ECOLOGICAL AND EVOLUTIONARY DIVERSIFICATION IN SEED

(COLEOPTERA: BRUCHINAE)

A thesis presented by

Geoffrey Easton Morse

to

The Department of Organismic and Evolutionary Biology

in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

in the subject of

Biology

Harvard University

Cambridge, Massachusetts

May, 2003

© 2003 Geoffrey Easton Morse

All rights reserved.

Thesis advisor: Prof. Brian D. Farrell Geoffrey Easton Morse

Ecological and Evolutionary Diversification in Seed Beetles (Coleoptera: Bruchinae)

The diversification of phytophagous is seen as one of the most fascinating

phenomena in the history of life. The goal of this thesis is to investigate the mode and

tempo of diversification in one group of these insects, the bruchine beetles (Coleoptera:

Chrysomelidae: Bruchinae). These beetles represent the largest diversification of insects

that use the embryonic tissue of . This thesis integrates multiple levels of analysis

in order to provide a thorough picture of diversification, from processes that govern

macroevolution across 80 million years to processes that create divergence within and

between populations.

The research first uses a phylogenetic framework based on mitochondrial and

nuclear loci to examine the link between phyletic and ecological diversification in

bruchines. Sister-group comparisons and ancestral state reconstructions indicate that

these beetles have diversified by treating individual as differentiable niches,

causing rates of diversification in the insects to closely correspond to those of their host

plants. Because this implies a role for specialization in the diversification of bruchines,

the research then examines the trajectory of diet breadth evolution based on a

phylogenetic reconstruction of the using mitochondrial and nuclear sequence data. Analyses of the trajectory of specialization suggest that a trend toward increased specialization is contingent upon the novelty of the host shift, behavior, and community ecology. This relationship between specialization and diversification is

iii examined more closely via interspecific phylogeographic and historical demographic analyses of the speciation of the specialist Stator beali from the generalist S. limbatus using mitochondrial sequence data. Phylogeographic analysis suggests a paraphyletic relationship and indicates a local geographic context for speciation, while demographic analysis suggests an important role for selection in a rapid speciation event, although elucidation of the specific mode of speciation is beyond the scope of this thesis.

This combination of microevolutionary and macroevolutionary analyses, and biogeographic and ecological information, provide a framework for investigating the processes and patterns of diversification. The insights that are gained from this integrative framework are more than the sum of their individual parts and provide a solid foundation on which to test specific phylogenetic, genetic, and ecological hypotheses.

iv

Table of Contents

List of Figures………………………………………………………..…………..vi

List of Tables………………………………………………….………..………..ix

Acknowledgments……………………….…………………………..………...... xi

I. Chapter 1……………………………………………………………………..……1

Adaptive radiation and ecological diversification in the bruchine seed beetles

(Coleoptera: Chrysomelidae: Bruchinae)

II. Chapter 2………………………………………………………………………..127

The evolution of specialization in the genus Stator Bridwell (Coleoptera:

Chrysomelidae: Bruchinae)

III. Chapter 3…………………………………………………………………….….245

Interspecific phylogeography of a paraphyletic species pair: The geographic

context of speciation and specialization

IV. Chapter 4…………………………………………………………………….….286

Patterns of population differentiation and demographic history in a sister-species

pair of seed beetles (Coleoptera: Stator): Implications for Speciation and

Specialization

V. Literature Cited…………………………………………………………………342

v

List of Figures

Chapter 1

Figure 1.1 Borowiec’s hypothesis of bruchid relationships 10 Figure 1.2 Saturation analysis, corrected vs. uncorrected distances 71 Figure 1.3 Saturation analysis, ti/tv ratio vs. transversions 72 Figure 1.4 Strict consensus of parsimony analysis 76 Figure 1.5 Burn-in generations for Bayesian analysis 79 Figure 1.6 Consensus Bayesian phylogeny 80 Figure 1.7 Consensus Bayesian phylogram 82 Figure 1.8 Sister groups on parsimony 84 Figure 1.9 Sister groups on Bayesian tree 88 Figure 1.10 Host plant clade on parsimony tree 92 Figure 1.11 Host plant clade on Bayesian tree 94 Figure 1.12 Maximum-likelihood reconstruction of host clade, Acanthoscelidini 97 clade 1 Figure 1.13 Maximum-likelihood reconstruction of host clade, Acanthoscelidini 98 clade 2 Figure 1.14 Maximum-likelihood reconstruction of host clade, Basal grade of 99 Bruchidae Figure 1.15 Maximum-likelihood reconstruction of host growth form 100 Figure 1.16 Host species recorded per bruchid host species 116

Chapter 2

Figure 2.1 ‘Grafen’ supertree of Stator host plants 149 Figure 2.2 ‘Minimal evolution’ supertree of Stator host plants 150 Figure 2.3 Supertree of Stator host plant lineages 176 Figure 2.4 Saturation analysis, corrected vs. uncorrected distances 183 Figure 2.5 Saturation analysis, ti/tv ratio vs. transversions 183 Figure 2.6 Strict consensus of parsimony analysis 186 Figure 2.7 Burn-in generations for Bayesian analysis 188 Figure 2.8 Consensus Bayesian phylogeny 189

vi

Chapter 2 (Continued)

Figure 2.9 Consensus parsimony phylogram 191 Figure 2.10 Consensus Bayesian phylogram 192 Figure 2.11 Histogram of phylogenetic distance (PD) of host species per Stator 193 species, ‘Grafen’ Figure 2.12 Histogram of PD of host species per Stator species, ‘Minimal 193 evolution’ trees Figure 2.13 Histogram of number of host species per Stator species 194 Figure 2.14 Histogram of number of host species per Stator species 194 Figure 2.15 Parsimony reconstruction of generalists and specialists 197 Figure 2.16 Parsimony reconstruction of oviposition guild 198 Figure 2.17 Parsimony reconstruction of host plant genera 199 Figure 2.18 Parsimony reconstruction of host plant hierarchical clade 202 Figure 2.19 Maximum likelihood reconstruction of generalists and specialists 204 Figure 2.20 Maximum likelihood reconstruction of host plant clade 206 Figure 2.21 Maximum likelihood reconstruction of of oviposition guild 207 Figure 2.22 GLS reconstruction of number of host species 208 Figure 2.23 GLS reconstruction of number of host genera 209 Figure 2.24 GLS reconstruction of PD of host species, ‘Grafen’ trees 210 Figure 2.25 GLS reconstruction of PD of host species, ‘Minimal evolution’ trees 211

Chapter 3

Figure 3.1 Distribution of Stator limbatus 256 Figure 3.2 Distribution of Stator beali 257 Figure 3.3 Genitalia of S. limbatus and S. beali 259 Figure 3.4 Stator beali eggs on 260 Figure 3.5 Stator limbatus eggs on tenuifolia 260 Figure 3.6 Map of sites sampled for S. limbatus and S. beali 263 Figure 3.7 Agreement topology between maximum-likelihood, Bayesian, and 272 parsimony analyses Figure 3.8 Phylogram from maximum-likelihood analysis 273 Figure 3.9 Phylogeography of monophyletic lineages 275

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Chapter 4

Figure 4.1 Sampling localities of S. beali and S. limbatus within the 303 Tamaulipan biogeographic province Figure 4.2 Genealogical relationships among Stator beali haplotypes 310 Figure 4.3 Genealogical relationships among Stator limbatus haplotypes in the 311 Mexican xerophytic province Figure 4.4 Frequency and distribution of haplotypes sampled for S. limbatus 315 within the Tamaulipan province Figure 4.5 Frequency and distribution of haplotypes sampled for S. beali 316

Figure 4.6 Plot of FST from pairwise population comparisons versus geographic 317 distance within the Tamaulipan geographic province for Stator limbatus and S. beali Figure 4.7 Mismatch distributions for Stator limbatus and S. beali 324 Figure 4.8 Observed and expected site frequency distributions for S. beali 324 given a coalescent model of rapid population expansion (Wakeley and Hey, 1997) Figure 4.9 Maximum likelihood estimation of θ and population growth rate (g) 325 for S. limbatus Figure 4.10 Maximum likelihood estimation of θ and population growth rates (g) 326 for S. beali Figure 4.11 Deterministic backward extrapolation of female effective population 327 sizes of S. limbatus and S. beali given the maximum-likelihood estimations of θ and g

viii

List of Tables

Chapter 1

Table 1.1 Classification and biogeography of the Bruchidae 6 Table 1.2 Species included in molecular phylogenetic analysis of the 29 Bruchidae Table 1.3 Sequences of primers used in molecular phylogenetic analysis of the 38 Bruchidae Table 1.4 Host growth form summarized by exemplar taxa 48 Table 1.5 Host plant clade summarized by exemplar taxa 53 Table 1.6 Ecological codings for macroevolutionary analyses of ecological 63 associations Table 1.7 Properties of gene subsets 70 Table 1.8 Tests of a priori hypotheses of monophyly 75 Table 1.9 Summary of sister-group diversity comparisons 86 Table 1.10 Results of analyses of phylogenetic constraint 91 Table 1.11 Results of the analysis of the apparency hypothesis 103

Chapter 2

Table 2.1 Host plant relationships of 22 species of Stator with established host 143 plant associations Table 2.2 Geographic distribution of 24 well-established species of Stator 146 Table 2.3 Specimens of Stator included in molecular phylogenetic analysis 156 Table 2.4 Sequences of primers used in this study 161 Table 2.5 Discrete ecological codings for analyses of character evolution 172 Table 2.6 Continuous ecological codings for analyses of character evolution 177 Table 2.7 Properties of gene subsets 184 Table 2.8 Results of analyses of phylogenetic constraint 200 Table 2.9 Results of analyses of trajectory of diet breadth 212 Table 2.10 Correlation between oviposition behavior and diet breadth 215

ix

Chapter 3

Table 3.1 Site localities, collection codes, and haplotypes sampled for S. beali 264 Table 3.2 Site localities, collection codes, and haplotypes sampled for North 265 American S. limbatus Table 3.3 Site localities, collection codes, and haplotypes sampled for South 266 American S. limbatus

Chapter 4

Table 4.1 Sampling localities with number of individuals sampled at each site 298 Table 4.2 Number of singletons and frequency of haplotypes from the 309 sampling localities where more than one individual was sampled Table 4.3 CO1 sequence diversity within and between populations of Stator 314 limbatus and Stator beali Table 4.4 Diversity estimates and effective population sizes for Stator 320 limbatus and S. beali Table 4.5 Estimates from models of size change in S. beali 323

x

Acknowledgments

I owe many people a great deal of thanks for their support, assistance, patience, and friendship over the course of my dissertation work. A special thanks to my advisor, Brian Farrell. Brian’s knowledge of natural history and evolutionary biology make his laboratory group function at a very high level and this research would have suffered without his guidance. He has always had an open door and a welcoming ear, and his patience as an advisor and friend during occasionally difficult times has been a tremendous aid over the years. Of course without Naomi Pierce I never would have been at Harvard in the first place. From the time I first met her in Australia she has been an enthusiastic and encouraging supporter of my research and progress. She also provided a great deal of support in the trials and tribulations that have occurred outside of my dissertation work and I owe her a great deal of gratitude for that. None of the work on speciation could have been possible without the guidance of John Wakeley. Through conversations, discussion groups, and his course on coalescence I managed to shift my perspective to look at diversification from the microevolutionary level as well as the macroevolutionary level. I also owe a great deal to Peter Stevens, whose incredible knowledge of and enthusiasm for plant systematics turned this entomologist into a pretty decent botanist My friends and colleagues in the Farrell lab have also been a source of great fun, information, and support. It was wonderful to share a lab with Ben Normark and Andrea Sequeira, who brought their wealth of knowledge into the lab and were more than happy to share it. I only hope I can set a similar example as a post-doc. It has been great fun to share interests, lab space, and time with Chris Smith, Chris Elzinga, Bruce Archibald, Catherine Linnen, Amanda Evans, Duane McKenna, Adriana Marvaldi, and Brian O’Meara. My thesis could not have been accomplished without the staff in the Department of Organismic and Evolutionary Biology and the Museum of Comparative Zoology. Phil Perkins, Stephan Cover, Emily Wood, and Pam Greene have been particularly helpful in allowing me to pursue a very rewarding dissertation. And, of course, other friends in the department have been wonderful, both socially and intellectually. Ellen Freund, Colin Meiklejohn, Amity Wilczek, Matt Thompson, Chris Dick, Rick Ree, Ryan Oyama, Kevin Boyce, Mike Shapiro, Kate Jackson, George Weiblen, Susanna Porter and many others made the last few years a very enjoyable experience. Most of all, I owe a great deal to my parents and my brother. My parents instilled in me a curiosity that has allowed me to thrive as a scientist and provided more support and encouragement to me than I would have thought possible. My brother has always been my best friend and his support, friendship, and encouragement have meant more than I can express. Wallace has been a wonderful companion and has been by my side (quite literally) throughout the entire writing process. I also must thank Christine for six years of patience, support, and friendship.

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Outside of Cambridge, C. Dan Johnson has been nothing short of a godsend. His knowledge of seed beetles, his enthusiasm for their study, and his willingness to share both of these was inspiring and incredibly helpful. Chuck Fox provided considerable encouragement, insight, and advice on the biology of Stator beetles. Susanna Muruaga de L’Argentier of Jujuy, Argentina has been both a great friend and colleague. She made research in Argentina a wonderful experience and I will always have a soft spot in my heart for her country as a result. Jesús Romero Nápoles made my experience in Mexico quite similar. I can only hope that this collaboration will continue for a long time to come. Terry Erwin of the Smithsonian has been a great source of support, as well as a wonderful resource to do research in Ecuador. Don Windsor of the Smithsonian’s Tropical Research Institute in Panama was a wonderful host and a great boon for my fieldwork there. Guido Pereira and José Clavijo Albertos of Venezuela made fieldwork in their country both enjoyable and successful. Felipe Noguera greatly assisted in fieldwork in western Mexico and Roger Blanco greatly facilitated my stay in Santa Rosa, Costa Rica. Numerous people provided specimens for the research, notably Midori Tuda of Kyushu, Japan, Steve Lingafelter of the Smithsonian, Betty Benrey of UNAM, and Masakazu Shimada of Tokyo, Japan. Dr. David Rockefeller has become a great friend and an inspiration as a coleopterist. It has been a wonderful experience getting to know him and to work on his magnificent collection. I owe a considerable debt of graditude to the generosity of Mr. George Putnam, whose support for field research at the Museum of Comparative Zoology made this research possible. I can only hope that my contributions to the entomology collections can express some of my gratitude. Lastly, I would also like to gratefully acknowledge financial support of this research by the National Science Foundation in the form of both a graduate research fellowship and a dissertation improvement grant (DEB-0073330), by a Smithsonian Tropical Research Institute short-term fellowship, and by two travel grants from the Department of Organismic and Evolutionary Biology.

xii

CHAPTER 1:

ADAPTIVE RADIATION AND ECOLOGICAL DIVERSIFICATION IN THE BRUCHINE SEED

BEETLES (COLEOPTERA: CHRYSOMELIDAE: BRUCHINAE)

INTRODUCTION

The rise of angiosperms in the late Cretaceous and early Tertiary fundamentally

altered the structure of terrestrial ecosystems, and their diversification since that time has

provided a wealth of ecological opportunities for those that rely on plants for a

living. Perhaps most notably, phytophagous insects have radiated evolutionarily and

diversified ecologically onto the ecological opportunities proffered by the diversification

of the angiosperms. The remarkable diversity of these interacting groups has generated

conjecture and research since the inception of evolutionary biology. After all, the

observation that these two groups contain more than one-half of all terrestrial

multicellular species diversity begs an explanation. The growth of applied phylogenetics

and advances in molecular systematics have allowed biologists to pursue these questions

more intensely and with greater scrutiny. In the most comprehensive study of this kind to date, Farrell (1998) showed quite conclusively that the remarkable diversity of the

Coleoptera is most readily explained by adaptive radiation within lineages that have shifted to feed on angiosperms. However, species diversity within the angiosperms is quite disparate, being highly concentrated in particular lineages and highly depauperate in others (Dodd et al., 1999; Eriksson and Bremer, 1992; Judd et al., 1994; Magallon et al.,

1999; Magallon and Sanderson, 2001; Ricklefs and Renner, 1994). One would like to know if such disparity in diversity is also reflected in the diversity of the Coleoptera that feed on these different lineages. Therefore, in order to further the study of this remarkable correlated diversification and to begin to examine causal features that might explain these shifts in diversification rates, we will need to increase the resolution of our

2 phylogenetic hypotheses to encompass phytophagous clades that have diversified

across some of the phylogenetic disparity of the angiosperms.

While to date there have been no comprehensive phylogenetic analyses of the

bruchid seed-beetles (Coleoptera: Chrysomelidae: Bruchinae1), traditional classification and intuitive phylogenetic schemes suggest that their diversity is highly concentrated within a small handful of lineages. For example, of the approximately 2,000 species, the majority of the species are included within the traditional ‘subfamily’ Bruchinae (see notes on bruchid below). In addition, bruchids are known to use some 30 angiosperm families as host plants (Borowiec, 1987; Johnson, 1981d; Johnson, 1989a), some more than others. Furthermore, different lineages attack either woody or herbaceous hosts2, and these types of plants may present distinctly different resources

with accompanying divergent selection regimes. Such a disparity in diversity, and

diversity in ecology, suggest that phylogenetic analysis of this clade may provide insight

into how intimately correlated angiosperm and insect diversification may be. This, in

turn, should provide specific and testable hypotheses concerning the cause(s) of this

correlation. This chapter therefore serves a dual purpose. The first is to examine the

pure phylogenetics of the bruchids—the relationships between major lineages that would

correspond to a phylogenetically-based classification system. The second is to examine

the applied phylogenetics of the bruchids—to use the phylogenetic hypotheses in order to

understand the ecological history of the group and to test specific hypotheses concerning

1 The Bruchinae are quite obviously nested within the Chrysomelidae and should be considered as a subclade of that group. However, they have traditionally been referred to as the family Bruchidae and I will refer to them as such through the remainder of this thesis so as to prevent confusion with the general literature and with the lower-level classification of the seed beetles. This matter is discussed further later in the chapter. 2 Host plant information has been compiled from approximately 600 literature sources, and the bruchid species-plant species associations are in a database maintained by the author. These references are not included in this thesis, but are available upon request.

3 the evolution of ecological interactions and their roles in diversification, phylogenetic

conservatism, and stasis.

PURE PHYLOGENETICS

Traditional classification of the Bruchidae has been hampered by an excessive

influence of ‘evolutionary taxonomy’, with current schemes focusing almost exclusively

on intuition, apomorphic characters, and groupings based on similarity, regardless of

homoplasy, symplesiomorphy, or synapomorphy. The first purpose of this chapter is to

evaluate the phylogenetics of the seed beetles using a rigorous, character-based,

phylogenetic analysis.

Bruchid systematics and taxonomy. Bruchidae, with slightly less than 2,000

described species, represents the largest clade of obligate endophagous seed predators

(Janzen, 1971b). Monophyly of the group is indicated by numerous morphological characters, both related and unrelated to their spermophagous habit (lists are given in

Borowiec, 1987; Kingsolver, 1995; Pfaffenberger and Johnson, 1976). The group has traditionally been treated as a separate family, the Bruchidae, based on arguments from evolutionary taxonomists who feel that this suite of autapomorphic characters earn them this elevated rank (Borowiec, 1987; Kingsolver, 1995; Verma and Saxena, 1996). While these numerous characters may indicate that bruchids form a natural and monophyletic group, research into their morphology (Crowson, 1946; Monros, 1955; Reid, 1995) as well as more recent research in molecular systematics (Farrell, 1998; Farrell and

Sequeira, in prep.) places them as a clade nested well within the -beetles (Coleoptera:

Chrysomelidae) and sister group to the Sagrinae. Therefore, recognition of their

4 relationships indicates that they should be nested within the Chrysomelidae and that their taxonomic rank should be subordinate to that of family. However, because most of the taxonomic research on this group has treated the Bruchidae as a family, the traditional nested ranks refer to subfamilies and tribes instead of tribes and subtribes. In order to avoid confusion I will follow with this convention in the following discussion. A summary classification of the Bruchinae is shown in Table 1.1 and is based on the classification schemes of Borowiec (1987) and Udayagiri and Wadhi (1989).

For nearly 180 years after the original description of Dermestes pisorum ( pisorum) by Linnaeus (Linné, 1758), the majority of systematic work on the Bruchidae focused on description of species and generic treatments. It was not until Bridwell’s

(1932) separation of the bruchids into subfamilies that an attempt at a higher classification of the "Bruchidae" was attempted. His classification and subsequent separation of numerous species groups into genera set the stage for the majority of systematic work that followed. New World researchers such as J.M. Bottimer, C.D.

Johnson, and J.M. Kingsolver and Old World researchers such as J. Decelle and L.

Borowiec have made significant contributions to our understanding of bruchid classification, largely following the lead provided by Bridwell. They have used a suite of external morphological characters and characters from the male genitalia to structure the classification and to develop hypotheses of relationships within and among taxonomic groups of the bruchids. Borowiec (1987) provided the most thorough treatment of bruchid relationships and phyletic evolution to date, developing phylogenetic hypotheses based upon his assessment of plesiomorphic and apomorphic characters. In general, he suggested that these 'sagroid' plesiomorphic characters are present to a greater or lesser

5 Table 1.1: Classification and biogeography of the Bruchidae based primarily on Borowiec (1987) and Udayagiri and Wadhi (1989). The separation of Bruchidiini and Acanthoscelidini is likely not justified (Borowiec, 1987), but is shown here to demonstrate the separation between Old World and New World taxa. Supplemental information for this classification is based on numerous revisionary works and the addition and description of genera since these publications (Anton, 1994a; Anton, 1999f; Johnson, 1990c; Johnson and Southgate, In press; Nilsson and Johnson, 1993b; Romero and Johnson, 2001). Supplemental information on species numbers since the publications of Borowiec (1987) and Udayagiri and Wadhi (1989) are found in (Ali and Al-Ali, 1987; Anton, 1993; Anton, 1994a; Anton, 1994b; Anton, 1996a; Anton, 1996b; Anton, 1998; Anton, 1999a; Anton, 1999b; Anton, 1999c; Anton, 1999d; Anton, 1999e; Anton, 1999f; Anton, 1999g; Anton, 1999h; Anton, 1999i; Anton, 2000a; Anton, 2000b; Anton, 2000c; Borowiec, 1990a; Borowiec, 1990b; Borowiec, 1991; Borowiec, 1994; da Silva and Ribeiro-Costa, 2001; Delobel, 1997; Johnson, 1990b; Johnson, 1990c; Johnson et al., 1989; Johnson and Southgate, In press; Karapetyan, 1989; Kingsolver, 1987; Kingsolver, 1988; Kingsolver, 1990; Kingsolver, 1999; Kingsolver et al., 1989; Kingsolver and Ribeiro-Costa, 2001; Lopatin and Chikatunov, 2000; Morimoto, 1990; Muruaga de L'Argentier and Kingsolver, 1994; Nilsson and Johnson, 1990; Nilsson and Johnson, 1993b; Ribeiro-Costa, 1992; Ribeiro-Costa, 1997; Ribeiro-Costa, 1999a; Ribeiro-Costa, 1999b; Ribeiro-Costa, 1999c; Ribeiro-Costa, 2000; Ribeiro-Costa and de Souza Costa, 2002; Ribeiro-Costa and Kingsolver, 1992; Ribeiro-Costa and Kingsolver, 1993; Ribeiro-Costa and Reynaud, 1998; Romero and Johnson, 1999; Romero and Johnson, 2000; Romero and Johnson, 2001; Romero et al., 1996; Savitsky, 2000; Singal, 1989; Singal and Pajni, 1989; Terán and Kingsolver, 1992; Tewari and Pajni, 1995; Wendt, 1995; Zampetti, 1992a; Zampetti, 1992b; Zampetti, 1997; Zhang and Liu, 1987). An ‘i' indicates that a member of the genus is introduced into that region.

Subfamily Tribe Genus gion ecies e p Number of s Neotropical Region Nearctic Region Palearctic Region Oriental Region Afrotropical Region Madagascar Australian R Rhaebinae 7 + Rhaebus 7 + Pachymerinae 110 + + + + + + Caryedontini 71 i i + + + + Afroredon 4 + + Caryedon 63 i i + + + + Caryotrypes 2 + + Exoctenophorus 1 + Mimocaryedon 1 + Caryopemontini 18 + + + + Caryopemon 12 + + + +

6 Table 1.1 (Continued) Pachymerinae (Continued) Diegobruchus 6 + + Pachymerini 21 + Caryoborus 3 + Caryobruchus 7 + + Pachymerus 7 + i Speciomerus 4 + Eubaptinae 4 + Eubaptus 4 + Kytorhininae 19 + + + Kytorhinus 19 + + + Amblycerinae + + Amblycerini 124 + + Amblycerus 124 + + Spermophagini 150 + + + + + + Pygospermophagus 1 + Spermophagus 121 + + + + Zabrotes 28 + + i i i Bruchinae Acanthoscelidini 599 + + i i i i Abutiloneus 1 + Acanthoscelides 326 + + i i i Algarobius 6 + + i Althaeus 3 + Bonaerius 1 + Caryedes 42 + Cosmobruchus 1 + Ctenocolum 8 + Dahlibruchus 5 + Gibbobruchus 13 + + Lithraeus 10 + Margaritabruchus 1 + Megasennius 1 + Meibomeus 28 + + 26 + + Mimosestes 19 + + Neltumius 3 + Palpibruchus 1 + Pectinibruchus 1 + Penthobruchus 2 + Pseudopachymerina 2 + Pygiopachymerus 2 + Rhipibruchus 7 + Scutobruchus 6 + Sennius 52 + +

7 Table 1.1 (Continued) Bruchinae (Continued) Acanthoscelidini (Continued) Spatulobruchus 1 + Stator 30 + + Stylantheus 1 + Bruchidiini 441 Acanthobruchidius 1 + Borowiecius 5 + + Bruchidius 337 i i + + + + + Callosobruchus 41 i i + + + i + Conicobruchus 11 + + Decellebruchus 1 + Horridobruchus 1 + Kingsolverius 1 + Megabruchidius 2 + Paleoacanthoscelides 2 + Parasulcobruchus 1 + Pygobruchidius 2 + Salviabruchus 1 + Specularius 12 + + Sulcobruchus 10 + + + + Tuberculobruchus 13 + + + Bruchini 196 i i + + + + i Bruchus 196 i i + + + + i Megacerini 55 + + Megacerus 55 + +

extent in a basal3 grade of bruchids, with various apomorphic characters appearing

gradually until the 'typical' bruchid morphology (characterized by the Bruchidae) is

reached.

Based on this scheme, Borowiec postulated a somewhat pectinate tree (Figure

1.1) of relationships between Bridwell's subfamilies. This phylogenetic scheme is based

largely on his intuition of plesiomorphic and apomorphic character states and does not

include the rigorous analysis of a data matrix or explicit comparisons to sister taxa.

Indeed, his claim that metallic coloration in the bruchid subfamily Rhaebinae is the

3 Throughout the course of this chapter ‘basal’ is used as shorthand to refer to lineages that are sister groups to the remainder of bruchid lineages.

8 plesiomorphic condition for Sagrinae + Bruchidae indicates a muddled phylogenetic

perspective in this analysis. The majority of sagrines have a non-metallic coloration,

similar to bruchids. The major exception is in the metallically-colored genus Sagra, a

group which is generally considered to have numerous autapomorphic characters and to

be nested within a clade of non-metallic genera.

Regardless, his grouping of taxa followed largely along traditional grounds and

has generally been accepted by systematists that specialize on bruchid taxonomy.

Rhaebinae, consisting of a handful of species within the genus Rhaebus, remains a

somewhat enigmatic taxon. Kingsolver and Pfaffenberger (1980) provided convincing

morphological evidence that Rhaebus Fischer von Waldheim formed a monophyletic

group with the rest of the bruchids, settling previous controversy on its broadscale

relationships. However, its relationships with other bruchids are difficult to determine.

While the assessment of metallic coloration as a symplesiomorphy with the Sagrinae does

not appear to be valid, it does share characters with other chrysomelids that are not found

elsewhere within the bruchids, suggesting an early split from other seed-beetles.

However, numerous Pachymerinae share potential symplesiomorphic characters with the

Sagrinae (particularly if the sagrine genus Carpophagus McLeay maintains symplesiomorphic characters with the bruchids (Kingsolver, 1995; Reid, 1995)), indicating that the placement of Rhaebus may not be as certain as Borowiec claimed.

The group is strictly Palearctic and appears to be limited to feeding within the seeds of species from the genus Nitraria L. (Zygophyllaceae).

The palm bruchids, subfamily Pachymerinae in Bridwell's (1932), Borowiec's

(1987), and Nilsson and Johnson's (1993b) classification scheme, represent a

9 conglomerate of Paleotropical and Neotropical genera united largely by plesiomorphic characters. As a result, there is fairly little morphological support for their unity as a monophyletic group. Apomorphic characters within the pachymerines indicate monophyly of particular groups (probably at the tribal level of Nilsson and Johnson's

1993 classification) within the subfamily and the variation of plesiomorphic and apomorphic characters may indicate that they form a paraphyletic grade early in the diversification of the bruchids, but their classification cannot be considered to be natural at the moment. Notably, the few described bruchid fossils, collected from the

Figure 1.1. Borowiec's (1987, p. 164) hypothesis of bruchid relationships.

10 Florissant beds in Missouri (Kingsolver, 1965) and from Dominican amber (Poinar,

1999a), have been placed in this subfamily. These and some recently discovered and as- yet-undescribed fossils collected by B. Archibald (Museum of Comparative Zoology,

Harvard University) actually share synapomorphic characters with members of the

Pachymerini (sensu Bridwell, 1929; Nilsson and Johnson, 1993b), indicating that this clade may have diverged as early as the early Eocene. A possible disparity between classification and phylogeny in the pachymerines is also indicated by ecological data: all

Neotropical species of pachymerines with known host associations (75% of species, including species from all genera) feed on seeds of palms (Arecaceae); on the other hand no species of Paleotropical pachymerines with confirmed host associations feed on palms. While bionomic information on Paleotropical bruchids significantly lags behind those from the Neotropics, the species of Caryedontini with known host associations

(48%) feed on either or (although there is an unconfirmed report that Pandanus (Arecoideae: Pandanaceae) may be the host of Caryotrypes pandani

(Blanchard, 1845)) and the only two species with known host associations from the

Caryopemontini (representing 11% of those described) feed on Fabaceae.

The genus Eubaptus Lacordaire is represented by a small number of species and specimens from South America. The genus has been traditionally placed into its own subfamily, the Eubaptinae, due to various distinctive and apomorphic morphological characters. Host associations give few cues as to potential relationships as the only host records are from the genus Ruellia L., the only known association in the bruchids with

Acanthaceae. Borowiec considered Eubaptinae to be sister to the Amblycerinae +

Kytorhininae + Bruchinae. The subfamily Amblycerinae consists of four recognized

11 genera, two with an exclusively Old World distribution (Spermophagus Schoenherr and the monotypic Pygospermophagus Pic), and two with an exclusively New World distribution (Amblycerus Thunberg and Zabrotes Horn). There appear to be numerous morphological characters distinguishing these genera and supporting the monophyly of each. While hosts are unknown for Pygospermophagus brevicornis Pic, legume feeding is found in all three other genera. However, the majority of species with known host associations from both Spermophagus (31% with known hosts) and Amblycerus (38% with known hosts) feed on non-legumes (Spermophagus 84%, Amblycerus 61%).

Monophyly of the Amblycerinae is generally accepted by bruchid taxonomists, although this view is based on very few characters and should not be taken as given.

The subfamily Kytorhininae is based on the single genus Kytorhinus Fischer von

Waldheim from East Asia and northwestern North America. All species with known host associations (50%) feed on legumes. Numerous synapomorphic characters appear to unite this genus as a monophyletic group, although there is some debate over its placement as separate from the Bruchinae. The Bruchinae represents the bulk of seed beetle diversity, with some 75% of described species being placed in this subfamily. In most phylogenetic representations or phyletic descriptions of the Bruchidae, this subfamily is considered to be of relatively recent origins, suggesting that bruchid diversity may be concentrated within this group. Traditionally this subfamily has been separated into four tribes, although the separation of the Acanthoscelidini and Bruchidiini is based exclusively on biogeographic grounds and is almost certainly unjustified.

Placement of the genus Megacerus F. into the tribe Megacerini is based on numerous morphological characters and this seems to form a fairly coherent New World group.

12 Clues into the relationship of this group to other bruchids are difficult to come by on numerous grounds. This is the only tribe placed within the Bruchinae that have no known leguminous hosts: all species feed in Convolvulaceae seeds. Furthermore, based on morphology it is not readily apparent why Megacerus has been placed within the

Bruchinae. The Old World genus Bruchus L. constitutes the tribe Bruchini. It is distinguished largely on apomorphic grounds, particularly on prominent secondary sexual characters. As a result, while it is likely a monophyletic group, its relationship to the more variable Acanthoscelidini (derived within, sister to, etc.) is open to question. All known host associations within the Bruchini are with species of papilionoid legumes.

The Acanthoscelidini (including the Bruchidiini) seems to be a fairly well supported group, although its relationship to other taxa, particularly Bruchini and Kytorhininae, is somewhat sketchy. This group includes numerous distinctive genera whose taxonomy has been quite well resolved, particularly in the New World. However, it also includes the genera Acanthoscelides Schilsky and Bruchidius Schilsky, which have served as

‘grab-bag’ genera for the New World and Old World, respectively. These two genera are almost certainly not monophyletic. Johnson (1989a; 1990c) has segregated the genus

Acanthoscelides into 14 species groups that appear to be well-supported and may end up as monophyletic groups. He is currently intending to separate these into separate genera

(personal communication). Comparatively little work has been done on the genus

Bruchidius, with nothing approaching the scope of the research of Johnson and

Kingsolver over the past 4 decades. Anton (1998) has begun a revision of this genus, but this research is only just beginning to be published. Legumes serve as the predominant host in this tribe, constituting the host species of 84% of the species with known host

13 associations (40% of all described species), with Malvaceae constituting 8% and other

families making up the remaining 8%.

In summary, there is a significant history of taxonomic work in the Bruchidae.

This has included not only alpha-taxonomic work, but also attempts at higher level

classification and phylogenetic hypotheses. However, evolutionary systematics has

pervaded the classification of the bruchids, with the majority of the classification and

phyletic hypotheses based on intuition and apomorphic characters. Almost none of this

work has been based on explicit phylogenetic analysis of a character-based data matrix.

To this point only two studies have been published that are based on molecular sequence

data or an explicit data matrix: Silvain and Delobel’s (1998) molecular and

morphological phylogenetic analysis of West African Caryedon and Romero et al.’s

(2002) morphological phylogenetic analysis of the Neotropical genus Amblycerus. In this

chapter, I examine evolutionary relationships of major lineages within the Bruchidae

using DNA sequence data. The sampling for this analysis was designed in part to enable

me to examine the relationships between the major taxonomic groupings, including

traditional subfamilies, tribes, genera, and Acanthoscelides Schilsky species groups; and

in part to allow me to apply this phylogenetic analysis to the evolution of ecological

associations and their correspondence to lineage diversification within the seed-beetles.

APPLIED PHYLOGENETICS

As noted earlier, the chrysomelid subfamily Bruchidae represents by far the largest single radiation of insects that obligately feed within the reproductive tissues of

14 plants. The vast majority of these species belong to the traditional subfamily Bruchinae

and most of these feed on seeds of legumes (Fabaceae). This has led multiple authors to

suggest that the ecological history of the bruchids is most readily explained via their

associations with legumes and that their relative phyletic success is the result of an

adaptive radiation onto the opportunity presented by this plant group (Borowiec, 1987;

Huignard et al., 1989; Janzen, 1969; Janzen, 1981a; Johnson, 1981d; Johnson, 1981e;

Johnson, 1989a; Johnson, 1989b; Johnson, 1990a; Southgate, 1979). After all, legumes

constitute one of the most noticeable features of many plant communities. This is true of

both the plants that belong to the bean family Fabaceae (common name legumes) and

their distinctive biloculicidally dehiscent fruits (legumes). It makes sense that these high-

quality protein/carbohydrate resources that display a wide diversity of secondary

defensive chemicals and defensive morphology (Harborne, 2000; Harborne et al., 1971;

Kass and Wink, 1995) provide a diverse, partitionable, and abundant ecological resource

as a substrate for ecological and evolutionary radiation. However, without reference to

explicit comparative analyses based on phylogenetic data, our understanding of how the

interaction between bruchids and their host plants has evolved and diversified remains

superficial and anecdotal.

Research into the macroevolution of interactions often proceeds along one of two

complementary themes. The first seeks to explain the disparity in diversity that is present

throughout the phyletic evolution of all of life. Whether this has been the result of

deterministic processes based on ecological interactions or of contingent differences fueled by stochastic events has sparked a considerable amount of research into adaptive radiation. The second seeks to explain the historical trajectories that underlie the patterns

15 of ecological associations that we see today. Whether or not these trajectories are largely

structured via specific selective regimes within particular ecological contexts, or proceed

along phylogenetically or developmentally constrained paths has been at the center of a

schism between ecologists and systematists for decades. While these themes are clearly

complementary and readily addressed within a comparative framework, one must take

slightly different approaches in order to address each. The second goal of this chapter is

to use the phylogenetic hypotheses derived in the previous section to (1) test whether

ecological associations affect rates of diversification in bruchids, and (2) to explain the

historical trajectories that this diversification has taken.

Macroevolutionary diversification. The theoretical underpinnings for the study

of adaptive radiation were developed throughout the New Synthesis and were perhaps

most clearly and strongly championed by Simpson (1953). He argued that “adaptive

radiation strictly speaking refers to more or less simultaneous divergence of numerous lines from much the same adaptive type into different, also diverging adaptive zones (p.

223)”. Inherent in this sentence are two main themes, those of evolutionary diversification (“simultaneous divergence of numerous lines”) and ecological diversification (“into different, also diverging adaptive zones”). While the comprehensive study of adaptive radiation requires integrating across multiple levels of analysis, the detection of macroevolutionary patterns of adaptive radiation are an essential first step. This requires the identification of significant asymmetries in diversification rates consistently associated with the entrance into a novel adaptive zone.

Ehrlich and Raven (1964) extended this hypothesis to phytophagous insects and

suggested that the coevolutionary dynamic between plants and their insect associates

16 drives an escape-and-radiation mode of mutual diversification: plants diversity as they

escape through the evolution of novel defensive chemistry into enemy-free-space while

herbivorous insects then diversify as they evolve counterdefenses that allow them to use

these now diverse resources. This model was supported via sister group comparisons by

Mitter et al. (1988) who showed that diversification in insects significantly and

predictably increases when they shift to feed on plants; and by Farrell et al. (1991) who

showed that the escape of plant lineages through the evolution of defensive latex

secretory canals predictably and significantly results in increases in plant diversification.

The observations of Ehrlich and Raven (1964) and the quantitative examination of

diversification rate by Mitter et al. (1988) and Farrell et al. (1991) support a causal

mechanism linking the co-diversification of insects and plants. These observations did

not provide direct evidence into the implicit assumption in Ehrlich and Raven’s

hypothesis that the numerous plant species produced through their radiation following

escape from herbivores would then serve as the partitionable niches that allow

phytophagous insects to then subsequently diversify. For this, the observation of more

direct correlations between insect and plant diversity are needed. To this end, Farrell

(1998) showed that the diversification of phytophagous insects is in large part explained by the diversification of the flowering plants. Indeed, the study of the diversification of the angiosperms is in and of itself a cottage industry. Until recently, forays into this area focused on those traits that separated the angiosperms from other tracheophytes, including the complex flower and its consequences for insect pollination, the post- fertilization shift in reproductive investment, increased rates of growth, and on the presence of an integumented indehiscent megasporangium, or enclosed seed (Bond,

17 1989; Doyle and Donoghue, 1986; Heywood, 1993; Regal, 1977; Stebbins, 1981). The

logic followed from fairly simple taxic comparisons: angiosperms as a clade are orders

of magnitude more diverse than any prospective sister taxa; therefore angiosperms per se

must share a key synapomorphy that can explain their diversity. However, the

application of phylogenetic approaches to the study of diversity has shown that the

remarkable radiation of the angiosperms is probably a product of diversification within subsets of the clade, and not diversification of the clade itself. Recent research has instead focused on (1) shifts in absolute rates of diversification in lineages within the angiosperms (Magallon and Sanderson, 2001), or (2) on characters that may be expected to cause, and are therefore phylogenetically correlated with increases in diversity within the angiosperms (Dodd et al., 1999; Eriksson and Bremer, 1992; Ricklefs and Renner,

1994; Silvertown and Dodd, 1996; Smith, 2001). The former approach is a highly informative heuristic device that may lead researchers to investigate the mechanisms that have promoted that diversification. By itself it does not lead us directly to those processes that may have contributed to the dominance of angiosperms in the biological diversity of terrestrial plants. The latter approach, on the other hand, is hypothesis-driven and gets at the very heart of those characteristics that are largely responsible for producing the bulk of angiosperm diversity.

Numerous traits have been put forward to explain concentrated diversity within the angiosperms, but three appear to have repeatable and significant explanatory power: insect pollination, herbaceousness, and shifts in defensive chemistry (Dodd et al., 1999;

Eriksson and Bremer, 1992; Farrell et al., 1991; Ricklefs and Renner, 1994; Silvertown and Dodd, 1996). Insect pollination as a significant factor is identified not so much by

18 the high levels of diversity in clades defined by it, but rather in low levels of diversity in clades that have secondarily lost it. Presumably it acts (1) by localizing and limiting gene flow, thereby promoting the reproductive isolation necessary for speciation and (2) by allowing fertilization to occur even when particular species occur in low density, thereby decreasing rates of extinction. Herbaceousness has evolved multiple times within angiosperms and other tracheophytes, and is yet highly correlated with surprising levels of diversification in the former. Therefore it appears that herbaceousness likely (1) increases speciation rates through ephemeral populations with short generation times; these increased speciation rates in angiosperms are preserved as macroevolutionary diversity via (2) decreased extinction rates as a result of the coupling of high colonization propensities with the advantages of having protected seeds. In regards to defensive chemistry, Farrell et al. (1991) showed that the acquisition of latex canals, which are known to defend plants against pathogens and phytophages results in predictable increases in diversification. Presumably this acts in the manner envisioned by Ehrlich and Raven (1964) by which plant lineages that are able to escape (at least temporarily) from their enemies are able to attain larger ecological and geographic ranges, providing increased opportunities for ecologically-driven or vicariant-driven speciation, while at the same time decreasing the risk of extinction through the maintenance of higher population sizes. While all host plants of bruchids are entomophilous, the rule of higher diversity of herbaceous lineages does seem to hold true for all host plant taxa that show variation in this life history character, most notably in the two most common hosts of bruchids, the

Fabaceae and Malvales. This allows the examination between the correlated diversification of bruchids and their host plants, using herbaceousness as a proxy for

19 plant diversity. At this point, a phylogenetic treatment of defensive syndromes

throughout the hosts of the bruchids is unavailable, precluding the examination of the

effects of evolved defensive chemistry on host plant diversification, and subsequently on

bruchid diversification. This is an obvious avenue of exploration, as one the most

convincing examples of biochemical coevolution is between the non-protein amino acid

defenses of the tropical liana Dioclea macrocarpa Rolfe and the ability of Caryedes brasiliensis (Thunberg) to use these in its own metabolism (Bleiler et al., 1988; Janzen,

1971a; Rosenthal, 1983; Rosenthal, 1989; Rosenthal and Janzen, 1983). Patterns of bruchine infestation throughout the legumes also suggests a potential role for defensive chemistry in diversification as a smaller proportion of the more complexly-defended

Papilionoideae are attacked than are the or (Bell, 1971;

Bell et al., 1978; Harborne et al., 1971; Janzen, 1981a; Johnson, 1981e; Johnson, 1990a;

Zarnowski et al., 2001).

The study of diversity and diversification within the insects has followed a trajectory similar to the exploration of diversification within the angiosperms. Early explanations focused on taxic differences in diversity and the traits that defined those differences (Borror et al., 1981). For example, compartmentalization and specialization of body segments and appendages was suggested as a key feature favoring the diversity of the Hexapoda; the evolution of the wing has long been cited as the key feature explaining the diversification of the Pterygota since their appearance in the Devonian; the evolution of distinct life stages that allow the exploration of multiple niches is argued as a key feature in the diversification of the Holometabola; the evolution of sclerotized forewings serving multiple roles including protection against desiccation, aids in flight,

20 and the ability to burrow into different substrates has been discussed as a key feature

explaining the remarkable diversity of the Coleoptera. However, within any of these

single clades there is significant disparity in species diversity in different lineages, and a

taxic approach fails at adequately explaining insect diversification. With the growth of phylogenetic approaches to the study of diversity, it has become clear that the impressive diversity of the insects is the result of impressive concentrations of diversity within particular clades of insects. It appears that in large part the concentrated diversity of the insects can be explained by numerous shifts in various lineages to a phytophagous habit

(Farrell, 1998; Mitter et al., 1988). In becomes a reasonable hypothesis, therefore, that the colonizations of the ecological opportunities presented by the rapid diversification of the angiosperms in the late Cretaceous represent adaptive radiations, and it is these radiations that explain the majority of the diversity within these phytophagous lineages.

As stated before, the diversification of the angiosperms is at least partly due to the repeated evolution of herbaceousness from otherwise woody ancestors. While this habit is certainly not novel within plants, its combination with the packaged and protected seed of angiosperms appear to have allowed herbaceous plants to diversify into novel habitats not otherwise occupied by woody plants. The evolution of herbaceousness in plant lineages also entailed the entrance into new selective regimes, particularly in terms of different life histories and defensive chemical strategies. These new circumstances presented both new opportunities and new barriers to phytophagous insects. For instance, these selective regimes appear to have resulted in the repeated increase in investment in reproductive tissues (Gadgil and Solbrig, 1972; Obeso, 2002; Silvertown and Dodd,

1996), and in a shift from quantitative defensive chemicals that consist of a limited

21 number of digestion-reducing metabolites to qualitative defense chemicals such as alkaloids or protein-based secondary metabolites that require specific adaptations to detoxify or avoid (Feeny, 1976; Strong et al., 1984), such as many of the defenses found in the seeds of herbaceous legumes (Janzen, 1981a; Rehr et al., 1973; Rosenthal, 1981).

The increased rate of diversification of herbaceous plants and their increased investment in reproductive output likely represented numerous ecological opportunities for seed predators, and I argue that their different set of defensive chemistry and distinctive life history characters indicate that herbaceous plants represent a distinct adaptive zone from woody plants. The question is, was the invasion of this adaptive zone accompanied by a radiation of spermophagous insects? A priori, it is not clear that this should be the case, as slightly more than half of bruchid species belong to lineages that feed on woody plants. However, it would appear that for certain host plant clades, particularly the

Fabaceae and the Malvales, the herbaceous habit evolved from considerably older woody ancestors (Alverson et al., 1998; Alverson et al., 1999; Doyle, 1994; Doyle et al., 1997;

Judd and Manchester, 1997; Kass and Wink, 1995; Kass and Wink, 1997; Laduke and

Doebley, 1995). As a result, it is absolutely necessary to control for the effects of age.

