The sex pheromone of the scarab beetle, Phyllophaga anxia, is a blend of the methyl esters of two amino acids, L-valine and L-isoleucine. A field trapping study was conducted, deploying different blends of the two compounds at 59 locations in the United States and Canada. More than 57,000 males of 61 Phyllophaga species (Coleoptera: Scarabaeidae: Melolonthinae) were captured and identified. Three major findings included: (1) widespread use of the two compounds [of the 147Phyllophaga (sensu stricto) species found in the United States and Canada, males of nearly 40% were captured]; (2) in most species intraspecific male response to the pheromone blends was stable between years and over geography; and (3) an unusual pheromone polymorphism was described from P. anxia. Populations at some locations were captured with L-valine methyl ester alone, whereas populations at other locations were captured with L-isoleucine methyl ester alone. At additional locations, the L-valine methyl ester-responding populations and the L-isoleucine methyl ester-responding populations were both present, producing a bimodal capture curve. In southeastern Massachusetts and in Rhode Island, in the United States, P. anxia males were captured with blends of L-valine methyl ester and L-isoleucine methyl ester.
La feromona sexual del escarabajo, Phyllophaga anxia, es una mezcla de los ésteres metílicos de dos aminoácidos, L-valina y L-isoleucina. Se condujo un estudio de campo usando diferentes mezclas de los dos componentes en 59 sitios de Estados Unidos y Canada. Más de 57,000 machos de 61 especies dePhyllophaga fueron capturados e identificados. Tres de los resultados más importantes incluyen: (1) el extenso uso de los dos componentes [de las 147 especies de Phyllophaga (sensu stricto), en Estados Unidos y Canada, fueron capturados machos de cerca del 40% de ellas.]; (2) para la mayoría de las especies, la respuesta intraespecífica de los machos a las combinaciones de los dos aminoácidos fue consistente entre años diferentes, y en todos los sitios geográficos; y (3) un inusual polymorfismo de la feromona fue descrito para P. anxia. Poblaciones de algunos sitios fueron atrapados sólo con valina, mientras que poblaciones de otros sitios fueron atrapados sólo con isoleucina. También se encontraron sitios donde las poblaciones responden a ambos componentes, valina e isoleucina, produciendo una curva de captura bimodal. En el sureste del estado de Massachusetts y en Rhode Island, en Estados Unidos, machos de P. anxia fueron atrapados en trampas con mezclas de valina e isoleucina.
Introduction
The scarab beetle genus Phyllophaga (sensu lato) is one of the largest genera of animals in the United States (Woodruff and Beck 1989), encompassing 203 described species in 8 subgenera, including 147 species (and 8 subspecies) in the subgenusPhyllophaga (sensu stricto), 39 species inListrochelus, 7 species in Phytalus, 3 species in Cnemarachis (all non-native and confined to south Florida), 3 species in Eugastra, 2 species in Tostegoptera, 1 species in Chlaenobia, and 1 species in Triodonyx (Evans 2003, Smith and Evans 2005). Their striking genitalic morphology, first described in the late 19th century (Smith 1888), continues to be the most important taxonomic character used to separate species in this group (Luginbill and Painter 1953). SeeWoodruff and Beck (1989) andWoodruff and Sanderson (2004) to view excellent scanning electron microscopy images ofPhyllophaga genitalia.
The economic importance of this genus relates principally to the root feeding habits of the larvae, commonly called white grubs. Larvae of various species ofPhyllophaga have been recorded feeding on crops that include, but are not limited to, nursery stock (Andre 1937), corn (Bessin 1999), commercial turfgrass (Brandenburg and Villani 1995; Vittum et al. 1999), cranberries (Dunn and Averill 1996; Eck 1990; Franklin 1950), sugarcane (Gordon and Anderson 1981), sweet potato (Hammond et al. 1997), and pasture (Luginbill and Painter 1953). True to the Greek origin of their generic name (Phyllo-leaf + phaga-eat), adult Phyllophaga in very large flights have been known to defoliate stands of trees. Although adults of some Phyllophaga species are apparently host specific, most are polyphagous (Luginbill and Painter 1953).
P. anxia (LeConte) is the most widely distributedPhyllophaga species in North America (Luginbill and Painter 1953; Woodruff and Beck 1989). Two genitalic morphs are described in this species, the northern form and the southern form (Luginbill and Painter 1953; Woodruff and Beck 1989). The first sex pheromone described from the genus Phyllophaga was identified from virgin P. anxia adults dug in mid-April from a cranberry bog in Carver, Massachusetts, before their May flight. The female-produced sex pheromone was determined to be a 75/25 blend of L-valine methyl ester and L-isoleucine methyl ester (Zhang et al. 1997). L-isoleucine methyl ester was first elucidated fromHolotrichia parallela (Leal et al. 1992), an Asian melolonthine species. Holotrichia is regarded by some as being inseparable taxonomically from the Nearctic Phyllophaga (Saylor 1937; Saylor 1939).
L-valine methyl ester and L-isoleucine methyl ester are unusual pheromone compounds in that their precursors are the essential amino acids, L-valine and L-isoleucine. These essential amino acids are available only via sequestration from food plants fed on by the larvae, or perhaps from endo-symbionts. Our future investigations will hopefully determine the source of these amino acids. For the most recent overview of beetle semiochemicals see Francke and Dettner (2005).
