PHYLOGENY AND FOUNDING STAGE OF (ACANTHOMYOPS) (: FORMICIDAE)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of

Philosophy in the Graduate School of The Ohio State University

By

Joseph Martin Raczkowski, M. S.

**** The Ohio State University

2008

Dissertation Committee: Approved by

Professor John W. Wenzel, Co-Advisor

Professor Steve Rissing, Co-Advisor ______

Professor Norman F. Johnson ______

Professor Joan Herbers Co-Advisors

Graduate Program in , Ecology, and Organismal Biology

ABSTRACT

The evolution of social in remains a controversial topic despite nearly 200 years of study and discussion. A lack of the details of socially parasitic behavior and phylogenies of socially parasitic groups are the greatest hindrances to understanding the evolution of this behavior. In this dissertation I address these problems for a group of North American ants, Lasius (Acanthomyops). I review social parasitism in ants, summarizing what we know and identifying the problems with the available data including distributional data. I present a study of socially parasitic behavior for my group of interest, as well as a phylogeny based on morphology and molecules. Finally, I discuss the implications of my findings on the evolution of social parasitism within the context of Emery's Rule.

ii

Dedicated to Michell, Ava, and Dean Raczkowski.

iii ACKNOWLEDGMENTS

This dissertation would not have been possible without the help of many people.

I would like to thank my committee members John Wenzel, Steve Rissing, Norm

Johnson, and Joan Herbers for improving my work. I thank all Wenzel Lab people, past and present, including: Kurt Pickett, Mary Daniels, Dave Rosenthal, Sibyl Bucheli,

Hojun Song, Todd Gilligan, Sarah Mominee, Gloria Luque, Giancarlo Lopez-Martinez,

Erin Morris, Ryan Caesar, and Glené Mynhardt. I am grateful to the following people for sharing their knowledge: Chris Johnson, Larry Phelan, Meg Daly Lab, John Freudenstein

Lab, and Hans Klompen. I thank all people of the Museum of Biological Diversity for providing a pleasant work environment. I thank the following people from outside Ohio

State University: Edward O. Wilson, Stefan Cover, Gary Alpert, Jim Carpenter, Roberto

Keller, Ted Schultz, John LaPolla, Mike Sharkey Lab, Earl Werner, Justin Congdon, and

Joshua Capps. I thank Michell, Ava, Dean, Russell, Carl, Paul, Mary, Bill, and Jody

Raczkowski for their support. I thank Ralph Martin, Gail Martin, and Brandon Martin for their support. Lastly I thank Robert Stieferman, Jeff Latham, Jonathon Tanner, Steve and Tiffany Graham, and John and Laura Riffle for their friendship.

iv TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... iii

Acknowledgments ...... iv

Vita ...... v

List of Figures ...... viii

List of Tables ...... xi

Chapters

1. Introduction...... 1

2. Review of social parasitism in Formicidae (Insecta: Hymenoptera) with an emphasis on the Lasius ...... 5

Introduction ...... 5

Associations of Ants ...... 6

Host characteristics in the different forms of social parasitism ...... 8

Temporary social parasitism in Lasius ants ...... 9

Dependence of measures of diversity upon taxonomic effort ...... 22

Predictors of richness among ants ...... 24

Limitations of the data ...... 26

Taxonomic effort, lumping and splitting ...... 27

v Importance of revisionary systematics ...... 29

Conclusion ...... 31

3. Founding Stage of Lasius (Acanthomyops) and the incidence of social

parasitism ...... 52

Introduction ...... 52

Methods ...... 55

Results ...... 58

Discussion ...... 63

Conclusions ...... 71

4. Phylogenetic Analysis of Lasius (Acanthomyops) ...... 90

Introduction ...... 90

Taxonomic history ...... 92

Materials and methods ...... 95

Results and discussions ...... 98

Conclusions ...... 111

5. Appendix A : Cytochrome Oxidase Subunit 1 Sequences Aligned by MUSCLE .... 123

6. Appendix B: wingless sequence data aligned by MUSCLE ...... 132

7. Appendix C : Taxonomic Descriptions...... 136

8. Bibliography ...... 161

vi LIST OF FIGURES

Figure Page

2.1 Number of taxa (species and subspecific ranks) described from different nations, according to Antbase. Nations specifically indicated are, by region, Canada (41), the United States (1013), Nicaragua (13), Costa Rica (248), Colombia (233), Brazil (1266), Switzerland (91), Algeria (206), Egypt (57), Congo (95), Zaire (455),South (515), Russia (237), China (169), India (505), Indonesia (903), New Guinea (657), and Papua New Guinea (41) ...... 42

2.2 Relationship between number of taxa described from a region and (a) the region’s area (square kilometers; r2= 0.201), (b) the mean temperature of the region (degrees Celsius; r2= 0.001), (c) the region’s average annual precipitation (millimeters; r2 = 0.059), and (d) the number of taxonomists describing taxa in the region (r2 = 0.592). The numbers in the figure panels refer to the following countries: (1) Brazil, (2) United States, (3) Indonesia, (4) New Guinea, (5) India, (6) Costa Rica, (7) Colombia, (8) Switzerland, (9) China, (10) Canada, (11) Russia, (12) South Africa, (13) Algeria, (14) Egypt, (15) Papua New Guinea, and (16) Zaire ...... 43

2.3 The number of taxa described from Colombia, by author ...... 48

2.4 Classifications of taxa Forel described from Colombia ...... 49

2.5 Number of taxa described from Congo, by author ...... 50

2.6 Classifications of taxa Santschi described from Congo ...... 51

3.1 Reorganization of Wing’s (1968) hypotheses for the founding stage of Lasius (Acanthomyops) according to overall strategy (non-parasitic v parasitic), number of queens founding a colony, and details of the non-parasitic and parasitic habit ...... 84

3.2 Grouping experiment arenas. Four L. interjectus gynes were placed in each arena. Each arena contained four perches (rocks), four shelters (5 x 5 cm piece of glass with one end propped up by a cap from a 1.5 mL plastic vial), and four sources of food (piece of tin foil with Bhatkar’s diet) ...... 85

vii 3.3 Survivorship of individual queens that started either singly (P < 0.03, Kaplan-Meier, Tarone-Ware), in groups of two (P < 0.04, Kaplan-Meier, Tarone-Ware) and in groups of four...... 86

3.4 Number of flagging events recorded within the first hour of observations in all trials. Number of flagging events = -0.190 x minutes + 16.033, r = -0.602, P < 0.0001 ...... 87

3.5 Number of interactions observed in the first hour of observations of all trials. Number of interactions = 0.044 x minutes + 4.342, r = 0.258, P = 0.047 ...... 88

3.6 Number of interactions = 0.783 + 0.388(minutes) - 0.006(minutes)2 r square = 0.329, P < 0.0001 ...... 89

4.1 Phylogeny from COI and wingless. Lasius (Acanthomyops) taxa in blue. The strict consensus of two most parsimonious trees ...... 99

4.2 Phylogeny from morphology and molecules. Lasius (Acanthomyops) in blue. Lasius (Acanthomyops) is not monophyletic ...... 101

4.3 Phylogeny of morphology and molecules with problematic taxa Lasius (Dendrolasius) spathepus removed. Lasius (Acanthomyops) is monophyletic ...... 103

4.4 Phylogeny from morphology and molecules. Both putative hybrids (latipes x and pogonogynous) were removed for this analysis. Lasius (Acanthomyops) taxa in blue ...... 105

4.5 Lasius murphyi gyne, illustrating the dense mats of hair on the head and thorax ...... 113

4.6 gyne, illustrating the large genual plates on the foreleg and the stout head and antennae ...... 114

4.7 gyne ...... 115

4.8 gyne ...... 116

viii 4.9 GCMS of mandibular gland contents of Lasius claviger gyne Peak at 6.61 is citronellal and the peak at 7.48 is citral ...... 117

4.10 GCMS of mandibular gland from Lasius latipes gyne. Note the absence of peaks at 6.61 (citronellal) and 7.48 (citral) ...... 118

C.1 Distribution of Lasius claviger (solid circles) according to Wing (1968) (based on samples). Distribution of Lasius coloradensis (hollow circles) according to Wing (1968) (based on 70 samples) ...... 154

C.2 Distribution of Lasius interjectus according to Wing (1968) (based on 337 samples) ...... 155

C.3 Distribution of Lasius latipes (solid circles) according to Wing (1968) (based on 448 samples). Distribution of Lasius latipes x claviger (hollow circles) according to Wing (1968) (based on 26 samples) ...... 156

C.4 Distribution of Lasius murphyi according to Wing (1968) (based on 49 samples) ...... 157

ix LIST OF TABLES

Table Page

2.1 Most prolific authors of ant systematic papers ...... 42

3.1 Wing’s (1968) summary of founding stage evidence for Lasius (Acanthomyops). The two Lasius claviger entries (marked with *) were considered unreliable by Wing (1968) ...... 75

3.2 Summary of Tanquary’s (1911) Introduction Experiments ...... 77

3.3 Forced association experiments for Lasius interjectus ...... 78

3.4 Description of behaviors observed in grouping experiments ...... 79

3.5 Summary of Introduction Experiments of current study ...... 80 HW = Host Worker, P = Parasite.

3.6. Results of G-test. MAX # DAYS = Maximum number of consecutive days gynes were grouped in the same site of the arena. # DAYS CONS = number of days gynes grouped in same site of arena as the previous census ...... 81

3.7 Chi-square test table for finding different group sizes in two-, three-, and four-gyne trials. Expected values from truncated Poisson distribution ...... 83

4.1 Characters and character states used in this analysis ...... 119

4.2 Morphological matrix used in this study ...... 121

4.3 Primers used in this analysis. All primers were used for both amplification and sequencing ...... 122

x CHAPTER 1

INTRODUCTION

Parasitism is a relationship between two organisms in which one organism benefits at the expense of another. Parasites affect most life on earth and do so in a variety of ways (Price 1980). For example, endoparasites such as bacteria and viruses live within their host while ectoparasites such as mistletoe or body lice live on their host.

Social parasitism is a special case of parasitism in which the beneficiary exploits the social system of the host. This takes the form of brood care or other socially managed resources (Schmid-Hemple 1998). Brood parasitism is found in birds (Hamilton and

Orians 1965, Rothstein 1994, Lotem 1995), spotted hyenas that steal the prey of African wild dogs (Carbone et al 1997, Gorman et al 1998), and thrips that exploit the galls of other thrip species (Bono 2007). Ant social parasitism has been studied more thoroughly than in other organisms and broad trends, such as the distribution of parasitic species and host characteristics, can be recognized (Hölldobler and Wilson 1990, Huang and

Dornhaus 2008). Despite nearly 200 years of study, the evolution of social parasitism in ants remains a controversial topic. A lack of both the details of socially parasitic behavior and phylogenies of socially parasitic groups are the greatest hindrances to resolving arguments on this topic (Janda et al 2004, Hung and Dornhaus 2008). In this

1 dissertation I address both of these issues through observations of socially parasitic behavior and a phylogenetic analysis of a group of temporary social parasites, Lasius

(Acanthomyops).

In Chapter Two, I review the ant social parasitism literature with special emphasis on temporary social parasitism in Lasius ants. I point out inconsistencies in the literature and discuss the limits of our knowledge including problems with distributional data. In

Chapter Three I report my findings on temporary social parasitic behavior in Lasius

(Acanthomyops) from the field and laboratory. These include new host-parasite records and details of parasitic gyne behavior. In Chapter Four, I present a phylogenetic analysis of Lasius (Acanthomyops) based on morphology and molecules. With the species level phylogeny in place, I discuss the evolution of social parasitism in the group including an evaluation of Emery's Rule (Emery 1909, Le Masne 1956).

2 Literature cited

Bono, J.M. 2007. Patterns of kleptoparasitism and inquilinism in social and non-social Dunatothrips on Australian Acacia. Ecological Entomology 32: 411-418.

Carbone, C., Du Toit, J.T., and I.J. Gordon. 1997. Feeding success in African wild dogs: does kleptoparasitism by spotted hyenas influence hunt group size? Journal of Ecology 66: 318-326.

Emery, C. 1909. Über den Ursprung der dulotischen, parasitischen und myrmekophilen Ameisen. Biologisches Centralblatt 29: 352-362.

Gorman, M.L., Mills, M.G., Raath, J.P., and J.R. Speakman. 1998. High hunting costs make African wild dogs vulnerable to kleptoparasitism by hyaenas. Nature 391: 479-481.

Hamilton , W.J. and G.H. Orians. 1965. Evolution of brood parasitism in altricial birds. Condor 67: 361-382.

Huang, M.H. and A. Dornhaus. 2008. A meta-analysis of ant social parasitism: host characteristics of different parasitism types and a test of Emery's rule. Ecological Entomology 33: 589-596.

Janda, M., Folkova, D. and Zrzavý. 2004. Phylogeny of Lasius ants based on mitochondrial DNA and morphology, and the evolution of social parasitism in the (Hymenoptera: Formicidae). Molecular Phylogenetics and Evolution 33:595-614.

Le Masne, G. 1956. Recherches sur les fourmis parasites: grassei et l'évolution des Plagiolepis parasites. Comptes Rendus de l'Acadé des Sciences, Paris 243: 673-675.

Lotem, A., and S.I. Rothstein. 1995. Cuckoo–host coevolution: from snap-shots of an arms race to the documentation of coevolution. Trends Ecology and Evolution 10: 436–437.

3

Price, P.W. 1980. Evolutionary biology of parasites. Princeton University Press, Princeton, New Jersey, USA.

Rothstein, S. I., and S. K. Robinson. 1994. Conservation and coevolutionary implications of brood parasitism by cowbirds. Trends in Ecology and Evolution 9:162–164.

Schmid-Hempel, P. 1998. Parasites in Social . Princeton University Press, Princeton. pp. 111-117.

4 CHAPTER TWO

REVIEW OF SOCIAL PARASITISM IN FORMICIDAE (INSECTA: HYMENOPTERA) WITH AN EMPHASIS ON THE GENUS LASIUS

Introduction

The first record of social parasitism in ants comes from Pierre Huber (1810). He discovered the relationship between and sanguinea known as piracy, dulosis, and slave-making. About this discovery Wheeler (1910) wrote, "this was so extraordinary that it engrossed the attention of observers for nearly a century." In their review of social parasitism in ants, Hölldobler and Wilson (1990) credited Wheeler

(1901, 1910) and Forel (1898, 1901) as important contributors to the study of social parasitism in ants. They summarized the literature in table form and discussed the classification of social parasitism in ants following the work of Wheeler and Forel. The

Hölldobler and Wilson (1990) review remains an important synthesis and is usually the starting point for discussions of social parasitism in ants (Schmid-Hemple 1998, Lenoir et al 2001, Janda et al 2004, Huang and Dornhaus 2008). In this chapter I review the literature on social parasitism in ants with a special emphasis on temporary social parasitism in Lasius ants, and discuss the limits of our knowledge including distributional data.

5 Associations of ants

Interspecific associations of ants are common and vary greatly in their details

(Wheeler 1910, 1933, Hölldobler and Wilson 1990). The two major categories are relationships found in compound and those found in mixed nests. Compound nests involve two or more species living in close proximity to each other but not mixing their brood. In mixed colonies, two species live in close proximity and mix their brood.

Mixed colonies usually involve social parasitism.

Compound nests

Wheeler (1910) described the following relationships found in compound nests:

1. Plesiobiosis involves different species nesting very close to one another, but engaging in practically no direct communication.

2. Cleptobiosis involves a small species building their nests near a of a larger species and feeding on refuse or stealing food from host workers.

3. Lestobiosis involves a small species living in the walls of a larger species and stealing food or eating brood.

4. Parabiosis involves two or more species that keep their brood separate but use the same nest and in some cases, odor trails.

5. Xenobiosis involves one species living in the walls or chambers of the nest of another species and moving freely among its hosts, obtaining food from them by one means or another, usually soliciting regurgitation. The brood is still kept separate.

6 Mixed nests

There are three main categories of social parasitism in ants, temporary, dulotic

(slave-making, piracy), and inquilinism. Wheeler (1910) summarized socially parasitic relationships in ants and subsequent authors have continued to use his categorization

(Hölldobler and Wilson 1990, Lenoir et al 2001, Huang and Dornhaus 2008).

Temporary social parasitism is usually described as an inseminated gyne entering a host colony and eventually being accepted by the host colony either through force or some sort of conciliation. The host queen is killed by either the parasitic gyne or her own workers, and the parasitic gyne lays eggs that are tended by the host workers. A mixed colony of parasitic and host workers exists until the host workers die (Wilson 1971,

Hölldobler and Wilson 1990, Lenoir et al 2001, Janda et al 2004, Huang and Dornhaus

2008).

Dulosis (slavery, piracy) is an obligate or facultative reliance on workers of other species for routine colony maintenance. The obligate parasites must raid host colonies for workers continually because the parasite workers are not suited for brood care and foraging. This form of social parasitism is relatively well-studied in Formica (Kutter

1969, Mori et al 2001, Savolainen and Deslippe 1996), Polyergus ( Topoff et al 1984,

1985, 1987) and Temnothorax and Protomognathus (Herbers and Foitzik 2002, Johnson and Herbers 2006, Johnson 2008).

Inquilinism is a permanent parasite-host association without slave raiding. In extreme cases the queen produces only a sexual caste and tolerates the host queen, which provides the inquiline with a continuous supply of workers. Workerless

7 inquilinism is viewed as the ultimate, degenerate stage of social parasitism in ants

(Hölldobler and Wilson 1990). Prominent examples of this type of social parasitism are

Teleutomyrmex (Kutter 1950) and Anergates (Forel 1874).

Host characteristics in the different forms of social parasitism

In their meta-analysis of ant social parasitism Huang and Dornhaus (2008) examined the literature (Hölldobler and Wilson 1990, Schultz et al 1998, Delabie et al

1993, Sumner et al 2004, Maschwitz et al 2000, Ward 1985, Ward 1996) for trends in host colony size, number of hosts, and relatedness of host and parasite. and temporary social parasites most often target hosts in their own genus, dulotic species generally target members in their tribe but different genera, and xenobiotic species tend to target hosts from different tribes. Inquilines usually have one host, temporary and dulotic species use multiple hosts, and xenobiotic species vary in this regard. Inquilines and temporary parasites target hosts with medium sized colonies (100 to 50,000 workers), dulotic species use hosts of various colony sizes, and xenobiotic species do not parasitize small (<100 workers) colonies. Overall, temporary social parasites are more similar to inquilines than dulotic species in the categories used. Temporary social parasites and dulotic species both tend to have multiple hosts but differ in other ways including host colony size and level of relatedness to the host. Huang and Dornhaus suggest that the ability to parasitize a wide range of host colony sizes might be attributed to the wide variety of strategies used by dulotic parasites (e.g. chemical mimicry, chemical repellency) but caution that invasion strategies are better known in dulotic species, which creates a study bias.

8 The details of temporary social parasitism are better known in Formica ants than

Lasius (Mori et al 2001, Janda et al 2004). The most recent synthesis of temporary social parasitism in Lasius (Janda et al 2004) included 19 parasitic species with details of the founding stage available for two. Here I provide my review of the literature concerning temporary social parasitism in Lasius ants. I include references not cited in

Hölldobler and Wilson (1990), Janda et al (2004), or Huang and Dornhaus (2008) and point out inconsistencies in the literature.

Temporary social parasitism in Lasius ants

Most host-parasite relationships are established by discovering colonies containing workers of two different species or observations of parasitic gynes inside or very near a host colony after the mating flights of the parasitic species. Knowing that a host-parasite relationship exists is useful information because, in the context of a phylogeny, this information allows for interpretations of number of origins and/or losses of the behavior especially if a group (i.e. subgenus) contains parasitic and independent founding species. If, as in the case of some groups of Lasius ants, social parasitism is relatively common, details of the founding stage, such as, how the parasitic gyne enters the host colony, what happens to the parasitic gyne after entering the colony, and the fate of the host queen, are needed to discuss the evolution of the behavior. Laboratory and field observations can provide details of the founding stage, but these types of studies are very rare in Lasius ants (Janda et al 2004).

In several publications, authors provide an example of a mixed colony or, more rarely, an observation of the founding stage of a parasitic species then offer other possible

9 hosts in sympatry with the parasitic species. Unfortunately, subsequent authors report these conjectures as fact (Hölldobler and Wilson 1990, Janda 2004), which skews what we think we know about the topic. I have organized this review according to subgenus and have not included the independent founding subgenera Lasius sensu stricto and

Cautolasius.

Of the six subgenera of Lasius ants (Acanthomyops, Austrolasius, Cautolasius,

Chthonolasius, Dendrolasius, Lasius) only Cautolasius and Lasius lack temporary socially parasitic species. In general, the parasitic species of Acanthomyops,

Austrolasius, Chthonolasius, and Dendrolasius target Lasius sensu stricto and

Cautolasius species. Notable for its departure from the general rule and as an example of hyperparasitism, is Lasius (Dendrolasius) fuliginosus exploiting the known social parasite, Lasius (Chthonolasius) umbratus.

Acanthomyops

My review of the literature on the Acanthomyops founding stage is found in

Chapter Three of this dissertation.

Austrolasius

This subgenus contains two western Palearctic species, Lasius reginae and carniolicus. Buschinger and Seifert (1997) report that, historically, Lasius carniolicus, was thought to be a social parasite because of the small body size of the queen (roughly the same size as a worker). Schmid's (1975) discovery of a mixed L. carniolicus and L. flavus colony in Switzerland was the first evidence to support this suspicion. More than

20 years later, during a vacation stay in France, Buschinger found two mixed colonies of

10 L. carniolicus and L. piliferus, a species that had not been reported from France up to that point (Buschinger and Seifert 1997). Colony foundation has never been directly observed for L. carniolicus.

Faber (1967) twice observed young L. reginae queens in L. alienus nests and discovered one mixed colony of L. reginae and L. alienus. Seifert (Buschinger and

Seifert 1997) found another L. reginae/alienus mixed colony in 1980. Faber (1967) introduced a L. reginae queen to a queen right L. alienus colony in what has become a famous example of temporary social parasitic behavior (Hölldobler and Wilson 1990,

Janda et al 2004). The L. reginae queen flipped the L. alienus queen on her back and bit her violently.