Therefore, I use the sister-group approach pioneered by Mitter et al. (1988) as means for identifying significant asymmetries in diversity in bruchid lineages that are associated with herbaceous plants.

Macroevolutionary trajectory. In his initial address as president of the Society for the Study of Evolution, Hutchinson (1959) argued that the high species-specificity and tissue-specificity of phytophagous insects has allowed them to effectively treat the highly diverse and morphologically differentiated species of angiosperms as separate,

22 available niches that can be partitioned through speciation and divergence. He was largely concerned with the role of ecological opportunities in the origins of diversity and the perspective was largely ahistorical. On the other hand, in their seminal paper on -plant coevolution, Ehrlich and Raven (Ehrlich and Raven, 1964) argue that available niches are filled by natural selection shackled by the constraints of evolutionary history, particularly as it relates to plant chemistry. As such they were largely concerned with the role of historical constraints in structuring diversification. While these ideas almost certainly represent complementary processes in the macroevolution of phytophagous insects, it is unclear at what levels and along what resource axes these influences exert their relative effects.

Plant chemistry, a feature fairly phylogenetically conserved in plants, served as the driving force behind Ehrlich and Raven’s model and has been thought of as the most important feature governing the macroevolution of insect-plant interactions in most research since (e.g. Becerra, 1997; Farrell and Mitter, 1990; Farrell et al., 1992; Futuyma et al., 1994; Janz and Nylin, 1998; Kelley and Farrell, 1998; Mardulyn et al., 1997;

Ronquist and Liljeblad, 2001; Termonia et al., 2001). This has resulted in a considerable amount of interest in the degree to which plant taxon use (with plant classification often used as a proxy for phylogeny) is phylogenetically constrained across phytophagous insect phylogenies, and an interest in understanding the historical trajectories (e.g. the ancestral hosts, convergent host shifts, etc.) of host use. However, other host related factors, either more or less correlated with plant phylogeny, may play more important roles in limiting or promoting host shifts, colonization, and diversification. As a fairly simple example, it is quite obvious that the tissue being attacked has limited the

23 ecological opportunities for bruchids, as all species feed exclusively on seeds. In a more

general sense, Marvaldi et al. (2002) and Farrell and Sequeira (in prep.) found that there

are considerably fewer shifts to different tissue types being attacked than any other

feature in both curculionoid and chrysomeloid beetles (respectively), indicating that

different plant tissue types may present greater barriers to host shifting than different

phytochemistry. In a study on the interactions between and their host plants,

Janz and Nylin (1998) found that shifts between hosts with different growth forms were also more conservative than shifts between different clades of host. Furthermore, they found that the propensity to shift host taxa was influenced by the life history of the host plants involved, suggesting that the resource presented by herbaceous hosts is fundamentally different than that presented by woody hosts (as was discussed above).

The apparency hypothesis elaborated by Feeny (1976) suggests that the quantitative toxins in woody plants present a more homogeneous defense than do the qualitative toxins present in herbaceous plants. This should influence the rate of host shifting, resulting in a higher degree of host use conservatism in clades associated with herbaceous plants than in those associated with woody plants, a result confirmed in Janz and Nylin’s study.

The type of phylogenetic conservatism predicted by Ehrlich and Raven’s escape- and-radiation hypothesis has often been assumed to be the case for bruchids, largely because some 75-80% of all known host plant associations are with plants in the family

Fabaceae. However, neither the degree to which these relationships are conserved nor other mitigating factors, such as host plant growth form, has been explicitly examined in a phylogenetic context. Bruchid seed beetles represent an opportunity to further the

24 research on the macroevolutionary patterns of host taxon use and the relevance of host growth form for the macroevolutionary trajectory of host use. Because all Bruchidae feed exclusively on seeds, I am able to control for the potential confounding effects of host tissue type on the interaction of the constraints imposed by host plant phylogeny and host plant growth form. Additionally, bruchids show considerable variation in both the growth form of the host plants that they use (about ½ of all species come from lineages that feed on woody plants) and in the host taxa that they use (about 30 different families), allowing for phylogenetic tests of both variables.

In addition to the hypothesis-testing described above, uncovering the evolutionary and ecological history for a group is informative for understanding macroevolutionary trajectories of ecological associations. By reconstructing this history for the bruchids, I can evaluate and develop hypotheses concerning the ecological situations that may have allowed entrance into the spermophagous niche. In particular I will examine three patterns of host use that have drawn considerable interest in explaining the ecological and evolutionary diversity of extant bruchids: (1) the ancestral plant association; (2) the growth form of the ancestral hosts; and (3) the origins of legume-feeding.

As I mentioned earlier, all of the published earliest appearances of bruchids in the fossil record are from the tribe Pachymerini. This group currently feeds exclusively within the seeds of palms (Arecaceae). Combined with the observations that (1) some of these fossils have been found near fossil palm beds, and that (2) the Pachymerini share a close resemblance to the presumably plesiomorphic sagrines (e.g. Carpophagus), this association has been assumed to be ancestral for the bruchids. If true, it would suggest that this ecological relationship has persisted largely unchanged for 55-85 million years

25 (Poinar, 1999a; Poinar, 1999b). It may even suggest that the dominance of palms (and their well-endowed seeds) in communities at the end of the Cretaceous provided a resource that allowed for the evolution of spermophagy by the ancestors of the bruchids.

The second question concerns the origins of the opportunities of bruchids to shift onto herbaceous plants. There are numerous examples of ancient herbaceous lineages of angiosperms—the paleoherbs (e.g. Piperales) appear to be one of the oldest lineages of flowering plants (Bremer et al., 1998). However, if the ecological associations of phytophagous insects are constrained by the associations of their ancestors, then we may assume that many of these lineages were not available to the bruchids. In fact, woodiness is the ancestral habit of the major lineages used by the Bruchidae, with the

Convolvulaceae being a major exception (Olmstead et al., 2000; Olmstead et al., 1992).

An ancestral reconstruction of feeding on woody plants by bruchids would agree well with recent advances in our understanding of plant phylogenetics (Alverson et al., 1998;

Alverson et al., 1999; Doyle et al., 1997; Kass and Wink, 1997; Olmstead et al., 2000;

Olmstead et al., 1992). Additionally, this could reconcile what would be apparently contradictory observations: (1) more than half of bruchid species are from lineages that feed on woody plants; with (2) evidence for accelerated bruchid diversification onto herbaceous lineages. There would simply have been more time for diversification onto woody hosts.

The final question concerns the observation that the majority of bruchid species feed on either legumes or Malvales. Evidence into the number of origins of either can lend us insight into the nature of bruchid diversification. Multiple origins would suggest that there may be something predictive about the use of those hosts—their chemical

26 attributes, their seed morphology, or the types of communities in which they occur, for

example (Becerra, 1997; Becerra and Venable, 1999; Futuyma et al., 1994; Menken and

Roessingh, 1998). Single origins would suggest that their diversification may rest on the

contingency of shifting onto the ancestral host at a single event in their history.

In the second part of this section I present the results of comparative analyses of the interaction between bruchids and their host plants. In addition to the more general question of elucidating the ecological history of the bruchids, I examine the history of host shifts in light of host tissue and host taxon use by asking the following questions: (1)

Are patterns of host taxon and/or host tissue use phylogenetically constrained in bruchids? (2) Does plant phylogeny constrain macroevolutionary trajectories of bruchids more than growth form or vice versa? (3) Was the ancestral host plant likely to have been herbaceous or woody? (4) Is there evidence of an ancestral relationship with palms? (5)

What is the history of legume-feeding? (6) Are there convergent patterns of host taxon use? (7) Do lineages of bruchids that are associated with woody taxa use more host plant clades than those associated with herbaceous plant clades?

By sampling widely across the evolutionary and ecological diversification present within the seed-beetles, the present study aims to examine the phylogenetic relationships of major lineages within the Bruchidae and to address specific hypotheses concerning the diversification of bruchids. To achieve this end, I have produced and analyzed partial

DNA sequences of two genes: the mitochondrial protein-coding gene Cytochrome

Oxidase 1 (CO1) and the D2 and D3 expansion segments of the nuclear ribosomal gene

28s. Using the phylogenetic analysis based on these sequences, I examine whether

27 bruchid diversification is (1) correlated with shifts to feeding on herbaceous host plants;

and (2) proceeds along phylogenetically constrained trajectories. I do this by addressing

whether or not clades of insects are limited to particular clades of host plants.

MATERIALS AND METHODS

TAXA EXAMINED

Ingroup taxa are included to maximize taxonomic coverage of the Bruchidae and to reflect the ecological diversity (in terms of host plant use) found in the group. While I strived to include as many genera as possible, sampling is considerably more intense for

New World genera than for Old World genera because I was able to collect specimens in the field from the eastern and southwestern U.S.A., throughout Mexico, Costa Rica,

Panama, Venezuela, Ecuador, Argentina, and Puerto Rico. These collections included four Old World genera that have been introduced in the New World. Additional Old

World specimens were supplied by colleagues (see Table 1.2 and Acknowledgments).

However, sampling from Old World taxa is very low as I received no response whatsoever from colleagues in either Africa, , or India. The current phylogenetic analysis must therefore be taken as provisional on this caveat.

In total, 33 of the currently recognized 66 genera of Bruchidae are sampled. This includes representatives of 4 of the 6 recognized subfamilies--unfortunately, I was unable to collect any specimens of the South American genus Eubaptus Lacordaire, the lone genus in the Eubaptinae; and I was unable to amplify successfully a large portion of COI for pinned specimens of Rhaebus solskyi Kraatz—and 9 of 11 tribes. 27 of 35 New

World genera are included in this analysis. Six of the eight New World genera not

28 29 30 31 32 33

34 included in this analysis (Abutiloneus Bridwell, Bonaerius Bridwell, Cosmobruchus

Bridwell, Margaritabruchus Romero and Johnson, Megasennius Whitehead and

Kingsolver, and Spatulobruchus Borowiec) are monospecific and known from very few

specimens. In addition to Eubaptus, I was unable to collect any specimens of the palm- feeding genus Caryoborus Schoenherr.

Sampling of Old World taxa is considerably less intense: of 31 Old World genera,

I am able to include only 8. This is problematic for the evaluation of the evolution of host use associations in the Old World Bruchidiini, a tribe with some 400 species with diverse host associations.

Many of the genera are represented by more than one species. This is particularly important for the large composite genus Acanthoscelides. 18 species are included from this genus and represent 11 of the 14 species groups.

Taxon sampling is also intended to cover a wide diversity of ecological associations in this group. As is the case with bruchid diversity in general, approximately

80% of the species are from Fabaceae, including 25% from Papilionoideae, 33% from

Mimosoideae, and 22% from Caesalpinioideae. The remaining 20% of the species were collected from 7 different host families. 68% of the species included in this analysis are from woody hosts; the remainder are from herbaceous hosts.

Outgroups were chosen largely based on Farrell’s (1998) molecular phylogeny of the Phytophaga, Reid’s (1995) morphological phylogeny of the Chrysomelidae, and results of a more extensive molecular phylogeny of the (Farrell and

Sequeira, in prep.). Two Sagrinae (Sagra femorata and Polyoptilus sp.) and one

Criocerinae (Crioceris sp.) are included as outgroups. Considerable taxonomic and

35 phylogenetic work provides strong support of the sagrines as sister group to the bruchids, with these two taxa sister to a Criocerinae + Donaciinae clade.

In total, sequences from ninety taxa are included in this analysis, including 87 ingroup and three outgroup taxa. Sequences will be submitted to GenBank prior to publication. Summary information on classification, host plant information, and collection information is included in Table 1.2.

DNA SEQUENCE DATA

Standard molecular systematic techniques were used to obtain partial sequences of two genes: CO1 and 28s. Details follow.

DNA Extraction

The majority of specimens used in this analysis were collected as larvae within seeds in the field, reared as adults in the laboratory, and killed and stored in a –80C freezer. Some field-collected adults were stored refrigerated in 95-100% ethanol. A single specimen of each species was used for sequencing and phylogeny reconstruction.

Voucher specimens are deposited in the Museum of Comparative Zoology at Harvard

University. Total nucleic acids were extracted from hind femora of larger specimens or from the whole insect for very small specimens. Ethanol-preserved individuals were rehydrated using three 5 minute washes in 10 mM Tris–HCl (pH 8) containing 100 mM

NaCl and 1 mM MgCl2 (Dowton and Austin, 1998). Specimens were ground to a fine powder in liquid nitrogen. Genomic DNA was then isolated using either a ‘salting out’

36 protocol (Sunnucks and Hales, 1996) or the Qiagen QIAamp Tissue Kit (Qiagen

Valencia, CA, USA).

DNA Amplification

Amplifications were performed in 50µl reactions to produce double-stranded product under the following conditions: 0.2µM each primer, 0.15mM each dNTP, 2.5µM

MgCl2, 1X buffer supplied by the manufacturer (Qiagen), and one unit of Taq

Polymerase (Qiagen). Reactions were brought up to 50µl using water and 1.5µl DNA genomic template.

Typical temperature profiles for amplification of CO1 segments consisted of 40 cycles of 30s at 95˚C, 30s at 50˚C, and 1.5 min at 72˚C, followed by a 5-min extension step at 72˚C. Considerably more success with 28s was achieved using a touch-down procedure in which the annealing temperature of 58˚C was decreased by 2˚C every 3 cycles until a final temperature of 42˚C was reached and held for 18 cycles. For CO1, sense primers s1460, s1541, s2191, and s2442, and antisense primers a1969, a2191, a2590, and a2963 were used for amplification; for 28s, primers s3660 and a335 were used. These were based on primers from Sperling and Hickey (1994), Simon et al.

(1994), Dowton and Austin (1998) and Whiting et al. (1997). Primer sequences are shown in Table 1.3. PCR products were checked on 1.5% agarose gels and products were purified directly or after gel extraction using QIAquick columns (Qiagen Valencia,

CA, USA).

37 Table 1.3: Sequences of primers used in this study. The direction of the primers is either sense (s) or antisense (a).

Primer Sequenceb Length From/alias a CO1: s1460 TACAATTTATCGCCTAAACTTCAGCC 26-mer Sperling & Hickey 1994: TY-J-1460 s1541 TGAKCYGGAATASTAGGANCATC 23-mer B. Crespi: Zeus s1859 GGAACIGGATGAACWGTTTAYCCICC 26-mer Simon et al. 1994: CI- J-1859 (alias RonII) s2183 CAACATTTATTTTGATTTTTTGG 23-mer Simon et al. 1994: CI- J-2183 (alias Jerry) s2441 CCTACWGGAATTAAAGTWTTTAGATGATT 32-mer Simon et al. 1994: CI- AGC J-2441 (alias Dick) (*) a1969 CCTTTAGGTCGTATATTAATTAC 23-mer a2191 CCCGGTAAAATTAAAATATAAACTTC 26-mer Simon et al. 1994: CI- N-2191 (alias Nancy) a2590 GCTCCTATTGATARWACATARTGRAAATG 29-mer a2963 AGGRAGTTCATTATAIGAATGTTC 24-mer rcJesse 28s: s3660 GAGAGTTMAASAGTACGTGAAAC 23-mer Dowton & Austin 1998: nrDNA 28s forward (*) s1 GACCCGTCTTGAAMCAMGGA 20-mer Whiting et al. 1997: 28s a (*) a1 TCCKGTKTTCAAGACGGGTC 20-mer Whiting et al. 1997: 28s a (*) a335 TCGGARGGAACCAGCTACTA 20-mer Whiting et al. 1997: 28s b (*) aNames refer to direction (s, sense; a, antisense) and to the position of the 3’-end. For CO1 names refer to position in the Drosophila yakuba mtDNA genome (Clary and Wolstenholme, 1985; Simon et al., 1994). S1541 was designed by B. Crespi (Simon Fraser University). bIUPAC ambiguity codes (Cornish-Brown, 1985) refer to equal mixtures of bases; I, inosine. *Modified slightly for use in this study.

DNA Sequencing

The amount of DNA from the purified PCR product was estimated by comparison to a Low Mass Ladder (Gibco) and 70-90ng (ABI370, 373) or 20-50ng

(ABI3100) was used for sequencing reactions. Sequencing primers were identical to those used in PCR reactions, with the addition of internal primers s1 and a1 for sequencing of 28s. Sequencing of this double-stranded produced was carried out using

38 25 PCR cycles of 96˚C for 30s, 50˚C for 15s and 60˚C for 4 min with a 2˚C increase per s

in a 10µl reaction. This reaction was carried out using dye terminator cycle sequencing

chemistry with the specified amount of template, 2.0µl of DyeDeoxy FS Terminator or

BigDye premix (PE Biosystems, Inc., Foster City, CA), 0.16µM primer, and water to

the final volume. Finished reactions were precipitated in 0.5mM MgCl2 in 70% ethanol, centrifuged, and dried under a vacuum. The majority of the sequencing was done using an ABI 370 or 373 automated DNA sequencer, with some sequencing done using an ABI

3100 automated DNA sequencer. Compiled segments resulted in 1113 base pairs of CO1 for all 90 species included in this analysis; alignable sequences ranging from 782-870 base pairs in length for 28s for all 90 species, depending on insertions/deletions. Both directions of the PCR product were sequenced and contigs were assembled and edited with Sequencher ver. 4.1 (GeneCodes, Ann Arbor, MI).

Sequence Alignment

There were no insertions or deletions in the protein-coding sequences of CO1, making alignment of these sequences unambiguous. Alignment of 28S was done in

ClustalX (Thompson et al., 1997) using a 28s structural model developed for

Chrysomelidae and kindly supplied by K. Kjer of Rutgers University, as well as the program default for the relative cost of gap openings vs. gap extensions. This alignment was compared to multiple alignment using the following as gap opening: extension cost parameters: 20:5, 15:3, 12:7, 10:5, 10:2, 8:3, 7:2, and 5:1. According to the criteria developed in Maddison et al. (1999), the alignment using the structural model is considerably better than any used without the model. As such these alternative

39 alignments were not considered in the phylogenetic analysis. For the final alignment, sequence positions containing internal indels of five or more contiguous gaps for any taxon were excluded from the analysis due to difficulties in assessing positional homology (Chalwatzis et al., 1996; Maddison et al., 1999). In order to avoid bias caused by missing data, both data sets were trimmed so that the beginning of all sequences includes actual data.

METHODS OF PHYLOGENETIC ANALYSIS

Saturation Levels

Between taxa that have diverged over relatively long periods of evolutionary time, certain types of molecular sites that are not under stringent selective constraints are expected to undergo some degree of multiple substitution events. As these events accumulate, it becomes more difficult to determine homologous from homoplasious character states and eventually any evolutionary information between the two sequences becomes saturated by noise. While some authors argue that this noise will be structured by other phylogenetic information and will therefore provide resolution for apical nodes

(Källersjö et al., 1999), there is a good argument that this noise will provide misleading phylogenetic signal by swamping out the information from more slowly evolving sites

(Dowton and Austin, 1998; Holmquist, 1983; Huelsenbeck and Nielsen, 1999). While the former is almost certainly true at some levels, the latter becomes a particular problem when evolutionary rates in some sites are extremely low (such as in the 1st and 2nd codon positions of a protein codon gene) while those in others are extremely high (such as in the

3rd codon position of a protein codon gene). This becomes even more problematic in loci

40 such as mitochondrial protein-coding genes where amino acid composition can be very

highly conserved, constraining evolution in nonsynonymous 1st and 2nd positions, while

the rate of evolution at neutrally-evolving sites, such as 3rd codon positions, is

considerably higher than in nuclear protein-coding genes due to the lack of DNA repair

enzymes in the mitochondria (Graur and Li, 2000). In order to judge whether or not

specific data partitions should be excluded from the analysis, I evaluated the levels of

saturation in three data partitions specified a priori (28s, CO1 1st and 2nd positions, and

CO1 3rd positions) using two methods.

In the first I compared uncorrected pairwise sequence differences with corrected

pairwise sequence differences using the HKY85 model (Hasegawa et al., 1985) for each data partition (Dowton and Austin, 1998). This method identifies both (a) character sets that are likely to have undergone multiple substitutions (and are therefore problematic for parsimony analysis), and (b) character sets that have diverged so much that model-based analysis is unlikely to correct adequately for saturation effects. The latter are problematic for both parsimony and model-based analyses such as maximum-likelihood or Bayesian inference.

In the second I compared the transition/transversion ratio against transversions for all pairwise sequence comparisons. This measure allows one to examine the amount of observed saturation in the dataset (Holmquist, 1983). Ideally, it should allow one to compare the observed ratio to that of the expected ti/tv ratio under complete saturation

(Holmquist, 1983). This measure, however, does not take into account heterogeneity in base pair frequencies across taxa and as such is likely to underestimate the expected ratio under complete saturation if there is heterogeneity in the data partition. Therefore, this

41 analysis follows traditional analyses in examining the proportion of the pairwise comparisons that plateau with time, thereby suggesting saturation. Based on the results of these two analyses, I assessed whether particular partitions should be excluded from the analysis in different reconstruction methods. I used PAUP*4.0b10 (Swofford, 2000) to calculate the pairwise transition, transversion, transition/transversion ratio, and total uncorrected substitution distance for all ingroup pairwise comparisons.

Phylogeny Reconstruction

Phylogenetic analyses were performed by maximum parsimony using version

4.0b10 of PAUP* (Swofford, 2000) and by Bayesian inference using MrBayes

2.01(Huelsenbeck and Ronquist, in Press). Because of the size of this dataset, a realistic search using maximum likelihood was deemed untenable due to time limitations and computational constraints. Indeed, in two separate aborted runs, all 90 taxa had not yet been added to the analysis after 2 weeks of continuous analysis on a Macintosh G4.

Using a total evidence approach, I combined the 28s and CO1 data sets, a procedure that maximizes the information internal to each dataset (Baker and DeSalle, 1997; Kluge and

Wolf, 1993; Remsen and DeSalle, 1998).

Under the maximum parsimony criterion I searched for shortest trees by performing 500 heuristic searches, each with starting trees based on random addition of taxa. The maximum number of trees kept in memory was not limited and the tree- bisection-reconnection heuristic algorithm was implemented. Bootstrap analyses were performed based on 250 replications, each with 100 random-addition starting trees. For reasons explained under the Results section, the 3rd codon positions of CO1 were

42 excluded. The decay index, or Bremer support (Bremer, 1988) was calculated using the program Treerot 2.0 (Sorenson, 1999) and PAUP*. Constraint trees were generated based on the strict consensus of the most parsimonious trees. The length of trees meeting these constraints were calculated in PAUP* using heuristic searches with 500 random- addition starting trees and the decay index was determined by examining the difference in length between these trees and the length of the most-parsimonious trees.

For the model-based Bayesian analyses, third positions were excluded for reasons discussed in the Results section. The program ModelTest 3.0 (Posada and Crandall,

1998) was used to determine the appropriate model of molecular evolution for the analysis based on Bayesian inference (Posada and Crandall, 2001b). This test performs a likelihood ratio test between increasingly complex models and determines whether or not increasing parameters significantly increases the fit of the model to the data. The model selected using this analysis was then incorporated into the phylogeny reconstruction using Bayesian inference in the program MrBayes 2.0. In addition, a more complex model that is not incorporated into ModelTest 3.0 was used and the results were compared based on the Akaike Information Criterion (Posada and Crandall, 2001b). All

Bayesian searches were run with four simultaneous chains for 1,000,000 generations, sampling every 100 generations and applying temperatures of 0.2 and 0.5. Heating the chains at these different temperatures influences the rate of how often chains are swapped, and using multiple temperatures increases the efficacy of the search. The burnin time (the number of generations before the tree-search reaches the optimum) was estimated by plotting the number of generations versus the ln Likelihoods (lnL). Trees in the burnin were discarded from the analysis. Posterior probabilities of nodes were

43 estimated based on the majority rule consensus of the trees that were found at

stationarity, and branch lengths for the phylogram are based on the mean branch lengths

from these trees. The model selected in ModelTest 3.0 is a General Time Reversible

Model, estimating the proportion of invariable sites and the shape of the gamma

parameter (GTR+I+G). However, subsequent analysis showed that the data were better

explained using site-specific models based on the 3 data partitions (CO1 1st positions,

CO1 2nd positions, 28s), and estimating the shape of the gamma parameter (SS+G).

Results from this model are used for all subsequent analyses.

I also examined the monophyly of the Pachymerinae (Caryobruchus,

Pachymerus, Speciomerus, and Caryedon) and the Amblycerinae (Zabrotes,

Spermophagus, and Amblycerus), two groups that have been suggested to be

monophyletic by Kingsolver (1970), Borowiec (1987), Nilsson and Johnson (1993b), and

Romero et al. (1996) but I do not find in any of the trees in this analysis. Additionally, I

examined whether the Megacerini are monophyletic with the traditional Bruchinae, and

whether the Bruchini are a clade outside of the Acanthoscelidini, as is also suggested by

the above authors but is not found in either analysis. To do this, I separately constrained

these nodes to be monophyletic and performed a parsimony search using the same

parameters as in the initial search. I then used Templeton’s Wilcoxon signed rank test

(Templeton, 1983) and Prager and Wilson’s (1988) winning sites test as implemented in

PAUP* in order to examine whether or not these relationships are significantly

contradicted by the data. Because tree length is dependent upon node resolution, I

randomly compared 10 most parsimonious trees from the constrained and the

unconstrained searches in order to avoid problems due to different resolution. In

44 addition, I examined the Bayesian posterior support for the monophyly of each of these

clades.

COMPARATIVE ANALYSES

Character assignment to taxa.

Because of the diversity of the Bruchidae, this analysis was by necessity based on

exemplar taxa. As a result, various assumptions were made concerning the evolution of

host use prior to the phylogenetic analysis. First, with the exceptions of Bruchidius and

Acanthoscelides (which are discussed later), there are very strong morphological grounds to assume that genera are monophyletic. Therefore, the included species were taken as place-holders of the phylogenetic placement and diversity of the genera that they represent. Furthermore, with very few exceptions, there is no variation within genera of either the growth form (herbaceous vs. woody) or the fruit type of their host plants.

Because of this, the placeholders were taken to represent the phylogenetic placement and diversity of the genera that they represent, as well as the growth form and taxonomic affiliation of the host plants of the genus, even when host plants are not known for every member of that genus. For example, the four species of Megacerus included in this analysis are taken as placeholders for all 55 species described in this genus. This makes the assumption that this genus is monophyletic, and that the 23 species with no known host associations feed on species of herbaceous Convolvulaceae in the same manner that do all 32 species with known hosts.

The genus Acanthoscelides is sampled extensively in order to assess its phylogenetic status and to adequately represent its diversity in both host growth form and

45 host taxon use. While the monophyletic status of this genus is certainly questionable

(Borowiec, 1987; Johnson, 1970; Johnson, 1983; Johnson, 1990c), there are very good

morphological reasons to assume that the species groups (and some combinations

thereof) distinguished by Johnson (Johnson, 1989a; Johnson, 1990c) are monophyletic.

Because these show a similar lack in variation of host use as genera, in the analysis of

diversification they were treated as was described above for genera. In an ideal world,

the same level of sampling would have been done for the Old World Bruchidius and its

close relatives in the Bruchidiini (sensu Udayagiri and Wadhi, 1989). However, time,

resources, and a lack of Old World collaborators did not allow extensive sampling of this

group. Additionally, the state of knowledge of host plants, taxonomy, and systematics of

these Old World species is poor in comparison to the state of knowledge of the

comparable New World Acanthoscelidini (sensu Udayagiri and Wadhi, 1989). As a

result, only Bruchidius ater (Marsham) and Specularius impressithorax (Pic) are included

and cannot be taken to represent the diversity of this very large clade.

Table 1.4 and Table 1.5 show those clades that are designated a priori as monophyletic. Each table includes a summary of taxa that are unsampled. Relationships for these are unclear, and thus they were not included in the analysis. Additionally, Table

1.4 provides a summary of the host growth form (woody or herbaceous) of each clade.

Woody taxa are defined as those that showed a perennial, consistent habit that includes the accumulation of lignified taxa. This definition is chosen rather than a definition based on height or status (e.g. herb, , treelet, tree, etc.), because it is the persistence of the host locally and temporally that is relevant to this discussion. Because of this definition, taxa often labeled as herbaceous, such as some members of the genera

46 and , are included as woody taxa. Woody lianas, represented almost

exclusively by the genus Bauhinia, are also included as woody taxa. Herbaceous taxa are

taken as annual species whose stems do not lignify and die at the end of the growing

season. Designations were compiled from throughout the plant systematic literature, in

particular from various plant taxonomy reference books (e.g. Judd et al., 2002;

Mabberley, 1997; Zomlefer, 1994). Table 1.5 provides a summary of the host taxa used by each clade. These were compiled from the literature and from personal rearing of bruchid specimens from host plants. There are few exceptions to the designation of each clade’s host affiliation and host growth form as represented by the exemplar taxa. All exceptions are listed in the appropriate table and all appear to represent novel departures from what is otherwise a fairly consistent habit. Because the resolution of this phylogenetic analysis is not at the species level and because the exceptions are few and minor, they were not factored into the analysis of diversification. However, the genus

Zabrotes deserves particular attention (Kingsolver, 1990; Romero and Johnson, 2000).

Because of the post-dispersal oviposition exhibited by members of this genus, host associations are not very well-known for many of its species. Those that are known fall into two camps—those that feed on the seeds of woody plants, particularly of species in the tribe Cassieae, and those that feed on the seeds of cultivated herbaceous legumes. As a result, it is difficult to score the host association of this genus. It is likely that the normal hosts are indeed woody legumes and that the shift to cultivated herbaceous legumes resulted from both an increase in their abundance as a resource and a decrease in the concentration of their defensive chemicals due to artificial selection during

47 48

49 50 51 52 53 54 55 56

57 cultivation. Because of this, the analyses were done with Zabrotes included as a woody- feeding clade.

Sister-group comparisons.

Macroevolutionary explanations of adaptive radiation have historically focused on the identification of asymmetries in species diversity and the subsequent designation of a hypothesis of key innovation. This key innovation is often equated with a synapomorphy that defines the more species-rich group and often appear to make sense in a narrative framework. This approach is unfortunately laden with pitfalls, not the least of which is this reliance on post hoc identifications of asymmetry in diversity and subsequent designation of key innovations. Also problematic is the reliance on taxic designations.

These tend to obfuscate the possibility that the disparity in diversity may actually occur within the more diverse taxon, instead of being defined between the two taxa in question.

Additionally, reliance on single-case instances of evolutionary radiations are not overly

convincing due to artifacts caused by historical contingency or due to the confounding

effects of other coeval synapomorphies. In order to test a causal hypothesis of increased

diversification, we must examine it within a repeatable statistical framework. This

approach allows us to test a priori hypotheses of adaptive radiation. It is also based on

explicitly phylogenetic hypotheses, examining character evolution and not clade

designation, and relies on multiple observations. Furthermore, the use of sister clades

takes into consideration divergent evolution prior to the origin of the two clades (there is

none), and the amount of time to accumulate diversity (by definition sister clades are the

same age). One potential problem comes when the primitively woody-feeding sister

58 group of an herbaceous-feeding clade includes an herbaceous-feeding component (Mitter et al., 1988). However, none of the comparisons in the following phylogenies shows this pattern, and thus this problem does not affect the analyses shown here.

There is some disagreement on the way in which sister-group analyses should be analyzed. Mitter et al. (1988), Farrell et al. (1991), and Wiegmann et al (1993) argue that a strict sign-test makes no assumptions about null models of speciation and is conservative with respect to Type II statistical error. However, Slowinski and Guyer

(1993) argue that a model-based method increases statistical power because the non- parametric method makes no distinction in the disparity in diversity between (for example) 4 vs. 5 species and 4 vs. 500 species. Their model incorporates a model of tree growth via random speciation and extinction. I used an Excel spreadsheet based on their equation 3 (p. 1022) to calculate the test statistic that was then evaluated using Fisher’s combined probability test (Sokal and Rohlf, 1995). I used both of these methods in examining diversification within the Bruchidae so that I can compare and contrast the results and examine their robustness to different methods of inference. The tests were performed on the strict consensus of the most-parsimonious trees from the parsimony analysis. There was no need to examine alternate resolutions of the most-parsimonious trees because all were in agreement with the sister-group comparisons present in the strict consensus.

Of perhaps greater concern to an analysis of diversification is the problem inherent in applying these statistical tests to a single phylogeny. Systematists in general know that a phylogenetic hypothesis is exactly that—an hypothesis. Various measures have been devised in order to assess the support or uncertainty in phylogenetic analyses.

59 Whatever the philosophical leanings of the researcher, the use of bootstrap support, decay

indices, branch lengths, confidence indices, retention indices, or even consensus trees in the publication of phylogenetic analyses is meant to convey the extent to which the data support the proposed phylogenetic tree. However, in analyses of character evolution, phylogenetic constraint, or diversification, these phylogenetic trees are often taken as given and this uncertainty is not incorporated into the final analysis. I examined the use of incorporating uncertainty estimates into the phylogenetic analysis by examining the credible set of trees found in the Bayesian search. Briefly, the 95% credible set of trees are determined from the post burnin sampling of phylogenies, and these 8645 trees are sampled for sister-group comparisons and weighted based on their posterior probabilities.

The test statistic is then determined by summing across these credible sets of trees.

Macroevolutionary trajectory.

Phylogenetic constraint. The degree to which host use is evolutionarily conservative in phytophagous insects depends to a large extent on the scale and resolution of both the insect and the plant phylogenies in question. For example, an analysis of phylogenetic constraint of a genus of bruchids will likely entail a different focus than one of the entire subfamily. Therefore it is important that the level of interest be explicitly stated. For the purpose of this analysis, I want to ask whether or not feeding on different major clades of angiosperms is phylogenetically conserved across the bruchid phylogeny, largely because the use of a small set of clades has been put forward as explaining much of bruchid diversification. Primarily this discussion has focused on the Fabaceae (the legumes), but other clades used by a significant number of bruchid

60 species include the Malvales and Arecaceae. By examining phylogenetic conservatism

across these few clades I can examine whether or not this diversification is constrained by

history. Tests of the correlation of major host clade with phylogeny were done based on the method of Maddison and Slatkin (1991) for examining phylogenetic inertia. These tests were done using the PTP utility in PAUP* as implemented by Kelley and Farrell

(1998) and followed two coding schemes. In the first scheme, all taxa included in the phylogeny are coded for the host clade that they use. Because this analysis could be biased by the dominance of the Fabaceae as host plants, the analysis is also done separately for each of the Fabaceae, Malvales, and Arecaceae, coded as binary characters

(e.g. Fabaceae-feeding, non Fabaceae-feeding). This final analysis is also done separately for two major clades of legumes (Mimosoideae and Papilionoideae) and for the basal grade of legumes collectively referred to as the Caesalpinioideae. One concern with an analysis such as this is the degree of sampling of ecological associations included in this analysis. For example, the genus Caryedon feeds on both Fabaceae and

Combretaceae, but the placeholder in this phylogeny (Caryedon serratus) feeds on

Fabaceae. For the majority of these circumstances, the placeholders were treated as polymorphic with the polarity of the shifts being unresolved. However, for the genus

Amblycerus, there is external information based on the cladistic analysis of Romero et al.

(2002) that Fabaceae are the ancestral host. In this case only the species A. cistelinus (the

non-legume placeholder) was treated as polymorphic. Unfortunately, there is insufficient

sampling and resolution in Silvain and Delobel’s (1998) cladistic analysis of the genus

Caryedon to provide similar information. Host clades known for single species not

sampled for the phylogeny are not included in the analysis. Tests of the correlation of

61 host growth form with phylogeny were similarly tested, with woody and herbaceous lineages defined as above.

Bruchidius ater and Specularius impressithorax were not coded in this analysis for the same reasons discussed in the previous section. Codings for the ecological characters are shown in Table 1.6.

These analyses were performed separately on the strict-consensus of the parsimony trees (10,000 PTP repetitions) and on each of the parsimony trees (1,000 PTP repetitions on each). For the Bayesian analysis, these analyses were performed on the consensus Bayesian topology (10,000 PTP repetitions) and on a subsample of 100 trees chosen at random (1,000 PTP repetitions on each).

History of associations. In order to estimate the sequence of bruchid-host plant associations, I used the same multistate character (with multiple host use coded as polymorphism) as in the first analysis of the previous section (second column in Table

1.6) for the parsimony analysis, and the binary codings for the different host clades

(columns 3-8 in Table 1.6) for the Bayesian analysis. In particular, I want to know: (1) if there is evidence of an ancestral relationship with woody plants, and are there single or multiple shifts to herbaceous hosts; (2) if there is evidence of an ancestral relationship with palms, as may be predicted by evidence from the fossil record; (3) if there is evidence of trajectory within the Fabaceae that is in accord with recent advances in legume phylogenetics (that is, of an ancestral relationship with the Caesalpinioideae with

62 63 64

65 shifts to the Mimosoideae and Papilionoideae); and (4) if there is evidence of multiple

origins of feeding on major plant clades.

For both the Bayesian and the parsimony analysis, ancestral states are estimated

on the strict consensus tree using the default settings as implemented in MacClade v4.05

(Maddison and Maddison, 2002). Host tissue type and the major clade used are coded as

in the 2nd and last columns of Table 1.6. Because the strict consensus in the parsimony analysis is used, only unequivocal reconstructions are examined because neither the

ACCTRAN nor DELTRAN optimization can be used.

In addition, ancestral states were estimated on the Bayesian consensus phylogram with branch lengths included using a likelihood approach based on a Markov model of binary character evolution (Pagel, 1999b). While the phylogram from the Bayesian analysis deviates significantly from a molecular clock, branch lengths are still used because they are assumed to reflect some degree of temporal separation and are likely better than giving each internode an arbitrarily equal length. The binary characters for growth form and plant clade use, in columns 3-8 and column 9 of Table 1.6 respectively, were used in this analysis and are reconstructed using the program Discrete v4.0 (Pagel,

1999b). Taxa using multiple host plant clades were coded as present in multiple analyses. While this results in a probability greater than 1.0 for some nodes, the relative proportion of those probabilities is assumed to contain information into the confidence placed in the reconstruction of such nodes. Additionally, both Bruchidius ater and

Specularius impressithorax were coded as having ‘character absent’ for all analyses, as the species that these two exemplars represent vary considerably in both growth form and

66 host plant use. This creates some nodes reconstructed with a probability less than 1.0. In these cases, the difference was included as an equivocal reconstruction.

Plant phylogeny and host plant growth form. To test whether plant phylogeny and plant growth form constrain macroevolutionary trajectories of bruchids to different extents, I assess the number of steps needed to trace each on the bruchid phylogeny. The multistate character coding for host plant is used, and host growth form is coded as woody or herbaceous. The numbers of steps are averaged over the most-parsimonious trees from the parsimony analysis and over a subsample of 100 trees from the Bayesian analysis. The results are tested using the chi-square distribution and the null hypothesis that growth form and host plant clade changes are equally phylogenetically constrained.

This analysis closely follows the tactic taken by Janz and Nylin (1998) in asking a similar question about the macroevolution of butterfly-host plant interactions. However, they condensed their host plant clades into three somewhat artificial groupings in order to create an equal number of characters with host growth form (their analysis included vines), and created a step matrix for both characters. While this method eliminates the problem of there being a larger pool of possible character states for changes to occur, it is unclear if this is relevant in an analysis of phylogenetic constraint when character ordering is unconstrained. Even if it is relevant, the question remains as to whether or not the artificial condensing of host clades renders the analysis biologically meaningless. For the purpose of this analysis, I want to know if the host clade used is more phylogenetically conserved among the bruchids than host tissue use, and the multistate option seems justified. However, in order to examine the role of the potential characters,

67 the analysis was also done using feeding on Fabaceae as a binary character. This is done

because most (but not all) of the transitions in host use on the phylogenies are between

the Fabaceae and non-Fabaceae clades.

The apparency hypothesis (Feeny, 1976) predicts that rates of change in host use

should be greater on lineages that feed on woody hosts than lineages that feed on

herbaceous hosts. I tested this possibility using a modification of Sillén-Tullberg’s

(1993) contingent states test. As designed, this test examines whether changes in one

binary character are contingent upon the state of a second contingent character, taking

into account the distribution of changes across the tree. This test in analogous to and is

similar in philosophy to Maddison’s (1990) concentrated changes test. However, the

latter test is confined to two binary characters and requires fully-resolved trees. Because artificially grouping the character ‘host clade use’ into two states would eliminate a considerable amount of information in the current context, it is not applied to this data set. Instead, I modified the contingent states test somewhat in order to fit the purpose of this analysis. The test as originally proposed examines the number of events (0→0,

0→1, 1→0, 1→1) that have occurred in the dependent character on branches reconstructed as either 0 or 1 in the independent character. Because I want to know the number of overall changes in host use on either branch, I modified the test such that any change in the multistate character of host use is counted as an event. In terms of Sillén-

Tullberg’s test, this functionally transforms any character to be coded as a 0 at the beginning of each internode. The events are then counted and tested in a 2 X 2 contingency table. This test was performed on all most-parsimonious trees from the parsimony analysis, and on a sample of 100 trees from the Bayesian analysis. This test

68 once again depends on reconstruction of ancestral states. Ancestral states were

reconstructed using both the ACCTRAN and DELTRAN optimizations in MacClade and

the results compared. It would be interesting to be able to use the information from the

Discrete analysis for the purposes of this test, but it is unclear how this would be done

and this is beyond the scope of this chapter.

RESULTS

Saturation levels.

The level of saturation due to multiple substitutions is low for 28s and for the first

two codon positions of CO1, as can be seen by the nearly linear relationship between

corrected and uncorrected distances for these partitions (Figure 1.2) and the sloped relationship between the transition/transversion ratio and transversions for these partitions (Figure 1.3). Overall transition/transversion ratios are quite high and base pair frequencies are not significantly heterogeneous across taxa for either of these data partitions (Table 1.7). On the other hand, the level of saturation is high for the third codon position of CO1. Correcting for multiple substitutions by using an HKY85 model of nucleotide substitution (Hasegawa et al., 1985) reveals that most sites contain numerous hidden substitutions, as can be seen by the divergence from the straight line in

Figure 1.2. In fact, the model was unable to correct for 10.2% of all sites because levels of saturation are too high. This indicates that even a model-based analysis would be unlikely to adequately correct for saturation effects. Additionally, it is clear from the analysis of the transition/transversion ratio that most of the sites are saturated (Figure

1.3). This can also be seen by the overall low transition/transversion ratio (Table 1.7) for

69

70

Figure 1.2. Saturation analysis of molecular character partitions. Pairwise uncorrected and corrected distances are estimated using PAUP v.4.0b10 (Swofford, 2000), using the HKY85 (Hasegawa et al., 1985) model to correct for multiple substitutions. Uncorrected distances are then plotted on the X-axis, corrected distances on the Y-axis. The extent of hidden substitutions is indicated by the distance of the points to the left of the diaganol line. The plot for 3rd codon positions in COI included numerous distances that the model is unable to correct, and are not shown here (10.2% of all pairwise comparisons).

71

Figure 1.3. Saturation analysis of molecular character partitions. Ti/Tv ratios and transversions are determined using PAUP v.4.0b10. Transversions are plotted on the X- axis, ti/tv ratios on the Y-axis. The plots for 1st and 2nd COI codon positions and 28s include large numbers of comparisons that have no transversions and are included at 10% above the maximum (38 in the former, 30 in the latter).

72 third codon positions. In addition, base pair frequencies are significantly heterogeneous across taxa (P<<<0.0001, Table 1.7), indicating that both parsimony and model-based analyses are likely to be misled by this data partition. While I agree with Källersjö et al.

(1999) that saturated sites can be given structure by unsaturated sites, the level of saturation is so high that only a very small number of pairwise comparisons contain information regarding relationships between apical nodes. As such, the benefits of including the thirds are minimal and are outweighed by the costs of including a large number of highly homoplasious characters that show distinct differences in base pair composition across taxa. For these reasons, third codon positions are excluded from the remainder of the analyses.

RESULTS OF PHYLOGENETIC ANALYSES

Results of the parsimony analysis.

Sixty-three most parsimonious trees of length 2621 were found in the analysis excluding third codon positions (Figure 1.4). Most intergeneric relationships in this analysis are only weakly resolved, and support along the ‘backbone’ of the phylogeny is particularly low. This is born out by both low bootstrap values and decay indices. On the other hand, there is high support for the monophyly of almost all genera that are represented by multiple species. The one exception is the broadly polyphyletic genus

Acanthoscelides, a result anticipated by most morphological systematic work on bruchids.

Many higher-level taxa proposed by taxonomists do not appear in the most parsimonious trees (Figure 1.4). The Pachymerinae and Amblycerinae are both

73 paraphyletic, the Megacerini do not resolve as being monophyletic with the rest of the traditional Bruchinae, and the Bruchini are nested within the traditional Acanthoscelidini

(* in Figure 1.4). However, due to the general lack of resolution along the backbone of the phylogeny, none of these relationships are significantly different than those proposed by taxonomists when considered under the criteria of Templeton’s (1983)Wilcoxon signed rank test and Prager and Wilson’s (1988) winning sites test (Table 1.8). The

Bruchidae as a clade are well-supported by both relatively high bootstrap support (91%) and decay index (12), and the traditional Acanthoscelidini are monophyletic, with the exception that the Bruchini are nested within this clade. In general, support is higher at the tips of the phylogenies, as indicated by both higher bootstrap values and decay indices, while more basal nodes have bootstrap values <50% and decay indices of 1.

This result is consonant with other recent intergeneric phylogenies of Coleoptera (e.g.

Jordal et al., 2000; Maddison et al., 1999) and is a common result for phylogenies with large numbers of species in general (Sanderson and Wojciechowski, 2000). Support for relationships relevant in the macroevolutionary trajectory of host clade and host tissue use will be discussed in those sections.

Results of the Bayesian analysis.

The burn-in for this analysis is conservatively estimated at 100,000 generations,

10% of the total length of the run. By this generation the log likelihood of the topologies accepted has reached a noticeable plateau (Figure 1.5). In the majority-rule consensus of the sampled 9100 trees (Figure 1.6, the values at each node indicate the percentage of

74

75

Figure 1.4. Strict consensus of 63 most-parsimonious trees from parsimony analysis (CO1 3rd positions excluded). Heuristic search using 500 replicates with a random addition sequence and tree-bisection-reconnection (TBR). Decay indices using same search strategy. Bootstrap proportions based on 250 replicates, each replicate consisting of a heuristic search strategy, with 100 random-addition sequences and TBR. Brackets indicate traditional classification of the Bruchids. Asterisk indicates the traditional Acanthoscelidini, excluding the Megacerini and Bruchini. Continued on next page.

76 Figure 1.4 (Continued)

those samples in which that node is present and can be thought of as being analogous to parametric bootstrap values (Huelsenbeck et al., 2002). This topology from this analysis contains considerably more support than the parsimony analysis, particularly along the backbone of the phylogeny. Low support is found primarily within the Acanthoscelidini

77 at five nodes subtending and resolving the clades included by Bruchus pisorum + Sennius

rufomaculatus. Cursory examination of the consensus phylogram from this analysis

(Figure 1.7) suggests that this lack of resolution could be the result of very fast diversification of this clade, although more data would provide more information in this regard. With the exception of the genus Acanthoscelides, all genera with multiple exemplars were well-supported as monophyletic. Aside from the genus Sennius (support for S. breveapicalis as monophyletic with the remainder is only 39%) all have posterior probabilities of 100%. Beyond this, monophyly of the Pachymerinae and Amblycerinae is significantly rejected, as are relationships that include the Megacerini as monophyletic with the Bruchinae and relationships that exclude the Bruchini from within the

Acanthoscelidini (Table 1.8). As with the parsimony analyses, relationships that are important for the study of macroevolutionary trends in ecological associations will be discussed in more detail in later sections.