Since P. anxia is a common species throughout the Northeast, the pheromone was deployed in the field near Geneva, New York, in 1996. P. anxia males were captured with this blend, as expected. However, another species of Phyllophaga, P. futilis, was also captured in the traps in much smaller numbers. Our interest was piqued by theP. futilis catches because sex pheromones are generally regarded as species-specific mate recognition signals, although studies have demonstrated varying degrees of pheromone specificity between closely related species (Roelofs 1978), as well as pheromone polymorphism in geographically separated conspecifics such asAgrotis segetum (Wuet al. 1999), Ostrinia nubilalis (Glover et al. 1991; Klun and Cooperators 1975), and Hemileuca eglanterina (McElfresh and Millar 2001). Since P. anxia is a common species throughout most of North America, this finding presented an opportunity to examine the response specificity of different populations of P. anxia, as well as responses of otherPhyllophaga species, over a large geographic region.
Material and Methods
Vane traps baited with various blends of L-valine methyl ester and L-isoleucine methyl ester were deployed at 59 different locations in the U.S. and Canada during the years 1996–2001 (Figure 1). At each of these locations, traps were maintained for one to four seasons. Tables 1a and 1b list the trap locations, years during which traps were deployed, and a brief note about the habitat. The trapping sites in Carver, Lakeville, and Plympton, Massachusetts; Chatsworth, New Jersey; Babcock, Wisconsin; Lincolnville Center, Maine; Aggasiz, British Columbia; and Bandon, Oregon, were chosen because the traps could be located adjacent to cranberry acreage. Researchers involved in studies of Phyllophaga infesting turf, pasture, nursery, or other commodities maintained some trapping sites. Other sites were selected because it was likely that they might harbor differentPhyllophaga species from those in other geographic areas and where the lures had never been tested.
Table 1a
Table 1b
When blends are referred to in this study, it is in the ratio of L-valine methyl ester/L-isoleucine methyl ester. In 1996, five blends were deployed, including 100% L-valine methyl ester, 65/35, 50/50, 35/65, and 100 % L-isoleucine methyl ester. In 1997 and 1998, 95/5 and 5/95 blends were added to the array. In 1999, 2000, and 2001, the eight blends tested included 100% L-valine methyl ester, 90/10, 80/20, 60/40, 40/60, 20/80, 10/90, and 100% L-isoleucine methyl ester. In 1996, the lures were produced in our own laboratory by loading 5 mm rubber stopper septa (Thomas Scientific, www.thomassci.com/index.jsp) with 3 mg each of various blends using hexane as the solvent. From 1997 to 2001, Dr. A.C. Oehlschlager of ChemTica Internacional S.A. (San Jose, Costa Rica, www.chemtica.com) generously supplied the rubber septa lures for the tests. During that time, the lures were loaded with 4 mg of the various blends. At each location a control trap with a blank septum also was deployed.
In 1996 and 1997, the traps used in the study were either Trécé Japanese beetle vane traps (Trécé Incorporated, www.trece.com/) or Fuji Flavor Company vane traps (Fuji Flavor Company, www.fjf.co.jp/). Beginning in the 1998 season, vane traps were fabricated in the laboratory from three-liter soda bottles and 4 mm white corrugated plastic (Figure 2). When removed from the field during the winter, these traps lasted up to three field seasons.
Traps were set in the field 15–20 meters apart and at heights of 1–2 meters. The traps were checked and re-randomized one to three times each week. Captured beetles were bagged and frozen, or infrequently preserved in ethanol. Plastic bags or bottles marked with the catch date and blend were shipped at the completion of the trapping period to Geneva, NY, for identification.
Phyllophaga species identifications were assigned using a number of published sources (Luginbill and Painter 1953;Ratcliffe 1991;Riley 1988, Saylor 1939; Saylor 1940; Woodruff and Beck 1989), comparisons withPhyllophaga species in the Cornell University insect collection, and consultations with and verifications by E. Richard Hoebeke (Cornell University, Ithaca, NY), Dr. Paul Lago (University of Mississippi, University, MS), Edward C. Riley (Texas A & M University, College Station, TX), and William B. Warner (Farnam Companies, Inc., Phoenix, AZ). In 1998, Dr. Robert Crocker (then at Texas A & M University, Dallas, TX) did identifications of the Texas catch and sent the results to Geneva NY. In 2000 and 2001, Dr. Robert Bauernfiend (Kansas State University, Manhattan, KS) did the same for the Manhattan, Kansas catches. Whenever possible, a series of each species from the various locations was pinned for later vouchering in the Cornell University insect collection.
Results and Discussion
General observations
The following outline condenses the large number of figures and tables found in this publication into general groupings for the convenience of the reader.
A total of 57,129 Phyllophaga individuals were examined and identified in the course of this study. The 215 captured females represented only 0.38% of the total catch, suggesting that the attractants do not function as aggregation pheromones in any of the species. A total of 145 males were counted from the control traps, amounting to only 0.25% of the total catch.
No Phyllophaga beetles were captured in Aggasiz, British Columbia; Bandon, Oregon; or Homestead, Florida. In British Columbia and Oregon, the traps were located at cranberry bogs. A light trap operated at the Oregon cranberry bog site also captured no Phyllophaga. Despite extensive sampling for root weevils in northwest commercial cranberry bogs, no white grubs have been reported (D. C. Weber, unpublished data). Moreover, when the Phyllophaga species distribution maps in Luginbill and Painter (1953) are examined, only four species of Phyllophaga are found listed from Oregon. Regarding the Homestead, Florida site, Woodruff (1961) indicates that very few of the Florida Phyllophaga species occur in Miami or in the Florida Keys. In the same publication, he points out that “The soil in the Miami area is extremely shallow and underlain by öolitic limestone and, in general, is not a good soil for white grubs.” Only 4 of the 42 species of Phyllophaga recorded from Florida have been collected in that area.
