Pauropsalta ayrensis Ewart, 1989
POPPLE, L. W., 2013, <p class = " HeadingRunIn " align = " left "> <strong> A revision of the <em> Pauropsalta annulata </ em> Goding & amp; Froggatt species group (Hemiptera: Cicadidae) based on morphology, calling songs and ecology, with investigations into calling song structure, molecular phylogenetic relationships and a case of hybridisation between two subspecies </ strong> </ p>, Zootaxa 3730 (1), pp. 1-102: 80-99
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|Pauropsalta ayrensis Ewart, 1989|
Material: Holotype: ♂ ‘Alva Beach/Ayr’, ‘ Jan.1981 ’, ‘ A. Ewart’ ( QM, reg. no. T.9189); other material: QUEENSLAND: 1♂ Bald Hills Stn, 4km N. of Isabella Falls Qld, 15°15'S 145°00'E, 29.xii.1984, m. v. lamp, Daniels & Daniels (MSM) ; 1♂ Balnagowan W. of Mackay Qld, 24.xii.1984, Adams ( MSM); 1♂ "Bedrock", Mt Surprise N. Qld, 18°08.64'S 144°19.26'E, to light, 19.i.2005, Ewart, recorded ( AE); 1♂ "Belwood", Edungalba C. Qld, 5.xii.1979, on brigalow, Adams ( MSM); 1♂ 1♀ Binjour, 25°31'S 151°27'E, 27.xi.2005, Popple & Finlay- Doney, 441-0032 & 441-0033 ( LWP); 1♂ same data as previous, xi.2005, 441–0048 ( LWP); 2♂ 1♀ Black Rock S. of Boonah [Qld], 7.i.1991. Burwell ( QM); 2♂ 1♀ Blencoe Ck Falls , Kennedy NE. Qld 7.iii.1960, Calder ( QM); 1♂ 1♀ Bowen Qld, 23.xii.1980, Moss, 441-0001 & 441-0002 ( LWP); 1♀ Bowen [Qld], 8.ii.1973, fruit trees in garden, Meurow ( MSM); 1♂ 1♀ Bowen Qld, 1917, Rainford ( QM); 1♂ Brookhill nr Townsville N. Qld, 11.xii.1988, RING ( MSM); 2♀ Calliope River , Bruce Hwy nr Gladstone Qld, 16.i.1989, Eastwood ( MSM); 2♂ Castle Hill, Townsville N. Qld, 18.xi.1988, Eastwood ( MSM); 8♂ 1♀ Cordelia Qld, 10.xii.1988, Ring ( MSM); 10♂ 3♀ Eidsvold, 25°22'6"S 151°7'25"E, 24–25.xi.2006, m. v. lamp, Popple & McKinnon , 441-0034 to 441-0047 ( LWP); 5♂ 6♀ Qld: 16°58'S 145°26'E, Emerald Hill, Mareeba, 6–10.ii.2003, Monteith, m. v. light (QM) ; 6♂ 3♀ Qld: 19°49.0'S 146°03.5'E, Fletcher Ck Rest Area , 280M, m. v. lamp , 12.ii.2007, QM Party ( QM); 1♀ 40.0km WNW. [of] Greenvale N. Qld, 18°56.73'S 144°38.68'E, 31.i.2005, Ewart ( AE); 1♂ 1♀ Herberton district , 17°23'22"S 135°21'2"E, 3.i.2007, Popple & McKinnon, 448-0001 & 448-0002 ( LWP); 1♂ same data as previous, 29.xii.2007, Popple & McKinnon, 448-0003 ( LWP); 1♂ Kuranda N. Qld, 1.i.1980, Wood ( MSM); 2♂ L[ake] Awoonga, 12km SE. of Calliope Qld, 11–14.xi.1985, R. Leggett ( AE); 7♂ 1♀ 1km N. of Maroon Dam, 28°10'44"S 152°39'21"E, 11.i.2003, Popple & MacSloy, 441-0005 to 441-0014 ( LWP); 1♂ same data as previous, on minidisc LWP, 441-0004 ( LWP); 4♂ 5♀ same data as previous, 15.i.2003, Popple & Popple, 441–0015 to 441–0023 ( LWP); 1♀ C. Qld: 22°03'S 148°04'E, 6km S. of Moranbah, 220m, 20.xii.1997 – 26.iv.2008, flight intercept, BoxFlat , Monteith , 5803 ( QM); 1♂ same data as previous, 31.x.1986 ( MSM); 17♂ 19♀ C. Qld: 20°07'S 147°46'E, 3km SE. of Mt Abbot , 200m, 7.xii.1996. at light, Monteith , Cook & Cook ( QM); 1♂ Mt Corbin , Townsville N. Qld, 26.xi.1988, Eastwood ( MSM); 1♀ Mount Surprise E. of Georgetown N. Qld, 1.xii.1982, Walford-Huggins ( MSM); 2♂ 1♀ Mt Surprise (caravan park) N. Qld, 18°08.61'S 144°19.30'E, open woodland, 7.i.2003, Ewart ( AE); 1♂ same data as previous, recorded ( AE); 2♂ 2♀ "Mourangee H[ome]s[tea]d nr Edungalba C. Qld, 15.xii.1979, Adams ( MSM); 6♀ Mundubbera district , 25°34'40"S 151°18'11"E, xi.2005, Papacek , 441-0025 to 441-0031 ( LWP); 2♂ 1♀ SEQ: 25°42’S 151°26’E, Nipping Gully site 6, 18–19.xii. 1998, 200m, Monteith , Gough & Maywald, 7532 ( QM); 1♂ 3km S. of Peak Crossing SE. Qld , 16.i.2001, Popple & Moss, recorded, 441-0003 ( LWP); 1♀ Qld : 23°49'S 150°04'E, "Pheasant Creek" W of Wowan, 30.ix.2003, Wallace , 9743 ( QM); 1♀ Rockhampton Qld , 16.xii.1977, Hiller ( MSM); 3♂ 1♀ Spear Creek , 8.5km N. of Palmer River x-ing Qld , 16°03'S 144°48'E, 26.xii.1984, m. v. lamp, Daniels & Daniels (MSM) ; 1♂ 2♀ Qld : 19°58.2'S 145°35.2'E, "Toomba" H.S., 390m, hand, pasture, 13–15Feb2007, QM party, 14798 ( QM); 2♂ 2♀ Qld : 19°58.0'S 145°34.9'E, "Toomba" site 3, 390m, 13–16.ii.2007, vinescrub edge/pasture, hand netting, QM party, 14777 ( QM); 1♂ Townsville N. Qld, 16.xi.1988, Eastwood ( MSM); 1♀ Townsville , 16.i.1969, Monroe (reg. no. T 11235 View Materials ) ( QM); 1♂ Yarrol Scrub, 24°53'13"S 152°19'14"E, 11.xii.2004, Beard, 441-0024 ( LWP); 2♂ 1♀ Yeppoon Qld, 28.i.1983, Eastwood ( MSM). GoogleMaps
Description. Male ( Figs 4Q–R View FIGURE 4 , 31H–I View FIGURE 31 , 39E–F View FIGURE 39 , 46E, 46G View FIGURE 46 ). Head. Dorsal surface black with small yellowbrown to dark brown triangular depression between the lateral ocelli, pointing anteriorly, with flat side against posterior margin of head; long silver pubescence behind eyes, with shorter yellow pubescence over remainder. Genae black, with long silver pubescence; vertex black, sometimes with yellow-brown to dark brown vertex lobes. Mandibular plate black; long silver pubescence. Antennae black, paler (dark brown) around apex. Ocelli pink. Eyes dark red in live insects, fading to dark reddish-brown or appearing black in preserved specimens; subtle furrow between eyes and pronotum. Postclypeus shiny black with reddish-brown extreme lateral margins and on midline (in pale specimens only), extending to dorsal surface; short yellow pubescence along posterior ventral surface. Anteclypeus black, with long silver pubescence. Rostrum dark brown grading to black apically; extending to mid coxae.
Thorax. Pronotum shiny black, with orange-brown to dark brown midline (absent in some specimens) not extending to proximal or distal margins; pronotal collar brown to orange-brown, paler laterally in some specimens; silver-yellow pubescence. Mesonotum, including submedian and lateral sigilla, shiny black, sometimes with orange-brown to dark brown dorsolateral and sometimes lateral fasciae present in some specimens; when present, dorsolateral fasciae narrowly longitudinal to triangular, never merging medially, positioned between submedian and lateral sigilla; cruciform elevation yellow-brown to orange-brown, extending to surrounding posterior mesonotum (in most specimens) and ridges between wing grooves, black anteriorly; silver-yellow pubescence mostly evident immediately posterior of cruciform elevation.
Legs. Coxae black, yellow-brown apically; femora dark brown, pale brown apically; fore and mid tibiae dark brown, paler at base; hind tibiae pale orange-brown to medium brown; fore tarsi medium brown; mid and hind tarsi pale brown; claws and spines dark brown.
Wings. Fore wing venation medium to dark brown, becoming darker towards apical cells and ambient veins; fore wing costal veins pale to medium brown; subtle angulation of fore wings at node; pterostigma dark brown; hind wing infuscation at the juncture of the anal lobe and wing margin, surrounding the distal termination of vein 2A, distinct.
Timbals. Long ribs 1–2 fused ventrally; long ribs 1–4 fused dorsally to basal spur. Long rib 5 typically extending ventrally up to the same extent as adjacent intercalary rib.
Opercula. Roughly sickle-shaped, obliquely elongated; central region domed, black; medial and lateral margins pallid; parallel to body axis.
Abdomen. Tergites mainly black with contrasting yellow-orange posterior margins, wider on tergite 8; narrow brown dorsolateral medial fascia present on paler specimens; silver short pubescence conspicuous laterally on some specimens. Sternites yellow to yellow-brown with a continuous black medial fascia.