78

Figure 1.5. Estimation of burn-in time for Bayesian analysis. The likelihood conservatively reaches a plateau by the 100,000th generation.

79

Figure 1.6. Consensus Bayesian phylogeny of the Bruchidae. Model incorporates site- specific rate variation based on three data partitions (28s, CO1 1st and 2nd positions), estimating the shape of the gamma parameter (SS+gamma). Analysis ran 4 simultaneous chains for 1,000,000 generations (first 100,000 generations discarded as burn-in), sampling every 100 generations and applying temperatures of 0.2 and 0.5. Numbers indicate Bayesian posterior support for individual clades. Brackets indicate traditional classification of the Bruchids. Asterisk indicates the traditional Acanthoscelidini, excluding the Megacerini and Bruchini. Continued on next page.

80 Figure 1.6 (Continued)

81

Figure 1.7. Consensus Bayesian phylogram of the Bruchidae. Based on site-specific rate variation and estimated gamma parameter (SS-gamma). Branch lengths are those lengths with highest posterior probability.

82 RESULTS OF COMPARATIVE ANALYSES

Results of sister group comparisons

Sister-group comparisons based on parsimony analysis. The strict consensus tree allows complete resolution of all sister-group comparisons between clades that feed on woody hosts and those that feed on herbaceous hosts (Figure 1.8). There are six unequivocal and resolved origins of herbaceous feeding across the phylogeny. The origin of feeding on herbaceous taxa by Sennius nov. sp. near willei is not considered within this analysis due to lack of resolution within the genus Sennius, and was excluded from sister- group comparisons. In addition there are, at minimum, two other origins of feeding on herbaceous taxa: at least one within the genus Kytorhinus and at least one within the very large Old World clade represented by Specularius impressithorax and Bruchidius ater.

Unfortunately, in neither of these cases is the resolution high enough to examine the consequences of this shift for rates of diversification. Of the remaining six independent origins of feeding on herbaceous taxa, all result in increased rates of diversification ranging in magnitude from 4.3X to 20.5X (Table 1.9; one-tailed sign test P = 0.016;

Slowinski and Guyer (1993) parametric test statistic 24.9, P = 0.015).

While support for the monophyly of the herbaceous clades is relatively high in all cases, support for sister group relationships in the comparisons concerning

Callosobruchus and Bruchus is relatively low. However, even if the sister group of

Bruchus were to include all of the other members in the clade that is sister to the

Bruchidius + Specularius clade, the diversity would still be higher for the herbaceous-

83

Figure 1.8. Diversity of groups monophyletic for feeding on either herbaceous or woody hosts, summarized on parsimony strict consensus tree. Species diversity is based on summing the diversity represented by the exemplars (Tables 1.4 and 1.5) included in each clade. Continued on next page.

84 Figure 1.8 (Continued)

feeding Bruchus (196 vs. 80). Similarly, if the sister group for Callosobruchus includes

both the Bixa feeding Stator species and the genus Gibbobruchus, diversification is still

higher for Callosobruchus (41 vs. 15). It is only when the genus Stator is added to the

woody clade that the relationship changes (41 vs. 43). Therefore, the preponderance of

the evidence supports the hypothesis that shifting to herbaceous plants results in higher

diversification rates within the Bruchidae.

85 Table 1.9: Summary of sister-group diversity comparisons for independent herbaceous- feeding lineages vs. woody-feeding sister groups. Analysis based on strict consensus of 63 most-parsimonious trees, or any one of these 63 trees individually.

Primitively herbaceous-feeding Sister-group lineage Clade* Approximate Clade* Approximate Sign of Magnitude number of number of difference in of difference species species diversity† in diversity (herbivorous (excluding feeders only) herbivorous feeders) Acanthoscelides 153 Acanthoscelides 29 + 5.3 manducus + puniceus + Stylantheus Scutobruchus macrocerus ceratioborus Bruchus 196 Rhipibruchus 36 + 5.4 atratus + Merobruchus insolitus Callosobruchus 41 Stator (Bixa 2 + 20.5 feeders) Caryedes 71 Ctenocolum 8 + 8.9 helvinus + Meibomeus surrubresus Megacerus 55 Pachymerus + 11 + 5 Speciomerus Spermophagus 121 Zabrotes 28 + 4.3 Note: Summary: the number of positive differences: 6; the number of negative differences: 0; sign test, one-tailed, P = 0.016; Slowinski and Guyer parametric test- statistic 24.9; P = 0.015. *Where clade includes multiple taxa, clade refers to the two species listed, their most recent common ancestor, and all the descendants of that ancestor. †The herbaceous-feeding clade minus the sister group.

Sister-group comparisons based on Bayesian analysis. The consensus phylogram from the Bayesian search provides an example of the sister-group

relationships sampled in the Bayesian analysis (Figure 1.9). The origins of feeding on

herbaceous hosts in Sennius nov. sp. near willei, in the genus Kytorhinus, and within the

large clade represented by Specularius impressithorax and Bruchidius ater are not

included in this analysis for the same reasons as given in the parsimony analysis above.

86 Across the 8645 sampled phylogenies, many of the sister group relationships are different than those shown in Figure 1.9. One advantage of Bayesian analysis in sister group comparisons is that it allows for the incorporation of uncertainties in the phylogenetic hypothesis. The 8645 trees represent 525 different combinations of sister group analyses and are present in proportions ranging from 12% of the sampled phylogenies down to a single sampled phylogeny. While some sister group pairs are present in all (Zabrotes +

Spermophagus) or almost all (99.2% for Megacerus + (Pachymerus + Speciomerus)) of the analyses, others vary in both the membership of the herbaceous clade or the woody clade. The number of comparisons, the sign of the difference for each comparison alone

(for the one-tailed sign test) and in combination with the magnitude of the difference (for the parametric test) are weighted by the proportion of times each of these combinations of sister groups is sampled. These are then summed in order to arrive at the appropriate test-statistic and P-value. Under both criteria, the data are able to reject the null hypothesis of no difference in diversity between clades that feed on herbaceous hosts and clades that feed on woody hosts (one-tailed sign test P = 0.022; Slowinski and Guyer

(1993) parametric test statistic 20.8, P = 0.036). As a weighted average, there are 5.6 sister group comparisons between clades feeding on herbaceous and clades feeding on woody hosts. In only 0.69% of the trees sampled is there a single comparison that goes against the general trend, and in no cases are there two. Therefore, as in the parsimony analysis, there is support for increased diversity in clades that shifted from feeding on woody hosts to feeding on herbaceous hosts.

87

Figure 1.9. Diversity of groups monophyletic for feeding on either herbaceous or woody hosts, summarized on Bayesian consensus tree. Note that in the diversity analysis, those clades sampled as sister groups across the 8,645 trees within the 95% credibility set of the most-likely tree may be different from those shown here. Continued on next page.

88 Figure 1.9 (Continued)

Macroevolutionary trajectory of host plant utilization

Phylogenetic constraint. While the majority of bruchids feed on species of legumes (Fabaceae), other clades of hosts are used by a significant number of bruchid species, particularly the Malvales, Solanales, and Arecaceae. In addition, roughly half of the species with known host associations feed on woody hosts with the other half feeding

89 on herbaceous hosts. Feeding on all of these major clades of hosts is significantly conserved (Table 1.10) across both the parsimony analysis (Figure 1.10) and the

Bayesian analysis (Figure 1.11). The host growth form of plant is also significantly conserved across hosts (Table 1.10) regardless of analysis (see Figures 1.8 and 1.9).

Indeed, it appears as though most cladogenesis within the bruchids occurs without major shifts in host plant associations.

History of associations.

Parsimony reconstructions. Most-parsimonious reconstructions of growth form on both the strict consensus tree and the Bayesian consensus tree indicate that feeding on woody hosts is indeed the ancestral condition for bruchids (Figures 1.8 and 1.9). As intimated in the section on sister-group comparisons, a minimum of eight origins of feeding on herbaceous hosts has occurred within the bruchids, a number in agreement between the two analyses.

The picture is much less clear in terms of reconstructing the evolutionary history of early bruchid associations. Due either to inadequate sampling or the vagaries of macroevolutionary history, it is unclear what the ancestral associations were, as all of the basal nodes are reconstructed as equivocal. If internodes leading up to the palm feeders

(New World pachymerines) can be reconstructed as palm feeding, then this association appears early in the evolutionary history of bruchids on either tree. This certainly makes sense, as fossils that are distinctly Pachymerinae appear early in the fossil record. While this is in agreement with data from the fossil record, it is unclear whether or not there are multiple origins. Further sampling, particularly of the interesting palm-feeding genus

Caryoborus and Old World Pachymerinae, could very well shed light on this issue.

90 Table 1.10: Results of analyses of phylogenetic constraint. PTP utility based on method of Kelley and Farrell (1998). Significant results indicate that ecological characters are distributed nonrandomly on the tree, given the topology of the tree and the distribution of the character states. Ecological codings are shown in Table 1.6.

Parsimony Bayesian Ecological Test using strict Tests on each of Tests using Tests using coding consensus tree 63 most Bayesian random sample (10,000 parsimonious consensus tree of 100 post burn- repetitions) trees (1,000 (10,000 in trees (1,000 repetitions repetitions) repetitions each) each)* Multistate P=0.0001 P=0.001 P=0.0001 P=0.001 Fabaceae P=0.0001 P=0.001 P=0.0001 P=0.001 Malvales P=0.0001 P=0.001-0.006 P=0.0001 P=0.001 Arecaceae P=0.002 P=0.002 P=0.0001 P=0.001 Caesalpinioideae P=0.0001 P=0.001 P=0.0001 P=0.001 Mimosoideae P=0.0001 P=0.001 P=0.0001 P=0.001 Papilionoideae P=0.0001 P=0.001 P=0.0001 P=0.001 Growth form P=0.0001 P=0.001 P=0.0001 P=0.001 *range is given if different trees showed different significant values, range is not given if they are identical.

Feeding on Caesalpinioideae is found throughout the tree, particularly within the

‘basal’ paraphyletic Amblycerinae (see also Romero et al., 2002), the most basal lineage

within the traditional Acanthoscelidini (represented by the genera Penthobruchus and

Pygiopachymerus), and the genus Sennius. While this provides reason to speculate that the lineages in the paraphyletic grade represented by the Caesalpinioideae were the first legumes to be colonized by the bruchids, the reconstruction of ancestral nodes is inconclusive. The distinct departure from clocklike evolution in this phylogeny precludes examining particular timing events in order to address this issue by using an absolute time scale. Feeding on mimosoid legumes appears throughout the derived and diverse tribe Acanthoscelidini, but is almost entirely absent outside of it, indicating that mimosoids may have been colonized more recently. Whether or not mimosoids have been colonized multiple times is beyond the resolving ability of these phylogenies.

91

Figure 1.10. Major clades of host plant traced onto strict consensus of most- parsimonious trees. Taxa with asterisks include species that feed on other hosts, see Table 1.5 for details. Only unequivocal groupings are reconstructed here. Continued on next page.

92 Figure 1.10 (Continued)

93

Figure 1.11. Major clade of host plant (Fabaceae split into Mimosoideae, Caesalpinioideae, and Papilionoideae) traced onto Bayesian consensus tree. Taxa with asterisks include species that feed on other hosts, see Table 1.5 for details. Note that transitions may be different in 100 randomly sampled topologies. Continued on next page.

94 Figure 1.11 (Continued)

Feeding on the diverse papilionoid legumes has arisen at least seven times independently from either mimosoid or caesalpinioid-feeding ancestors, and appears to be generally derived within legume-feeding. A more comprehensive analysis of the history of the ecological associations between bruchids and legumes will require more comprehensive

95 sampling of the bruchids and robust phylogenetic analyses of the major lineages of the

Fabaceae.

The Malvales have been colonized multiple times throughout the evolutionary

history of the bruchids, at least four of which are represented in the sampling for this

phylogenetic analysis. There is not enough information to examine other ecological

associations, particularly because sampling within the Amblycerini, Spermophagini, and

(in particular) the Old World Bruchidae is relatively low.

Maximum-Likelihood Reconstructions. Maximum-likelihood reconstructions for

ancestral states of host affiliation based on the program Discrete (Pagel, 1999b) are

shown for Acanthoscelidini clade 1 (Figure 1.12), Acanthoscelidini clade 2 (Figure 1.13),

and for the remainder of the bruchids (Figure 1.14). As in the parsimony-based

reconstructions, the majority of the internodes along the ‘backbone’ of the phylogeny are reconstructed with low resolution. For example, a single origin of palm-feeding at the base of the tree cannot be ruled out or supported based on the information currently present in the dataset. Similarly, there is no support for or against an older or ancestral relationship with caesalpinioid legumes than with other legumes. There is some support for a derived shift to mimosoid legumes within the traditional Bruchinae soon after the divergence of the lineage leading up to Caryedes, Meibomeus, and Ctenocolum. There is considerably more support when the relationships with the genus Prosopis are ignored for the different species of Amblycerus (reasons for this are given in the Discussion). There is very strong support for multiple origins of feeding on Papilionoideae and Malvales.

Reconstructions for ancestral states of host growth form are shown across the entire phylogeny (Figure 1.15). With the exception of three nodes, all nodes were

96

Figure 1.12. Maximum likelihood reconstruction of ancestral states on bayesian consensus phylogram, clade 1 ('top' clade in Figure 1.11) of Acanthoscelidini clades. All reconstructions are based on the "local" approach to ancestral reconstruction (Pagel 1999). Proportions based on multiple analyses with host plants coded as binary.

97

Figure 1.13. Maximum likelihood reconstruction of ancestral states on bayesian consensus phylogram, clade 2 (sister clade to Figure 1.12) of Acanthoscelidini clades. All reconstructions are based on the "local" approach to ancestral reconstruction (Pagel 1999). Proportions based on multiple analyses with host plants coded as binary. Legend as in Figure 1.12.

98

Figure 1.14. Maximum likelihood reconstruction of ancestral states on bayesian consensus phylogram, basal clades of Bruchidae. All reconstructions are based on the "local" approach to ancestral reconstruction (Pagel 1999). Proportions based on multiple analyses with host plants coded as binary. Legend as in Figure 1.12.

99

Figure 1.15. Growth form of host plant (woody v. herbaceous) traced onto bayesian consensus topology. Woody lianas in the genus Bauhinia are treated as woody plants. All ML reconstructions are above 95% confidence intervals, except for those shown with pie graphs. Continued on next page.

100 Figure 1.15 (Continued)

reconstructed at levels above the 95% confidence limits. The three nodes that are reconstructed with less confidence do not change the conclusions that feeding on herbaceous plants is derived from feeding on woody plants in all instances (with the exception of the shift within the Acanthoscelides species group Aequalis to feeding on

Guazuma sp.). They only change the possible number of shifts to herbaceousness through adding a possible convergent shift by both Meibomeus and Caryedes instead of a

101 single shift leading up to the origin of the clade. In general, the conclusions drawn from

the maximum likelihood ancestral reconstructions are identical to those drawn from the

parsimony-based ancestral reconstructions.

Plant phylogeny and host plant growth form.

Relative levels of constraint. The mean number of steps to trace the character

‘host clade’ onto the 63 most-parsimonious trees is 7.4, while it is 7 on every most-

parsimonious tree for the character ‘growth form’, indicating that there is no significant difference between the level of phylogenetic constraint on these two ecological variables

(χ2 goodness-of-fit test not significant, average P = 0.92, range 0.80-1.0). The result

holds true for the 100 randomly sampled Bayesian phylogenies as well (average steps for

‘host clade’ = 7.0, growth form = 7.3; χ2 goodness-of-fit test not significant, average P =

0.93, range 0.62-1.0). This indicates that at this level of sampling and resolution that the

rates of change in host use and growth form are indistinguishable.

Janz and Nylin (1998) suggested that having different numbers of character states

for the ecological characters being compared could bias the results of this analysis.

Because of this I also compared the number of steps that it takes to trace the

presence/absence of legume-feeding on the phylogenies versus the number of steps it

takes to trace the character ‘growth form’ on the phylogenies. As suspected, the results

remain largely unchanged. The average number of steps to trace the character ‘Fabaceae’

onto the 63 most parsimonious trees is 5.4, 2.0 lower than that in the previous analysis.

This is because (1) the transition between Arecaceae (Speciomerus + Pachymerus) and

Euasteridae (Megacerus) are ignored and (2) the dual transition to the Euasteridae and

102 Malvales are lumped together within Spermophagus. The difference, again, did not approach significance (χ2 goodness-of-fit test not significant, average P = 0.64, range

0.56-0.78). The results are in accord with the 100 randomly sampled Bayesian phylogenies (average steps for ‘Fabaceae’ = 6.3, growth form = 7.3; χ2 goodness-of-fit test not significant, average P = 0.78, range 0.41-1.0).

The apparency hypothesis. This application of Sillén-Tullberg’s (1993) contingent-states test shows no support for the hypothesis that colonization of new host plants is facilitated by feeding on woody plants. Results are not significant for any of the reconstructions in either the parsimony or Bayesian analysis (Table 1.11). While it is possible that this is an artifact of sampling (of lineages or ecological associations) or inadequate phylogenetic resolution of host plant clades, in the context of the current analysis there is no support for the apparency hypothesis.

Table 1.11: Results of the analysis of the apparency hypothesis, using a slight modification of Sillén-Tullberg’s contingent states test. Analyses on parsimony trees were done on all 63 trees, on Bayesian trees analyses were performed using 100 randomly sampled trees. Average number of changes are shown, with the range in parentheses if there is variation. P-values indicate results of chi-square goodness of fit analyses.

0Æ0 0Æ1 1Æ1 1Æ0 Parsimony Change 8 1 5 0 ACCTRAN No change 129 8 34 1 P = 0.50 Parsimony Change 8 1 5.37 (5-6) 0 DELTRAN No change 129 8 33.7 (33-34) 1 Average P = 0.41, range 0.27-0.50 Bayesian Change 7.9 (7-8) 1 5 0 ACCTRAN No change 129.1 (127-134) 8 34.6 (34-36) 1.3 (1-3) Average P = 0.50, range 0.39-0.57 Bayesian Change 8 1 5 0 DELTRAN No change 129.6 (127-132) 8.1 (8-9) 33.3 (30-35) 1.1 (1-2) Average P = 0.47, range 0.37-0.52

103 DISCUSSION

Phylogeny and classification of the Bruchidae4.

The higher-level classification of the Bruchidae has been somewhat muddled ever

since the first attempts were made by Bridwell (1932). For the most part, ‘higher’

classification simply involved the placement of species into their appropriate subfamily.

Attempts at determining relationships between these subfamilies were neglected, based

on intuition, or were hypothesized based on single characters. The strict adherence of

many bruchid systematists to the separation of the bruchids as a family distinct from the

Chrysomelidae indicated an underlying distrust in phylogenetics as a tool for evaluating

classification. Furthermore, it became plainly obvious that determining the higher-level

relationships within the bruchids was going to be a difficult task—Borowiec published

two very different phylogenetic trees in two successive articles (Borowiec, 1987;

Borowiec, 1988), and the latter was meant to be a Polish-language translation of the

former! In addition to these difficulties in determining relationships between higher-level

groups, there is considerable confusion as to relationships between lower-level groups.

Even in terms of placement, many species of the derived Bruchinae have been simply

thrown into the genera Acanthoscelides and Bruchidius because it is too difficult to

determine how they might be segregated into other genera. Even these two genera cannot

be separated morphologically and are dependent exclusively on biogeography (Borowiec,

1984; Borowiec, 1987). Clearly, an intense character-based phylogenetic assessment of

the group is needed to bring order to the chaos, and this study is the first to attempt that.

4 The use of the familial designation “Bruchidae” is not meant to imply that the seed beetles should be kept as a family separate from the Chrysomelidae. It is only used in the current context so as to avoid confusion when discussing lower-level relationships. It would be particularly cumbersome to modify all of the traditionally applied subfamilies and tribes, and is beyond the scope of this chapter.

104 The Bayesian and parsimony analyses result in similar, but distinct trees. In

general, the Bayesian analysis results in nodes with higher support (as measured by

posterior probabilities) than the parsimony analysis (as measured by bootstrap

proportions or decay indices), a common result that has been noted and discussed by

Huelsenbeck et al. (2002). From either analysis it is clear that the classification of the bruchids needs to be reconsidered. Of particular note are the potential non-monophyly of the Pachymerinae and Amblycerinae, and the potential basal position of the

Amblycerinae (Figures 1.4 and 1.6). This last relationship was perhaps anticipated in the more recent dendogram published by Borowiec (1988), even though most systematists assume that the Amblycerinae were closely related to, and probably sister to the

Bruchinae. Each of these relationships must be examined more thoroughly, particularly with more extensive sampling of Old World pachymerines and sampling of the small and presumably basal subfamilies Rhaebinae and Eubaptinae. A basal position of the

Amblycerinae is interesting in that it suggests that feeding on legumes evolved early

within the group, before herbaceousness had evolved within any of the legume

subfamilies. Placement of the Kytorhininae is considerably different depending upon the

two methods used. In the parsimony analysis, it is sister to the pachymerine Caryedon,

albeit with low support. If this is the case, it could indicate a basal relationship of the

Caryedontini with legumes, a prospect neither confirmed nor refuted by the study of

Silvain and Delobel (1998). In the Bayesian analysis, it is highly supported as sister to

the Amblycerini (represented by the genus Amblycerus), a relationship that would further

support Romero et al.’s (2002) conclusion that legume-feeding is the ancestral condition

for the Amblycerini. This also suggests that phylogenetic work on the genus Kytorhinus

105 itself may elucidate whether or not there is a general trajectory to shift from woody to herbaceous legumes, as members of the Amblycerini feed exclusively on woody hosts while different species of Kytorhinus feed on either woody or herbaceous legumes.

Of considerable interest is the lack of monophyly in both analyses of the

Pachymerinae in general, and the New World Pachymerini in particular. While monophyly of the Pachymerinae cannot be rejected in the parsimony analysis, it is significantly rejected in the Bayesian analysis (Table 1.8). This result has never been suggested in the literature, as the monophyly of the Pachymerini has always been assumed (e.g. Nilsson and Johnson, 1993b). This certainly warrants further investigation, particularly through the inclusion of the genus Caryoborus and the addition of more representatives of the Old World pachymerine tribes Caryedontini and Caryopemontini.

This, in combination with the accurate placement of the Amblycerinae, would be particularly applicable in assessing the hypotheses that the ancestor of sagrines and bruchids fed on a monocot host (Farrell, 1998; Farrell and Sequeira, in prep.), and that the bifid tarsi shared by the sagrines, bruchids, criocerines, and donaciines indicate their shared ancestral heritage through common ecological associations (Reid, 1995). As mentioned before, the placement of lineages leading to the pachymerines at the base of the phylogeny is suggested by the only currently classified bruchid fossils from the

Florissant bed in Missouri, as well as fossilized pachymerines recently collected by B.

Archibald (Museum of Comparative Zoology, Harvard University) in even older shale beds in British Columbia (unpubl. data). However, other bruchids found in both of these beds (Scudder, 1876; Wickham, 1912; Wickham, 1913; Wickham, 1914; Wickham,

1917) are clearly not pachymerines and appear to show affinities with amblycerines or

106 even bruchines (personal observation). These fossils, combined with an obvious mistake

(Reid, 2000) of a bruchid for a sagrine from 70 million year old Canadian Cretaceous

amber by Poinar (1999b), suggest that the bruchids could be considerably older than their scant fossil record suggests. As such, simple presence in the fossil record at a seemingly old age cannot be taken as prima facie evidence that those lineages are the oldest.

The observation in both analyses of a sister relationship between the Megacerini and one of the clades of the Pachymerini is suggestive of an interesting host shift from the woody palms to the herbaceous Convolvulaceae. It also warrants a review of the conventional wisdom among bruchid systematists that the Megacerini should be included within, or sister to, the recently-derived and highly diverse Bruchinae (Borowiec, 1987;

Bridwell, 1946; Terán and Kingsolver, 1977). In the only comprehensive treatment of the Megacerini, Terán and Kingsolver (1977) suggest reasons for including the

Megacerini within the Bruchinae, but also caution that this classification is nebulous at best.

Monophyly of the highly diverse Bruchinae, with the exception of Megacerini, is supported in both analyses, as is monophyly of the traditional tribe Bruchini. However,

Bruchini is nested within the Acanthoscelidini, a relationship rendering the latter clade paraphyletic. As noted in the introduction, this is hardly surprising given that the

Bruchini are separated almost exclusively on the basis of a small suite of secondary sexual characteristics. At a broader level, this clade appears to be broken into four fairly well supported lineages. The first consists of the woody caesalpinioid-feeding genera

Penthobruchus and Pygiopachymerus; the second consists of the genera Caryedes,

Meibomeus, Ctenocolum and probably Margaritabruchus; and the third and the fourth

107 clades split the remaining genera of the Bruchinae. The third clade (the Acanthoscelidini

clade 1 of Figure 1.12) includes various bruchine genera as well as six species groups of the genus Acanthoscelides. The fourth clade (the Acanthoscelidini clade 2 of Figure 1.13) includes the Bruchini, the only sampled members of the controversial (Borowiec, 1987;

Bridwell, 1946; Udayagiri and Wadhi, 1989) tribe Bruchidiini, and four species groups of the genus Acanthoscelides. Genera held together as monophyletic throughout, with the exception of the genus Acanthoscelides. This exception was widely anticipated by systematists on the basis of morphological systematic evidence (Johnson, 1983; Johnson,

1990), as discussed in the introduction.

The resolution between lineages within these third and fourth clades of the

Bruchinae have an interesting juxtaposition: while a distinct lack of resolution occurs between major lineages within these clades, good resolution can be found within these major lineages (this effect is particularly noticeable in clade 4, see Figure 1.7).

Preliminary investigation of these relationships when including a third molecular marker

(EF1-α) fails to increase this resolution to any considerable extent (results not included).

This type of pattern may well be the signature of rapid lineage diversification—the lack of resolution is a hard polytomy resulting from diversification without sufficient time for the buildup of apomorphies, thereby precluding resolution (Jackman et al., 1999; Jordal et al., 2000; Lovette and Bermingham, 1999; von Dohlen and Moran, 2000). There are, of course, other causative explanations for this sort of pattern (Jordal et al., 2000), and one must be cautious in drawing conclusions based on star phylogenies. However, a slowdown in rate during the diversification of these lineages is unlikely as speciation is actually likely to preserve polymorphisms that are segregating between diverging

108 populations (Avise, 1994; Avise, 2000; Futuyma, 1987; Harrison, 1991). This pattern could be the result of insufficient character sampling, as there are only slightly less than four times as many taxa as there are informative characters (Table 1.7) in this analysis.

Addition of a third gene fails to markedly improve this situation, particularly for the fourth clade (see Figure 1.13), as expected when diversification is rapid (Otto et al.,

1996). To this point, however, preliminary sampling of this gene, EF-1α, is not extensive enough to directly address whether or not it would rectify this.

Shallow internodes may reflect rapid diversification between these two clades.

While it is difficult to discern what would cause this rapid diversification, it is notable that the majority of this diversification is onto those lineages that feed on the shrubby and herbaceous legumes and Malvales that themselves diversified rapidly during the aridification that occurred during the late Oligocene and early Miocene (Axelrod 1972).

If these rapidly diversifying host plants provided opportunities for ecological and evolutionary differentiation within the bruchids, then the rapid diversification of the bruchids may well be a direct result of an adaptive radiation of certain plant lineages into newly xeric areas. Fossil dates and temporal information on these diversifications is needed in order to test this hypothesis. Unfortunately, the non-clocklike behavior of the molecular datasets in this analysis precluded such a test. Additional evidence from taxa that use hosts that similarly diversified as a result of this diversification (e.g. lycaenid butterflies) would provide further comparative data to test this hypothesis.

109 Adaptive radiation and host use in the Bruchidae.

Farrell (1998) was the first to produce phylogenetic evidence that the rise and

rapid diversification of angiosperms resulted in the adaptive radiation of those

phytophagous insects that first managed to invade such a burgeoning resource. The

implication of such a study is that the diversification of the angiosperms resulted in the

generation of numerous partitionable ecological niches that phytophagous insects could

exploit. In such a case, host plant species are hypothesized to have diverged significantly

in characters that were relevant to the ability of insects to use them as food. Such

divergence would therefore have permitted character displacement and competitive

coexistence for a diverse array of phytophagous insects (Ehrlich and Raven, 1964;

Hutchinson, 1959; MacArthur and Levins, 1967; Strong et al., 1984). In this case, plant

species and plant tissues functionally interact to serve as distinct niches for phytophagous

insects (Farrell and Sequeira, in prep.; Marvaldi et al., 2002), resulting in the

diversification of phytophagous insects as their host plants diversify.

The observation that the vast majority of these insects are specialists (Futuyma

and Moreno, 1988; Jaenike, 1990; Mitter et al., 1988; Thompson, 1994) certainly

supports this explanation, but one must bear in mind that alternative explanations could

also explain the increased diversity of phytophagous insects when they shift onto

angiosperms. For example, the increased diversity could be the direct result of something

about one or numerous synapomorphies of the angiosperms perse that causes increased

diversification in phytophagous insects, regardless of niche differentiation. For example, the evolution of an indehiscent megasporangium may allow host plants to persist in local viable populations through ecological fluctuations (Judd et al., 2002), decreasing overall

110 extinction rates of the insects that depend upon them. The increased diversity could be

due simply to the fact that angiosperms dominate in the tropics, and only give way in

biomass as one moves away from the equator. The increased diversity of those insects

that happen to feed on angiosperms could simply be the result of any number of

hypotheses unrelated to niche partitioning that have been put forward to explain increased

diversity in the tropics, including their increased area, a longer time since devastation by

climatic events, increased productivity, a glacial refugia species pump, or a mid-domain

effect (Brown, 1988; Colwell and Lees, 2000; Haffer and Prance, 2001; Hewitt, 1996;

Prance, 1981; Rosenzweig, 1995).

Because angiosperm diversity is so disparate between lineages (Dodd et al., 1999;

Eriksson and Bremer, 1992; Judd et al., 1994; Magallon et al., 1999; Magallon and

Sanderson, 2001; Ricklefs and Renner, 1994), one observation that would support the view that angiosperm species serve as partitionable niches for phytophagous insects is that this disparity in diversity is correlated between angiosperm lineages and the phytophagous insects that feed on them. The results of this analysis provide significant support for this prediction. Because the disparity of diversity between angiosperms is largely explained by growth form in both observed pattern and theorized process (Dodd et al., 1999; Eriksson and Bremer, 1992; Ricklefs and Renner, 1994; Silvertown and

Dodd, 1996), the observation that shifts to herbaceous plants within bruchids resulted in a significant increase in their diversity supports the suggestion that it is the ecological opportunity provided by the species diversity of the angiosperms that has resulted in the

diversification of the bruchids.

111 The shift to feeding on herbaceous hosts that led to the significant increase in

bruchid diversity almost certainly required the successful clearance of numerous

biological hurdles. While these were not as drastic as those required for the shift from an

aphytophagous to a phytophagous habit (Farrell and Mitter, 1994; Mitter et al., 1988;

Strong et al., 1984), it is possible that they are more meaningful than any hurdles

presented by the initial appearance of the angiosperms. The successful colonization of

herbaceous hosts likely requires changes in host-searching behavior, the

counteradaptation to different types of defensive plant chemicals, and changes in life history that are associated with a more ephemeral and unpredictable resource (Bernays and Chapman, 1994; Bernays and Funk, 1999; Feeny, 1976; Levins and MacArthur,

1969). The observation that host growth type is significantly conservative in the bruchids

(and others; see Janz and Nylin, 1998) also suggests that shifting between hosts of different growth type is under considerable phylogenetic constraint. At the same time, the appearance of herbaceous angiosperms likely provided a substantial opportunity for the obligately spermophagous bruchids due to their increased investment in reproductive tissues (Gadgil and Solbrig, 1972; Obeso, 2002; Silvertown and Dodd, 1996). This combination of (1) barriers to colonization, (2) ecological opportunity, (3) partitionable resources, and (4) multiple increases in rates of diversification indicate that the shift from

feeding on woody plants to herbaceous hosts is resulted in the adaptive radiation of the

bruchid seed beetles (Schluter, 2000; Strong et al., 1984).

There are possible explanatory alternatives to those suggested above, but I feel

that they have weaker support than the hypothesis of adaptive radiation onto herbaceous

plants. First, herbaceous plants may have additional intrisic features that cause increased

112 rates of speciation and/or decreased rates of extinction in bruchids and are therefore

neutral with regards to niche partitioning. For example, the increased population structure found in herbaceous plants is presumably caused by the ephemeral and patchy nature of their populations. This could translate into increased rates of speciation in bruchids, without speciation and differentiation in their host plants. Two observations argue against this hypothesis, however. First, while seeds may appear to be an even more temporally ephemeral resource than the host plants themselves, bruchids have population sizes probably orders of magnitude higher than their host plants and they appear to be

good dispersers (unpublished data). Second, there is no known case of a host plant

having different species of bruchids attacking geographically different wild populations

of a host plant species.

A second alternative would be that herbaceous plants are correlated with another

factor that would cause increased diversity, such as a presence in the tropics beyond that of woody plants. For reasons discussed above, this could result in increased diversity of their associated insects without relying on the idea of niche partitioning. There appears to be no support for such a supposition, however, because if anything herbaceous plants tend to become more diverse and prominent in temperate zones (Judd et al., 1994). In fact, most of the woody hosts of bruchids are tropical or subtropical trees. This includes some of the most prominent woody hosts such as Acacia, Prosopis, Mimosa, and the

Cassia alliance. In general, this appears to be an insufficient explanation in that the majority of bruchid species are either tropical or subtropical in distribution, whether they feed on woody or herbaceous species (Borowiec, 1987; personal database information).

113 A third alternative is the possibility that bruchids that feed on herbaceous plants have higher levels of specialization. This would not directly support the idea that it is the increased diversity of herbaceous plants that provide more partitionable niches for bruchids. Rather it would support the idea that the bruchids treat herbaceous plant species as more differentiable entities than they do woody plants. The apparency

hypothesis (Feeny, 1976) lends credence to such an idea—the qualitative defenses of

herbaceous plants may cause host species to be considerably distinct when it comes to the

bruchids’ detoxification mechanisms; on the other hand, the quantitative defenses of

woody plants may not prevent competitive exclusion on species that feed on different

hosts, because the resource may be functionally the same when it comes to the bruchids’

detoxification mechanisms. However, at least for bruchids, this does not appear to

explain the increased diversity with feeding on herbaceous hosts. When tabulating the

numbers of hosts for species of bruchids with known hosts that feed on either woody or herbaceous plants (Figure 1.16), there is no significant difference between the proportions that feed on different numbers of hosts (2-factor ANOVA without replication, P = 0.45, SS = 3.4, 1 degree of freedom). This indicates that there is no direct evidence for a higher degree of specialization on herbaceous versus woody plants.

Second, there is no significant support for the apparency hypothesis, as examined using the modified Sillén-Tullberg (1993) contingent-states test, suggesting that there is no support for the hypothesis that there is a higher degree of phylogenetic constraint on herbaceous versus woody hosts. This indicates that bruchids partition woody and herbaceous plants along similar axes—at the species level. This negative result may be due to a qualitative difference between the tightly packaged and ephemeral resource of

114 the seed versus the persistent and replaceable nature of foliage. The apparency

hypothesis itself may not predict such a difference in toxic compounds between the seeds

of woody and herbaceous hosts in the same way that it does for the of woody and

herbaceous hosts, although this remains to be seen.

A fourth alternative is that the increased diversity of species feeding on

herbaceous plants is an artifact of field collecting. This is highly unlikely, however, in

that it is considerably easier to collect seeds from woody plants because they produce

larger numbers of seeds than herbaceous plants, are easier to find in the field than

herbaceous plants, and are considerably more predictable from year-to-year, which is

especially relevant if one is searching for potential hosts based on herbaria locality labels

(personal experience). If anything, a bias in collecting would probably underestimate the

number of species of bruchids that feed on herbaceous plants.

Another argument against the hypothesis that shifting to herbaceous hosts resulted

in the adaptive radiation of the bruchids would be if diversity within those host plant clades on which bruchids feed is not correlated with growth form, in contrast to the general trend for angiosperms. If this were the case, then equating ‘host plant growth form’ with increased diversity would not be justifiable. However, in all of the bruchid host plant clades that include herbaceous lineages, diversity is correlated with herbaceousness. This is true in the , the Malvales, the Asterales, and the

Solanales (Dodd et al., 1999; Doyle, 1994; Judd et al., 2002; Mabberley, 1997; Magallon et al., 1999; Sanderson and Wojciechowski, 1996; Silvertown and Dodd, 1996; Zomlefer,

1994). This suggests that there really are more potential host species that are herbaceous than there are potential hosts that are woody.

115

Figure 1.16. The number of recorded hosts for species of Bruchidae that feed on either herbaceous or woody hosts. Host records are from the literature and total 763 bruchid species. There is no significant difference between the distribution of numbers of hosts for herbaceous or woody-feeders (ANOVA 2-factor without replication, P = 0.45, 1 degree of freedom).

One final argument is that competition is not an important process in structuring the evolutionary trajectories of those lineages that feed on herbaceous plants (but is for lineages feeding on woody plants), thereby allowing considerable niche overlap between species from these lineages. A general argument in the literature is that many phytophagous insect communities in general are not structured by competition (Hairston et al., 1960; Lawton and Strong, 1981; Strong et al., 1984). However, there is no suggestion nor is there any obvious reason as to why this would be different for lineages that feed on herbaceous plants versus those that feed on woody plants. Some of the best examples of competition in insect communities come from bruchids in general (Delgado et al., 1997; Fox et al., 1999; Fox et al., 1996b; Savalli and Fox, 1999; Shimada et al.,

2001; Strong et al., 1984), and lineages that feed on herbaceous legumes in particular

116 (Colegrave, 1995; Eady, 1994; Giga and Smith, 1991; Kawecki, 1995; Kawecki, 1997a;

Messina, 1991; Szentesi et al., 1996; Toquenaga, 1993; Toquenaga and Fujii, 1990).

Furthermore, at a qualitative level, there is more overlap in host use in species from genera that feed on woody plants such as Stator (Johnson and Kingsolver, 1976; Johnson et al., 1989; Johnson and Siemens, 1992), Sennius (Johnson and Kingsolver, 1973;

Johnson and Siemens, 1992), Algarobius (Johnson and Siemens, 1997a; Kingsolver,

1986), Scutobruchus (Kingsolver, 1968; Kingsolver, 1983), Rhipibruchus (Kingsolver,

1967; Kingsolver, 1982), Mimosestes (Johnson, 1987; Kingsolver and Johnson, 1978),

Merobruchus (Kingsolver, 1980; Kingsolver, 1988), and species groups from

Acanthosceldies (Johnson, 1970; Johnson, 1981d; Johnson, 1983; Johnson, 1989a;

Johnson, 1990c) than there is in lineages that feed on herbaceous plants.

The preponderance of evidence therefore suggests that bruchids have diversified largely as a function of the diversification of their host plants. This supports the hypothesis of Ehrlich and Raven (1964) and earlier work by Mitter et al. (1988) and

Farrell (1998) that plant species in some way define the axis along which phytophagous insects partition resources. Tests of this hypothesis in bruchids are facilitated by the independent colonizations of herbaceous host plants, allowing for the comparative study of diversification based on sister groups. However, seed beetles may well treat plants in a fundamentally different way then do phytophagous insects that feed on plant vegetative tissue, and/or on external parts of plants (Denno et al., 1995; Janzen, 1971b; Janzen,

1981a; Southgate, 1979; Strong et al., 1984). Therefore, further comparisons are needed using phylogenies of different guilds of phytophagous insects to determine whether or not this is a pattern that helps explain the preponderance of phytophagous insects in general.

117

Macroevolutionary trajectory of host use in the Bruchidae.

Hutchinson (1959) argued that phytophagous insects have diversified by

partitioning the highly diverse and morphologically differentiated species of angiosperms

into distinct niches. His argument that ecological opportunities are the driving force

behind diversification is born out by this analysis of the bruchids. Clearly, however, this

diversification has not been unshackled by the constraints of history—bruchids feed on only a limited number of angiosperm clades, and they are completely limited to feeding internally on the reproductive organs of these plants. The significant degree of phylogenetic conservatism in host use during the diversification of the bruchid seed beetles (Table 1.10) is consistent with the argument of Ehrlich and Raven (1964) that niches are only available given the evolutionary history of the organisms involved. Their argument that phylogenetic constraint directs the trajectory of diversification is therefore also born out by this analysis. As a result, the history of bruchid diversification can best be described as an intertwining between the seemingly contrary concepts of opportunity and constraint.

Opportunity and constraint exert their effects on bruchid diversification along different axes, however. For example, it has become clear in general that the host tissue attacked is generally more conserved phylogenetically that host taxon is, whether the organism in question are viruses (Bowen et al., 1997; Taber and Pease, 1990), plant viruses (Dewey et al., 1997), or phytophagous insects (Farrell and Sequeira, in prep.; Marvaldi et al., 2002). This is plainly obvious in the Bruchidae, which are exclusively and obligately internal seed-feeders. Nor is this surprising—turning the

118 material of a structure that was intended to produce an individual plant into an individual

beetle (from the inside-out, no less) almost certainly requires a suite of complicated

adaptations that are difficult to reverse. These adaptations are evident in larval

morphology (Arora, 1978; Lee, 1993; Pfaffenberger, 1981; Pfaffenberger and Johnson,

1976) and larval metabolism (Applebaum, 1964; Janzen, 1977; Rosenthal, 1981;

Rosenthal, 1983; Rosenthal, 1989; Rosenthal et al., 1978).

Interestingly there is no significant difference in the phylogenetic conservatism

between the growth form of the host attacked and the taxonomic affiliation of the host

plants, nor were the two significantly correlated. This result is in contrast with that found

by Janz and Nylin (1998) for butterflies. However, there is something qualitatively

different between these two characters. There is only one case in which a shift from

woody (the ancestral state) to herbaceous hosts has been reversed: the association within

the Aequalis species group of Acanthoscelides with the malvaceous tree Guazuma. On

the other hand, there are numerous derived shifts within leguminous or malvaceous

lineages (within the genera Amblycerus, Caryedon, Neltumius, Spermophagus, species

groups of Acanthoscelides, and probable other instances not included in the phylogeny such as in the genera Bruchidius, Lithraeus, and Salviabruchus). In fact, treating these

two ecological characters as ‘irreversible’ results in a statistically significant difference in

the number of steps in the parsimony tree and the Bayesian consensus tree (P < 0.05). It

appears that either: (1) the adaptations required to feed on herbaceous hosts (listed

earlier) constrain subsequent evolution more than detoxification adaptations to new plant

defensive chemistry; and/or (2) that feeding on herbaceous hosts confers such a strong

selective advantage in the community context in which it occurs that the opportunity to

119 reverse has never presented itself, while changes in community context may favor the

colonization of different host taxa enough to overcome phylogenetic constraint.

The highly constrained habit of internal seed-feeding by ancestral bruchids

probably originated with the emerging dominance of the angiosperms in the late

Cretaceous (Borowiec 1987; Farrell 1998; Reid 2000). The radical change in community

structure that accompanied the rise of flowering plants has resulted in an interesting

history of persistent associations coupled with shifts onto newly emerging resources. The

significant levels of phylogenetic constraint in all host associations suggests that there is

a clear signal of the actual evolutionary history in the phylogeny of the bruchids, and that

the associations are not simply the result of persistent and convergent host shifts onto

particular taxa. Therefore, examining the phylogeny of the bruchids provides a context

within which to examine the trajectory of their ecological associations over the course of

such drastic changes in community structure.

This analysis shows that the first bruchids likely fed on woody hosts (Figure

1.15), despite the presence of herbaceous paleoherbs at the time of bruchid origins. This

likely reflects the predominance of forested terrestrial habitats at that time, as open grasslands did not begin to appear for another 40-50 million years during the aridification that accompanied the Oligocene/Miocene warming (Axelrod, 1972; Bredenkamp et al.,

2002; Stromberg, 2002). This observation reconciles the observation that more than half of bruchine species are from lineages that feed on woody plants with the evidence for accelerated bruchine diversification on herbaceous lineages. The lineages on woody plants simply had more time to accumulate species.

120 If the original bruchid hosts are ancestors of bruchid hosts that occur today, it is

difficult to determine which lineage would be descended from the ancestral host given

the current analysis. The parsimony analysis suggests an association with either palms or

tree legumes. Although further sampling of basal lineages could shed more light on this

subject, palms are perhaps the most likely candidate based on two observations. First is

the ancestral association with monocots of all the clades of Chrysomelidae that are

closely related to the bruchids—the Sagrinae, the Donaciinae, the Criocerinae, and the

Hispinae (Farrell 1998; Reid 2000). Second is the observation that palms were well- established and were dominating many terrestrial communities by the late Cretaceous

(Crabtree, 1987; Daghlian, 1981), while legumes are not known in the fossil record until the Paleocene some 57 million years ago (Herendeen and Crane, 1992). Both palms and tree legumes produce large, nutrient-rich seeds and either likely would have provided a substantial ecological opportunity for the already endophagous (Farrell 1998, Farrell and

Sequeira in prep.) ancestors of the bruchids.

The maximum-likelihood analysis using the Bayesian consensus phylogram is considerably more unclear about the ancestral reconstruction, allowing for the possibility that the ancestral host could have been a progenitor of any of the three subfamilies of legumes, the Malvales, the Arecaceae, or the Euasteridae (Figure 1.12). However, consideration of external evidence provides the possibility of eliminating the mimosoid or herbaceous papilionoid legumes, as neither appear to have been present at the end of the Cretaceous (Herendeen and Crane, 1992). The possibility of a euasterid ancestor is almost certainly the result of the grouping of the Convolvulaceae (the host of the genus

Megacerus) and the Boraginaceae (the host of some species of Amblycerus) as a single

121 character. Neither of these families appear in the fossil record until the Eocene

(Chandler, 1964; Collinson et al., 1993). Additionally, cladistic analysis of the genus

Amblycerus (Romero et al., 2002) suggests that the evidence for Malvales as a possibility

is due almost certainly to low sampling in the current analysis. This leaves, again, only

the Arecaceae and early caesalpinioid or papilionoid tree legumes as the likely hosts.