Tables 2a and 2b list alphabetically thePhyllophaga species captured, the number of males captured in each species, the number of discrete locations at which they were captured, and the total number of site-years for each species [site-years = (site A x number of years that species was captured at that site) + (site B x number of years that species was captured at that site) + .......]. Tables 3a and 3b are sorted by descending catch numbers, beginning with thePhyllophaga species caught in the greatest number. Tables 4a and 4b are sorted by site, listing in descending order the number of sites at which a particular species was recorded.
Table 2a
Male catches, sites, and sites-years sorted by species.
Table 2b
Male catches, sites, and sites-years sorted by species.
Table 3a
Male catches, sites, and sites-years sorted by n.
Table 3b
Male catches, sites, and sites-years sorted by n.
Table 4a
Male catches, sites, and sites-years sorted by sites.
Table 4b
Male catches, sites, and sites-years sorted by sites.
Of all site-years when and where beetles were captured, only two (NJ Chatsworth #2, 1999 Table 59 and UT Salt Lake City, 1999, Table 91) recorded a single species of Phyllophaga during a flight season. The average number of species captured during a site-year was 4.15. Three sites (AL Auburn, 1998, Table 6; KS Manhattan #1, 2000, Table 27; and KS Manhattan #2, 2001,Table 28) recorded more than 10 species of Phyllophaga captured during the flight season.
Table 59
New Jersey, Chatsworth #2 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 91
Utah, Salt Lake City 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 6
Alabama, Auburn 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 27
Kansas, Manhattan #1 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 28
Kansas, Manhattan #2 2001
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
The species that was taken in the greatest number (20,480), in the greatest number of sites (33), and in the greatest number of site-years (54) wasP. anxia. This is not surprising given that, as was indicated previously, it is the most widespread species ofPhyllophaga in North America. Luginbill and Painter (1953) list it as occurring in every state in the United States except Arizona, California, Florida, Nevada, West Virginia, and Wyoming. They also report P. anxia from all ten Canadian provinces. Woodruff and Beck (1989) have subsequently reported it from Florida. W. B. Warner (personal communication) has examined a specimen of P. anxia from California and has reports ofP. anxia in Arizona.
Males of ten other Phyllophaga species were also captured in numbers >1000. These include P. futilis, P. congrua, P. crassissima, P. praetermissa, P. gracilis, P. hirtiventris, P. ephilida, P. rubiginosa, P. rugosa, and P. fusca. Males of 13 species were captured in numbers >100 but <1000 and males of 15 species were captured in numbers >10 but <100 (Tables 3a and 3b). Eighteen species ofPhyllophaga were recorded at ≥5 locations and 8 species were recorded at ≥11 locations (Tables 4a and 4b).
Geographical distributions and range extensions
Geographical distribution maps of Phyllophaga species captured during this study are found in Figures 3–63, arranged alphabetically by species. Shaded areas in these figures indicate the geographical ranges conveyed by Luginbill and Painter (1953) or Woodruff and Beck (1989) [P. (Phytalus)georgiana and P. (Phytalus)obsoleta only].
Most captures of Phyllophaga species recorded during this study were found within the geographical species distributions reported by Luginbill and Painter (1953). There are, however, several range extensions to report. The following species were found in locations in addition to those reported by Luginbill and Painter:P. curialis (Figure 14), P. drakei (Figure 17), P. forbesi (Figure 20), P. foxii (Figure 22),P. futilis (Figure 25), P. gracilis (Figure 29), P. gracilis var.angulata (Figure 30), P. hirtiventris (Figure 33), P. longispina (Figure 40), P. lota (Figure 41),P. marginalis (Figure 43), P. (fraterna)mississippiensis (Figure 46), P. postrema (Figure 50), P. praetermissa (Figure 51), P. quercus (Figure 53), and P. taxodii (Figure 59). Riley (1988) had previously noted range extensions of P. forbesi, P. quercus, andP. taxodii into Louisiana.
Changes in population levels from year to year within sites
Numbers of beetles of a particular species changed dramatically from year to year at the same trapping location. For example, traps were maintained at the Auburn, Alabama site during the 1997, 1998, and 1999 seasons for approximately the same time period each year. The numbers of P. gracilis captured declined from 1123 in 1997, to 710 in 1998, and to 98 in 1999 (Tables 5, 6, 7). In Lexington, Kentucky, from 1999 to 2000, the numbers of each of the six species captured more than doubled (Tables 29, 30).
Table 5
Alabama, Auburn 1997
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 7
Alabama, Auburn 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 29
Kentucky, Lexington 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 30
Kentucky, Lexington 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Factors affecting the size of the population include not only weather during the flight period, but soil moisture and tilth conditions suitable for oviposition, egg hatch, and growth of larvae during the previous year or years that it takes for development to adults. Quality and quantity of larval host plants, soil textural characteristics, and species’ preferences for soil types also factor into population size and distributions (Katovich et al. 1998). Changes in population size of other species at other trapping sites may be seen in Tables 5–97. Species captured at a particular site changed from year to year as well, indicating that multiyear studies will yield a more realistic picture of population sizes and species distributions than will a single year of captures.
Table 97
Wisconsin, Verona 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Male captures with sex attractants
The most important finding of the present study was the demonstration of the extensive use of the methyl esters of L-valine and L-isoleucine as sex attractants in the mate recognition systems of the Phyllophaga. In 56 discrete locations across the US and Canada, during 94 observation periods (some locations were trapped for multiple years), 61 species ofPhyllophaga were captured. The overwhelming majority (58) of these species are found in the Phyllophaga (sensu stricto) subgenus. Since there are 147 species in this subgenus in America north of Mexico (Evans 2003; Smith and Evans 2005), 39% of the species in this group were captured in traps during the course of this study.