Genitalia. Pygofer yellow-brown or dark brown, grading to black posteriorly; upper lobes prominent, erect, with somewhat hooked termination; lower lobes distinct, with an inner tooth weakly developed on each lobe or absent; inner lobes enlarged, elongated, posteriorly tapering; claspers with a pair of hooked processes; aedeagus with dorsal pseudoparameres that bifurcate and join theca near gonocoxite IX; pseudoparameres apically curved, tapering; theca strongly sclerotized, conspicuously widened apically; apex conspicuously of greater vertical width than shaft, finely ornamented, with extreme dorsal edge extending into a small spine that projects further posteriorly than ventral edge, and with a large spine on ventral edge.
Female ( Figs 46F, 46H View FIGURE 46 ). Closely similar in colouration and patterning to male. Head similar in colour to that of male. Pronotum black; central fascia (when present) conspicuous, orange-brown to medium brown, forming a smear from behind the head that tapers distally. Mesonotum similar in colour to that of male, sometimes with medium to dark brown dorsolateral fasciae between submedian and lateral sigilla, and additional fasciae along lateral margins; when present, dorsolateral fasciae longitudinal to triangular; lateral fasciae narrow. Legs similar in colour to those of male. Abdomen with tergites similar in colour to those of male; abdominal segment 9 medium brown to orange-brown with a pair of longitudinal, near-dorsal, black fasciae that extend to the anterior edge and ventrolaterally to some extent; sternites yellow-brown to medium brown with a diffuse black fascia distributed medially along sternites II to VII; ovipositor sheath extending approximately 0.5 mm beyond apex of abdomen.
Distinguishing features. Male specimens of P. ayrensis differ from all species in the P. annulata group, apart from P. simplex , by their dark brown, hooked upper pygofer lobes. They can be distinguished consistently from P. simplex in the shape of the theca, which is conspicuously wider at the apex than along the shaft ( Fig. 39E–F, c.f. P View FIGURE 39 . simplex: Fig. 22B View FIGURE 22 ). Females that are predominantly black (the dominant colour form), are easily distinguished from others in the P. annulata group by their obvious yellow-brown to orange-brown cruciform elevation (a feature also apparent in males of the same colour form). Females of P. inversa may be superficially similar, but in Queensland they are matte black on the pronotum rather than shiny black. Females that are predominantly brown may be distinguished from all species in the complex, apart from P. simplex , by a combination of their dull, brown and black appearance and ovipositor that extends only 0.5 mm beyond the apex of the abdomen. They cannot be reliably distinguished from females of P. simplex .
Notes on geographical variation. Some specimens of P. ayrensis on the southern and western edge of the Atherton Tableland (e.g. west of Herberton, Dimbulah) and all specimens in the Mount Surprise region of northern Queensland are much paler than those found across the remainder of the distribution. Both males and females are generally browner in colouration, with distinct fasciae present on the mesonotum between the submedian and lateral sigilla. These specimens account for the paler extremes of variation given in the species description. More typical specimens are extensively shiny black and orange-brown or yellow-brown. Their mesonotum is often entirely shiny black, but occasionally has contrasting, narrow fasciae between the submedian and lateral sigilla.
Measurements. N= 30 ♂ 18 ♀. Ranges and means (in parentheses), mm; BL: ♂ 11.7–15.0 (13.0); ♀ 11.3–14.1 (12.9); FWL: ♂ 14.0–17.5 (15.5); ♀ 14.8–17.2 (15.9); FWB: ♂ 4.9–5.7 (5.3); ♀ 4.9–5.8 (5.4); HW: ♂ 3.7–4.2 (3.9); ♀ 3.7–4.3 (4.0); PW: ♂ 2.9–3.6 (3.2); ♀ 3.0–3.6 (3.3); AW: ♂ 3.2–4.1 (3.7); ♀ 3.4–4.0 (3.7); OL: ♀ 3.8–4.8 (4.2).
Distribution and Ecology. Eastern Queensland from near Cooktown in the far north to the Fassifern Valley in the far south ( Fig. 51 View FIGURE 51 ). Generally found to the east of the Great Dividing Range, with records from Millmerran
( Ewart, 1989), Porcupine Gorge, Blackbraes National Park and localities around Mt Surprise being notable exceptions. Adults may be present from October to May, but are most conspicuous between November and February. They occur in open grassland and grassy woodland and emergence seems to be dependent upon rainfall. Numbers can be very high with the adults conspicuous for 1–2 weeks after emergence, and with numbers falling substantially about four weeks later. Females have been observed ovipositing in the branches of young eucalypts and other woody shrubs. Oviposition has been associated with fatal damage to citrus seedlings in the Mundubbera district, which apparently followed a large emergence in a newly grassy field followed by a spill over into a citrus orchard (L. W. Popple, unpublished field data, 2005).
Geological and Pedological Associations. Adults are generally associated with plants that occur in silt dominated alluvial soils of variable depth or in areas of weathered laterite. The former soils are found almost exclusively on flats and lower slopes near water level, while the latter soils are associated with plateaus on rises.
