Biochemical Systematics and Ecology, Vol. 17, No. 7/8, pp. 595-601, 19~9. 0305-1978/89 $3.00 + 0.00 Printed in Great Britain. © 1989 Pergamon Press plc.

A Biochemical Approach Toward the Systematics of the "muscorum" Group in (" Formicidae)

JORGEN HEINZE Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138, U.S.A.*

Key Word Index--Leptothorax "muscorum"-complex; Leptothoracini; Formicidae; Hymenoptera; genetic variability; electro- phoresis. Abstract--Enzyme patterns have been used to distinguish among a number of morphologically very similar belonging to the Leptothorax "muscorum" group in North America. Not counting the already described species L. retractus, L. sphagnicolus and L. crassipilis, the complex apparently consists of at least three or four more different taxa.

Introduction As part of a taxonomic revision of this com- Ever since Provancher described the myrmicine plex, two species have recently been described: ant Leptothorax canadensis about 100 years ago, L. retractus, a common ant throughout boreal taxonomists have disagreed on the systematic North America, and L. sphagnicolus, known only position of this and related taxa from North from some spruce-bogs in Quebec [5]. In most America. belonging to the myrmicine areas of North America at least two additional genus Leptothorax, subgenus Leptothorax (s.str.) taxa can be distinguished, which have been M. R. Smith (=Myrothorax Ruzsky) are rather referred to as "small brown" and "large black" inconspicuous and small in size. They nest in Leptothorax "muscorum" in Alberta [3, 8] or preformed cavities in decaying wood, in bark, Leptothorax spp. A and B in Quebec and New under stones, and in moss in boreal and alpine, England [6, 7]. It is still unknown whether large mostly coniferous forests. Because intraspecific black Leptothorax from Quebec are conspecific variablity is often high, making distinctions with the large black ants from Alberta and between species based on morphological Colorado, or whether small brown specimens characteristics is frequently difficult. from Alaska belong to the same taxon as small In , four non-parasitic species are brown L. "muscorum" from New Hampshire. known: Leptothorax acervorum (Fabricius), L. Material has almost exclusively been collected muscorum Nylander, L. gredleri Mayr and L. in the Rocky Mountains and in Northeast scamni (Ruzsky). Specimens from North America, and records from the connecting America have been treated as subspecies of areas, e.g. the Canadian Northwest Territories or palaearctic L. acervorum and L. muscorum by northern Ontario, are rare [1, 2]. some authors, as varieties of L. canadensis, or as Though the characterizations "large black" or additional species by others (for a detailed "small brown" suggest reliable differences in review see [1]). Brown's synonymization of all size and color, more intensive morphological North American taxa with the palaearctic L. and ethological studies indicate that some large muscorum [2] certainly does not reflect the black ants actually are "small brown" L. actual situation. In recent publications several muscorum or that bicolored, small black, and authors have agreed that the subgenus Lepto- large brown "Leptothorax" (s.str.) from thorax (s.str.) comprises a number of different Northeast America usually can be sorted into species in North America [3-7]. one of the two taxa. In a number of ant genera biochemical markers have been used for the definition of *Present address: Zool. Inst. I1., D8700 WLirzburg, F.R.G. species in sibling complexes such as Irido- (Received 13 June 1989) myrmex "humilis" [9], Aphaenogaster "rudis"