Male trap captures are graphically illustrated in Figures 64–126. The figures are arranged alphabetically byPhyllophaga species. Within each species figure, graphs are arranged alphabetically by state or province abbreviation. The graphs demonstrate three general patterns of species-specific male responses to a particular blend or group of blends. The three general patterns are displayed inFigure 127. First, some species, such as P. vehemens, flew primarily to the 100/0 L-valine methyl ester/L-isoleucine methyl ester lure and were sensitive to increasing amounts of L-isoleucine methyl ester in the other blends, its presence significantly reducing captures1. P. congrua, however, had a broader response profile and was captured not only with the 100 % L-valine methyl ester lure, but also with blends containing 10%–20% L-isoleucine methyl ester (Figure 71). A similar situation was seen in responses of male P. hirtiventris (Figure 96). The second case (Figure 127) involved species such as P. forbesi that were captured primarily with the 100% L-isoleucine methyl ester lure. Increasing titers of L-valine methyl ester resulted in reduced or no captures2. In the third case (Figure 127), some species ofPhyllophaga, such as P. glabricula, required the presence of both compounds before captures occurred3. An examination of these male- response curves reveals that, whereas certain species had a rather broad response range to the L-valine methyl ester/L-isoleucine methyl ester blends,4 others exhibited response curves occupying a narrower range of blends5.
Specificity of response over time and space
A striking aspect of the intra-specific male flight responses to the sex attractants was their consistency between years and across geographic locations. Several species were recorded at only one site, but were captured at that site for two consecutive years6. The response profiles for each species in both years at those sites were nearly identical.
The majority of Phyllophaga species were recorded at more than one site (Tables 4a and 4b). As with the across-years comparison above, the intra-specific male-response curves from different geographic locations are similar as well. For instance, only five specimens ofP. balia were captured during this study, but they were captured at three different locations (Figure 6) and all with the 100% L-isoleucine methyl ester lure (Figure 68). Nearly 7000 P. congrua were captured at eight different sites (Figure 9) and all sites exhibited similar response curves (Figure 71). Comparable results are seen when other species are examined7.
Some species were captured at only one location and only during a single year, but multiple catches over time in the same or nearby blends furnish a series of independent observations that support responses to a particular lure despite the small numbers. For instance, six specimens of P. davisi Langston were captured in Marion Junction, Alabama in 1999, all in traps baited with the 100% L-isoleucine methyl ester lure (Figure 77) on six different dates between 4/5 and 4/28. P. davisi is a species that Luginbill and Painter (1953) indicate is rare, with “Only a few specimens seen.”
In Auburn, AL in 1998 two specimens of P. diffinis (Blanchard) were captured with the 100% L-valine methyl ester lure (Figure 78) - one taken on 4/16 and one taken on 4/21.
Another uncommon species, P. (Phytalus)georgiana Horn, was captured in Tifton, Georgia, in 2000. Two individuals of this species flew to the 100% L-valine methyl ester lure (Figure 89), one on 7/17 and one on 7/21. Woodruff and Beck (1989) report that no adult host plants are recorded, the larva is undescribed, and the life cycle is unknown. This species is one of seven North American species in the subgenus Phytalus. Members of this subgenus can be discriminated from the Phyllophaga (sensu stricto) by their cleft tarsal claws.
P. mariana is reported as a very rare species (Luginbill and Painter 1953). Four specimens were captured in Tifton, Georgia, in 2000 and 2001, on four different dates, in traps baited with the 90/10 or 80/20 L-valine methyl ester/L-isoleucine methyl ester blends (Figure 107).
Two specimens of P. taxodii Langston were captured in Louisiana in 1997 in the 35/65 L-valine methyl ester/L-isoleucine methyl ester blend (Figure 122), one on 7/3 and the other on 8/15. Luginbill and Painter (1953) list this species as uncommon, having been captured only in AL and MS. However, Riley (1988) reports that this species is not frequently taken at lights and that fair numbers have been captured in flight-intercept traps 50 feet above the ground in cypress stands. Riley concludes that a lack of light trap catches gives the impression of rarity but that the method of collection may be more important.
Similarly, R. J. Bauernfiend in Manhattan, Kansas indicated that in many years of light trapping he had never captured P. sylvatica, and was surprised to capture 104 individuals of this species during two years of sex attractant trapping (Figure 121).
Of some interest is the capture of both P.gracilis andP.gracilis variety angulata in traps baited with 100% L-isoleucine methyl ester (Figures 92 and 93). Woodruff and Beck (1989) indicate that “The exact status of this form (angulata) awaits further study”. The different forms of the male genitalia easily separate these populations ofP. gracilis. Photos of the two genitalic forms can be seen in Luginbill and Painter (1953). The two forms are sympatric over a large range (Figures 29 and 30). Langston (1927) provides illustrations of the genitalia indicating what appears to be a form intermediate between P. gracilis and P. gracilis varietyangulata.
Intra-location species interactions
The location tables (Tables 5–97) list the blends presented at each site in a particular trapping year and the numbers of Phyllophaga captured with each blend. At each site, the listed species are, by definition, sympatric. At some sites, sympatric species displayed asynchronous flight patterns, including species captured days, weeks, or in some cases, months apart. At other sites, different species ofPhyllophaga were synchronic as well as sympatric, but may or may not have been captured with the same sex attractant blends. The distinction of whether males of different species were or were not captured in the same blends is important because it can aid in identifying locations where inter-specific competition for pheromonal space may be occurring. Congeneric males flying to the same sex attractant blends indicate situations where there is potential for inter-specific mating interactions that afford opportunities for investigation into reproductive isolation involving additional species-specific sex attractant compounds, close-range mating behavior, and/or mating at different times.
Each of the three scenarios (1. sympatric and synchronically flying species captured using different blends; 2. sympatric and asynchronically flying species captured using the same blend; 3. sympatric and synchronically flying species captured using the same blend) were encountered in the course of this study, individually as well as in combination at different study sites. The following examples illustrate the three scenarios.
