Figure 3). Such an intermediate and overlapping phenotype is not surprising in the geographically most isolated member of a convergent species trio: volley period overlap will not compromise reproductive isolation between species that can never encounter each other in nature. Furthermore (and hypothetically), past or occa- sional contact between C. shahrudensis and C. bolti in Asia could have enhanced song differences between them through reproductive character displacement (Pfennig and Pfennig 2009;Gerhardt2013). Clearly, this is not parallel speciation of the kind meant by Schluter (1995), which assumes the primacy of adaptive, ecological processes of phenotypic convergence. Instead, we suggest that non-adaptive, random events are responsible for the origin of highly similar duetting songs in allopatric populations of carnea-group lacewings (Henry et al. 2013). Convergent songs originate in separate geographic regions because the acoustic information space available for clear propagation and discrimination of such very low frequency songs can support only a limited number of truly distinctive types (Henry and Wells 2010). That will lead to the establishment of a larger number of valid species of carnea-group lacewings than there are highly distinctive songs available to delineate them. It follows that any future breakdown of the geographic barrier JOURNAL OF NATURAL HISTORY 1623 between two valid species sporting songs that are too much alike will trigger reverse speciation through hybridization, as has been documented in several classic examples of parallel ecological speciation (Turner 2002; Seehausen 2006; Kleindorfer et al. 2014; Rudman and Schluter 2016). In summary, parallel speciation in lacewings should be thought of as the independent origin of the ‘same’ song in, e.g. populations on different continents, creating the illusion of a single wide-ranging or intercontinental species when in fact two (or more, as in this study) are present.

Adult morphology Chrysoperla shahrudensis is a typical cryptic species of the Chrysoperla carnea-group, and as such is extremely diﬃcult to distinguish morphologically from other members of the complex (see Taxonomy). The symmetrical, broadly rounded apex of the fore wing is conspicuous, although this character state is shared with C. carnea s. str. and C. ‘carnea- K.’ The aspect ratio of the fore wing is typical of several species in the C. carnea-group and is not diagnostic. Lastly, the relative size of the basal dilation of the metatarsal claw is intermediate among other Eurasian (and global) species and therefore of little value for discriminating C. shahrudensis from such commonly co-occurring species in Iran as C. carnea, C. agilis and C. heidarii (Table 6). Chrysoperla bolti is also a truly cryptic member of the carnea-group (see Taxonomy). Its wings are narrower than those of C. shahrudensis (aspect ratio of C. bolti = 3.12 ± 0.17, C. shahrudensis = 2.89 ± 0.12), but its aspect ratio still overlaps the range found in other carnea-group species. Darker-green body colouration, extensive yellow pigment on the lower frons, and less intense black markings on the crossveins also distinguish C. bolti from C. shahrudensis and several other species, but again those qualities are not truly diagnostic character states of C. bolti. Finally, the relative size of the basal dilation of the