595 596 JORGEN HEINZE

[10], and Rhytidoponera "irnpressa" [11 ]. Heinze colonies could be collected, there is a chance and Buschinger [7] have shown that esterase that some principally detectable variation has stains of crude Leptothorax homogenates, been overlooked. The probability (P) of missing separated by IEF, can help to distinguish a rare allele with the frequency a in a small between morphologically similar taxa such as L. sample of n diploid individuals is muscorum and L. gredlerL The esterase "finger- P= (l--a) 2n [13]. prints" of species belonging to the North Ameri- can L. "muscorum "/L. canadensis complex, However, if n whole colonies rather than n however, are very similar and differences are individuals are used to collect data, the expo- difficult to interpret. In addition, reliable results nent becomes 3n. The probability of an allozyme could be obtained only with white pupae, which with a frequency of 0.2 to remain undetected if a are not always available. sufficient number of workers from five colonies To find enzyme systems that can be visualized is studied, therefore, is about 3.5%, and the per- in tissue-homogenates from adult workers and centage is even smaller if the colonies contain that can be used for the distinction between several queens (polygyny). Though additional different taxa of L. "muscorum", samples of allozymes might have been missed in some Leptothorax (s.str.) from various populations in populations in this study, it is improbable that North America have been studied with electro- the observed specific differences of IDH (isocitric phoresis in cellulose acetate plates. dehydrogenase, EC 1.1.1.42), MDH-1, and PGD (6- phosphogluconic dehydrogenase, EC 1.1.1.44) in Results and Discussion the various taxa are accidental. In a sample of 20 The results of this study demonstrate that colonies the chance that rare allozymes with a enzyme data can be used to define taxa of Lep- frequency of less than 0.05 are not found is tothorax (s. str.); they also show, however, that below 5%. AIIozymes that occur only with fre- this subgenus is far more complex in North quencies less than 0.05 have often been America than in Europe. neglected in comparative studies and have been Of about ten loci that could be reliably stained defined as "absent" [14]. in homogenates of individual adult Leptothorax If data from all populations are compared, it workers, only ME (malic enzyme, EC 1.1.1.40) becomes likely that in spp. A and B, and prob- and MDH-2 (malic dehydrogenase, EC 1.1.1.37) ably also in additional species, MDH-1, IDH and showed considerable variation within species. In PGD are almost monomorphic within species, as a sample of about 20 colonies of large black L. they are in L. acervorum from southern Sweden "muscorum" (sp. B) from Mount Monadnock, (Douwes, personal communication). PGI (phos- New Hampshire, the most extensively studied phoglucose isomerase, EC 5.3.1.9) is known to population, two different electromorphs of be polymorphic in L. acervorum [15]; however, MDH-2 and three, perhaps four of ME could be in various taxa from North America, variability in separated by electrophoresis in cellulose acetate this enzyme could not be found. plates. Similar variability was found in small The low gene diversity in social Hymenoptera brown L. "muscorum" (sp. A) from Mount was explained by the influence of sociality on Monadnock and from Tadoussac, Quebec. Addi- the effective population size (e.g. [16]). In some tional data indicate that these two enzymes are species, such as the sweat-bee Lasioglossum polymorphic also in other taxa of the subgenus zephyrum [17] or the parasitic ant Epirnyrma Leptothorax (s.str.). In sublaevis, a kraussei (Jessen and Klinkicht, in preparation), palaearctic slave-making ant closely related to no variation at all could be detected by electro- this subgenus, MDH-2 and ME have also been phoresis, and in numerous species belonging to found to be variable [12], and at least MDH-2 is the subgenus Leptothorax (Myrafant) most of the variable in L. acervorum (Douwes, personal com- five examined loci were found to be mono- munication). morphic within species [18]. It thus is not Other enzyme systems show little or no surprising that most of the studied enzymes are intraspecific variability in Leptothorax (s.str.). more-or-less constant either within taxa (MDH-1, Since in a number of populations, only few PGD, IDH and, perhaps, PGI), or within the SYSTEMATICS OF THE LEPTOTHORAX "MUSCORUM" GROUP IN NORTH AMERICA 597