- In Lexington, Kentucky, in 1999, both P. futilis andP. rugosa flew synchronically between 5/10 and 7/7, but were captured in traps baited with different blends of L-valine methyl ester/L-isoleucine methyl ester (Figure 128).
- In Lincoln, Nebraska, in 1999, both P. vehemens andP. crenulata were captured in the trap baited with the 100% L-valine methyl ester, but their flight periods were separated by 18 days during which no males of either species were captured. P. vehemens flew from 5/16 through 5/25, whereas P. crenulata flew from 6/12 through 7/5 (Figure 129).
- In Amherst, Massachusetts, in 1999, both P. anxia andP. longispina flew to the 100% L-isoleucine methyl ester lure during the period between 5/18 and 6/15 (Figure 130). This latter scenario is the most engaging because it is in this case that the potential for conflict in terms of pheromonal space arises.
From the male capture data at the various trapping sites, many examples of different species flying synchronically, sympatrically, and to the same blends or blend groupings have been documented. However, nothing is known about whether these species fly at different times of the night or how close-range courtship behaviors are involved in mate recognition. Figures 131–133 display detailed information relating male response specificity curves and time of flight from three sites that demonstrate the complexity of interactions that can occur over a season. Figure 131 (KS Manhattan #2, 2001), demonstrates that P. inversa andP. vehemens flew synchronically to the 100% L-valine methyl ester lure. Similarly, there is potential for interaction between P. rubiginosa and P. sylvatica (middle L-valine methyl ester/L-isoleucine methyl ester blends), P. crassissima and P. fusca (lower L-valine methyl ester/L-isoleucine methyl ester blends), and P. futilis andP. bipartitia (100% L-isoleucine methyl ester lure). In the July and August flights at the same site, P. glabricula, P. affabilis, and P. ephilida were captured synchronically, but with different blends. Figure 132 (KY Lexington, 2000) shows that P. futilis and P. hirticula flew synchronically to the 100% L-isoleucine methyl ester lure, whereasP. ephilida was captured with the same lure, but later in the season. In Figure 133 (MA Amherst, 1999), synchronic species P. forsteri, P. fraterna, and P. fusca displayed overlapping male response curves to L-valine methyl ester/L-isoleucine methyl ester mixes, whereas P. anxia, P. longispina, andP. drakei also flew synchronically to the 100% L-isoleucine methyl ester lure.
Table 98 outlines, in a briefer format, other trapping locations and the species involved where inter-specific attraction might occur as a result of competition for pheromonal space. However, the possibility of interactions postulated by the overlap of the male response curves in time and space may not necessarily reflect the reality in the field in that females may or may not have a narrower range of sex pheromone blend production than is suggested by what the males are capable of responding to. The potential for inter-specific interactions might be predicted with greater accuracy by combining analysis of blend ratios produced by a number of individual females with knowledge of male captures by various blends and determining if male response curves overlap congeneric female production curves.
Table 98a
Synchronic species captured by the same or nearby sex attractant blends, by state.
Interspecific copulation between Phyllophaga species has been reported in the literature. Fattig (1944) reports (p. 26) that he collected a male P. hirticula copulating with a female P. anxia. Referencing the data from the present study, P. hirticula males were captured in traps baited with the 100% L-isoleucine methyl ester lure (Figure 95).P. anxia males of both the northern and the southern genitalic form were also captured in traps baited with the 100% L-isoleucine methyl ester lure (Figures 66a, 66b, 66c, 66d, 66e and 67). It is likely that the male P. hirticula flew upwind following an L-isoleucine methyl ester plume to find not a conspecific female P. hirticula, but a congeneric female P. anxia. P. hirticula and P. anxia males possess genitalia whose cuticular structures are exceedingly different (see images in Woodruff and Beck, 1989). Their soft sac structures are very different as well (P.S. Robbins, personal observation). Their vestitures also differ, P. anxia being glabrous and P. hirticula being hirsute. Although interspecific genitalic differences could and sometimes do play a role in reproductive isolation of some taxa (Eberhard 1985, Sota and Kubota 1998), genitalic differences clearly did not prevent copulation from taking place in this case.
Copulation does not inevitably lead to fertilization or production of offspring (Eberhard 1996), and similarly, attraction to a congeneric Phyllophaga female does not inevitably lead to copulation. In a study from Costa Rica, Eberhard (1993), reporting on the copulatory behavior of several species of Phyllophaga, states of P. vicina, that “Early in the evening solitary females rested immobile and apparently emitted an attractant, as males arrived in flight from downwind. The pheromone apparently also attracted males of P. valeriana, as on three occasions I saw one or more males of this species hover near a female P. vicina (beetles were captured to verify their species identity). One P. valeriana male landed on the female, then immediately took flight and left, suggesting that a second cue, possibly on the beetle’s surface, was used to distinguish species identity. Contact pheromones may be used in the melolonthine genus Macrodactylus (Eberhard 1992).” These observations indicate that more studies are needed to clarify the role of morphology and/or contact pheromones in close-range mate recognition in Phyllophaga. Eberhard (1993) reports that “secondary sexual modifications of the sculpturing of the front legs, the ventral bristles, and the overall leg length of maleMacrodactylus may function as courtship devices prior to and during copulation”. Although the Phyllophaga are renowned for their extravagant and often asymmetric genitalic morphology, male hind tibial spurs also often assume unique configurations that provide excellent taxonomic characters for species assignments (Luginbill and Painter 1953; Woodruff and Beck 1989; Woodruff and Sanderson 2004). The role tibial spurs play in close-range mate recognition or copulatory courtship in the Phyllophaga is unclear. Males of somePhyllophaga species also have extensive ventral bristles or possess unique morphological characters on the venter, similar toMacrodactylus (P.S. Robbins, personal observation).