Dendrolasius

Two species of Lasius (Dendrolasius), L. fuliginosus and teranishii, are temporary social parasites, while another, L. spathepus, is suspected to be despite a lack of evidence. has a Palearctic distribution while L. teranishii and spathepus are known only from Japan. In response to Stumper's (1920) paper suggesting that Lasius fuliginosus founded colonies independently, Donisthorpe (1922) summarized the work of previous authors, including 11 of his own papers. Also included in

Donisthorpe's review was W. M. Wheeler's (1910) discussion on the founding stage of L. fuliginosus, in which he describes a temporary parasitic habit for the species.

Donisthorpe reports L. fuliginosus/umbratus and L. fuliginosus/mixtus mixed colonies and describes introduction experiments with L. fuliginosus queens and L. umbratus, in which L. fuliginosus gynes were accepted and the host workers cared for their brood.

11 Stärcke (1944) introduced Lasius fuliginosus queens to L. alienus, niger and rabaudi colonies and the three species accepted the queens. Furukawa (1953) reported a mixed

Lasius fuliginosus/umbratus colony that was still mixed a year later as well as his discovery of a L. fuliginosus queen side by side with a L. niger queen in a L. niger nest.

Wilson (1955) considered Donisthorpe' (1920) review as proof that the species was a temporary social parasite on L. umbratus and mentioned that, because of the species definitions at that time, L. rabaudi was a strong candidate for a host species.

Yamauchi and Hayashida (1968) offered their own summary of the L. fuliginosus research as a preface to their findings on L. teranishii. Yamauchi discovered two mixed

Lasius teranishii/flavus colonies (Yamauchi and Hayashida 1968). The first discovery involved L. teranishii "dwarf workers" with full sized L. flavus workers. The pupae

Yamauchi brought back to the laboratory were "distinctly larger" L. teranishii workers.

The second mixed colony contained a L. teranishii queen. The pupae he collected contained L. teranishii workers. Yamauchi and Hayashida (1968) pointed out the morphological similarities (head broader than thorax) of L. spathepus and crispus queens to those of L. teranishii and fuliginosus and suggested those species were also social parasites.

Chthonolasius

The earliest reports on temporary social parasitism in Chthonolasius come from

W.M. Wheeler's (1917) descriptions of L. subumbratus in the Sacramento Mountains at

Cloudcraft, New Mexico. Wheeler observed many dealated L. subumbratus gynes

12 running around a rocky slope where many L. neoniger colonies lived. He turned over many of the stones and reported: "In one area, about 200 feet in diameter, nearly every nest had from one to five dealated subumbratus queens running about in the large shallow superficial chambers." The gynes ran in and out the chambers and when attacked by L. neoniger workers made "supplicatory movements with antennae" similar to the behavior of Formica consocians gynes entering F. incerta nests. The L. neoniger workers bit the antennae and legs of the L. subumbratus gynes but did not hold on for long periods.

After escaping, the gynes left the nest and hid under nearby stones or hid in another chamber of the nest. Wheeler observed L. subumbratus gynes pick up L. neoniger workers, carry them out of the nest, and then release them. The workers were not injured during this process.

The day after he made the above observations, Wheeler went to a different rocky slope and found many dealate L. subumbratus gynes under stones that covered L. neoniger and sitkaensis colonies. He offered three cases that indicated the gynes were accepted into the nest: (1) L. sitkaensis nest with a couple of subumbratus queens,

"lurking quietly in a little cavity at the edge of a mass of cocoons nearly filling a large, shallow superficial chamber."; (2) L. subumbratus gynes under stones that covered a L. sitkaensis colony. Again, the gynes were near the pupae. ; (3) L. subumbratus gynes surrounded by dozens of workers in the center of a pile of worker and female pupae.

Wheeler's interpretation of his findings was that L. subumbratus gynes had to acquire the brood odor to gain acceptance. After gaining the brood odor the gynes were "immune to attack by the host workers." Not suprisingly, he called for an investigation into the

13 chemical makeup of L. subumbratus, L. (Acanthomyops), and L. fuliginosus. In addition to these relatively detailed observations of the L. subumbratus founding stage, Wheeler also found two mixed L. subumbratus/sitkaensis colonies in the same area.

Wilson (1955) summarized the knowledge of the founding stage of L. umbratus and mixtus that Donisthorpe (1927), Gösswald (1938), and Hölldobler (1953) had accumulated. He reported that the normal hosts were L. niger and alienus and described details of how L. umbratus gynes enter a host colony. In both the field and lab, gynes attacked host workers outside of the nest and carried them in their mandibles as they moved around in the vicinity of the host nest. The host worker was usually killed and sometimes eaten by the gyne. Hölldobler (1953) interpreted this behavior to be an attempt on the part of the gyne to gain the host odor before attempting to enter the host colony. In some cases, host workers still attacked the gyne before she was accepted.

Wilson (1955) also described his introduction experiments with dealate L. umbratus gynes. He introduced the gynes to colony fragments of L. sitkaensis, alienus, and neoniger. He never observed an acceptance of the gynes by these species and did not observe a gyne carrying a host worker. To test if L. umbratus gynes join L. neoniger gynes after mating flights (mating flights of these species overlap) he put dealate gynes of both species together. All of his attempts failed. Wilson (1955) also reported two nest series that contained L. umbratus and niger workers collected in Ute Park, New Mexico and a nest series that contained both L. umbratus and alienus workers collected in Beatty,

PA. The series contained L. umbratus minims.

In his study of bog ants in southeastern Michigan, Kannowski (1959) observed

14 founding stage behaviors of L. minutus and speculiventris. He describes dealate L. minutus gynes in small chambers in loose soil just beneath the surfaces of mounds that were already occupied by large L. minutus colonies. He suggested that the L. minutus gynes were targeting other L. minutus colonies or possibly L. sitkaensis.

Kannowski saw L. speculiventris gynes forming chambers in the top layer of loose soil of L. minutus colonies and then seal themselves in. He interpreted this behavior as the gyne's attempt to gain the host colony odor so that she could enter the nest later without being attacked by host workers. L. speculiventris gynes that tried to enter through nest entrances instead of forming chambers were attacked by host workers.

The gynes "appeared to feign death and allowed themselves to be moved about on the nest surface." When released by host workers the gynes attempted to enter the nest. The gynes did not retaliate when bitten by host workers. Kannowski saw several of these gynes carried into the nests by host workers while others stopped trying to enter the nest and hid nearby in the vegetation. He did not see those that hid in the vegetation again.

Kannowski also described L. speculiventris alates leaving a mixed nest of L. speculiventris and minutus. Some gynes flew away while others mated on the nest surface and kicked off their wings. Other gynes exited the nest wingless, which

Kannowski assumed meant they had mated in the nest. Some of the gynes that mated on the surface or emerged wingless tried to form chambers on the surface of neighboring L. minutus nests and on their maternal colony.

Kannowski felt that L. speculiventris probably have hosts other than L. minutus because the species was discovered in habitats that L. minutus does not occupy (upland

15 forest and dry pasture). He suggested L. umbratus as an alternative host.

Kannowski also drew similarities between the L. speculiventris behavior and what

Wheeler (1917) observed with L. subumbratus. Specifically, gynes of both species will try to enter the colony directly or hide in some portion of the nest, presumably to gain the host colony odor.

In her summary of social parasitism at the Edwin S. George Reserve in southeastern Michigan, Talbot (1979) reported finding one mixed L. umbratus/alienus colony. She also offers that though she suspected L. minutus as a temporary social parasite, but there was no evidence offered for this.

Janda et al (2004) erroneously cite Collingwood (1979) as reporting that L. bicornis is a parasitic species. L. bicornis is described in the publication, but the species founding stage is not mentioned. On the other hand, Collingwood (1979) reported that single L. umbratus gynes found colonies by invading L. niger, alienus, or occasionally brunneus nests. He also reports that L. umbratus gynes "often wander over the surface of

L. niger nests, sometimes carrying a dead worker as a prelude to securing adoption."

Collingwood (1982) suggested that L. crinitus was parasitic and founded colonies like other members of L. (Chthonolasius), specifically, "by securing adoption by niger or allied species." He went on to say there was no evidence for this idea. Regarding , Collingwood (1982) reported that dealate gynes were often found wandering alone above ground in spring and "were thought" to found colonies by parasitizing L. alienus or niger. He then offered that "actual recorded instances are very few or dubious."

16 In his revision of European Chthonolasius Seifert (1988) stated: "All species are, as far as we know, temporary social parasites securing colony foundation in nests of the subgenus Lasius sensu stricto." He reported three known cases of parasitic behavior, proposed possible hosts for two species, and offered no comment on the other six species.

For known cases of social parasitism, Seifert offered the following: (1) the only known host of L. meridionalis is L. alienus; (2) the main host species of L. umbratus is L. niger and more rarely L. alienus, but also L. emarginatus is suspected and Collingwood (1979) names L. brunneus as occasional host species; and (3) three direct observations proved that L. alienus is the host species of L. jensi jensi. The species composition in other jensi sites suggest that alienus is the main and possibly the only host. In Bobrostan, Bulgaria,

September 10, 1982, a dealate L. jensi queen was found in the middle of an L. alienus colony on, which indicates that colony foundation occurred immediately after a mating flight.

Seifert's suggestions for possible host-parasite relationships involve L. mixtus and distinguendus. He offered the following: (1) the main and possibly only host of L. mixtus is probably L. niger. This is based on species composition in mixtus habitats. Beginning from September, many dealate queens may be found under stones and in other hidden places, but they are frequently observed to move above ground beginning from warm

November and winter days until spring. That could mean that mixtus gynes will hide after mating flight and will begin to search for a host colony while most ants are less active; and (2) the major host species of L. distinguendus is probably L. alienus which is often the dominant Lasius species at the L. distinguendus sites.

17 In a supplement to the revision, Seifert (1990) described a new species, Lasius viehmeyeri, but did not include information about the founding stage. He proposed that

L. citrinus probably uses L. brunneus as a host based on species compositions in L. citrinus territory and one museum specimen of a dealate L. citrinus gyne with a L. brunneus worker attached to one of her antennae.

Seifert (1996) made several statements about parasite-host relationships without providing citations or reasons for his statements. These statements included novel relationships for ten parasitic species listed here (parasite listed first): L. reginae - flavus; carniolicus - flavus and alienus; fuliginosus - umbratus, mixtus, brunneus, and niger; meriodionalis - psammophilus and niger; jensi - umbratus; bicornis - Lasius sensu stricto; citrinus - brunneus; umbratus - psammophilus; sabularum - niger; and mixtus - niger.

Janda et al (2004) used nine of Seifert's 1996 statements as the sole citation for the relationships they report. These relationships are used in the context of a phylogeny to discuss the evolution of social parasitism. Given the lack of citations for these relationships, the discussion of the evolution of social parasitism is flawed.

Seifert and Buschinger (2001) described pleometrotic colony foundation by L. meridionalis using L. paralienus as a host. This was the first time pleometrotic colony foundation was observed in any Lasius (Chthonolasius) species. The authors observed eight dealate gynes of L. meridionalis in a L. paralienus nest in northern in August

1998. This was the first direct evidence of L. paralienus as host to L. meridionalis. Prior to this discovery L. psammophilus was considered the main host.

18 Seifert and Buschinger (2001) suggested that pleometrosis might increase the colony foundation success of L. meridionalis. They also considered that the pleometrotic colony foundation might be the initial step for the development of oligogyny

(multiple queens that live apart from each other in the colony). In addition they offered that the L. (Chthonolasius) gyne head and mandible morphology is adapted for lethal biting and the enlarged Dufour's gland is probably the source for propaganda pheromones emitted to influence host workers.

Schlick -Steiner et al (2002) suggested that L. (Chthonolasius) gyne morphology indicates temporary social parasitism and report one observation of a dealate L. distinguendus cutting up a L. niger worker with her mandibles prior to colony foundation.

The gyne had caught the host worker in front of the host worker's nest just before entering the nest. The authors also described a mixed L. platythorax and distinguendus colony in Austria. The colony was under a log and when the nest was exposed both species' workers moved larvae into the nest. This was the first observation of L. platythorax as a host and supported the idea that L. distinguendus has low host specificity. The authors suggested that, given the distribution of L. distinguendus, other possible hosts are L. paralienus and alienus.

Schlick-Steiner et al (2003) stated that the 12 European species of Lasius

(Chthonolasius) are social parasites, apparently citing Seifert (1988). They reported the occurrence of L. bicornis in Slovenia for the first time and suggested that the most probable host is L. brunneus. No evidence for this is offered.

Dekoninck et al (2004) reported three mixed nests of L. umbratus and platythorax

19 in the Netherlands and one in Slovakia. They also reported observations of L. umbratus gynes carrying dying L. platythorax workers in their mandibles prior to entering the host colony. The authors suggested that L. platythorax serve as host to other Lasius

(Chthonolasius) species such as L.sabularum, meridionalis, and psammophilus.

Stemming from the fact that L. platythorax is an aggressive species of ant, Dekoninck et al (2004) discussed possible mechanisms of parasitic gyne entry. They offered two mechanisms: (1) chemical camouflage and (2) low temperature strategy. They suggested the chemical camouflage strategy for species whose mating flights overlap. workers are aggressive towards their own alates at the time of the mating flight so the authors suggested that the parasitic gynes could attempt to enter at the time when the host workers were preoccupied with their own gynes. Picking up a host worker would transfer the colony odor to the parasite and she could then enter. Apparently, L. meridionalis gynes do this when they enter psammophilus colonies in the Dutch and

Belgian dunes. For species whose flights do not overlap, the low temperature strategy is suggested. In this case, the parasitic gyne enters the host colony when worker activity is low. For example, L. mixtus gynes seek shelter after their mating flight, wait for the temperature to drop, then look for a suitable host. The authors suggest that L. sabularum could use this strategy with L. platythorax.

Out of all the literature reviewed, there are a total of six reports of the details of the founding stage of Lasius ants for three of the eighteen species mentioned. We lack details of temporary social parasite behavior in ants partly because it is difficult to observe the behavior in the field or laboratory. The parasite's entry into a host colony

20 happens relatively quickly so one has to be present at that moment. This is difficult because mating and the subsequent founding stage often relies on exact environmental cues (e.g. temperature, relative humidity, and wind speed) that are not easily predicted.

Host-parasite relationships can be established in the field by thoroughly examining an area (i.e. turning over rocks, rotting logs, digging into nests, etc.), which is labor intensive. Bringing the host-parasite system into the laboratory gives us an opportunity to observe interactions between host and parasite species. Unfortunately, this can also be time consuming. For example, a host colony must be in culture and a parasitic gyne collected at the time of the founding stage to observe the parasitic habit in the laboratory.

Collecting parasitic gynes at the time they normally behave as parasites relies on the same environmental conditions mentioned above and culturing a specific species of ants in the lab can require a great deal of time. Learning about social parasitism usually involves a considerable time investment, so it is not surprising to find a correlation between discoveries of social parasitism and the time spent searching a specific area.

Wilson and Hölldobler (1990) summarize this idea well with the following passage,

Most known parasitic species have been recorded exclusively from the temperate

areas of North America, , and South America. Almost certainly this

reflects at least in part the strong bias of ant collectors, most of whom reside in

these areas and devote a large part of their lives to a meticulous examination of

local faunas. Switzerland is the present capital of parasitic ants because Forel

and Kutter lived there. One-third of Swiss species are parasitic (Kutter 1969).

Forel and Kutter were both prolific ant biologists. The idea that discoveries of biological

21 phenomena happen in areas where researchers look is not foreign to field biologists. This concept is relevant to many areas of biology including the distribution of taxa and its implications on studies.

Dependence of measures of diversity upon taxonomic effort

Biodiversity hotspots are of particular importance because they may represent places where evolutionary processes generate species at high rates (Myers et al. 2000).

Discussions of regions of endemism or modes of speciation often rely on counting closely related species (or other taxa) in a region. Historically, species richness has been attributed to abiotic and biotic factors, but it has been widely suspected that species counts may reflect formalities of nomenclature and activities of taxonomists as much as actual biological phenomena (Issac 2004). Ease of travel to a region and a hospitable work environment might also lead to greater research efforts in some places than in others--inaccessible or inhospitable places are simply less well known (see BBC News

2006). Yet although we know that some areas are better studied than others, we do not know how the uneven distribution of taxonomic effort relates to concepts concerning the baseline of biological diversity in different locales.

The number of species found in an area relies implicitly on the crucial step of determining what constitutes a species. The task of defining species themselves falls to relatively few individuals. For example, at the end of 2001, William Morton Wheeler,

Felix Santschi, Auguste-Henri Forel, and Carlo Emery were responsible for 28 percent of the 2588 primary publications in ant systematics from which 11,079 species of ants were named (Table 1; Agosti and Johnson 2003). Defining species may be the most

22 challenging part of assessing species richness (Grissell 1999, Alonso and Agosti 2000), and factors such as a taxonomist’s personal philosophy can play a large role in the number of species described (Agapow 2004). The number of species concepts (22 found by Mayden [1997]) and disagreements over character choice commonly found in the literature suggest that personal philosophies may have a large effect on this assessment.

In addition, diagnostic characters may provide practical utility, but they have no necessary relationship to underlying philosophical principles of species identity (Hey

2006). Practical characters such as those found in a key are rarely the foundation of species descriptions. For example, distinctive coloration can be useful for recognition of a given species in the field (“there is the blue one”), but variation in color may be ignored when defining species taxonomically (“the six species of this genus differ in genital morphology”). Nonetheless, details about a taxonomist’s decisions regarding species identity are generally ignored when other researchers count those species for ecological purposes.

Here I examine how the number of taxa described in a region compares with the number of taxonomists describing that region’s taxa, as well as with abiotic factors

(precipitation, temperature, and area in square kilometers (km2). Because species themselves are not uniformly distinguished, criteria for identifying species are necessarily variable. I therefore also discuss the importance of an alpha taxonomist’s personal philosophy regarding species concepts and primary descriptions.

23 Predictors of species richness among ants

The Social Insects World Wide Web (www.antbase.org) provides a list of species and subspecific taxa described in countries or regions of the world; it also lists the author of the description. I counted the number of taxa described in a region (usually a nation), the number of authors working in the region, and the number of taxa described by each author within each region. I included subspecific ranks because I wanted to examine the importance of the effect of “lumpers” (those who tend to have broader, more inclusive species concepts) versus “splitters” (those with a tendency toward narrower, more restrictive concepts) on the primary foundation of biodiversity studies. The number of ants described from a region provides an indication of biodiversity that corresponds loosely in many cases to what conservation biologists see as general hotspots (Figure

2.1). I gathered annual average precipitation data and mean temperature data from the

Climatic Research Unit at the University of East Anglia in Norwich, ; the area (km2) of regions or countries is from a world gazetteer (Parsons 1982). I ran a stepwise multiple regression to examine the predictive power of area, average precipitation, mean temperature, and number of taxonomists describing species in a region. I included only those regions with at least 25 described taxa, resulting in a total of

84 regions and 13,857 taxa.

Area has some predictive power (r2 = 0.201; Figure 2.2a). Outliers in Figure 2.2a indicate the limitations of the environmental data for explaining observed diversity. For example, China (167 described taxa over 9,574,479 km2), Canada (41 described taxa,

9,976,137 km2), and Russia (237 described taxa, 17,075,400 km2) appear rather

24 depauperate, especially in comparison with smaller regions such as Costa Rica (248 described taxa, 51,090 km2). Some of this discrepancy might reasonably be explained by differences in mean annual temperature (-5 degrees Celsius [°C]) for Russia and Canada, for example, and more than 20 °C for Costa Rica. However, although a prediction based on area can be improved by adding mean temperature, the explanatory power is still weak

(r2= 0.282). Whereas Russia has a few taxa per unit area (Figure 2.2a), it has relatively many for its average temperature (Figure 2.2b). China remains low on both plots. New

Guinea and Indonesia appear to have high taxa counts relative to all three geographic variables. Temperature and precipitation (Figure 2.2b, 2.2c) alone were not useful.

The best single predictor for the number of taxa described in a region is the number of taxonomists studying in that region (Figure 2.2d; F< = 118, r2= 0.591, P <

0.0005). Taking this information into account, all reference points are more aligned with the pattern; departures from expectation are much more modest for Russia, Canada,

China, New Guinea, and Indonesia (Figure 2.2d). It is interesting that Switzerland, which is so prominent in myrmecology in general, is never an outlier in any plot. After accounting for the taxonomists themselves, the next best predictor is temperature, confirming the assumption that warm areas are more species rich than cold areas. Adding the average temperature of the region improves the model (r2= 0.629), as does using a stepwise procedure to add area (r2 = 0.657). Precipitation does not add significantly to the model. Area explains 20% percent of the variance when considered alone, but more taxonomists work in larger areas (r2= 0.26, p < 0.05), and area contributes only about 3 percent to the three-variable model. Indeed, area and temperature together contribute less

25 than 7 percent more to the predictive power of the number of taxonomists alone. The effect of taxonomic effort is apparent in a comparison of New Guinea (the island) and

Papua New Guinea (the country), which appear high and low, respectively, relative to the expected value (Figure 2.2c), unless the number of taxonomists is taken into account

(Figure 2.2d).

Limitations of the data

I chose to focus on ants because the relevant data are easily available. Of course, ants are not an unbiased indicator of biotic diversity, often being speciose in environments that may be depauperate in other ways, such as xeric habitats. The study I conducted could be repeated with other taxa (birds come quickly to mind; see especially

Orme et al. [2005]), and I would expect that different groups might show different absolute patterns, although all would reflect at some level the hotspots already identified by various researchers (Myers et al. 2000). The point of this study is that the intensity and quality of the community of taxonomists’ concentration in a particular region will bias any data set. Data on some taxa may be less biased than others because more people are competent at identifying species and general coverage of areas is good (as would be true for birds), but even these data sets will depend ultimately on the decisions made by the taxonomists who study the species of a region.

The data I present have limitations. Geographic data like those reported here capture large areas poorly. Political units are not homogenous and may not be comparable. Average annual temperature and precipitation data may be misleading, especially for large regions with many different habitats (e.g., the United States). Also,

26 counting species described from a region may not reflect closely the number of species present in that region. For example, describing species from Costa Rica does not mean they do not occur in Nicaragua, and the original description often includes a range, whereas a holotype is unique. Nonetheless, the data I used were the best available at the scale at which this study was conducted, and it is evident that measures of biodiversity are indeed confounded by sociological features of the scientists themselves.