The subsequent divergence of the Bruchidae occurred against an ever-changing

ecological landscape. The gradual warming of the earth during the Oligocene and

Miocene paved the way for the appearance of grasslands and other vegetation types that

were evolving in response to the resultant aridification (Axelrod 1972). The current

evidence supports a hypothesis of a more recent divergence onto the thornscrub

mimosoid, herbaceous papilionoid, and Malvales that serve as the host of many of the

derived and diverse Bruchinae. While a lack of clock-like molecular evolution in this

phylogeny precludes the possibility of dating divergence events, the phylogenetic

topology alone is suggestive of a macroevolutionary trajectory of host associations in line

with current hypotheses of plant community evolution during the Tertiary. This is particularly true in the parsimony analysis. And while the Bayesian analysis appears more equivocal, this almost certainly due to the consideration of relationships within the genus Amblycerus with the mimosoid genus Prosopis. This is the only mimosoid host for

any non-bruchine species and can perhaps justifiably be ignored: Prosopis are

notoriously free of defensive chemicals, appearing to use a predator satiation strategy

instead (Kingsolver et al., 1977). As such, numerous bruchine lineages have

convergently shifted to feed on this genus and many of the more generalist bruchid

species include Prosopis in their diet.

122 For the most part, bruchid diversification has followed along phylogenetically- constrained trajectories. According to the coevolutionary hypothesis put forward by

Ehrlich and Raven (1964) this is due to the importance of plant chemistry in determining the evolution of host use in phytophagous insects, as closely related plants can be expected to have similar defensive chemistry profiles. While phylogenetic constraint is certainly a predominant theme in most phylogenetic studies of insect-plant interactions, almost equally predominant are the prevalence of host shifts more dramatic than would be accounted for by phylogeny alone (Becerra and Venable, 1999; Farrell, 1998; Funk et al., 1995; Janz and Nylin, 1998; Kelley and Farrell, 1998; Mardulyn et al., 1997;

Termonia et al., 2001). The uniqueness of many host shifts leaves the circumstances of their origins shrouded in historical contingency. However, multiple colonizations of the same taxa suggest that these shifts may in some way be predictable (Becerra and

Venable, 1999; Mardulyn et al., 1997). Bruchids appear to have colonized the Malvales at least four times independently, and with increased sampling that number would almost certainly increase. The herbaceous papilionoid legumes have been colonized at least seven times independently from either mimosoid or caesalpinioid-feeding ancestors. In addition, bruchids appear to have colonized the Convolvulaceae, the Combretaceae, and the Rhamnaceae at least twice. Other multiple origins are likely as well, given the taxonomic distinctness of species that feed on (), Asteraceae, and

Anacardiaceae. Such a general theme of convergent host shifts suggests that bruchids may be constrained in some degree when they do colonize new host plant lineages.

Becerra (1997) and Becerra and Venable (1999) found that such a pattern was almost certainly due to convergent similarities in plant chemistry. However, there is no obvious

123 commonality in the defensive chemistry of the seeds of the above groups (Amorprats and

Harborne, 1993; Harborne et al., 1971; Hylin and Watson, 1965; Janzen, 1981a;

Rosenthal, 1981; Rosenthal, 1986). On the other hand, all of these plant groups are

common components of dry tropical and subtropical communities. Probably correlated

with this are the dry, often dehiscent nature of the fruits (Judd et al., 2002; Zomlefer,

1994) that make the seeds available to bruchids. It appears, then, that colonization of novel hosts in bruchids may be directed and limited by the community context in which the ancestral hosts occur as well as the morphological similarities of fruits that are likely adaptations to similar ecosystems. While further investigation is needed to examine if the subset of plant clades that bruchids feed on is non-random given the their morphology and geographic distribution, these factors have been put forward as being highly relevant in directing host plant use in phytophagous insects (Atsatt and O'Dowd, 1976; Becerra

and Venable, 1999; Janzen, 1969).

These results suggest that the history of bruchid macroevolution is a history of the

interaction between opportunity and constraint. The ecological opportunities presented

by the diversification of lineages of herbaceous angiosperms provided the basis for a

significant increase in the diversification rate of the bruchids, but this diversification

proceeded along the constraints imposed by their evolutionary history and is limited to

endophagous seed-feeding of particular host-plant clades. It is clear that the changes in

community and ecosystem structure that have occurred as a consequence of both the rise

of the angiosperms and the climatic changes over the subsequent millennia have

structured the diversification of the bruchids. The signature of these changes is still

124 present in the macroevolution of ecological associations that we can discern from the reconstructed phylogeny of the Bruchidae. As a result, this analysis and others have shown that the historical examinations of ecological associations provide insight to the processes that create the extant diversity that we see around us.

The goals of this chapter were to study the pure and the applied phylogenetics of the Bruchidae. It is clear that considerable revisionary work must be done in order for the classification to accurately reflect relationships within the group. The influence of evolutionary taxonomy in bruchid systematics must be discarded in order to gain an appreciation of the history of adaptations that have resulted in the morphological and ecological diversity of the bruchids. Considerable fieldwork and museum work is still

desperately needed for this group, as probably less than 10% of world bruchid species are

currently described (C.D. Johnson and J.M. Kingsolver, pers. comm.) and only 30% of

the described species have known host associations. This work would greatly facilitate

our understanding of bruchid relationships as well as provide more accurate assessment

of the ecological and evolutionary history of the group.

The applied phylogenetics has explicitly focused on the macroevolutionary

trajectory of ecological associations and their resultant implications for rates of

diversification. The results suggest that niche partitioning is an important ecological

determinant of bruchid diversification and that the axes along which this partitioning

occur appear to be defined largely by characteristics that differentiate host plant species.

This niche diversification has proceeded along phylogenetically, morphologically, and

ecologically constrained pathways, however. In addition to the rapid diversification of

lineages associated with herbaceous plants, other bruchid lineages have maintained

125 persistent affiliations over relatively long periods of bruchid history. These patterns are consistent with Ehrlich and Raven’s (1964) coevolutionary model that explains the correlation between phylogenetic diversification and ecological diversification in phytophagous insects.

The present analysis refines the suggestion by Farrell (1998) that the success of the Coleoptera seems to have been enabled by the rise of flowering plants. The success, at least of the Bruchidae, seems to have been enabled by the rise of particular lineages of flowering plants. Given the underlying process of niche diversification proposed by

Farrell (1998), a similar pattern is expected from other higher resolution phylogenies of phytophagous insects.

126

CHAPTER 2:

THE EVOLUTION OF SPECIALIZATION IN THE GENUS STATOR BRIDWELL

(COLEOPTERA: CHRYSOMELIDAE: BRUCHINAE)

INTRODUCTION

The impressive species diversity of phytophagous insects is likely a consequence

of both the number of host-plant species available as food resources and specialization by insect species onto these diverse food resources (Ehrlich and Raven, 1964; Farrell, 1998;

Hutchinson, 1959; Mitter et al., 1988; Strong et al., 1984). Resource specialization is nearly ubiquitous in phytophagous insects (Bernays and Chapman, 1994; Futuyma and

Moreno, 1988; Jaenike, 1990; Mitter et al., 1988; Thompson, 1994), despite the apparent advantages of being able to use multiple hosts when resources are temporally or spatially patchy (Futuyma and Moreno, 1988; Mopper et al., 1995). Ecological research has generated considerable insight into the generation and maintenance of resource specialization in phytophagous insects. The potential importance of generalist predators, competition, host-plant defenses, local adaptation, and trade-offs in performance in generating and maintaining resource specialization in ecological time is well-documented

(Bernays and Graham, 1988; Denno et al., 1995; Rausher, 1988; Schultz, 1988; Strong et al., 1984; Van Zandt and Mopper, 1998; Via, 1986; Via, 1991), but how variation in diet breadth evolves at a macroevolutionary level is poorly understood.

In order to contribute to our knowledge of the long-term evolution of resource specialization and niche differentiation, this chapter explores the macroevolutionary pattern of host use in the seed beetle genus Stator Bridwell (Coleoptera: Chrysomelidae:

Bruchinae). This clade of seed beetles shows considerable interspecific variation in diet

breadth and in life history traits linked to host use. By using a phylogenetic analysis of

mitochondrial and nuclear DNA sequence data, I address hypotheses concerning the

phylogenetic trajectory of resource specialization, host affiliation, and life history, and

128 how these three interact. Specifically, I pose the following questions: (1) What is the

polarity of changes in host range (breadth of diet)? (2) How do host affiliation and diet

breadth interact in the direction and rate of the evolution of host use? (3) How does

oviposition guild differentiation evolve at the macroevolutionary level, and how does this

differentiation affect the macroevolutionary trajectories of host affiliation and diet

breadth?

Polarity of Evolution of Diet Breadth. The ecological theory of adaptive

radiation (Schluter, 2000; Simpson, 1944) suggests that diversification begins as a

lineage enters a previously unoccupied adaptive zone and gives rise to species that

diverge and subsequently partition the newly available resources. There is considerable

evidence that supports this model (summarized in Schluter, 2000), including evidence

from phytophagous insects (Farrell, 1998; Farrell and Mitter, 1994; Mitter et al., 1988;

Strong et al., 1984). For example, phytophagous insect clades are consistently more diverse than their primitively aphytophagous sister clades (Mitter et al., 1988), speciation is associated with changes in host use in many groups of insects (Funk et al., 1995; Mitter et al., 1991; Strong et al., 1984), and increases in diversification rates of host-plants can be correlated with increases in diversification rates of insects (Farrell, 1998). The approximately 260,000 species of tracheophytes, most of which are used as food by phytophagous insects, provide a vast diversity of resources that differ chemically, morphologically, and phenologically. These observations led Ehrlich and Raven (1964) to suggest that the diversification of both plants and herbivorous insects was in large

129 measure due to the coevolution between plant defenses and the counter-adaptations of insect species as they adapted to these diversifying defenses.

If the high diversity of phytophagous insects is indeed due in large measure to specialization via the partitioning of a diverse resource along phylogenetically constrained trajectories, as these hypotheses suggest, then we must attempt to explain the patterns that underlie that diversity. These hypotheses assume that selection favors specialization, and it is often assumed that resource generalists give rise to specialized descendant species more often than the reverse occurs (Schluter, 2000; Thompson, 1994).

Specialization may be favored because of competition or predator-prey interactions

(Bernays and Graham, 1988; Denno et al., 1995), because of trade-offs in adaptations to use alternative host-plants (Dethier, 1954; Fry, 1993; Futuyma and Moreno, 1988;

Jaenike, 1990; Via, 1990), or simply due to a selective advantage to using a single host without the necessity for trade-offs (Bernays and Funk, 1999; Kawecki, 1994; Kawecki,

1998; Whitlock, 1996). Furthermore, selection for host specialization might include a correlated selection for specialized host preferences, resulting in the origin (sympatrically or allopatrically) of host-specialized species in those insects that mate on the host-plant

(Diehl and Bush, 1989; Via, 1986; Via, 2001). In this case speciation and the evolution of specialization may be causally coupled in phytophagous insects.

While selection may favor specialization in the short term, the enormous temporal and spatial variation in the availability of host-plants over evolutionary time suggests that this selection for increased performance may not enable the species to persist as a specialist in the long run. Indeed, specialization may entail the consequent loss of the ability to use alternate hosts, via (1) direct selection in the form of performance trade-

130 offs, (2) through the loss of genetic variation to use alternate resources through persistent directional selection, or (3) by the capacity to use alternate hosts becoming a nearly neutral character that is lost by mutation and genetic drift (Futuyma et al., 1995; Futuyma et al., 1993; Futuyma and Moreno, 1988; Hougen-Eitzman and Rausher, 1994; Via,

1991). As a result, it has been suggested that specialization should result in a greater likelihood of extinction (Mayr, 1963; Simpson, 1953). Therefore, the predominance of specialists seems paradoxical given both the likelihood of extinction of specialists over ecological time and the argument that generalists should have an advantage because of their ability to deal with the often patchy and unpredictable nature of resources (Bernays and Minkenberg, 1997; Futuyma, 1983a; Jaenike, 1978; Kawecki, 1998; McPeek, 1996;

Price, 1983; Spitze and Sadler, 1996).

One reconciliation of this apparent contradiction is that specialist lineages are frequently derived from generalist lineages but persist only briefly, an hypothesis known as Cope’s rule (Cope, 1896; Thompson, 1994). The resultant phylogenetic prediction is that specialists should appear at the tips of trees as sister species to generalists, but should not persist through evolutionary time. Such a lack of persistence would result in the reconstruction of ancestral character states as being generalists. This would explain both the predominance of specialists in terms of extant diversity and their inability to persist through long periods of time. While this idea of specialization as a “dead end” has held a dominant place in macroevolutionary thought for over 100 years (Berenbaum, 1996;

Thompson, 1994), only in the last few years have phylogenies of phytophagous insects and other organisms provided evidence on the direction of niche width. Thompson

(1994) challenged the hypothesis and cited studies (e.g. insect/plant, cowbird/host) that

131 argued that resource specialization is phylogenetically labile—degree of specialization can either increase or decrease across a phylogeny. Studies since that publication have largely corroborated this view and led Schluter (2000) to state that phylogenetic results

“provide little support for the generalists-to-specialists hypothesis (p. 48)”. Instead, it may appear that specialization is not necessarily a dead end. Alternative hypotheses suggest that this may be because genetic variation in defense-related characters within plant species maintain sufficient genetic variation in host-use characters to allow host shifts (Hawthorne, 1997; Rausher, 1984; Thompson, 1994); or that closely related host- plants may actually be ‘available’ for colonization due to similarities in characters relevant to host use and given changes in selection regime caused by shifts in community structure (Becerra and Venable, 1999; Futuyma et al., 1995; Johnson and Siemens,

1991a; Mitter et al., 1991; Wahlberg, 2001). However, so few phylogenies are available that the macroevolutionary trajectory of diet breadth remains a largely open question.

Furthermore, a handful of somewhat disparate analyses do not enable us to examine those conditions that might alter the long-term outcomes of specialization. In order to understand the evolution of resource specialization in phytophagous insects, it will be necessary to characterize the rate and direction of evolutionary change in specialization within different guilds of herbivores (e.g. seed-feeders, foliage feeders, stem borers, etc.) in which the selection pressures exerted by hosts, predators, and competitors may be different (Denno et al., 1995; Hawkins, 1994). Because of new conceptual and technical advances in systematics, we are now poised to investigate whether the variation in the rate and direction of shifts in resource specialization reflect lineage-specific, guild-specific, host-specific, or habitat-specific syndromes. Certainly,

132 addressing the macroevolution of resource specialization in phytophagous insects is in its

nascent stage, but four recent phylogenetic analyses do suggest that specialization is

commonly derived in phytophagous insects: in sap-sucking aphids (Moran, 1988), in wood-mining bark beetles (Kelley and Farrell, 1998), and to a mixed extent in root- feeding beetles (Dobler and Farrell, 1999) and leaf-feeding beetles (Funk et al., 1995).

However, Dobler et al. (1996) found very little evidence that specialists tend to be derived in the external leaf-feeding chrysomelid beetle genus Oreina Chevrolat; Brown

(1994) found very little evidence of an association between resource specialization and

phylogenetic position in pollinating seed parasitic moths in the prodoxid genus Greya

Busck, although phylogeographic analysis suggests that this may be an artifact of the exemplar approach to species-level phylogenetics (Brown et al., 1997); Janz et al. (2001) found no support for this hypothesis in their study of the butterfly tribe Nymphalini, although this was not a species-level phylogeny and may not have the appropriate level of resolution to directly address this hypothesis; and Termonia et al. (2001) found strong support for the biochemical mechanism that allowed for an escape from specialization in

Chrysomelina leaf beetles.

The current study of host use evolution in the seed beetle genus Stator is the first to explicitly examine the macroevolution of diet breadth in the guild of internal seed- feeders. The genus shows considerable interspecific variation in diet breadth. S. pruininus (Horn), S. limbatus (Horn), and S. sordidus (Horn) have some of the largest host ranges known for bruchine seed beetles (Janzen, 1980a; Johnson, 1981c), further species are oligophagous on a small number of mimosoid genera, and the remaining species are monophagous on single species of Acacia Miller or species in the mimosoid

133 tribe Ingeae (Johnson, 1963; Johnson, 1967; Johnson, 1981b; Johnson, 1981c; Johnson,

1982; Johnson, 1984; Johnson, 1995; Johnson, 1998; Johnson and Kingsolver, 1976;

Johnson et al., 1989; Johnson and Lewis, 1993; Johnson and Siemens, 1995). This

interspecific variation suggests that the genus Stator is a promising candidate for the

study of the phylogenetic trajectory of specialization.

Trajectory of Evolution of Host Affiliation. To explain the adaptive radiation of

phytophagous insects, we must not only understand the macroevolution of ecological

specialization, but we must also understand how it evolves against a background of

phylogenetic constraint in host use. The phylogenetic examination of host use evolution

has had a significant impact on our understanding of the coeval diversification of insects and plants, resulting in the remarkably consistent observation that host use evolution is phylogenetically constrained (Farrell, 1998; Farrell and Mitter, 1993a; Farrell and Mitter,

1994; Farrell et al., 1992; Janz et al., 2001; Janz and Nylin, 1998; Kelley and Farrell,

1998; Mitter et al., 1991). This is one of the main predictions of Ehrlich and Raven’s

(1964) seminal hypothesis on insect-plant coevolution. Because it has also become clear

from these studies that parallel cospeciation is not likely generally to explain the co-

diversification of insects and plants (but see Farrell and Mitter, 1990; Farrell and Mitter,

1998), detailed phylogenetic analysis of the interconnected roles of host shifts and

specialization are more likely to provide insight into the patterns and processes

underlying the adaptive radiation of phytophagous insects. The majority of phylogenetic

studies have focused on the evolution of host affiliation or the evolution of diet breadth

134 (even separately within the same paper), not on the combination of host affiliation and

diet breadth, but the two may strongly interact.

Examining the interaction between specialization and phylogenetic constraint may

yield insight into the diversification of phytophagous insects that separate study may not

provide. For example: (1) Does diet expansion in a generalist follow phylogenetically

constrained pathways in a similar manner to that displayed in host shifting by specialists?

Or does specialization itself, while perhaps not leading to a dead end, significantly

decrease the avenues for future ecological colonizations? (2) Can the evolution of generalism open pathways for future colonizations, thereby functionally serving as a limited release from phylogenetic constraint? (3) Can certain types of host shifts, such as those that involve the colonization of more phylogenetically distant hosts, result in

different evolutionary trajectories? One can envision that the sensory, biochemical,

phenological, and other adaptations required for more distant host shifts may be more

profound than for closer host shifts, thereby preventing the recolonization of ancestral host-plants (Futuyma et al., 1995). Likewise, more distant host shifts could serve as key innovations and open the pathway for new opportunities for diversification.

All Stator species show affiliations with legumes in the Fabaceae subfamily

Mimosoideae, suggesting that there is some level of constraint in host associations in the genus. However, numerous species also affiliate to a lesser degree with

Caesalpinioideae, and two species feed on plants in the subfamily Papilionoideae. In addition, while many species of Stator are associated, either exclusively or not, with plants in the genus Acacia, others associate exclusively with legumes in the mimosoid tribe Ingeae. Such interspecific variation in host use begs the question as to whether or

135 not host use is free to evolve within a particular subset of mimosoid legumes—the

Acacieae/Ingeae alliance. This study explicitly examines this question, as well as whether or not this phylogenetic constraint is dependent upon resource specialization and whether or not the trajectory of resource specialization is dependent upon phylogenetic constraint.

Oviposition Guild Differentiation and the Evolution of Host Use. The study of the diversification of insects onto plants has largely focused on the species diversity of the plant resources involved. However, it has not escaped the attention of many researchers that diversification along other niche axes has been important as well. For example, it is quite clear that the evolution of tissue specialization in tracheophytes opened opportunities for niche partitioning, and insects have specialized to use particular tissues at a much greater extent and under much stronger phylogenetic constraint than they have onto particular plant species (Farrell, 1998; Farrell and Mitter, 1994; Farrell and Sequeira, in prep.; Janz and Nylin, 1998; Marvaldi et al., 2002; Strong et al., 1984).

Such specialization has allowed for an accumulation of diversity at a greater rate than would be allowed for by only the number of species of tracheophytes.

Plant phenology also provides another axis along which phytophagous insects might partition host-plants. For example, one of the most convincing examples of ecologically-based or sympatric speciation is that of the Rhagoletis pomonella complex, and it appears that a great deal of the original differentiation in this case is due to phenological differences between host-plants (Feder, 1998; Feder and Filchak, 1999;

Feder et al., 1997a; Feder et al., 1997b; Feder et al., 1999; Filchak et al., 1999). Seed

136 beetles also appear to phenologically partition the hosts that they use. Johnson (1981a)

described three phenologically separated oviposition guilds in seed beetles. Species in

guild A, the mature pod guild, oviposit directly onto the integument of the pod usually

while it is still on the host-plant. Species in guild B, the mature seed guild, oviposit

directly onto the seed while they are still in the pod. These species therefore must find

either partially dehisced fruits or enter into exit holes left by species from guild A.

Species in guild C, the scattered seed guild, oviposit onto seeds after they have been

exposed on the substrate. These three guilds are clearly separated along the temporal axis

of fruit development from an indehisced fruit, to a partially dehisced fruit, to dispersed

seeds.

This phenological partitioning could also result in different selection regimes for

using particular hosts by species in the different guilds. For example, differential

allocation of quantity or class of defensive chemicals between the integument of the pod

and the testa of the seed could present very different biochemical hurdles for members of

guild A versus members of guilds B and C (Janzen, 1981a; Johnson and Siemens, 1991a;

Siemens et al., 1992). If nothing else, guild A must penetrate two separate protective

tissues (pod integument and seed testa) as a first instar , while those in guilds B and

C need only to penetrate through the seed testa. The three guilds may also experience

significantly different parasitoid regimes. Larvae feeding within pods still on trees are

almost certainly easier targets for parasitoids than those feeding within scattered seeds

(Gratton and Welter, 1999), particularly since many parasitoids use plant volatile chemicals to locate their insect hosts (Kessler and Baldwin, 2001). Finally, searching for scattered seeds may require more specialized foraging behavior by gravid females in

137 guild C, particularly if there are neural limits to the amount of spatial information that

these insects can process (Bernays, 1998; Bernays and Funk, 1999; Bernays and Wcislo,

1994).

The macroevolutionary trajectory of such resource partitioning is wholly

unexplored in seed beetles, and the implications for competitive coexistence have been

largely ignored through the bruchine literature (but see Johnson, 1981a; Traveset, 1990;

Traveset, 1991). This behavioral differentiation could also have implications for the

evolution of host use and/or resource specialization, particularly if the three guilds

experience the different selection regimes mentioned above. While most genera of

bruchines can be characterized as being monomorphic for this oviposition behavior

(Johnson, 1981a), Stator is notable in that it includes multiple species in each of these

guilds. This makes it a good candidate for exploring the macroevolutionary

consequences of this phenological differentiation.

In this chapter, I use a phylogenetic analysis of the seed beetle genus Stator to test hypotheses regarding the macroevolutionary trajectory of host-plant specialization and affiliation. In addition to the more general descriptive investigation of the ecological and biogeographic history of the seed beetles, I examine the history of host shifts and host

specialization by asking the following questions: (1) Is host use significantly

phylogenetically constrained, and does this differ between specialists and generalists? (2)

Is diet breadth significantly phylogenetically constrained? (3) Is there a bias toward

reconstructing ancestral states as being generalists, as would be predicted if Cope’s rule

holds? (4) Is there a correlation between the phylogenetic relatedness of newly colonized

138 host-plants and phylogenetic position? (5) Is oviposition guild phylogenetically

conserved? (6) Does oviposition guild affect macroevolutionary trends in constraint or host use?

STUDY SYSTEM: THE GENUS STATOR

Information on the taxonomy, distribution, and host-plants of the genus Stator are

based on personal collection information, museum specimen information, and published

records (Bottimer, 1973; Johnson, 1963; Johnson, 1967; Johnson, 1981b; Johnson, 1984;

Johnson, 1995; Johnson, 1998; Johnson and Kingsolver, 1976; Johnson et al., 1989;

Johnson et al., 1991; Johnson and Lewis, 1993; Johnson and Siemens, 1992; Johnson and

Siemens, 1995; Kingsolver, 1972), all of which I have recorded and maintained in a

relational database.

Taxonomy and distribution. The genus Stator is a member of a clade of

Bruchinae (Coleoptera: Chrysomelidae) that appears to have diversified largely onto the

shrub and treelet legumes that diversified in the tropics and subtropics during the

aridification of the Oligocene and Miocene (Axelrod, 1972). The genus itself is found

exclusively in the New World and is distributed from Argentina and Chile in South

America to the southwestern in North America, and throughout the

Caribbean. Previous morphological research suggested that the genus Sennius Bridwell

(Johnson and Kingsolver, 1976; Johnson et al., 1989) is most closely related to Stator.

There is little support for such a sister-group relationship in my phylogenetic analysis of

the bruchines (Chapter 1), and it is possible that the reasons to unite the two are largely

139 due to the overall gestalt of the two genera. Nevertheless, genera in this clade of the bruchine tribe Acanthoscelidini seem to have appeared very quickly, as judged by the very short internodes in the phylogenetic analyses, and it is difficult to determine which genus or genera are most closely related to Stator. Candidate genera include

Gibbobruchus Pic, Callosobruchus Pic, Merobruchus Bridwell, Mimosestes Bridwell,

Sennius, Bruchus L., and the mexicanus species group of Acanthoscelides Schilsky.

The genus Stator nominally includes 30 species, although the analysis in the previous chapter shows that the Bixa (Malvales: Bixaceae) feeders S. championi (Sharp) and S. bixae (Drapiez) are not monophyletic with the rest. In addition, given the species criteria for the genus provided by Johnson (1995), S. exturbatus Johnson, Kingsolver and

Terán is probably a synonym of S. limbatus. In addition, more specimens of S. harmonicus Johnson, Kingsolver and Terán are needed in order to determine whether or not it should be placed as separate from S. pacarae Johnson, Kingsolver and Terán; and it is quite possible that S. coconino Johnson is based on a male of either S. sordidus, S. pygidialis (Schaeffer), or S. chihuahua (Johnson and Kingsolver) that had underdeveloped genitalia (C.D. Johnson, pers. comm.). Whether or not S. dissimilis

Johnson and Kingsolver should really be included within the genus Stator is a matter of debate as well, given that it and the Bixa feeders are the only species in the genus that lack the complete lateral prothoracic carina that is characteristic of the genus. Of the 24 remaining species of Stator, S. biplagiatus (Gyllenhal) and S. postumus Johnson,

Kingsolver, and Terán are known only from their type specimens.

The distributions of species of Stator are largely dependent upon the individual plant species and the number of those species they feed on (Tables 2.1 and 2.2). Almost

140 all species occur in dry deciduous or thornscrub forests. The lone exception is S.

aegrotus (Sharp), a specialist on one of the few wet tropical species of Acacia, the woody

liana Acacia hayesii Benth. Interestingly this species has the widest range of all of the specialists, probably because its host is distributed throughout the wet tropical forests from Guatemala to the Amazon Basin. S. trisignatus (Sharp) is also found in wet tropical forests of Panama where it feeds on the same species, but elsewhere it is found in thornscrub or dry deciduous forest.

Six species show exceptionally broad geographic distributions. S. pruininus, S.

limbatus, S. sordidus, and S. vachelliae Bottimer are all found almost continuously at

lower elevations from the southwestern United States to northern South America. The

distributions of the first three are likely due to the fact that they show the widest host

range in the entire genus. The distribution of S. vachelliae is almost certainly due to the

widespread distribution of its main host, Acacia farnesiana (L.) Willd. S. vittatithorax

(Pic) and S. monachus (Sharp) have almost identical disjunct distributions to the north

and south of the Amazon basin, occurring on the west coast of central Mexico south to

northern Venezuela and in southern Brazil, Argentina, and Uruguay. These two species

also have relatively broad host ranges, a fact contributing to their widespread distribution.

The remaining species show much more restricted geographic ranges. The

species-specific specialists are completely restricted to the range of their host-plant, and

sometimes are restricted within that range itself (Johnson, 1982; Johnson and Janzen,

1982). The species that feed on multiple species of Acacia appear restricted to particular

biogeographic regions that are defined largely by contiguous distributions of thornscrub

or dry deciduous forests.

141

Host-plant affiliations. Known host-plant relationships, as well as some

taxonomic notes, are included in Table 2.1 and are based on numerous publications

(Bottimer, 1973; Johnson, 1963; Johnson, 1981b; Johnson, 1984; Johnson, 1995;

Johnson, 1998; Johnson and Kingsolver, 1976; Johnson et al., 1989; Johnson and Lewis,

1993; Johnson and Siemens, 1992; Johnson and Siemens, 1995; Kingsolver, 1972) and

personal collection records. Like many Bruchinae, species in the genus Stator are

restricted to feeding on legumes (Fabaceae). The 22 species with known host

associations encompass a wide range of resource specialization, ranging from specialists

that feed on a single host species to relative generalists that feed on hosts from multiple

legume subfamilies. All of the species in the genus include plants in the legume

subfamily Mimosoideae in their diet, and the majority are restricted to this subfamily.

Three species, S. limbatus, S. pruininus, and S. sordidus consistently include

Papilionoideae and/or Caesalpinioideae in their diets, as well. These are also the most

generalist of the species, being known to use at least 59, 52, and 15 species of native

hosts from 18, 15, and 11 genera, respectively.

Five other species include multiple genera in their diet. S. vittatithorax and S.

chihuahua are both restricted to the mimosoid tribes Acacieae and Ingeae, which together

form a monophyletic clade (Chappill and Maslin, 1995; Miller and Bayer, 2001;

Robinson and Harris, 2000). Interestingly, S. vittatithorax uses only species in the genus

Acacia in the southern part of their disjunct distribution. This may well be due to the

distribution of potential Ingeae hosts with thin integuments (it is in guild A), which are mostly limited to the Amazon basin and northward to the southwestern United States

142 Table 2.1: Host plant relationships of 22 species of Stator with established host plant associations. Two species are known only from their type specimen(s). Four are probably not legitimate species, and taxonomic notes of these are included. Oviposition guild: (A) mature pod guild; (B) mature seed guild; (C) scattered seed guild. The subgenera of Acacia are treated as separate genera in column 2 because they may not form a monophyletic group. Host associations and guilds are from (Bottimer, 1973; Johnson, 1963; Johnson, 1981b; Johnson, 1984; Johnson, 1995; Johnson, 1998; Johnson and Kingsolver, 1976; Johnson et al., 1989; Johnson and Lewis, 1993; Johnson and Siemens, 1992; Johnson and Siemens, 1995; Kingsolver, 1972) and from personal collection records.

Species # hosts Species recorded/Taxonomic notes Oviposition spp./genera Guild S. aegrotus (Sharp) 1/1 Mimosoideae: Acacia (Aculeiferum) hayesii A Benth. S. beali Johnson 1/1 Mimosoideae: Ebenopsis ebano (Berland.) B Barneby & J.W. Grimes S. biplagiatus Unknown Distinctive species known only from (Gyllenhal 1839) lectotype female and cotype male specimens. S. bottimeri 2/1 Mimosoideae: Acacia (Acacia) farnesiana C Kingsolver (L.) Willd., A. (Acacia) pinetorum F.J. Herm. S. chalcodermus 1/1 Mimosoideae: unguis-cati C Kingsolver (L.) Benth. S. chihuahua 4/4 Mimosoideae: Acacia (Aculeiferum) C Johnson and angustissima (Mill.) Kuntze, A (Acacia) Kingsolver1 constricta Benth. ex A. Gray, Calliandra eriophylla Benth., Lysiloma microphyllum Benth. S. coconino Johnson Unknown Possibly underdeveloped males of S. and Kingsolver chihuahua, S. pygidialis, or S. sordidus, known only from 3 specimens. S. dissimilis Unknown Probably not monophyletic with Stator. Johnson and Kingsolver S. exturbatus Unknown Probable junior synonym of S. limbatus. Johnson, Kingsolver, and Terán S. furcatus Johnson, 6/2 Mimosoideae: Acacia (Acacia) aroma B Kingsolver, and Gillies ex Hook. & Arn., A. (Acacia) caven Terán (Molina) Molina, A. (Aculeiferum) bonariensis Gillies ex Hook. & Arn., A. (Aculeiferum) furcatispina Burkart, A. (Aculeiferum) praecox Griseb., A. (Aculeiferum) visco Lorentz ex Griseb. S. generalis 1/1 Mimosoideae: Enterolobium cyclocarpum C Johnson and (Jacq.) Griseb. Kingsolver

143 Table 2.1 (Continued) S. harmonicus Unknown Specific separation from S. pacarae Johnson, debatable, known only from holotype Kingsolver, and specimen. Terán S. limbatus (Horn) 59/18 + 9 Caesalpinioideae: moschata Kunth., B introduced Cercidium (5 spp.), (2 spp.) Mimosoideae: Acacia (Aculeiferum) (18 spp.), (4 spp.), Calliandra (3 spp.), Chloroleucon mangense (Jacq.) Br. & R., Ebenopsis (2 spp.), Havardia (2 spp.), Leucaena (3 spp.), Lysiloma (5 spp.), Piptadenia (2 spp.), Pithecellobium (6 spp.), Pseudosamanea guachapele (Kunth) Harms, Samanea saman (Jacq.) Merr., Sphinga platyloba (DC) Barneby & Grimes, Zapoteca portoricensis (Jacq.) H.M. Hern.. Also 8 spp. introduced Australian Acacia (Phyllodineae) and 1 spp. Introduced Asian Albizia. Papilionoideae: Sesbania sp. S. maculatopygus 1/1 Mimosoideae: Acacia (Aculeiferum) A (Pic) bonariensis S. mexicanus 1/1 Mimosoideae: Acacia (Acacia) cornigera C Bottimer (L.) Willd. S. monachus 8/1 Mimosoideae: Acacia (Aculeiferum) (8 spp.) A (Sharp) S. pacarae Johnson, 1/1 Mimosoideae: Enterolobium C Kingsolver, and contortisiliquum (Vell.) Morong Terán S. postumus Unknown Distinctive species known only from type Johnson, specimen. Kingsolver, and Terán S. pruininus (Horn) 52/15 + 6 Mimosoideae: Acacia (Acacia) (6 spp.), B introduced Acacia (Aculeiferum) (4 spp.), Calliandra (2 spp.), Desmanthus (8 spp.), Havardia pallens (Benth.) Br. & R., Lysiloma sp., Mimosa (20 spp.), Neptunia plena (L.) Benth., Zapoteca portoricensis. Also 9 spp. introduced Australian Acacia (Phyllodineae). Papilionoideae: Coursetia glandulosa A. Gray, Marina scopa Barneby, Olneya tesota A. Gray, Robinia pseudoacacia L., Sesbania (4 spp.) S. pygidialis 1/1 Mimosoideae: Calliandra humilis Benth. C (Schaeffer) S. rugulosus 1/1 Mimosoideae: Abarema glauca (Urb.) Kingsolver Barneby & Grimes

144 Table 2.1 (Continued) S. sordidus (Horn) 15/11 Caesalpinioideae: Caesalpinia (2 spp.), C Parkinsonia aculeata L. Mimosoideae: Acacia (Acacia) (2 spp.), Acacia (Aculeiferum) (3 spp.), Albizia sp., Lysiloma divaricatum (Jacq.) Macb., Mimosa laxiflora Benth., Piptadenia (2 spp.), Pithecellobium dulce (Roxb.) Benth., Samanea saman S. subaeneus 1/1 Mimosoideae: Acacia(Acacia) collinsii C (Schaeffer) Safford, A. (Acacia) cornigera, A. (Acacia) farnesiana S. testudinarius 3/2 Mimosoideae: A. (Acacia) farnesiana, A. C (Erichson) (Acacia) macracantha, Samanea saman S. tigrensis (Pic) 2/2 Mimosoideae: Acacia (Acacia) caven, A. B (Aculeiferum) visco S. trisignatus 5/1 Mimosoideae: Acacia (Aculeiferum) A (Sharp) articulata Ducke, A. (Aculeiferum) hayesii, A.(Aculeiferum) polyphylla DC., A. (Aculeiferum) tamarandifolia Griseb., A. (Aculeiferum) tenuifolia (L.) Willd. S. vachelliae 7/2 Mimosoideae: Acacia (Acacia) (6 spp.), C Bottimer2 Samanea saman S. vittatithorax (Pic) 3/14 Mimosoideae: Acacia (Aculeiferum) (12 A spp.), Lysiloma acapulcense (Kunth) Benth., Zapoteca formosa. 1Stator chihuahua has also been reared on a single occasion from Mimosa biuncifera (Johnson and Kingsolver, 1976). However, considerable rearing attempts from field collected seeds by both myself and C.D. Johnson (pers. comm.) have not resulted in any further collections and this plant is unlikely to normally support development of S. chihuahua, much less support populations. 2Stator vachelliae has also been reared from Parkinsonia aculeata. However, this should not be counted as a normal host for this species. The record is based on a single location in which the seeds of this host plant were mixed in with seeds of a normal Acacia host of S. vachelliae (Johnson and Siemens, 1991a). Females apparently did not discriminate between the dispersed seeds in their oviposition behavior. However, of 1,118 eggs laid on Parkinsonia aculeata, only one produced an adult, suggesting that this plant species is not within the ecological repertoire of S. vachelliae.

145 Table 2.2: Geographic distribution of 24 well-established species of Stator. Geographic ranges are based on more 3200 specimen locality records from published sources (Bottimer, 1973; Johnson, 1963; Johnson, 1981b; Johnson, 1984; Johnson, 1995; Johnson, 1998; Johnson and Kingsolver, 1976; Johnson et al., 1989; Johnson and Lewis, 1993; Johnson and Siemens, 1992; Johnson and Siemens, 1995; Kingsolver, 1972), personal collection records, and museum specimens. These records are maintained in a relational database by the author.

Species Distribution S. aegrotus (Sharp) Bolivia, Brazil, Guatemala, Panama, Peru, Suriname, Venezuela: In wet tropical rainforest. S. beali Johnson USA () to east-central Mexico: In Gulf of Mexico coastal plain. S. biplagiatus (Gyllenhal 1839) Brazil. S. bottimeri Kingsolver Northern Caribbean: Bahamas and Florida Keys to Cuba. S. chalcodermus Kingsolver Southern Caribbean: Cuba, Hispañola, Jamaica, Puerto Rico. S. chihuahua Johnson and Mexico, USA ( and Texas): Chihuahuan and Kingsolver Sonoran deserts. S. furcatus Johnson, Kingsolver, Argentina, Bolivia, Brazil, Uruguay: Thornscrub forests. and Terán S. generalis Johnson and Colombia, Panama, Venezuela: Dry deciduous forests. Kingsolver S. limbatus (Horn) Northern South America to southwestern United States and Caribbean. S. maculatopygus (Pic) Argentina, southern Brazil, Uruguay: Thornscrub forests. S. mexicanus Bottimer Mexico, Guatemala: Thornscrub forests. S. monachus (Sharp) Argentina to Mexico (disjunct distribution excludes Amazon basin): Thornscrub and dry deciduous forests. S. pacarae Johnson, Kingsolver, Argentina: Dry deciduous and thornscrub forests. and Terán S. postumus Johnson, Kingsolver, Northern Venezuela. and Terán S. pruininus (Horn) Northern Venezuela to southwestern United States: thornscrub forests. S. pygidialis (Schaeffer) USA (Arizona): Mountains ringing . S. rugulosus Kingsolver Cuba. S. sordidus (Horn) Northern Venezuela to southwestern United States. S. subaeneus (Schaeffer) USA (Texas) to Yucatán peninsula of Mexico: Gulf of Mexico coast. S. testudinarius (Erichson) Ecuador and Peru: thornscrub forests. S. tigrensis (Pic) Argentina and Chile: thornscrub forests. S. trisignatus (Sharp) Panama to northern Brazil: thornscrub, dry tropical, and wet forests (Panama only). S. vachelliae Bottimer USA (Texas) to northern Venezuela: thornscrub forests. S. vittatithorax (Pic) Argentina to Mexico (disjunct distribution excludes Amazon basin): Thornscrub and dry deciduous forests.

146 (Barneby, 1998; Barneby and Grimes, 1996; Barneby and Grimes, 1997; Thompson,

1980). One species, S. vachelliae, normally includes only species of Acacia in its diet.

However, at the southernmost extent of its large geographic distribution it also uses the

Rain Tree, Samanea saman (Jacq.) Merr., as a host. S. testudinarius (Erichson), the only

species endemic to the west of the Andes, has also been reared from S. saman although A.

macracantha Humb. & Bonpl. ex Willd. appears to be its main host. All seven of these

species include a large number of Acacia species in their diet, and nine of the remaining

species are either restricted to a single Acacia species or multiple Acacia species.

However, six species (S. beali Johnson, S. chalcodermus Kingsolver, S. generalis

Johnson and Kingsolver, S. pacarae, S. pygidialis, and S. rugulosus Kingsolver) are specialists on single species in the tribe Ingeae.

One potential consideration when undertaking a study of host affiliation, especially one including diet breadth, is the depth of knowledge of host plant associations. While this may well be a problem in many groups of Bruchinae, as only around 35% have known host records (see Chapter 1, this thesis), this is much less the case in the genus

Stator. While this genus of seed beetle does not have the advantage of including a large number of economically important species, its host relationships are remarkably well known. This is in large measure due to the considerable efforts and interests in this genus of C. Dan Johnson, the world’s foremost expert in bruchine biosystematics; the efforts of

Daniel H. Janzen, a leading researcher into the ecology of insect-plant interactions; and to

the collecting and rearing efforts I expended during my dissertation research. As a result,

there is fairly strong confidence in the relative delimitation of specialists and generalists

in this group. The host lists of the generalists continue to expand with additional field

147 research effort, but this underrepresentation creates a conservative bias in distinguishing

specialists from generalists. This is almost certainly true for S. sordidus, as multiple

hosts for this species were unknown until it was realized that the genus Stator includes

large numbers of species in guild C (Johnson, 1981c; Johnson and Lewis, 1993; Johnson

and Siemens, 1995).

The host lists of species that feed on multiple genera within the Mimosoideae are

also likely to expand, but there is no indication that they will expand beyond the

Acacieae/Ingeae clade. The difficult delimitation, rapid diversification, and broad

sympatry of Acacia species (Clarke et al., 1989; Clarke et al., 1990; Janzen, 1974; Lee et al., 1989; Miller and Bayer, 2001; Robinson and Harris, 2000; Seigler and Ebinger, 1995) suggests that host lists of species that feed on multiple Acacia species are likely to grow as well, but this is unlikely to affect conclusions based on the current analysis. The same is potentially true of those species that feed on single species of Acacia, with the exception of S. aegrotus on A. hayesii. The geographic overlap of these two is so exact,

and the biology of A. hayesii so distinct from all other species of Acacia, that this is likely

to be confirmed as a one-to-one association. Extreme specialization is almost certainly

the case for four of the six species that feed on single species of Ingeae for similar

reasons: S. beali on Ebenopsis ebano (Berland.) Barneby & J.W. Grimes, S. generalis on

Enterolobium cyclocarpum (Jacq.) Griseb., S. pacarae on E. contortisiliquum (Vell.)

Morong, and S. pygidialis on Calliandra humilis Benth. I established Pithecellobium unguis-cati (L.) Benth. as the only known host for S. chalcodermus, but it is possible that other species of the Pithecellobium complex present in and south of Cuba are also hosts.

I found no evidence that Acacia served as hosts for this species, despite collecting a large

148 number of pre and post-dispersal seeds on Puerto Rico. The Cuban endemic species S.

rugulosus is reported in the literature (Kingsolver, 1972) to have been reared from

Pithecellobium discolor Britton, a junior synonym of Abarema glauca (Urban) Barneby

& Grimes. The host affiliation and range of this species should be confirmed, although it

is not included in this analysis.

It is possible that there are undescribed species of specialized Stator in existence.

Bruchines are quite easily collected using specific techniques, most notably rearing from

host plant material. However, they are rarely collected by generalist beetle collecting

techniques such as sweep-netting, light-trapping, bait-trapping, or flight-intercept

trapping. Therefore, undersampling may be particularly acute of species that would

occur in the Brazilian caatinga or cerrado, two areas that are diverse in both species of

Acacia and Ingeae, and two areas that have not been subject to specialized bruchine collecting. While it is highly unlikely that any generalist species have escaped sampling, the current analysis must be interpreted under its present level of sampling and our current understanding of Stator species diversity.

Finally, another matter of consideration in attempting to study the macroevolution of resource specialization is the notion that generalist species may actually be the result of a geographic mosaic of specialized populations (Fox and Morrow, 1981; Thompson,

1994; Thompson, 1999). It is unclear how this pertains directly to our understanding of the long-term evolutionary consequences of species-level specialization. While it is clearly important from a metapopulation standpoint of ecological evolution, and for inferring the microevolutionary processes that may lead to specialization, it is only relevant in a macroevolutionary context if such intraspecific variation becomes

149 partitioned via speciation, and therefore isolated from the effects of gene flow, into

interspecific variation. Regardless, this appears to be a somewhat moot argument in the

current context in that research has shown that Stator generalists appear to be generalists

at the intra-individual level, much less the interpopulation level (Fox et al., 1999; Fox et

al., 1996a; Fox et al., 1997; Fox and Savalli, 2000; Fox et al., 1994; Fox et al., 1995;

Johnson, 1981c; Siemens and Johnson, 1990; Siemens et al., 1991), and are regularly able

to use hosts in laboratory settings that are well outside of their natural host range

(Bridwell, 1918; Bridwell, 1919; Johnson, 1981c; Johnson and Kingsolver, 1976).

Related to this is the similar difficulty in differentiating between fundamental and

realized niches when identifying specialists. Janz et al. (2001) show that butterflies capable of a polyphagous diet (their fundamental niche) are often times limited to a monophagous diet (their realized niche), possibly by ecological factors (Fox and Morrow,

1971) or uncorrelated female oviposition preferences (Thompson, 1988a). For the most part, I am interested in the trajectory of specialization at the evolutionary level and am interested in those specialist species whose fundamental and realized niches are largely overlapping. This appears to be the case in most bruchines, as they are largely limited to a single or to a small handful of closely related species (Huignard et al., 1989; Johnson,

1981e; Johnson, 1990a). There is little direct evidence for this in the genus Stator, although there is substantial indication that the specialists are largely restricted in their performance abilities to their host plants (Fox et al., 1996b; Fox and Mousseau, 1995b;

Johnson, 1981b; Johnson, 1981c; Johnson and Siemens, 1991a).

150 Diet breadth—defining specialists and generalists. One of the most difficult aspects of studying the macroevolutionary trajectory of resource specialization is actually defining and quantifying what we mean by diet breadth. Futuyma and Moreno (1988, p.

208) state that “specialization must lie in the eye of the beholder”, and it is most certainly a term that depends on being relative to something else. In some groups relative specialists are those that feed on single families of plants, a common criterion amongst those that study butterflies (Janz et al., 2001; Wahlberg, 2001). At other extremes, beetles in the genus Tetraopes Schoenherr (Coleoptera: Cerambycidae) are largely restricted to feeding on a single species of Asclepias L. milkweed (), and relative generalists are those like T. annulatus LeConte that feeds on three species of

Asclepias (Chemsak, 1963; Farrell, 2001; Farrell and Mitter, 1998). As a result, relative levels of diet breadth must be defined within the context of the phylogenetic and ecological history of the group under study.