Table 6. Relative size, expressed as a ratio, of the basal dilation of the metatarsal claw measured in 18 species and subspecies of the global Chrysoperla carnea-group. Species Range Mean SD n C. pallida 1.67–2.14 1.94 0.15 39 C. nigrocapitata 2.02–2.52 2.18 0.16 32 C. carnea 1.98–2.76 2.23 0.19 39 C. agilis 1.94–2.53 2.23 0.15 26 C. nipponensis 1.79–2.56 2.28 0.23 34 C. johnsoni 2.10–2.82 2.32 0.29 5 C. shahrudensis sp. nov. 2.28–3.17 2.50 0.22 20 C. adamsi – 2.54 – 1 C. mediterranea (east) 2.04–4.0 2.65 0.40 36 C. lucasina 2.30–3.10 2.71 0.24 55 C. heidarii 2.40–3.30 2.71 0.21 37 C. bolti sp. nov. 2.47–3.13 2.81 0.22 16 C. plorabunda 2.40–3.09 2.83 0.28 5 C. calocedrii 2.92–4.67 3.81 0.41 23 C. z. sillemi 3.50–5.10 4.20 0.50 121 C. downesi 3.70–5.17 4.28 0.59 5 C. z. zastrowi 3.60–4.90 4.40 0.58 20 C. mediterranea (west) 4.27–9.80 6.28 1.49 19 C. renoni (Lacroix) – 10.25 – 1 Results for C. shahrudensis sp. nov., C. bolti sp. nov. and C. adamsi are highlighted in bold. Smaller ratios indicate larger basal dilations. SD = standard deviation, n = number of adult specimens measured. Data from (Brooks 1994; Henry et al. 1996; Henry, Brooks et al. 1999, 2002, 2003, 2006, 2010, 2012, 2014; and, 2015). 1624 C.S. HENRY ET AL. metatarsal claw is broader than found in C. shahrudensis but nonetheless falls within the midrange of carnea-group values (Table 6). In both C. shahrudensis and C. bolti a small red spot is present laterally on the frons below the outer margin of the antennal scape. This spot appears to be characteristic for these two species and is not known in any other Old World C. carnea-group species. Initially, we thought our specimens of C. shahrudensis might be Chrysoperla rotundata (Navás 1929), described and discussed in detail by Brooks (1994)asChrysoperla iranica (Hölzel) and reported as occurring in Iran, Turkey and Mongolia (Hölzel 1967a, 1967b) (C. iranica was rendered a junior synonym of C. rotundata by Aspöck et al. 2001). However, the key features of C. rotundata – a particularly broad fore wing (aspect ratio = 2.7–3.1, Brooks 1994), and in the male an extraordinary protrusion of the lip on sternite 8 + 9 (ﬁg. 42 in Brooks 1994) – are either unremarkable (for aspect ratio) or not present (for genital protrusion) in our specimens of C. shahrudensis. Furthermore, neither of the rotundata-associated character states are found in adults of C. bolti, C. ‘carnea-K’ or C. ‘downesi-K’ from Kyrgyzstan, even though the putative geographic range of C. rotundata includes that part of Asia where those three species are found (Hölzel 1967a, 1967b). This all points to the presence of an additional species in western and central Asia, one that is presumably characterized by the diagnostic morphological features of rotundata plus a distinctive duetting song. Such a unique song was indeed recorded from three males and three females raised from one gravid adult collected by H. Heidari in croplands near Shahrud, Iran in July 2002 and informally named CcY (C.S.H., unpublished data). Unfortunately, specimens of CcY that should have been preserved have been lost. However, the validity of CcY as a new species would confer upon Iran the distinction of having the highest diversity yet documented of co-occurring carnea- group cryptic species – at least seven, and possibly (if, as expected, C. mediterranea and C. lucasina are eventually conﬁrmed to be present there) as many as nine.

