The Paper Wasps and Hornets of Florida (Hymenoptera: Vespidae: Polistinae & Vespinae) By Hugo Kons Jr. & Rex Rowan

APPENDIX E: Use of Phallus Characters to Separate Polistes (Fuscopolistes) Species with Similar or Indistinguishable Patterns

Version 2018.1

This Appendix shows males side by side of species that are similar or indistinguishable in pattern, along with the phallus of each species in the orientation(s) that will enable species separation.

Many males of southeastern Polistes (Fuscopolistes) species are so similar in pattern to other species that identifications based on pattern alone are not reliable. However, all Fuscopolistes males can be sorted into three categories based on three characteristics of the phallus, and these characters can be examined under a microscope simply by pulling the genitalic capsule out of the apex of the metasoma, without needing any dissection or KOH treatment. Fortunately, most of the species with similar pattern have a different type of phallus from the species they are similar to, and some of the exceptions can be distinguished by other structural characters. Buck et al. (2012) used all three of these characters in their "Preliminary key to eastern Nearctic species of Polistes (Fuscopolistes) based on male genitalia" (page 26). Furthermore, after examining over 700 males collected on nests for these characters, HLK found the form of each of these three characters is always uniform among nest mates.

Extracting the male genitalia capsule: For specimens that have been freshly collected or stored in 70% alcohol, the male genitalic capsule can be removed from the metasomal apex by reaching in with a fine forceps and gently pulling it out by the apex of the phallus. For specimens in 99% alcohol or specimens that have dried out, the apex of the metasoma needs to be softened with water. This is achieved by dipping the metasomal apex into water and holding it there for about two minutes, after which time the capsule can be extracted as indicated above. After the capsule has been extracted, dip the portion of the metasoma that was exposed to water in 99% IsOH or ethanol for about a minute, and then allow it to dry before returning the specimen to a tight box or drawer. This will remove any water that could lead to mould, rot, or discoloration.

Unless one plans to do a KOH treated dissection, it is best to leave the male genitalia capsule attached to the specimen. Once the capsule is extracted from the metasoma there is still soft tissue connecting it to the metasoma, which will harden and dry after a few hours. If the capsule is detached, it can be stored in a vial of alcohol with a unique code number matching the same code number pinned under the specimen.

If the characters of the phallus are obscured by the parameres, brace the dorsal side of the parameres with one forceps and gently pull the apex of the phallus up (dorsally) with another. Once the phallus is in a good position it can be held there with forceps for several minutes, after which time it usually retains this position long enough to study or photograph the phallus characters.

The three types of Fuscopolistes male genitalia based on three characters of the phallus: The three characters to examine on the phallus include (1) the stoutness of the apical portion of the phallus posterior to the edge of the median process, (2) if the teeth on the sides of the posterior portion of the phallus extend nearly to the apex of the median process or terminate distinctly basal of this apex, and (3) the size of the teeth on the phallus. The differences are shown in Figure C4 below.

Figure C4: Three types of male genitalia that occur in Fuscopolistes, based on three characters of the phallus.

1. Stoutness of the apical portion of the phallus: We define the stoutness of the apical portion of the phallus as the ratio between the red and yellow lines shown in Figure E1 (below), although precise measurements are only possible if the phallus is dissected out of the capsule and placed in a fixed position where it is perfectly flat. This is the case for the ventral phallus images from KOH treated dissections in Appendix C, which were taken with the phallus flat against the bottom of a petri dish and held in place by a piece of glass. Any tilt to the phallus will make it appear stouter than it really is, and it is difficult to view or photograph the phallus without some tilt when it is still attached to the capsule. However, the difference between species with a Type 1 phallus versus one with a Type 2 or 3 phallus is sufficiently large that even with some tilt the differences should be visually clear from comparisons with the photos shown in this Appendix. When the phallus is still attached to the capsule, the dorsal side is examined for this character (as in Figure E1 below) because the ventral side tends to be obscured by the volsella and membranous tissue.

Figure E1: Comparing the stoutness of the apical portion of the phallus between Type 1 (left) and Type 2 or 3 (right) genitalia.