Intraspecific variation in male response: P. anxia
The consistency of intra-specific male responses over space and time that was discussed earlier contrasts with the extensive variation noted in the male-response curves of P. anxia to the various blends of L-valine methyl ester/L-isoleucine methyl ester sex attractants (Figures 66a, 66b, 66c, 66d, and 66e). The variations demonstrated in the male-response profiles ofP. anxia are of four general forms:
- Those profiles from locations where the males flew primarily to L-valine methyl ester alone (Figure 134 and Figures 66a, 66b, 66c, 66d, and 66e including CT Vernon, 1999; NY Bellona, 1999 and 2000; NY Saratoga Springs, 1999 and 2000; NY Warwick, 1999 and 2000; NY Waterloo #1, 1998 NY Waterloo #1, 1999 and 2000; NY Waterloo #2, 1998; ON Woodlawn, 1999 and 2000; PA State College, 1999 and 2000, WI Babcock, 1999 and 2000)
- Those profiles from locations where the males primarily flew to L-isoleucine methyl ester alone (Figure 135 and Figures 66a, 66b, 66c, 66d, and 66e including MA Amherst, 1999 and 2000; NH Madbury, 1999 and 2000).
- Those profiles from locations where males flew to L-valine methyl ester alone or L-isoleucine methyl ester alone on the same night in the same place (Figure 136 andFigures 66a, 66b, 66c, 66d, and 66e, ME Lincolnville Center, 1999 and 2000; NY Franklinville, 1999; VT Burlington, 1999 and 2000).
- Those profiles from locations where the males were mainly captured in traps baited with blends of L-valine methyl ester and L-isoleucine methyl ester (Figure 137 and Figures 66a, 66b, 66c, 66d, and 66e, MA Carver, 1996, Figure 137 and Figures 66a, 66b, 66c, 66d, and 66e, MA Carver, 1998, Figure 137 and Figures 66a, 66b, 66c, 66d, and 66e, MA Carver, 1999 and 2000; MA Lakeville, 1999 and 2000; MA Plympton #1, 1996; MA Plympton #2, 1996; RI Kingston, 1999 and 2000).
All the P. anxia males from Figures 134 to 137, as well as all other P. anxia males in Figures 66a, 66b, 66c, 66d, and 66e are of the northern genitalic form (Luginbill and Painter 1953;Woodruff and Beck 1989). Male P. anxia of the southern genitalic form (Luginbill and Painter 1953;Woodruff and Beck 1989) were captured exclusively with L-isoleucine methyl ester alone (Figure 67). They were captured in both a smaller number of locations (8 vs.25) and in much smaller numbers (20 vs. 20,640) (Table 2a) thanP. anxia males of the northern genitalic form. Unpublished data from studies conducted at the Franklinville, NY, site suggest that L-isoleucine methyl ester responding P. anxia males (both northern and southern genitalic forms) are more sensitive to the presence of L-valine methyl ester than are L-valine methyl ester responding males to the presence of L-isoleucine methyl ester. Male captures in the L-isoleucine methyl ester-baited traps may have been suppressed by the presence of the L-valine methyl ester at the trap sites.
The manner in which the different forms of the P. anxia male sex pheromone response profiles are distributed across North America reveals important information. The five locations yielding response profiles (10 site-years) from P. anxia males that were captured in blends of both L-valine methyl ester and L-isoleucine methyl ester are found only in southeast Massachusetts and Rhode Island (see Figures 66a, 66b, 66c, 66d, and 66e including all the MA Carver, MA Lakeville, MA Plympton #1, MA Plympton #2 and RI Kingston sites). Surrounding these five locations are trapping sites to the west (as far west as Wisconsin) and to the north (as far north as the provinces of Nova Scotia and Ontario) that represent those populations of P. anxia males that responded to only L-valine methyl ester or to only L-isoleucine methyl ester, but did not require a blend of the two for capture (Figure 138). The male response curves generated by the beetle captures at those trapping sites yield a distribution map that is patchy in terms of unequal distributions of the two populations. Some sites harbor only one of the two populations, while other sites hold both, thus generating a bimodal distribution curve.
A detailed examination (including the soft sacs) of male individuals of the L-valine methyl ester responding populations, the L-isoleucine methyl ester responding populations, and the blend responding populations of P. anxia revealed no character that could be used to differentiate the populations. Further work is planned that will use DNA sequence data from both mitochondrial and nuclear genes to generate gene genealogies with which to document genetic relationships within and among the three races across the US. Microsatellite markers will also be used to characterize allele frequencies in natural populations and consequently estimate the extent of gene exchange among different pheromone races where they occur together and between geographically isolated populations of single pheromone races.
Intra-specific variation in male response: P. fraterna
A second instance of intra-specific variation in male response to blends presented was noted at the State College, Pennsylvania, trapping site in 1999 and 2000. Male individuals of a species determined as P. fraterna were captured by two blend groupings in both years (Tables 80 and 81 and Figures 85 a, 85b, and 86). In 1999, 18 males were captured with the 0/100 L-valine methyl ester/L-isoleucine methyl ester blend, whereas 13 males were captured with the L-valine methyl ester/L-isoleucine methyl ester mixtures. In 2000, 19 males were captured with the 0/100 L-valine methyl ester/L-isoleucine methyl ester blend whereas 5 males were captured with the L-valine methyl ester/L-isoleucine methyl ester mixtures. Phyllophaga fraterna from locations other than Pennsylvania were captured exclusively in the blends of L-valine methyl ester/L-isoleucine methyl ester (Figures 85a and 85b).