whole subgenus (SOD, superoxide dismutase, Quebec (B), Alberta (D), Colorado and New EC 1.15.1.1; LDH, lactic dehydrogenase EC Mexico (E), nor between small brown Lepto- 1.1.1.28). thorax A from Northeast America, C from The low variability of MDH-1, IDH and PGD in Alberta and H from Colorado. Identity of enzyme Leptothorax (s.str.) from North America makes it patterns, especially when based on a small possible to define taxa by species-specific sets amount of data, does not necessarily imply that of enzymes. The results of isoenzyme studies two taxa are conspecific. It still remains to be strongly support provisional distinctions based settled whether large black B from Northeast on morphological, ethological and karyological America is the same species as large black D characters. and E from the Rocky Mountains. The three taxa As can be seen in Table 1, taxa are charac- are very similar in morphology and biology too. terized by one of four different combinations of Both Leptothorax A and B can be found in coni- fast (f), slow (s), and very slow (v) migrating ferous forests throughout Northeast America. MDH-1, IDH and PGD electromorphs. Though Whereas in New Hampshire and Massachusetts each taxon does not have its own specific com- they are common only in the mountains (Mount bination, the following distinctions become Greylock, Mount Monadnock), in Quebec or possible: northern Maine they nest at lower elevations or (1) L. retractus (f/s/f) can be easily distin- directly at the coast, too (Tadoussac, Harpswell, guished from the morphologically similar small Reid State Park). Leptothorax C and D have been brown Leptothorax (f/f/s) of Northeast America collected at various elevations (1000-2000 m) in and the Canadian Rocky Mountains. Only the open pine forests in the Canadian Rocky Moun- large brown Leptothorax F from ponderosa pine tains (D, in addition, in similar habitats at lower forests in Arizona (elevation 2500 m) shows the elevations on Vancouver Island). Colonies of same combination of electromorphs; however, large black Leptothorax E were found in alpine it is morphologically different from L. retractus. meadows in Colorado (3450 m) and coniferous (2) L. crassipilis (f/f/s), a species which is very forests in New Mexico (2600-2800 m). Lepto- similar morphologically to the large black form thorax A and C are known to differ in chromo- (s/s/f) [19], differs from the latter in all three some number and colony structure: whereas A enzymes; it is indistinguishable from small usually has 2n=30 chromosomes and colonies brown L. "muscorum" in enzyme patterns, regularly contain only one functional queen [20], though easily distinguished by a number of C has 2n=34 chromosomes and is facultatively morphological characters such as pilosity. L. polygynous [3]. Workerlike, intermorphic crassipilis has been collected at relatively low queens, common in A [6], have never been elevations (2200-2500 m) in warmer habitats found in C. However, in one of three colonies of such as ponderosa pine or cottonwood forests, the small brown L. "muscorum"H from lodge- surrounded by sage brush desert or pinyon- pole pine forests in Colorado (2750 m), the juniper woodland (S. Cover, personal communi- queen was intermorphic. This taxon differs mor- cation). phologically only little from Leptothorax sp. A (3) In all populations, large black (s/s/f) and and might well belong to the same species. small brown (f/f/s) L. "muscorum"could reliably (5) Leptothorax G, a "large red" form from be distinguished by isoenzyme analysis. Colorado, differs from other taxa in its MDH (4) The large black L. "muscorum" differ from electromorph, as well as in morphology and all other taxa by its slower migrating MDH-1 habitat. The colonies nested in cottonwood electromorph. Large black and small brown branches and stumps at an elevation of 2350 m. Leptothorax, in addition, are distinguished by (6) Of more than 500 colonies collected in their IDH and PGD isoenzymes. At least in Quebec and New England, only five could be Northeast America, the results of biochemical identified as neither spp. A nor B, even in inten- studies correspond to differences in chromo- sive morphological studies, but the ants in each some number and colony structure of A and B. had a somewhat intermediate morphology. Enzyme studies did not show any differences Workers from all five colonies showed a unique between large black L. "muscorum" from MDH-1 pattern, consisting of the slow migrating 598 JORGEN HEINZE

TABLE 1. ALLOZYME PATTERNS IN DIFFERENT TAXA OF NORTH AMERICAN AND PALAEARCTIC ANTS BELONGING TO THE SUBGENUS LEPTO- THORAX (S.STR.) AND RELATED PARASITIC GENERA. x, f, m, c AND v STAND FOR VERY FAST, FAST, MEDIUM, SLOW AND VERY SLOW MIGRATING ALLOZYMES. In some cases, PGI could not be studied in all collected colonies. This is indicated by parentheses

Taxon Population Number of colonies MDH-1 IDH PGD PGI

Nearctic species LeptobSorax "muscorum'A (small brown) Quebec: Tadoussac 13 f f s (s) St Simeon 6 f f s (s) Rouyn-Noranda 5 f f Aiguebelle 3 f f Shawinigan 2 f f Maine: Mt Desert Island 1 f f Reid State Park 3 f f New Hampshire: Mt Monadnock 8 f f (s, vs) L. "muscorum'B (large black) Quebec: Tadoussac 11 s s (f) Sept lies 1 s s Petit Lac Ha!Hat 1 s s La Bale 2 s s Mt du Lac des Cygnes 3 s s f f Bic 6 s s f New Brunswick: Lavillette 1 s s f Moncton 1 s s f Nova Scotia: MacKenzie Mountain 5 s s f f Maine: Mt Desert Island 1 s Harpswell 1 s s Wesley 1 s s Madawaska Lake 1 s s Reid State Park 5 s s New Hampshire: Mt Monadnock 22 s s (f) Massachusetts: Mt Greylock 2 s s Vermont: Mt Mansfield 5 s, fs s L. "muscorum " A X B ? Quebec: Chapais 1 fs fs Tadoussac 1 fs fs fs St Simeon 2 fs s fs Mt du Lac des Cygnes 1 fs f, fs fs L. "muscorum" C (small brown) Alberta: Banff 4 f f s s Maligne Canyon 6 f f, s s s Kananaskis 1 f f s British Columbia: Manning Prov. Park 1 f s s s L. "muscorum" D (large black) Alberta: Cavell Lake 2 s s f f Maligne Lake 3 s s f f British Columbia: Mystery Lake 3 s s f f Sutton Pass 1 s s f L. "muscorum" E (large black) Colorado: San Isabel Nat. Forest 4 s s x,f Rico 2 s s f Priest Gulch Trail 3 s s f Fossett Gulch Road 3 s s f New Mexico: Santa Fe Nat. Forest 4 s s x,f Carson Nat. Forest 1 s s f Borrego Trail 3 s s f Utah : Monticello 1 s s f L. "rnuscorurn" F (small brown) Arizona : Chiricahua Mts 4 f s f, s (s) L. "muscorum " G (large red) Colorado: Curecanti 1 f, m s SYSTEMATICS OF THE LEPTOTHORAX "MUSCORUM" GROUP IN NORTH AMERICA 599