2. Extent of the phallus teeth on the median process. In figure E2 below (and subsequent figures) the red arrow indicates the apex of the median process, whereas the yellow arrow indicates the anterior extent of the teeth on the median process. In species with Type 3 genitalia, the teeth extend nearly to or all the way to the apex of the median process, as shown by the bottom image in Figure E2 below. For species with Type 1 (top image) or Type 2 (middle image) genitalia there is a distinctly larger gap between the anterior extent of the teeth and the apex of the median process.

Figure E2: Phallus apex in lateral aspect for Fuscopolistes species with Type 1 (top), Type 2 (middle), and Type 3 (bottom) genitalia.

3. Relative size of the phallus teeth: Species with Type 3 genitalia have distinctly larger teeth on the phallus relative to species with Type 1 or Type 2 genitalia, as shown in Figure E2 above.

The following images of Fuscopolistes males are pairs of species which are similar or indistinguishable in pattern, but which can be separated by characters of the phallus shown with the specimens. While we discuss tendencies in size or pattern that may give insight into separating these pairs of species, the phallus (and/or other structural characters mentioned) should be examined for reliable identifications between these pairs.

Note many Polistes are sexually dimorphic. The below discussions of pattern characters may only apply to males. Sexual dimorphism and female characters are covered in the species accounts.

Figure E3: Polistes bellicosus versus Polistes fuscatus Species 2.

Males of both species have similar color patterns and we found no way to separate them from pattern. Males of each species may have either a predominately ferruginous or predominately black mesopleuron, as documented from nest mates. Polistes bellicosus often has the dorsum of the apical flagellomere orange whereas it is often black in P. fuscatus species 2, but this character is not reliable as either species may have the apical flagellomeres orange or black. Males of either species may have a pair of yellow dots on tergum III; however, the majority of P. bellicosus lack these dots whereas the majority of P. fuscatus species 2 have them. Buck et al. (2012) state they examined two specimens with a bellicosus pattern that had a stout aedeagus, but do not suggest these specimens had the characteristics of the fuscatus species group (i.e. Type 3 genitalia). Thus, there may be another species we did not examine with Type 2 genitalia and a similar pattern to P. bellicosus and P. fuscatus species 2.

Figure E4: Polistes bellicosus versus Polistes fuscatus species 2.

See comments under Figure E3 (above).

Figure E5: Polistes hirsuticornis (or species near) versus Polistes fuscatus species 2.

We have only examined one Florida male of Polistes hirsuticornis, so we do not know the range of pattern variation, but the pattern of the one known specimen is very similar to Polistes fuscatus species 2 and P. bellicosus. Polistes fuscatus species 2 can be distinguished by having Type 3 genitalia, but both P. bellicosus and P. hirsuticornis have Type 1. However, males Polistes hirsuticornis can be distinguished from all other species by the abruptly narrowing propodeal orifice, as shown in Appendix G.

Figure E6: Polistes dorsalis versus Polistes bellicosus.

Both of these species have Type 1 genitalia, but the apex of the phallus is more slender in P. dorsalis than P. bellicosus. In addition, P. dorsalis differs from all other Fuscopolistes by having a notch in the antereoventral lobe of the digitus (Figure C29). Furthermore, P. dorsalis has a more prominent ventral turbercle on sternum VIII+IX relative to P. bellicosus (Figure C 56; P. carolina species 1 is shown in this figure, which has a small ventral turbercle similar to P. bellicosus). One of these structural characters should be examined to reliably separate these two species. While P. dorsalis averages as the smallest Fuscopolistes, runt P. bellicosus are the typical size of P. dorsalis and the largest P. dorsalis are the size of a typical P. bellicosus. Males of P. dorsalis apparently always have the mesopleuron predominately black, whereas in P. bellicosus it may be predominately orange, black, or intermediate.

Figure E7: Polistes carolina species 2 versus Polistes fuscatus species 6.

We found no way to reliably separate males of these species from pattern, although the females of these sexually dimorphic species are easily distinguished from each other but confused with other species. Polistes carolina species 2 is usually larger, but there is at least some overlap in size due to runt specimens of P. carolina species 2.

Figure E8: Polistes Phenotype 2.2 versus P. fuscatus species 3.