The quantification of diet breadth should take into account the ‘availability’ of potential hosts, both in terms of the phylogenetic constraint in host use present in the clade of interest and the availability of those hosts to particular species given their biogeographic range (Colwell and Futuyma, 1971; Futuyma and Gould, 1979; Kelley and

Farrell, 1998). Kelley and Farrell (1998) addressed this issue in the bark-beetle genus

Dendroctonus Erichson by (1) determining that the level of phylogenetic constraint appeared to be at the level of host plant genus (that is, even generalists were largely restricted to multiple species within a single genus) and then (2) determining both the number of conifer species within a beetle species’ range that are congeneric with its host(s) and the proportion of those species that are actually used by that species. As a

151 result, they were able to identify those hosts that were likely to be ‘available’ in both a phylogenetic and ecological context. A somewhat different tactic must be taken for the current study of the genus Stator, however.

First of all, Dendroctonus is limited to four genera of relatively species-poor

Pinaceae, making the task of identifying potential hosts and range overlap fairly straightforward. On the other hand, Stator uses 29 genera of legumes, many of which

(e.g. Acacia and Mimosa L.) are quite species-rich. Therefore, identifying phylogenetically ‘available’ hosts is not as straightforward in this clade. Indeed, generalists use as many as 18 different genera of host plants from three different subfamilies of legumes, and the specialists collectively use six different genera of mimosoid hosts. As such a more traditional measure such as ‘number of species or genera used’ or a phylogenetic diversity index of diet breadth (e.g. Symons and

Beccaloni, 1999), instead of the proportion of available hosts, is a more tenable approach.

While this precludes the possibility of incorporating ecological opportunity into the analysis, this necessity may be an acceptable sacrifice in the genus Stator. With the possible exception of the northern Caribbean endemic species S. bottimeri Kingsolver, there are numerous Acacia and Ingeae hosts available throughout the range of every species of Stator. Indeed, all of the species (again with S. bottimeri being the lone exception) show a considerable degree of overlap with other species of Stator, including sympatric distributions between specialists and relative generalists. This suggests that all species have the opportunity to use multiple hosts within their geographic distribution.

Therefore, while taxonomic or phylogenetic indices of host dispersion may not capture the full extent of the boundaries of specialization, they are likely to be an accurate

152 approximation for the genus Stator. Once relative generalists and specialists have been identified, I use a phylogenetic approach to examine whether or not there is a tendency for specialists to be more often derived from generalist ancestors; that is, found at the tips of phylogenetic trees. I will also examine whether this tendency is dependent upon the background context of host affiliation.

Oviposition guild association. As noted earlier, the genus Stator is somewhat unique amongst the Bruchinae in that it includes species from all three oviposition guilds

(Johnson, 1981a; Johnson and Siemens, 1995). One advantage of the interest taken in the genus Stator over the years is that oviposition guilds are as well-established as host plant associations. This is particularly important for those that oviposit on dispersed seeds.

For the most part, this behavior characterizes genera with notoriously poorly known host distribution affiliations, such as the genus Zabrotes Horn (Romero and Johnson, 2000).

However, oviposition guilds are well-established for all species of Stator with known host associations. S. aegrotus, S. maculatopygus (Pic), S. monachus, S. trisignatus, and

S. vittatithorax can all be characterized as belonging to guild A, ovipositing on the integument of the pod (the first two through personal observation, the last three as published in Johnson and Siemens, 1995, and confirmed by personal observation). S. beali, S. furcatus Johnson, Kingsolver, and Terán, S. limbatus, S. pruininus, and S. tigrensis (Pic) can all be characterized as belonging to guild B, ovipositing on predispersal seeds (the first two through personal observation, the last three as published in Johnson and Siemens, 1995, and confirmed by personal observation). And S. bottimeri, S. chalcodermus, S. chihuahua, S. generalis, S. mexicanus Bottimer, S.

153 pacarae, S. pygidialis, S. sordidus, S. subaeneus (Schaeffer), S. testudinarius, and S. vachelliae can all be characterized as belonging to guild C, ovipositing on postdispersal seeds (the first four through personal observation, the last six as published in Johnson and

Siemens, 1995, and confirmed by personal observation).

The straightforward nature of the characterization of this phenological differentiation in oviposition behavior allows a standard character coding of this behavior. This behavior is remarkably consistent within species, but it is unknown whether such consistency carries across phylogenetic boundaries as well. If it does, it may affect the evolutionary signatures of host use across the genus Stator and may structure the component communities of these seed predators accordingly.

METHODS

SPECIMENS EXAMINED

Fresh material of 21 of 22 species of Stator with known host affiliations was collected during fieldwork in northern Argentina, Ecuador, Venezuela, Panama, Costa

Rica, Mexico, the southwestern United States, and three locations in the Caribbean.

Stator rugulosus, a Cuban endemic, was not collected as fieldwork in Cuba presented too many logistic hurdles. S. postumus and S. biplagiatus remain known only from their type specimens. Host plant seeds were field-collected and transferred to cloth-covered jars in an environmental chamber at the Museum of Comparative Zoology, Harvard University.

Adults were removed as they emerged from seeds and were immediately frozen and stored in a -80°C freezer. I collected multiple populations from throughout the geographic range of most species.

154 Multiple individuals from each species were reared from seeds from each locale and a portion were mounted and labeled for identification. I identified all specimens included in the analysis using the keys in Johnson and Kingsolver (1976) and Johnson et al. (1989). Due to the interspecifically distinctive and intraspecifically uniform genitalia of the different species, I was able to positively confirm all identifications through genitalic dissections and preparations.

When possible, multiple specimens from each species were included from several geographic regions. In total, 50 specimens representing 22 species were included in the analysis (Table 2.3). Sampling was particularly intense for the species S. limbatus for three reasons: (1) preliminary analysis suggested a paraphyletic relationship between it and S. beali; (2) because of its extremely wide geographic distribution; and (3) because of the historical separation of South American populations as a separate species, S. cearanus

(Pic), which is now treated as a synonym of S. limbatus (Johnson, 1995; Johnson and

Kingsolver, 1976; Johnson et al., 1989).

Outgroups were chosen based on the phylogenetic analysis of the first chapter.

Due to difficulties in obtaining Elongation Factor 1-α for Gibbobruchus and

Callosobruchus, I was forced to use somewhat more distantly related outgroups: two species of Sennius (S. morosus (Sharp) and S. breveapicalis (Pic)), Bruchus rufimanus

Boheman, and Merobruchus insolitus (Sharp). The slightly less closely related outgroups are unlikely to unduly affect the phylogenetic analysis, given the rapid origins of all of the genera in this clade of Acanthoscelidini.

155 Table 2.3: Specimens of Stator included in molecular phylogenetic analysis, including exact locality information and host plant species.

Species ID # Collection information Host plant species S. aegrotus 133-00 Panama, Panamá: Parque Nacional Acacia hayesii Benth. (Sharp) Soberana, Sendero de Plantación; 9°5.811’N, 79°39.097’W; 18 March 2000 S. beali Johnson 441-98 USA, Texas: Edinberg, corner of Cisner & Ebenopsis ebano Ebano; 26°17.455’N, 98°09.473’W; 24 (Berland.) Barneby & October 1998 J.W. Grimes S. bottimeri 331-00 USA, Florida: Watson’s Hammock, Big Acacia pinetorum F.J. Kingsolver Pine Key; 24°42.529’N, 81°23.022’W; 19 Herm. May 2000 S. chalcodermus 340-99 Puerto Rico: San Juan, near airport; 27 Pithecellobium dulce Kingsolver December 1999 (Roxb.) Benth. S. chihuahua 452-98 USA: Texas; 1 km NW Ryan, US20; Acacia angustissima Johnson and 30°26.382’N, 104°18.683’W; 26 October (Mill.) Kuntze Kingsolver 1998 S. furcatus 39-98 Argentina, Catamarca: Entre Rios; Acacia visco Lorentz Johnson, 26°50.190’S, 65°2.160’W; 1 March 1998 ex Griseb. Kingsolver, and Terán S. furcatus 50-98 Argentina, Santiago del Estero: Rt. 64 Acacia furcatispina between Lavalle & Santa Catalina; Burkart 28°08.160’S, 64°48.495’W; 3 March 1998 S. generalis 93-99 Venezuela, Aragua: 0.5 km E La Victoria; Enterolobium Johnson and 10°14.135’N, 67° 17.654’W; 5 March 1999 cyclocarpum (Jacq.) Kingsolver Griseb. S. generalis 282-99 Panama, Los Santos: 10 km N Tonosí; Enterolobium 7°27.021’N, 80° 31.033’W; 21 April 1999 cyclocarpum S. limbatus (Horn) 213-98 Mexico, Jalisco: 5 km on road to coast from Lysiloma sp. Hwy 200 to Tenacatita (km #27); 3 June 1998 S. limbatus 272-98 Mexico, Nayarit: Tepic, 28 km NW, Hwy Acacia sp. 15 Libre; 6 June 1998 S. limbatus 322-98 Mexico, Oaxaca: 74 km S. Oaxaca, Hwy Acacia sp. 175; 13 June 1998 S. limbatus 415-98 Mexico, : 8 km S. Benjamin Hill; Acacia angustissima 30°02.621’N, 111° 06.156’W; 18 October 1998 S. limbatus 436-98 USA, Texas: 5 km W Brownsville on TX Parkinsonia aculeata 281; 25°57.299’N, 97° 33.820’W; 23 L. October 1998 S. limbatus 440-98 USA, Texas: Outside entrance to Bentsen Acacia greggii var. State Park; 26°10.141’N, 98°22.899’W; 24 wrightii (Benth. ex A. October 1998 Gray) Isely S. limbatus 452-98 USA, Texas: 1 km NW Ryan, US20; Acacia angustissima 30°26.382’N, 104°18.683’W; 26 October 1998

156 Table 2.3 (Continued) S. limbatus 6-99 Ecuador, Guayas: Salinas; 2°12.074’S, Pithecellobium 80°58.522’W; 5 February 1999 excelsum (Kunth) Mart. S. limbatus 8-99 Ecuador, Guayas: Salinas; 2°12.427’S, Pithecellobium 80°58.245’W; 5 February 1999 excelsum S. limbatus 61-99 Ecuador, Manabí: 20 km NW Jipijapa; Pithecellobium 1°10.073’S, 80°34.911’W, 13 February excelsum 1999 S. limbatus 78-99 Venezuela, Nueva Esparta: Playa El Agua, Pithecellobium 1 block west of beach; 11°08.562’N, unguis-cati (L.) Benth. 63°52.072’W; 7 February 1999 S. limbatus 101-99 Venezuela, Carabobo: Camaca; Calliandra sp. 10°17.191’N, 67°56.595’W; 6 March 1999 S. limbatus 155-99 Venezuela, Falcón: 60 km SW Dibajiero; Acacia polyphylla DC 10°55.355’N, 70°46.888’W; 9 March 1999 S. limbatus 177-99 Venezuela, Barinas: Barinas, Avenida Pithecellobium dulce Ribereña; 8°38.167’N, 70°12.588’W; 13 March 1999 S. limbatus 316-99 Panama, Herrera: Chitré; 7°57.169’N, Samanea saman 80°25.861’W; 27 April 1999 (Jacq.) Merr. S. limbatus 346-99 Martinique: Between St. Anne & Marin; Acacia tamarindifolia 14°26.942’N, 60°51.823’W; 30 December Griseb. 1999 S. limbatus 447-00 Mexico, Nuevo León: 10 km N Paras; Acacia greggii A. 26°31.921’N, 99°26.206’W; 4 August 2000 Gray S. limbatus 1-01 Mexico, Quintana Roo: Xcaret; 28 February Pithecellobium dulce 2001 (Roxb.) Benth. S. limbatus 18-01 Mexico, Veracruz: 2.5 km NE Tantoyuca; Acacia angustissima 21° 23.002’N, 98°14.507’W; 9 March 2001 (Mill.) Kuntze S. maculatopygus 107-98 Argentina, Entre Rios: La Virgen, Just Acacia bonariensis (Pic) before, Rta 5 at bridge crossing Rio Gillies ex Hook. & Gualeguay Paso Gallo; 30°56.417’S, 58° Arn. 28.212’W; 25 March 1998 S. mexicanus 340-98 Mexico, Oaxaca: Puerto Escondido, 78 km Acacia cornigera (L.) Bottimer NW, 3 km on road to Cecyte off of hwy 200 Willd. at km 66; 15 June 1998 S. monachus 126-99 Venezuela, Falcón: 51 km NW Churuguara; Acacia tamarindifolia (Sharp) 10°59.884’N, 69°42.377’W; 8 March 1999 S. monachus 272-98 Mexico, Nayarit: Tepic, 28 km NW, Hwy Acacia sp. 15 Libre; 6 June 1998 S. pacarae 88-01 Argentina, Jujuy: 10 km W La Mendieta; Enterolobium Johnson, 24°19.336’S; 65°03.857’W; 16 September contortisiliquum Kingsolver, and 2001 (Vell.) Morong Terán S. pruininus 209-98 Mexico, Jalisco: 7km NW of Estación Mimosa sp. (Horn) Biologica de Chamela. Off dirt track W of Rd, left fork; 3 June 1998

157 Table 2.3 (Continued) S. pruininus 301-98 Mexico, Oaxaca: 163 km SE Oaxaca, hwy Mimosa sp. 190; 9 June 1998 S. pruininus 419-98 USA, Arizona: Colossal Cave Road, 1 km S Acacia constricta Colossal Cave; 32°03.313’N, Benth. ex A. Gray 110°40.312’W; 19 October 1998 S. pygidialis 429-98 USA, Arizona: Mt. Elden base, South Calliandra humilis (Schaeffer) Slope, Flagstaff; 35°13.447’N, humilis Benth. 111°36.855’W; 20 October 1998 S. sordidus (Horn) 231-98 Mexico, Jalisco: 3 km from Hwy 200 on Pithecellobium dulce road to Arroyo Seco, near km #35; 4 June 1998 S. sordidus 207-99 Venezuela, Anzoateguí: 33 km SE El Tigre; Piptadenia obliqua 8°44.747’N, 63°58.720’W; 15 March 1999 (Pers.) J.F. Macbr. S. subaeneus 3-01 Mexico, Yucatán: 43 km N Tizimín; Acacia collinsii (Schaeffer) 21°31.305’N, 88°8.425’W, 4 March 2001 Safford S. testudinarius 34-99 Ecuador, Loja: 21 km S Catamayo; 4° Acacia macracantha (Erichson) 08.240’S, 79°22.712’W; 9 February 1999 Humb. & Bonpl. ex Willd. S. tigrensis (Pic) 17-98 Argentina, Salta: Near El Carril; Acacia caven (Molina) 25°02.463’S, 65°29.788’W; 27 February Molina 1998 S. trisignatus 214-99 Venezuela, Bolivár: 73 km SE Upata; 7° Acacia polyphylla (Sharp) 42.038’N, 61°57.388’W; 16 March 1999 S. trisignatus 133-01 Panama, Panamá: Parque Nacional Acacia hayesii Benth. Soberana, Sendero de Plantación; 9°5.811’N, 79°39.097’W; 18 March 2000 S. vachelliae 262-98 Mexico, Nayarit: Las Lomas, hwy 200; 6 Acacia pennatula Bottimer June 1998 (Cham. & Schltdl.) Benth. S. vachelliae 438-98 USA, Texas: At entrance to Santa Ana Acacia farnesiana NWR; 26°04.992’N, 98°08.110’W; 23 (L.) Willd. October 1998 S. vachelliae 106-99 Venezuela, Carabobo: 4 km W Puerto Acacia tortuosa (L.) Cabello; 10°28.676’N, 68°05.373’W; 6 Willd. March 1999 S. vachelliae 161-99 Venezuela, Mérida: 2 km S Lagunillas; Acacia farnesiana 8°29.109’N, 71°23.256’W; 11 March 1999 S. vittatithorax 213-98 Mexico, Jalisco: 5 km on road to coast from Lysiloma sp. Hwy 200 to Tenacatita (km #27); 3 June 1998 S. vittatithorax 294-98 Mexico, Oaxaca: 75 km SE Oaxaca, hwy Lysiloma sp. 190; 9 June 1998

158 DNA SEQUENCE DATA

Standard molecular systematic techniques were used to obtain partial sequences

of two genes: CO1 and EF1-α. Details follow.

DNA Extraction

All specimens used in this analysis were collected as larvae within seeds in the

field, reared as adults in the laboratory, and killed and stored in a –80C freezer. Voucher

specimens are being deposited in the Museum of Comparative Zoology at Harvard

University. Total nucleic acids were extracted from individuals ground to a fine powder with a melted pipette tip and Eppendorf tube in liquid nitrogen. Genomic DNA was then isolated using either a ‘salting out’ protocol (Sunnucks and Hales, 1996) or the Qiagen

QIAamp Tissue Kit (Qiagen Valencia, CA, USA).

DNA Amplification

I amplified a ~1300 base-pair fragment of the mtDNA Cytochrome Oxidase I

(COI) and a 550 base-pair fragment of the nuclear protein-coding gene Elongation Factor

1-α (EF1α) using the polymerase chain reaction (PCR). Longer amplifications of EF1α were not possible due to a 450-1100 base pair intron that displayed considerable length heterozygosity. Therefore, only this smaller fragment that contained no introns was amplified. Amplifications were performed in 50µl reactions to produce double-stranded product under the following conditions: 0.2µM each primer, 0.15mM each dNTP, 2.5µM

MgCl2, 1X buffer supplied by the manufacturer (Qiagen), and one unit of Taq

Polymerase (Qiagen). Reactions were brought up to 50µl using water and 1.5µl DNA

genomic template for COI and 2.5µl DNA for EF1α.

159 Typical temperature profiles for amplification of fragments consisted of 40 cycles of 30s at 95°C, 30s at 50°C, and 1.5 min at 72°C, followed by a 5-min extension step at

72°C. Lower annealing temperatures had to occasionally be used when using the COI primer s1460 and in EF1α amplifications. For CO1, sense primers s1460, s1541, s2191, and s2442, and antisense primers a1969, a2191, a2590, and a2963 were used for amplification; for EF1α, primers s149 and a692 were used. These were based on primers from Sperling and Hickey (1994), Simon et al. (1994), and (Normark et al., 1999) and from one sequence designed specifically for bruchines. Primer sequences are shown in

Table 2.4. PCR products were checked on 1.5% agarose gels and products were purified directly or after gel extraction using QIAquick columns (Qiagen Valencia, CA, USA).

DNA Sequencing

The amount of DNA from the purified PCR product was estimated by comparison to a Low Mass Ladder (Gibco) and 70-90ng (ABI370, 373) or 20-50ng (ABI3100) was used for sequencing reactions. Sequencing primers were identical to those used in PCR reactions. Sequencing of this double-stranded product was carried out using 25 PCR cycles of 96°C for 30s, 50°C for 15s and 60°C for 4 min with a 2°C increase per second in a 10µl reaction. This reaction used dye terminator cycle sequencing chemistry with the specified amount of template, 2.0µl of DyeDeoxy FS Terminator or BigDye premix (PE

Biosystems, Inc., Foster City, CA), 0.16µM primer, and water to the final volume.

Finished reactions were precipitated in 0.5mM MgCl2 in 70% ethanol, centrifuged, and dried under a vacuum. The majority of the sequencing was done using an ABI 370 or 373 automated DNA sequencer, with some sequencing done using an ABI 3100 automated

DNA sequencer. Compiled segments resulted in 1294 base pairs of CO1 for all 54

160 Table 2.4: Sequences of primers used in this study. Primers were used for both PCR amplification and sequencing.

Primera Sequenceb (5’ to 3’) Length From/alias CO1: s1460 TACAATTTATCGCCTAAACTTCAGCC 26-mer Sperling & Hickey (1994): TY-J-1460 s1541 TGAKCYGGAATASTAGGANCATC 23-mer B. Crespi: Zeus s1859 GGAACIGGATGAACWGTTTAYCCICC 26-mer Simon et al. (1994): CI-J-1859 (alias RonII) s2183 CAACATTTATTTTGATTTTTTGG 23-mer Simon et al. (1994): CI-J-2183 (alias Jerry) gms2441 CCTACWGGAATTAAAGTWTTTAGATGATTAGC 32-mer redesigned for bruchines from Simon et al. (1994): CI-J-2441 (alias Dick) a1969 CCTTTAGGTCGTATATTAATTAC 23-mer a2191 CCCGGTAAAATTAAAATATAAACTTC 26-mer Simon et al. (1994): CI-N- 2191 (alias Nancy) a2590 GCTCCTATTGATARWACATARTGRAAATG 29-mer a2963 AGGRAGTTCATTATAIGAATGTTC 24-mer rcJesse EF1α: s149 GARAARGARGCNCARGARATGGG 23-mer Normark et al.1999 a692 GGTGGGAGGATGGCATCAAGAG 22-mer Designed for bruchines a Names refer to direction (s, sense; a, antisense) and to the position of the 3’-end. For EF1α names refer to position in the coding sequence only, as in Danforth and Ji (1998); for mitochondrial DNA (mtDNA), names refer to position in the Drosophila yakuba mtDNA genome (Clary and Wolstenholme, 1985; Simon et al., 1994). S1541 was designed by B. Crespi (Simon Fraser University). b IUPAC ambiguity codes (Cornish-Brown, 1985) refer to equal mixtures of bases; I, inosine.

species included in this analysis and 556 base pairs of EF1α. Both directions of the PCR product were sequenced and contigs were assembled and edited with Sequencher ver. 4.1

(GeneCodes, Ann Arbor, MI). Because both fragments consisted of protein-coding sequences with no insertions or deletions, alignment of both loci was unambiguous.

161

METHODS OF PHYLOGENETIC ANALYSIS

Saturation Levels

While problems due to saturation are likely to be far less problematic in an interspecific phylogenetic analysis than in a deeper-level phylogenetic analysis such as the one in Chapter 1, one must still be aware of the problems that saturated sites could present for phylogeny reconstruction (Dowton and Austin, 1998; Holmquist, 1983). For this reason, the I use the same strategy used in Chapter 1 to examine whether or not any of the relevant data partitions (COI 1st and 2nd; COI 3rd; EF1α 1st and 2nd; EF1α 3rd) displays saturation in the current analysis.

The first strategy compares uncorrected pairwise sequence distances with sequences distances corrected for multiple substitution using the HKY model (Hasegawa et al., 1985) of substitution for each data partition (Dowton and Austin, 1998). This method identifies both (a) character sets that are likely to have undergone multiple substitutions (and are therefore problematic for parsimony analysis), and (b) character sets that have diverged so much that model-based analysis is unlikely to adequately correct for saturation effects. The latter are problematic for both parsimony and model- based analyses such as maximum-likelihood or Bayesian inference.

The second strategy compares the transition/transversion (ti/tv) ratio against transversions for all pairwise sequence comparisons. This measure allows one to examine the amount of observed saturation in the dataset (Holmquist, 1983). Ideally, it should allow one to compare the observed ratio to that of the expected ti/tv ratio under complete saturation (Holmquist, 1983). This measure, however, does not take into

162 account heterogeneity in base pair frequencies across taxa and as such is likely to

underestimate the expected ratio under complete saturation if there is heterogeneity in the

data partition. Therefore, this analysis follows traditional analyses in examining the

proportion of the pairwise comparisons that plateau with time, thereby suggesting

saturation. Based on the results of these two analyses, I assessed whether particular

partitions should be excluded from the analysis in different reconstruction methods. I

used PAUP*4.0b10 (Swofford, 2000) to calculate the pairwise transition, transversion,

transition/transversion ratio, and total uncorrected substitution distance for all ingroup

pairwise comparisons.

Phylogeny Reconstruction

Phylogenetic analysis was performed by maximum parsimony using version

4.0b10 of PAUP* (Swofford, 2000), and by Bayesian inference using MrBayes 2.01

(Huelsenbeck and Ronquist, in Press). Using a total evidence approach, I combined the

EF1α and CO1 data sets, a procedure that maximizes the information internal to each dataset (Baker and DeSalle, 1997; Kluge and Wolf, 1993; Remsen and DeSalle, 1998).

Under the maximum parsimony criterion I searched for shortest trees by

performing 1000 heuristic searches, each with starting trees based on random addition of

taxa. The maximum number of trees kept in memory was not limited and the tree-

bisection-reconnection (TBR) heuristic algorithm was implemented. Bootstrap analyses

were performed based on 500 replications, each with 250 random-addition starting trees.

The decay index, or Bremer support (Bremer, 1988) was calculated using the program

Treerot 2.0 (Sorenson, 1999) and PAUP*. Constraint trees were generated based on the

163 strict consensus of the most parsimonious trees. The length of trees meeting these constraints were calculated in PAUP* using the same search criteria as the original search

(e.g. heuristic searches with 1000 random-addition starting trees, TBR) and the decay index was determined by examining the difference in length between these trees and the length of the most-parsimonious trees.

I used the program ModelTest 3.0 (Posada and Crandall, 1998) to determine the appropriate model of molecular evolution for the analysis based on Bayesian inference.

This test performs a likelihood ratio test between increasingly complex models and determines whether or not increasing parameters significantly increases the fit of the model to the data. The model selected using this analysis was then incorporated into the phylogeny reconstruction using Bayesian inference in the program MrBayes 2.0. In addition, a more complex model that is not incorporated into ModelTest 3.0 was examined and the results were compared based on the Akaike Information Criterion

(Posada and Crandall, 2001b). All Bayesian searches were run with four simultaneous chains for 1,000,000 generations, sampling every 100 generations and applying temperatures of 0.2 and 0.5. Heating the chains at these different temperatures influences the rate of how often chains are swapped, and using multiple temperatures increases the efficacy of the search. The burnin time (the number of generations before the tree-search reaches the optimum) was estimated by plotting the number of generations versus the ln

Likelihoods (lnL). Trees in the burnin were discarded from the analysis. Posterior probabilities of nodes were estimated based on the majority rule consensus of the trees that were found at stationarity, and branch lengths for the phylogram are based on the mean branch lengths from these trees. The model selected in ModelTest 3.0 was a

164 General Time Reversible Model, estimating the proportion of invariable sites and the shape of the gamma parameter (GTR+I+G). However, subsequent analysis showed that the data were better explained using site-specific models based on the 6 data partitions

(CO1 1st positions, CO1 2nd positions, CO1 3rd positions, EF1α 1st positions, EF1α 2nd positions, and EF1α 3rd positions), and estimating the shape of the gamma parameter

(SS+G). Of note, this model was also significantly better than a simpler model based on just codon position, regardless of locus. Results from this model were used for all subsequent analyses.

COMPARATIVE ANALYSES

All comparative analyses were performed on the species exemplar phylogeny unless otherwise noted in the results section. Branch lengths were based on the minimum branch lengths from the MRCA to the species tip when species are represented by multiple specimens. In cases in which widespread species were found to be paraphyletic to more narrowly distributed species, that structure was maintained by leaving in two members of the widespread species. In the case of S. limbatus, the exemplars are based on the monophyletic North and South American clades, and branch lengths are based on the mean path lengths from the MRCA to the clade’s tip.

Defining diet breadth.

As noted in the introduction, defining and quantifying diet breadth for insect species is a daunting task both conceptually and pragmatically. An ideal definition would

165 incorporate the ecological availability of potential host plants (Colwell and Futuyma,

1971; Kelley and Farrell, 1998), the relative abundance of the insect species on different

hosts (Futuyma and Gould, 1979), and the phylogenetic disparity of hosts (Beccaloni and

Symons, 2000; Symons and Beccaloni, 1999). Unfortunately, the amount of information

required for the first two measures is simply not practical across a geographic distribution that spans South and North America and an ecological distribution that includes 136 native species of Fabaceae from all three of its subfamilies. Therefore, I have concentrated on measures of phylogenetic diversity alone for this analysis.

The prevailing methods for quantifying diet breadth along something that would approximate a phylogenetic index have focused on counts of numbers of species or counts of higher taxa within a diet (Dyer and Floyd, 1993; Fiedler, 1998; Novotny, 1994).

I used the number of species and the number of genera in the host profile (with the

Acacia subgenera separated) as a starting point for assessing diet breadth. However, focusing on number of species alone ignores phylogenetic disparity between those species. In this sense, an insect feeding on four closely related species of Acacia would have the same diet breadth as an insect feeding on four phylogenetically unrelated host plants. And focusing on higher taxonomic levels is problematic because taxa within distinct ranks are not phylogenetically equivalent unless they happen to be sister taxa

(Felsenstein, 1985; O'Hara, 1997; Ronquist and Nylin, 1990). Therefore, we can more accurately represent diet breadth by taking an explicitly phylogenetic perspective

(Symons and Beccaloni, 1999).

I used the Phylogenetic Diversity (PD) index of Faith (1992; 1996) for the phylogenetic quantification of diet breadth in the genus Stator. Other methods are

166 deemed inappropriate for this analysis. The Clade Dispersion index of Symons and

Beccaloni (1999) functionally measures the amount of phylogenetic constraint within the

diet of a phytophagous insect species, and not necessarily the diet breadth. For example,

this measure would result in a more narrow diet for a species that feeds on all 10

members of a pectinate clade than for a species that feeds on only the first to diverge and the last to diverge. The node counting method of Webb (2000) is similar in that it more accurately measures phylogenetic relatedness than it does phylogenetic diversity. I decided that an index such as this that is based on node-counting across minimum- spanning trees would be far too sensitive to taxon sampling to be useful in this case. For quantification of the PD, the index for specialists was taken as the length of the terminal node leading to their host. While Symons and Beccaloni (1999) claim that the PD index cannot take into account single-species specialists, they do not hesitate to use terminal nodal lengths in calculating the PD for other species, and therefore this does not seem like a valid criticism.

For construction of the host phylogeny, I used two published phylogenies of the

Acacieae (Miller and Bayer, 2001; Robinson and Harris, 2000), and a published phylogeny of the Ingeae (Grimes, 1999) for the topology estimate. These three phylogenies differ significantly in both their focus and their taxonomic sampling.

However, at deeper phylogenetic levels they are in complete agreement except for the rooting of the Acacieae/Ingeae clade. For this reason, the node leading to Acacia subgenus Acacia, A. subg. Aculeiferum, and Ingeae is treated as a polytomy.

Relationships within A. subg. Acacia are based on recent revisions and phenetic assessments of the species groups (Clarke et al., 1989; Clarke et al., 1990; Ebinger et al.,

167 2000; Lee et al., 1989; Seigler and Ebinger, 1988; Seigler and Ebinger, 1995).

Relationships outside of the Acacieae/Ingeae are based on recent molecular phylogenies of the Fabaceae (Doyle, 1994; Doyle, 1995; Kajita et al., 2001). The host plant phylogeny was pruned to include only those species that are known hosts for the genus

Stator. Including all members that would be derived from the most recent common ancestor (MRCA) of all the hosts would have been entirely too cumbersome. When host plants are not included as exemplars in these phylogenetic analyses, they are placed in their genus as an unresolved polytomy.

The host plant phylogeny in this analysis does not have branch lengths or support values because it is the supertree amalgamation of multiple phylogenetic hypotheses.

However, the PD index requires branch length information to estimate the length of the minimum spanning trees of the hosts in the diet of an insect species. I therefore modelled branch lengths in two ways using the program CACTUS 1.13 (Schwilk and Ackerly,

2001, available at http://www.pricklysoft.org/software/cactus.html). In order to avoid problems with unbalanced sampling on the phylogeny, both methods set the length from the root to any tip to be constant. This seems justifiable as a proxy for time since all taxa are derived from a single MRCA. The first estimate is based on Grafen’s (1989) method that sets node height from the tip proportional to the number of descendent terminal node

(taxa) minus one. The tree based on this method (Figure 2.1) is referred to as the ‘Grafen tree’ and analyses are based on ‘Grafen branches’. The second method, the minimal extension (ME) method, makes node height proportional to the daughter clade with the most daughter nodes plus one. Because of this all internodes are in a constant ratio to each other. The tree based on this method (Figure 2.2) is

168

Figure 2.1. Phylogeny of Stator host plants, constructed from published trees and phenetic arrangements (Clarke et al., 1989; Clarke et al., 1990; Ebinger et al., 2000; Lee et al., 1989; Grimes, 1999; Miller and Bayer, 2001; Robinson and Harris, 2000; Seigler and Ebinger, 1988; Seigler and Ebinger, 1995). The tree is pruned to include only host plants, then host plants are added in polytomies based on their classification. Branch lengths are modelled based on the Grafen (1989) algorithm.

169

Figure 2.2. Phylogeny of Stator host plants, constructed from published trees and phenetic arrangements (Clarke et al., 1989; Clarke et al., 1990; Ebinger et al., 2000; Lee et al., 1989; Grimes, 1999; Miller and Bayer, 2001; Robinson and Harris, 2000; Seigler and Ebinger, 1988; Seigler and Ebinger, 1995). The tree is pruned to include only host plants, then host plants are added in polytomies based on their classification. Branch lengths are modelled based on the minimal evolution model discussed in the text.

170 referred to as the ‘ME tree’ and analyses are based on ‘ME branches’. While the topologies of the two trees are identical, the branch lengths differ, allowing me to determine how sensitive this analysis is to differences in branch length assessment.

Using this method, all branch lengths were scaled based on a root to tip length of 1.

These metrics were used for continuous trait analysis and to examine whether or not there are distinct categories of resource specialization that allow for the reconstruction of discrete ancestral states.

Phylogenetic constraint.

Character mappings of transitions between generalists and specialists, between different host plant clades, and between different oviposition guilds was performed using

MacClade 4.05 (Maddison and Maddison, 2002). All analyses were rooted using the genus Merobruchus. This is largely because the maximum-likelihood analysis in the previous chapter reconstructed the ancestral state of the genus Stator, as well as the node subtending the genus Stator as feeding on mimosoid legumes. Of the three outgroups included here, only Merobruchus feeds on mimosoid legumes. It is also representative of both the degree of specialization and the oviposition guild (guild A, the mature pod guild) that all closely related species of Bruchinae display. Ecological codings are shown in

Table 2.5.

Tests of the correlation of these ecological characters with phylogeny were done using the method proposed by Maddison and Slatkin (1991) to test for phylogenetic inertia. These tests were done with the PTP utility in PAUP* as implemented by Kelley and Farrell (1998) and in Chapter 1. Following Kelley and Farrell (1998), character

171 states are randomized 10,000 times while holding the tree topology constant. This results in the distribution of the frequencies of the tree lengths of the randomly permuted character to test if the non-permuted tree length is significantly shorter than expected under a random model. Significantly shorter tree lengths allow one to reject the null hypothesis that characters are not phylogenetically constrained. Constraint analyses

Table 2.5: Discrete ecological codings for analyses of character evolution. Codings for life history information are based on the information summarized in Tables 2.1 and 2.2. Species Generalist/ Oviposition Host Host Clade use6 Specialist1 guild2 genus/genera3,4 tribe(s)5 S. aegrotus 1 0 1 1 0 S. beali 1 1 5 2 1 S. bottimeri 1 2 0 0 0 S. chalcodermus 1 2 4 2 1 S. chihuahua 1 2 0,1,2,6 0,1,2 2 S. furcatus 1 1 0,1 0,1 0 S. generalis 1 2 3 2 1 S. limbatus 0 1 1,2,4,6,7,8,9 1,2,3,4,5 3 S. maculatopygus 1 0 1 1 0 S. mexicanus 1 2 0 0 0 S. monachus 1 0 1 1 0 S. pacarae 1 2 3 2 1 S. pruininus 0 1 0,1,2,6,7,9 0,1,2,3,5 3 S. pygidialis 1 2 2 2 1 S. sordidus 0 2 0,1,4,6,7,8 0,1,2,3,4 3 S. subaeneus 1 2 0 0 0 S. testudinarius 1 2 0,6 0,2 2 S. tigrensis 1 1 0,1 0,1 0 S. trisignatus 1 0 1 1 0 S. vachelliae 1 2 0,6 0,2 2 S. vittatithorax 1 0 1,6 1,2 2

1Generalist =0; Specialist = 1. 2Guild A = 0; Guild B = 1; Guild C = 2. 3Acacia (Acacia) = 0; Acacia (Aculeiferum) = 1; Calliandra = 2; Enterolobium = 3; Pithecellobium = 4; Ebenopsis = 5; other Ingeae = 6; Mimoseae = 7; Caesalpinioideae = 8; Papilionoideae = 9. 4Only character states 0-4 were examined for phylogenetic constraint. All other genera were either autapomorphic (Ebenopsis) or shared only across generalists. 5Acacia (Acacia) = 0; Acacia (Aculeiferum) = 1; Ingeae = 2; Mimoseae = 3; Caesalpinioideae = 4; Papilionoideae = 5. 6Acacieae = 0; Ingeae = 1; Acacieae + Ingeae = 2; Acacieae + Ingeae + other legumes = 3.

172

were done only on the ingroup taxa, Merobruchus is treated as missing data. These analyses were performed using both the parsimony exemplar tree and the Bayesian exemplar tree.

In a meta-analysis using phylogenies from different insect groups, Farrell and

Mitter (1993a) showed that the evolution of host affiliation in phytophagous insects is often quite phylogenetically conservative with respect to host taxon (family, tribe, or genus). If classification can be taken as predictive of or as a proxy for phylogeny, then it is suggested that these taxonomic groupings represent disparity in plant traits (especially those relevant to defense against herbivores such as secondary chemistry or defensive morphology) that present obstacles to host shifting. While it is clear that host use in the genus Stator is taxonomically constrained at particular levels (e.g. they all feed on legume seeds), I tested the hypothesis that host affiliations are constrained at the level at which there appears to be phylogenetic variation in host use in the group. Nominally, this appears to be at the level of genus. However, the genus Acacia is paraphyletic with respect to the Ingeae and shows major and obvious distinctions between monophyletic groups. Therefore, I used combined phylogenetic estimates of the tribes Acacieae and

Ingeae (Grimes, 1999; Miller and Bayer, 2001; Robinson and Harris, 2000) in order to identify monophyletic groupings that correspond to major phylogenetic disjunctions within the tribes. Generally these group according to the generic criteria set forward for the Pithecellobium complex in the tribe Ingeae (Barneby, 1998; Barneby and Grimes,

1996; Barneby and Grimes, 1997). However, I split the genus Acacia into its three monophyletic subgenera, Acacia subgenus Acacia, Acacia subgenus Aculeiferum, and

173 Acacia subgenus Phyllodineae. Only the first two are relevant in this analysis, as

subgenus Phyllodineae is restricted to Australia and is not a natural host for species in the

genus Stator (although it is used by two of the generalists in areas where it has been

planted as an ornamental). Monophyly of the remaining two subgenera conflicts between

the two phylogenetic analyses of the genus. In the analysis of Miller and Bayer (2001),

these form a paraphyletic basal group within this clade: (A. subg. Acacia (A. subg.

Aculeiferum (remainder of Ingeae + Acacia subg. Phyllodineae))). The lack of

monophyly of the genus has only moderate nonparametric bootstrap support, however.

In the analysis of Robinson and Harris (2000), these two subgenera form a monophyletic

group that is the sister clade to the remainder of the Ingeae + other Acacia. Again, the

support for this grouping is only moderate. Because of this, analyses of constraint were

done separately for the two subgenera. However, the two were grouped when

reconstructing ancestral states due to the necessity of limiting the number of parameters

being estimated. This appeared to be the most justifiable grouping given the current

status of mimosoid phylogenetics.

The potentially large number of character states required in a multi-state coding of

host genus would render the interpretation of a PTP analysis of host use difficult.

Therefore, tests of the hypothesis that host affiliations show phylogenetic constraint took

two forms: (1) separate analyses of each of the host genera used by at least two species in

the phylogeny, one of which must be a specialist (the two subgenera of Acacia,

Calliandra, Enterolobium, and Pithecellobium); and (2) an analysis that examines the level of constraint more broadly by examining whether there is phylogenetic constraint for using clades that are more deeply defined than the level of genus for the tribe Ingeae.

174 For both of these codings, the two subgeneric groups of Acacia are treated as separate,

and the tribe Ingeae is coded as a single character. In essence, the latter coding is

analogous to a coding based on host tribe, except that it more closely corresponds to a

character-based phylogenetic hypothesis of the group (Figure 2.3).

Taxa used only by the generalists were not included in this analysis because the

three generalists appear to be independently derived. Therefore, it can be assumed that

the origins of feeding on these hosts is independently derived and not structured by

macroevolutionary history.

I also examined whether diet breadth, when treated as a continuous character

(Table 2.6), is phylogenetically constrained. Because the character permutation test

described above is not applicable to continuous data, I assessed the significance based on a model of evolution using a generalized least squares (GLS) approach for the analysis of comparative data (Pagel, 1997; Pagel, 1999a). Using this approach, trait correlation due to phylogenetic relationship is controlled for by reference to a matrix of the expected covariances among species that is based on a random walk model of evolution. I examined how much diet breadth in Stator has followed the phylogenetic topology or branch lengths during its evolution by examining whether or not the maximum likelihood estimation of the scaling parameter lambda (λ) significantly differs from zero. This

scaling parameter allows for the deviation from a random walk of character evolution

dependent upon the degree that the phylogeny correctly predicts the patterns of

covariance among species. Lambda takes the value zero when trait evolution is

effectively independent of phylogeny, and increases toward one, when the trait is

completely correlated with phylogeny. I examinee the significance of any deviation from

175

Figure 2.3. Supertree of Stator hosts. Mimosoideae relationships based on phylogenies of Miller and Bayer (2001), Robinson and Harris (2000), and Grimes (1999). The two analyses differed in the placement of the root, hence the lack of resolution at the base of the Acacia + Ingeae clade. Support for monophyly of Mimosoideae is from Kajima et al. (2001). Asterisk indicates placement of Acacia subgenus Phyllodineae within the Ingeae. Because this is a supertree, information on support is incorporated only in the agreement of the phylogenetic topologies.

176 Table 2.6: Continuous ecological codings for analyses of character evolution. The genus Acacia is separated into two genera based on the subgenera Acacia and Aculeiferum. The length in the spanning trees of hosts in the diet of each species of Stator are given as a percentage of the total spanning tree shown in Figures 2.1 and 2.2. Hosts used are summarized from information and references summarized in Table 2.1 Species # host # host species Grafen spanning Minimal evolution genera1 tree2 spanning tree2 S. aegrotus 1 1 1.1498029 0.4693676 S. beali 1 1 0.065703 0.4693676 S. bottimeri 1 2 1.2483574 0.9387352 S. chalcodermus 1 1 0.2299606 0.4693676 S. chihuahua 4 4 10.315375 11.215415 S. furcatus 2 6 10.446781 8.8685771 S. generalis 1 1 0.065703 0.4693676 S. limbatus 18 59 56.438896 55.237154 S. maculatopygus 1 1 1.1498029 0.4693676 S. mexicanus 1 1 0.2299606 0.4693676 S. monachus 1 8 8.0486202 3.2855731 S. pacarae 1 1 0.065703 0.4693676 S. pruininus 15 52 52.759527 47.035573 S. pygidialis 1 1 0.131406 0.4693676 S. sordidus 11 15 29.467806 29.001976 S. subaeneus 1 3 1.1169514 2.3468379 S. testudinarius 2 3 6.5045992 6.5464427 S. tigrensis 2 2 6.6031537 6.5217391 S. trisignatus 1 5 5.7490145 2.3468379 S. vachelliae 2 7 7.9500657 9.3626482 S. vittatithorax 3 14 19.710907 13.117589

1The genus Acacia is separated into two genera based on the subgenera Acacia and Aculeiferum. 2Spanning trees are given as a percentage of the total spanning tree.

zero using the likelihood ratio (LR) statistic: LR = -2ln [H0/H1], which is evaluated based on the chi-squared distribution with a single degree of freedom. These analyses were done using the program Continuous v1.0d13 (Rambaut and Pagel, 2001) and the topology and branch lengths from the consensus Bayesian phylogram.

177 Reconstruction of ancestral states.

For both the Bayesian and the parsimony analysis, ancestral states were estimated

on the pruned exemplar tree using the default settings as implemented in MacClade v4.05

(Maddison and Maddison, 2002). Results based on unequivocal reconstructions were

examined, as there is no real reason to select between accelerated or delayed transitions.

This type of character reconstruction is somewhat problematic for the reconstruction of ancestral host associations because so many species of Stator are polymorphic for the codings used in Table 2.5. Unfortunately, this method will attempt to reconstruct ancestral nodes as monomorphic, while the very real possibility exists that the ancestors fed on multiple hosts. In order to address this issue, I scored host use based on both the hierarchical relationships of host plants used throughout the genus and the extent of variation that is seen within the different individual species of Stator. In order to incorporate both host plant affiliation and diet breadth, I scored as separate character states feeding on Acacieae only, feeding on Ingeae only, feeding on both of these, and feeding on these as well as other legumes. These codings are shown in the last column of

Table 2.5. While these delimitations can be criticized as being somewhat subjective (e.g.

I could have chosen to code other clade levels instead), they do appear to represent the variation within and between species of Stator. Because of the hierarchical nature of phylogenetics, clades do represent discrete entities, and treating them as such seems a reasonable starting point. Transitions between generalists and specialists and between the three oviposition guilds were also reconstructed using this optimization method. Discrete ecological codings are shown in Table 2.5.

178 In addition, ancestral states were estimated on the Bayesian consensus phylogram

with branch lengths included using a likelihood approach based on a Markov model of

binary character evolution (Pagel, 1997; Pagel, 1999a; Pagel, 1999b). While the

phylogram from the Bayesian analysis deviates significantly from a molecular clock,

branch lengths were still used because they are assumed to reflect some degree of

temporal separation and are likely better than giving each internode an arbitrary equal

length. The binary characters for diet breadth, in column 2 of Table 2.5, were used in this

analysis and were reconstructed using the program Discrete v4.0 (Pagel, 2001). The

multistate character of oviposition guild, in column 3 of Table 2.5, was used and

ancestral states reconstructed using the program Multistate v0.6 (Pagel, 2002). Multistate was also used for reconstruction of host plant use, but only a three character state of host clade (Acacieae, Ingeae, multiple) is used in order to limit the number of parameters being estimated. The proportional support for each different character state (i of n) at each node are determined based on the equation (EXP [LnL (state xi)])/ Σ EXP [LnL (state

xn)]). Greater than 95% support was taken as significant, and is roughly equivalent to a

difference in 2 log-likelihoods between the most likely state and the next most-likely

state (Edwards, 1972).

Ancestral states for diet breadth, when treated as continuous characters, are

estimated on the Bayesian consensus phylogram with branch lengths included via a

maximum-likelihood approach based on a generalized least squares (GLS) model for the

across-species analysis of comparative data similar to the one discussed above, but as

implemented by Martins and Hansen (1997). The trait values listed in Table 2.6 were

analyzed using the program COMPARE ver 4.4 (Martins, 2001). Because this

179 quantitative data is based on species-level estimates, standard errors were not

incorporated into the analyses.

Trajectory of specialization

I tested the trajectory of specialization using the continuous characters in Table

2.6 within a likelihood framework based on the same GLS model as above, again as

implemented by Pagel (1997; 1999a). In order to test whether there is a general trend on

the phylogeny toward specialization, I examined whether or not a model that incorporates

a directional parameter into the random walk model is significantly more likely than one

that does not. These likelihoods were compared based on the likelihood ratio (LR)

statistic discussed above, which is evaluated based on the chi-squared distribution with a

single degree of freedom. These analyses were done using the program Continuous

(Rambaut and Pagel, 2001) and the topology and branch lengths from the consensus

Bayesian phylogram.