Taxonomic effort, lumping and splitting

Some correlation between species diversity and number of taxonomists is to be expected, because taxonomists tend to gravitate to regions of high diversity. However, we must ask ourselves if the difference between numbers of taxa described in Costa Rica

(248 taxa of ants described) and in Nicaragua (13) is attributable mostly to biological differences between the two countries. Tourism and research institutions permit hundreds of biologists to go to lowland Costa Rica annually, increasing the species counts, whereas relatively few biologists go to Nicaragua, whose lowland landmass is much more extensive but much less well known. Indeed, once a region has been identified as being of special interest, it is likely to attract a great deal more attention than a comparable, but lesser known region, amplifying the false impression of a difference between the two.

There is no clear way to correct for this “time of effort,” as ecologists often do before interpreting their sampling results (Delabie et al. 2000).

In many regions, a majority of the taxa were described by only a few people

(particularly Emery, Forel, Santschi, and Wheeler). Thus, an individual researcher may greatly influence the primary data of diversity, depending on the manner in which he or

27 she distinguishes new species. One way to assess an individual’s personal philosophy is to examine the number of taxa (species, subspecies, race, and variety) he or she described. For example, naming 45 varieties in a region probably indicates a tendency to

“split” (at least in ants), while never dropping below the species level indicates a tendency to “lump.” Differences can be extreme: among North American mammals, every population of grizzly bears has been designated as its own subspecies (Hall 1981), even those known from a single specimen. It is hard to think of a more generous foundation for studies of biodiversity.

Differences in species concepts appear not only between taxonomists but also between phases of a taxonomist’s career. For example, Forel, Emery, and Wheeler became more exacting about minor differences in specimens later in their careers, when they relied more on specimens from museum cabinets and less on field studies of live in their natural habitat (Creighton 1950, Buhs 2000). Examining Forel’s history, we see that he described the majority of taxa in Colombia (Figure 2.3), and his use of ranks below species was prolific (Figure 2.4). While Colombia is recognized as a very diverse region (Myers et al. 2000), the number of taxa described from there may be inflated because Forel’s species concepts led to finer distinctions than other taxonomists would make (Creighton 1950). The problem is not unique to Forel's descriptions. If we examine Santschi's treatment of Congo, the same difficulty is apparent (Figures 2.5, 2.6).

It is not yet clear how regions would fare if they could be evaluated on similar taxonomic criteria.

Often, a researcher is more conservative in designating varieties when he or she

28 has direct experience in the field, apparently discounting small differences if the ecological milieu, behavioral repertoire, and other factors are consistent from one location to another. As some researchers become limited to working from preserved specimens, they are more attentive to small differences they would have discounted if they had field experience with the animals. Creighton (1950) wrote of Emery and Forel:

“Both men became increasingly absorbed with the study of cabinet specimens from exotic sources. As a result the recognition of subspecies came to depend less and less on the behavior of the in the field and more and more on the structural characteristics which it showed.” Buhs (2000) wrote, “Later in his career W.M. Wheeler began using the same pentanomial naming system he once opposed. This change in his practices coincided with a shift in his studies from North American ants to exotic species.”

Importance of revisionary systematics

Of course, the solution to problems produced by noncomparable species concepts is reexamination and revision by other, later authorities. In taxonomic work, the best platform is not the static shoulders of a few giants, but a pyramid of ordinary researchers revising each other’s work. Revisionary work adjusts earlier concepts, sometimes dramatically. Creighton revised Smith’s (1947) 742 North American taxa, dissolving into synonymy 22 percent of them. Later workers are likely to validate some of these and elevate them again. With successive efforts, a general community standard will eventually take shape, and the tally of taxa should converge on a reliable estimate. This means that large-scale ecological agendas need to support not only alpha but also revisionary systematics on taxa of special interest.

29 It is widely reported that we have a general crisis in the training of new systematists (Celebuki and Farris 1990, Wheeler and Cracraft 1997, Wheeler 2004). Not enough people are qualified to interpret classical scholarship, which means that we will not be able to revise our taxonomy soon, leaving us with whatever baseline the original authors described. It would serve us well to have a new researcher reevaluate Forel’s

Colombian ants, as Creighton did for Smith. It might seem that removing the personality of the taxonomist altogether would be a good idea, and we have tools that might permit that. Modern DNA taxonomy is useful for labeling specimens whose names are already known, and DNA sequencing is met with much enthusiasm today (Tautz et al. 2003).

However, such data are not likely to completely substitute for formal revisionary systematics for a number of fundamental reasons (see Lipscomb et al. 2003). For example, molecular studies rely on exemplars of species described through traditional means, and do not reexamine the original designations, and rarely do they include as many individuals as contributed to the original description (but see Pons et al. [2006] for an example of a traditional type of sampling in a DNA study). Indeed, it is possible that as we increasingly use DNA sequencing to enhance the power to discover small differences among species, we will also amplify problems associated with oversplitting and lose track of the more holistic species concepts that are the foundation for all the rest of biology. Our best future lies in advancing classical systematics hand in hand with modern DNA methods.

30 Conclusion

Studies of biodiversity rely on lists of species found in various regions. Regional lists may differ, in part, because taxonomic effort is spread unevenly across regions and across individual researchers. Defining species remains a challenging task, especially in groups that are not well known, and species concepts are necessarily tailored for specific groups. Because taxonomists’ personal philosophies differ about what constitutes a species, it is critical that well-trained systematists revisit earlier work. Modern tools of

DNA analysis are helpful in many ways, but they do not replace classical scholarship.

Revisionary systematics is rarely considered to be an important part of studies of biodiversity. However, our counts of species may very well be unreliable without frequent reevaluation and expansion of the character and distributional data upon which the taxonomic concepts are based.

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40 Author Number of publications

Wheeler WM 200 Forel A 176 Santschi F 176 Emery C 163 Brown WL Jr 104 Donisthorpe H 94 Smith MR 58 Kempf WW 58 Menozzi C 55 Mayr G 43 Karavaiev V 36 Bolton B 34 Smith K 36 Taylor RW 32 Cole AC Jr 31 Weber NA 31 Baroni Urbani C 30 Cagniant H 30 Borgmeier T 27 Ruzsky M 27

Table 2.1. Most prolific authors of ant systematic papers

41

Figure 2.1. Number of ant taxa (species and subspecific ranks) described from different nations, according to Antbase. Nations specifically indicated are, by region, Canada (41), the United States (1013), Nicaragua (13), Costa Rica (248), Colombia (233), Brazil (1266), Switzerland (91), Algeria (206), Egypt (57), Congo (95), Zaire (455), South Africa (515), Russia (237), China (169), India (505, Indonesia (903), New Guinea (657), and Papua New Guinea (41).

42 Figure 2.2. Relationship between number of taxa described from a region and (a) the region’s area (square kilometers; r2= 0.201), (b) the mean temperature of the region, (degrees Celcius; r2= 0.001), (c) the region’s average annual precipitation (millimeters; r2 = 0.059), and (d) the number of taxonomists describing taxa in the region (r2 = 0.592). The numbers in the figure panels refer to the following countries: (1) Brazil, (2) United States, (3) Indonesia, (4) New Guinea, (5) India, (6) Costa Rica, (7) Colombia, (8) Switzerland, (9) China, (10) Canada, (11) Russia, (12) South Africa, (13) Algeria, (14) Egypt, (15) Papua New Guinea, and (16) Zaire

43

(Figure 2.2 continued)

44

(Figure 2.2 continued)

45

(Figure 2.2 continued)

46 (Figure 2.2 continued)

47

Figure 2.3. The number of taxa described from Columbia, by author.

48

Figure 2.4. Classifications of taxa Forel described from Columbia.

49

Figure 2.5 Number of taxa described from Congo, by author.

50

Figure 2.6. Classifications of taxa Santschi described from Congo.

51 CHAPTER 3

FOUNDING STAGE OF LASIUS (ACANTHOMYOPS) AND THE INCIDENCE OF SOCIAL PARASITISM.

Introduction

Colony foundation is a critical and dangerous stage of the ant colony life cycle

(Hölldobler & Wilson 1990, Bourke and Franks 1995). The most basic categorization of ant colony founding strategies is independent versus dependent. Independent colony founding involves one or more foundresses rearing their first cohort of workers without the help of a worker force. Dependent colony founding involves one or more foundresses rearing their first cohort of workers with the aid of a mature worker force. The mature worker force can be from the maternal colony or, in the case of social parasitism, from a different colony of the same or a different species. Foundresses must navigate , desiccation, mate location, mating, and nest site location to begin a new colony. High foundress mortality is the rule regardless of strategy and a variety of modes is found among ants (Hölldobler & Wilson 1990).

Pirate (also known as dulotic or slave-making) species rely permanently on a host species’ work force. Pirate workers raid host colonies for larvae and pupae that subsequently eclose and perform ordinary colony tasks (e.g. nest maintenance, tending

52 brood). Temporary parasitism involves a relatively short period of reliance on the host, always at the founding stage (Hölldobler and Wilson 1990).

Details of temporary social parasitism vary among ant species. In all cases, a fertilized foundress is adopted by a different species’ workers. Gaining entry to the host colony may involve some form of force or a conciliation strategy. The host queen is usually killed by the parasitic foundress or possibly by host workers. At some point after gaining acceptance by the host workers, the parasitic foundress will lay her own eggs and rear them to maturity with the help of the host workers. The host workers will die without replacement, but for some period the nest will contain both host and parasitic workers (Hölldobler and Wilson 1990).

Lasius sensu lato is comprised of 116 species in six subgenera and is one of the most abundant terrestrial in the north temperate zone. Four Lasius subgenera

(Acanthomyops, Austrolasius, Chthonolasius, Dendrolasius) contain parasitic species

(Janda et al. 2003). The free-living subgenera (Lasius sensu stricto and Cautolasius) are commonly hosts of the parasitic Lasius species. A few studies of social parasitism in

Lasius suggest a variety of modes of entry into the host colony (Faber 1967, Rigato and

Sciaky 1987, Sciaky and Rigato 1987). Lasius (Austrolasius) reginae gynes use a conciliation strategy (approach host colony and initiate contact with workers; Faber

1967) while Lasius (Chthonolasius) umbratus gynes are more aggressive (kill host worker and rub corpse on her body apparently resulting in transfer of the host colony odor; Sciaky and Rigato 1987, Rigato and Sciaky 1987).

53 Lasius (Acanthomyops) species have long been suspected as temporary social parasites of other Lasius ants based on evidence from only two of the 16 species (Wing

1968). For a summary of Wing’s reports on mixed colonies see Table 3.1. Details of L.

(Ac.) founding stage are sparse and have not been investigated except for two studies

(Tanquary 1911, Cover and Sanwald 1988.) Lasius (Ac.) murphyi is considered a parasite of L. neoniger based on mixed colonies and observations of L. murphyi gynes entering L. neoniger nests (Talbot 1963, Sanwald 1964-1965). In addition, L. murphyi gynes located and killed L. neoniger queens in 15 of 37 laboratory introduction experiments (Cover and Sanwald 1988). Lasius (Ac.) latipes have been found in mixed colonies with Lasius (Lasius) neoniger (Wing 1968) and have been observed entering neoniger colonies (Gary Umphrey personal communication). Lasius (Ac.) latipes have also been found in mixed colonies with L. alienus and in Tanquary’s (1911) introduction experiments, L. alienus workers accepted L. latipes gynes in 2 of 14 trials. Tanquary also observed L. latipes gynes accepted by L. interjectus workers in the laboratory (1 of 2 trials) (Table 3.2 for all species used in Taquary's trials). Lasius (Ac.) claviger was considered socially parasitic by Talbot (1979) due to reports of gynes overwintering above ground in groups (Wing 1968); however she did not report mixed colonies or observe the parasitic habit of this species. Although two authors (Smith 1934, Wilson

1955) collected L. claviger near other Lasius species, Wing (1968) did not consider these

54 cases as evidence for mixed colonies. No observations of the founding stage of Lasius

(Ac.) interjectus are published and due to the species’ habit of nesting near buildings, mating flight data are skewed by mid-winter flights occurring in warm buildings (Wing

1968).

Wing (1968) proposed eight non-mutually exclusive ways that new Lasius

(Acanthomyops) colonies are established. What follows is my interpretation of Wing's ideas (Figure 3.1). Acanthomyops foundresses are either parasitic on other ants or non- parasitic and might work alone or in groups. If not parasitic, foundresses found claustrally, forage during the founding stage (= semiclaustral), or start colonies near a population of for access to their honeydew. If parasitic, foundresses might enter host colonies during the early spring or soon after mating flights in summer - fall.

Foundresses might cooperate with a host queen, carry a host worker in her mandibles to gain host odor then enter, or lure away host workers to begin a colony with her.

Here, I investigate the founding stage of Lasius (Acanthomyops) latipes (alpha and beta forms, claviger, and interjectus via field observations and laboratory experiments.

Methods

Collecting gynes

I spent the following dates at the Edwin S. George Reserve, Livingston County,

MI (approximately 42° 28' N, -84° 00' W): 2005; 20 – 22 May, 9 July, 15 August - 16

September. 2006; 2 - 4 July, 18-23 and 28-30 June, 1-6 July, 7-11 and 14 – 31 August,

55 1-19 September. 2007; 23 -25 May, 1 September – 13 October. In 2005 I located many

Lasius colonies on the 1,800 acre reserve and I monitored these colonies for mating flight activity and searched for dealate Lasius gynes walking on the soil surface. I continued this protocol in 2006 and 2007, discovering more Lasius colonies and many dealate gynes subsequently used in introduction experiments.

Introduction experiments

Dealate Lasius interjectus, latipes, and claviger gynes were introduced to arenas containing putative host workers, brood, and, in some cases, a host queen of L. neoniger, alienus, claviger, latipes, murphyi, umbratus, or minutus. Of the 117 introduction experiments, dealate gynes were used once each except one L. latipes gyne that was used twice. Host colonies were used once each except a L. neoniger colony that was used three times with different gynes (L. latipes, murphyi, and latipes) and a separate L. neoniger colony that was used twice with different gynes (L. latipes and interjectus). The dealate gynes were placed into the host arena using featherweight forceps (BioQuip). I used square 9 x 9 cm Petri dishes lined with moist paper towel when only host workers and brood were available and 14 x 14 cm plastic Tupperware containers lined with moist soil when workers, brood, and a host queen were available. In all cases, the host species settled in a place in the arena (chamber in soil, under paper towel) prior to introduction of the parasite. The arenas were monitored for host aggression (host workers biting, pulling, and spraying body parts of the parasitic gyne), gyne aggression (parasitic gyne biting or biting and spraying the host workers), and acceptance (host workers tend parasitic gyne) during the first hour of introduction and at least once a day subsequently.

56

Forced associations

Lasius interjectus gynes were held in solitary confinement or in groups of 2, 3, or

4 and monitored for survivorship to investigate potential benefits of grouping I observed in the field. Gynes were housed in 9 cm diameter circular Petri dishes lined with moist paper towel. Half of the gynes were fed while the other half were not to test whether L. interjectus is capable of claustral founding (see Table 3.3).

Overwintering

To test if Lasius interjectus is an independent, claustral founding, species, I used a protocol that induces egg-laying in a known independent claustral species, (Stefan Cover personal communication). Forty Lasius interjectus gynes were cooled in a refrigerator for 10 weeks then returned to room temperature and monitored for the presence of eggs until gyne death.

Grouping Experiments

Initial Hour of Gyne Behavior

Four Lasius interjectus gynes were placed in arenas that contained four shelters, four sources of food (Bhatkar and Whitcomb 1970), and four platforms (rocks) (see

Figure 3.2). Arenas were soil-lined 35 x 51 cm Sterilite plastic containers (30 L), and shelters were 5 x 5 cm pieces of glass covered with red cellophane and propped on a 1.5 mL vial cap. Gynes (N = 108) were used in only one trial each (N = 27).

I observed the initial hour of the experiments and recorded gyne-gyne interaction

57 (antennation, short and long exploration), location of gyne-gyne interaction, movements of gynes around the arena, climbing (wall of arena, shelter, perch), antennal flagging, and cleaning (see Table 3.4 for description of behaviors). At a minimum, I recorded observations every two minutes. After one hour of observation, the gynes were provided food (Bhatkar and Whitcomb 1970) and then every other day for the duration of the experiment.

Daily Monitoring

I observed 18 of the original 27 arenas once a day, each day at a different time

(from 08:00 to 20:00), to monitor whether gynes were grouped or solitary as well as their location in the arena. Each of the 18 arenas had two, three, or four gynes when daily monitoring began. I stopped observing an arena when only one gyne was alive. Trials began on different days, therefore I have unequal number of days censused per trial

(Maximum number of observations/arena = 31, Minimum number of observations/arena

= 7) .

Results

Field Results

Mixed interjectus/claviger colony

I found an Lasius (Acanthomyops) colony nesting in a 2.5 meter tall stump on 30

August 2006 on the ESGR and identified workers as L. (Ac.) claviger. Subsequently I observed the colony during a mating flight involving males and females. The alate gynes were L. (Ac.) interjectus and the workers L. (Ac.) claviger (Stefan Cover Museum of

58 Comparative Zoology verified my identifications). This marks the first mixed colony involving two Lasius (Ac.) species.

Mixed Lasius claviger/alienus colony

On 13 October 2007 I discovered a mixed colony of Lasius (Lasius) alienus and

L. (Acanthomyops) claviger at the edge of a swamp on the ESGR. Workers from both species moved brood from the light when uncovered. Both species worked quickly and showed no aggression towards each other or the brood.

Lasius interjectus entering Lasius latipes colony

On 19 September 2007, I discovered many Lasius (Acanthomyops) interjectus gynes entering a L. (Ac.) latipes colony that had released alates earlier in the day. The gynes ran to the nest where they encountered L. latipes workers that attacked them.

Workers held the gynes down by biting and pulling their legs. Gynes struggled against the attacking workers and some broke free and ran into the L. latipes nest. I surveyed the area the next morning and found many dead L. interjectus gynes lying on and around the nest. This marks the first account of L. interjectus founding behavior from the field.

Four parallel Lasius claviger/alienus colonies

On four occasions I found Lasius (Acanthomyops) claviger and L. (Lasius) alienus workers under the same log. The workers were not interacting; however they were within 1 cm of each other. It seemed they were living in parallel, in some cases separated only by a thin layer of galleried decayed log.

Laboratory Results

59 Introduction experiments

A total of 117 introductions resulted in 39 acceptances (33%) of a foreign gyne by putative host workers (Table 3.5). The greatest percentage of acceptances (84%, 16 of

19) was Lasius interjectus gynes by L. claviger workers. All host species had at least one acceptance of Lasius interjectus gynes except L. neoniger. All host species were aggressive toward the foreign L. interjectus gynes (biting and pulling legs/antennae/mandibles and curling up to spray), and the gynes retaliated with attacks on the host workers (biting and spraying) of all species except L. claviger.

Both the alpha and beta forms of Lasius latipes were accepted by L. neoniger workers, and both forms killed L. neoniger queens (64%, 9 of 14 introductions). Host worker aggression (same form as seen with L. interjectus) was not returned by the gynes.

L. alienus did not accept L. latipes in any case.

One Lasius claviger gyne was accepted by L. alienus workers after they initially avoided her. When the host workers and L. claviger gyne encountered each other, the host workers would run away. I observed host worker aggression in one trial; however the L. claviger gyne was not aggressive in return. Host worker avoidance of the L. claviger gyne was much more common.

Forced associations

Gynes that did not eat died within 20 days of the start of the experiment while fed gynes lived up to 253 days. Gynes that started in groups of four outlived those that started alone (Figure 3.3, p < 0.03, Kaplan-Meier [1958]) or in groups of 2 (Figure 3.3 p

< 0.04, Kaplan-Meier[1958]).

60 Overwintering experiment

No gynes laid eggs after the 10 week cooling period.

Grouping Experiments

One hour observations

The most frequent behaviors observed during the first hour were flagging and cleaning with a total of 331 and 142 events recorded respectively (flagging = 13 ± 9 events per trial; cleaning = 6 ± 4 events per trial). Also, gynes were frequently observed climbing on rocks or on the walls of the arena (102 and 33 times respectively) but rarely hid under the shelters or any other object in the trial. Another behavior frequently observed was the egg laying position (94 times). Interactions between two or more gynes, including antennation and exploration, were less frequent than other behaviors; however they occurred at 12.2 ± 11.1 minutes from the beginning of the observations.

Flagging events were frequent early in the trial and decreased over time (Number of flagging events = -0.019 x minutes + 16.033, r = - 0.602, model F1,58 = 32.99, df = 1, p

< 0.0001, Figure 3.4). The decrease in flagging events over time coincided with an increase in the number of interactions over time (Number of interactions = 0.044 x minutes + 4.342, r = 0.258, F1,58 = 4.136, df = 1, p = .047, Figure 3.5). Although interactions increased over time, the estimated curve in Figure 3.6 suggests a decrease in interactions 30 minutes (Number of interactions = 0.783 + 0.388*minutes -

0.006*minutes2, r square = 0.329, p < 0.0001

61 Daily monitoring

Gynes tended to group. The number of trials per day with no groups (Mean = 5.4

± 1.2 trials) was significantly lower than the number of trials with queens grouped (Mean

=7.8 ± 1.3 trials; Wilcoxon matched pairs test, p=0.0001). Groups of gynes tended to stay in the same location for many consecutive days (7 ±4 days) (Table 3.6).

Gynes used different sites of the arenas to form groups, including under rocks, under shelters, under pieces of aluminum foil used to place food, and in the corners of the arena. As there were four of each of these, there were 16 different sites where gynes could be found, so the probability of finding them grouped in one of these sites would be

1/16. I used this figure to test for the probability of finding them two consecutive days grouped in the same place using a G-test (Table 3.6). In most cases, gynes were grouped in the same site for more consecutive days than was expected by chance (Table 3.6, p <

0.001 in all cases but one). However, gynes grouped in many sites (Table 3.6), and all sites were used at least once for some of the queens of some trial.