Calling Song ( Figs 2P View FIGURE 2 , 52–53 View FIGURE 52 View FIGURE 53 ). The calling song of P. ayrensis has two components, buzzing and lilting, and is largely dominated by the lilting component. The lilting component comprises a long echeme (12–73 syllables, 0.195 –0.979 s) followed by a short silence (0.035 –0.062 s), a short echeme (1–3 syllables, 0.014 –0.040 s) and another silence (0.045 –0.216 s). On occasions the last silence is the followed by an additional 1–4 short echemes (varying within individuals and each comprising 1–3 syllables, 0.013 –0.037 s, n=24) each punctuated by silences (0.021 –0.083 s; with those following the last short echeme being 0.003 –0.026 s), immediately preceding the long echeme of the next phrase. As found in most other species, the buzzing component is emitted occasionally and is simply a sustained version of the long echeme from the lilting component (above). The song has a pulse repetition rate of 0.434 –1.149 s, a syllable repetition rate of 80–100 Hz, a frequency plateau of ~7.0–11.5 kHz and a highest amplitude dominant frequency of 8.5–10.2 kHz. No instances of male–female duets have been heard or recorded.
Considerable variation exists in the calling song of this taxon, which is demonstrated most apparently in Figure 53 View FIGURE 53 . Further investigation may reveal geographically distinct subspecies, or even species, as apparent for the P. annulata species complex. However, at this juncture, there are not enough recordings available to establish the underlying cause of this song variation.
Statistical analyses of song specificity in the Pauropsalta annulata species complex
Methodology. All recordings were made between 04. xi.2002 and 10. i.2011. A total of 532 individuals of the P. annulata species complex, including P. annulata , P. notialis notialis , P. notialis incitata , P. n. notialis x incitata hybrids and P. tremula , were recorded at sites across their geographical distributions in eastern Australia ( Table 1; Figs 7 View FIGURE 7 , 11 View FIGURE 11 and 19 View FIGURE 19 ). A wide sample was taken across both sites of allopatry (i.e. where only one species occurred) and also sites of sympatry (where two or more species were present). Each individual cicada recording was identified a priori by initial aural diagnosis in the field and later confirmed based upon a visual inspection of calling song structure. All recordings of individual P. notialis cicadas at sites dominated by calls that were intermediate between P. n. notialis and P. n. incitata or contained song characters of both subspecies within recordings of a single individual were identified as P. n. notialis x incitata hybrids. All taxa exhibit similar phenologies and were present and calling together on each recording occasion at the sites of sympatry. The procedures for ensuring that recordings were not taken from the same individual cicadas at each site are described by Popple et al. (2008). From each recorded song, 12 replicate measures were taken for separate instances of each of the four song segments of the lilting component ( Fig. 54 View FIGURE 54 ). This is the component to which the female responds in acoustic duets, which allows the calling male to locate the female and engage in copulation.
The calling song data were averaged for each song segment of each individual and entered into a matrix with individuals as objects and the four song segments as attributes. All data were analysed in PC-Ord 5 ( McCune and Mefford, 2006). Examination of the cumulative variance within objects and attributes, combined with an outlier analysis, identified a modest proportion of outliers in the data. Removal of these outliers had no effect on the allocation of individuals in clustering procedures, so no relativisation, transformation and/or data exclusion was necessary.
A single cluster analyses was performed on the data, using the Relative Sørensen distance measure as per Popple et al. (2008). This approach compares individuals on the basis of the four song segments ( Fig. 54 View FIGURE 54 ) being treated as relative (proportion-based) values, thus removing individual differences in total phrase length ( Pelton and Conran, 2002) and also excluding rate-based temperature effects. A flexible beta algorithm (ss=-0.25) was used to cluster the data. This provides optimal arrangement of ordinations with low chaining and no reversals, whilst also being space conserving ( Legendre & Legendre 1998). The data were also subject to a Non-metric Multidimensional Scaling (NMDS) using the same distance measure above. The procedure was run initially with step-down dimensionality (beginning with four axes, followed by runs in reduced dimensions) to determine the most appropriate number of dimensions for the ordination procedure. Evaluation of the scree plot of stress versus dimensionality revealed that the stress reduced to acceptable levels (<15%) at two dimensions. For each distance measure the NMDS was then rerun at two dimensions ( McCune et al. 2002).
Results and Discussion. The cluster analysis revealed five clusters within the rhythmic song structure data, as shown on the NMDS ordination presented in Figure 55 View FIGURE 55 . The composition of the clusters emerged as follows:
Cluster 2 comprised the remainder of the P. annulata individuals (8%, n=136), most of the P. n. incitata individuals (57%, n=102) and more than one third of the P. n. notialis x incitata hybrid individuals (37%, n=138);
Cluster 3 contained the rest of the P. n. incitata individuals (43%, n=102), a large portion of the P. n. notialis individuals (42%, n=108) and the majority of P. n. notialis x incitata hybrid individuals (53%; n=138);
Cluster 5 housed all of the representatives of P. tremula ; five individuals within this cluster appeared as outlier anomalies on the NMDS (including 2/12 from Lake Broadwater, 2/12 from Benarkin and 1/12 from Eidsvold; Table 1).