I also tested the hypothesis that the trajectory of specialization is dependent upon

host taxon using the program Discrete. For this I coded Stator species as specialists on

Acacia, specialists on Ingeae, or generalists in separate analyses. I then constrained the

forward and backward transition rates to be equal versus when they are unconstrained

and examined the difference in likelihood scores between the two using the LR statistic with one degree of freedom. This tests separately if there is a bias in the direction of evolution of Acacia specialists or Ingeae specialists. Again, this analysis was based on the consensus Bayesian phylogram.

180 Character correlation.

I tested whether or not diet breadth as a continuous character is significantly

correlated with oviposition behavior using a GLS approach (Pagel, 1997; Pagel, 1999a)

as implemented in the computer program Continuous (Rambaut and Pagel, 2001). I

examined each oviposition behavior separately as a binary character; otherwise, the program treats the character states as continuous. If an oviposition behavior is significantly correlated with diet breadth, this suggests that the mean value of diet breadth differs for those species that differ in oviposition behavior even when taking phylogeny into account.

RESULTS

Saturation levels.

The level of saturation due to multiple substitutions is low for both EF1-α partitions and for the first two codon positions of COI, as can be seen by the nearly linear relationship between corrected and uncorrected distances for these partitions (Figure 2.4) and the sloped relationship between the transition/transversion ratio and transversions for these partitions (Figure 2.5). Overall transition/transversion ratios were quite high and

base pair frequencies were not significantly heterogeneous across taxa for these three data

partitions (Table 2.7). The level of saturation is higher for the third codon position of

CO1. Correcting for multiple substitutions by using an HKY85 model of nucleotide

substitution (Hasegawa et al., 1985) reveals that many sites contain numerous hidden

substitutions, as can be seen by the divergence from the straight line in Figure 2.4.

However, the model was able to correct for all but 3.2% of these pairwise comparisons,

181 suggesting that a model-based analysis is likely to adequately correct for many of the

effects of saturation. Given that COI 3rd positions account for 69% of the total parsimony

informative sites (Table 2.7), this seems like an adequate sacrifice, particularly if these

somewhat saturated sites are structured by phylogenetic relationships (Källersjö et al.,

1999). It also appears from the analysis of the transition/transversion ratio that a large

number of the sites are not saturated (Figure 2.5). While there does appear to be a plateau at the tail of this distribution, a large number of pairwise comparisons appear to maintain phylogenetic signal. In fact, a partition homogeneity test (Farris et al., 1994) of first and second codon positions versus third codon positions supports no significant incongruence between the datasets (100 replicates, 100 RAS per replicate; P = 0.80). A somewhat more worrisome observation is that base pair frequencies are significantly heterogeneous across taxa for the third codon position (P<<<0.0001, Table 2.7). While this test does not take into account phylogenetic autocorrelation in base pair changes, it is indicative that third positions are likely to be homoplasious across the phylogeny because of shifts in base codon bias. Nevertheless, given the amount of phylogenetic information present in the third codon positions and the only modest levels of saturation the third position is included in the analysis.

182

Figure 2.4. Saturation analysis of molecular character partitions. Pairwise uncorrected and corrected distances were estimated using PAUP v.4.0b10 (Swofford, 2000), using the HKY85 (Hasegawa et al., 1985) model to correct for multiple substitutions. Uncorrected distances are then plotted on the X-axis, corrected distances on the Y-axis. The extent of hidden substitutions is indicated by the distance of the points to the left of the diaganol line. The plot for 3rd codon positions in COI includes some distances that the model is unable to correct, and are not shown here (3.2% of all pairwise comparisons).

Figure 2.5. Saturation analysis of molecular character partitions. Transition/transversion (ti/tv) ratios and transversions were determined using PAUP v.4.0b10. Transversions are plotted on the X-axis, ti/tv ratios on the Y-axis. The plots for all partitions includes comparisons that had no transversions and are included at 10% above the maximum.

183

184

RESULTS OF PHYLOGENETIC ANALYSES

Results of the parsimony analysis.

Eight most parsimonious trees of length 3147 were found in the parsimony analysis (Figure 2.6). Bootstrap support is relatively high (>65%) for most of the interspecific nodes in the tree, with some exceptions in more basal divergences.

Intraspecific nodes within S. limbatus are generally less well-resolved. There is high support for a monophyletic S. limbatus + S. beali. However, there is also high support for a paraphyletic S. limbatus, with S. beali forming a clade (100% bootstrap support, decay index 15) with North American S. limbatus, and South American S. limbatus forming a separate monophyletic clade (100% bootstrap support, decay index 25). The widespread S. sordidus also is paraphyletic with respect to the more narrowly restricted S. chihuahua + S. pygidialis, and S. furcatus is paraphyletic with respect to S. tigrensis.

For the most part, the clades correspond to the four species groups of Johnson et al.(1989), with only two exceptions. They place S. bottimeri, S. chalcodermus, S. mexicanus, S. subaeneus, and S. pruininus in the Subaeneus species group. In this phylogenetic analysis, S. pruininus is not monophyletic with the rest. This is perhaps not surprising given that S. pruininus was considered only a peripheral member of the group and was actually included in a different species group in an earlier publication (Johnson and Kingsolver, 1976). However, monophyly of this group cannot be rejected based on

Templeton’s (1983) signed-rank test nor Prager and Wilson’s (1988) winning sites test (P

= 0.26-0.29 and 0.27-0.32, respectively). They also placed S. vachelliae in the Aegrotus species group, which also includes S. aegrotus, S. maculatopygus, S. monachus, and

185

Figure 2.6. Strict consensus of 8 most-parsimonious trees of length 3147 from parsimony analysis. Heuristic search using 1000 replicates with a random addition sequence and tree-bisection-reconnection (TBR). Decay indices using same search strategy. Bootstrap proportions based on 500 replicates, each replicate consisting of a heuristic search strategy, with 250 random-addition sequences and TBR. Numbers following species names refer to collection reference numbers in Table 2.3.

186 S. trisignatus, a relationship not supported in this phylogenetic analysis. While the remainder of these species are monophyletic, monophyly including S. vachelliae can be significantly rejected by both Templeton’s Wilcoxon signed-rank test and Prager and

Wilson’s winning sites test (P < 0.05 in both cases).

Results of the Bayesian analysis.

The burn-in for this analysis is conservatively estimated at 65,000 generations,

6.5% of the total length of the run. By this generation the log likelihood of the topologies accepted have reached a noticeable plateau (Figure 2.7). In the majority-rule consensus of the sampled 9351 trees (Figure 2.8), the values at each node indicate the percentage of those samples in which that node is present and can be thought of as being analogous to parametric bootstrap values (Huelsenbeck et al., 2002). The topology from this analysis contains considerably more support than the parsimony analysis. This is a common result for Bayesian analyses vs. parsimony analyses in general (Huelsenbeck et al., 2002), and could be due to the fact that this model-based approach is able to correct more adequately for multiple substitutions in the third codon position of CO1.

As in the parsimony analysis, the topology does not support the placement of S. vachelliae within the Aegrotus species group, nor the placement of S. pruininus within the Subaeneus group. In fact, the posterior probability of both of these relationships is

0.00%. The Bayesian topology is in general agreement with the parsimony topology.

Only two specific relationships are different—the placement of S. testudinarius as sister to a clade containing S. pacarae, S. generalis, S. furcatus, and S. tigrensis instead of as sister to only S. furcatus + S. tigrensis; and the placement of S. aegrotus as sister to S.

187 trisignatus + S. maculatopygus instead of as sister to S. monachus + S. vittatithorax.

While some of the intraspecific relationships of S. limbatus differ between the two methods, the Bayesian analysis also strongly supports a North and South American clade of S. limbatus, with S. beali monophyletic with the North American clade.

Because of the general agreement between the two phylogenetic analyses, I will regularly refer to three clades throughout the remainder of this paper. I will refer to the

Limbatus group or clade as the monophyletic assemblage of S. beali, S. furcatus, S. generalis, S. limbatus, S. pacarae, S. testudinarius, and S. tigrensis. I will refer to the

Aegrotus group as the clade including S. aegrotus, S. maculatopygus, S. monachus, S. trisignatus, and S. vittatithorax. The Subaeneus group will include the species in the

remaining clade: S. bottimeri, S. chalcodermus, S. chihuahua, S. mexicanus, S. pruininus,

S. pygidialis, S. sordidus, S. subaeneus, and S. vachelliae.

Figure 2.7. Estimation of burn-in time for Bayesian analysis. The likelihood conservatively reaches a plateau by the 65,000th generation.

188

Figure 2.8. Consensus Bayesian phylogeny of the genus Stator. Model incorporates site- specific rate variation based on six data partitions (CO1 1st, 2nd, and 3rd positions; EF1- a 1st, 2nd, and 3rd positions), estimating the shape of the gamma parameter (SS + gamma). Analysis ran four simultaneous chains for 1,000,000 generations (first 65,000 generations discarded as burn-in), sampling every 100 generations and applying temperatures of 0.2 and 0.5. Numbers indicate bayesian posterior support for individual clades. Numbers following species names refer to collection reference numbers in Table 2.3.

189 RESULTS OF COMPARATIVE ANALYSES

Throughout the comparative analyses, I will be using phylogenies that have been pruned to include the specific exemplars. Multiple specimens from species are only included in the three cases (S. limbatus, S. sordidus, and S. furcatus) where the phylogeny suggests a paraphyletic relationship. Where branch lengths are incorporated into the analysis, I use the mean path lengths from the MRCA to the species ‘tip’ of the parsimony consensus phylogram (Figure 2.9) and the Bayesian consensus phylogram

(Figure 2.10).

Quantification of diet breadth.

Phylogenetic diversity (PD) indices of host use for each species of Stator are calculated based on both the Grafen phylogeny (Figure 2.1) and the ME phylogeny

(Figure 2.2) and are listed in columns 3 and 4, respectively, of Table 2.6. This table also summarizes the number of genera and species known to be used by each species. S. limbatus, S. pruininus, and to a lesser extent S. sordidus are distinctly separated from the other species of Stator in their diet breadth when using the PD index based on either the

Grafen tree (Figure 2.11) or the ME tree (Figure 2.12). The separation of all three is even more clear when examining the simple tallies of host species (Figure 2.13) or host genera

(Figure 2.14). While the exact indices are used in analyses based on continuous data, these histograms formed the basis for delineating these three species as generalists and the remainder as specialists. The four quantifications of diet breadth are highly correlated with each other (r = 0.93- 0.99; P<<<0.001), suggesting that number of species or number of genera is an adequate proxy for the phylogenetic diversity of the host plants.

190

Figure 2.9. Phylogram of strict consensus of 8 most-parsimonious trees of length 3147 from parsimony analysis. Heuristic search using 1000 replicates with a random addition sequence and tree-bisection-reconnection (TBR). Decay indices using same search strategy. Bootstrap proportions based on 500 replicates, each replicate consisting of a heuristic search strategy, with 250 random-addition sequences and TBR. Numbers following species names refer to collection reference numbers in Table 2.3.

191

Figure 2.10. Consensus Bayesian phylogram of the genus Stator. Based on site-specific rate variation and estimated gamma parameter (SS-gamma). See methods for analysis details. Branch lengths are those lengths with highest posterior probability. Numbers following species names refer to collection reference numbers in Table 2.3.

192

Figure 2.11. Histogram of the Phylogenetic Distance of hosts included in the diet of each species of Stator, given as the percentage of the total distance on the tree. Branch lengths modelled based on Grafen's (1989) method.

Figure 2.12. Histogram of the Phylogenetic Distance of hosts included in the diet of each species of Stator, given as the percentage of the total distance on the tree. Branch lengths modelled based on minimal evolution method.

193

Figure 2.13. Histogram of the number of species of hosts included in the diet of each species of Stator.

Figure 2.14. Histogram of the number of species of hosts included in the diet of each species of Stator.

194

Phylogenetic constraint.

The results of the permutation based analysis of phylogenetic constraint are

summarized in Table 2.8. Analysis reveals that diet breadth (Figure 2.15) is

phylogenetically labile (PTP test, both topologies; P = 1.000). This is perhaps not all that

surprising given that the three generalists are independently derived across the phylogeny

and that there is subsequent speciation and specialization within two of the generalist

species. On the other hand, oviposition guild (Figure 2.16) is highly significantly

constrained (PTP test, both topologies; P = 0.0001). The results for whether or not host

plant genus is significantly correlated with the Stator phylogeny are dependent upon both

the host taxon and the inclusiveness of the host clade being examined (Figure 2.17).

At the generic level, host use is only significantly conservative for those species that feed

on Acacia subg. Acacia (PTP test; parsimony topology P = 0.0009; Bayesian topology P

= 0.0100) and for Enterolobium feeders (PTP test; parsimony topology P = 0.0283;

Bayesian topology P = 0.0321). The latter is not surprising given that feeding on

Enterolobium is limited to two sister species. Feeding on Acacia subg. Aculeiferum is only marginally correlated with phylogeny (PTP test; parsimony topology P = 0.0905;

Bayesian topology P = 0.0897), while feeding on either Calliandra (PTP test; parsimony topology P = 0.3002; Bayesian topology P = 0.2990) or Pithecellobium (PTP test, both topologies; P = 1.000) appears largely unconstrained by phylogenetic history. When all

Ingeae genera are grouped into a single clade, host use is significantly phylogenetically constrained (PTP test; parsimony topology P = 0.0194; Bayesian topology P = 0.0190).

195 This suggests that while related species of Stator tend to feed on closely related plants

and oviposit upon those hosts in a similar manner, changes in diet breadth may not be

constrained by historical circumstances.

This could be due to the treatment of diet breadth as a binary character. If, for

example, diet breadth changed gradually over the phylogeny then this analysis cannot

capture this information. However, the generalized least squares analyses based on the

continuous quantification of diet breadth show that differences in diet breadth are not

structured phylogenetically, as the parameter lambda is not significantly different from

zero (no phylogenetic structure) for the number of genera or species fed upon, or for the

PD index based on either the Grafen or Minimal Evolution trees (P>0.99 for all cases).

Reconstruction of ancestral states.

Of particular note in the following analyses are issues concerning the incorporation

of the three paraphyletic assemblages. An argument might be made that all species

should be trimmed to exemplar level, and that the paraphyletic relationships should be

ignored. However, this approach would ignore the polarity inherent in the paraphyletic

assemblages. Ignoring the observation that there are reproductively isolated specialist

lineages derived within generalist species does not take all evidence into account and would bias these estimates. Because these relationships are well-supported and do provide considerable information on evolutionary transitions they are included in all analyses. At a more philosophical level, there are those that would argue that there is no such entity as a paraphyletic species (Baum and Donoghue, 1995; Baum and Shaw, 1995;

Cracraft, 1989) and that these entities should be subsumed under one exemplar. While

196

Figure 2.15. Most-parsimonious reconstruction of the transitions between generalists and specialists (treated as a binary character) in Stator (see Results; Figures 2.11-2.14). In both the Bayesian and parsimony phylogeny, the generalist habit originated 3 separate times.

197

Figure 2.16. Most-parsimonious reconstruction of the transitions between the three ovipositioning guilds in Stator on both the Bayesian (top) and parsimony (bottom) topology. The reconstruction clearly suggests that the ancestor of Stator oviposited directly onto scattered seeds.

198

Figure 2.17. Most-parsimonious reconstruction of the transitions between host plant genera in Stator on both the Bayesian (top) and parsimony (bottom) topology. Both topologies suggest an ancestral association with Acacia subgenus Aculeiferum. If the genus Acacia is monophyletic, then all of the basal nodes are reconstructed as being affiliated with this host plant group.

199

Table 2.8: Results of analyses of phylogenetic constraint. PTP utility based on method of Kelley and Farrell (1998). Significant results indicate that ecological characters are distributed nonrandomly on the tree, given the topology of the tree and the distribution of the character states. Ecological codings are shown in Table 2.5. All analyses are based on 10,000 replicates. * indicates significant difference.

Ecological character Parsimony Analysis Bayesian Analysis Generalist/Specialist P = 1.000 P = 1.000 Oviposition guild P = 0.0001* P = 0.0001* Acacia subg. Acacia P = 0.0009* P = 0.0100* Acacia subg. Aculeiferum P = 0.0905 P = 0.0897 Calliandra P = 0.3002 P = 0.2990 Enterolobium P = 0.0283* P = 0.0321* Pithecellobium P = 1.000 P = 1.000 Ingeae P = 0.0194* P = 0.0190*

this may be a convenient way to proceed for one interested strictly in classification, it

fully ignores the reality, complexity, and historically extended nature of the speciation

process (Harrison, 1998; Hey, 2001; Kliman et al., 2000; Wang and Hey, 1996; Wang et

al., 1997), thereby leading to a deficient study of the macroevolution of ecological associations in this genus.

Parsimony reconstructions. Most-parsimonious reconstructions of diet breadth

(when treated as a dichotomous character) on both the parsimony tree and the Bayesian tree indicate that the ancestral condition for the genus Stator was to be relatively

specialized (Figure 2.15). In both analyses, generalists evolved from more specialized

ancestors on three separate occasions—in S. pruininus, S. limbatus, and S. sordidus.

Interestingly, there appear to have been two speciation events that have been

accompanied by specialization within the latter two widespread and ecologically diverse

species: S. beali within S. limbatus; and S. chihuahua + S. pygidialis within S. sordidus.

200 Treating specialization as a dichotomous character in this manner decreases the

resolution of the analysis, as diet breadth is not readily split dichotomously and is a

function of both host affiliation and the phylogenetic distribution of those hosts. A more

directed analysis that looks at the evolution of the nested subsets of host plant clade

(column 6, Table 2.5) suggests that diet breadth and host affiliation evolve rapidly on the

phylogeny, as none of the ancestral nodes are unequivocally reconstructed (Figure 2.18).

Because of this it is difficult to say with any confidence that the ancestral association in

Stator was as a specialist on Acacieae, Ingeae, or on both. Based on the previous

analysis, a broad generalist as the ancestor is most readily rejected.

Most-parsimonious reconstructions of host plant use per se, without regard to diet

breadth, do provide a more resolved picture of the history of host associations (Figure

2.17). The associations with host plants that are exclusively limited to the generalists are

independently derived in each of those lineages (the codings ‘other Ingeae’ and ‘other

While only 11 of 23 nodes are reconstructed with significant support, there is a non-

significant trend of reconstructing all of the basal nodes as being somewhat more

generalized. In this case there are six origins of specialization, although I must reiterate

that the ancestral reconstructions are not statistically significant.

Reconstructing diet breadth while incorporating the axis of host taxon affiliation could lend greater insight into diet breadth evolution, as the previous analysis treating diet breadth as a binary character may be too blunt of a tool. When nested subsets of host plant clade (column 6, Table 2.5) are reconstructed using maximum likelihood, it again appears as though diet breadth and host affiliation evolve rapidly on the phylogeny, as only 9 of 23 nodes are reconstructed with significant support (Figure 2.20). While there

201

Figure 2.18. Most-parsimonious reconstruction of the transitions between the clades of host plants used by Stator on both the Bayesian (top) and parsimony (bottom) topology. Clades are based on the nested subsets of hosts used by different species of Stator.

is a trend toward feeding on multiple host plant clades in the ancestors of the species of

Stator, this reconstruction is not significantly better than feeding on a single host plant clade.

202 Like the parsimony-based reconstructions, oviposition behavior is the most readily reconstructed using maximum likelihood. 17 of the 23 nodes are reconstructed with 95% confidence or better, and two more are within 93% confidence intervals for being significantly reconstructed (Figure 2.21) The maximum-likelihood reconstructions support the conclusions based on the parsimony that there has been a single origin of ovipositing directly onto the integument of the pod (guild A). However, the reconstruction of guilds B and C differ slightly between the two analyses. The parsimony reconstruction postulates that there have been two origins of guild B within the Limbatus clade, and a third by S. pruininus, with guild C being plesiomorphically homologous throughout the phylogeny (Figure 2.16). The ML reconstruction suggests a single origin of guild B within the Limbatus clade that is convergent with the origin in S. pruininus, and three parallel shifts to guild C, two of which are nested within the Limbatus clade

(Figure 2.21). The unique host association of S. pacarae + S. generalis with the indehiscent genus Enterolobium, and the secondarily derived association of S. testudinarius with indehiscent are quite readily reconciled with a higher probability of multiple origins of guild C within the Limbatus clade. The convergent oviposition guild reconstruction would parallel the convergence in feeding on indehiscent host plants, and in fact becomes an equally parsimonious solution when the outgroup state of guild A is included in the analysis. This seems a reasonable tactic as any of the potential sister clades of Stator, and indeed the majority of seed beetles, belong to guild

A.

Generalized least squares reconstructions of the continuous quantifications of diet breadth on the Bayesian topology support similar conclusions to the parsimony and

203

Figure 2.19. Maximum likelihood reconstruction of ancestral states for diet breadth when coded as a binary (specialist/generalist) character. All reconstructions were based on the "local" approach to ancestral reconstruction (Pagel 1999), using branch lengths based on maximum likelihood analysis. No scaling transformations were applied to the data as the added parameter did not increase in a significant increase in likelihood score. The nodes nested within the paraphyletic S. limbatus and S. sordidus were fixed as generalist, given the external information of the paraphyletic relationship. Proportions indicated with a star are significantly reconstructed based on a difference of 2 log likelihoods.

204 maximum likelihood analyses of discrete quantifications of diet breadth—that this character evolves very rapidly on the tree. There appears to be no trend of increasing or decreasing specialization when quantified based on the number of species in the diet

(Figure 2.22), as 13 of the 43 transitions increase in diet breadth, and the remainder decrease. Almost identical conclusions can be drawn when reconstructions are based on the number of genera used (12 of 43 transitions; Figure 2.23) the PD index for hosts used based on Grafen branches (14 of 43 transitions; Figure 2.24) or the PD index for host used based on minimal evolution branches (13 of 43 transitions; Figure 2.25).

Trajectory of Specialization

The trajectory of specialization was tested with a maximum-likelihood framework using the continuous characters in Table 2.6 and a Generalized Least Squares approach.

In no case is a model of evolution which incorporates a directional trajectory of host use significantly more likely than a random walk model of evolution when applied to the untransformed data set and phylogeny (Table 2.9). While in all cases the trajectory is assessed as decreasing diet breadth toward the terminal nodes of the phylogeny, the standard errors encompass positive values as well. These results indicate that when treated independent of host use, diet breadth does not significantly decrease toward terminal nodes.

However, the evolution of diet breadth has not necessarily followed the topology or branch lengths of phylogenetic relationships, and therefore a model of evolution described by the constant-variance random walk model (the default setting in

Continuous) may not accurately estimate the true phylogenetic parameters of the data set.

205

Figure 2.20. Maximum likelihood reconstruction of ancestral states using Multistate (Pagel 2002) for clade use treated as feeding on only Acacia, only Ingeae, or multiple clades. All reconstructions were based on the "local" approach to ancestral reconstruction (Pagel 1999), using branch lengths based on the Bayesian analysis. Fixing the forward and backward rates to be equal did not significantly decrease the likelihood score, therefore the number of parameters was decreased from six independent parameters to two. A scaling transformation was also applied to the data as it significantly increased the likelihood score. Proportions indicated with a star are significantly reconstructed based on a difference of 2 log likelihoods.

206

Figure 2.21. Maximum likelihood reconstruction of ancestral states for oviposition behavior. All reconstructions were based on the "local" approach to ancestral reconstruction (Pagel 1999), using branch lengths based on maximum likelihood analysis. Fixing the forward and backward rates to be equal did not significantly decrease the likelihood score, therefore the number of parameters was decreased from six independent parameters to two. However, no scaling transformations were applied to the data as they did not significantly increase the likelihood score. Proportions indicated with a star are significantly reconstructed based on a difference of 2 log likelihoods.

207

Figure 2.22. Reconstruction of ancestral states of number of species used as hosts based on the Generalized Least Squares approach of Martins and Hansen (1997). Standard errors are shown in parentheses. Because these are species totals, no standard errors for species tallies (shown in boxes at tips) were used.

208

Figure 2.23. Reconstruction of ancestral states of number of genera used as hosts based on the Generalized Least Squares approach of Martins and Hansen (1997). Standard errors are in parentheses. Because these are species totals, no standard errors for species tallies (shown in boxes at tips) were used.

209

Figure 2.24. Reconstruction of ancestral states of diet breadth based on the Generalized Least Squares approach of Martins and Hansen (1997). Reconstructions are based on the quantification of the phylogenetic diversity of hosts based on the Grafen tree. Standard errors are shown in parentheses. Because these are species totals, no standard errors for species tallies (shown in boxes at tips) were used.

210

Figure 2.25. Reconstruction of ancestral states of diet breadth based on the Generalized Least Squares approach of Martins and Hansen (1997). Reconstructions are based on the quantification of the phylogenetic diversity of hosts based on the minimal evolution tree. Standard errors are shown in parentheses. Because these are species totals, no standard errors for species tallies (shown in boxes at tips) were used.

211

Table 2.9: Results of analyses of trajectory of diet breadth using the program Continuous. Untransformed data indicate estimates based on the default settings of a standard constant-variance random walk model of evolution. Transformed data indicate estimates based on scaled transformations of this model of evolution when a standard model does not accurately explain the data on the phylogenetic hypothesis. Scaled parameters are estimated for the null model of no significant trajectory to evolution. β (with standard errors) refers to the estimated directional change in diet breadth. Κ refers to the transformation required to take into account the relatively punctuational mode of evolution of diet breadth. λ refers to the transformation required to take into account the relative independence of diet breadth from the phylogenetic topology. Significance values are based on the likelihood ratio test using the chi-squared distribution and one degree of freedom. * indicates significant difference.

Diet Likelihood Likelihood P- Estimate of β Estimate of Estimate of breadth of H0 of HA value (S.E.) Κ λ measure Untransformed data: # genera -81.380 -81.352 0.82 -17.09 (51.14) NA NA # species -108.37 -108.30 0.72 -67.09 (157.2) NA NA PD -109.94 -109.88 0.73 -69.98 (167.9) NA NA (Grafen) PD (ME) -109.41 -109.37 0.78 -59.00 (164.3) NA NA Transformed data (Κ and λ jointly estimated): # genera -76.534 -74.385 0.04* -12.31 (5.88) 0.42 0.0 # species -104.78 -101.46 0.01* -43.91 (16.49) 0.39 0.0 PD -104.67 -101.62 0.01* -72.06 (28.34) 0.55 0.0 (Grafen) PD (ME) -103.68 -101.03 0.02* -40.82 (17.39) 0.41 0.0 Transformed data (λ estimated): # genera -77.069 -76.413 0.25 -26.73 (23.44) NA 0.0 # species -105.42 -104.12 0.11 -119.6 (74.37) NA 0.0 PD -104.99 -103.94 0.15 -106.6 (73.80) NA 0.0 (Grafen) PD (ME) -104.22 -103.31 0.18 -96.50 (71.88) NA 0.0 Transformed data (Κ estimated): # genera -78.018 -77.061 0.17 -4.73 (3.44) 0.17 NA # species -106.14 -105.33 0.20 -17.36 (13.74) 0.22 NA PD -106.65 -105.62 0.15 -18.40 (12.94) 0.20 NA (Grafen) PD (ME) -105.29 -104.26 0.15 -11.07 (7.74) 0.09 NA

One of the useful features of the program Continuous is that it allows scaling the

parameters of the model of evolution to correct for deviations from this generalized least-

squares model. One can correct for significant deviations from a purely gradual mode of

212 evolution (estimated using the parameter kappa, Κ), a constant rate of change of evolution (estimated using the parameter delta, δ), or the dependence of trait of evolution on phylogeny (estimated using the parameter lambda, λ). If these parameters differ significantly from the default values, incorporating their estimation into the analysis of the data can significantly improve the fit of the data to the model. For this reason I examine deviation of these values from the default values and examined the trajectory of specialization using the scaled parameters. Parameters were tested and scaled based on the null model of no directional change in order to take a conservative approach to examining significant trends in specialization. In all four metrics, both Κ and λ are significantly different than one. They are therefore estimated jointly for the standard constant-variance random walk model, and these estimates are incorporated into the likelihood analysis examining if there is a significant trend to specialization. When these corrections are applied to the datasets, there is a significant trajectory toward specialization in all four metrics (Table 2.9). Unfortunately, interpretation of this result is not straightforward because in all cases λ was estimated as zero. This indicates that internodal corrections are not necessary and instead implies that lower diet breadth values are found on species whose root to tip lengths are longer, a result interesting in and of itself. The interpretation becomes particularly difficult given that Κ is also not estimated appropriately by a simple random walk model of evolution. This suggests that speciation is significantly correlated with diet breadth change and argues for a punctuational model of evolution in this trait. Therefore, the number of internodes leading to a terminal taxon also affects the estimate of a trajectory toward specialization, with a significant result suggesting that lower diet breadth values are found on species whose root to tip path

213 traverses more internodes. In fact, the significant trajectory toward increased

specialization is the result of synergistic effect between both longer path lengths from

root to tip for specialists and more internodes from root to tip for specialists. Estimation

of these parameters individually did not result in a significant trajectory toward

specialists (Table 2.9).

I also examine the trajectory of specialization in combination with the taxon being

specialized upon in order to address host-lineage-specific effects. For both specialists on

Acacia and specialists within the tribe Ingeae (the two classes of specialists observed

within the genus Stator), I examine the trajectory of specialization by setting the forward

(α) transition rates as equal to the backward (β) transition rates in a continuous time

Markov model on the phylogram from the Bayesian analysis and compare the log- likelihood scores to an unrestricted model (in which α and β are allowed to vary considerably). For both analyses, adding an estimate of the scaling parameter Kappa does not significantly improve the log-likelihood. In the case of Acacia specialists, there is no significant difference in log-likelihood scores between the unrestricted model and the restricted model (lnL = -25.11 vs. lnL = -25.34; P = 0.50), supporting no significant trajectory toward specialization on Acacia. This is also seen in the similar maximum likelihood estimates of the forward and backward transition rates (α = 3.04, β = 4.51). In stark contrast is the analysis of specialization on hosts in the tribe Ingeae. In this case the unrestricted model is significantly better than the restricted model (lnL = -23.96 vs. lnL =

-29.24; P = 0.001), supporting a significant trajectory in Ingeae specialization.

Interestingly, the model estimates the backward transition rates as being roughly five times higher than the forward transition rates (α = 11.48, β = 50.00). The implications of

214 this given the distribution of Ingeae specialists on the tree are addressed in the Discussion section.

Correlation between diet breadth and oviposition behavior

Treating each oviposition guild as separate binary characters reveals that diet breadth is significantly associated with oviposition guild when diet breadth is quantified using a phylogenetic index (P = 0.05), and approaches significance when measured based on number of species (P = 0.06) or number of genera (P = 0.07) in the diet (Table 2.10).

This is true even when taking into account corrections due to phylogenetic

Table 2.10: Correlation between oviposition behavior and diet breadth. All correlations were examined with each oviposition guild separately. The scaling parameter Kappa was estimated for the null hypothesis of no correlation and incorporated into all analyses. Estimation of neither delta nor lambda resulted in significant departure from a Markov model of character change. Correlations with the four quantifications of diet breadth in Table 2.6 were examined. Significance values are based on the likelihood ratio test using the chi-squared distribution and one degree of freedom. * indicates significant difference at the 0.05 level.

Diet breadth measure Likelihood of no Likelihood with P-Value correlation (H0) correlation (HA) Oviposition Guild A # genera -66.605 -66.416 0.54 # species -94.112 -94.027 0.68 PD (Grafen) -95.207 -95.093 0.63 PD (ME) -94.078 -93.886 0.54 Oviposition Guild B # genera -85.485 -83.864 0.07 # species -113.25 -111.48 0.06 PD (Grafen) -114.03 -112.13 0.05* PD (ME) -113.08 -111.18 0.05* Oviposition Guild C # genera -85.537 -84.808 0.23 # species -113.21 -112.31 0.18 PD (Grafen) -114.08 -113.02 0.14 PD (ME) -113.17 -112.23 0.17

215 autocorrelation by using a maximum likelihood treatment of continuous characters. In

particular, increased diet appears to be correlated with oviposition guild B, ovipositing

directly onto the seed while it is still inside the pod on the plant.

DISCUSSION

The history of host shifts and host specialization in the genus Stator appear to be structured by a complex interaction between host taxon use, diet breadth, and oviposition behavior. When considered separately, the macroevolution of each of these aspects of host plant affiliation provides a limited picture of host use evolution within the genus

Stator. When considered jointly, it is clear that the macroevolution of ecological associations is contingent upon the emergent properties of these variables acting together.

The geographic context of diversification.

Phylogenetic reconstruction of the genus Stator suggests that the deeper-level macroevolutionary diversification within this genus has been distinctly structured by geographic history. There are two major geographic clades within the genus: one represented by the Subaeneus clade; the second represented by the Limbatus and

Aegrotus clades. The former clade is present almost exclusively within North America

and the Caribbean, with three widespread species only reaching into very northern South

America (S. pruininus, S. vachelliae, and S. sordidus). The latter clade is present almost

exclusively within South America and tropical Central America, with only S. beali

having originated within North America from a more recent invasion northward by S.

limbatus. However, there is no clear geographic structure to more recent diversifications,

216 with broad sympatry exhibited by some sister species (S. furcatus + S. tigrensis; S. beali

+ S. limbatus; S. monachus + S. vittatithorax; S. vachelliae + S. pruininus), nearly strict

allopatry being held by others (S. generalis + S. pacarae; S. trisignatus + S.

maculatopygus; S. bottimeri + S. chalcodermus; S. chihuahua + S. pygidialis), and partial range overlap displayed by S. mexicanus + S. subaeneus. Only S. bottimeri (in the northern Caribbean), S. chalcodermus (in the southern Caribbean), S. testudinarius (west

of the Andes in South America), and S. pygidialis (in the mountainous areas flanking the

Sonoran desert) have distributions largely isolated from other Stator species, although the

second two do overlap to a large extent with S. limbatus which has secondarily expanded

into Ecuador and the southern Caribbean.

The communities of Stator species are therefore structured to some extent by the

geographic context of diversification in this group. For the most part, the species in the

Subaeneus clade do not form a constituent of the South American seed beetle

communities, and to a large extent the species in the other two clades do not form a

component of North American communities. This distinction breaks down from northern

Venezuela to southern Mexico as the faunas appear to have mixed more recently in these

areas. Despite this caveat, the composition of Stator communities at more local levels

appears to be the result of ecological processes such as host plant taxon and phenological

partitioning.

History of host associations.

Like in most phytophagous insects, the genus Stator exhibits moderately strong

levels of phylogenetic constraint in host taxa used (Farrell and Mitter, 1993b), but this is

217 highly dependent on the inclusiveness of the host clade being considered (Funk et al.,

1995). At the most obvious level is an association with either the Acacieae or Ingeae by

every species in the genus, and the exclusive association with these by all of the specialist

species. In stark contrast to this, the three generalists appear to have independently

broken this constraint and secondarily invaded hosts (to one degree or another) in one of the two remaining tribes of Mimosoideae, the Mimoseae, and in the two remaining subfamilies of legumes, the Papilionoideae and Caesalpinioideae. This suggests that for some reason the colonization of novel hosts in these species is not coincident with cladogenesis nor is it limited by the level of phylogenetic constraint which characterizes the macroevolutionary diversification of more specialized lineages. Why these generalists appear to have escaped the shackles of the phylogenetic constraint that limits

ecological diversification throughout the rest of the genus is not an obvious question to

address. All three species occur sympatrically with closely related specialists. As such,

they do not appear to occur in notably more extreme or heterogeneous climates than the

specialists, one ecological circumstance that has been suggested to promote higher levels

of resource generalism (Funk and Bernays, 2001; Futuyma and Moreno, 1988; Jaenike,

1978; Jaenike, 1990). Furthermore, the three species do not appear to be comprised of

populations that are specialized (Fox et al., 1999; Fox et al., 1996a; Fox et al., 1997; Fox

et al., 1994; Johnson, 1981c). They could differ significantly in their population genetic

structure, have independently evolved detoxification or host-searching mechanisms that

allow such distinctive host expansion, or they could use hosts in a manner that permits

them to expand their host use. While these and other explanations are beyond the scope

of the current research, such comparative study between specialists and generalists within

218 this phylogenetic context should be highly illuminating for studying the processes leading to such divergent ecological habits.

At higher levels of host clade resolution, feeding on the two subgenera of Acacia is most highly conserved across the phylogeny, although feeding on subgenus Acacia is significantly correlated with phylogeny, while feeding on subgenus Aculeiferum only approaches statistical significance at the standard 0.05 level. In addition, feeding on the genus Enterolobium is also significantly conserved; although this habit is only present in the sister species pair S. generalis + S. pacarae. Feeding on any one of the remaining genera within the Acacia + Ingeae alliance is not significantly conserved, as these arise multiple times either as the constituent of a specialist’s diet or as part of a generalist’s diet.

Interestingly, while the Ingeae taxa that serve as hosts for Stator form no coherent grouping according to the cladistic analysis of Grimes (1999) or to recent revisionary work on these taxa (Barneby and Grimes, 1996; Barneby and Grimes, 1997; Grimes,

1995), feeding on Ingeae is significantly conserved across the Stator phylogeny. This suggests that not only is there a proclivity for Stator species to shift either to use host plants within this clade or to include them in their diet as they broaden their host range, there is also a tendency for such relationships to be carried across speciation events.

Unlike associations with the genus Acacia there is only one instance—the association with Enterolobium—in which an association with Ingeae is carried across a speciation event that separates specialist taxa. The remainder of the constraint appears to be due to the close relationship between generalists and Ingeae specialists. In fact, when the generalists are removed from the analysis, this significant level of phylogenetic constraint

219 disappears. It therefore appears that this significant constraint may be due to two separate explanations: (1) generalists may serve as a stepping stone for the colonization and subsequent specialization onto Ingeae; and (2) the methods were flawed by not incorporating the ancestral associations within the generalist species into the analysis of constraint. The first explanation could apply to: (1) the paraphyletic relationship of S. limbatus with S. beali, in which the shift to specializing on Ebenopsis ebano by the latter was facilitated by the expanded host range of the former; and (2) the paraphyletic relationship of S. sordidus with S. chihuahua + S. pygidialis, with the recent specialization of S. pygidialis on Calliandra humilis facilitated by the initial dietary expansion by S. sordidus. The second explanation is entirely dependent upon the reconstruction of the ancestral state for each of the generalists. For example, if the ancestors of these generalist species fed on Acacia, then there would be evidence for multiple independent colonizations of Ingeae, not for phylogenetic constraint in this character.

The parsimony reconstructions of host use do appear to provide information regarding the ancestral associations of the generalists before they independently increased their diet, and support the contention that multiple independent colonizations of species of Ingeae have occurred. All of the ancestral nodes are unequivocally reconstructed as feeding on Acacia, although the reconstruction of the host plant of the ancestor of the genus Stator is equivocal as to the subgenus of Acacia if Merobruchus is excluded from the reconstruction. It therefore appears that Stator originally diversified onto the ecological opportunity provided by the diversification of the New World acacias. This early diversification then set the stage for subsequent colonization of other lineages of

220 woody legumes, predominantly those in the closely related Ingeae. In addition, the major

hosts of all three generalists are acacias. This observation in and of itself is not

necessarily informative as to their ancestral relationship. But within the context of both

the levels of phylogenetic constraint across the genus and the ancestral state

reconstructions it does support the suggestion that the majority of evolutionary

diversification in the genus occurred against a background of feeding on the genus

Acacia (see similar argument in Schluter, 2000).

The analyses of both phylogenetic constraint and ancestral reconstructions suggest

that there have been multiple independent colonizations of host plants in the tribe Ingeae.

Why this is the case is an interesting question to ponder. One possibility is that plants

bearing the distinctive morphology of the Ingeae are of much more recent origin than

Acacia. If the phylogram in Robinson and Harris (2000) is an approximation of the relative divergence times of the Acacieae and the Ingeae, then this is a plausible suggestion. As a result, lineages of Ingeae would have been available for colonization for a much shorter period of time. Such a scenario would suggest that future diversification of Stator may result in the appearance of lineages that are restricted to particular clades of

Ingeae, as appears to be happening for the lineage currently feeding on Enterolobium.

Testing of this hypothesis would require adequate estimation of divergence dates for both the beetles and the hosts, information that is currently not available. An additional hypothesis is that feeding on acacias provides a preadaptation for feeding on the morphologically and phenologically similar fruits of the Ingeae. If this is the case, then shifting to these hosts would not require overcoming significant developmental, physiological, or behavioral barriers. Testing of this hypothesis could certainly take the

221 form of assessing Acacia specialists for genetic variation to use Ingeae host species (e.g.

Funk, 1998; Futuyma et al., 1995). Lastly, such a homoplasious distribution is often suggested as a signature for the role of natural selection in shaping ecological traits

(Coddington, 1988; Losos and Miles, 1994). Therefore, this pattern may indicate significant selection pressure for colonization of the Ingeae that is imposed by the ecological opportunity they present in various communities. Testing of this hypothesis in general is difficult because it relies on the historical reconstruction of host colonization and community structure. However, specific cases could be examined given an extensive phylogeographic analysis in combination with an adequate paleontological record of the history of floral communities. None of these hypotheses are mutually exclusive and could certainly be acting either in combination or separately throughout the phylogeny.

One interesting observation regarding these hypotheses is that two other genera of

Bruchinae have overlapping host assemblages. The genus Merobruchus shows almost identical taxonomic patterns of host use across almost identical species diversities within almost identical geographic ranges. It includes a large number of species that are specialists on Acacia (all in the subgenus Aculeiferum), some that are specialists on species of Ingeae, and some generalists that include species from both taxa. In contrast, species in the mostly North American genus Mimosestes are primarily specialists on species of Acacia subg. Acacia. And while one species is a specialist on the genus

Enterolobium, the remaining specialists are associated with other mimosoids (Mimoseae:

Prosopis L. or Parkieae: Parkia R. Br.) or caesalpinioids (Cercidieae: Cercidium Tul. +

Parkinsonia L.), and the generalists broaden their diet along these trajectories. While there are no phylogenetic analyses of either of these genera, comparative analysis

222 including these could reveal convergent or divergent patterns in host use diversification and provide some indication about how communities of bruchines are formed.

Trajectory of diet breadth evolution.

One of the goals of this chapter is to assess whether there is a phylogenetic trajectory to diet breadth evolution per se, without reference to any other ecological or behavioral characters. In other words, is there something unique about the macroevolution of resource specialization that results in a tendency for the derivation of specialized lineages?

Diet breadth within the genus Stator appears to evolve quickly at the macroevolutionary level, similar to the observation by Kelley and Farrell (1998) for the bark-beetle genus Dendroctonus. No metric of phylogenetic constraint (parsimony or maximum likelihood, discrete or continuous) even approaches significance for this character. This agrees well with the arguments by Thompson (1994) that diet specialization is highly labile in a phylogenetic context.

The different analytical methods used to examine the evolution of diet breadth in the genus Stator provide somewhat different results. A quick perusal of the parsimony- based analysis of diet breadth as a discrete character shows that almost all trajectories of host evolution are possible: specialists can give rise to specialists, specialists can give rise to generalists, and generalists can give rise to specialists. The only transition not observed is that between generalists and generalists. Perhaps this is not surprising given that there is no longer an isolating role for host plant use. A cladogenetic event in this case would likely require geographical boundaries more pronounced than those required

223 when ecological specialization is involved in limiting the geographic distribution of a

Stator species.

The likelihood analysis of diet breadth as a binary character is somewhat more agnostic on the types of transitions. While there is significant support for transitions from specialists to specialists and generalists to specialists, there is no significant support for a transition from specialist to generalist because of uncertainly in the reconstruction of ancestral nodes. The ancestral node is reconstructed as being more likely to have been a generalist, although this likelihood is not significant. Therefore, while the reconstructions are not statistically significant, the maximum likelihood analysis is suggestive of generalism as a plesiomorphic condition for the genus. This result was affected somewhat by the inclusion of two exemplars for the paraphyletic species.

Including a single exemplar did alter the results slightly. In this analysis, the ancestral nodes joining S. limbatus + S. beali and S. sordidus + (S. chihuahua + S. pygidialis) were fixed as generalists so as to maintain the information of the paraphyletic relationship.

This resulted in small quantitative changes in the reconstruction of many nodes, but only qualitatively changed the reconstruction at the node subtending the clade including S. bottimeri and S. pygidialis. This node was significantly reconstructed as a specialist.

Therefore, including only species-level exemplars resulted in the unequivocal reconstruction of at least two independent origins of the generalist.

The lack of phylogenetic constraint in diet breadth causes the ancestral state reconstructions of the continuous measures of diet breadth using the GLS approach to have large and overlapping standard errors, making the interpretation of this analysis difficult because there are virtually no significant increases or decreases in diet breadth.

224 Taking the values at face value suggests that the ancestral state was to be slightly less

specialized than the distribution of diet breadth used by the specialist species (Grafen:

17.8 on Figure 2.11; ME: 16.2 on Figure 2.12; number of species: 16.0 on Figure 2.13;

5.4 on Figure 2.14), but rather less generalized than the three generalists. Overall,

dramatic increases and decreases in diet breadth both occur using this measure, providing

a similar result to that of the binary quantification of diet breadth. However, the

quantification for diet breadth for all three generalists lies outside of the standard errors

of the reconstruction of the root node, and for all three it lies outside of the standard error

of the immediate subtending node. This supports the parsimony-based analysis that the

generalist habit has been independently derived three separate times.

The majority of species of Stator are relatively specialized in their host associations. When looked at from the standpoint of the parsimony reconstructions of both diet breadth as a binary character or of the GLS reconstructions of diet breadth as a continuous character the distribution of this specialization across the Stator phylogeny is the consequence of both a plesiomorphic homologous association with a small number of hosts, and the homoplasious rederivation of a specialist habit on two separate occasions.

This result is neither supported nor refuted by the maximum-likelihood reconstructions.

On the other hand, notable increases in diet breadth have arisen independently on three separate occasions. This is probably not simply the result of an expanded geographic range (Fox and Morrow, 1981) for two reasons: (1) the generalists use multiple hosts even at local geographic levels; and (2) there are numerous specialist species with ranges that are similarly broad or broader than those of the generalists. S. monachus and S. vittatithorax are both found from Argentina to west-central Mexico, excluding the

225 Amazon basin, S. vachelliae is found almost entirely sympatrically with all three

generalists, and S. aegrotus has one of the largest inferred distributions in the genus. In

regard to this, in a separate project I have examined the mode of speciation using the interspecific phylogeny of Stator. This uses the correlation between genetic distance and geographic overlap to test specific hypotheses concerning the macroevolutionary signatures of speciation (unpublished data, methodology based on Barraclough and Nee,

2001; Barraclough and Vogler, 2000). An ancillary result from this analysis is that there is no correlation between diet breadth and geographic range.

While it is not surprising that shifts between generalists and specialist occur in both directions, what is of interest is whether or not specialization is more often phylogenetically derived (Janz et al., 2001; Kelley and Farrell, 1998). This tendency

would be consistent with the hypothesis that specialists evolve often but go extinct

quickly. This means that it is necessary to examine, in a statistical framework, whether

there is a bias toward derived specialization. It is not enough to simply compare the

reconstructed ancestral state for the clade with the overall tip values (sensu Schluter,

2000). Kelley and Farrell (1998) introduced a permutation based statistical test of the

‘tippiness’ of a character on a phylogenetic tree. According to this measure, neither

specialists nor generalists are significantly derived across the Stator phylogeny. This is

perhaps not surprising given that the ancestral states are reconstructed as being specialist,

and that there are only three generalists (at least 5 independent origins are required for a

significant result).