The propensity to aggregate with regard to the number of gynes in a trial was also considered. When only two gynes were in an arena, they grouped more than expected but not significantly (Table 3.7) (tests based on truncated Poisson distribution,

Chi-Square = 0.462, df = 1, p = 0.4965). In three-gyne trials, they were found solitary more than expected, formed groups of two less than expected, and formed groups of three more than expected (Table 3.7) (tests based on truncated Poisson distribution, Chi-Square

= 11.415, df = 2, p < 0.003). Although not significant (tests based on truncated Poisson

62 distribution, Chi-square = 7.521, df = 3, p =0.057), four-gyne trials showed fewer solitary gynes than expected and more grouping than expected especially groups of four (Table

3.7).

Discussion

Field and laboratory observations of Lasius interjectus, claviger, and latipes gynes and workers revealed previously undocumented host-parasite relationships and founding stage details of Lasius (Acanthomyops). These findings doubled the knowledge of social parasitism in L. (Acanthomyops) and allow a much more thorough discussion of the evolution of social parasitism in an ecologically dominant genus of ants, Lasius.

Findings are discussed in the context of Wing's (1968) hypotheses for the founding stage of Lasius (Acanthomyops).

Non-parasitic

Claustral or Non-claustral

High mortality of unfed Lasius interjectus gynes indicates that this species requires nourishment during the founding stage and does not found claustrally contrary to Wing's hypothesis. Because fed gynes lived much longer, but failed to oviposit, it is unlikely that L. interjectus is a semi-claustral founding species. The hypothesis that gynes burrow down to an colony to receive nourishment was not tested and has not been observed in the field.

63

Parasitic

Number of queens

Preference for groups in Lasius interjectus

Grouping experiment results of frequent flagging, cleaning, and climbing onto elevated surfaces by L. interjectus gynes matched my observations in the field. Hundreds of gynes alternated between running on the soil surface, climbing up blades of grass, flagging their antennae, and cleaning. The gyne-gyne interactions in the laboratory were also similar to my field observations. The gynes would encounter each other, antennate/interact then continue running, in some cases, one gyne following close behind another. The laboratory results support my interpretation that the gynes I observed in the field were seeking each other. Gynes began flagging at high rates, apparently seeking other gynes, but this rate decreased as interactions (discoveries) increased (Figure 3.4).

As gynes remained in associations, new interactions eventually declined (Figure 3.5 and

3.6). Figure 3.6 clearly shows an increase in interactions followed by a decrease in new interactions as gynes remained in associations. Note the much greater fit of Figure 3.6 curve (r square = 0.329, p < 0.0001) compared to Figure 3.5 (r = 0.258, p = 0.047

Although groups of gynes show some site fidelity (stayed in the same location over several days), they were also found grouped in other locations. This indicates they formed groups again even after departing the original site.

The lack of grouping in two-gyne trials indicates that gynes prefer more than one partner. In the three-gyne trials, only groups of three were observed more than expected,

64 indicating a preference for larger groups. Similarly, the four-gyne trials suggest a preference for larger groups, especially groups of four. Perhaps finding a single gyne (as in two-gyne trials) is not enough to stop searching, whereas larger groups are more satisfactory.

These results suggest that gynes prefer to form groups with more than one other gyne. In two-gyne trials, ants were not found together significantly more than expected, suggesting that the ants continued to look for others. In contrast, three-gyne trials suggest that the ants stopped looking for others when two were located. Similarly, the four-gyne trials further suggest more grouping than expected, especially groups of four. Lasius interjectus gynes need nourishment during the founding stage, and there is an advantage to grouping as shown by Figure 3.3. The gynes could gain nourishment either by foraging or via host worker trophollaxis. The increased survivorship associated with grouping (Figure 3.3) and the preference for grouping (Table 3.6) suggests that, in the field, groups of gynes enter a host colony, as observed with L. interjectus entering L. latipes, or they group together during the winter and enter a host colony in the spring as hypothesized for L. claviger (Wing 1968). The lack of oviposition by the cold-treated gynes further suggests that L. interjectus is not an independent claustral founding species.

These findings refute Wing's hypothesis of independent claustral founding.

Field observations of many Lasius interjectus gynes entering a L. latipes colony, greater survivorship of groups of gynes and the preference for grouping seen in the laboratory indicates that this species founds a new colony with multiple gynes. Although an individual gyne was accepted by a small group of host workers in the laboratory,

65 colony founding by one gyne seems unlikely when all evidence is considered.

Lasius latipes

Single L. latipes gynes gained acceptance in small laboratory host colonies and in several cases found and killed a host queen. On the other hand, field observations of groups of gynes entering L. neoniger colonies (Umphrey personal communication) suggests this species also founds colonies with multiple gynes.

Lasius claviger

Introduction experiments indicate a single L. claviger gyne can be accepted by L. alienus workers. Alternatively, L. claviger gynes overwintering in groups (Wing 1968) suggests a group founding strategy. Further data are required to address this question adequately.

Timing of entry to host colony

Field observations of Lasius interjectus and latipes gynes indicate these species enter a host colony soon after mating. Field observations of L. claviger gynes overwintering in groups suggests an early spring entry, however data are lacking.

Details of parasitic habit

Gyne carries host worker in her mandibles

I did not observe any species' gynes carrying a host worker in her mandibles prior to entering a field colony or during a laboratory introduction experiment.

Gyne cooperates with host queen

In one introduction experiment a Lasius latipes gyne located the host queen (L. neoniger), did not attack her, and then stood next to her for 15 days until she died. The L.

66 latipes gyne never exhibited any aggressive behaviors toward the host queen. Due to the short duration of the trial, it is difficult to consider this one case as support for Wing's hypothesis of a parasitic gyne cooperating with a host queen.

Gyne lures host workers away

A Lasius (Acanthomyops) gyne luring away host workers from their colony has not been observed in the field. In the laboratory, L. latipes and murphyi gynes locate and kill a host queen and do not interact with host workers unless attacked. Given these findings, luring host workers away from the maternal nest is unlikely for L. murphyi and latipes. Observations of L. interjectus gynes fighting their way into a L. latipes colony and gyne aggression toward host workers suggests that L. interjectus does not use a luring strategy. The discovery of four L. claviger/alienus parallel colonies, the lack of L. claviger gyne aggression towards L. alienus host workers and the eventual acceptance of a L. claviger gyne by L. alienus workers in the laboratory suggests that a luring strategy is possible for L. claviger. Alternatively, the laboratory observations could indicate a strategy similar to L. reginae , in which the parasitic gyne has non-aggressive interactions with host workers outside the nest then enters and kills the host queen (Faber 1967).

Host-parasite relationships

The L. claviger/alienus mixed colony confirms Talbot's (1973, 1979) assertion of this relationship. Wing (1968) considered two putative pieces of evidence from pinned specimens as unconvincing for a host-parasite relationship between L. claviger and alienus. He also reported large groups of L. claviger dealate gynes overwintering above ground until April when they "suddenly disappeared" and suggested the gynes' relatively

67 high cold tolerance might allow them to gain acceptance in a lethargic host colony during the early spring. Wing (1968) reported E.O. Wilson and W.L. Brown attempted introduction experiments with L. claviger independently, both trying artificially cooled and room temperature trials. Neither observed acceptance of L. claviger by L. alienus, neoniger, nearcticus, or flavus. Of my five L. claviger/alienus trials attempted, one L. claviger gyne was fully accepted by L. alienus workers. This required no cooling of the host workers. The gyne was collected with many others prior to the mating flight. When used for the experiments gynes were dealate. Although it is possible dealates mated with their brothers at the nest and were inseminated, I believe they lost their wings during collection. Regardless, Jemielity et al (2006) induced post-mating behavior, oviposition, in virgin Lasius niger gynes, corroborating Wheeler's (1906) opinion that dealation switches gynes into founding behavior Tanquary's (1911) experiments were conducted mostly with artificial dealates because of Wheeler's (1906) assertions. The combined evidence of this study firmly places L. claviger in the parasite category and refutes the notion that the gynes rely on a lethargic host colony for entry.

L. interjectus can now be considered a parasite of L. claviger due to the discovery of a mature mixed colony containing hundreds of L. interjectus alates and L. claviger workers. Supporting these findings are the results of introduction experiments in which

L. interjectus gynes were not aggressive towards attacking (biting and pulling foreign gyne legs, antennae, mandibles; and curling up gaster to spray) L. claviger workers and an exceptionally high percentage (82%) of acceptance. These findings place L. interjectus in the socially parasitic category and leave open the possibility of Wing's (1968)

68 "essentially parasitic" mode of a parasitic gyne cooperating with a host gyne.

The Lasius interjectus / claviger mixed colony is an interesting relationship because it suggests a long-term social parasitism rather than a temporary social parasitism as has been hypothesized for Acanthomyops. The presence of L. interjectus alates and L. claviger workers suggests an L. interjectus queen living in a queen-right L. claviger nest.

This could be the result of an L. interjectus foundress being accepted by a group of L. claviger workers without killing the host queen. The L. interjectus gyne could remain away from the L. claviger queen in a separate part of the nest or could share a chamber.

Given that only four L. (Acanthomyops) queens in mature colonies have been collected

(EO Wilson personal communication), including one L. claviger by Raczkowski in MN in 2005), definitive statements about the number of queens in Acanthomyops nests are impossible. However, if multiple queens exist in claviger nests, another scenario for the mixed interjectus /claviger colony is that an interjectus gyne enters the claviger colony and kills one queen, is accepted, and then relies on the workforce generated by a different queen. Regardless of mode of acceptance the foundress will lay eggs: the caste of these eggs could be sexual and/or worker. In this case, the solely claviger workforce suggests the interjectus queen is not producing a worker caste or maintains workers in the interior of the nest.

Lasius interjectus can now be considered a parasite of L. latipes due to the discovery of L. interjectus gynes entering a L. latipes colony and acceptance of L. interjectus gynes by L. latipes workers in introduction experiments despite both host and gyne aggression (biting and curling up gaster to spray). The field discoveries support

69 Wing's (1968) hypothesis that several parasitic gynes could enter the same host colony after their mating-flight.

Overall, the introduction experiments indicate that L. interjectus has a potentially broad host range within its own genus. Of the seven species used as a host in the introduction experiments, only L. neoniger did not accept the L. interjectus gynes. Host worker aggression toward the foreign gyne and gyne aggression toward the host workers was consistent with what was described above for other hosts and parasites. In no cases did I observe a parasitic gyne pick up a host worker and carry her around as Wing hypothesized.

Lasius latipes alpha and beta forms

Lasius latipes introduction experiments with L. neoniger revealed that both forms

(alpha and beta) seek out a host queen and kill her. The alpha form, considered a claviger x latipes hybrid by Wing (1968) and Umphrey and Danzmann (1998), behaves as a parasite refuting the hypotheses by those authors that the alpha form is rare because the gynes lack the behavioral specializations required to enter a host colony. Lasius latipes

(beta) gynes were not aggressive when attacked while the alpha form retaliated against host worker aggression 33% of the time. The beta form behavior was very similar to the strategy that Cover and Sanwald (1988) observed with murphyi gynes entering a L. neoniger colony. Although L. alienus workers did not accept L. latipes gynes, the documented cases of L. latipes/alienus mixed nests (Sanwald 1964-1965) illustrates that they may do so in the field, at least occasionally.

70 Conclusions

The Lasius (Acanthomyops) species studied are parasites of other Lasius ants and include two cases of hyperparisitism (L. claviger and latipes are parasites and are parasitized. The host range is broader than suspected, particularly in the case of Lasius interjectus. Field evidence suggests that L. interjectus may be involved in a long-term social parasitism with L. claviger. Lasius claviger is a parasite of alienus and either exists in parallel or as a true mixed colony. Lasius interjectus and latipes enter the host colony immediately after mating while L. claviger probably waits until early spring to enter

Lasius interjectus gynes prefer to group, suffer lower mortality in groups, and were observed entering a host colony in large numbers. Lasius latipes alpha form behaves as a parasite in a similar fashion to the beta form, refuting the hypothesis that the gynes fail as parasites. Lasius latipes beta was not aggressive towards host workers while Lasius interjectus was aggressive towards six of the seven host species to which it was introduced. No evidence of a parasitic gyne carrying a host worker in its mandibles was found. No evidence for a parasitic gyne luring host workers away from their colony was found. Given the similar morphology of other Lasius (Acanthomyops) to the studied species, it is likely that all species of this group are social parasites.

71 Literature Cited

Bhatkar, A. P. and W. H. Whitcomb. 1970. Artificial diet for rearing various species of ants. Florida Entomologist. 53: 229-232.

Bourke, A. F. G. and N. R. Franks. 1995. Social evolution in ants. Princeton, Princeton University Press.

Cover, S. P. and R. Sanwald 1988. Colony founding in Acanthomyops murphyi, a temporary social parasite of Lasius neoniger. Advances in myrmecology. J. C. Trager. Leiden, E. J. Brill: 405-417.

Faber, W. 1967. Beiträge zur Kenntnis sozialparasitischer Ameisen. I. Lasius (Austrolasius n. sg.) reginae n. sp., eine neue temporär sozialparasitische Erdameise aus Österreich (Hym. Formicidae). Pflanzenschutz Ber. 36: 73-107.

Hölldobler, B. and E. O. Wilson 1990. The ants. Cambridge, Mass., Harvard University Press.

Janda, M., Folkova, D., and J. Zrzavy. 2003. Phylogeny of Lasius ants based on mitochondrial DNA and morphology, and the evolution of social parasitism in the Lasiini (Hymenoptera : Formicidae) Molecular Phylogenetics and Evolution. 33: 595-614.

Jemielity , S., Graff, J. and L. Keller. 2006. How to fool a virgin: Artificial dealation trigger oviposition in Lasius niger queens. Insectes Sociaux. 53: 323-325

Kaplan, E. L. & Meier, P. 1958. Nonparametric estimation from incomplete data. Journal of the American Statistical Association 53: 457-481.

Rigato, F., Sciaky, R. and F. LeMoli. 1987. Preliminary studies on interspecific interactions within the genus Lasius. Pubbl. Ist. Entomol. Univ. Pavia 36: 35-38.

Sanwald, R. 1964/1965. Mixed colonies of ants on Long Island, N.Y. Bull. Brooklyn Entomol. Soc. 59/60: 81.

72

Sciaky, R. and F. Rigato 1987. Studies on the behaviour of the parasite queens of the genus Lasius (Hymenoptera Formicidae). Pubbl. Ist. Entomol. Univ. Pavia 36: 39- 41.

Smith, M. R. 1934. Two new North American ants. Psyche 41: 211-213.

Talbot, M. 1963. Local distribution and flight activities of four species of ants of the genus Acanthomyops Mayr. Ecology 44: 549-557.

Talbot, M. 1973. Five species of the ant genus Acanthomyops (Hymenoptera: Formicidae) at the Edwin S. George Reserve in southern Michigan. Great Lakes Entomol. 6: 19-22.

Talbot, M. 1979. Social parasitism among ants at the E. S. George Reserve in southern Michigan. Great Lakes Entomol. 12: 87-89.

Tanquary, M. C. 1911. Experiments on the adoption of Lasius, Formica and Polyergus queens by colonies of alien species. Biol. Bull. 20: 281-308.

Tarone, R., and J. Ware. 1977. On distribution free tests for equality of survival distributions. Biometrika, 64, 156-160.

Umphrey, G. J. and R. G. Danzmann (1998). Electrophoretic evidence for hybridization in the ant genus Acanthomyops (Hymenoptera: Formicidae). Biochem. Syst. Ecol. 26: 431-440.

Wheeler, W. M. 1906. On the founding of colonies by queen ants, with special reference to the parasitic and slave-making species." Bull. Am. Mus. Nat. Hist. 22: 33-105.

Wilson, E. O. 1955. A monographic revision of the ant genus Lasius. Bull. Mus. Comp. Zool. 113: 1-201.

73 Wing, M. W. (1968). Taxonomic revision of the Nearctic genus Acanthomyops (Hymenoptera: Formicidae), Mem. Cornell Univ. Agric. Exp. Stn. No. 405,173 p.

74 Table 3.1. Wing’s (1968) summary of founding stage evidence for Lasius (Acanthomyops). The two Lasius claviger entries (marked with *) were considered unreliable by Wing (1968).

75 PARASITE HOST DATE COLLECTOR CITY COUNTY STATE latipes neoniger 3.ix.1946 Wing Freeport Cumberland Maine latipes alienus 21.iv.1910 Wheeler WM Ellisville Massachusetts (Tanquary) (=americanus) latipes alienus 23.viii.1904 Wheeler WM Colebrook Connecticut

latipes alienus 1910 a, Wheeler Colebrook Connecticut (=americanus) WM 1910 b latipes alienus 1910a, Wheeler Colebrook Connecticut (=americanus) WM 1910b latipes alienus 1910a, Wheeler Colebrook Connecticut (=americanus) WM 1910b latipes neoniger 27.vi.1952 Kannowski P Inverness Cheboygan MI Twp. Co. latipes neoniger 11.vii.1947 Kennedy C Pigeon Cheboygan MI River Co. latipes crypticus 30.iii.1941 Leech 8 MI N of British Keremeos Columbia latipes alienus 1963 Gregg CO latipes neoniger 22.viii.1961 Talbot ESGR MI murphyi neoniger 1964-1965 Sanwald Medford Suffolk New York murphyi neoniger 1964-1965 Sanwald Medford Suffolk New York murphyi neoniger 1964-1965 Sanwald Medford Suffolk New York murphyi neoniger 1963 Gregg Falcon CO *claviger “L niger var” 1934 Smith (Frison Herrin Illinois and Ross) *claviger umbratus 1950 Wilson Alabama

76

PARASITE HOST ACCEPTANCE TRIALS NOTES latipes alienus 2 14 beta and (=americanus) alpha latipes nearcticus 0 4 latipes claviger 0 4 latipes subglaber 0 1 latipes brevicornis 0 1 latipes interjectus 1 2 alpha latipes minutus 0 1 (=umbratus var. minutus) latipes latipes 0 1

Table 3.2. Summary of Tanquary’s (1911) Introduction Experiments.

77

1 GYNE 2 GYNES 3 GYNES 4 GYNES

Fed 10 10 10 10

Not Fed 10 10 10 10

Total # 20 40 60 80 gynes

Table 3.3. Forced association experiments for Lasius interjectus.

78

BEHAVIOR DESCRIPTION flagging Moving both extended antennae back and forth in unison. Often associated with side to side movement of the head and whole body. antennation In general, extending an antenna to probe an object. In this study, two ants stand head to head and extend antennae to other ant then move antennae rapidly over other ant. short exploration Generally begins with antennation head to head then a movement to a side to side position and antennation of thorax and gaster. Behavior lasts < 20 seconds long exploration Similar to short but last >1 min cleaning Pulling own femur and/or tarsi through mandible, pulling own antennae through tibial spur egg-laying position Gyne rests on gaster supported by mid and hind legs and curls tip of gaster up in a C-posture. She bends her head down toward the tip of the gaster and appears to lick it.

Table 3.4. Description of behaviors observed in grouping experiments.

79 PARASITE HOST # TRIALS # # TIMES HW # TIMES P AGGR. ACCEPT-AGGR. TOWARD HW ANCES TOWARD P interjectus neoniger 32 0 13 13

interjectus alienus 27 5 22 22

interjectus claviger 19 16 17 0

interjectus latipes 4 3 4 4

interjectus murphyi 3 1 3 3

interjectus umbratus 7 2 7 6

interjectus minutus 2 1 2 2

alpha latipes neoniger 6 4 5 1

alpha latipes alienus 4 0 4 2

beta latipes neoniger 8 6 6 0

beta latipes alienus 1 0 1 0

claviger alienus 4 1 1 0

Total 117 39

Table 3.5. Summary of Introduction Experiments of current study. HW = Host Worker, P = Parasite. 80

TRIAL # DAYS # DAYS MAX # # CENSUSED GYNES # DIFFERENT DAYS GROUPED DAYS SITES CONS. G-TEST P 1 31 21 7 4 10 27.80 <<0.001 9 31 11 5 5 5 13.34 <<0.001 11 31 27 4 7 11 26.56 <<0.001 12 31 28 4 10 12 30.36 <<0.001 13 31 11 6 4 6 18.75 <<0.001 14 31 26 5 11 9 18.55 <<0.001 15 31 4 2 3 1 1.43 >0.1 16 31 21 12 3 16 66.31 <<0.001 19 13 11 10 2 9 39.73 <<0.001 20 21 12 4 4 6 17.40 <<0.001 21 31 12 5 5 5 12.32 <<0.001 22 31 12 2 4 4 7.93 <0.005 23 7 4 4 1 3 12.26 <<0.001 24 22 10 4 4 6 20.32 <<0.001 25 12 11 10 1 9 39.73 <<0.001 26 10 8 5 4 4 11.60 <<0.001 27 9 8 2 4 2 2.86 <<0.001

Table 3.6. Results of G-test. MAX # DAYS = Maximum number of consecutive days gynes were grouped in the same site of the arena. # DAYS CONS = number of days gynes grouped in same site of arena as the previous census.

81

2 QUEENS OBSERVED EXPECTED O-E (O-E)2/E

Solitary 63 66.6 -3.6 0.194594595

Two Grouped 52 48.4 3.6 0.267768595

Sum 115 0.46236319

3 QUEENS OBSERVED EXPECTED O-E (O-E)2/E

Solitary 95 83.6 11.4 1.554545455

Two Grouped 53 76 -23 6.960526316

Three Grouped 58 46.4 11.6 2.9

Sum 206 11.41507177

Table 3.7. Chi-square test table for finding different group sizes in two-, three-, and four- gyne trials. Expected values from truncated Poisson distribution.

82 Table 3.7 continued.

4 QUEENS OBSERVED EXPECTED O-E (O-E)2/E

Solitary 10 20.50 -10.50 5.37804878

Two Grouped 31 27.30 3.70 0.501465201

Three Grouped 26 24.10 1.90 0.149792531

Four Grouped 21 16.10 4.90 1.491304348

Sum 88 7.520610861

83

Figure 3.1. Reorganization of Wing’s (1968) hypotheses for the founding stage of Lasius (Acanthomyops) according to overall strategy (non-parasitic v parasitic), number of queens founding a colony, and details of the non-parasitic and parasitic habits.

84

perch food shelter

Figure 3.2. Grouping experiment arenas. Four L. interjectus gynes were placed in each arena. Each arena contained four perches (rocks), four shelters (5 x 5 cm piece of glass with one end propped up by a cap from a 1.5 mL plastic vial), and four sources of food (piece of tin foil with Bhatkar’s diet).