Overall, the cluster analysis agreed with the original designations based on aural identification and visual examination of song structure ( Table 1), of P. annulata , P. notialis and P. tremula , with only 14 individuals (3%, n=532) not being assigned to clusters containing the same species as the original designation. The vast majority of P. n. notialis x incitata (hybrids) (98%, n=138) were divided among clusters dominated by P. n. notialis and P. n. incitata, and these hybrids dominated the vector space between the two subspecies as would be expected ( Fig. 55 View FIGURE 55 ). The apparent disjunction on the NMDS ordination ( Fig. 55 View FIGURE 55 ) in cluster 5 ( P. tremula ), where two individuals from Lake Broadwater, two from Benarkin and one from Eidsvold clustered separately from the other individuals, is likely to be an artefact of data visualisation in a two dimensional vector space rather than a meaningful separation. In any case, no such pattern emerged in the cluster analysis.
The only residual anomaly in the otherwise straightforward results from this analysis concerns the unexpected allocation of 14 individuals to adjacent clusters. Eight of the above are P. annulata individuals recorded from Carina Heights (2), Maroon Dam (4), Peak Crossing (1) and Eidsvold (1) (locality details in Table 1) that appear to show the typical structure of P. annulata , despite being placed in a cluster otherwise dominated by P. n. incitata and P. n. notialis x incitata hybrid individuals (Cluster 2). All of these, with the exception of the Eidsvold individual, are positioned adjacent to Cluster 1 and it may be reasonable to postulate that these are the result of minor misclassifications during the cluster analysis. The remaining six individuals include three of P. annulata and three of P. n. notialis x incitata from Upper Yarraman ( Table 1). Close examination of the structure of these calls cannot rule out the possibility that at least some of these may be examples of hybridisation between P. annulata and P. n. notialis x incitata at this location.
P. annulata is rare at Upper Yarraman (only five recordings were obtained in total) by comparison with P. n. incitata (n=85; same location). The occurrence of these species at this location is restricted to a narrow 300 m long strip of roadside vegetation surrounded by cleared paddocks, which forces these two species into close proximity. This aspect, combined with the higher relative abundance of P. n. notialis x incitata could make P. annulata somewhat prone to hybridisation with P. n. notialis x incitata at this location. Hybridisation due to rarity in modified habitats has been reported in other systems ( Rhymer & Simberloff, 1996; e.g. Clarke et al., 2001).
Nevertheless, the potential example of hybridisation between P. annulata and P. notialis described above is exceptional. Otherwise, P. annulata and P. notialis (including P. n. notialis , P. n. incitata and P. n. notialis x incitata) have calling songs that cluster discretely in relation to one another, including where both occur together in sympatry ( Fig. 55 View FIGURE 55 ). They also differ in dominant frequency of the calling song, as well as plant associations and geographical distributions ( Popple et al., 2008; Popple & Walter, 2010).
By contrast, almost no examples exist where P. n. notialis and P. n. incitata have been found in sympatry and display song structures that cluster discretely. Instead, where the two subspecies have been predicted to overlap ( Popple & Walter, 2010), P. n. notialis x incitata hybrid populations are nearly always found. The only exception is at Possum Park, where four P. n. notialis and two P. n. incitata were recorded and the two subspecies clustered separately in the analysis (with all four P. n. notialis individuals restricted to Cluster 4 and the two individuals of P. n. incitata split across Clusters 2 and 3). Interestingly, this is also the only known example where the distributions of P. n. notialis and P. n. incitata coincide within remnant vegetation. Areas where P. n. notialis x incitata hybrid populations occur are characterised by landscapes that have been extensively cleared of the former habitats suitable for both P. n. notialis and P. n. incitata. It would make an interesting future study to explore the hypothesis that hybridisation / introgression between these subspecies (and also the potential case involving P. annulata and P. notialis at Upper Yarraman) has been influenced by widespread vegetation clearing.
Molecular phylogenetic relationships and divergence time estimates in the Pauropsalta annulata species group.
Data sampling and preparation. Cicada specimens were collected in the field and stored in either 100% ethanol or 30% polyethylene glycol. Ninety-four specimens were included in the phylogenetic reconstruction of the P. annulata species complex ( Table 2). These comprised 83 individuals from the P. annulata species complex itself (including all (sub)species redescribed or newly described in this study apart from P. rubristrigata and P. blackdownensis , for which no fresh material was available. The remaining 17 individuals represent outgroups, including five individuals from three congeneric species ( P. mneme (Walker) , P. castanea Goding and Froggatt (presently a junior synonym of Yoyetta abdominalis (Distant)) and P. sp. nr corticinus Ewart), two individuals from an allied genus ( Palapsalta virgulatus Ewart ), four individuals from three species within the same tribe ( Birrima varians (Germar) , B. sp. nr varians and Ewartia sp. nr oldfieldi (Distant); all tribe Cicadettini , subfamily Cicadettinae . Specimens of each taxon in the P. annulata species group were sourced from multiple localities, to include intraspecific variation where possible ( Table 2).
* Individuals labelled P. tremula ? were collected at light by A. Ewart and no calling song recording was taken. In morphology they match P. tremula in the distinctive shape of the male aedeagus and colouration of the body (see species description), but whether they represent true P. tremula or another as yet undescribed species, with a different calling song, remains uncertain.