Larval morphology Larvae of C. shahrudensis (see Taxonomy section) are typical of the C. carnea-group (Tauber 1974; Henry et al. 1996;Henry,Brooksetal.1999, 2002, 2003), exhibiting head markings dominated by a pair of dorso-lateral stripes (Figure 5). The cream-coloured body is also typical of larvae of the group examined to date (Figure 5,insert).However,unlikewhatis seen in some species (e.g. Henry et al. 2015), head markings in C. shahrudensis are limited and simple, most closely resembling populations of C. mediterranea (Hölzel) from the Mediterranean coast of France, Italy and Greece (Henry, Brooks et al. 1999, fig. 10). An apparently distinctive feature of the third-instar larva of C. shahrudensis is the dorso-medial brown spot on the antennal scape (Figure 5). This spot can separate C. shahrudensis larvae from those of potentially sympatric C. heidarii (Henry et al. 2014)andC. pallida (Henry et al. 2002). Furthermore, the remaining head markings are much more extensive in C. pallida than in C. shahrudensis,andthird-instarC. heidarii larvae display extensive brown infusca- tion between the eye and dorso-lateral stripe and 52% bear a fronto-medial spot. On the other hand, C. pallida can sometimes share with C. shahrudensis the latter’s small, pale markings on the pronotum and inconspicuous or absent markings on the posterior body segments – a suite of features also found in North American C. calocedrii (Henry et al. 2012). Nevertheless, simplified post-cephalic markings, in conjunction with the limited, relatively JOURNAL OF NATURAL HISTORY 1625 pale head markings and presence of a dorso-medial spot on the scape form a distinctive gestalt that should generally delineate C. shahrudensis larvae from other known Chrysoperla larvae. However, as noted above, the small number of C. shahrudensis specimens examined and the limited geographic range represented by these specimens together reduce confidence in species identifications based on larval features. Contributing additional uncertainty to the diagnosis of C. shahrudensis and C. pallida is lack of larval specimens of the latter from the area of actual sympatry between the two species: the nearest larvae of C. pallida that have been examined are from a population in Georgia (Duelli et al. 2015), nearly 1000 km from Shahrud, Iran. Finally, the Chrysoperla fauna east of Iran is poorly known at best. Thus, further study could require modification of the current larval description and diagnosis of C. shahrudensis. As is the case in C. shahrudensis,thelarvaeofC. bolti are unexceptional representatives of the carnea-group ground plan (see Taxonomy, and Figure 6). Also, like the larvae of C. shahrudensis but unlike what is seen in some other species (e.g. Henry et al. 2015), head markings in C. bolti are limited and simple, most closely resembling populations of C. mediterranea from Austria and Slovakia (Henry, Brooks et al. 1999, fig. 10) and C. pallida from Caslano, Switzerland (Henry et al. 2002). An apparently distinctive feature of third-instar C. bolti is the complete or nearly complete pigmentation of the scape (Figure 6). The dark scape can separate C. bolti larvae from those of C. pallida (Henry et al. 2002)andC. heidarii (Henry et al. 2014), both of which have ranges that extend into Asia but have not yet been foundclosertonorthernKyrgyzstanthanIran (2100 km away) and the Caucasus (2400 km away). The serially graded extent of femoral pigmentation also seems to be unique to third- instar C. bolti larvae.However,asistrueofC. shahrudensis, the small number of C. bolti specimens examined and the limited geographic range represented by them together reduce confidence in species identifications based on larval features. Adding further uncertainty is our poor understanding of the larvae of the two undescribed carnea-group species that co- occur with C. bolti in Kyrgyzstan: C. ‘carnea-K’ and C. ‘downesi-K.’ Consequently, new information will probably necessitate revision of the current larval description and diagnosis of C. bolti.

Life history and ecology The typical habitat for C. shahrudensis in Iran (western Asia) appears to be fields, croplands, herbaceous plants, bushes and the lower branches of small deciduous trees at the edges of fields (Table 1). Those ‘generalist’ preferences are shared with many other species in the carnea-group (Henry et al. 2013), including five of the seven taxa most likely to live in sympatry with C. shahrudensis in Iran. Thus niche differences are not sufficient to distinguish C. shahrudensis from most other carnea-group species, except from ecological specialists like woodland-dwelling C. pallida or conifer-associated C. mediterranea. In contrast to C. shahrudensis, the new central Asian species C. bolti is a habitat specialist rather than a generalist: in Kyrgyzstan, it is consistently found at high elevations in sunny fields of tall grasses and herbs. Despite this association with alpine meadows, C. bolti was occasionally collected with both of the two other undescribed species found in Kyrgyzstan, C. ‘carnea-K’ and C. ‘downesi-K,’ which are separable from it by their generalist and conifer niches, respectively. On the other hand, distinguishing C. bolti from C. shahrudensis using habitat differences is moot, because the geographical ranges of these two species do not overlap. 1626 C.S. HENRY ET AL.