The small series of males examined for P. fuscatus species 3 have the lateral side of the head concolorous or with a less contrasting pale patch in the ventro-anterior region relative to the one specimen examined of Polistes phenotype 2.2. However, given the limited amount of material studied this may not hold up as a consistent pattern difference. Lighter specimens of Polistes carolina phenotype 3 (below) are also similar in pattern, but have Type 1 genitalia. Thus, there is one species with this type of pattern for each of the three Fuscopolistes genitalia types, even though it is unclear if phenotype 2.2 is a poorly known species or a rare aberration of one of the species with Type 2 genitalia.

Figure E9: Polistes carolina Phenotype 3 versus Polistes fuscatus species 3.

We found no way to distinguish these species from pattern, although there is little overlap in size. Only uncommon runts of Polistes carolina phenotype 3 are comparable in size to specimens we studied of P. fuscatus species 3. However, there is also an unplaced phenotype in the fuscatus species group (phenotype 3.1) that is the same size as a typical P. carolina Phenotype 3, and patterned like one of the darker examples than the one shown above (see the comparison in Figure E12 below).

Figure E10: Polistes carolina Phenotype 3 versus Polistes fuscatus species 5.

Polistes carolina phenotype 3 males are variable in pattern and can be confused with both P. fuscatus species 3 and species 5. Specimen HLYHYM:804 (above) is somewhat atypical for P. fuscatus species 3, but it is from a locality where many females of species 5 have been collected and where no females or typical males of species 3 were ever collected. It has the broad pale band on the anterior edge of the mesopleuron characteristic of species 5 and not species 3, but the metasomal coloration is the darker orange-brown more typical of species 3 than the lighter orange more typical of species 5. P. carolina phenotype 3 metasomal coloration varies from lighter orange to darker orange-brown to blackish. Polistes carolina phenotype 3, P. fuscatus species 3, and P. fuscatus species 5 always have broad black bands on the anterior edges of metasomal segments II-VII.

Figure E11: Polistes carolina species 1 versus Polistes carolina species 4.

Polistes carolina species 4 has a less stout phallus than other species with Type 2 genitalia, but it is still clearly more stout than Polistes carolina species 1. The one male examined of Polistes carolina species 4 is distinctly larger than any males examined of P. carolina species 1 (as is the case for a small series of species 4 females (from the same locality as the male) versus a large series of species 1 females).

Figure E12: Polistes carolina phenotype 3 versus Polistes fuscatus phenotype 3.1.

The one specimen examined of phenotype 3.1 has the typical size and pattern of P. carolina phenotype 3 males, but the Type 3 genitalia place it in the fuscatus species group.

Figure E13: Polistes carolina Phenotype 3 versus Polistes metricus Phenotype A.

While the overall pattern is quite similar between a typical P. metricus phenotype A and forms of P. carolina phenotype 3 with a predominately dark metasoma, as far as we know even the darkest males of P. carolina phenotype 3 have some ferruginous markings on tergum VIII, whereas P. metricus has this tergum black with at most some brown markings. Also, the second metasomal segment (=third abdominal segment) is usually distinctly more robust in lateral aspect in P. metricus phenotype A than in P. carolina phenotype 3.

Figure E14: Polistes carolina Phenotype 3 versus Polistes metricus Phenotype B.

Polistes metricus phenotype B is even more similar to dark forms of P. carolina phenotype 3 than is the case for P. metricus phenotype A (discussed above). In P. metricus phenotype B males the second metasomal segment is not proportionately more robust in lateral aspect, unlike P. metricus phenotype A. Either P. carolina phenotype 3 or P. metricus phenotype B may have an ivory-pale yellowish band on the posterior edge of tergum II, a condition never found among over 250 male nest mates examined of P. metricus phenotype A. Also, P. metricus phenotype B may have lighter brown on the terminal tergum than phenotype A, such that the absence of ferruginous markings on this tergum (a consistent character for P. metricus phenotype A) becomes more ambiguous.

Figure E15: Polistes carolina Phenotype 3 versus Polistes Phenotype 2.3.

Specimen HLKHYM:601 is unique singleton with some pattern elements of P. metricus and some of P. carolina species 2, and it could be a rare aberration of either species as these two species cannot be separated by male genitalia. Like P. metricus the metasoma is predominately black and lacks ferruginous markings on tergum VIII, whereas like P. carolina species 2 there is a broad pale band on the anterior mesopleuron and a prominent ivory band on the posterior edge of tergum 2. The specimen is large with metasomal segment II robust in lateral aspect, unlike males P. metricus phenotype B, and furthermore it lacks the wider black bands along mesopleural sutures typical of P. metricus phenotype 3. The pattern is much like the darkest specimens of P. carolina phenotype 3.