Because diet breadth is not readily categorized into two discrete states, a

continuous quantification is a more realistic metric for this trait. The analysis of the

226 trajectory of specialization using this metric in a maximum-likelihood framework also showed that there is no significant macroevolutionary trajectory toward increasing specialization (Table 2.9). Interestingly, when the data set is transformed to correct for deviations from a random-walk model, there is a significant trajectory toward increasing specialization (Table 2.9). However, this requires correcting for the fact that there is no phylogenetic structure to diet breadth and that diet breadth appears to change in a punctuational manner. The somewhat superficial interpretation of this analysis is that given enough time and speciation events, the generalists will become specialists as well.

Note that neither transformation independently results in a significant trajectory toward specialization in the phylogeny, although there is a trend in that direction.

One of the problems with the interpretation of the analyses that use branch lengths to inform the estimation of ancestral states or other aspects of macroevolutionary patterns is the significant departure from a molecular clock in the phylogenies from both the parsimony and Bayesian analyses. As a result, branch lengths are not an accurate approximation of time, an important assumption for the assessment of the opportunity for character transitions along a phylogenetic tree. Interestingly, it appears that branch length is slightly (although not significantly) negatively correlated with diet breadth.

Why there could be different rates of nucleotide substitution in specialist lineages could depend on differences in population size, differences in generation time, or differences in

DNA repair efficiency (Cherry, 1998; Li et al., 1987; Nei and Graur, 1984; Ohta, 1995;

Ohta and Ina, 1995; Sharp, 1991). The last is perhaps least likely given the relatively close relationships between all of the species in this genus. The remaining two explanations have some biologically plausible explanations.

227 If population sizes are consistently smaller for specialists or if dramatic changes in

population size occur more frequently in specialists, then substitution rate could be

elevated in specialist lineages (Cherry, 1998; Li et al., 1987; Ohta, 1992; Ohta, 1995;

Ohta and Ina, 1995). There are at least two contingencies for this hypothesis to have any explanatory power. The first is that the population dynamics of specialists are significantly different from generalists so as to cause such a difference. This in fact would be the population biology corollary of the macroevolutionary hypothesis of specialization as a ‘dead end’ (Futuyma and Moreno, 1988; Thompson, 1994). Second, the population size or changes in population size should have no effect on the substitution rate of neutrally evolving sites (Graur and Li, 2000; Kimura, 1962; Nei and Graur, 1984), only on non-synonymous or nearly neutral sites (Ohta, 1992; Ohta, 1995). However, the majority of changes in the phylogeny of Stator in both Cytochrome Oxidase I and

Elongation Factor 1α are in neutral characters (third positions and toggle first positions).

This could suggest that these presumptively neutral characters actually evolve under some selective constraints, such as availability of tRNA codons or nucleic acids. The presence of severe codon bias in the third codon position in the mitochondrial genome of insects in general, and the heterogeneity in the third position of COI in this phylogeny suggest that this may indeed be the case.

A second explanation would be a shorter generation time or more generations per year for the specialists (Li et al., 1987). This would appear to contradict the biology of specialists and generalists. While capable of being continuous breeders (Fox and

Mousseau, 1995b; Johnson, 1981b; Johnson, 1982), the majority of Stator specialists probably reflect the annual phenology of their hosts and are largely univoltine or

228 bivoltine. On the other hand, the more generalist species have potential year-round hosts and can be reared from seeds at many locales throughout the year. This results in a multivoltine, continuous breeding population with many more generations per year than specialists.

Sequence data from more loci would need to be collected to determine if there really is a diet breadth dependent rate of molecular evolution, as well as to demonstrate that such dependence is the result of population-level processes that affect the entire genome and not the result of female-specific, organelle-specific, or gene-specific processes (Kliman et al., 2000; Wakeley and Hey, 1997). Research on the historical demography of specialist and generalist species could also examine whether or not there is a bias in the population biology of specialist toward smaller population sizes or more dramatic fluctuations in population size. Finally, research into the population biology of generalists and specialists to more adequately estimate such parameters as standing population size and voltinism in the natural environment would shed considerable light on this issue.

The preponderance of the data and analyses of phylogenetic trajectory do not support the hypothesis that specialists occupy more derived positions in the phylogeny.

This result is in agreement with other recent studies that have found a similar result

(Brown, 1994; Brown et al., 1997; Dobler et al., 1996; Janz et al., 2001; Termonia et al.,

2001), but is in contrast with others (Dobler and Farrell, 1999; Funk et al., 1995; Kelley and Farrell, 1998; Moran, 1988). Explanations for the absence of such a pattern include the possibility that specialists maintain genetic variation in host-use characters to allow host shifts either through genetic variation in defense-related characters within host plant

229 species (Hawthorne, 1997; Rausher, 1984; Thompson, 1994) or in similarity in defense-

related characters between closely related species (Becerra and Venable, 1999; Ehrlich and Raven, 1964; Futuyma et al., 1995). The latter seems a highly likely explanation for the persistence of specialists that are limited to feeding on Acacia. First of all, the degree of phylogenetic constraint for feeding on this taxon suggests that closely related species of Stator often feed on species of Acacia. Second, the genus Acacia in the New World is considered to be highly problematic taxonomically because of the extreme similarity of species, the apparent overlap of variation in numerous morphological characters between species, and the high levels of hybridization that occur in the genus (Clarke et al., 1989;

Clarke et al., 1990; Janzen, 1974; Lee et al., 1989; Seigler and Ebinger, 1988). This may provide opportunities to shift between numerous host Acacia species given changes in the frequency of particular Acacia species within a given community. On the other hand, the pod and seed morphology within the Ingeae show considerable disparity, and there is considerably less geographic overlap in congeneric species in this group than in Acacia

(Barneby, 1998; Barneby and Grimes, 1996; Barneby and Grimes, 1997). This combined with the differences in phylogenetic constraint in species that use different genera of

Ingeae (discussed previously) could well suggest that in macroevolutionary sense the genus Stator uses Acacia differently than it does Ingeae.

Diversification and oviposition behavior.

The most phylogenetically conservative aspect of host use in the genus Stator is the oviposition behavior displayed by females (Figures 2.16 and 2.21). While behavioral characters are often assumed to evolve too quickly to contain historical evidence of

230 relationships, this result agrees with arguments to the contrary (Brooks et al., 1995;

Lorenz, 1965; Miller and Wenzel, 1995; Wenzel, 1992). It appears that this phenological

partitioning of their host plants may have allowed parallel radiations onto either different

or overlapping resources. The Aegrotus clade shows the least degree of host shifting or

of variation in diet breadth, and also includes the only species restricted to ovipositing

directly onto the integument (or valve) of the pod of their host plants. This appears to

limit these species to those legumes with pods that have notably thin integuments,

particularly Acacia subg. Aculeiferum. The only shifts away from this clade of Acacia are by S. vittatithorax to Lysiloma acapulcense (Kunth) Benth. and Zapoteca formosa

(Kunth) H.M. Hern., two species with similar, paper-thin valves (Barneby and Grimes,

1996; Hernández, 1986; Hernández, 1989; Thompson, 1980). Depositing eggs on

scattered seeds (guild C) is the rarest strategy of the three guilds throughout the

Bruchinae, yet may be the only of the three behaviors that has originated multiple times

within the genus Stator. It appears that the evolution of this strategy has allowed the

Subaeneus clade to diversify onto indehiscent and/or animal dispersed legumes (Acacia

subg. Acacia and Pithecellobium) and explosively dehiscent legumes (Calliandra). It has

also permitted two subsequent shifts from using strictly dehiscent legumes to using

indehiscent legumes within the Limbatus clade. Guild B, ovipositing directly onto seeds

before they have been dispersed, appears to have evolved twice: once by the ancestor of

the Limbatus clade and a second time by S. pruininus within the Subaeneus clade.

It appears that the transition between ovipositing on pods and ovipositing directly

on seeds may be more constrained than the transition between ovipositing on pre-

dispersal versus post-dispersal seeds. If one separates oviposition behavior into these two

231 characters, then there is only a single transition between oviposition substrate while there are three transitions between the dispersal status of the host seed. This could suggest that the barriers presented by the initial tissue encountered by the first instar larva (whether chemical or structural) constrain the macroevolution of host use more than do the barriers presented by the changes in host-searching strategy likely required by shifting to oviposit on scattered seeds. One possible explanation of this is that the latter barriers have historically not been as stringent as they may seem at face value. The indehiscent fruits used by all members of guild C (with the exception of S. pygidialis, which uses an explosively dehiscent host) appear to be adaptations for dispersal by vertebrate herbivores. Pods of these plants are consumed as fodder by cows, goats, sheep, donkeys, horses, and (among other things) throughout the Neotropics (Barneby and Grimes,

1996; Janzen, 1969; Mesquita, 1990; Traveset, 1990), and likely evolved in the context of being dispersed by a now extinct vertebrate megafauna (Janzen, 1981b; Janzen, 1981c;

Janzen and Martin, 1982; Siemens and Johnson, 1996). The seeds of these plants pass through the guts of these dispersal agents in tact but now released from the indehiscent pods. They are then deposited along with the dispersers’ dung in great numbers and high concentrations at some distance from the parent plant. The species of Stator that attack these seeds are collected in greatest numbers by interested humans by locating large piles of dung, and a similar strategy could be at work in the search strategy of the female beetles. Given that the search strategy may not be as limited as searching for a single seed in a figurative haystack of an environment, the multiple transitions between guilds B and C are perhaps not all that surprising.

232 In addition, the phenological differentiation involved between ovipositing on pre-

dispersal versus post-dispersal seeds has probably allowed Stator to diversify as guild C

onto host plants that would not otherwise be available. Stator larvae do not appear to

have the ability to penetrate the thick integument of most animal-dispersed pods (Center

and Johnson, 1974; Janzen, 1969), therefore this phenological stage of potential host

plants are probably not available from a strictly morphological standpoint. More than

that, however, is the observation that the majority of these animal dispersed pods are used

by more robust species of Bruchinae that oviposit directly onto the integument of the pod,

particularly the genera Merobruchus and Mimosestes and certain species of

Acanthoscelides (Johnson, 1981d; Johnson, 1983; Johnson and Siemens, 1991b; Johnson

and Siemens, 1996; Johnson and Siemens, 1997b; Kingsolver, 1988; Kingsolver and

Johnson, 1978). Evolving the ability to use post-dispersal seeds probably served as a key

innovation to exploit this abundant resource of seeds that had been saved by vertebrate

dispersers from the pre-dispersal seed predation of other bruchine species. They

therefore exploited a new resource and escaped competition in a single step.

While the convergent shifts by S. testudinarius and S. generalis + S. pacarae to indehiscent host plants may have been facilitated by having ancestors that already oviposited directly onto seeds, it is possible that there are other factors that contributed to this transition in host use and oviposition behavior. First is the community context within which these shifts occurred. S. testudinarius is the only species limited to the west of the

Andes in South America. While few species of dehiscent Acacia occur in this area, the xeric landscapes are dominated by indehiscent species of Acacia, most notably A. macracantha and A. farnesiana. Because members of guild C are completely absent

233 from this area, these likely presented available ecological resources that S. testudinarius

was able to exploit. Trees in the genus Enterolobium present a massive resource for seed

beetles to exploit. They dominate many of the savanna landscapes of the Neotropics and

produce an extraordinarily large number of seeds per tree (Janzen, 1969; Johnson and

Janzen, 1982; Mesquita, 1990; Siemens and Johnson, 1996). In the face of competition

on ancestral legumes, any genetic variation to use this unoccupied resource would likely

have been under strong directional selection. The second factor is the possibility of

entrance into enemy-free space. As noted earlier, many parasitoids use volatile

compounds to locate the plant hosts of their insect hosts. Shifting to oviposit on seeds

that have already been dispersed away from the parent hosts is likely to result in the

initial escape from such parasitoids and could be an important selective force in such a

transition (Gratton and Welter, 1999; Strong et al., 1984). No parasitoids have been

reported in the literature for S. testudinarius, S. pacarae, or S. generalis, and in

collections that resulted in rearing at least 1,000 individuals from each of these species, I

did not rear a single parasitoid wasp.

The phenological differentiation in oviposition behavior between species of Stator

may also permit overlap of host use between the various clades. For the most part, it

appears as though indehiscent fruits are excluded from the diet of species in both guild A

and B. For example, S. limbatus has the widest diet breadth of any species in the genus, and yet is not known to attack any species in Acacia subg. Acacia. The fact that they do not attack the dehiscent members of this subgenus, Acacia constricta Benth. ex A. Gray and its close relatives, may indicate that there may be other factors causing this pattern such as biochemical considerations or competition from S. pruininus (Siemens et al.,

234 1991). However, there is considerable overlap in host use between members of guild A and guild B. It is quite common to rear members of both guilds from pods collected at the same locality, in fact. The only species of guild B that does not overlap with Stator species in guild A to some degree is S. beali, a strict specialist on the Texas ebony tree

Ebenopsis ebano. However, this also supports the suggestion that guild differentiation allows species coexistence, as E. ebano is the only host plant for Merobruchus major

(Fall), which is in guild A. Apparently, enough resources are still available by the time that the pods of the host plants of the Aegrotus clade dehisce to support the successful development of species in guild B, suggesting that there may be density-dependent regulation of populations of species in guild A. The possibility that population sizes in guild A are thus limited is also supported by the observation that different Stator species of guild A are often reared from the same host plant pod, much less the same host plant species. For example, it is quite common to rear both S. vittatithorax and S. monachus together throughout their range; to rear S. vittatithorax from the pods of the single host

(A. bonariensis Gillies ex Hook. & Arn.) of S. maculatopygus; to rear S. trisignatus from the single host (A. hayesii) of S. aegrotus; and to rear S. trisignatus and S. vittatithorax together from their hosts in northern South America. Such coexistence of species that clearly use local resources in an almost identical manner suggests that interspecific and/or intraspecific competition is not limiting (Denno et al., 1995; Hairston et al., 1960;

MacArthur and Levins, 1967). Three testable hypotheses can contribute to this observation: (1) members of guild A vary considerably in their ability to use alternate host plants (Fox and Morrow, 1981), and the hosts on which they have a competitive advantage serve as a source for contributing to other hosts on which they could not

235 maintain persistent populations (Thompson, 1988b; Thompson, 1994; Thompson, 1999);

(2) populations of species in guild A are limited by plant defenses to a greater extent than

are members of other guilds (Hairston et al., 1960; Strong et al., 1984), preventing

competitive exclusion; and (3) populations of species in guild A are controlled by

parasitoids to a greater extent than are species in other guilds due to both the presence of their eggs on the host plant (Kessler and Baldwin, 2001) and the exposure of their eggs to the external environment (Hawkins, 1994; Marvaldi et al., 2002), preventing competitive exclusion (Paine, 1966; Paine, 1971). It is quite possible that each of these is affecting the ability of species in guild A to coexist.

Oviposition guild diversification has clearly structured the macroevolution of host use of the genus Stator. It has allowed the diversification onto the otherwise unavailable resource of indehiscent fruits, and has allowed significant partitioning along the phenological axis of resource use. It is also clear that shifts in diet breadth and host plant taxon can affect evolutionary trajectories. It therefore seems quite plausible that these

factors interact to affect patterns of diversification in the genus Stator.

Taxon-specific effects on dietary specialization.

Selection for resource specialization in bruchines would appear to be very strong, given their life history and the intensity of the relationship with their host. The fact that they feed obligately within a self-contained resource places them in an ideal situation to experience the selective force of competition (Denno et al., 1995); their intense, parasitoid-like predation on the offspring of their host plants is likely to result in a constant coevolutionary arms race (Janzen, 1971b; Janzen, 1980a; Janzen, 1980b; Janzen,

236 1981a); and the ephemeral nature of seeds and the particular fruiting phenologies of host

plants may entail specialization in mechanisms for host searching in order to improve

decision-making efficiency (Bernays, 1998; Bernays and Funk, 1999; Bernays and

Wcislo, 1994). This appears to be born out by the fact that the majority of bruchines are

specialists on one to a small number of closely related plants (Janzen, 1969; Janzen,

1971b; Janzen, 1980a; Johnson, 1981a; Johnson, 1981e; Johnson, 1989a). The

diversification of bruchines suggests that specialization is probably not generally an

evolutionary ‘dead end’, and taxonomic patterns of host use associations suggest that

specialists maintain the genetic variation to use closely related host plants. This is in fact

probably generally true, as witnessed by the numerous phylogenetic studies of

phytophagous insects in which all members of the clade at issue are specialists (Farrell

and Mitter, 1990; Farrell and Mitter, 1998; Funk, 1998; Scheffer and Wiegmann, 2000;

Silvain and Delobel, 1998), an observation somewhat overlooked in the debate on

whether or not specialists tend to be derived. The phylogeny of Stator supports such a model. While this clade is unusual in that it even has notable variation in diet breadth

(Janzen, 1980a; Johnson, 1981c), the generalists are actually derived from more specialized ancestors. Additionally, Acacia specialists are closely related and evolutionary diversification in these lineages appears to be facilitated by a relative lack of ecological diversification. This raises the possibility that these species of Stator are more accurately described as Acacia generalists, with fundamental niches and realized niches not completely overlapping. Indeed, this could be true at a more general level for the phylogenetic studies of clades of specialists that were previously mentioned.

237 The hypothesis of specialization as a dead end entails the supposition that the

narrowing in diet breadth occurs along the fundamental niche of the species. Therefore, a

more accurate test of the hypothesis should examine the situation in which host shifts are

distinct enough to require the evolution of more complex adaptations, including sensory,

physiological, phenological, chemical, or behavioral adaptations. If the characters in

plants that exert this directional selection are correlated with the evolutionary history of

the host plants, one may expect that phylogenetic position could serve as a proxy for such

distinctiveness. In contrast to the Acacia specialists in the genus Stator, the host plants of

the Ingeae specialists form no coherent phylogenetic group; the lone exception being the

speciation of S. generalis and S. pacarae against a background of feeding on species of

Enterolobium.

Specialization onto these Ingeae host plants appears to have fundamentally

different macroevolutionary consequences than specialization onto Acacia. First, not

only do the host plants of the Ingeae specialists not form a coherent phylogenetic group,

but there is also no evidence of phylogenetic constraint in the use of these host plants.

The result of significant phylogenetic constraint in the use of Ingeae in general appears to

be an artifact of the tendency for generalist species to use Ingeae host plants secondarily.

Second, the phylogenetic trajectories of specialization on Acacia and specialization on

Ingeae are distinctly different. A quick perusal of ancestral state reconstructions (Figures

2.17, 2.18, and 2.20) suggests that Acacia was either the ancestral host plant or at least a component of the ancestor’s diet. This association was then carried through multiple cladogenetic events and was always a part of the diet when host ranges were broadened.

The Ingeae specialists on the other hand appear separately derived on four different

238 occasions. While species of Ingeae are also independently included in the diet of generalists, there is no continuity between different hypothesized monophyletic plant groups across this diversification in diets of Stator. This observation is born out by the lack of a trajectory of specializing on Acacia and a highly significant trajectory of specializing on Ingeae in the maximum-likelihood analysis. However, the maximum- likelihood analysis estimates a rate of decreasing specialization on Ingeae five times higher than the rate of increasing specialization on Ingeae, despite the lack of direct observation on the phylogeny of any transitions away from specialization on Ingeae.

Superficially, the interpretation is that given enough time, the Ingeae specialists will disappear by becoming resource generalists. However, there is an alternative interpretation of this result. The result actually estimates a reasonably high forward transition rate toward specialization on Ingeae, something in agreement with the independent origins of this character on the phylogeny. To reconcile this, it also estimates a high transition rate away from specialization on Ingeae. In other words,

Ingeae specialists evolve often, but do not persist long before returning back to the ancestral state. This lack of persistence can also be interpreted as extinction. This fits well with the hypothesis that selection for specialization in the fundamental niche of a species happens often, but the trait does not persist and the lineage goes extinct.

The hypothesis of specialization as a dead end therefore appears to be contingent upon the distinction between specialization as an ecological strategy in the face of competition (the realized niche) versus specialization as the result of intense directional selection for the increased ability to use a particular resource (the fundamental niche). In the genus Stator, this clarification supports the ecological model of adaptive radiation in

239 which species specialize to fill unoccupied niches (Schluter, 2000; Simpson, 1953).

Indeed, the derived evolution of ecological specialization within the species genealogy of

two of the most extreme generalists in the clade could suggest that the secondary

derivation of generalism in Stator may be setting the stage for an adaptive radiation following the invasion of the largely unoccupied Ingeae. There may be evidence of this already happening in the colonization of Enterolobium and the subsequent ecologically local speciation event. Is this the sign of macroevolutionary trends to come? Unlike the

remaining groups of Mimosoideae (Acacieae, Parkieae, and Mimoseae), the genera of

New World Ingeae are only attacked by a scattering of bruchids, and the majority of the

phylogenetically incoherent genera that do serve as hosts are attacked by generalists in

the genera Stator, Merobruchus, and Mimosestes. There are only a handful of Ingeae

specialists (6 in Stator; 8 in Merobruchus, and 1 in Mimosestes), and these may all be

relatively independently derived within clades of Acacia oligophages. This could be the

signature of impending radiations onto the seeds of hosts in the Ingeae.

Interestingly, the one generalist that does not use a large number of Ingeae species

does not appear to be closely related to any specialists. S. pruininus is only known from

5 species of Ingeae, and records from these are few and far between. For example, while

S. pruininus is known to feed on Havardia pallens (Benth.) Britton & Rose, Grimm

(1995) reported no attack by these species in a study of seed predation on this species,

despite reported high levels of attack by Merobruchus insolitus and moderate levels by S.

limbatus. In collecting H. pallens throughout its range along the Gulf Coast of Mexico I

reared over 10,000 specimens of S. limbatus and fewer than 10 of S. pruininus. S.

pruininus also appears to use Calliandra eriophylla Benth. in a similarly consistent yet

240 rare manner. Even less can be said for the association of S. pruininus with Calliandra rubescens (M. Martens & Galeotti) Standl., Lysiloma sp., and Zapoteca portoricensis

(Jacq.) H.M. Hern. All are known from single records and the last is the result of a mutual introduction to Hawaii. S. pruininus appears to have broadened its diet along a very different axis from S. limbatus and S. sordidus, principally expanding its diet to include species of Mimoseae and Papilionoideae instead of species of Ingeae.

The effect of oviposition behavior on dietary specialization.

We have already seen that oviposition behavior appears to be important in determining the taxa of host plants that are available for macroevolutionary diversification. Furthermore, it appears that the different guilds can experience different selection pressures. Because the phenological partitioning of resources affects the type of host plants that can be used and perhaps even the degree of host overlap observed, it is possible that evolution in diet breadth variation could be affected by oviposition behavior. For example, an even temporary escape from plant defenses could release species from the constraints of specialization (Cates, 1980; Ehrlich and Murphy, 1988;

Ehrlich and Raven, 1964; Futuyma, 1983b; Futuyma and Moreno, 1988), as could an escape from generalist insect parasitoids (Bernays and Graham, 1988; Lawton, 1986;

Strong et al., 1984). Indeed, diet breadth is either significantly positively correlated or approaches significance (Table 2.10) with ovipositing directly on seeds while they are still on the host plant (guild B), even when correlations due to phylogeny are taken into account. This appears to be due to the fact that the two most extreme generalists in the

241 phylogeny, S. limbatus and S. pruininus, are in guild B and are part of separate

derivations of the behavior. Furthermore, only one species in guild B is strictly

monophagous (S. beali) while the remainder are oligophagous on multiple species of

Acacia.

This association between guild B and increased diet breadth has three probably

interacting explanations. (1) They are able to escape the defensive properties in the

integument of the pod and have only to deal with a single defensive tissue, the seed testa.

(2) They are able to ‘hide’ from parasitoids by ovipositing within the pod. This would

increase the searching time and probably the handling time for parasitoids, making them

a less preferred food type for generalist foraging wasps than eggs laid by species in guild

A (Gratton and Welter, 1999; MacArthur and Pianka, 1966). And (3) they can use host

plants to locate available seeds and need not evolve specialized foraging mechanisms for

finding scattered seeds (Bernays and Funk, 1999; Bernays and Wcislo, 1994; Futuyma,

1983b; Menken and Roessingh, 1998). This may not be as relevant given the fact that

these scattered seeds are often present in large numbers in the dung of their vertebrate

dispersers.

The evolution of ovipositing directly on seeds may also facilitate the colonization

of hosts in the Ingeae. As noted earlier, species in guild A are most limited in their forays

away from the Acacia. This is likely because the fruits of numerous Ingeae species are specialized for animal dispersal in one way or another (Barneby, 1998; Barneby and

Grimes, 1996; Barneby and Grimes, 1997). Numerous species have a thickened and pulpy endocarp apparently adapted to dispersal by large vertebrates and are indehiscent or tardily dehiscent. As a result, these species are not available to Stator species in guild

242 A although they are used by more robust Merobruchus and Mimosestes in guild A. These

species appear to be readily available to species in guilds B and C. Other species are

precociously dehiscent, with the seeds remaining attached to the pod by a spongy arillate

funicle. These ‘monkey’s earring’ pods are largely bird-dispersed and also are not

available to species in guild A as the seed does not remain in contact with the integument

of the valve. Finally, most members of Zapoteca and Calliandra are explosively

dehiscent (Barneby, 1998; Hernández, 1986; Hernández, 1989) and probably do not

provide a suitable substrate for oviposition by species in guild A. A single report exists

of S. vittatithorax using Z. portoricensis, but this is not surprising given that many of the

pods of this species fail to dehisce. Furthermore, the use of these two genera by members

of guild B is limited to the few seeds that are not dispersed at dehiscence.

Conclusions.

The results of this analysis suggest that the macroevolution of host plant use in

general and diet breadth in particular is contingent upon numerous factors including

geography, community structure, and behavior. The monophyletic genus Stator is particularly interesting within the Bruchinae for its variation in both diet breadth and oviposition behavior. These two aspects of host use do not appear to vary to a considerable extent in other groups of seed beetles.

The argument that specialists should occupy derived positions in a phylogeny is not immediately supported by this analysis. Specialists on Acacia are present throughout the phylogeny and the behavior is often carried through multiple speciation events.

However, this lack of support may be due to a lack of distinguishing between a species’

243 fundamental and realized niches. When examined from this point of view, those specialists that are the result of more phylogenetically radical host shifts do occupy derived positions in the phylogeny. Whether or not these species retain considerably less genetic variation to use alternative hosts because of intense directional selection (sensu

Futuyma et al., 1995) would provide evidence to support or reject this hypothesis.

I hypothesize that the independent derivation of generalism in the genus Stator may be setting the stage for a new radiation onto the largely unoccupied resource of the

Ingeae, and has been facilitated by the origins of ovipositing directly on seeds within the genus Stator. The appearance of these three generalists against a background of feeding on species of Ingeae, as well as the appearance of similar generalists in a similar context in the genus Merobruchus, is suggestive of a recent invasion of a previously unoccupied resource. Given this, the ecological theory of adaptive radiation suggests that as these niches become filled there should be specialization and speciation that adequately partitions these niches. Such a pattern is suggested by the paraphyletic relationships of S. limbatus and S. sordidus with species that specialize on Ingeae. While it appears that S. limbatus and S. sordidus are generalists at local levels when they have been studied, a research program that examines the mosaic of host use throughout the geographic range of the two could be highly illuminating as to whether there is a bias in localized specialization when hosts include species of Ingeae.

244

CHAPTER 3:

INTERSPECIFIC PHYLOGEOGRAPHY OF A PARAPHYLETIC SPECIES PAIR: THE

GEOGRAPHIC CONTEXT OF SPECIATION AND SPECIALIZATION

INTRODUCTION

The role that ecological specialization plays in speciation, and by extension in diversification, has a long history in evolutionary theory. According to the ecological theory of adaptive radiation, diversification begins as a lineage enters a previously unoccupied adaptive zone and gives rise to species that, perhaps due to competition, diverge and partition resources (Schluter, 2000; Simpson, 1944). Considerable evidence supports of this model (Schluter, 2000), including evidence from phytophagous insects

(Ehrlich and Raven, 1964; Farrell, 1998; Mitter et al., 1988; Strong et al., 1984). For example, phytophagous insect clades are consistently more diverse than their primitively aphytophagous sister clades (Mitter et al., 1988), and speciation is associated with changes in host use in many groups of insects (Strong et al. 1984, Mitter and Farrell

1991). Furthermore, most of the ca. 260,000 species of tracheophytes are used as food by phytophagous insects, and provide a vast variety of resources that differ in chemical, morphological, and phenological features. The observation that the majority of these species serve as hosts to insect species that are specialized to one degree or another

(Bernays and Chapman, 1994; Futuyma and Moreno, 1988; Jaenike, 1990; Thompson,

1994) has been advanced as supporting the idea that the divergent evolution of plant defenses and subsequent divergence of insect species as they adapted to the diverse defenses (Ehrlich and Raven, 1964).

This hypothesis assumes that selection favors specialization, and it is often assumed that resource generalists give rise to specialized descendant species more often that the reverse occurs (see Schluter, 2000). Specialization might evolve because of competition or predator-prey interactions (Bernays and Graham, 1988; Denno et al.,

246 1995; Holt and Lawton, 1993), ecological availability (Thompson, 1994), or because of intrinsic physiological trade-offs in adaptation to chemical or other differences between host plants (Futuyma and Moreno, 1988; Jaenike, 1990). Such trade-offs, moreover, might constitute disruptive selection for specialized host preferences, resulting in the origin (sympatrically or allopatrically) of host-specialized species in those insects that mate on the host plant (Diehl and Bush, 1989). Thus speciation and the evolution of specialization may be causally coupled in phytophagous insects.

These ideas are appealing, but are by no means necessary. For example, a broad diet can obviously be advantageous in ecological or temporally heterogeneous environments (Futuyma and Moreno, 1988), and density-dependent resource limitation is unfavorable for the evolution of specialization from generalism (Rausher, 1993).

Evidence for physiological trade-offs has been found in a few instances, but not in others

(Feder and Filchak, 1999; Fox and Caldwell, 1994; Fox et al., 1994; Fry, 1993; Futuyma and Moreno, 1988; Jaenike, 1990). Thus, the hypothesis must be entertained that in a population that adapts to a new host where its ancestral host is uncommon, the capacity to use the ancestral host may be a nearly neutral character that is lost by mutation and genetic drift—thereby rendering the population a host-shifted specialist rather than a versatile oligophagous generalist. Evaluating these hypotheses will require evidence on the polarity of change in host use, on the population structure and mechanisms of speciation, and on the geographic context of speciation.

Only in the last few years have phylogenies of phytophagous insects and other organisms provided evidence on the direction of evolution of niche width. Although some clades consist entirely of specialists with different host associations, others include

247 species that vary in “niche width”. Some such clades support the hypothesis that specialists are derived (Kelley and Farrell, 1998; Moran, 1988), but others present no significant trend in diet breadth (Dobler et al., 1996; Termonia et al., 2001). At this time, it may be said that phylogenetic results “provide little support for the generalists-to- specialists hypothesis” (Schluter 2000, p. 48), but so few phylogenies are available that it remains a largely open question. Moreover, exemplar phylogenies (phylogenies in which species are represented by single specimens) may not always be appropriate for determining if specialist species often originate from within generalist lineages. For example, paraphyletic patterns of mitochondrial haplotype relationships were used to infer an ancestral generalist in two sister species pairs of Greya moths (Brown et al.,

1994); and similar patterns of mitochondrial haplotype and nuclear gene relationships supported a similar conclusion in two separate situations in the genus Stator (Chapter 2).

In all of these studies, an exemplar phylogenetic analysis would reconstruct the ancestor at these nodes as either equivocal or as a specialist.

Phylogeographic analyses of gene genealogies in recently diverged species may be more suitable than exemplar phylogenies for addressing the origins of diet breadth evolution for at least four reasons:

(1) A species-level phylogeny represented by a simple bifurcating tree with species names at the tips may ignore information useful for reconstructing the ancestral character. In the studies described above, wide geographic sampling of species functionally shifted the outgroup to more distantly related populations of the generalist species.

248 (2) Older divergences may no longer contain information on the origins of

character states. Suppose two or more specialist species form from localized populations

of a widespread generalist. At first, the generalist will be genetically paraphyletic with

respect to the specialists, and the pattern of shared haplotypes may show the history of

divergence. But after about 2 Nf generations (for a mitochondrial marker) to 4Ne – 7Ne generations (for a nuclear marker) this information will be lost, because the loss of gene lineages by genetic drift makes the species reciprocally monophyletic (Avise, 2000;

Hudson and Coyne, 2002; Moore, 1995; Neigel and Avise, 1986).

(3) An exemplar phylogeny may not adequately capture the geographic context of speciation as information from extant geographic ranges becomes uninformative regarding initial divergences with increasing genetic distance (Barraclough and Nee,

2001; Barraclough and Vogler, 2000).

(4) The observation that diet breadth appears to be a fairly labile trait at the phylogenetic level (Janz et al., 2001; Kelley and Farrell, 1998; Scheffer and Wiegmann,

2000; Termonia et al., 2001) suggests that studies of niches and species may provide a more appropriate framework for addressing the role of specialization in diversification.

One corollary to the hypothesis that natural selection for specialization allows the

diversification of lineages via the partitioning of resources is that speciation is fast and

often quite geographically local (Bush, 1993; McCune and Lovejoy, 1998; Schluter,

2000). Three observations have led to an increase in the popularity of the point of view that sympatric speciation, or at least ecologically driven speciation, may be the dominant form of lineage diversification in phytophagous insects. First, there is a growing body of

249 empirical evidence and theoretical work that supports the assertion that adaptations to

feed on different host plant species may cause population differentiation, the evolution of

assortative mating, and ultimately speciation (Feder et al., 1990; Feder et al., 1997a;

Feder et al., 1999; Funk, 1998; Kondrashov et al., 1998; Rice and Salt, 1990; Via, 2001).

Second, an insect’s host plant may largely define its niche space and the diversity of those hosts its niche breadth (Bush, 1993; Strong et al., 1984). Because of this, the observation that closely related species in many taxa of phytophagous insects often have different host affiliations suggests that speciation is causally associated with host shifts.

Third, a strictly allopatric, selectively neutral mode of speciation may not be able to account for the incredible diversity of phytophagous insects (Bush, 1994; Bush and

Smith, 1997).

However, few topics remain more controversial in evolutionary biology than the mode of speciation in adaptive radiations, and how this relates to specialization. Many authors maintain that the primary mode of speciation must be allopatric, emphasizing the theoretical obstacles to sympatric speciation and that as a result sympatric speciation bears the burden of evidence (Coyne and Orr, 1997; Coyne and Price, 2000; Futuyma and

Mayer, 1980; Mayr, 1963; Turelli et al., 2001). While even some of the more skeptical opponents of sympatric speciation have begun to yield their stance for particular cases and taxa (Coyne and Price, 2000), examining the geographic context of speciation remains a largely unexplored task.

The task for the study of diversification, then, is to understand the circumstances that lead to a causal link between specialization and speciation. This has been addressed to some degree with reference to interspecific phylogenies (Barraclough and Nee, 2001;

250 Barraclough and Vogler, 2000; Coyne and Price, 2000; Ribera et al., 2001), but this level of resolution may not provide an adequate picture for the reasons mentioned above.

When reduced to causality, the topic of diversification in phytophagous insects appears to be best addressed by examining the role of host shifts in speciation. Therefore, examining the phylogeographic context of speciation between closely related species that have diverged ecologically may provide different and complementary insights into understanding the link between ecological diversification and evolutionary diversification.

The recently emerged field of phylogeography provides a means to evaluate the combined roles of geographic history, evolutionary history, and ecological associations in the structuring of populations and the process of speciation (Avise, 2000). This field is based chiefly on inferences from gene genealogies and networks and coalescent theory

(Avise, 2000; Hudson, 1990; Posada and Crandall, 2001a; Templeton, 1998b; Templeton,

2001; Templeton et al., 1995). Applied phylogeographic methods (Excoffier et al., 1992;

Templeton, 1998a; Templeton, 1998b; Templeton et al., 1995) provide evidence of disjunction or reduced gene flow among populations (Althoff et al., 2001; Brown et al.,

1996; Funk et al., 1995; Mardulyn, 2001; Segraves and Pellmyr, 2001), the region of origin of clades of haplotypes (Holder et al., 1999; Holder et al., 2000; Irwin, 2002), range expansion (Gómez-Zurita et al., 2000; Hewitt, 2001; Taberlet et al., 1998), and gene flow (Beerli and Felsenstein, 1999; Hudson et al., 1992; Nielsen and Wakeley,

2001). These approaches have been used to infer histories of population size, subdivision, and range expansion for many species, most famously Homo sapiens (Avise,

251 2000), but they have been applied to the origin of species of phytophagous insects in few

cases. The most comprehensive analyses of the phylogeographic patterns of speciation

have been studies of several species groups of Drosophila (Hilton et al., 1994; Kliman et

al., 2000; Wang et al., 1997), which provided examples of reduced Ne and of

introgression between species. In these and many other instances of recent speciation,

sister species display genetic polyphyly or paraphyly (Avise, 2000; Harrison, 1998;

Kliman et al., 2000), the latter providing evidence of differences in Ne between sister

species. While these studies exemplify the utility of Drosophila as a model organism in

studying the molecular genetics of speciation, their utility in studying the role of ecology

in speciation is highly suspect given that the natural history of most of these species is

largely unknown.

Applications of these methods to host-specific phytophagous insects provides

considerable information regarding the link between ecology and population

differentiation (Althoff et al., 2001; Brown et al., 1996; Brown et al., 1997; Knowles et

al., 1999; Leebens-Mack et al., 1998), but applications to speciation in phytophagous

insects have been few (see Groman and Pellmyr, 2000; Knowles et al., 1999).

The interaction between seed beetles in the genus Stator and their leguminous

host plants is a system in which widespread, generalist species appear to have given rise

to geographically localized specialist species on at least three separate occasions (Chapter

2). In South America, the widespread species S. furcatus shows a paraphyletic

relationship with the narrowly distributed S. tigrensis. This contrast suggests the least

amount of ecological differentiation involved in such a relationship, as both species feed

252 on Acacia. In North America, the widespread and highly generalist species S. sordidus shows a paraphyletic relationship with a sister species pairing of S. chihuahua + S. pygidialis, two geographically localized species with limited host ranges. The paraphyletic relationships of these two lineages are based on limited sampling, however, and must be currently taken as provisional. On the other hand, more extensive sampling strongly supported a paraphyletic relationship between the most generalized species of

Stator, S. limbatus, and the highly specialized S. beali. The geographic range of the latter is entirely nested within that of the former, suggesting that speciation could have occurred within a highly localized geographic context.

The extreme specialization of S. beali also suggests that natural selection could have played a role in the speciation event. It is particularly notable that all of the proposed mechanisms that would select for specialization appear to be important for seed beetles in general: (1) the intense intimacy of the relationship between bruchines and their host plants—a bruchine larva basically converts the resources intended by the parent plant to produce a new plant into a beetle, while at the same time killing the plant’s offspring—has the possibility to drive a strong coevolutionary arms race that results in the production of diverging defensive chemicals and the creation of strong disruptive selection to use single hosts (Bleiler et al., 1988; Ehrlich and Murphy, 1988; Huignard et al., 1989; Janzen, 1971b; Johnson, 1989b; Rosenthal et al., 1982); (2) competition is prevalent because bruchine larvae are largely immobile consumers of a discretely packaged resource; this creates the conditions for selection for niche differentiation

(Denno et al., 1995; Fox et al., 1996b; MacArthur and Levins, 1967); (3) bruchines are attacked by generalist hymenopteran parasitoids, and escape into enemy-free space may

253 select for specialization (Bernays and Graham, 1988; Hetz and Johnson, 1988; Lawton,

1986; Thompson, 1986); and (4) the ephemeral and patchy nature of seeds as resources

could select for more efficient neural capabilities to search for particular hosts (Bernays,

1998; Bernays and Funk, 1999; Bernays and Wcislo, 1994).

These three observations—paraphyly, sympatry, and ecological differentiation—

all superficially support the model of adaptive divergence via specialization described

earlier. However, the results of the phylogenetic analysis were limited in their ability to

discern the geographic context of the divergence of S. beali from S. limbatus as sampling

was not sufficient to adequately address the combined intra- and interspecific

phylogeography necessary for this purpose.

The goal of this chapter is to examine the phylogeographic structure of this

species pair using mtDNA sequence variation in Cytochrome Oxidase I with the intention

of understanding the geographic context of speciation. In particular, I will address three

questions: (1) Does the phylogeography of S. limbatus reflect historical boundaries to dispersal? (2) Is there evidence of recent colonization of the areas of sympatry by either species, a result that would be indicative of secondary sympatry? (3) Are the nearest S. limbatus relatives of S. beali localized to a particular geographic area, and what are the implications of such localization for understanding the geographic context of speciation?

The purpose of this chapter is therefore to establish whether genealogical history reflects widespread barriers to dispersal and to understand the geographic context of speciation.

While this is certainly part of the equation in examining the mode and tempo of speciation, the sampling in this chapter was not designed to address the demographic

254 history or population structure that are important in assessing the likelihood of alternative

scenarios. These topics are addressed directly in Chapter 4.

STUDY SYSTEM

Species of Stator are obligate endophagous seed parasitoids of legumes

(Fabaceae) and show considerable variation in host plant specialization (Chapter 2).

Molecular phylogenetic analysis of both a mitochondrial locus (Cytochrome Oxidase

subunit 1) and a nuclear protein-coding locus (Elongation Factor-1α) shows strong support (100% bootstrap in parsimony analysis and 100% posterior probability in

Bayesian analysis) for the sister relationship of S. limbatus + S. beali as well as for paraphyly of the former with respect to the latter.

S. limbatus (Horn) feeds on about 70 species of legumes throughout a range that extends from northern South America to the southwestern United States (Figure 3.1). S. limbatus does not appear to consist of specialized local populations, as rearing experiments and quantitative genetic analyses have shown it to be generalized at the intra-individual level, much less at the interpopulation level (Fox et al., 1996a; Fox et al.,

1997; Fox and Savalli, 2000; Fox et al., 1994; Fox et al., 1995; Siemens and Johnson,

1990). While this could be an artifact of generalized populations in Arizona and Texas

(the source populations for these experiments) and not a species-wide phenomenon (Funk and Bernays, 2001), S. limbatus is readily reared from multiple hosts in numerous areas throughout its range (Janzen, 1980a; Johnson, 1995; Johnson and Kingsolver, 1976;

Johnson and Lewis, 1993; Johnson and Siemens, 1995).

255

Figure 3.1. Distribution of Stator limbatus (Horn) in South America, North America, and the Lesser Antilles. Locations are from GPS coordinates recorded during collection of specimens, are inferred from collection locales on museum specimens, or are inferred from published locality records (Bottimer, 1973; Johnson, 1963; Johnson, 1981b; Johnson, 1984; Johnson, 1995; Johnson, 1998; Johnson and Kingsolver, 1976; Johnson et al., 1989; Johnson and Lewis, 1993; Johnson and Siemens, 1992; Johnson and Siemens, 1995; Kingsolver, 1972). S. limbatus is found throughout the Neotropics, except at high altitudes or moist tropical rainforest. The box indicates the approximate area of sympatry with S. beali.

S. beali Johnson is restricted to the Texas ebony tree Ebenopsis ebano (Berl.)

Barneby & Grimes below about 500m in the coastal flood plain of the Gulf of Mexico.

Its range is completely coincident with that of its host (Barneby and Grimes, 1996): it is bounded on the south by an arm of the Sierra Madre Oriental which reaches the coast near Xalapa, Veracruz; on the west by the main north-south trajectory of the same mountain range; and on the north by an increasingly seasonal climate (Figure 3.2). The host plant is one of the dominant trees in this lowland and can form dense stands in

256 particular areas (Barneby and Grimes, 1996). A single tree can produce as many 1,500

pods (personal observation), with each pod containing 5-12 seeds. The seeds are notably

large for the synandrous Mimosoideae and I have reared, as a record, 14 individuals of S.

beali from a single seed. The range of S. beali is nested completely within that of S.

limbatus (see box, Figure 3.1), and it is common to collect seeds with larvae of S.

limbatus from host plants that are growing under the canopy of infested E. ebano.

Figure 3.2. Distribution of Stator beali Johnson in the Gulf Coast of northeastern Mexico and southeastern Texas. Locations are from GPS coordinates recorded during collection of specimens, are inferred from collection locales on museum specimens, or are inferred from published locality records (Bottimer, 1973; Johnson, 1963; Johnson, 1981b; Johnson, 1984; Johnson, 1995; Johnson, 1998; Johnson and Kingsolver, 1976; Johnson et al., 1989; Johnson and Lewis, 1993; Johnson and Siemens, 1992; Johnson and Siemens, 1995; Kingsolver, 1972).

257 The ecological difference between the two species is also apparent within the area

of sympatry. S. limbatus uses 15 native and one introduced species of legume within the

confines of the Gulf Coast: Acacia acatlensis Benth., A. angustissima (Mill.) Kuntze, A. berlandieri Benth., A. greggii A. Gray, A. roemeriana Scheele, A. wrightii Benth. ex

Gray, Albizia lebbeck (L.) Benth., Havardia pallens (Benth.) Br. & R., Leucaena pulverulenta (Schlecht.) Benth., Lysiloma acapulcense (Kunth) Benth., Painteria elachistophylla (S. Wats.) Br. & R., Parkinsonia aculeata L., P. macra texana (I.

Johnst.) Isely, Pithecellobium dulce (Roxb.) Benth., P. lanceolatum (Willd.) Benth., and

P. unguis-cati (L.) Benth.. The two species differ consistently in external morphological characters, most notably in the golden pubescence covering the body of S. beali, versus whitish pubescence covering S. limbatus. They also differ in characters that are highly relevant in sexual selection in bruchine beetles, particularly in the armature of the male genitalia (Johnson and Kingsolver, 1976)—S. beali lacks the recurved serrations extending from the apical sclerite of the median lobe that characterize S. limbatus throughout its range (Figure 3.3). Finally, the two beetle species exhibit highly divergent oviposition behavior. S. beali uses very large seeds and oviposits eggs in clutches that can exceed 20 eggs (Figure 3.4), although they may manipulate this clutch size depending on the size of the host seed (Fox et al., 1996b; Fox and Mousseau, 1995b). S. limbatus oviposits eggs singly, generally depositing one egg per seed unless seeds are limiting (Fox et al., 1996b). Even when multiple eggs are deposited on a single seed, they are not oviposited in arranged clutches (Figure 3.5).

These consistent phenotypic differences between S. limbatus and S. beali are certainly concordant with the idea that the evolution of barriers to gene exchange has

258 occurred and that they belong to separate species according to the biological species

concept (Coyne and Orr, 1998; Coyne et al., 1988; Mayr, 1963; Turelli et al., 2001). The

observations of either a complete lack of successful fertilization when S. beali females are mated to S. limbatus males, or sterile hybrids when the occasional mating of S. limbatus females with S. beali males actually produces a developing egg support the conclusion that reproductive isolation has evolved between these lineages (Fox and

Mousseau, 1995a; Nilsson and Johnson, 1993a).