As yet, we cannot determine whether either C. shahrudensis or C. bolti qualifies as a nomadic species, as defined by Duelli (2001) and applied to highly migratory taxa that include C. carnea, C. plorabunda, C. agilis, C. zastrowi (both subspecies), C. heidarii and Chrysoperla nipponensis (Okamoto). Because C. bolti has a small geographic range limited to just one part of Kyrgyzstan and occupies a specialized alpine niche, it is unlikely to be nomadic. However, a single female of C. shahrudensis collected at 2267 m on the mountain pass between Qazvin (1300 m) and the Alamut Valley (1000 m) was so far away from crop fields that it could easily have been a migrating individual, indicating the possibility of a nomadic life cycle in the new Iranian species. Similar to the six above-mentioned nomadic species, C. shahrudensis is able to change colour in the laboratory under short day conditions, most commonly acquiring light sandy-brown body pigmentation. The irrigated parts of the cultivated landscape in northern Iran are green in both summer and winter, which does not seem like an appropriate background for effective crypsis of the diapausing adults of this species. Especially around Shahrud, however, the pale sandy or rocky desert is reminiscent of the most likely overwintering conditions for C. shahrudensis that existed in pre-agricultural Iran. As for the absence of a colour change to brown in diapausing C. bolti, we suggest that this species overwinters in nearby conifers at the edges of alpine meadows and therefore retains its green body colour for crypsis. Diapause enhancement of red facial pigmentation in C. bolti is shared with several other permanently green carnea-group species, including C. calocedrii (Henry et al. 2012), C. mediterranea, C. lucasina and C. ‘downesi-K’ (P.D., unpublished data), but its significance in those taxa is unknown.

Phylogenetic considerations Using mitochondrial DNA markers, it has been impossible reliably to infer phylogenetic relationships among the species of the carnea-group. For example, well-delineated and widespread Eurasian species such as C. agilis, C. mediterranea, C. lucasina and C. heidarii are clearly polyphyletic in published mitochondrial trees (Henry et al. 2012, 2014;Priceetal.2015). AddingDNAfromthetwonewspeciestothemitochondrialanalysisdoesnotsolvethat problem: the updated tree (Figure 7)placesC. shahrudensis close to several individuals of C. agilis and C. mediterranea,andputsC. bolti closest to C. carnea and two other individuals of C. agilis. Phylogenetic studies targeting commonly used nuclear coding genes have also failed to resolve evolutionary relationships within the carnea-group (Winterton and De Freitas 2006; Haruyama et al. 2008; Duelli, Duelli et al. 2014a;Mochizukietal.2017). Progress in the future will require a different approach, probably using nuclear DNA markers (single nucleotide polymorphism) generated by sequencing methods for investigating restriction site-associated DNA markers (Rubin et al. 2012;Wagneretal.2013). Nevertheless, mitochondrial sequence divergences (Table 5) do reveal that neither C. shahrudensis nor C. bolti is more or less similar to one another than to several other species in the carnea-group. Each is also relatively dissimilar to North American C. adamsi, the third member of the convergent song trio. These findings, reinforced by the topology of the mitochondrial phylogenetic tree (Figure 7), support the independent origins of C. shahrudensis, C. bolti and C. adamsi, mirroring earlier phylogenetic results that confirmed three other cases of parallel song evolution between lacewing species on different continents (Henry, Wells et al. 1999, 2012, 2014). Therefore the JOURNAL OF NATURAL HISTORY 1627 hypothesis of repeated allopatric parallel speciation in the carnea-group receives additional confirmation from the discovery of these two new Asian species.

Conclusions Results from analyses of song phenotype, adult and larval morphology, life history and ecology, and molecular sequencing all strongly support the validity of C. shahrudensis and C. bolti as distinct cryptic species of the carnea-group. For each of the two species, a larger geographic range is likely, considering how poorly we know the carnea-group fauna of central and western Asia. Chrysoperla shahrudensis has evolved acoustically in parallel with the North American species C. adamsi and the central Asian species C. bolti, providing additional examples of previously documented parallel speciation in this actively evolving species complex.