Table 80
Pennsylvania, State College 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 81
Pennsylvania, State College 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Summary and conclusions
This study demonstrates the extensive use of the methyl esters of L-valine and L-isoleucine in the mate recognition systems of a widely distributed and speciose taxon. Since each trapping site is a snapshot of activity over a very restricted area, it is compelling that nearly 40% of thePhyllophaga (sensu stricto) species in America north of Mexico were captured, despite all the possible locations that were not trapped.
Consistency in male response among geographically separated populations of conspecifics was demonstrated in numerous examples. This was useful to document in itself because it provided information concerning sex attractant use in a taxon that had never before been investigated in this fashion. However, this information also served, perhaps more importantly, to contrast and highlight the unusual geographic variation in male response to sex attractants between various populations ofP. anxia and in a more minor way, P. fraterna.
This study also documents the interspecific interactions betweenPhyllophaga species, through both literature citations and capture results. These interactions may be as benign as attraction to congeneric females (Eberhard 1993), with presumably little or no accompanying loss of fitness, or may involve copulation and perhaps exchange of genetic material (Fattig 1944), resulting in a more expensive or even fatal conclusion. Interactions between species are of great interest because of their connection to both species concepts and hybridization studies (Arnold 1997, Claridge et al. 1997, Rand and Harrison 1989).
Since this trapping study was terminated in 2001, additional research on sex pheromones of the Phyllophaga has been accomplished that extends the understanding of mate finding in this large genus. This research will have bearing on the phylogenetic relationships of this group as well.
The sex pheromone of Phyllophaga crinita was identified as methyl 2-(methylthio)benzoate (Robbinset al. 2003). Interestingly, Coca-Abia (2002) has recently resurrected the genusTrichesthes and removed P. crinita as well as a number of other species from the Phyllophaga (sensu stricto) to Trichesthes. Among the species moved toTrichesthes were P. lenis and P. tristis, two species that have also been captured in large numbers using methyl 2-(methylthio)benzoate (P.S. Robbins, unpublished data).
The sex pheromone of Phyllophaga (Tostegoptera)lanceolata was identified as L-leucine methyl ester (Nojima et al. 2003). Tostegoptera is a small sub-genus of the Phyllophaga consisting of only two species, Phyllophaga (Tostegoptera) lanceolata andPhyllophaga (Tostegoptera)squamipilosa. Both species have been captured using L-leucine methyl ester and field tests have demonstrated that L-valine and L-isoleucine methyl esters function as antagonists to Phyllophaga (Tostegoptera) lanceolata males.
Table 10
Canada, Nova Scotia, Kentville Center 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 11
Canada, Nova Scotia, Kentville Center 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 12
Canada, Ontario, Woodlawn 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 13
Canada, Ontario, Woodlawn 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 14
Connecticut, Vernon 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 15
Connecticut, Vernon 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 16
Florida, Holiday 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 17
Georgia, Athens 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 18
Georgia, Athens 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 19
Georgia, Griffin 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 20
Georgia, Tifton 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 21
Georgia, Tifton 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 22
Iowa, Ames, East Reactor Woods 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 23
Iowa, Ames, East Reactor Woods 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 24
Indiana, West Lafayette 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 25
Kansas, Greensburg #1 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 26
Kansas, Greensburg #2 2001
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 31
Louisiana, Chase 1997
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 32
Massachusetts, South Amherst 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 33
Massachusetts, South Amherst 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 34
Massachusetts, Carver 1996
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 35
Massachusetts, Carver 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 36
Massachusetts, Carver 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 37
Massachusetts, Carver 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 38
Massachusetts, Lakeville 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 39
Massachusetts, Lakeville 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 40
Massachusetts, Plympton #1 1996
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 41
Massachusetts, Plympton #2 1996
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 42
Maine, Lincolnville Center 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 43
Maine, Lincolnville Center 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 44
Minnesota, St. Paul #1 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 45
Minnesota, St. Paul #1 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 46
Minnesota, St. Paul #2 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 47
Mississippi, Leroy Percy State Park 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 48
Mississippi, Sharkey County, 5 miles SE of Anguilla 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 49
Mississippi, Stoneville 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 50
Nebraska, Lincoln 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 51
Nebraska, Lincoln 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 52
Nebraska, Lincoln 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 53
New Hampshire, Madbury 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 54
New Hampshire, Madbury 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 55
New Jersey, Chatsworth #1 1996
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 56
New Jersey, Chatsworth #1 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 57
New Jersey, Chatsworth #1 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 58
New Jersey, Chatsworth #2 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 60
New Jersey, Chatsworth #2 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 61
New Jersey, Hammonton 1996
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 62
New Jersey, New Brunswick 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 63
New York, Bellona 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 64
New York, Bellona 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 65
New York, Bellona 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 66
New York, Franklinville 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 67
New York, Riverhead 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 68
New York, Saratoga Springs 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 69
New York, Saratoga Springs 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 70
New York, Warwick 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 71
New York, Warwick 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 72
New York, Waterloo #1 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 73
New York, Waterloo #1 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 74
New York, Waterloo #1 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 75
New York, Waterloo #2 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 76
North Carolina, Raleigh #1 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 77
North Carolina, Raleigh #2 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 78
Ohio, Columbus 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 79
Ohio, Wooster 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 8
Alabama, Marion Junction 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 82
Rhode Island, Kingston 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 83
Rhode Island, Kingston 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 84
South Carolina, Anderson County 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 85
Texas, Dallas #1 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 86
Texas, Dallas #1 1999
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 87
Texas, Dallas #1 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 88
Texas, Dallas #2 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 89
Texas, Dallas #3 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 9
Alabama, Marion Junction 2000
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 90
Texas, Dallas #4 1998
Blends indicate the ratio of the methyl esters of L-valine/L-isoleucine
Table 98b
Synchronic species captured by the same or nearby sex attractant blends, by state.