Genomic DNA was extracted from either thoracic tissue or legs using a Qiagen DNeasy Blood & Tissue kit and stored at -20°C in the prescribed elution buffer. Oligonucleotide primers were used to amplify specific gene regions ( Table 3) by means of standard polymerase chain reaction (PCR) using Biotech® PCR master mix (CO1) or Invitrogen Platinum Taq DNA polymerase (dynamin). Dynamin was targeted specifically using an initial PCR with the primers (3006F1.1 and 3006R2.1) designed by Hardy (2007), followed by a nested PCR with a new internal forward primer (DYNCICF; designed in this study) and the same reverse primer ( Table 3). All PCR amplifications included a negative control to detect possible contamination. Amplified PCR products were visualised using 1–2% agarose gels, purified using ammonium acetate precipitation and stored in Milli-Q purified water.
All sequencing reactions were performed by Macrogen ( Korea). For CO1, most individuals were sequenced in both the forward and reverse directions (81%, n=94), to compare TAQ readings in initial sequences. The remaining individuals were sequenced only with the forward primer ( Table 3). For dynamin, sequences were obtained in both directions for all individuals to enable alignment on either side of a long repeat region in the amplified product. Three individuals were amplified and sequenced once with Hardy's (2007) primers (and Qiagen gel purification kit), and a second time using nested PCR as described above, to ensure that both procedures amplified a homologous copy of dynamin. Where sequencing reactions gave unexpected results, all steps, including DNA extraction and PCR were performed a second time to confirm the repeatability of each result.
Sequences were sourced only from individual cicadas that were collected during the course of this study, including all ingroups and outgroups. General sequence editing and alignment were performed manually using Geneious (©2005–2008 Biomatters Ltd). The two alignments included 646 base pairs (bp) of the CO1 gene and 769–1141 bp of the dynamin intron (variable among individuals and species, with a long repeat region). For dynamin, a number of individuals displayed a signal that was too weak when sequenced in the reverse direction (possibly through bad primer matching for those individuals) and the forward direction was too weak to obtain a complete reading through to the exon in sequences over 950 bp. Consequently, the ~100 bp exon (coding region) at the 3' end of the dynamin gene was not included in the alignment.
Individual sequences in alignments of both loci were examined for pseudogenes, to eliminate anomalous phylogenetic reconstruction ( Pamilo et al., 2007). CO1 sequences were translated into amino acids in Geneious to visually check for stop codons and anomalous rates of amino acid substitution. The nucleotide alignment was also subjected to tests of substitution pattern homogeneity with a disparity index test, implemented in MEGA 4.0 ( Tamura et al., 2007). Amplification of identical sequences using two different sets of primers gave an indication that the copies were homologous rather than paralogous.
All sequences were submitted to Genbank under the voucher codes given in Table 2.
Phylogenetic analysis methodology. To test the monophyly of each species in the P. annulata species complex and to explore divergence times within the group, the data were subjected to four analyses. These were (1) maximum likelihood reconstruction, (2) Bayesian inference with unconstrained branch lengths, (3) maximum parsimony, and (4) Bayesian inference with branch lengths constrained by a relaxed molecular clock. For the first, second and third analyses, each gene region was analysed independently, whereas in the fourth analysis, both gene regions were incorporated into a single analysis. Partition schemes and partition-specific substitution models for CO1, based on the three codon-position categories, as well as substitution model selection for the dynamin locus, were tested using the 'all' algorithm of the program PartitionFinder v1.1.1 ( Lanfear et al., 2012) and the BIC criterion, with the branch lengths of alternative partitions linked and with the software set to evaluate the full substitution model set.
Maximum likelihood reconstructions were performed in using RAxML via the CIPRES Gateway Version 3.3 ( Stamatakis et al., 2008), allowing a GTR + I + G model of substitutions for CO1 and a GTR + G substitution model for dynamin. For CO1, the alignment was partitioned by codon position to enable each position to be modelled independently (based on the scheme recommended in the PartitionFinder analysis output). Duplicate sequences were pruned from each alignment prior to running the analyses. Totals of 400 and 700 bootstrap replicates were performed for CO1 and dynamin respectively.
Bayesian inferences with unconstrained branch lengths were performed in MrBayes 3.2.1 ( Ronquist et al., 2012), with the same substitution models and partitioning specified for the maximum likelihood analysis. Chains were run for 12 million generations, sampling every 10000th tree, in two parallel runs to check for convergence. The analyses were terminated when the standard deviation of split frequencies plateaued at approximately 0.009 and 0.008 for CO1 and dynamin respectively. Convergence was assessed using the 'scale' and 'compare' plots in the AWTY Web Tool ( Nylander et al., 2008). For both CO1 and dynamin, the first 900 trees were discarded as burn-in, leaving 302 post burn-in trees. The remaining trees were used to create a majority rule consensus tree.
Maximum parsimony analysis was conducted using PAUP* 4.0 ( Swofford, 2002). This involved heuristic searches that comprised 1000 random addition starting trees, TBR branch swapping, and no maxtrees restrictions. These were followed by 100 bootstrap replicates (each consisting of 100 heuristic search replicates), with the same parameters described above, to assess node support. A weighting scheme for codon position (1st: 2nd: 3rd = 4: 4: 1) was used for the CO1 protein coding gene ( Kergoat et al., 2004). To explore patterns of haplotype divergence among species and subspecies of the P. annulata species complex in more detail, statistical parsimony networks were constructed for the two loci using TCS 1.2.1, under the default (95%) connection limit ( Clement et al., 2000 – 2005).