85

Figure 3.3. Survivorship of individual queens that started either singly (p < 0.03, Kaplan-Meier [1958], Tarone-Ware [1977]), in groups of two (p < 0.04, Kaplan-Meier [1958], Tarone-Ware [1977]) and in groups of four.

86

Figure 3.4. Number of flagging events recorded within the first hour of observations in all trials. Number of flagging events = -0.190 x minutes + 16.033, r = -0.602, p < 0.0001

87

Figure 3.5. Number of interactions observed in the first hour of observations of all trials. Number of interactions = 0.044 x minutes + 4.342, r = 0.258, p = 0.047.

88

Figure 3.6. Number of interactions = .783 + 0.388(minutes) - 0.006(minutes)2 r square = 0.329, p < 0.0001.

89 CHAPTER 4

PHYLOGENETIC ANALYSIS OF LASIUS (ACANTHOMYOPS) : AN EVALUATION OF

EMERY'S RULE

Introduction

The evolution of socially parasitic behavior is intriguing because the very characteristics that are believed to make social animals successful are those that the parasite exploits. Ants are an important system in the study of social parasitism because all ants are social and many are social parasites. In addition, the forms of social parasitism in ants vary from permanent relationships, sometimes involving extreme morphological variation of the parasite, to temporary relationships associated with the founding stage. The gynes of temporary parasitic species in Lasius (Acanthomyops) vary in their mode of entry into the host colony, morphologically, and in their ability to produce pheromones. These characteristics make Lasius (Acanthomyops) useful for evaluating Emery's Rule (Emery 1909, Le Masne 1956), which states that social parasites target their closest relatives.

Temporary social parasites are particularly important in the study of social parasitism because their behaviors are viewed as a transition from independent living to

90 parasitism. Despite this, temporary social parasitism is not as thoroughly studied as other forms (e.g. inquilinism, dulosis), particularly in the Lasius ants. Janda (2004) pointed to a lack of knowledge about the Lasius founding stage particularly Lasius (Acanthomyops) as a major hindrance to a thorough discussion of the evolution of social parasitism in

Lasius ants. Similarly, Huang and Dornhaus (2008) suggest a need for studies of the mechanisms of ant social parasitism as well as phylogenies of socially parasitic groups to understand the evolution of the behaviors. Most ant biologists familiar with Lasius

(Acanthomyops) species suspect that all members of the group are temporary social parasites (Wheeler and McClendon 1903, Wheeler 1906, Creighton 1950, Wing 1968,

Cover and Sanwald 1988). These authors point to the bizarre morphology of some of the queens (e.g. dense mats of setae on the head or thorax, plates extended from the femur) as evidence for the species' probable parasitic habit. Usually, the suggestion that all species are parasitic is followed with the caveat that there is very little evidence for this claim. In

Chapter Three I synthesized what we know about the socially parasitic habit and reported discoveries of varying modes of social parasitism from the field and laboratory. With this new evidence of host-parasite relationships and details of the parasitic habit in place, a phylogeny of the group will provide us with the framework to discuss the evolution of social parasitism in Lasius (Acanthomyops).

In this chapter I provide a phylogeny of Lasius (Acanthomyops) based on morphological and molecular characters and use this as a basis to evaluate the strict and loose versions of the so-called Emery's Rule. The Lasius (Acanthomyops) phylogeny also provides the basis for evaluating Wing's (1968) hypotheses of relationship in Lasius

91 (Acanthomyops) including putative hybrids, and for interpreting the evolution of different mechanisms of the socially parasitic habit.

Taxonomic history of Lasius (Acanthomyops)

Generic status

Mayr (1862) erected the genus Acanthomyops in 1862 based on the single species

Formica clavigera. In 1866, before the other subgenera of Lasius were erected, Mayr changed Acanthomyops to a subgenus of Lasius. At that time, Lasius was a loosely defined genus, containing members of the modern and Melophorus.

Adding Acanthomyops as a subgenus of Lasius did not substantially increase the broad scope of the genus. Emery created Pseudolasius in 1887 and moved other species to

Melophorus in 1895 but left Acanthomyops as a subgenus. This was puzzling because

Pseudolasius was erected to hold species with three-segmented maxillary palps, which is one of the defining characters of Acanthomyops. In 1913 Ruzsky created the Lasius subgenera Dendrolasius and Chthonolasius based on the proportions of the six- segmented maxillary palpi. Creighton believed that the subgeneric divisions of Lasius were inconsistent due to the three-segemented maxillary palps of Acanthomyops in comparison to the six-segmented maxillary palps in the other subgenera. He elevated

Acanthomyops to generic status in 1950. In his revision of Lasius, Wilson (1955) offered that Acanthomyops was closely related to Lasius, particularly the subgenus

Chthonolasius based on similarities of habitat and pilosity. He did not change the taxonomic status of Acanthomyops and still holds this position (personal communication). Wing (1968) revised Acanthomyops in 1968 and concurred with

92 Creighton's earlier decision to elevate Acanthomyops to generic status. Wing provided the followig nine characteristics he felt made Acanthomyops distinct from other Lasius:

1. Maxillary palpus in all castes. Acanthomyops: 3-segmented and short. Lasius: 6- segmented and moderately to very long.

2. Mandibular gland of living worker. Acanthomyops much larger than Lasius (average linear measurement about 3 times that of Lasius).

3. Products of mandibular glands of workers. Acanthomyops: citral and citronellal; most

Lasius: no citral or citronellal (L. umbratus a possible exception).

4. Relative dimension of eye length and head width in workers (Eye length/Head width).

Acanthomyops ranges smaller than other Lasius.

5. Profile of propodeum of workers. Acanthomyops length of declivitous face of propodeum usually not much greater than that of dorsum, which is usually flat or evenly rounded. Lasius: declivitous face long relative to dorsum, which is often angular, showing abrupt changes of contour.

6. Shape of propodeal spiracle in all castes. Acanthomyops: more or less circular.

Lasius: more or less elliptical in queens and males. Shape in workers ranges from circular to elliptical

7. Mesopropodeal spiracle of workers. Acanthomyops: often prominent,; Lasius: usually less prominent.

8. Maximum dimension of metapleural gland opening (M) perpendicular to its long axis in relation to maximum dimension of propodeal spiracle(S), in all castes. Lasius

(Acanthomyops): In queens, ranging from M = 2S to M = S/2 in latipes in which M is

93 greatly reduced in size. In males, M equal to or less than S. In workers, M approximately equal to S. Other Lasius: In queens, M greater than S in L. (Dendrolasius) and L. (Chthonolasius). In males, the same inequalities hold as for the queens. In workers, M greater than S for all subgenera except L. (Chtonolasius) where M is less than

S.

9. Presence, relative number, and position of guard hairs with respect to opening of metapleural glands, in all castes. Acanthomyops: always present, but usually less numerous and having their origin farther from the opening of the metapleural gland, which is often not covered. Lasius: If present, usually more numerous and arising close to a covered gland opening.

Savolainen's (IUSSI 2002 oral presentation) unpublished molecular phylogeny of

Lasius and close relatives supposedly indicates that Acanthomyops was nested within

Lasius. Janda et al (2004) phylogeny of Lasius and close relatives using molecules and morphology also suggested that Acanthomyops was nested within Lasius. The authors suggested a taxonomic change was in order. Although Bolton (2003) made several taxonomic changes in his synopsis of Formicidae, he left Acanthomyops at generic status. Subsequently, Ward (2005) synonomized Acanthomyops based on Janda (2004) and the unpublished Savolainen phylogeny.

Subspecies and species

The first species described was Formica clavigera by Roger (1862). It was then the basis of establishing the genus Acanthomyops. Individual authors then added species or subspecies to the genus. W.M.Wheeler (1914, 1917) authored four subspecies of Lasius interjectus, L. interjectus mexicanus, californicus, coloradensis, and arizonicus.

94 Creighton (1950) disagreed with Wheeler's decisions and shifted his four subspecies from

Lasius interjectus to Lasius claviger. Creighton admitted that he was uncertain of some of his decisions, however, he felt his placement was far better than Wheeler's. Wing

(1968) revised the group and elevated the subspecies of Wheeler and Creighton to species level. He also proposed five hybrids from pinned specimens. No new species have been proposed since Wing's revision.

Materials and Methods

Morphology

Taxon sampling

I studied specimens of Lasius (Acanthomyops) housed at the Ohio State Charles

A. Triplehorn Insect Collection, the University of Michigan Exhibit Museum, The

American Museum of Natural History (AMNH), The Museum of Comparative Zoology

(MCZ) the National Museum of Natural History (NMNH) and my personal collection.

(arizonicus, latipes, murphyi, interjectus).

Wing (1968) revised Lasius (Acanthomyops) based on 1414 nest samples, an estimated 30,900 individuals. He studied 24,600 workers, 4100 queens, and 220 males.

He deposited a collection at the MCZ on which he writes "To make available a comprehensive reference collection for future students of Acanthomyops, representative duplicates from many widely distributed nest series were segregated. This collection, which contains many types was deposited at the Museum of Comparative Zoology."

The USNM collection contained holotypes of Lasius bureni, mexicanus, plumopilosus and pubescens. The collection also housed specimens of Lasius arizonicus,

95 californicus, claviger, coloradensis, interjectus, latipes, mexicanus, occidentalis, pogonogynous, subglaber, and the latipes x claviger hybrid).

Characters

I used characters of Wing (1968), Wilson (1955), and Janda (2004) for the morphological matrix. All characters were coded as non-additive with equal weighting (Tables 4.1 and

4.2).

Molecules

Extraction

I extracted DNA from whole ants that had been stored in 95% alcohol. I used a

Qiagen DNeasy Tissue Kit (Qiagen Inc. USA) and followed the protocol provided in the kit.

PCR Amplification

Wingless

I used the primers Wg 578- Wg1032 (see Table 4.3 for details). For amplification of the wingless genes I used 28µL reactions: 12.5 µL Qiagen Master Mix (1.5 mM

MgCl2, 2.5 units TAQ, 200 mM of dNTPs), 8.5 µL RNase free H2O, 2 µL of each primer, and 3µL of template. For the Wingless gene I used the protocol of Ward and Downie

(2005) (40 cycles of 95° C for 30 seconds, 54° C for 30 seconds, 72° C for 1 minute 30 seconds, preceded by 95° C for 1 minute, and followed by 72°C for three minutes) and

Moreau (personal communication - very similar to Moreau et al 2006) (30 cycles of 94°C

1 min, 54°C 1 min, 72°C 2 min, preceded by 94°C 2min, and followed by 72°C 10 min).

COI

96 I used the primers 1751-2191 and 2083-3014 (see Table 4.3). For amplification of Mitochondrial DNA I used 32µL volume reactions: 25µL Master Mix, l µL each primer, and 5µL template. I used the following protocol for amplification of COI: 94°C 2 min, 80°Cv 15 min, 94°C 2 min, 94°C 1 min, 45°C 1 min, 65°C 1 min, 65°C 6 min.

Sequencing

All sequencing was done at the Plant-Microbe Genomic facility at the Ohio State

University using Applied Biosystems 3730 DNA Analyzer and BigDye Terminator Cycle

Sequencing chemistry.

Alignment

I assembled sequences in Sequencher. I used MUSCLE and TCOFFEE for alignment. MUSCLE gave the same results as TCOFFEE in less time. Sequence data are provided in Appendices A and B.

Phylogenetic analysis

Data were analyzed by running NONA (Goloboff 1999) in Winclada (Nixon

2002). Generally, commands were ">rs 0; hold 500; mult*20; max*;". Such searches were repeated several times, and the maximum number of MPT trees returned from analysis were saved in a treefile with ">ksv*" commmand.

GCMS

Samples were run at the Ohio State Campus Chemical Instrument Center. I ran hexane extracts with a 2-nonanone internal standard on a Finneagan Trace GCMS with a

5% diphenyl, 95% dimethyl polysiloxane column and helium as the carrier gas. I ran the extracts with a split flow of 10µL/min at a temp of 200 with a split ratio of 10. I ran the samples for 15 minutes with the temperature ramping up from 40°C to 175°C in ramp 97 one at 20°C/minute and up to 300°C at 30°C/minute in ramp two. The detector was electron impact and I identified compounds by automated comparison to a known compound library.

Results and Discussion

I fused outgroup species mexicanus with M. semirufus because in these I had good morphological specimens of semirufus and good DNA sequence from mexicanus. The role of these taxa as an outgroup for Lasius should not be compromised at all by the composite nature of the data in the matrix. Similarly, I fused Lasius

(Cautolasius) flavus (morphology) and L. (C.) nearcticus (DNA) for the same reason, and these serve as an outgroup for L. (Acanthomyops).

CO 1 gene (Appendix A) was more informative (230 bases from among about

1300) than wingless (seven informative bases from about 400), but wingless was retained in all analyses (see Appendix B) . Running the molecular data alone gave two trees, with a well resolved consensus and Bremer supports on the consensus of mostly greater than

5. (Figure 1). Lasius (Dendrolasius) spathepus appears rather apical in the tree, as sister to L. (A.) murphyi, which is most unexpected because L. spathepus is in a different subgenus and known only from Japan.

98

Figure 4.1. Phylogeny from COI and wingless. Lasius (Acanthomyops) taxa in blue.

The strict consensus of 2 most parsimonious trees.

99 Removing spathepus from the analysis results in the same topology (but for the absence of spathepus, of course) with similar high support,. Removing murphyi yields a different topology, with spathepus as the sister of the coloradens-latipes component, with Bremer support of 2 for its placement there. This implies that the unexpected position of spathepus might be due to long branch attraction between spathepus and murphyi, according to the method of Siddall and Whiting (1999), by which reciprocal exclusion indicates the degree to which one taxon is placed correctly and the other is placed erroneously (see below).

Morphological data alone provided 238 trees of 67 steps (average CI=0.42,

RI=0.65), the consensus of which dissolves 13 nodes (to produce a length of 84) and preserves the monophyly of Acanthomyops, but shows little else. This indicates that there are several topologies that are in conflict rather than that there is no signal in the data.

Combined analysis with morphology and both genes produced 22 trees at length

670, with a consensus dissolving 12 nodes and producing a poorly resolved tree of length

884. Acanthomyops is not monophyletic (Figure 2).

100

Figure 4.2. Phylogeny from morphology and molecules. Lasius (Acanthomyops) in blue. Lasius (Acanthomyops) is not monophyletic. 101 Examining trees individually demonstrates that Lasius (Dendrolasius) spathepus, was a potentially problematic taxon (as indicated above). I suspected long-branch attraction between L. spathepus and murphyi and performed the Siddall-Whiting test.

When L. spathepus was removed from the matrix L. murphyi remained in the same position. When L. murphyi was removed from the combined morphology and molecular matrix, L. spathepus moved basally to a position consistent with the matrix of morphology only. Eliminating spathepus from the combined analysis produced 20 trees at length 658, which reduce to 14 with the ">best" command. The consensus of these is length 715 (Figure 3), and is much better resolved than in the consensus including spathepus. Because the remaining outgroups should serve adequately to test the monophyly of the ingroup, and root the ingroup, additional analyses were performed without spathepus.

102

Figure 4.3. Phylogeny of morphology and molecules with problematic taxa Lasius

(Dendrolasius) spathepus removed. Lasius (Acanthomyops) is monophyletic.

103 Effect of hybrids

Returning to the combined matrix, without spathepus, the individual solution trees show ambiguity in the regions of the tree that encompass the putative hybrid taxa. The latipes x claviger clusters with latipes. Because the DNA data are mostly mitochondrial, this implies that the maternal ancestor of the hybrids is latipes (as proposed by Wing).

L. pogonogynous is supposedly a cross of latipes and murphyi, and these three species are always clustered together, although the exact relationship among them is unclear. Thus, the placement of the putative hybrids is consistent with proposals of their origins.

However, including these hybrid taxa in the analysis appears to contribute to the ambiguity among solutions regarding the other species. Eliminating both hybrids produces only 6 solution trees at length 651, with a consensus that is better resolved

(Figure 4.4), though with low Bremer support on most nodes.

104

Figure 4.4. Phylogeny from morphology and molecules. Both putative hyrbrids (latipes x claviger and pogonogynous) were removed for this analysis. Lasius (Acanthomyops) taxa in blue.

105

This topology differs from what obtains as any of 57 MPTs at length 63 (not shown) when the hybrids are eliminated from a morphological only matrix. For example, californicus, which is in a polytomy just inside the ingroup node in the combined data tree (Figure 4.3) nests consistently with plumopilosus and creightoni when the morphology with no hybrids is run alone. In addition, californicus, plumopilosus and creightoni can be either a clade in several positions, or a comb along the spine of the morphology MPTs, whereas they are not a clade in the combined tree (Figure 4.3). Third, colei jumps across most of the ingroup in several positions with morphology alone, whereas it is placed unambiguously rather apically in the combined analysis. Thus, the

DNA data do contribute to resolving ambiguity in the morphology-only MPTs, but the

DNA solution is not among the morphology-only MPTs. This will contribute to low

Bremer support because competing topologies are not many steps away from the optimal, combined matrix MPTs.

106

Morphological Evolution.

Wing (1968) relied heavily on setal characters in his revision. He used distribution of setae, length of setae, and density of setae . Length of setae was particularly problematic because Wing used differences of 1/100 of a mm to differentiate species. Setal characters were unreliable in my view. Wing also used pubescence characters such as distribution and density. The shape of the petiole was reliable character for all castes. Overall, queen morphology was more useful than worker or male characters.

Evaluating Wing's and Wilson's hypotheses of relationship in a cladistic framework

Wilson (1955) drew a phylogeny Lasius subgenera and provided additional comments in the text. His idea that L. (Chthonolasius) was the closest relative to L.

(Acanthomyops) was supported in this study. Wing did not draw a diagram to summarize his thoughts on the evolutionary relationships of Lasius (Acanthomyops) species but he stated that some species were "closely related", "very similar", or

"difficult to separate." In this section I evaluate Wing's statements of relationship in a cladistic framework.

Lasius (Acanthomyops) claviger-coloradensis

Wing described Lasius coloradensis as closely related to L. claviger based on the similar appearance of workers and queens. Workers of both species vary in setal and pubescence characters as well as color. The coloradensis queen is very similar to

107 claviger except smaller. Wing suggested a band in western Minnesota where the western species L. coloradensis overlaps with the predominantly eastern L. claviger.

The combined analysis (Fig 4.4) phylogeny suggests that L. claviger and L. coloradensis are not closely related. The species are nested in distinct clades on opposite sides of a major branch in the phylogeny.

Lasius (Acanthomyops) bureni - pubescens

Wing wrote that the two rare species, Lasius bureni and pubescens, were closely related and very "Lasius like." He based this on the appearance of the queens and workers as there is no known specimen of a L. pubescens male. My analysis does not indicate a close relationship between these two species. Lasius bureni is in a polytomy with L. californicus and occidentalis while Lasius pubescens is most closely related to L. murphyi.

Lasius (Acanthomyops) subglaber

Wing viewed Lasius (Ac.) subglaber and occidentalis workers as being very similar and "difficult to separate". In addition, he characterized the males of these two species as similar other than the larger size of L. occidentalis. He also mentioned that L. subglaber queens resemble L. claviger queens. My phylogenetic analysis refutes this idea because L. occidentalis is in a polytomy near the base of the tree while L. subglaber is nested within one of the two major clades. Lasius subglaber is most closely related to

L. coloradensis.

Lasius (Acanthomyops) mexicanus

Wing wrote that L. mexicanus and L. occidentalis were closely related. Lasius mexicanus males are larger than L. occidentalis males but are very similar otherwise.

108 The queens of the two species are also very similar except that L. mexicanus lacks the emarginate head of L. occidentalis. Workers of the two species are also closely similar except for a broader petiolar scale in L. mexicanus L. occidentalis.

In this analysis, L. mexicanus is alone at the base of the tree and sister to a polytomy of L. bureni, californicus, occidentalis. Although the polytomy prevents a detailed hypothesis of relationship, Wing's hypothesis that L. mexicanus is closely related to L. occidentalisi is not refuted. On the other hand, modern cladistic perspectives imply that their "similarity", if based on pleisiomorphic characters, not be used to indicate relationship relative to more apical species.

Lasius (Acanthomyops) californicus - colei

Wing felt that L. californicus was closely related to L. colei based on workers and males. He offered that discovery of the L. colei queen may shed light on the relationship.

The L. colei queen is now known and was included in this analysis. L. californicus and colei are not closely related.

Emery's Rule

Emery (1909) generalized that inquilines, dulotic (slave, pirate) species, and temporary social parasites are parasitized by close relatives. This became known as

"Emery's Rule" (LeManse 1956) and was later divided into strict and loose versions by

Ward (1989, 1996). In the strict version, hosts are parasitized by their sister species while the loose version requires only that hosts are parasitized by a close relative.

Predictably, the strict version has been refuted many more times than the loose version

(Agosti 1994, Ward 1996). Despite its shortcomings, Emery's rule is the framework for

109 most discussions on the evolution of social parasitism in ants (Ward 1989, 1996, Janda

2004, Savolainen 2003). The results of my phylogenetic analysis allow for an evaluation of the strict and loose versions of "Emery's rule".

Emery's rule, loose

My findings support a loose version of Emery's Rule because parasite-host relationships are between species in the genus Lasius. Lasius (Acanthomyops) species parasitize inside and outside their subgenus.

Emery's rule, strict

The strict version is refuted in the case of L. latipes and murphyi because the host is

Lasius (Lasius) neoniger and not their sister species. The L. claviger - interjectus relationship does not refute the strict version because these two species are in a polytomy with L. arizonicus. For example, L.(Ac.) interjectus parasitizes L. (Ac) claviger and latipes (field and laboratory observations) and show a potentially broad host range in laboratory introduction experiments. Lasius (Ac.) interjectus gynes were accepted by L.

(Chthonolasius) umbratus and minutus. Further support for the loose version comes from other Lasius ants. Lasius (Chthonolasius), (Austrolasius), and (Dendrolasius) ants are known to parasitize outside their own subgenus, usually targeting Lasius (Lasius) and to a lesser degree Lasius (Cautolasius).