Taxonomy

Order NEUROPTERA Family CHRYSOPIDAE Subfamily CHRYSOPINAE Genus ChrysoperlaSteinmann, 1964 Type Species: Chrysoperla carnea (Stephens, 1836) Chrysoperla shahrudensis sp. nov. (Figures 1(a), 2(a), 4(a,b) and 5; Table 1, 2, 3)

Holotype. NHMUK010729670. Male: IRAN: Shahrud (36.37°N, 54.99°E), 1296 m, 7 August 2014, coll. P. Duelli, A. Mirmoayedi and D. Bolt, BMNH. Paratypes. Same data as holotype, 4 ♂,8♀ (specimens NHMUK010729671, NHMUK010729672, NHMUK010729673, NHMUK010729674, NHMUK010729678, NHMUK010729679, NHMUK010729680, NHMUK010729681, NHMUK010729682, NHMUK010729683, NHMUK010729684 and NHMUK010729685). IRAN: Shahrud (36.60°N, 54.98°E), 1286 m: 3 ♂,1♀, coll. same as holotype, BMNH (specimens NHMUK010729675, NHMUK010729676, NHMUK010729677 and NHMUK010729686); Pass Qazvin to Alamut Valley (36.38°N, 50.21°E), 2267 m, 5 August 2014: 3 ♀, coll. same as holotype, BMNH (specimens NHMUK010729687, NHMUK010729688 and NHMUK010729689); 6 ﬁrst-instar, 6 second-instar and 12 third-instar larvae, reared from parents pooled from Shahrud paratypes, WFBM.

Etymology Named for the type locality: Shahrud, Iran.

Description Adult. Ground colour uniform bright or light green on upper surface, paler green on lower surface, pale yellow-white medial dorsal stripe running along rear of head and length of thorax and abdomen. Stipes with apical 25–75% marked dark brown or black. Maxillary palp unmarked or basal segment marked dark brown dorsally. Gena marked 1628 C.S. HENRY ET AL. black, but sometimes red-brown or red. Clypeus lateral edge marked with black-brown stripe, occasionally red. Frons marked laterally with small red spot below outer margin of antennal base, ground colour entirely green. Scape, pedicel and postocciput unmarked. Pronotal setae pale in most specimens, occasionally with some black setae. Pronotum marked with indistinct greyish lateral spots. Metatarsal claw basal dilation ratio 2.50 (2.28–3.17). Forewing apically symmetrical, broadly rounded, and not tapered; length 11.7–15.75 mm; length : breadth ratio 2.89 (2.59–3.12); venation entirely green except basal vein of intramedian cell, the two Cu crossveins, and ends of costal and subcostal crossveins black; costal setae short, inclined towards wing apex; basal Rs-M crossvein usually at right angles to Rs but occasionally obtuse. Abdominal segments 1–3 marked with dorso-lateral rectangular black spots; abdominal sternites clothed by ﬁne, blond setae, occasionally 50% black setae on sternite 8; pleural membrane of second segment usually unmarked but rarely showing a faint band; lip of sternite 8 + 9 in male broad and short above a small chin; tignum as in other carnea-group species.

Courtship song (25°C). Substrate-borne vibrational signals produced by abdominal oscillations; nearly identical in males and females. Shortest repeated unit usually disyllabic and iterative, always beginning with single primary volley of abdominal vibration of ≈ 0.75-s duration, usually followed (> 80% of the time) by atonal rumble of ≈ 0.50-s duration. In solo ‘calling’ context, SRU repeated every 2.40 s. Frequency structure of each volley is complex (bipartite), starting at ≈ 76 Hz, falling quickly to ≈ 35 Hz, rising again to ≈ 60 Hz at end. Heterosexual duets consist of each individual repeatedly and politely answering single SRUs (volley-plus-rumble units) of its partner without overlapping; duets have slightly slower repetition rates than solos.

Larva, third-instar. Background of head, body and legs cream-coloured. Dorsum of head with pair of relatively narrow pale- to medium-brown longitudinal, dorso-lateral stripes with narrow baso-lateral expansions extending towards eyes and medium-brown lateral stripe of variable width immediately behind the eye; head surface rarely (17% of larvae) exhibiting a fronto-medial spot, but nearly always bearing a brown spot dorso- medially on scape. Pronotum usually with narrow, pale brown, longitudinal dorso-lateral stripes; meso- and metanota usually, and abdomen always, unmarked. Coxa bearing two small, medium-brown spots laterally; distal femoral band and base of tibia pale brown.