TABLE 1--CONTINUED

Taxon Population Number of colonies MDH-1 IDH PGD PGI

L. "muscorum" H (small brown) Colorado: San Isabel Nat. Forest 3 f f s f Leptothorax crassipilis Colorado: Curecanti 3 f f s (s) Gobel Trail 3 f f f? Rico 1 f f f? Fossett Gulch Road 2 f f s New Mexico: Santa Fe 5 f f s Borrego Trail 6 f f s Cibolo Nat. Forest 7 f f s Jemez Spring Trail 2 f f s Jemez Canyon 4 f f s Leptothorax retractus Quebec : Rouyn-Noranda 4 f s Aiguebelle 2 f s Alberta: Athabaska Falls 1 f s f s Maligne Canyon 5 f s f s Kananaskis 3 f s f (v, s) Banff 1 f s f s New Brunswick: Moncton 2 f s f Maine: Wesley 1 f s f

Palaearctic species Leptothorax aceryorum Sweden: Nyehusen 3 f f Germany: N0rnberg 5 f f s Pipplinger Au 1 f f Japan: Hokkaido 3 f f s Leptothorax muscorum Sweden : Nyehusen 3 f f France: Brian,:on 3 f f Germany: Nernberg 5 f f Turkey: ~,ami~i 1 f f Leptothorax gredleri Sweden: Nyehusen 1 f f Germany: Wi3rzburg 4 f f

Parasitic species Leptothorax kutteri Sweden: Nyehusen 1 f f Leptothorax goesswaldi France: Brian~:on 3 f f Germany: Ni~rnberg 5 f s Harpagoxenus canadensis Quebec : Rouyn-Noranda 2 f s Tadoussac 5 f s f, m (s) St Simeon 1 f s f s Nova Scotia: MacKenzie Mountain 1 f s f s Doronomyrmex pocahontas Alberta: Maligne Canyon 3 f f v

allozyme found in B, the fast migrating allozyme for example, seems to be variable in Leptothorax of A, and a hybrid band. PGD and IDH patterns B from Mount Mansfield, Vermont), the occur- also indicated a hybridization between the two rence of hybrid patterns in three enzymes within taxa A and B. Though all three enzymes might a single colony is quite unlikely. Instead, they show some rare intraspecific variability (MDH-1, may be the result of hybridizations between A 600 JORGEN HEINZE