Modes of parasitic invasion: Armor vs. chemistry

Based on field and laboratory observations, I find that Laisus (Acanthomyops) shows two distinct modes of parasitic invasion that relate to morphology for withstanding the attacks of host workers while the other relies on chemistry to repel or confuse host workers. For example, Lasius murphyi (Figure 4.5) gynes possess dense mats of hair on

110 their head and thorax, which prevents host workers (Lasius neoniger) from injuring the exoskeleton. Lasius latipes gynes (Figure 4.6) have stout heads and antennae and possess what Wing termed genual plates on the forelegs. Lasius latipes gynes enter L. neoniger colonies with brute force. Host workers attacks seem to have little or no effect on the gyne. In both the L. latipes and L. murphyi case the parasitic gyne is not aggressive toward the attacking host workers. In contrast, L.interjectus and claviger gynes are slightly built and do not have unusual amounts of setae on their bodies (Figures 4.7 and

4.8). When attacked by host workers, L. interjectus gynes are aggressive towards their attackers and release citronella from their mandibular glands. Lasius claviger gynes also release citronella when encountering host workers but are not as aggressive as L. interjectus gynes. Host workers are repelled by the L. claviger gyne and tend to avoid it in the early stages of entry to the host nest. GCMS analysis of the mandibular glands of gynes indicate that L. claviger (Figure 4.9) and interjectus produce citronella while L. latipes does not (Figure 4.10). These alternative strategies appear to relate to the two clades of L. (Acanthomyops). Field observations (Wing 1968, Raczkowski unpublished) indicate Lasius umbratus produce small amounts of citronella when disturbed, I conclude that the morphological modifications of L. murphyi and latipes are derived as a further specialization toward parasitism, and the lack of a citronella production represents a loss of character.

Conclusion

In this study I used morphological and molecular data to provide the first species- level phylogenetic analysis of Lasius (Acanthomyops). Lasius (Acanthomyops) was 111 shown to be monophyletic. Some earlier assertions are validated, such as Wilson's idea that Chthonolasius is the closest relative to Acanthomyops and Wing's hypotheses regarding hybrids. Emery's Rule in the strict sense was refuted. Lasius latipes robust morphology and lack of citronella production represents a loss of character.

112

Figure 4.5. Lasius murphyi gyne, illustrating the dense mats of hair on the head and thorax.

113

Figure 4.6. Lasius latipes gyne, illustrating the large genual plates on the foreleg and the stout head and antennae.

114

Figure 4.7. Lasius interjectus gyne.

115

Figure 4.8. Lasius claviger gyne.

116

Conclusions

Figure 4.9. MS of mandibular gland contents of Lasius claviger gyne. Peak at 6.61 is citronellal and the peak at 7.48 is citral.

117

Figure 4.10. MS of mandibular gland from Lasius latipes gyne. Note the absence of peaks at 6.61 (citronellal) and 7.48 (citral).

118 Table 4.1 Characters and character states used in this analysis.

0. Queen pubescence on gaster: dilute = 0; dense = 1; absent = 2.

1. Queen pubescence on head: dilute = 0; dense = 1.

2. Queen gular hair: entire surface = 0; less than entire surface = 1.

3. Queen petiolar crest: straight = 0; emarginate = 1; curved = 2.

4. Queen setae on dorsum of gaster : posterior margins of tergites = 0;

entire surface = 1; absent = 2.

5. Queen head: without supra-antennal ridge = 0; supra-antennal ridge = 1.

6. Queen genual plate: absent = 0; present = 1.

7. Queen femur width: 0.80mm or greater = 0; 0.73 mm or less = 1.

8. Male crest of petiole: sharp = 0; blunt = 1.

9. Worker pubescence on scape: appressed = 0; decumbed to suberect = 1;

erect = 2.

10. Worker petiole in frontal view sides: parallel = 0; convex = 1;

diverging dorsad = 2.

11. Worker petiole dorsal crest: straight = 0; emarginated = 1; curved = 2.

12. Worker occipital border: straight = 0; feebly convex = 1;

strongly convex = 2.

13. Worker number maxillary palp segments: 3 = 0; 6 = 1.

14. Worker crest of petiolar Scale: sharp = 0; blunt = 1.

15. Worker hair type: plumose tips = 0;

simple or weakly to strongly barbulate = 1.

16. Worker gular hair: absent = 0; present = 1.

119 Table 4.1 continued.

17. Worker shape of propodeal spiracle: circular = 0; elliptical = 1.

18. Worker impression anterobasally on gaster: absent = 0; present = 1.

19. Worker dorsum of propodeum: flat or evenly rounded = 0; angular = 1.

20. Worker activity: epigeic = 0; hypogeic = 1.

21. Worker setae on gaster: posterior margins of tergites = 0;

entire surface = 1.

120

Table 4.2 Morphological matrix used in this analysis.

0 5 10 15 20 | | | | | Myrmecocystus semirufus 0000100010110101111100 Lasius (Chthonolasius) umbratus 1110100001111101111111 Lasius (Austrolasius) carniolicus 1111100012121111111111 Lasius (Dendrolasius) spathepus 2011200010112111111101 Lasius (Cautolasius) flavus 1111100001201111111111 Lasius (Lasius) alienus 1111100001110101111101 Lasius (Acanthomyops) claviger 0001000002111001100011 Lasius (Acanthomyops) coloradensis 0011000001110001100011 Lasius (Acanthomyops) californicus 0011000001010001100011 Lasius (Acanthomyops) colei 0010000000100001100010 Lasius (Acanthomyops) arizonicus 0011000000011001000010 Lasius (Acanthomyops) interjectus 0111000002111001100010 Lasius (Acanthomyops) latipes 1002111111221011100011 Lasius (Acanthomyops) murphyi 1102210010221011100011 Lasius (Acanthomyops) pogonogynous 1000011011221011100011 Lasius (Acanthomyops) subglaber 0011000001111011100011 Lasius (Acanthomyops) plumopilosus 0011100000010010100011 Lasius (Acanthomyops) bureni 1111000000210001100011 Lasius (Acanthomyops) pubescens 10100000?0120011100011 Lasius (Acanthomyops) creightoni 0011100001010001100011 Lasius (Acanthomyops) occidentalis 1111000000111001100011 Lasius (Acanthomyops) mexicanus 1111100000110001100011 Lasius (Acanthomyops) latipes x claviger -0020-0111221110100011

121

Table 4.3. Primers used in this analysis. All primers were used for both amplification and sequencing.

Primer Primer Sequence Primer Location Name Citation mtDNA C1-J-1751 5' GGA TCA CCT Simon et GAT ATA GCA al 2004 TTC CC-3' C1-N- 5'-CCC GGT Simon et 2191 AAT ATT AAA al 2004 ATA TAA ACT TC-3' C1-J-2183 5'-CAA CAT TTA Simon et TTT TGA TTT al 2004 TTT GG-3' TL2-N- 5'-TCC AAT Simon et 3014 GCA CTA ATC al 2004 TGC CAT ATT A-3' nDNA Wg 1032 5'-ACY TCG Abouheif Wingless CAG CAC CAR and Wray (Wg) TGG AA-3' 2002 Wg 578 R 5'-TGC ACN Ward and GTG AAR ACY Downie TGC TGG ATG 2004 CG - 3'

122

APPENDIX A

CYTOCHROME OXIDASE SUBUNIT 1 SEQUENCES ALIGNED BY MUSCLE (MULTIPLE SEQUENCE COMPARISON BY LOG EXPECTATION)

123

0 5 10 15 20 25 30 35 40 45 50 55 | | | | | | | | | | | | tagcttacccacgaataaataatataagattttgacttcttcccccatcaatttcc Lasius umbratus ------atttct Lasius spathepus tagcattccctcgtataaataatataagattctgacttttacctccttcaatctct ------cgtataaataatataagattctgacttttacctccttcgatttct tagcttaccctcgtataaataatataagattttgacttctacctccctcaatttct Lasius claviger tagcattccctcgcataaataatataagattctgactcttacctccttc-atctct Lasius coloradensis tagcattccctcgtataaataatataagattctgacttttacctccctctatctct Lasius californicus ------ataaataatat?agattttgact?ttwcccccctctattata Lasius colei tagcattcccccgcataaataatataagattctgactcttacctccttc-atctct Lasius arizonicus tagcattccctcgcataaataatataagattctgactcttacctccttc-atctct Lasius interjectus tagcattccctcgcataaataatataagattctgactcttacccccttc-atctct Lasius latipes tagcattccctcgtataaataatataagattctgacttttacctccctctatctct Lasius murphyi tagcattccctcgtataaataatataagattctgacttttacctccttcaatctct Lasius creightoni tagcattccctcgtataaataatataagattctgactcttacctccttc-atctct Lasius occidentalis ------tattacccccctctattata Lasius lat x claviger tagcattccctcgtataaataatttaagattctgacttttacctccctctatctct

56 61 66 71 76 81 86 91 96 101 106 111 | | | | | | | | | | | | Myrmecocystus mexicanus -ttacttctcctaagaaattttattaatgatggtgttggtacaggttgaactgttt Lasius umbratus -ctccttctattaagaaattttattaatgatggggttggaacaggatgaactgttt Lasius spathepus -ctcctcctcttaagaaattttattaatgatggagttggaacaggatgaaccattt Lasius nearcticus -cttcttcttttaagaaattttattaatgaaggagcaggaacaggatgaactattt Lasius alienus -ctccttcttttaagaaattttattaatgacggagtcggaacaggatggactgttt Lasius claviger -ctactcctcttaagaaattttattaatgacggagttggaacaggatgaaccattt Lasius coloradensis -ctcctcctcttaagaaattttattaatgatggaactgggacaggatgaactattt Lasius californicus tctattaatttcaag?agaattgt?gaaaatggagcaggaac?ggatgaacagt?t Lasius colei -ctactcctcttaagaaattttattaacgacggagttggaacagggtgaaccattt Lasius arizonicus -ctactcctcttaagaaattttattaatgacggagttgggacaggatgaaccattt Lasius interjectus -ctgctcctcttaagaaattttattaatgacggagttggaacaggatgaaccattt Lasius latipes -ctcctcctcttaagaaattttattaatgatggaactggaacaggatgaactattt Lasius murphyi -ctcctcctcttaagaaattttattaatgatggagttggaacaggatgaaccattt Lasius creightoni -ctactcctcttaagaaattttattaatgacggagttggaacaggatgaaccattt Lasius occidentalis -ctattaatttcaagtagaattgtagaaaaggargcaggaacaggatgaacagttt Lasius lat x claviger -ctcctcctcttaagaaattttattaatgatggaactggaacaggatgaactattt

112 117 122 127 132 137 142 147 152 157 162 167 | | | | | | | | | | | | Myrmecocystus mexicanus atccaccattagcttctaatattttccataatgggccttcagtagatttagctatt Lasius umbratus accccccattagcctcaaatatttttcataatggcccctcagttgatttaactatt Lasius spathepus atcccccattagcttcaaatatttttcataatggtccctcagttgatttaactatt Lasius nearcticus atcccccattagcttcaaatatttttcatagtggttcctcaattgatttaactatt Lasius alienus atcccccgttagcttctaatatctttcataatggcccttcagttgatttaactatt Lasius claviger accccccattagcttcaaatatttttcataatggcccctcagttgatttaactatt Lasius coloradensis accccccattagcctcaaatatttttcataatggcccctcagttgatttaactatt Lasius californicus acccccc?ctatcatcaaatattgc?catagaggaagatcagtagattt??ctatt Lasius colei accccccattagcttcaaatatttttcataatggtccctcagttgatttaactatt Lasius arizonicus accccccatta?cttcaaatatttttcataatggcccctcagttgatttaactatt Lasius interjectus accccccattagcttcaaatatttttcataatggtccctcagttgatttaactatt Lasius latipes accccccattagcctcaaatatttttcataatggcccctcagttgatttaactatt Lasius murphyi atcccccattagcttcaaatatttttcataatggtccctcagttgatttaactatt Lasius creightoni accctccattagcttcaaatatttttcacaatggcccctcaattgacttaactatt Lasius occidentalis accccccactttcttcaaatattgcccacagaggaagatctgtagatcttgctatt Lasius lat x claviger accccccattagcctcaaatatttttcataatggcccctcagttgatttaactatt

124 168 173 178 183 188 193 198 203 208 213 218 223 | | | | | | | | | | | | Myrmecocystus mexicanus ttttctttacatattgctggaatatcttccatcctaggagccattaattttatttc Lasius umbratus ttctcccttcatatcactggaatctcctctattttaggagctattaattttatttc Lasius spathepus ttttctcttcatattgcagggatatcttctattttaggagctattaattttatttc Lasius nearcticus ttttccttacatattgccggaatatcttctatcttaggggctatcaattttatttc Lasius alienus ttctctctacatattgctggtatatcttctatcctaggggctatcaattttatctc Lasius claviger ttttctctacatattgctgggatatcttctattttaggggctattaattttatctc Lasius coloradensis ttttctcttcatattgcagggatatcttctattttaggagctattaattttatttc Lasius californicus ttttccttacatttagc?ggaatttcatcaattttaggagctattaattttattac Lasius colei ttttctctacatattgctgggatatcttctattttaggggctattaattttatctc Lasius arizonicus ttttctctacatattgctgggatatcttctattttaggggctattaattttatctc Lasius interjectus ttttctctacatattgctgggatatcttctattttaggggctattaattttatctc Lasius latipes ttttctcttcatattgcagggatatcttctattttaggagctattaattttatttc Lasius murphyi ttttctcttcatattgcagggatatcttctattttaggagctattaattttatttc Lasius creightoni ttttctctacatattgccggaatatcttctattttaggggctattaattttatctc Lasius occidentalis ttttctttacatttggctggaatttcatcaattttaggagctattaaytttattac Lasius lat x claviger ttttctcttcatattgcagggatatcttctattttaggagctattaattttatttc

224 229 234 239 244 249 254 259 264 269 274 279 | | | | | | | | | | | | Myrmecocystus mexicanus tactattataaatatacaccacaaaaatatctctattgataaaattcccttacttg Lasius umbratus aaccattataaatatacaccataaaaatttttctatcgataaaatacctttacttg Lasius spathepus aaccattataaatatacatcataaaaatttttctatcgacaaaatacccctacttg Lasius nearcticus aaccattataaatatacatcacaaaaatttttctatcgataaaattcccttacttg Lasius alienus aactattataaatatacatcataaaaatttttctattgataaaatccctttacttg Lasius claviger aaccattataaatatacatcataaaaatttttctgtcgataaaatacccttacttg Lasius coloradensis aaccatcataaatatacatcataaaaatttttctatcgacaaaatacccttacttg Lasius californicus aactattattaatatacgacc?aataa?atatccttagatcaaataccc?tatttg Lasius colei aaccattataaatatacatcataaaaatttttctatcgacaaaatacctttacttg Lasius arizonicus aaccattataaatatacatcataaaaatttttctgtcgataaaatacccttacttg Lasius interjectus aaccattataaatatacatcataaaaatttttctgtcgataaaatgcccttacttg Lasius latipes aaccatcataaatatacatcataaaaatttttctatcgacaaaatacccttacttg Lasius murphyi aaccattataaatatacatcataaaaatttttctatcgacaaaatacccctacttg Lasius creightoni aaccattataaatatacatcataaaaatttttctattgacaaaatacccttacttg Lasius occidentalis aactattattaatatacgacctaataatatatcattagaycaaatacctytatttg Lasius lat x claviger aaccatcataaatatacatcataaaaatttttctatcgacaaaatacccttacttg

280 285 290 295 300 305 310 315 320 325 330 335 | | | | | | | | | | | | Myrmecocystus mexicanus tttgatcaattctaattactgcaattttattacttttatctcttcctgtacttgca Lasius umbratus tatgatcaattttaattactgcaattttattacttttatctcttccagttcttgca Lasius spathepus tatgatcaatcttaatcactgcaattttattacttttatctcttcccgttcttgca Lasius nearcticus tatggtcaattttaattactgcaattttattgcttttatctcttcctgttcttgct Lasius alienus tatggtcaattttaattactgcaattttattactcctatcccttccagttcttgca Lasius claviger tatgatcgattttaatcactgcaattttacttcttctatctcttcccgttcttgca Lasius coloradensis tatggtcaattttaatcactgcaattttactacttttatctcttcccgttcttgca Lasius californicus t?tgagcagt?ggaattac?gctttattactt?t?ctttctttacctgtattagc? Lasius colei tatgatcaattttaatcacagcgattttacttcttctttctcttcccgttcttgca Lasius arizonicus tatgatcgattttaatcactgcgattttacttcttctatctcttcccgttcttgca Lasius interjectus tatgatcgattttaatcactgcgattttacttcttctatctcttcccgttcttgca Lasius latipes tatggtcaattttaatcactgcaattttgctacttttatctcttcccgttcttgca Lasius murphyi tatgatcaatcttaatcactgcaattttattacttttatctcttcccgttcttgca Lasius creightoni tatgatcgattttaatcactgcgattttacttcttctatctcttcccgttcttgca Lasius occidentalis tttgagctgttggaattacagctttattattattactttctttaccggtattagct Lasius lat x claviger tatggtcaattttaatcactgcaattttactacttttatctcttcccgttcttgca

125 336 341 346 351 356 361 366 371 376 381 386 391 | | | | | | | | | | | | Myrmecocystus mexicanus ggagccatcactatacttctaactgatcgtaatcttaatacttcattttttgaccc Lasius umbratus ggagctattacaatacttctaactgaccgtaatcttaatacttcattttttgatcc Lasius spathepus ggagctatcactatacttttaactgatcgaaatcttaatacttcattttttgatcc Lasius nearcticus ggtgctattactatacttttaactgaccgtaaccttaatacttcattttttgaccc Lasius alienus ggagcaattactatacttctaactgaccgtaatcttaacacttcattttttgaccc Lasius claviger ggagctatcaccatacttttaactgatcgtaatcttaatacttctttttttgaccc Lasius coloradensis ggagctatcactatacttttgactgatcgaaatcttaatacttcattttttgaccc Lasius californicus ggagc?attac?atayttytaacagatcgaaatctwaatacatcattttttgatcc Lasius colei ggagctatcaccatacttttaactgatcgtaatcttaatacttctttttttgaccc Lasius arizonicus ggagctatcaccatacttttaactgatcgtaatcttaatacttctttttttgaccc Lasius interjectus ggagctatcaccatacttttaactgatcgtaatcttaatacttctttttttgaccc Lasius latipes ggagctatcactatacttttgactgaccgaaatcttaatacttcattttttgaccc Lasius murphyi ggagctatcactatacttttaactgatcgaaatcttaatacttcattttttgatcc Lasius creightoni ggagctatcaccatacttttaactgatcgtaatcttaatacttctttttttgaccc Lasius occidentalis ggtgctattacaatattattaacwgatcgaaatcttaatacatcattttttgatcc Lasius lat x claviger ggagctatcactatacttttgactgaccgaaatcttaatacttcattttttgaccc

392 397 402 407 412 417 422 427 432 437 442 447 | | | | | | | | | | | | Myrmecocystus mexicanus at-caggaggtggagatccaattttatatcaacatcttttttgattttttg----- Lasius umbratus at-caggtgggggagatcctattttatatcaacatcttttctgatttttcg----- Lasius spathepus ct-caggaggaggagatcctattttataccaacatcttttttgattttttg----- Lasius nearcticus at-caggaggaggagaccctattttatatca????tawttt?tattttt?g?a??? Lasius alienus at-caggtggtggggatcctattttatatcaacacctcttctgattttttg----- Lasius claviger tt-caggagggggagaccctattttatatcaacatcttttttgattttttg----- Lasius coloradensis ct-caggaggtggagaccctattttataccaacatcttttttgattttttg----- Lasius californicus ctgctggaggaggagatcc?attttatatcaacatttattttgatttttyg----- Lasius colei tt-caggagggggggaccctattttatatcaacatcttttttgattttttg----- Lasius arizonicus tt-caggagggggagaccctattttatatca?c?tcttttttgattttttg----- Lasius interjectus tt-caggagggggggaccctattttatatcaacatcttttttgattttttg----- Lasius latipes ct-caggaggtggggaccctattttataccaacatcttttttgattttttg----- Lasius murphyi ct-caggaggaggagatcctattttataccaacatyttttttgattttttg----- Lasius creightoni tt-caggaggaggggaccctattttataccaacatcttttttgattttttg----- Lasius occidentalis tg-ctggtggaggagatccaattttatatcaacatttwttttgattttttg----- Lasius lat x claviger ct-caggaggtggggaccctattttataccaacatcttttttgattttttg-----

448 453 458 463 468 473 478 483 488 493 498 503 | | | | | | | | | | | | Myrmecocystus mexicanus ------gccatcctgaagtttatattttaattcttcctggatttggatt Lasius umbratus ------gacatccagaagtttatattttaattcttcctggatttggatt Lasius spathepus ------gtcacccagaagtttatattttaathytaccvggatttggatt Lasius nearcticus ccattgatttt??g?catccagaagtttatattttaattctacctggatttggact Lasius alienus ------ggcatcctgaagtttatattttaattcttcctggatttggatt Lasius claviger ------gacacccagaagtttatattttaattcttcctggatttgggtt Lasius coloradensis ------gtcaccctgaagtttatattttaatcctacccggatttggatt Lasius californicus ------g?catccagaagtttatattttwattcttcctggatttgggtt Lasius colei ------gacacccagaagtttatattttaattcttcctggatttgggtt Lasius arizonicus ------gacacccagaagtttatattttaattcttcctggatttgggtt Lasius interjectus ------gacacccagaagtttatattttaattcttcctggatttgggtt Lasius latipes ------gtcaccctgaagtttatattttaatcctacccggatttggatt Lasius murphyi ------gtcacccagaagtttatattttaatcctacccggatttggatt Lasius creightoni ------gacacccagaagtttatattttaawtyttcctggatttgggtt Lasius occidentalis ------gwcaycctgaagtttatattttawtcytacccggatttggatt Lasius lat x claviger ------gtcaccctgaagtttatattttawtcctacccggatttggatt