Larva, earlier instars. Stripes, lateral expansions, and spots on head, thorax and abdomen less sharply delineated but often more extensive than in third-instar (mature) individuals. Femoral and tibial markings remain well-deﬁned or are often darker.

Life cycle and ecology A habitat generalist, associated broadly with crop ﬁelds and ﬁeld margins within primarily deciduous rather than coniferous niches, at elevations greater than 1000 m. Number of generations per year unknown. Stalked eggs oviposited singly rather than in groups. Adults turn light sandy-brown colour in winter diapause. Possibly a nomadic (migratory) species. JOURNAL OF NATURAL HISTORY 1629

Diagnosis/discussion. Chrysoperla shahrudensis sp. nov. can be most reliably separated from other species in the carnea-group by courtship song analysis. However, the presence of two small red spots on the frons is a diagnostic morphological trait that will usually delineate C. shahrudensis from other species in this genus, except C. bolti. Additional characters that help to distinguish this species from other carnea-group species, but which are not diagnostic, include relatively pale green body colouration; a frons that is mostly or entirely green; symmetrical, broadly rounded apex of the fore wing; intermediate size of the basal dilation of the claw; and C. carnea-like proportions of the abdominal chin and lip. The simple SRU of the C. shahrudensis vibrational song, consisting of just one relatively long volley (or volley plus rumble), superficially resembles that of both C. bolti sp. nov. in Kyrgyzstan (Asia) and C. adamsi in western North America. However, the C. shahrudensis primary volley has a complex, bipartite frequency structure that differentiates it not only from C. bolti and C. adamsi but also from all other species of the carnea-group. Limited and simple head markings characterize the larva of C. shahrudensis, but do not distinguish it absolutely from any other Eurasian species of the carnea-group. Only the dorso-medial brown spot on the antennal scape is potentially diagnostic, although this needs to be verified using larger larval sample sizes of all species. Species of the Chrysoperla carnea-group that have been collected in sympatry with C. shahrudensis include C. carnea, C. agilis, C. pallida, C. zastrowi sillemi and C. heidarii.Of these, only pale green C. pallida is relatively easy to separate from C. shahrudensis based on overall appearance, although all can be unambiguously assigned to species based on song phenotype.

Distribution. Northern Iran (western Asia) at elevations ≥1000 m.

Chrysoperla bolti,sp. nov. (Figures 1(b), 2(b), 4(c) and 6; Table 1, 3, 4)

Holotype. NHMUK010729838. Male: KYRGYZSTAN: Alamüdün District (42.58°N, 74.48°E), 1950–2056 m, 12 July 2016, coll. P. Duelli, BMNH. Paratypes. Same data as holotype, 7 ♂,6♀, BMNH (specimens NHMUK010729839– NHMUK010729851, inclusive), 2 ♀, UCMS; same locality as holotype 25 June 2016: 4 ♂, 1 ♀, UCMS; Chichkan Valley, (42.12°N, 72.80°E), 1750 m, 1 June 1995: 6 ♂,4♀, coll. P. Duelli, UCMS; 30 ﬁrst-instar, 8 second-instar, and 18 third-instar larvae, reared from parents pooled from Alamüdün paratypes, WFBM.

Etymology Named for our colleague and fellow insect collector, Mr Daniel Bolt.