Acknowledgements
This study would not have been accomplished without the participation of my co-authors. I am deeply grateful for their persistence and attention to detail. Donna Boyce, Joe Ogrodnick, and Rob Way of Communication Services here at the New York State Agricultural Experiment Station in Geneva provided the artistic guidance and technical input to produce the excellent figures seen in this publication. Thanks go to Nancy Consolie, my lab partner in the Villani soil insect lab at Cornell University for more than 13 years. Her research suggestions and invariable good humor added to this project. Marta Luisa del Campo, Anuar Morales, and Rebecca Smyth of Cornell University and Miguel Ángel Morón of the Instituto de Ecologia in Xalapa, Mexico, all contributed to making the Spanish abstract clear and understandable. My dissertation committee at Cornell University, consisting of Drs. Cole Gilbert, Rick Harrison, Charlie Linn, Wendell Roelofs, and Mike Villani, were patient and encouraging during this long study. A special note of thanks to Mike Villani who did not live to see this work published. Thanks, Mike, for encouraging me to follow the muses.
References
Notes
[1] Cite this paper as:
Robbins PS, Alm SR, Armstrong CD, Averill AL, Baker TC, Bauernfiend RJ, Baxendale FP, Braman SK, Brandenburg RL, Cash DB, Couch GJ, Cowles RS, Crocker RL, DeLamar ZD, Dittl TG, Fitzpatrick SM, Flanders KL, Forgatsch T, Gibb TJ, Gill BD, Gilrein DO, Gorsuch CS, Hammond AM, Hastings PD, Held DW, Heller PR, Hiskes RT, Holliman JL, Hudson WG, Klein MG, Krischik VL, Lee DJ, Linn Jr. CE, Luce NJ, MacKenzie KE, Mannion CM, Polavarapu S, Potter DA, Roelofs WL, Royals BM, Salsbury GA, Schiff NM, Shetlar DJ, Skinner M, Sparks BL, Sutschek JA, Sutschek TP, Swier SR, Sylvia MM, Vickers NJ, Vittum PJ, Weidman RB, Weber DC, Williamson RC, Villani MG. 2006. Trapping Phyllophaga spp. (Coleoptera: Scarabaeidae: Melolonthinae) with sex attractants in the United States and Canada. XXXpp. Journal of Insect Science X: XX, available online: insectscience.org/X.XX.
[2] see Figure 99, P. inversa; Figure 102, P. latifrons; Figure 106, P. marginalis;Figure 126, P. vehemens
[3] see Figure 68, P. balia; Figure 69,P. bipartita; Figure 74, P. crenulata; Figures 79a and 79b, P. drakei;Figure 82, P. forbesi; Figures 88a, 88b, 88c, and 88d, P. futilis; Figure 92, P. gracilis; Fig. 94, P. hirsuta; Figure 95,P. hirticula; Figure 103, P. longispina; Figure 113, P. postrema; Figure 124, P. ulkei
[4] see Figure 64, P. aemula; Figure 65,P. affabilis; Figure 72, P. corrosa; Figures 73a and 73b, P. crassissima; Figures 83a, 83b, and 83c, P. forsteri; Figures 85a and 85b, P. fraterna; Figures 87a, 87b, 87c, and 87d, P. fusca; Figure 90, P. glaberrima; Figure 91, P. glabricula; Figure 104, P. lota; Figure 107, P. mariana; Figure 108, P. micans; Figure 112, P. perlonga; Figure 114,P. praetermissa; Figure 116, P. quercus; Figure 117, P. rubiginosa;Figures 118a and 118b,P. rugosa; Figure 121, P. sylvatica; Figure 123, P. torta; Figure 125, P. uniformis
[5] see Figures 73a and 73b,P. crassissima; Figures 87a, 87b, 87c, and 87d, P. fusca; Figure 117, P. rubiginosa
[6] see Figure 64, P. aemula; Figure 65,P. affabilis; Figures 83a, 83b, and 83c, P. forsteri; Figures 85a and 85b, P. fraterna; Figures 118a and 118b, P. rugosa; Figure 125, P. uniformis
[7] see Figure 64, P. aemula; Figure 65,P. affabilis; Figure 69, P. bipartita; Figure 72, P. corrosa; Figure 91, P. glabricula; Figure 107, P. mariana; and Figure 125, P. uniformis
[8] see Figures 11, 73a and 73b, P. crassissima; Figures 12 and 74, P. crenulata; Figures 17, 79a and 79b,P. drakei; Figures 20 and 82,P. forbesi; Figures 21, 83a, 83b, and 83c, P. forsteri; Figures 23, 85a and 85b, P. fraterna; Figures 24, 87a, 87b, 87c, and 87d, P. fusca; Figures 25,88a, 88b, 88c, and 88d,P. futilis; Figures 27 and 90,P. glaberrima; Figures 29 and 92,P. gracilis; Figures 31 and 94,P. hirsuta; Figures 32 and 95,P. hirticula; Figures 33 and 96,P. hirtiventris; Figures 36 and 99,P. inversa; Figures 40 and 103, P. longispina; Figures 41 and 104, P. lota; Figures 43 and 106, P. marginalis; Figures 45 and 108, P. micans; Figures 47 and110, P. nitida; Figures 49 and112, P. perlonga; Figures 50 and 113, P. postrema; Figures 51 and 114, P. praetermissa; Figures 53 and 116, P. quercus; Figures 54 and 117, P. rubiginosa; Figures 55, 118a and 118b,P. rugosa; Figures 58 and 121, P. sylvatica; Figures 60 and 123, P. torta; Figures 61 and 124, P. ulkei; and Figures 63 and 126, P. vehemens