126 504 509 514 519 524 529 534 539 544 549 554 559 | | | | | | | | | | | | Myrmecocystus mexicanus aatttctcatatcattataaatgaaagaggaaaaaaagaaacatttggatctcttg Lasius umbratus aatttcccatatcattataaatgaaagtgggaaaaaagaaacatttggatctttag Lasius spathepus aatytcycatattattataaatgaaagaggaaaaaaagaaacatttggrtcyttag Lasius nearcticus aatttctcacattattataaatgaaagaggaaaaaaagaaacatttggatccttag Lasius alienus aatttctcatattatcataaatgaaagaggaaaaaaagaaacatttggatctttag Lasius claviger aatttctcatattattataaatgaaagggggaaaaaagaaacattcggatctttag Lasius coloradensis aatttctcatattattataaatgaaagaggaaaaaaagaaacattcggatccttag Lasius californicus aatttctcatattattataaa--aaagrggaaaaaaagaaacattcggatctttag Lasius colei aatttctcatattattataaatgaaaggggaaaaaaagaaacattcggatccttag Lasius arizonicus aatttctcatattattataaatgaaagagggaaaaaagaaacattcggatccttag Lasius interjectus aatttctcatattattataaatgaaagggggaaaaaagaaacattcggatccttag Lasius latipes aatttctcatattattataaatgaaagaggaaaaaaagaaacattcggatccttag Lasius murphyi aatttctcatattattataaatgaaagaggaaaaaaagaaacatttggatccttag Lasius creightoni aatttctcatattattataaatgaaaggggaaaaaaagaaacattcggatccttag Lasius occidentalis aatttctcatattattataaatgaaagaggaaaaaaagaaacattcggatccttag Lasius lat x claviger aatttctcatattattataaatgaaagaggaaaaaaagaaacatt?ggatccttag

560 565 570 575 580 585 590 595 600 605 610 615 | | | | | | | | | | | | Myrmecocystus mexicanus gtataatttacgcattaatagcaattggatttttaggatttgttgtttgagctcat Lasius umbratus gaataatttatgcccttatagcaattggatttttaggatttgttgtttgagctcat Lasius spathepus gaataatttatgcwctwatagcaattggatttttaggatttgttgtttgagctcay Lasius nearcticus gaataatttatgctcttatagcaattggatttttaggatttgttgtttgagctcat Lasius alienus gaataatttatgctctaatagcaattggatttttaggatttgtcgtatgagctcac Lasius claviger gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccat Lasius coloradensis gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccac Lasius californicus gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccat Lasius colei gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccat Lasius arizonicus gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccat Lasius interjectus gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccat Lasius latipes gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccac Lasius murphyi gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagctcac Lasius creightoni gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccac Lasius occidentalis gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagcccac Lasius lat x claviger gaataatttatgcacttatagcaattggatttttaggatttgttgtttgagctcac

616 621 626 631 636 641 646 651 656 661 666 671 | | | | | | | | | | | | Myrmecocystus mexicanus catatatttactattggattagatgttgatacccgagcttattttacttcagctac Lasius umbratus catatatttactattggcctagatgtcgatactcgtgcatattttacttcagcaac Lasius spathepus catatatttacyattggattagaygttgatactcgwgcataytttacttcygcmac Lasius nearcticus cacatatttacaattggattagatgttgatacccgcgcatattttacttctgcaac Lasius alienus catatatttactattggtttagatgttgatactcgagcatacttcacttctgcaac Lasius claviger catatatttactattggattagatgttgatactcgcgcatattttacttctgcaac Lasius coloradensis catatatttactattgggttagatgttgatactcgagcatattttacttctgcaac Lasius californicus catatatttactattggattagatgttgatactcgcgcatattttacttctgcaac Lasius colei catatatttactattggattagatgttgatactcgcgcatattttacttctgcaac Lasius arizonicus catatatttactattggattagatgttgatactcgcgcatattttacttctgcaac Lasius interjectus catatatttactattggattagatgttgatactcgcgcatattttacttctgcaac Lasius latipes catatatttactattgggttagatgttgatactcgagcatattttacttctgcaac Lasius murphyi catatatttaccattggattagatgttgatactcgtgcatattttacttccgcaac Lasius creightoni catatatttactattggattagatgttgatactcgcgcatattttacttctgcaac Lasius occidentalis catatatttactattgggttagatgttgatactcgagcatattttacttctgcaac Lasius lat x claviger catatatttactattgggttagatgttgatactcgagcatattttacttctgcaac

127 672 677 682 687 692 697 702 707 712 717 722 727 | | | | | | | | | | | |

Myrmecocystus mexicanus tataattattgctattccaacaggaatcaaaatttttagatgaattactacccttc Lasius umbratus aataattattgccattccaacaggaattaaaatttttagatgaattaccactcttc Lasius spathepus tataattattgcyatyccaacwggaatyaaaatttttagatgaattactactcttc Lasius nearcticus tataattattgcaattcctacaggaattaaaatttttagatgaattactactcttc Lasius alienus tataattattgctattccaactggaatcaaaatttttagatgaattactacccttc Lasius claviger tataattattgccatccctacaggaattaaaatttttagatgaattaccactcttc Lasius coloradensis tataattattgctatcccaacaggaatcaaaatttttaggtgaattaccactcttc Lasius californicus tataattattgccatccctacaggaattaaaatttttagatgaattaccactcttc Lasius colei tataattattgccattcctacaggaattaaaatttttagatgaattactactcttc Lasius arizonicus tataattattgccatccctacaggaattaaaatttttagatgaattaccactcttc Lasius interjectus tataattattgccatccctacaggaattaaaatttttagatgaattaccactcttc Lasius latipes tataattattgctatcccaacaggaattaaaatttttagatgaattaccactcttc Lasius murphyi tataattattgccatcccaacaggaattaaaatttttagatgaattactactcttc Lasius creightoni tataattattgccatccctacaggaattaaaattttcagatgaattaccactcttc Lasius occidentalis tataattattgctatcccaacaggaattaaaatttttagatgaattaccactcttc Lasius lat x claviger tataattattgctatcccaacaggaattaaaatttttagatgaattaccactcttc

728 733 738 743 748 753 758 763 768 773 778 783 | | | | | | | | | | | | Myrmecocystus mexicanus atggaacaaaaatcaacaataattcttccttatgatgagcaataggatttattttt Lasius umbratus atggaacaaaaatcaataataattcttctttatgatgaacaataggatttattttt Lasius spathepus atggwacaaaaatcaataataattcytcwttatgatgawcaataggatttatttty Lasius nearcticus atggaacaaaaatcaataataattcctccttatgatgatctataggatttattttt Lasius alienus atggtacaaaaatcaataataactcttccttatgatgagcaataggatttatcttc Lasius claviger atgggacaaaaattaataataattcttctctatgatgatctataggattcattttt Lasius coloradensis atggaacaaaaatcaataataattcttctttatgatgaacaataggattcattttt Lasius californicus atgggacaaaaattaataataattcttctctatgatgatctataggattcattttt Lasius colei atggaacaaaaattaataataattcttctctatgatgatctataggattcattttt Lasius arizonicus atgggacaaaaattaataataattcttctctatgatgatctataggattcattttt Lasius interjectus atggaacaaaaattaataataattcttctctatgatgatctataggattcattttt Lasius latipes atggaacaaaaatcaataataattcttctttatgatgaacaataggattcattttt Lasius murphyi atggaacaaaaatcaataataattcttctttatgatgatcaataggatttattttt Lasius creightoni atggaacaaaaattaataataattcttctctatgatgaactataggattcattttt Lasius occidentalis atggaacaaaaatcaataataattcttctttatgatgaacaataggattcattttt Lasius lat x claviger atggtacaaaaatcaataataattcttctttatgatgaacaataggattcattttt

784 789 794 799 804 809 814 819 824 829 834 839 | | | | | | | | | | | | Myrmecocystus mexicanus ctatttactataggaggtctaactggagtaatactttctaattcatcaattgatat Lasius umbratus ctattcactataggaggtcttacaggagtaatactttctaactcatcaattgatat Lasius spathepus ttattcactataggaggacttacaggagtwatactttcwaattcatcaattgayat Lasius nearcticus ttattcactatggggggtctcacgggagtaatactctcaaattcatcaattgatat Lasius alienus ttattcactataggaggtttaacaggagtaatactttcaaattcatcaattgatat Lasius claviger ctattcactatagggggacttacgggagtaatactttctaattcatcaattgacat Lasius coloradensis ctattcactataggaggacttacaggagttatactttctaattcatcaattgacat Lasius californicus ctattcactatagggggacttacgggagtaatactttctaattcatcaattgacat Lasius colei ctattcactatagggggacttacgggagtaatactttctaattcatcaattgacat Lasius arizonicus ctattcactatagggggacttacgggagtaatactttctaattcatcaattgacat Lasius interjectus ctattcactatagggggacttacgggagtaatactttctaattcatcaattgacat Lasius latipes ctattcactataggaggacttacaggagttatactttctaattcatcaattgacat Lasius murphyi ttattcactataggaggacttacaggagtaatactttctaattcatcaattgacat Lasius creightoni ctattcactataggaggacttacgggagtaatactttctaattcatcaattgacat Lasius occidentalis ctattcactataggaggacttacaggagttatactttctaattcatcaattgacat Lasius lat x claviger ctattcactataggaggacttacaggagttatactttctaattcatcaattgacat

128 840 845 850 855 860 865 870 875 880 885 890 895 | | | | | | | | | | | | Myrmecocystus mexicanus tattctt------Lasius umbratus catccttcatgatacctattacgtagttgctcattttcattatgtattatcaatag Lasius spathepus tattcttcatgayacmtattatgtagtwgcycattttcattatgtattatcaatag Lasius nearcticus tatccttcatgatacttattatgtagtagctcattttcattatgtattatcaatag Lasius alienus tattctt------Lasius claviger cattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius coloradensis tattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius californicus cattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius colei cattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius arizonicus cattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius interjectus cattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius latipes tattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius murphyi tattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius creightoni cattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius occidentalis tattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag Lasius lat x claviger tattcttcatgatacctattatgtagttgctcattttcattatgtattatcaatag

896 901 906 911 916 921 926 931 936 941 946 951 | | | | | | | | | | | | Myrmecocystus mexicanus ------Lasius umbratus gagcagtatttgctattattgctagatttatccattgatttcctttaataactggt Lasius spathepus gagcwgtwtttgctattattgctagattyattcactgattcccyttaataactgga Lasius nearcticus gagcagtatttgctattattgctagatttattcactgattccctctaataactggt Lasius alienus ------Lasius claviger gagcagtatttgctattatcgctagatttattcactgatttcctttaataactgga Lasius coloradensis gggcagtatttgctattattgctagatttatccattgattccctttaataactggc Lasius californicus gagcagtatttgctattatcgctagatttattcactgatttcctttaataactgga Lasius colei gggcagtatttgctattatcgctagatttattcattgatttcctttaataactgga Lasius arizonicus gagcagtatttgctattatcgctagatttattcattgatttcctttaataactgga Lasius interjectus gagcagtatttgctattatcgctagatttattcattgatttcctttaataactgga Lasius latipes gagcagtatttgctattattgctagatttatccattgattccctttaataactggc Lasius murphyi gagcagtatttgctattattgctagatttattcactgattccctttaataactggt Lasius creightoni gagcagtatttgctattatcgctagatttattcattgatttcctttaataactgga Lasius occidentalis gagcagtatttgctattattgctagatttatccattgattccctttaataactggc Lasius lat x claviger gagcagtatttgctattattgctagatttatccattgattccctttaataactggc

952 957 962 967 972 977 982 987 992 997 1002 1007 | | | | | | | | | | | | Myrmecocystus mexicanus ------Lasius umbratus ttctctttaaacaatttttttctaaatattcaatttatttgtatattcacaggagt Lasius spathepus ttttctttaaataatttytttytaaatattcaattcatytgtatatttwttggagt Lasius nearcticus ttctctttaaataatttctttttaaatattcaatttatctgcatattttttggagt Lasius alienus ------Lasius claviger ttttctttaaataatttttttctaaatattcaattcatttgtatatttattggagt Lasius coloradensis ttttctttaaataatttttttctaaatattcaattcatctgtatatttattggagt Lasius californicus ttttctttaaataatttttttctaaatattcaattcatttgtatatttattggagt Lasius colei ttttctttaaataatttttttctaaatattcaattcatttgtatatttattggagt Lasius arizonicus ttttctttaaataatttttttctaaatattcaattcatttgtatatttattggagt Lasius interjectus ttttctttaaataatttttttctaaatattcaattcatttgtatatttattggagt Lasius latipes ttttctttaaataatttttttctaaatattcaattcatctgtatatttattggagt Lasius murphyi ttttctttaaataatttttttctaaatattcaattcatttgtatatttattggagt Lasius creightoni ttttctttaaataatttttttctaaatattcaattcatttgcatatttattggagt Lasius occidentalis ttttctttaaataatttttttctaaatattcaattcatctgtatatttattggagt Lasius lat x claviger ttttctttaaataatttttttctaaatattcaattcatctgtatatttattggagt

129 1008 1013 1018 1023 1028 1033 1038 1043 1048 1053 1058 1063 | | | | | | | | | | | | Myrmecocystus mexicanus ------Lasius umbratus aaatttaacattttttcctcaacattttttaggtttaagaggtatgccccgtcgtt Lasius spathepus aaatttaacattttttcctcaacattttttaggtttaagaggyatacctcgwcgwt Lasius nearcticus aaatttaacattttttcctcaacactttttaggtttaagtggtatacctcgtcgtt Lasius alienus ------Lasius claviger aaatttaacattttttccacaacatttcttaggtttaagaggcatacctcgccgtt Lasius coloradensis aaatttaacattttttccacaacattttttaggtttaagaggtatgcctcgtcgtt Lasius californicus aaatttaacattttttccacaacatttcttaggtttaagaggcatacctcgccgtt Lasius colei aaatttaacattttttccacaacatttcttaggtttaagaggcatacctcgccgtt Lasius arizonicus aaatttaacattttttccacaacatttcttaggcttaagaggtatacctcgccgtt Lasius interjectus aaatttaacattttttccacaacatttcttaggtttaagaggtatacctcgccgtt Lasius latipes aaatttaacattttttccacaacattttttaggtttaagaggtatgcctcgtcgtt Lasius murphyi aaatttaacattttttccacaacattttttaggtttaagaggcatacctcgtcgtt Lasius creightoni aaatttaacattttttccacaacatttcttaggtttaagaggcatacctcgccgtt Lasius occidentalis aaatttaacattttttccacaacattttttaggtttaagaggtatgcctcgtcgtt Lasius lat x claviger aaatttaacattttttccacaacattttttaggtttaagaggtatgcctcgtcgtt

1064 1069 1074 1079 1084 1089 1094 1099 1104 1109 1114 1119 | | | | | | | | | | | | Myrmecocystus mexicanus ------Lasius umbratus attcagattacccagatacatatctttcatgaaatattatttcttctatcggatct Lasius spathepus aytcwgactayccagatwmmtayytttcatgraatattatttcttctattggatcy Lasius nearcticus actcagattatccagacacatatctttcatgaaatattatttcttctattggatct Lasius alienus ------Lasius claviger attcagattatccagatacatacctctcatgaaatattatttcttccattggatcc Lasius coloradensis attcagattatccagattcatacctttcatggaatattatttcttctattggatct Lasius californicus attcagattatccagatacatacctctcatgaaatattatttcttccattggatcy Lasius colei attcagattatccagatacatacctctcatgaaatattatttcttccattggatcc Lasius arizonicus attcagattatccagatacatacctctcatgaaatattatttcttccattggatcc Lasius interjectus attcagattatccagatacatacctctcatgaaatattatttcttccattggatcc Lasius latipes attcagattatccagattcatacctttcatggaatattatttcttctattggatct Lasius murphyi attcagactacccagattcatacctttcatggaatattatttcttctattggatct Lasius creightoni attcagattatccagatacatacctctcatgaaatattatttcttccattggatcc Lasius occidentalis attcagattatccagattcatacctttcatggaatattatttcttctattggatct Lasius lat x claviger attcagattatccagattcatacctttcatggaatattatttcttctattggatct

1120 1125 1130 1135 1140 1145 1150 1155 1160 1165 1170 1175 | | | | | | | | | | | | Myrmecocystus mexicanus ------Lasius umbratus ttaatttcaatacttagacttatttttttaatatatctaatctgagaatccttatc Lasius spathepus ttaatttcaattatwagaytwatttttttaatatacttaatttgagartctytatc Lasius nearcticus ttaatctcaattattagacttatttttttaatatacttaatttgagaatctttatc Lasius alienus ------Lasius claviger ttaatttcaataattagacttgtttttctaatatacttaatttgagaatccttatc Lasius coloradensis ttaatttcaattattagacttatttttttaatattcttaatttgagaatccctatc Lasius californicus ttaatttcaataattagacttgtttttctaatatacttaatttgagaatcyttatc Lasius colei ttaatttcaataattagacttgtttttctaatatacttaatttgagaatccttatc Lasius arizonicus ttaatttcaataattagacttgtttttctaatatacttaatttgagaatccttatc Lasius interjectus ttaatttcaataattagacttgtttttctaatatacttaatttgagaatccttatc Lasius latipes ttaatttcaattattagacttatttttttaatattcttaatttgagaatccctatc Lasius murphyi ttaatttcaattattagacttatttttttaatatacttaatttgagaatctctatc Lasius creightoni ttaatttcaataattagacttatttttctaatatacttaatttgagaatccttatc Lasius occidentalis ttaatttcaattattagacttatttttttaatattcttaatttgagaatccctatc Lasius lat x claviger ttaatttcaattattagacttatttttttaatattcttaatttgagaatccctatc

130 1176 1181 1186 1191 1196 1201 1206 1211 1216 1221 1226 1231 | | | | | | | | | | | | Myrmecocystus mexicanus ------Lasius umbratus ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius spathepus ytccaaacgaataattttaaatytattttttttaaattcatctcttgaatgattaa Lasius nearcticus ttccaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius alienus ------Lasius claviger ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius coloradensis ttctaaacgaataattttaaatctattttttttaaattcatctcttgaatgattaa Lasius californicus ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius colei ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius arizonicus ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius interjectus ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius latipes ttctaaacgaataattttaaatctattttttttaaattcatctcttgaatgattaa Lasius murphyi ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius creightoni ttctaaacgaataattttaaatttattttttttaaattcatctcttgaatgattaa Lasius occidentalis ttctaaacgaataattttaaatctattttttttaaattcatctcttgaatgattaa Lasius lat x claviger ttctaaacgaataattttaaatctattttttttaaattcatctcttgaatgattaa

1232 1237 1242 1247 1252 1257 1262 1267 1272 1277 | | | | | | | | | | Myrmecocystus mexicanus ------Lasius umbratus gactctattatcccccattaaatcacagatataatgaaattccttcaat Lasius spathepus gayy---wtacccyccattaaatcatagatataacgaaattccctcaat Lasius nearcticus gaac---ttatcctccattaaatcatagatataacgaaattccctcaat Lasius alienus ------Lasius claviger gact---ttaccccccattaaatcatagatataatgaaatcccatcaat Lasius coloradensis gact---ttatcccccattaaatcatagatataatgaaattccgtcaat Lasius californicus gact---ttaccccccattaaatcatagatataatgaaatcccatcaat Lasius colei gact---ttaccccccattaaatcatagatataatgaaattccatcaat Lasius arizonicus gact---ttatcccccattaaatcatagatataatgaaattccatcaat Lasius interjectus gact---ttatcccccattaaatcatagatataatgaaattccatcaat Lasius latipes gact---ttatcctccattaaatcatagatataatgaaattccgtcaat Lasius murphyi gact---ttaccctccattaaatcatagatataatgaaattccatcaat Lasius creightoni gact---ttaccccccattaaatcatagatataatgaaattccatcaat Lasius occidentalis gact---ttatcctccattaaatcatagatataatgaaattccgtcaat Lasius lat x claviger gact---ttatcctccattaaatcatagatataatgaaattccgtcaat

SUMMARY PERCENTAGES

MISSING (?): 41 cells, 0 percent of matrix. DASHES (-): 1325 cells, 6 percent of matrix. TOTAL POLYMORPHISM ($ , *): 88 cells, 0 percent of matrix. TOTAL FULL AMBIGUITY (? , -): 1366 cells, 6 percent of matrix. TOTAL FULL + PARTIAL AMBIGUITY (? , -, *, $): 1454 cells, 7 percent of matrix. STATE (0): 5742 cells, 28 percent of matrix. STATE (0) EMBEDDED IN POLYMORPHISM: 41 cells, 0 percent of matrix. STATE (1): 3203 cells, 15 percent of matrix. STATE (1) EMBEDDED IN POLYMORPHISM: 53 cells, 0 percent of matrix. STATE (2): 2273 cells, 11 percent of matrix. STATE (2) EMBEDDED IN POLYMORPHISM: 6 cells, 0 percent of matrix. STATE (3): 7824 cells, 38 percent of matrix. STATE (3) EMBEDDED IN POLYMORPHISM: 78 cells, 0 percent of matrix.

CHARACTER LIST (only named characters listed!)