Description Adult. Ground colour uniform dark green on upper surface, paler green on lower surface, pale yellow-white dorsal stripe running along rear of head and length of thorax and abdomen. Stipes marked entirely dark brown or black. Maxillary palp with apical segment yellowish-brown, other segments marked dark brown dorsally. Gena marked 1630 C.S. HENRY ET AL. dark brown or black. Clypeus lateral edge marked with black-brown stripe, occasionally reddish. Frons marked laterally with reddish stripe and sometimes with small red spot below the outer margin of the antennal base, ground colour of upper half green, lower half yellowish. Scape, pedicel and postocciput unmarked. Pronotal setae mostly pale in females, predominantly black in males. Pronotum marked with indistinct greyish lateral spots. Metatarsal claw basal dilation ratio 2.81 (2.47–3.13). Forewing apically symmetrical, narrow and rounded; length 11.55–14.40 mm; length : breadth ratio 3.12 (2.79–3.33); venation entirely green except basal vein of intramedian cell, the two Cu crossveins, and ends of costal and subcostal crossveins faint black; costal setae short, inclined towards wing apex; basal Rs-M crossvein at right angles to Rs. Abdominal segments 1–3 unmarked; abdominal sternites clothed by ﬁne, blond setae, but usually black setae on sternite 8; pleural membrane of second segment usually bearing a thin brown stripe; lip of sternite 8 + 9 in male broad and short above a small chin; tignum as in other carnea-group species.

Courtship song (25 degrees C). Substrate-borne vibrational signals produced by abdominal oscillations; nearly identical in males and females. The SRU usually monosyllabic, always beginning with single volley of abdominal vibration of nearly 1 s duration, sometimes followed (≈ 20% of the time) by atonal rumble of variable duration. In solo ‘calling’ context, SRU repeated every 3.8 s. Vibrational frequency of each volley simple, linearly falling from ≈ 80 Hz at start to ≈ 40 Hz at end. Heterosexual duets consist of each individual repeatedly and politely answering single SRUs of its partner without overlapping; duets have slightly slower repetition rates than solos.

Larva, third-instar. Background of head, body and legs cream-coloured. Dorsum of head with pair of moderately narrow medium to dark brown longitudinal, dorso-lateral stripes with baso-lateral expansions extending towards eyes and a medium to dark brown lateral stripe of variable width immediately behind the eye; head surface typically showing a darker spot in the dorso-lateral stripe mesad of the eyes, but lacking separate fronto-medial spot. Antennal scape pale to medium brown, sometimes darker medially and occasionally exhibiting a pale longitudinal stripe dorso-laterally; pedicel and basal flagellomere brown in 30% of specimens. Extensive red suffusion present dorso-laterally and laterally on all thoracic and abdominal segments; thorax and first several segments of the abdomen bearing narrow, brown, dorso-lateral longitudinal stripes (prothorax) or spots (meso- and metathoraces and abdomen). Base of coxa and associated pleuron medium brown; distal femoral band broad and brown, darkest apically and paler basad, successively narrower on more posterior legs; tibial base brown.

Larva, earlier instars. Stripes, lateral expansions, and spots on head and prothorax less sharply delineated but often more extensive than in third-instar (mature) individuals. Scape always (second-instars) or often (first-instars) brown; pedicel and first flagellomere brown in 12% of second-instars but unpigmented in first-instars. Meso-and metanota unmarked with brown and showing reduced area and intensity of red suffusion. Red suffusion similarly reduced on abdomen. Femoral pigmentation limited to distal one- third to one-half on all legs. JOURNAL OF NATURAL HISTORY 1631

Life cycle and ecology A habitat specialist, found in alpine meadows of tall grasses and herbs at elevations greater than 1000 m. Number of generations per year unknown. Stalked eggs oviposited singly rather than in groups. In winter diapause, adults remain dark green but typically acquire enhanced reddish suﬀusion on genae and frons. Apparently not a nomadic (migratory) species.