Figure E16: Polistes carolina Phenotype 3 versus Polistes fuscatus species 4.

The darkest specimens of P. carolina phenotype 3 are patterned much like dark males of P. fuscatus species 4 (and species 1). However, most males of P. carolina phenotype 3 are larger than P. fuscatus species 4 (or 1), although runts of P. carolina phenotype 3 are similar in size.

Figure E17: Polistes Phenotype 1.1 versus Polistes fuscatus Species 4.

Polistes phenotype 1.1 is patterned like the darkest specimens of P. carolina phenotype 3, P. fuscatus species 4, and P. fuscatus species 1. While P. fuscatus complex species are easily separated by the Type 3 phallus, both P. carolina phenotype 3 and Polistes phenotype 1.1 have a Type 1 phallus. However, phenotype 1.1 has the terminal tergum emarginate, versus narrowly convex in other Fuscopolistes species (Figure C55). Polistes phenotype 1.1 is smaller than any specimens of P. carolina phenotype 3 examined, but it is within the normal range of variation for P. fuscatus species 4 and P. fuscatus species 1.

Figure E18: Polistes metricus Phenotype B versus Polistes fuscatus species 4.

Some individuals of these two species may be indistinguishable by pattern, although there may be little overlap in the ranges. Polistes metricus phenotype B is very rare in peninsular Florida and may occur there only as a stray, whereas Polistes fuscatus species 4 may be endemic to peninsular Florida. These two species (provisionally including P. metricus phenotype A) are the only two Fuscopolistes species we examined where males lack ferruginous markings on the terminal tergum (although we did not examine any P. parametricus males). Unlike P. metricus phenotype A, P. metricus phenotype B males do not have the second metasomal segment relatively robust in lateral aspect; instead, it is comparable to the fuscatus species group. The first metasomal segment may or may not have a pale ivory band on the on the distal side in both P. metricus phenotype B and P. fuscatus species 4. Polistes fuscatus species 4 males have the mesopleuron and metapleuron predominately black, whereas P. metricus phenotype B usually has more of a mix of red-orange and black (as in the specimen above), but occasional P. metricus males of both phenotypes have the mesopleuron and metapleuron predominately black.

Figure E19: Polistes fuscatus species 1 versus Polistes fuscatus species 4. While both of these species have a type 3 phallus, the phallus of species 1 (and nominate fuscatus) averages stouter than species 4 (See Figure E20 below). In species 1 males the mesopleuron and metapleuron vary from predominately ferruginous to mixed ferruginous and black to predominately black, whereas in species 4 the mesopleuron and metapleuron are always predominately black. However, both species have a ferruginous and/or cream colored band on the anterior edge of the mesopleuron, and both often have a mesopleural spot on the anterior edge of the verticle suture line (this spot is ferruginous and/or cream colored in both species). The number of cream colored metasomal bands is variable in both species, but the metasomal ferruginous markings are more extensive in species 1. In species 4 males there are variably sized paired ferruginous markings on the first two metasomal tergites, the third metasomal tergite often lacks ferruginous markings but may have small paired markings, and the terminal tergite is typically black with some brown markings although there may be some ferruginous coloration (particularly in freshly emerged individuals). Ferruginous markings are lacking on the remaining metasomal tergites. Species 1 males may have ferruginous markings on all metasomal tergites, and always have ferruginous markings on both the terminal and penultimate metasomal tergites.

Figure E20: Phallus comparison between Polistes fuscatus species 1, P. fuscatus, and P. fuscatus species 4. On average P. fuscatus species 1 (and P. fuscatus) have a stouter phallus apex than P. fuscatus species 4. However, these photos were taken with the phallus still attached to the genitalia capsule, so precise measurements are not possible because the phallus is not completely flat or at exactly the same angle between specimens. KOH treated dissections with the phallus extracted and placed completely flat against the bottom of a petri dish would enable precise measurements, and these are needed to determine if there is a true gap or areas of non-overlapping variation between these species. However, based on studies of nest mates, the combination of pattern characters noted under Figure E19 above should enable reliable separation of males of P. fuscatus species 1 and species 4.