131

APPENDIX B

WINGLESS SEQUENCE DATA ALIGNED BY MUSCLE (MULTIPLE SEQUENCE

COMPARISON BY LOG EXPECTATION)

132

0 5 10 15 20 25 30 35 40 45 50 55 | | | | | | | | | | | | Myrmecocystus mexicanus -----gatgcaggcgcacctctcgaccaccagcacctcctgcgtcttgtagcctct Lasius umbratus tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius spathepus ------tct Lasius nearcticus tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius alienus tggaagatgcaggcgcacctctcgaccaccagcacctcctgcgtcttgtagcctct Lasius claviger tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius coloradensis ------tcttgtagcctct Lasius californicus tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius colei tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius arizonicus tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius interjectus tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius latipes tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius murphyi tggaagatgcaggcgcacctctcgaccaccagcacctcctgcgtcttgtagcctct Lasius creightoni tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct Lasius lat x claviger tggaagatgcaggcgcacctctcgaccaccagcacctcctgtgtcttgtagcctct

56 61 66 71 76 81 86 91 96 101 106 111 | | | | | | | | | | | | Myrmecocystus mexicanus gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius umbratus gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius spathepus gccgcagcacatcagatcgcagccgwcgacgccg-aggctggtgtcgttgcactgt Lasius nearcticus gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius alienus gccgcagcacatcagatcgcagccgtctacgccg-aggctggtgtcgttgcactgt Lasius claviger gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius coloradensis gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius californicus gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius colei gccgcagcacatcagatcgcagccgtcgacgccg?aggctggtgtcgttgcactgt Lasius arizonicus gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius interjectus gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius latipes gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius murphyi gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius creightoni gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt Lasius lat x claviger gccgcagcacatcagatcgcagccgtcgacgccg-aggctggtgtcgttgcactgt

112 117 122 127 132 137 142 147 152 157 162 167 | | | | | | | | | | | | Myrmecocystus mexicanus cgcccgtgagtgcccaggataccgagcgtgggattcttctcgcagaacgccggcga Lasius umbratus cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius spathepus cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius nearcticus cgcccgtgggtgcccaggataccgagcgtgggattcttctcgcagaacgccggcga Lasius alienus cgcccgtgggtgcccaggataccgagcgtgggattcttctcgcagaacgccggcga Lasius claviger cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius coloradensis cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius californicus cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius colei cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius arizonicus cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius interjectus cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius latipes cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius murphyi cgcccgtgggtgcccaggataccgagcgtgggattcttctcgcagaacgccggcga Lasius creightoni cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga Lasius lat x claviger cgcccgtgggtgcccaggataccgagggtgggattcttctcgcagaacgccggcga

133

168 173 178 183 188 193 198 203 208 213 218 223 | | | | | | | | | | | | Myrmecocystus mexicanus ctgctccaggtagacgagatctttcggcccgggcggcttgtgctccggattgtacg Lasius umbratus ctgctcgaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius spathepus ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius nearcticus ttgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius alienus ctgctccaggtagacgaggtccttcggcccgggcggcttgtgctccggattgtacg Lasius claviger ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius coloradensis ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius californicus ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius colei ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius arizonicus ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius interjectus ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius latipes ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius murphyi ctgctccaggtagacgaggtccttcggcccgggcggcttgtgctccggattgtacg Lasius creightoni ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg Lasius lat x claviger ctgctccaggtagacgaggtctttcggcccgggcggcttgtgctccggattgtacg

224 229 234 239 244 249 254 259 264 269 274 279 | | | | | | | | | | | | Myrmecocystus mexicanus gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctgatgc Lasius umbratus gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius spathepus gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius nearcticus gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgg Lasius alienus gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius claviger gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius coloradensis gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius californicus gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius colei gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius arizonicus gcttcagctgaaagttgtaacgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius interjectus gcttcagctgaaarttgtarcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius latipes gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius murphyi gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius creightoni gcttcagctgaaagttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc Lasius lat x claviger gcttcagctgaaarttgtagcgttgccggcgcgcgagaccgtcgcgatgctggtgc

280 285 290 295 300 305 310 315 320 325 330 335 | | | | | | | | | | | | Myrmecocystus mexicanus acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattact Lasius umbratus acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius spathepus acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius nearcticus acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius alienus acggagttgctcgccgagttggcgtgcacattgctgcgcacgcgatccgagttgct Lasius claviger acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius coloradensis acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius californicus acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius colei acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius arizonicus acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius interjectus acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius latipes acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius murphyi acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius creightoni acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct Lasius lat x claviger acggagttgctcgccgagttgacgtgcacattgctgcgcacgcgatccgaattgct 134

336 341 346 351 356 361 366 371 376 381 386 391 | | | | | | | | | | | | Myrmecocystus mexicanus gaccattactcgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius umbratus gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius spathepus gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius nearcticus gaccataacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius alienus gaccattacccgggagg-cgccgt-cgaagcgatccttcagattgtcgc-cgacca Lasius claviger gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius coloradensis gaccattacccgggaggccgccgt-cgaagcgatcc-tcagat------Lasius californicus gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius colei gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius arizonicus gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgctcgacca Lasius interjectus gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius latipes gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius murphyi gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius creightoni gaccattacccgggagg-cgccgt-cgaagcgatccttcagattatcgc-cgacca Lasius lat x claviger gaccattacccgggagg-cgccgt?cgaagcgatccttcagattatcgc-cgacca

392 397 402 407 412 417 | | | | | | Myrmecocystus mexicanus cgcggaaacttggcagc------Lasius umbratus cgcggaaacttggcaaccgcatccagc Lasius spathepus cgcggaaacttggcaaccgcatccagc Lasius nearcticus cgcggaaacttggcagccgcatccagc Lasius alienus cgcggaaactcggcagccgcatccagc Lasius claviger cgcg?aaacttggcaaccgcatccagc Lasius coloradensis ------Lasius californicus cgcggaaacttggcaaccgcatccagc Lasius colei cgcg------Lasius arizonicus cgcggaaacttggcaaccgcatccagc Lasius interjectus cgcggaaacttggcaaccgcatccagc Lasius latipes cgcggaaacttggcaaccgcatccagc Lasius murphyi cgcggaaactcggcagccgcatccagc Lasius creightoni cgcggaaacttggcaaccgcatccagc Lasius lat x claviger cgcggaaacttggcaaccgcatccagc

SUMMARY PERCENTAGES

MISSING (?): 3 cells, 0 percent of matrix. DASHES (-): 230 cells, 3 percent of matrix. TOTAL POLYMORPHISM ($ , *): 4 cells, 0 percent of matrix. TOTAL FULL AMBIGUITY (? , -): 233 cells, 3 percent of matrix. TOTAL FULL + PARTIAL AMBIGUITY (? , -, *, $): 237 cells, 3 percent of matrix. STATE (0): 1024 cells, 16 percent of matrix. STATE (0) EMBEDDED IN POLYMORPHISM: 4 cells, 0 percent of matrix. STATE (1): 1895 cells, 30 percent of matrix. STATE (2): 1918 cells, 30 percent of matrix. STATE (2) EMBEDDED IN POLYMORPHISM: 3 cells, 0 percent of matrix. STATE (3): 1211 cells, 19 percent of matrix. STATE (3) EMBEDDED IN POLYMORPHISM: 1 cells, 0 percent of matrix.

135

APPENDIX C

TAXONOMIC DESCRIPTIONS

136 Taxonomic descriptions

Wing (1968) used the unusual term "digm" in his species descriptions. I interpreted the term as analogous to "type" for the putative hybrids he proposed. I consulted ant taxonomists Stefan Cover, Phil Ward, and Barry Bolton about this issue and they agreed that the unusual term was probably intended as a substitute for "type" but not a valid ICZN term. The term is repeated here for the verbatim transcript of label data and taxonomic description.

The distributional maps in this section come from Wing's 1968 revision. He provided distributional maps of the most commonly collected species but did not provide distributional data elsewhere in the revision. Without those data available, these maps provide the best representation of distributional data.

Lasius (Acanthomyops) claviger

Formica clavigera Roger ,1862: queen, p. 241, pl. 1, fig. 13.

Acanthomyops claviger (Roger). Mayr, 1862: queen, p.700.

Lasius claviger (Roger). Mayr, 1870: worker and male, p. 950.

Lasius (Acanthomyops) parvula M. R. Smith, 1934: worker, p. 213. Synonym.

Acanthomyops claviger (Roger). Creighton, 1950: p.426

Lasius (Acanthomyops) claviger (Roger). Ward, 2005: p. 13

Type locality: Pennsylvania

Location of type: Institut fur spezielle Zoologie und Zoologisches Museum der

Humboldt-Universitat Berlin.

Distribution: Fig C.1

137 Queen: gaster pubescence dilute; head pubescence dilute; gular setae on entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence erect; petiole in frontal view sides convex; dorsal crest emarginated; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) coloradensis

Lasius (Acanthomyops) interjectus subs. coloradensis Wheeler, 1917: worker, queen, and male p.532.

Acanthomyops claviger subsp. coloradensis (Roger). Creighton, 1950: pp. 429-430.

Acanthomyops coloradensis (Wheeler) Wing, 1968: p. 78.

Lasius (Acanthomyops) coloradensis. Ward, 2005: p. 13.

Type locality: Manitou, El Paso Co., Colorado.

Location of types: Syntypes in the MCZ.

Distribution: Fig C.1

Queen: gaster pubescence dilute; head pubescence dilute; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

138 Male: crest of petiole sharp.

Worker: scape pubescence decumbed to suberect; petiole in frontal view sides convex; dorsal crest emarginated; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) californicus

Lasius (Acanthomyops) interjectus subspecies californicus Wheeler, 1917: worker and queen, p. 531.

Acanthomyops claviger subspecies californicus (Wheeler). Creighton, 1950: p.430.

Acanthomyops californicus (Wheeler). Wing, 1968: pp. 85-88.

Lasius (Acanthomyops) californicus (Wheeler). Ward, 2005: p.13.

Type locality: Palmer's Canyon, San Gabriel Mts., near Clarmont, Los Angeles Co.,

California.

Location of types: one queen and four workers are in the MCZ.

Distribution: California. Los Angeles County: Altadena, Arcadia, Palmer's Canyon (San

Gabriel Mountains), Mescal Creek (San Gabriel Mountains). San Bernadino County:

Camp Baldy; San Diego County: Julian.

Queen: gaster pubescence dilute; head pubescence dilute; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

139 Male: crest of petiole sharp.

Worker: scape pubescence decumbed to suberect; petiole in frontal view sides parallel; dorsal crest emarginated; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface

Lasius (Acanthomyops) colei

Acanthomyops colei Wing, 1968: worker. pp. 88-89.

Lasius (Acanthomyops) colei Wing. Ward, 2005: p.13

Type locality : Cochise Stronghold, Dragoon Mts., Cochise Co., Arizona.

Location of types: Holotype worker and 3 paratype workers in the MCZ, 2 paratypes in the USNM, 2 paratypes in the Cornell collection, and 10 paratypes in the collection of

A.C. Cole.

Distribution: Arizona. Cochise County: Dragoon Mountains. Graham County: Post

Creek Canyon, Pinaleno Mountains; Post Canyon, Pinaleno Mountains. Pima County:

Apache Camp, South Catalina Mountains; Stratton, South Catalina Mountains. New

Mexico. Grant County: Black Mountain, Gila National Forest.

Queen: gaster pubescence dilute; head pubescence dilute; gular setae on less than entire surface; petiolar crest straight; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

140 Worker: scape pubescence appressed; petiole in frontal view sides convex; dorsal crest straight; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on posterior margins of tergites.

Lasius (Acanthomyops) arizonicus

Lasius (Acanthomyops) interjectus subspecies arizonicus Wheeler, 1917: worker, p.532.

Lasius (Acanthomyops) arizonicus (Wheeler). Buren, 1950: pp. 184, 186.

Acanthomyops interjectus subspecies arizonicus (Wheeler). Creighton, 1950: p.431.

Acanthomyops arizonicus (Wheeler). Wing, 1968: pp. 90-92.

Lasius (Acanthomyops) arizonicus (Wheeler). Ward, 2005: p.13.

Type locality: Huachuca Mts., Cochise Co., Arizona.

Location of types: syntypes in the MCZ.

Distribution: Cochise Co., Arizona.

Queen: gaster pubescence dilute; head pubescence dilute; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence appressed; petiole in frontal view sides parallel; dorsal crest emarginated; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae absent;

141 propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on posterior margins of tergites.

Lasius (Acanthomyops) interjectus

Lasius interjectus Mayr, 1866: queen, p.888, pl. 20, fig 3.

Acanthomyops interjectus (Mayr). Creighton, 1950: p. 430.

Lasius (Acanthomyops) interjectus (Mayr). Ward, 2005: p.13

Type locality: New Jersey

Location of type: Naturhistoriska Riksmuseum, Stockholm.

Distribution: Figure C.2

Queen: gaster pubescence dilute; head pubescence dense; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence erect; petiole in frontal view sides convex; dorsal crest emarginated; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on posterior margins of tergites.

142 Lasius (Acanthomyops) latipes

Formica latipes Walsh, 1862: queen, worker, and male, p. 311.

Lasius (Acanthomyops) latipes (Walsh). Mayr, 1866: queen, p.889, pl. 20, figs. 4a and

4b.

Lasius (Acanthomyops) latipes beta-form: Wheeler and McClendon, 1903.

Acanthomyops latipes (Walsh). Creighton, 1950: p.431.

Lasius (Acanthomyops) latipes (Walsh). Ward, 2005: p.13.

Type locality: Edwin S. George Reserve, Livingston Co., MI.

Location of types: Neotype in the MCZ.

Distribution: Fig C.3

Queen: gaster pubescence dense; head pubescence dilute; gular setae on entire surface; petiolar crest curved; setae on dorsum of gaster on entire surface; head distorted; genual plate present; femur width 0.73 mm or less.

Male: crest of petiole blunt.

Worker: scape pubescence decumbed to suberect; petiole in frontal view sides diverging dorsad; dorsal crest curved; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale blunt; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

143 Lasius (Acanthomyops) murphyi

Lasius (Acanthomyops) Murphii Forel, 1901: worker, p. 367, queen, p. 368, male, p. 369.

Acanthomyops murphyi (Forel). Creighton, 1950: p.113-117.

Lasius (Acanthomyops) murphyi (Forel). Ward, 2005: p. 13.

Type locality: Morganton, Burke Co., North Carolina.

Location of types: Syntypes in the MCZ, AMNH, and Forel Collection, Museum d'Histoire Naturelle, Geneva.

Distribution: Figure C.4

Queen: gaster pubescence dense; head pubescence dense; gular setae on entire surface; petiolar crest curved; setae on dorsum of gaster absent; head distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole blunt.

Worker: scape pubescence appressed; petiole in frontal view sides diverging dorsad; dorsal crest curved; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale blunt; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

144 Lasius (Acanthomyops) pogonogynous

Lasius (Acanthomyops) pogonogynous Buren, 1950: queen and worker, p.186.

*Wing proposed that pogonogynous was a hybrid of Lasius murphyi and latipes in 1968, p. 117-119.

Lasius (Acanthomyops) pogonogynous Buren. Ward, 2005: p.13.

Type locality: Red Feather Lakes, Larimer Co. Colorado.

Location of types: Holotype, female paratype, females, and workers in the USMN, paratype female in the MCZ.

Distribution: Red Feather Lakes, Larimer County, Colorado. Moscow, Latah County,

Idaho. Ames, Story County, Iowa.

Queen: gaster pubescence dense; head pubescence dilute; gular setae on entire surface; petiolar crest straight; setae on dorsum of gaster on posterior margins of tergites; head distorted; genual plate present; femur width 0.80mm or greater.

Male: crest of petiole blunt.

Worker: scape pubescence decumbed to suberect; petiole in frontal view sides diverging dorsad; dorsal crest curved; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale blunt; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

145 Lasius (Acanthomyops) subglaber

Lasius claviger var. subglaber Emery, 1893: worker, queen, and male, p.642.

Lasius clavigeroides Buren, 1942: worker, p.406, queen and male, p. 407.

Acanthomyops subglaber: Creighton, 1950: worker, p.433. (clavigeroides synonymized).

Lasius (Acanthomyops) subglaber (Emery). Ward, 2005: p.13.

Type locality: Washington, D.C.

Location of types: A queen, a worker, and a male in the AMNH.

Distribution: Figure C.5

Queen: gaster pubescence dilute; head pubescence dilute; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence decumbed to suberect; petiole in frontal view sides convex; dorsal crest emarginated; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale blunt; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) plumopilosus

Lasius (Acanthomyops) plumopilosus Buren, 1941: queen, p.231; worker and male, p.

232, fig.1.

Acanthomyops plumopilosus (Buren). Creighton, 1950: p.433.

Lasius (Acanthomyops) plumopilosus (Buren). Ward, 2005: p.13.

146 Type locality : Backbone State Park Delaware Co. Iowa.

Location of types: Holotype female USNM: paratypes in several collections including the MCZ.

Distribution: Backbone State Park, Iowa. Madison Co., Iowa, Crow Wing County,

Minnesota. Washtenaw County, Michigan. Rowan County, North Carolina.

Queen: gaster pubescence dilute; head pubescence dilute; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on entire surface; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence appressed; petiole in frontal view sides parallel; dorsal crest emarginated; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale blunt; setae with plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) bureni

Acanthomyops bureni Wing, 1968: pp.135-138.

Lasius (Acanthomyops) bureni (Wing). Ward, 2005: p.13.

Type locality: Comstock, Barron Co. Wisonsin

Location of types: Holotype queen, paratype queen, 2 paratype males, and 4 paratype workers in the MCZ. Paratypes, 2 of each caste in both the USNM and Cornell

Collections.

Distribution: Comstock, Barron County, Wisconsin.

147 Queen: gaster pubescence dense; head pubescence dense; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence appressed; petiole in frontal view sides diverging dorsad; dorsal crest emarginated; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) pubescens

Lasius (Acanthomyops) pubescens Buren, 1942: worker and female, p.405.

Acanthomyops pubescens (Buren). Creighton, 1950: p.433.

Lasius (Acanthomyops) pubescens (Buren). Ward, 2005: p. 433.

Type locality: Jenkins, Crow Wing Co., Minnesota.

Location of types: Holotype queen in the W. F. Buren Collection; paratypes in the collections of the MCZ, USNM, and Iowa State College.

Distribution: McGrath, Aitkin County, Minnesota. Jenkins, Crow Wing County,

Minnesota.

Queen: gaster pubescence dense; head pubescence dilute; gular setae on less than entire surface; petiolar crest straight; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: no specimen available

148 Worker: scape pubescence appressed; petiole in frontal view sides convex; dorsal crest curved; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale blunt; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) creightoni

Acanthomyops creightoni Wing, 1968: p. 141.

Lasius (Acanthomyops) creightoni. Ward, 2005: p.13.

Type locality: Warner Ranger Station, La Sal Mts., Moab, Grand Co., Utah.

Location of types: Holotype female, 2 paratype females, 3 paratype males, and 3 paratype workers in the MCZ.

Distribution:

Queen: gaster pubescence dilute; head pubescence dilute; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on entire surface; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence decumbed to suberect; petiole in frontal view sides parallel; dorsal crest emarginated; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

149 Lasius (Acanthomyops) occidentalis

Lasius (Acanthomyops) occidentalis Wheeler, 1909: worker, queen, and male p.83.

Acanthomyops occidentalis (Wheeler). Creighton, 1950: p. 432.

Lasius (Acanthomyops) occidentalis (Wheeler). Ward, 2005: p. 13.

Type locality: Colorado Springs, El Paso Co., Colorado.

Location of types: Syntypes in the MCZ.

Distribution: Figure C.4.

Queen: gaster pubescence dense; head pubescence dense; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on posterior margins of tergites; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence appressed; petiole in frontal view sides convex; dorsal crest emarginated; occipital border feebly convex; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) mexicanus

Lasius (Acanthomyops) interjectus subspecies mexicanus Wheeler, 1914: worker and queen, p. 55 male p. 56.

Lasius (Acanthomyops) mexicanus (Wheeler). Buren, 1950: 185-186.

150 Acanthomyops mexicanus (Wheeler). Creighton, 1950: p. 426.

Lasius (Acanthomyops) mexicanus (Wheeler). Ward, 2005: p.13.

Type locality: Guerrero Mill, State of Hildago, Mexico.

Location of types: Syntypes in the MC Z.

Queen: gaster pubescence dense; head pubescence dense; gular setae on less than entire surface; petiolar crest emarginate; setae on dorsum of gaster on entire surface; head not distorted; genual plate absent; femur width 0.80mm or greater.

Male: crest of petiole sharp.

Worker: scape pubescence appressed; petiole in frontal view sides convex; dorsal crest emarginated; occipital border straight; number of maxillary palp segments 3; crest of petiolar scale sharp; setae without plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

Lasius (Acanthomyops) latipes x claviger

Lasius (A.) latipes alpha-form: Wheeler and McClendon (1903) and subsequent authors.

Digm* locality: Selden Suffolk Co. Long Island New York

Location of digms: A typical queen, male, and 2 workers from a single colony collected at the above locality by R. Sanwald are in the MCZ.

Distribution: Figure C.3

151 Queen: gaster pubescence ambiguous(-); head pubescence dilute; gular setae on entire surface; petiolar crest curved; setae on dorsum of gaster on posterior margins of tergites; head ambiguous(-); genual plate absent; femur width 0.73 mm or less.

Male: crest of petiole blunt.

Worker: scape pubescence decumbed to suberect; petiole in frontal view sides diverging dorsad; dorsal crest curved; occipital border feebly convex; number of maxillary palp segments 6; crest of petiolar scale blunt; setae with plumose tips; gular setae present; propodeal spiracle circular; impression anterobasally on gaster absent; dorsum of propodeum flat or evenly rounded; activity hypogeic; gaster setae on entire surface.

latipes x coloradensis hybrid

Lasius latipes: Weber, 1935: alpha female, p. 200.

Acanthomyops latipes x coloradensis Wing 1968

Types: None but 1 digm specimen, a dealate queen in the MCZ.

Distribution: Towner, McHenry County, North Dakota.

murphyi x subglaber hybrid:

Acanthomyops murphyi x subglaber Wing, 1968.

Digm locality: Medford, Suffolk Co., New York.

Location of digms: a queen and 2 workers in the MCZ.

Distribution: Medford, Suffolk County, Long Island

152 subglaber x plumopilosus hybrid

Acanthomyops subglaber x plumopilosus Wing, 1968.

Digm locality: Selden, Suffolk Co., New York.

Location of digms: a queen and 2 workers in the MCZ.

Distribution: Seldon and N.E. Patchogue, Suffolk County, New York.

153

Fig C.1. Distribution of Lasius claviger (solid circles) according to Wing (1968) (based on 486 samples). Distribution of Lasius coloradensis (hollow circles) according to Wing

(1968) (based on 70 samples).

154

Figure C.2. Distribution of Lasius interjectus according to Wing (1968) (based on 337 samples).

155

Figure C.3. Distribution of Lasius latipes (solid circles) according to Wing (1968) (based on 448 samples). Distribution of Lasius latipes x claviger (hollow circles) according to

Wing (1968) (based on 26 samples).

156

Fig C.4 Distribution of Lasius murphyi according to Wing (1968) (based on 49 samples).

157

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