Diagnosis/discussion. Chrysoperla bolti sp. nov. can be most reliably separated from other species in the carnea-group by courtship song analysis. However, C. bolti differs from C. shahrudensis and several other Eurasian species in its darker-green body colouration, extensive yellow pigment on the lower frons, and less intense dark markings on the crossveins, but those traits are not absolutely diagnostic. Additional non-diagnostic characters helping to distinguish this species from several other common carnea-group species include a relatively narrow but apically rounded fore wing, intermediate size of the basal dilation of the claw, C. carnea-like proportions of the abdominal chin and lip, and the presence of a thin ‘lucasina stripe’ on the pleural membrane of the second abdominal segment (Henry et al. 1996). Of the two undescribed species that live in sympatry with C. bolti in Kyrgyzstan, C. ‘downesi-K’ is morphologically more similar to it than is C. ‘carnea-K.’ In particular, C. ‘downesi-K’ is darker green and has a yellow frons and relatively narrow wings, whereas C. ‘carnea-K’ has broad wings and prominent black tips on the crossveins, two traits shared with C. shahrudensis (S.J.B., unpublished data). The simple SRU of C. bolti’s vibrational song, consisting of just one relatively long volley (or volley plus rumble), superficially resembles that of C. shahrudensis in Iran and more exactly resembles that of C. adamsi in western North America. However, the C. bolti volley has a simple, smoothly descending frequency structure that differentiates it clearly from C. shahrudensis,whileC. bolti’s longer volley period (≈ 3.8 s) consistently separates it from true C. adamsi (≈ 3.2 s). There are no known members of the global carnea-group other than C. shahrudensis and C. adamsi that have duetting songs similar to the song of C. bolti. Limited and simple head markings characterize the larva of C. bolti, but do not distinguish it absolutely from any other Eurasian species of the carnea-group. However, the brown antennal scape and serially graded extent of femoral pigmentation are potentially diagnostic traits, although they need to be veri fied using larger larval sample sizes of all species. Species of the Chrysoperla carnea-group that have been collected in sympatry with C. bolti include only the two undescribed species informally known as C. ‘carnea-K’ and C. ‘downesi-K,’ each of which has acoustical, morphological and ecological characteris- tics that delineate it from C. bolti.

Distribution. Northern Kyrgyzstan (central Asia) at elevations >1000 m. Taxon abbreviations: C., Chrysoperla; z., zastrowi; shahrud, shahrudensis; nippon, nipponensis Locality abbreviations: RSA, Republic of South Africa; C, central; N, northern; S, southern; W, western; SE, southeastern; SW, southwestern; Switz, Switzerland; Pfyn, Pfynwald region of Switzerland; CA, state of California, USA; WA, state of Washington, USA; OR, state of Oregon, USA; ID, state of Idaho, USA; Inv, Inverness, CA, USA (San Francisco Bay area). 1632 C.S. HENRY ET AL.

Acknowledgements

Dr H. Heidari (Centre for Sustainable Development, Iran) and Mr Daniel Bolt (MTM Consulting, Switzerland) helped collect and rear living lacewing specimens over the time period covered by this study. We also thank our valued associates, Drs Masayuki Hayashi and Masashi Nomura (Laboratory of Applied Entomology, Chiba University) and Dr Hideshi Naka (Tottori University, Torrori City, Japan) for DNA extraction, ampliﬁcation and Sanger sequencing of C. shahrudensis specimens. Finally, we are indebted to Dr Elizabeth Wade (United States Department of Agriculture, Agricultural Research Service, Gainesville, FL, USA) for setting up and running Bayesian phylogenetic analyses of mitochondrial sequence data. We sincerely thank (1) the Ministry of the Environment of Iran and (2) the Kyrgyz Ministry of Environmental Conservation and Natural Resources, for granting us the necessary permits for carrying out ﬁeldwork in Iran (including Golestan National Park) and the Kyrgyz Republic, respectively. The 2014 trip was an approved activity within the project ‘Chrysopidae of Iran’ directed by Professor A. Mirmoayedi at Razi University, Kermanshah, Iran. Copies of all collecting permits are available on request. Laboratory facilities, support and supplies for this study were provided through the University of Connecticut, Storrs, CT, USA; the Natural History Museum, London, UK; the Department of Plant Protection of Razi University, Kermanshah, Iran; the Institute for Agro-Environmental Sciences NARO, Tsukuba City, Japan; and the Laboratory of Applied Entomology, Chiba University, Matsudo City, Japan. We must also acknowledge here that we are deeply saddened by the death on 11 November 2017 of our cherished colleague and co-author from Japan, Atsushi Mochizuki, after a long struggle with cancer. This paper is written and published to honour and celebrate his memory. His knowledge, wisdom, energy and humanity will be greatly missed.

Disclosure statement

No potential conﬂict of interest was reported by the authors.

References

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