To estimate divergence times within the P. annulata species complex, a single clock analysis was performed with each locus treated separately. Bayesian inferences with branch lengths constrained by relaxed molecular clock were conducted using *BEAST ( Heled & Drummond 2010) in the BEAST 1.5.4 software package ( Drummond & Rambaut 2007), with lognormal rate variation and birth-death tree prior ( Gernhard, 2008). For CO1, each codon position was modelled independently and a substitution rate of 0.0115 substitutions per site per million years was applied, with ucld.stdev set to 0.3 and with the operations for ucld.mean and ucld.stdev deactivated, as recommended by Marshall et al. (2012). For dynamin, the rate was left equal to units of substitution/site, so that the rate prior for CO1 would have the greatest influence on divergence estimates. Chains were run for 40 million generations, sampling every 4,000th tree. The analysis was run six times, with the six runs assessed for convergence, examined for appropriate effective sample sizes, and combined using Tracer 1.6.1 ( Rambaut and Drummond, 2009). For each run, the first 2.5–95% of trees were discarded as burn-in, leaving 27193 post burn-in trees. The remaining trees were used to create a maximum clade credibility tree in TreeAnnotator (included in BEAST software). Tree editing was performed in FigTree 1.4.0 and TreeGraph 2.0.47-206 beta ( Stöver and Müller, 2010). It should be noted that *BEAST assumes a monophyletic origin for all species, regardless of how individuals would otherwise cluster in any given locus.
Assessment of phylogenetic relationships. Examination of amino acid composition and nucleotide substitution patterns (CO1) revealed no evidence of pseudogenes among the individual sequences. Statistics for the sequence data of both loci are provided in Table 4.
Phylogenetic reconstruction of the CO1 alignment ( Fig. 56 View FIGURE 56 ) provided strong support (BPP=1.00) for the monophyly of most species within the P. annulata species complex, including P. simplex , P. ganiticus , P. subtropica , P. torrensis , P. decora , P. kobongoides , P. corymbiae , P. inversa and P. ayrensis . The reconstruction based on the Dynamin gene ( Fig. 57 View FIGURE 57 ) also provided strong support (BPP=1.00) for the reciprocal monophyly of majority of these species (including P. simplex , P. ganiticus , P. decora , P. corymbiae , P. inversa and P. ayrensis ); however, as expected, this locus displays a considerably more conserved rate of evolution compared with CO1 and also fewer individuals were sequenced for comparison. Among these species, only one strongly supported sister relationships was evident, between P. subtropica and P. torrensis (BPP=1.00 in CO1).
In contrast, P. annulata , P. notialis (including P. n. notialis , P. n. incitata and P. n. notialis x incitata hybrids) and P. tremula emerged non-monophyletic in both loci ( Figs 56 View FIGURE 56 , 57 View FIGURE 57 ). For CO1, the clustering of P. tremula individuals was particularly extreme with clusters emerging among several crown groups, including one cluster (from Cooyar–Benarkin region in south-east Queensland) sister to P. corymbiae ( Fig. 56 View FIGURE 56 ). In dynamin, the clustering of P. tremula was restricted to a clade (BPP=0.82) also containing P. annulata and P. notialis and the individuals of P. tremula from Cooyar and Benarkin showed no such association with P. corymbiae ( Fig. 57 View FIGURE 57 ).
Statistical parsimony networks showing possible paths of nucleotide substitution for CO1 and dynamin loci in the P. annulata species complex are illustrated in Figure 58 View FIGURE 58 . The connectivity among individual sequences within the CO1 network was lower than that derived from the dynamin data because a greater degree of genetic divergence between crown groups was apparent in the former gene (compare Figs 56 View FIGURE 56 and 57 View FIGURE 57 ). In the largest CO1 network, the main shared haplotypes were positioned close to the estimated root of the crown group (estimated from the strict consensus tree in PAUP, not shown). Fewer shared haplotypes were present in the dynamin network, but most haplotypes from species that emerged non-monophyletic were positioned internally within the network ( Fig. 58 View FIGURE 58 ). Pauropsalta tremula and P. annulata occurred in multiple clusters (with the former grouped according to site) among individuals of the different species that emerged non-monophyletic, with both also represented in disconnected haplotype groups in CO1 ( Fig. 58 View FIGURE 58 ). In both loci, P. notialis x incitata clustered close to the estimated root of the tree ( Fig. 58 View FIGURE 58 ).
In general, the different species sampled from the same site do not cluster together in either of the networks ( Fig. 58 View FIGURE 58 ). Examples include (1) P. annulata , P. n. notialis and putative P. tremula from Mt Moffatt; (2) P. annulata , P. tremula and P. notialis x incitata hybrids from Cooyar; (3) P. decora and P. kobongoides from St George; (4) P. annulata , P. inversa and P. ayrensis from Eidsvold; (5) P. simplex and P. ayrensis from Herberton; and (6) P. inversa and P. ayrensis from Mundubbera. An exception is found in the CO1 network where P. annulata and P. n. incitata individual sequences from Eidsvold are clustered together, including one shared haplotype. This pattern is not reflected in dynamin, where the same individuals are clustered separately ( Fig. 58 View FIGURE 58 ).