and B. In the field, the two species are isolated Experimental by different hours of sexual activity; in the Complete colonies of Leptothorax (s.str.) were collected in laboratory, however, it was possible to mate a B Alberta (Banff; Tunnel Mountain; Jasper N.P.: Athabaska female with an A male. The resulting hybrid Falls, Maligne Canyon, Maligne Lake, Edith Cavell Lake; Kana- naskis: Wasootch Creek), British Columbia (Manning Prov. workers showed the same MDH and IDH pat- Park; Mt Seymour Prov. Park: Mystery Lake; Vancouver terns as observed in the five colonies. Hybridiza- Island: Sutton Pass), New Brunswick (Northumberland Co.: tion between other species of ants has only Laviilette; Westmoreland Co.: Moncton), Nova Scotia (Inver- occasionally been reported from the field [21, 22, ness Co.: MacKenzie Mountain), Quebec (Abitibi Co.: Aigue- 23]. As recent studies in the parasitic genus belle; Abitibi Terr.: Chapais; Chicoutimi Co.: La Baie, Petit Lac Ha!Ha!; Charlevoix-Est Co.: St Simeon, Mt du Lac des Epimyrma show, in some cases the fertility of F1- Cygnes; Rimouski Co.: Bic; Saguenay Co.: Tadoussac, Sept hybrids appears to be similar to that of the lies; St Maurice Co.: Shawinigan; Temiscamingue Co.: parental species (Jessen and Klinkicht, in pre- Rouyn-Noranda), Arizona (Cochise Co.: Chiricahua Mts), paration). Colorado (Archuleta Co.: Fossett Gulch Road; Chaffee Co.: San Isabel Forest; Dolores Co.: Rico; Gunnison Co.: Curecanti (7) Doronomyrmex pocahontas, known only Nat. Recr. Area; Montezuma Co.: Gobel Trail, Priest Gulch from the type locality at Maligne Canyon in Trail), Maine (Aroostook Co.: Madawaska Lake; Cumberland Jasper National Park and thought to be a Co.: Harpswell; Hancock Co.: Mt Desert Island; Sagadahoc workerless parasite living in colonies of the Co.: Reid State Park; Washington Co.: Wesley), Massachu- setts (Berkshire Co.: Mt Greylock), New Hampshire (Cheshire small brown Leptothorax C [8], differs from Lep- Co.: Mt Monadnock), New Mexico (McKinley Co.: Cibolo Nat. tothorax "muscorum"in its PGI and PGD pattern. Forest; Santa Fe Co.: Borrego Trail; Sandoval Co.: Jemez Apparently Doronomyrmex pocahontas has its Spring Trail, Jemez Canyon; Taos Co.: Santa Fe Nat. Forest, own workers, who are biochemically and Carson Nat. Forest), Utah (San Juan Co.: Monticello), and morphologically different from those of C. The Vermont (Chittenden Co.: Mt Mansfield). They were kept in the laboratory in artificially shortened breeding cycles with life history of this rare ant still in not clear. daily temperature and light rhythms [25]. Single workers were (8) Harpagoxenus canadensis, a parasitic ant crushed in a drop of tray buffer (0.01 M sodium phosphate/ enslaving several species of the Leptothorax citrate-buffer, pH 6.4 for esterases, MDH-1, ME, IDH, PGI, PGD, "muscorum" complex in Northeast America, SOD, LDH: 0.01 M phosphate buffer, pH 7.8 for MDH-2). Enzymes were separated on cellulose acetate plates (Helena was included in this study. Its IEF-obtained Laboratories, Beaumont, Texas), a method that recently has esterase fingerprint had been reported to be been shown to be valuable for the separation of protein almost indistinguishable from that of L. samples from and other small invertebrates [26]. acervorum, one of the hosts of the palaearctic Proteins were separated at 200 V for about 20-90 min. congener H. sublaevis [7]. Both H. canadensis Enzymes were visualized using standard histochemical stains. and H. sublaevis differ from L. acervorum in their Acknowledgements--The studies on Leptothorax in North IDH and PGD atlozyme. Emery suggested that America were supported by a grant of the NATO Science parasitic ants are derived from the same stock Council through the DAAD. Colonies from Colorado, Arizona as their hosts [24]. Though esterase, MDH and and New Mexico were provided by Stefan Cover, MCZ, Cam- bridge, Massachusetts, who also commented on the manu- IDH patterns support "Emery's rule" in the work- script. Dr Peter Frumhoff and Leeanne Tennant, MCZ, helped erless parasites Leptothorax kutteri and L. goess- to collect colonies in New England and Quebec. Leptothorax waldi, it is not yet possible to calculate related- acervorum and L. goesswaldiwere provided by Dr Alfred Bus- ness between the slave-makers Harpagoxenus chinger, TH Darmstadt (F.R.G.). Jennifer Dougherb/, Newton, and their various Leptothorax hosts. The data Massachusetts, improved the English. suggest, however, that the two species of Harpa- References goxenus are closer related to their Leptothorax 1. Creighton, W. S. (1950) Bull~ Mus. Comp. Zoo/. Mass. 104, s.str, hosts than to other leptothoracines. 274. Similarly, preliminary results, based on small 2. Brown, W. L. (1955) Entomol. News 66, 43. sample sizes and five enzymes, indicate that the 3. Buschinger, A. (1982) Psyche 89, 197. 4. Stuart, R. J. (1984) Can. J. Zoo/. 62, 1995. nearctic parasites Leptothorax rninutissirnus, L. 5. Francoeur, A. (1986) Can. Ent. 118, 1151. duloticus, and Protomognathus arnericanus 6. Heinze, J. and Buschinger, A. (1987) Ins. Soc. 34, 28. (=Harpagoxenus americanu~ are closely related 7. Heinze, J. and Buschinger, A. (1988) Biochern. Syst EcoL to their hosts of the Leptothorax subgenus Myra- 16, 217. 8. Bushinger, A. (1979) Ins. Soc. 26, 216. fant (J. Heinze, R. J. Stuart and S. 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