An explanation for the lack of reciprocal monophyly between P. annulata , P. notialis and P. tremula requires consideration of two alternative scenarios, including (1) hybridisation, and (2) incomplete lineage sorting (an apparently common phenomenon; see Funk and Omland, 2003); or a combination of both. Lineage sorting occurs as a result of the loss of shared ancestral traits between two taxa, leading to reciprocal monophyly. Where retained ancestral polymorphisms occur, taxa tend to exhibit non-monophyly purely as a by-product of lineage sorting being incomplete ( Funk and Omland, 2003). Within the Pauropsalta annulata species complex, shared haplotypes at terminal branches might have indicated contemporary hybridisation, whereas the presence of internal haplotypes (which included most of those that are shared between species; Fig. 58 View FIGURE 58 ) strongly suggests that incomplete lineage sorting, rather than hybridisation, is the principal contributor to this pattern. Under such an interpretation, it remains acceptable to treat P. annulata , P. notialis and P. tremula as separate species, despite the apparent retention of shared ancestral genetic variation within each of them.
Nevertheless, the molecular data do also indicate that some instances of hybridisation occur or have occurred between some of these taxa. For instance, the terminal clustering of P. annulata and P. n. incitata individuals together at Eidsvold is suggestive of potential gene flow between these species ( Fig. 58 View FIGURE 58 ). However, the separate clustering of these same individuals in dynamin ( Fig. 58 View FIGURE 58 ) indicates that this may be the result of incidental or past hybridisation rather than introgression. Mitochondrial DNA is considered to be more sensitive for detecting hybrid events than nuclear DNA, because of maternal inheritance and lack of recombination obscuring the signal ( Funk and Omland, 2003). In the case of P. notialis , the hybrid individuals (P. n. notialis x incitata) cluster only in crown groups that also contain P. n. notialis or P. n. incitata individuals, as would be expected ( Figs 56 View FIGURE 56 , 57 View FIGURE 57 ). Haplotypes of the P. n. notialis x incitata hybrid individuals are clustered only around internal nodes ( Fig. 58 View FIGURE 58 ), which makes it difficult to distinguish among the alternatives of retained ancestral polymorphism and hybridisation based solely on the molecular data. In any case, these data do not provide an alternative to the interpretation from the morphological, ecological and calling song data, which indicates that these individuals are of hybrid origin.
Levels of genetic variation within and between species and subspecies of the P. annulata complex varied widely in the CO1 data. Genetic distances between species that were supported to be monophyletic ranged from 0.082 –0.174 substitutions/site. For species that were not supported to be monophyletic, levels of haplotype variation within species ranged from 0.027 substitutions/site in P. notialis , 0.065 substitutions/site in P. annulata , and 0.132 substitutions/site in P. tremula . This contrasted with the levels of haplotype variation in dynamin, which were found to be relatively uniform across all three of those species (0.017 substitutions/site in P. notialis , 0.016 substitutions/site in P. annulata , and 0.012 substitutions/site in P. tremula ). Also, in CO1 the large haplotype diversity in P. tremula was not reflected in amino acid translations, which were heavily conserved, with a maximum of two substitutions (across 215 sites) between individuals.
The case of such large mitochondrial divergence within P. tremula warrants further investigation, despite the same degree of variation not being present in dynamin. For example, no song recordings were available for the individuals labelled in Figures 56 View FIGURE 56 and 57 View FIGURE 57 as P. tremula ? from Mt Moffatt ( Table 2), so their allocation to this species must be considered tentative. However, for the remaining populations, no currently available ecological or morphological evidence indicates that what is recognised here as P. tremula is, in itself, a complex of species. Such genetic diversity could potentially occur in a single species as a result of a large gene pool being maintained through time, with populations varying in genetic structure across a landscape and partially isolated by distance. Alternatively, or additionally, the pattern could be suggestive of one or more introgression events in the history of this taxon.
Divergence time estimates. A chronogram showing divergence time estimates within the Pauropsalta annulata species group based on CO1 and dynamin loci is presented in Figure 59 View FIGURE 59 . The divergence time estimations produced here are entirely dependent on the assumed rate of molecular evolution (0.0115 substitutions per site per million years for CO1). Under this assumption, the phylogeny suggests that most of the extant diversity within this group emerged primarily as a result of a radiation that began in the mid Miocene between about 10 and 15 million years ago (mya). This estimate is significant because it occurs where a warm wet climate peaks prior to a period of gradual cooling and drying (from 10–15 mya) and later more rapid aridification (circa 8–10 mya) ( Byrne et al., 2008).
Other more recent divergences evidently occurred between P. subtropica and P. torrensis (somewhere between 5 million years ago and just prior to the present day) and within the common ancestor of P. annulata , P. notialis and P. tremula , which is estimated to have split quite recently (Pleistocene). The estimate in the later example is presumably due to the presence of shared haplotypes between these taxa.
Tavera, Department of Geology and Geophysics
Marine Science Museum, Tokai Univ.
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