Figure 3.3. Genitalia of S. limbatus (top) and S. beali (bottom). S. beali has lost the serrations on the apical spine in the median lobe. Reproduced with permission from original drawings by C.D. Johnson.

259

Figure 3.4. Stator beali eggs on Ebenopsis ebano. S. beali lays its eggs in clumps of often more than 20 eggs on the large seeds of its host plant.

Figure 3.5. Stator limbatus eggs on Acacia tenuifolia. S. limbatus lays its eggs singly even when it oviposits multiply on single seeds.

260 METHODS

SPECIMENS EXAMINED

I collected host seeds from numerous collection localities throughout the range of both species during collecting trips between 1998 and 2001. Infested host plant seeds were collected in the field and transported to an environmental chamber at the Museum of Comparative Zoology, Harvard University. They were then transferred into cloth- covered jars and adult specimens were removed as they emerged from seeds and were immediately frozen and stored in a -80°C freezer. Emerging adults were not allowed to begin a second generation; therefore all specimens included in this analysis were field collected.

Multiple individuals from each population were reared from seeds from each locality and a portion were mounted and labeled as voucher specimens. I identified all specimens included in this analysis using the characters discussed in Johnson and

Kingsolver (1976). All S. beali adults were reared from E. ebano, while no S. limbatus were reared from this host plant. In total, 108 specimens of S. beali from 18 different localities were included in this study (Table 3.1). Eight of these localities are represented by single specimens.

S. limbatus specimens were reared from numerous different host plants throughout its range. South American specimens were sampled less extensively because the phylogeny of the genus showed such strong support for a separate clade of South

American feeders. Therefore, I made a decision motivated in part by economic factors to sample North American specimens more extensively. In total, 13 specimens from different localities in Ecuador (3), Venezuela (8), Martinique (1), and Panama (1) were

261 included in this analysis (Table 3.2). North American specimens were sampled much more extensively, particularly in the area of sympatry and nearby geographic areas in

Mexico and the United States. 149 specimens of North American S. limbatus from 28 different localities were included in this study (Table 3.3). Eleven of these localities are represented by single specimens. In total 270 specimens of both species ranging from northern South America to Arizona are included in this analysis (Figure 3.6).

Initially, two specimens of S. testudinarius, two specimens of S. generalis, and two specimens of S. furcatus were included as outgroups. Analyses including all outgroup species or any of the three individually all supported a sister relationship between the South American and North American specimens (100% bootstrap for both nodes in all cases). Rooting in a phylogeographic analysis is quite difficult because the ratio of divergence within the ingroup to any outgroup species is often quite low

(Castelloe and Templeton, 1994; Routman et al., 1994; Templeton, 1998a). It is also important given that it structures the patterns of synapomorphy throughout the rest of the topology, and therefore should be based on closely related outgroups. For this reason, I used the information from the preliminary analyses to root the phylogenetic analyses with the South American clade of specimens.

262

Figure 3.6. Map of sites sampled for S. limbatus (filled circles) and S. beali (open circles). Site numbers correspond to the names in Tables 3.1-3.3. The inset shows the degree of sampling for the area of sympatry.

263

264 265 266 MOLECULAR METHODS

Frozen whole individuals were flash-frozen in liquid Nitrogen and immediately

ground to a fine powder with a melted pipette tip in an Eppendorf tube. Total genomic

DNA was then isolated using the ‘salting out’ protocol of Sunnucks and Hales (1996). I

amplified a ~700 base pair fragment of 3’ end of the mtDNA gene Cytochrome Oxidase I

(COI) using the polymerase chain reaction (PCR). Amplifications were performed in

50µl reactions to produce double-stranded DNA product under the following conditions:

0.2µM each primer, 0.15mM each dNTP, 2.5µM MgCl2, 1X buffer supplied by the

manufacturer (Qiagen, Valencia, CA), and one unit of Taq polymerase (Qiagen).

Reactions were brought up to 50µl using water and 1.5µl DNA genomic template.

Typical temperature profiles for amplification of fragments consisted of 40 cycles of 30s at 95°C, 30s at 50°C, and 1.5 min at 72°C, followed by a 5 min extension at 72°C.

Primer sequences were s1859: (5’-GGAACIGGATGAACWGTTTAYCCICC-3’) and a2590: (5’-GCTCCTATTGATARWACATARTGRAAATG-3’). Numbers refer to the nucleotide positions in the complete mtDNA sequence of Drosophila yakuba (Clary and

Wolstenholme, 1985; Simon et al., 1994). Non-standard nucleotide designations are

IUPAC ambiguity codes (Cornish-Brown, 1985) and refer to equal mixtures of bases. ‘I’ refers to inosine. PCR products were checked on 1.5% agarose gels and products were purified directly or after gel extraction using QIAquick columns (Qiagen).

The amount of DNA from the purified PCR product was estimated by comparison to a Low Mass Ladder (Gibco/Invitrogen, Carlsbad, CA) and 70-90ng (ABI373) or 20-

50ng (ABI3100) was used for sequencing reactions. Sequencing primers were identical to those used in PCR reactions. Sequencing of this double-stranded product was carried

267 out using 25 PCR cycles of 96°C for 30s, 50°C for 15s and 60°C for 4 min with a 2°C increase per second in a 10µl reaction. This reaction used dye terminator cycle sequencing chemistry with the specified amount of template, 2.0µl of Dye Deoxy FS

Terminator or BigDye premix (PE Biosystems, Inc., Foster City, CA), 0.16µM primer,

and water to the final volume. Finished reactions were precipitated in 0.5mM MgCl2 in

70% ethanol, centrifuged, and dried under a vacuum. The majority of sequencing was done using an ABI 373 automated DNA sequencer, with some sequencing done using an

ABI 3100 automated sequencer (PE Biosystems, Inc.). Compiled segments resulted in

665 base pairs of CO1 for all 273 specimens included in this analysis. Both directions of

the PCR product were sequenced and contigs were assembled, edited, and aligned with

Sequencher ver. 4.1 (GeneCodes, Ann Arbor, MI). Because the fragment consists of

protein-coding sequences with no insertions or deletions, alignment was unambiguous.

DATA ANALYSIS

Identical haplotypes were condensed into representative haplotypes and

phylogenetic analyses were performed on these using maximum parsimony, maximum-

likelihood, and Bayesian inference. I used Fitch parsimony, with heuristic searches

implemented using the parsimony ratchet algorithm (Nixon, 1999) implemented in Paup*

(Swofford, 2000) as a batch file (PAUPrat) written by Sikes and Lewis (2001). This is a

highly efficient search algorithm that is based on reweighting a proportion of the

characters in each reiteration. Three separate searches of 1000 iterations were employed,

with reweighting of 10%, 15%, and 20% of the characters in the separate searches. The

resulting 3000 trees were then compiled into a single file, duplicates were discarded, and

268 trees were filtered to include only the most-parsimonious trees. The program ModelTest

3.0 (Posada and Crandall, 1998) was used to determine the appropriate model of

molecular evolution for the maximum likelihood analysis. The model selected was a

variant of the HKY85 model (Hasegawa et al., 1985) with rates estimated based on a gamma distribution (α-parameter estimated from the data) with an estimated proportion of sites assumed to be invariable (HKY85 + I + G). The parameters estimated from the

ModelTest algorithm were incorporated into the searches in order to decrease the search to a reasonable time limit. The maximum likelihood analysis was implemented using

PAUP*, and consisted of 50 heuristic searches from the random addition of taxa, with each search allowed to examine 1,000,000 trees. The Bayesian search was implemented using MrBayes 2.01 (Huelsenbeck and Ronquist, in Press). The search was run with four simultaneous chains for 1,000,000 generations, sampling every 100 generations and applying temperatures of 0.2 and 0.5. The burnin time (the number of generations before the tree-search reaches the optimum) was estimated by plotting the number of generations versus the ln Likelihoods (lnL). Trees in the burnin were discarded from the analysis.

Posterior probabilities of nodes were estimated based on the majority rule consensus of the trees that were found at stationarity, and branch lengths for the phylogram are based on the mean branch lengths from these trees. The analysis used a model of evolution estimating different gamma distributions for the site partitions based on codon position

(SS + G).

Bootstrap analyses with all representative haplotypes included were prohibitively time-consuming because of low levels of divergence within major lineages. Therefore, a modified approach similar to that taken by Althoff et al. (2001) was used in order to

269 estimate bootstraps. Clades that were present in the strict consensus tree of the

parsimony analysis, that had greater than 95% posterior probabilities in the Bayesian

analysis, and were present in both the neighbor-joining and maximum-likelihood

analyses were each represented by five randomly chosen specimens and bootstrap

support for these clades were then estimated based on 100 bootstrap replicates, each

using starting trees based on 100 random addition sequences. The analyses agreed

between 5 different clades, as well as one individual that did not associate with any of the clades in the Bayesian analysis. Because all of the outgroup specimens were included, each of the bootstrap replicates required 42 taxa. This procedure was repeated 50 times, and the bootstrap values were added together and divided by 50 (total number of replicates).

RESULTS

DNA sequencing resulted in 665 consistently readable base pairs of CO1 for each

individual. For the ingroup, 21% (138) of sites are variable, although 33% (45) of these

variable sites are unique to particular haplotypes. Changes at third codon sites account

for 85% (117) of variable sites, and 94% (87) of parsimony-informative sites. Changes at

first codon sites accounte for 11% (15) of variable sites, and 6% (6) of parsimony-

informative sites. Changes at second codon sites account for 4% (6) of variable sites,

with none of them being parsimony-informative. There are 124 unique haplotypes

among the 270 specimens included in the analysis. For S. beali, there are 39 distinct

haplotypes among the 108 specimens included, 22 of which are found in single

specimens (Table 3.1). For North American S. limbatus, there are 74 distinct haplotypes

270 among the 150 specimens included, 54 of which are found in single specimens (Table

3.2). For South American S. limbatus, there are 11 distinct haplotypes among the 13 specimens included, 10 of which are found in single specimens (Table 3.3).

All analyses support six major clades of haplotypes (Figures 3.7 and 3.8): (1) a

South American S. limbatus clade accounting for 13 specimens and 11 haplotypes, including a well supported clade west of the Andes (Ecuatorial coastal region); (2) a clade found in the Madrean + Mesoamerican biogeographic regions (Rivas-Martínez et al., 1999) accounting for 17 specimens and 16 haplotypes; (3-5) three clades from the

Mexican xerophytic biogeographic region (Rivas-Martínez et al., 1999), the first accounting for 20 specimens and 5 haplotypes, the second for 7 specimens with unique haplotypes, and the third for 106 specimens and 44 haplotypes; and (6) a S. beali clade in the Tamaulipan province of the Mexican xerophytic biogeographic region (Brown et al.,

1998; Rivas-Martínez et al., 1999). There is not significant resolution between the four clades (3-6) found in the Mexican xerophytic region in the strict consensus of the parsimony tree, although the three clades of S. limbatus formed a monophyletic clade sister to S. beali in both the Bayesian and maximum-likelihood analyses. This resolution is also present in 85% of the 1265 most parsimonious trees (MPTs).

These clades correspond to divisions between the major biogeographic regions of

North and South America (Figure 3.9; Brown et al., 1998; Rivas-Martínez et al., 1999).

Clade 1 is restricted to the Colombian-Venezuelan region of South America, with a distinct clade occurring to the west of the Andes in the Ecuatorial coastal province.

Clade 2 is restricted to the Mesoamerican region, with extension into the western

271

Figure 3.7. Agreement topology between maximum-likelihood, Bayesian, and parsimony analyses. This tree is identical to the strict consensus of 1265 most parsimonious trees of 311 steps (CI = 0.521; RI = 0.900), with the exception of the two nodes indicated with * (present in 85% and 92% of the MPTs, read left to right. Letter codes refer to haplotypes that are represented by multiple individuals (see Tables 3.1-3.3), unique haplotypes are represented by the number codes that refer to the localities in Tables 3.1-3.3.

272

Figure 3.8. Phylogram from maximum-likelihood analysis based on HKY85+G+I model. See Methods for search parameters. The results agree with the six clades present in all analyses. Letter codes refer to haplotypes that are represented by multiple individuals (see Tables 3.1-3.3); unique haplotypes are represented by the number codes that refer to the localities in Tables 3.1-3.3.

273 lowlands of the Madrean province. Clades 3-5 form a clade limited to the Mexican

xerophytic region. Only clade 3 is limited to a particular province (the Tamaulipan

province) within this geographically diverse region. Both clades 4 and 5 include

haplotypes that are found in the Tamaulipan, the Chihuahuan, and the Sonoran provinces.

Clade 5 also includes a single haplotype found within the range of the Mesoamerican

clade in the Madrean region. This represents the only area of overlap between the 3

major phylogroups of S. limbatus. S. beali is completely limited to the Tamaulipan province of the Mexican xerophytic region and is found in sympatry with all three

Mexican xerophytic clades of S. limbatus.

The deepest divergence is between the North and South American clades, with the pairwise divergence between North and South America taxa averaging 10.2% (S.D.

1.4%) when estimated using the Hasegawa et al. (1985) model of evolution. Sequence variation was greatest within the Mexican xerophytic phylogroup of S. limbatus, with sequence divergence ranging between 0.2% and 5.4% (mean = 1.9%). The South

American and Mesoamerican phylogroups showed more moderate levels of sequence divergence, with divergences between 0.2% and 3.1% (mean = 1.6%) and 0.2% and 2.5%

(mean = 2.5%) respectively. S. beali showed the least diversity of any of the phylogroups, with a sequence diversity between 0.2% and 1.7% (mean = 0.7%). The phylogram of the maximum-likelihood analysis gives a graphical representation of the partitioning of haplotypic diversity within and between regions and clades (Figure 3.8).

274

Figure 3.9. Map of phylogeographic clades of monophyletic lineages of S. limbatus and S. beali as determined by the mtDNA phylogeny. While the open circles are difficult to see because they overlap with closed circles, S. beali is entirely within the Tamaulipan region in northeastern Mexico. All potential outgroups are from the South American Limbatus clade of Stator. The only area of overlap between S. limbatus clades is in western Mexico.

DISCUSSION

Stator limbatus has historically been separated into two separate species: a North

American S. limbatus and a South American S. cearanus (Johnson et al., 1989).

However, Johnson et al. (1991) and Johnson (1995) showed quite conclusively that the

morphological variation that was used to characterize these taxa transitions continuously

across Latin America. This combined with the consistency of genitalia between both taxa

and the ecological observation that both use numerous host plants led Johnson (1995) to

275 consider them to belong to a single evolutionary entity and he synonymized them under

the name S. limbatus. At first glance, the distinct divergence in mitochondrial sequences

between the reciprocally monophyletic South American and North American clades of S. limbatus would support the initial designation of these lineages as distinct species. Upon further consideration, however, this probably more accurately stems from the fact that the phylogeny of major lineages in S. limbatus generally reflects disjunctions between

biogeographic provinces (Figure 3.9). In fact, if one applies a standard rate of 2.3%

mitochondrial sequence divergence per million years, as estimated for numerous groups

of recently derived taxa by Brower (1994), the split between the North and

South American lineages is estimated as 3.4mya (SD 0.6mya) and corresponds roughly to

the appearance of the Panamanian land bridge connecting the two continents around 3

million years ago (Bermingham and Lessios, 1993; Keigwin, 1982). While this gene split

likely overestimates the population divergence (Edwards and Beerli, 2000), and is subject

to large stochastic variation because it is estimated from a single non-recombining locus

(Edwards and Beerli, 2000; Hudson and Turelli, 2003), it is does follow a biologically-

likely scenario. The remainder of the lineages of S. limbatus also correspond to major

biogeographic delimitations, including a distinctive clade from west of the Andes

Mountains in Ecuador, a Mesoamerican clade, and a clade that is found almost

exclusively within the Mexican xerophytic region, suggesting that geography plays an

important role in structuring lineages in this species (type IV concordance; Avise, 2000).

Such geographic structuring could be due to the sundering of previously

established widespread populations or to the dispersal of a species into geographically

separated areas (Avise, 1994; Avise, 2000). In the context of S. limbatus, it appears that

276 the latter explanation is preferable for two reasons: (1) this species belongs to a group of species that are limited to South America (Chapter 2); and (2) the apparent correspondence between a connection (as opposed to a disjunction) of North and South

America with the most basal genetic disjunction in the species. If this is the case, then the species then spread northward, with major haplotypic lineages diversifying in allopatry first in Mesoamerica and then in northern Mexico. Such a correspondence between geography and phylogeny can also be caused by low dispersal rates and rapid coalescence in haplotypic markers, and not reflect actual geographic barriers to dispersal

(Irwin, 2002). However, the correspondence between biogeographic regions and the phylogenetic structure of the haplotypes suggests that the colonization northward has been limited by the geographic boundaries that separate these regions and that gene flow between them is likely to be fairly limited. These barriers appear to be: (1) the Andes

Mountains in South America, (2) the Isthmus of Panama, and (3) the Sierra Madre

Oriental and Cordillera Transvolcanica in Mexico. S. limbatus is absent from the

Altiplano of Mexico and is not found in any montane regions throughout its range, suggesting that there is no possibility of continuous populations permitting stepwise gene flow across the barriers. In North America, the arm of the Sierra Madre Oriental that reaches the coast in Veracruz appears to separate the Mesoamerican and Mexican xerophytic clades in the east, while the Cordillera Transvolcanica appears to separate them in the west. The only area of overlap between phylogroups occurs near where this last mountain range approaches the coast in Jalisco and Nayarit. This is likely to be the result of secondary sympatry and not a coincidence of sorting of ancestral haplotypes in sympatry. I hypothesize that this is the result of the colonization of this area from

277 opposite directions. The Mesoamerican clade appears to have colonized this area from

the south using the dry deciduous forests characteristic of the southern Mexico coast.

Because there is no opportunity for the Mexican xerophytic clade to colonize the west

coast via the Sierra Madre Oriental, the Altiplano, and the , this

haplotype probably arrived via the relatively accessible habitat of the Chihuhuan and

Sonoran deserts and colonized this area via the Sinaloan biogeographic province.

However, considerably more sampling is necessary along the western coast of Mexico

and through the Chihuahuan desert to address this hypothesis adequately.

The Mexican xerophytic clade was the most extensively sampled and it appears

that this entire biogeographic region provides few barriers that structure the phylogenetic

lineages within this area. Of the three well-supported clades from this area, only clade 3

(Figures 3.7 and 3.8) is completely limited to one area, the Tamaulipan region. Clades 4

and 5 include haplotypes from the Tamaulipan, Chihuahuan, and Sonoran regions, with

clade 5 including a haplotype found in the Sinaloan region. Indeed, different specimens

reared from the same host plant individual at sites within both the Sonoran desert (site

#19) and Chihuahuan desert (site #23) are found in both of these clades; and specimens

reared from the same host plants within the flood plain of the Gulf of Mexico are found

in clades 3 and 5 or 4 and 5, although never in both 3 and 4. Given that S. limbatus is almost continuously distributed up the Rio Grande valley, through the Chihuahuan desert and into the Sonoran desert, the opportunity for persistent gene flow connecting these areas appears to exist (Figure 3.1).

Therefore, the phylogeography of S. limbatus reflects well-known historical boundaries to dispersal, with the well-supported lineages being separated by distinct

278 geographic boundaries. This is in stark contrast to the lineage leading to S. beali. While the remainder of well-supported lineages throughout this clade appear to be separated geographically, this morphologically, behaviorally, and ecologically distinct lineage is found in sympatry with haplotypes from all of the closely related lineages of S. limbatus

(clades 3-5; Figures 3.7 and 3.8). Indeed, divergence of S. beali within S. limbatus appears to have occurred within a biogeographic region with no known or hypothesized barriers to dispersal—the coastal floodplain of the Gulf of Mexico.

S. beali is completely restricted to this coastal floodplain in the Tamaulipan province of the Mexican xerophytic region. One possibility is that this divergence occurred in allopatry with the generalist species S. limbatus and that their current distributions are the result of secondary sympatry. However, there is no evidence that the occurrence of S. limbatus in the Tamaulipan province is the result of a recent colonization of the area. First, this is the only province in the Mexican xerophytic region with haplotypes from all three S. limbatus clades that occur in the area, and includes the only clade of the three that is restricted to a particular biogeographic province (clade 3).

Second, S. limbatus appears to have colonized Mexico from the south, with the

Mesoamerican clade moving up the west coast and the Mexican xerophytic clade moving up the east coast. Given that the Mexican xerophytic clade is absent from the Altiplano, the species would have had to move through the Tamaulipan province in order to colonize the Chihuahuan and then the Sonoran provinces. Combining these two observations suggests that the presence of S. limbatus in the Gulf Coast is not the result of an invasion into the area after the colonization, ecological specialization, and speciation of S. beali in the area.

279 The possibility remains that the current sympatric distribution is the result of an invasion into the area by S. beali after it diverged in allopatry. For example, if the origin and early distribution of its host plant, E. ebano, were vastly different from its current distribution; and if this early distribution were allopatric from the distribution of the numerous hosts of S. limbatus, then one would have a possible scenario for the allopatric origin of S. beali. This is virtually impossible to test using phylogeographic data, but evidence from paleobotanical studies of plant communities could provide insight into this possibility. Using the same molecular clock calibration cited earlier (with the same caveats), the split of S. beali from S. limbatus is estimated to have occurred during the early Pleistocene (1.4 mya; S.D. 0.25my), a period marked by the first of the Pleistocene glaciations (Graham, 1999). During the glaciations of the Pliocene and Pleistocene, the distribution of the subtropical flora was pushed southward and a temperate flora stretched into northern Mexico, while in the interglacial periods the reverse occurred. During these cycles of glaciation sea levels fluctuated by as much as 150m, alternatively exposing large portions of the now submerged shelf of the Gulf Mexico and submerging the currently exposed coastal flood plain. These oscillations would have provided numerous opportunities for the range of E. ebano to change dramatically, and for the community to which it belonged to alter significantly (Graham, 1976; Graham, 1987; Graham, 1988;

Graham, 1997; Graham, 1999). However, it is questionable that it would have been completely isolated from any of the numerous host plants of S. limbatus. S. limbatus is a generalist throughout its range, therefore it is unlikely that this is a recently evolved phenomenon. Furthermore, it uses numerous hosts that are currently in complete sympatry with E. ebano, as well as hosts distributed to the north and south of E. ebano.

280 Range fluctuations by E. ebano would likely then to have been either coincident with range fluctuations in host plants of S. limbatus, or would have moved this species into the range of other host plants. Indeed, hosts of S. limbatus are one of the few persistent and regular features of paleoecological communities in Mexico and the Neotropics throughout the climatic fluctuations of the Neogene (Graham, 1976; Graham, 1987;

Graham, 1988; Magallón and Cevallos F., 1994).

Therefore, while the phylogeographic pattern of relationships between the sympatric lineages of S. beali and S. limbatus is proposed by Avise (2000) to be the signature of secondary sympatry, there are reasons to suspect that the phylogeographic pattern in the mtDNA haplotypes may be the result of ecological differentiation within geographic areas of at least close geographic proximity. To summarize, these include:

(1) the observation that S. limbatus phylogeography generally reflects well-established biogeographic boundaries; with the lone exception being the appearance of S. beali in sympatry with the Mexican xerophytic clade of S. limbatus; (2) the observation that S. beali is most closely related to a clade of S. limbatus that includes diverse haplotypes within the area of sympatry, an area with no known or hypothesized geographic boundaries to dispersal; and (3) the ecological generalism of S. limbatus suggests that climatic fluctuations affecting the distribution of the single host of S. beali, E. ebano, would not likely have removed it from sympatry with at least some hosts of S. limbatus.

This result is in contrast to a recent phylogeographic study of the bogus yucca moth

Prodoxus quinquepunctellus (Prodoxidae) in which it was found that biogeographic distributions were better predictors of differentiation than host use patterns (Althoff et al.,

2001); but it agrees with the result found by Brown et al. (1996) for the goldenrod ball

281 gallmaker, Eurosta solidaginis (Diptera: Tephritidae) in which host use is a predictor of haplotype differentiation for a derived host association.

The interspecific phylogeography of mitochondrial haplotypes in these sister species of beetles suggests that the divergence of S. beali in ecological associations, behavioral adaptations for host use, morphology of the genitalia, and in the ability to form viable offspring occurred within a limited geographic context. In the absence of biogeographic evidence to support a conventional model of allopatric speciation, some other process such as natural and/or sexual selection or a ‘genetic revolution’ in a peripherally isolated population may account for the localized speciation of S. beali from

S. limbatus.

The extreme divergence in ecological associations between these two species, accompanied by evidence for behavioral and life history adaptations in S. beali to increase performance on the seeds of E. ebano (Fox et al., 1996b; Fox and Mousseau,

1995b) is certainly suggestive of a role for natural selection in this speciation process.

Additionally, the difference in armature in the genitalia of the males (Figure 3.3) is indicative of divergence due to sexual selection (Eberhard, 1985; Eberhard, 1993).

Because this type of armature is known to be important in intersexual conflict in other bruchines (Crudgington and Siva Jothy, 2000), divergence in intersexual conflict may have contributed to this speciation as this type of sexual selection can drive sympatric reproductive isolation (Gavrilets and Waxman, 2002; Kondrashov and Shpak, 1998) or accelerate reproductive isolation in allopatry (Gavrilets, 2000). Indeed, this diverging sexual conflict may be the direct result of divergence in host use; and these two factors,

282 natural selection and sexual selection, may be acting in tandem to rapidly drive speciation

in this group. E. ebano produces large seeds that can support numerous larvae of S. beali, while S. limbatus uses much smaller seeds that can generally only support 1-4 individuals

per seed. As a result, the offspring of any given S. beali female are less likely to compete

one with another than are the offspring of any given S. limbatus female (Fox et al.,

1996b; Fox and Mousseau, 1995b). Decreasing the levels of competition between

offspring also decreases the amount of conflict between the interests of the paternal

genome and the maternal genome if females can be multiply mated (Haig, 1999). The

loss of spines on the median sclerite of the male genitalia of S. beali is in accordance with

a decrease in intersexual conflict in this species, as it is the spines in this organ that have

been shown to cause genital damage in females of Callosobruchus maculatus

(Crudgington and Siva Jothy, 2000). Therefore the host shift by S. beali to the large-

seeded E. ebano may have set a separate course for both natural selection for

specialization and sexual selection due to decreased levels of intersexual conflict. An interesting avenue of research would include more intensive sampling of South American

S. limbatus and S. generalis and S. pacarae. The latter two species are very closely related to S. limbatus and are both specialists on large-seeded host plants. While they both possess spines on median sclerite of the male genitalia, these are not nearly as pronounced nor as numerous as those in S. limbatus. Research into the functional effects of this would be needed to determine whether or not this is a relevant observation, however.

The data presented here do not provide the information necessary to distinguish the historical demographic scenarios that different modes of speciation predict (Wakeley

283 and Hey, 1998). While the phylogeographic pattern may be suggestive of sympatric

speciation, various alternatives cannot currently be rejected. For example, transient

allopatry can permit the initiation of localized adaptation without the saturating effects of gene flow from a large source population (Futuyma, 1987; Futuyma, 1989). Subsequent natural and/or sexual selection can then drive reproductive isolation (Knowles, 2001b;

McCune and Lovejoy, 1998; Orr and Smith, 1998; Schluter, 1998), perhaps aided by reinforcement (Kelly and Noor, 1996; Noor, 1995; Noor, 1997; Noor, 1999). Such speculation requires evidence of gene flow during the initial speciation stages, information that can only be gleaned by examining both multiple neutral loci and loci responsible for reproductive isolation or ecological diversification (Kliman et al., 2000;

Wakeley and Hey, 1998). A severe bottleneck in a peripherally isolated population,

perhaps in a community in which only E. ebano occurs, could result in a ‘genetic revolution’ and the reproductive isolation of said population (Carson and Templeton,

1984; Mayr, 1963; Templeton, 1980; Templeton, 1996). Distinguishing this scenario requires examining the historical demography of the diverging populations at the time of speciation (Knowles, 2001b; Wakeley and Hey, 1997; Wakeley and Hey, 1998). Finally, habitat fidelity combined with low dispersal distances could result in micro-allopatric speciation in which an extrinsic barrier to dispersal is present although perhaps not immediately obvious (Mayr, 1947; Mayr, 1963). Distinguishing this scenario requires evidence into the nature of dispersal and gene flow in populations of S. limbatus and S. beali as well as concordance across the genome to such a selectively-neutral divergence.

284 In conclusion, the phylogeographic history of Stator limbatus and S. beali is highly informative into the role that biogeography plays in structuring large scale lineages of the former and into the geographic context of speciation of the latter. This combination certainly sets the stage for the possibility of speciation through specialization in S. beali, and future insights based on historical demography and population genetics promise to be highly informative in this circumstance.

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CHAPTER 4:

PATTERNS OF POPULATION DIFFERENTIATION AND DEMOGRAPHIC HISTORY :

IMPLICATIONS FOR SPECIATION AND SPECIALIZATION IN A SISTER-SPECIES PAIR OF

SEED BEETLES (COLEOPTERA: STATOR)

INTRODUCTION

Phylogenetic analysis has shown that the origins of feeding on plants by insects has been a key innovation that has predictably resulted in increased diversification rates

(Mitter et al., 1988). According to many authors, these phytophagous insects are extraordinarily diverse because both their host resources are diverse and because they tend to specialize on their host plants (Farrell, 1998; Mitter et al., 1988; Strong et al.,

1984). While this pattern does not explain the process, the suggestion is that there is both selection for specialization and that speciation is often accompanied by host shifts. Few topics in evolutionary biology have been more controversial than the role of natural selection in driving speciation; and the study of the causal connection between specialization and speciation in phytophagous insects fits this mold.

Recent theoretical and empirical work is beginning to demonstrate that ecologically-driven speciation can play a prominent role in diversification, particularly in the diversification of phytophagous insects (Barraclough and Vogler, 2000; Diehl and

Bush, 1989; Feder and Filchak, 1999; Kawecki, 1997b; Kondrashov and Kondrashov,

1999; Kondrashov and Shpak, 1998; Kondrashov et al., 1998; Rice and Salt, 1990; Via,

2001). Supporters of such a model hypothesize that (1) disruptive selection to use alternate hosts is strong; and (2) assortative mating is ecologically determined when mating occurs on the host plant. These appear to be the conditions under which sympatric selection is most likely. Critics argue that very low levels of migration would swamp natural selection to use an alternate host plant, and point out that the trade-offs expected from disruptive selection have only rarely been demonstrated (Coyne and Price,

2000; Futuyma and Mayer, 1980; Futuyma and Moreno, 1988; Turelli et al., 2001).

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If sympatric speciation is not a major force in the diversification of insects, the

correlation between host-shifting and speciation must still be explained. One model that

has been historically popular in the explanation of the evolution of numerous specialists

from generalist ancestors is that of peripatric speciation. The empirical observation that

many groups of species are composed of (1) geographically widespread species; and (2)

species closely related to these that occupy narrower ecological habitats in small ranges

along the periphery of the widespread species’ range initially led Mayr (1954; 1963) to

propose that peripatric speciation was a dominant form of species diversification. The

proposed mechanism is through a ‘genetic revolution’ during a severe population

bottleneck in a peripherally isolated population whereby genetic drift can alter the allele

frequencies of the peripheral populations significantly; as the population then recovers in

size natural selection can act and allow the population to climb adaptive peaks different

from the parent population (Carson and Templeton, 1984; Templeton, 1980). There is

considerable debate as to whether such a mode of speciation is likely: some authors

consider the combination of a genetic revolution, adaptive shift, and reproductive

isolation theoretically untenable (Barton, 1996; Barton and Charlesworth, 1984;

Charlesworth, 1995; Charlesworth, 1997; Coyne et al., 2000; Turelli et al., 2001); while

other models with different assumptions suggest that it is at least a credible mode of

speciation (Gavrilets and Boake, 1998; Gavrilets and Hastings, 1996; Rundle et al., 1999;

Slatkin, 1996; Whitlock, 1997).

Finally, allopatric or microallopatric speciation is often considered the least

restrictive process of reproductive isolation. Indeed reproductive isolation is a foregone conclusion given barriers to gene flow over long enough periods of time (Orr, 2001;

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Turelli et al., 2001), and divergent natural selection or sexual selection can facilitate this process (Gavrilets, 2000; Knowles, 2000; Schluter, 2001; Turelli et al., 2001).

Furthermore it has become clear that peripherally-isolated populations need not experience extreme bottlenecks, but that the divergence in conspicuous ecological or other phenotypic traits in these populations may be the result of selection alone. In these cases of microallopatric speciation, isolation from the homogenizing effects of unidirectional gene flow from the source ancestral population permits the fixation of alleles that are locally adaptive, permitting rapid anagenesis in peripheral populations

(Futuyma, 1987; Schluter, 2001).

It has traditionally been very difficult to distinguish between these different modes of speciation, as the predominant sources of information have been correlations between phenotypic differences and geographic range; and conclusions differed based on the philosophical leanings of the individuals interpreting the data. More recently, however, predictions and techniques based on patterns of molecular genetic variation have begun to illuminate the role of these different modes of speciation in generating biotic diversity. In particular, phylogeographic methods (Avise, 2000; Avise et al., 1987) and the advancements enabled by the development of coalescent theory (Hudson, 1990;

Kingman, 1982; Templeton, 2001; Wakeley and Hey, 1998) provide a conceptual framework to test whether observed patterns of genetic variation are consistent with the particular geographic and demographic predictions generated by these alternative hypotheses.

These methods are most directly insightful into the mode of speciation when species are very recently diverged—recently enough that they are still expected to share

289 ancestral polymorphisms (Edwards and Beerli, 2000; Hudson and Turelli, 2003; Wakeley and Hey, 1998). This gives insight into the historical demography and geographic context of speciation at the time of speciation. Furthermore, these approaches are most useful when multiple loci can be examined (Edwards and Beerli, 2000; Hare, 2001;

Hudson and Turelli, 2003; Wakeley and Hey, 1998). This allows for inferences based on between-locus variance that permit distinguishing between processes that affect the entire genome (such as changes in population size or selectively-neutral migration) versus processes that affect loci differently (particularly natural selection or locus-specific introgression). These applications to speciation have been most comprehensively applied to studies of several species groups of Drosophila (Hey and Kliman, 1993; Hilton et al.,

1994; Kliman et al., 2000; Wang et al., 1997), which provided examples of reduced Ne, selection, and of introgression between species. While this demonstrates the utility of

Drosophila as a model organism, it is very difficult to connect these molecular genetic results with ecological diversification as the natural history of these species is largely unknown.

Unfortunately, most phytophagous insects do not fall under the category of

‘model organism’. As a result, genomic information is not sufficient for the efficient development of dozens of independently-segregating nuclear loci, and many researchers are restricted to studying molecular genetic sequence variation from the (relatively) easily obtainable mitochondrial genome due to economic and time constraints. Further compounding this issue is the fact that many speciation events of interest to researchers are old enough that mitochondrial variation in a species at demographic equilibrium is likely to have completely coalesced after the time of speciation, even if there is a high

290

probability that nuclear loci are still segregating due to their larger effective population

size (Hudson and Turelli, 2003; Palumbi et al., 2001). In this case, information regarding

the speciation event itself is no longer present in the mitochondrial genome.

Regardless, there is historical information within the mitochondrial genome even

if it is a single estimate of the history of females in the population (Avise, 2000).

Applications of phylogeographic and coalescent methods to phytophagous insects have

been few and with the exception of Davies et al’s (1999) research on the estimation of

source populations of the medfly (Ceratitis capitata), they have all used mitochondrial

loci. The majority have focused on phylogeographic analyses of population disjunction

(e.g. Althoff et al., 2001; Brown et al., 1996; Davies et al., 1999; Gómez-Zurita et al.,

2000; Mardulyn, 2001; Segraves and Pellmyr, 2001). More recently, however, various researchers have begun to apply these methods to the examination of modes of speciation in phytophagous insects. Groman and Pellmyr (2000) found that recent adaptation of a yucca moth to a novel host was accompanied by a population bottleneck. In a series of papers, Knowles (Knowles, 2000; Knowles, 2001a; Knowles, 2001b) found paraphyletic relationships between numerous pairs of widespread versus local species of montane grasshoppers, suggesting that historically effective population sizes were smaller for the derived species versus the ancestral species. However, there is no evidence for a population bottleneck in these speciation events. The timing of divergence and the geographic distribution of haplotypes suggested that these were microallopatric speciation events resulting from the local isolation of habitats via glaciations. She also concluded that the apparent rapid speciation was due to sexual selection within the isolated populations. Finally, research on the genus Ophraella (Funk et al.,

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1995; Knowles et al., 1999) supports a paraphyletic relationship between the widespread

O. communa and the peripherally isolated species O. bilineata. The extreme specialization of the latter with respect to the former led Knowles et al. (1999) to hypothesize that this was a case of peripatric (founder) speciation. Population structure was very high in O. communa but showed no indication of isolation by distance, suggesting that it has been influenced by historical factors but not by current limits to gene flow. In addition, the historical demography of O. bilineata did not deviate from that expected under demographic equilibrium. These two observations are suggestive of microallopatric speciation instead of peripatric speciation. Phylogeographic structure argues against sympatric speciation in this case.

These studies indicate the utility of phylogeographic and coalescent methods for the study of the speciation of phytophagous insects using mitochondrial DNA, with the caveat that the results indicate only one of numerous possible realized evolutionary histories. These studies also indicate that information can be gleaned into the speciation process from the current demographic and geographic history of species even when they may not provide information directly concerning the demography at the time of speciation. For example, peripatric speciation is generally considered to be more likely when a species displays high levels of interpopulation differentiation and isolation-by- distance (Harrison, 1991; Palumbi, 1996). If populations are somewhat isolated to begin with due to limited gene flow, a rare founding event at the periphery of a species range need not be all that dramatic to allow a localized population to become completely isolated. In the analysis of Ophraella cited above, the combination of a paraphyletic relationship between the generalist and the specialist and the phylogeographic

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distribution of O. bilineata at the periphery of the range of O. communa led the

researchers to suspect either a microallopatric or peripatric speciation event. They also

argued that microallopatric speciation was more likely than peripatric speciation given

the lack of evidence of a population bottleneck and a population genetic structure lacking

isolation-by-distance, although peripatric speciation could not be rejected by the data.

While this did not address the occurrence of a population disjunction at the time that

reproductive isolation occurred, it does make reference to the current biology of a species

to indicate the likelihood of different models of speciation.

These researchers have understandably focused on the demographic and

genealogical histories of their study organisms in order to gain information regarding the

mode of speciation. Somewhat lost in this is the fact that this same information has

implications regarding the maintenance of the diversity of phytophagous insects. It is

generally assumed that ecological specialists are at a greater risk of extinction given the vagaries of ecological change through geographic timescales (Futuyma and Moreno,

1988; Thompson, 1994). However, the examples above from montane grasshoppers and the genus Ophraella show no evidence of population bottlenecks in their genealogical history. This is perhaps quite remarkable for O. bilineata given a divergence time from

O. communa of 1.3 million years ago, and a current distribution in the northern United

States and southern Canada that has experienced numerous dramatic ecological fluctuations during the glaciations of the Pleistocene (Graham, 1999). Such results indicate that speciation may not be as risky a strategy for phytophagous insects as general arguments might suggest. Indeed, the small body size and small necessary home range

293 for many phytophagous insects supports the idea that rather large effective populations could be contained in fairly small geographic areas.

In Chapter 3, I presented results from an interspecific phylogeographic analysis of the obligate seed predators Stator limbatus (Horn) and S. beali Johnson. This analysis supported a paraphyletic relationship of the widespread generalist S. limbatus with derivation of the extreme specialist and Gulf Coast endemic S. beali 1.2-1.6 million years ago (based on the average pairwise difference corrected for multiple hits and an estimated divergence rate of 2.3% per million years; Brower, 1994). The analysis also showed that while the phylogeography of S. limbatus in general corresponds very well to distinct biogeographic boundaries, this is not reflected in the divergence of S. beali. Instead, haplotypes from this species are most closely related to a clade of haplotypes of S. limbatus within the same geographic region. This suggests that while biogeographic boundaries play a large part in structuring S. limbatus genotypes, the ecological differentiation of S. beali much more readily explains its diversification than does its geographic distribution. The conclusion that diversification occurred in a very local geographic context is highly relevant and enables the rejection of broad allopatry of the kind envisioned by Mayr (1963) as the causal mechanism of speciation. However, that analysis was not intended to elucidate the demographic history of the speciation event or of the diversification of the populations since their diversification.

The goal of this chapter is to use the population structure and historical demography of S. beali and its sister taxon, the Mexican xerophytic clade of S. limbatus to examine the circumstances of speciation and the population biology of the two lineages since they split. Because these mitochondrial haplotype clades are reciprocally

294 monophyletic, they do not contain direct information regarding the demography of speciation. I therefore examine their population genetic structure and historical demography in the context of comparative inferences regarding particular predictions from different modes of speciation. Based on the phylogeographic structure, the generalist phenotype is considered the ancestral form throughout this analysis and the specialist phenotype is considered to have been derived either at or after speciation.

Comparing S. beali with its sister clade instead of S. limbatus as a whole is justified by the phylogeographic reconstruction of the relationships between the two species.

Because the two are hypothesized to share a common origin with the invasion of the

Mexican xerophytic biogeographic region, limiting the comparison to this clade focuses the contrast on their demographic histories since this geographically localized diversification event and removes the confounding effects of ancient phylogeographic population structure in estimates for S. limbatus based on the coalescent. As an example, if the invasion of this biogeographic area were based on a very small number of colonists

(and this information were still present in the data), then both this clade of S. limbatus and S. beali would show demographic histories of rapid population growth. However, including all populations throughout S. limbatus would neglect this and would result in the appearance that only S. beali experienced a bottleneck.

In the following study, I compare the population genetic structure and historical demography of the two species. In particular, I examine the presence of current versus historical boundaries to gene flow by examining both population genetic structure and evidence for isolation-by-distance. This analysis is buoyed by evidence from genealogical relationships between haplotypes. In general I am working under the

295 following framework: If population genetic structure at the time of speciation were similar to that currently observed, what would the implications be for the likelihood of different models of speciation? I then examine evidence of a population bottleneck in S. beali versus S. limbatus. Evidence in both would indicate either that they both reflect a recent invasion by only a few colonists into the Mexican xerophytic province or that they have both experienced bottlenecks due to living under similar ecological circumstances.

Evidence limited to S. beali would either support the peripatric model of speciation or suggest that as a specialist it has been more influenced by ecological fluctuations caused by the climatic changes during the Pleistocene glaciations. The timing of the bottleneck(s) is (are) assessed in order to address this question.

METHODS

STUDY SYSTEM

The general biology, ecology, and geographic distributions of Stator limbatus

(Horn) and S. beali Johnson are discussed in detail in Chapter 3. Summarizing briefly, S. limbatus is a generalist seed predator that has been reared from approximately 70 species of leguminous plants throughout its range (Table 2.1). Specimens of this species have been collected from northern South America to the southwestern United States (Figure

3.1). S. beali is a species-specific specialist that feeds exclusively on the Texas Ebony tree (Ebenopsis ebano (Berl.) Barneby & Grimes) below about 500m in the coastal flood plain of the Gulf of Mexico. Its range is nested completely within that of S. limbatus

(Figures 3.1 and 3.2).

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Phylogenetic analysis of mitochondrial and nuclear DNA sequence data of the genus Stator strongly supports a paraphyletic relationship of the widespread generalist S. limbatus with respect to the narrowly distributed specialist S. beali (Chapter 2). Detailed phylogeographic analysis based on mtDNA sequence data of the species pair shows strong support that S. beali and its sister haplotype clade of S. limbatus both diversified within the Mexican xerophytic biogeographic province. Furthermore, all three major haplotypic divisions of this clade of S. limbatus are found in sympatry with S. beali in the

Tamaulipan biogeographic region along the coast of the Gulf of Mexico (Figures 3.8 and

3.9). Within this area of sympatry S. limbatus is known to feed on at least 15 native species of host plant. The significant divergence in ecological associations, diet breadth, behavioral characters, morphology, male genital armature, and size of the seeds of their host plants imply the possibility of a substantial role for adaptation in the origin of S. beali.

SPECIMENS EXAMINED AND MOLECULAR METHODS

This analysis is based on the specimens included in Chapter 3 that form the

Mexican xerophytic clade of the S. limbatus + S. beali assemblage (Figure 3.9). Table

4.1 provides a summary of the sampling localities, the abbreviations used throughout the paper, the host plants for S. limbatus, and the number of specimens from each population.

In total 132 individuals of S. limbatus from 19 localities were included. Five localities were represented by single individuals; the remainder were represented by 5-10 individuals. 108 individuals of S. beali from 11 localities were included in this analysis.

10 populations were represented by 10 specimens. The remaining population is an

297 aggregate of 8 very closely spaced collecting localities in southern Texas and are listed in

Table 4.1 as TX3 (Cameron Co.). Collection sites from Arizona (AZ1-3), Sonora (SO),

Jalisco (JA), and Ryan, TX (CH) are the only sites from outside of the Tamaulipan biogeographic region.

Table 4.1. Sampling localities with number of individuals sampled at each site (n). All S. beali were reared from Ebenopsis ebano. Host plants for S. limbatus: (a) Acacia greggii; (b) Cercidium microphyllum; (c) A. angustissima; (d) Parkinsonia aculeata; (e) A. wrightii; (f) Havardia pallens; (g) Lysiloma acapulcense.

Species Localities n a S. limbatus AZ1 Colossal Cave, AZ 5 a AZ2 Tucson, AZ 1 b AZ3 Black Canyon City, AZ 1 SO Benjamin Hill, Sonora, Mexicoc 1 CH Ryan (Chihuahuan Desert), TXc 10 d TX1 Brownsville, TX 6 e TX2 Mission, TX 1 f NL1 Paras, Nuevo León, Mexico 10 f NL2 SW Doctor González, Nuevo León, Mexico 10 f NL3 NE Doctor González, Nuevo León, Mexico 10 f TS1 N. Soto La Marina, Tamaulipas, Mexico 9 f TS2 San Fernando de Presas, Tamaulipas, Mexico 10 f TS3 W. Soto La Marina, Tamaulipas, Mexico 9 f TS4 Ciudad Victoria, Tamaulipas, Mexico 10 c TS5 Manuel, Tamaulipas, Mexico 9 f TS6 Antigüo Morelos, Tamaulipas, Mexico 9 f VC1 S. Tantoyuca, Veracruz, Mexico 10 c VC2 N. Tantoyuca, Veracruz, Mexico 10 JA Lázaro Cárdenas, Jalisco, Mexicog 1 S. beali TX3 Cameron Co., TX 8 TX4 Falfurrias, TX 10 NL4 Doctor González, Nuevo León, Mexico 10 TS7 Valle Hermosa, Tamaulipas, Mexico 10 TS8 N. San Fernando de Presas, Tamaulipas, Mexico 10 TS9 S. San Fernando de Presas, Tamaulipas, Mexico 10 TS10 Ciudad Victoria, Tamaulipas, Mexico 10 TS11 Soto La Marina, Tamaulipas, Mexico 10 SL Ebano, San Luis Potosí, Mexico 10 VC3 El Higo, Veracruz, Mexico 10 VC4 Alamo, Veracruz, Mexico 10

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I collected host seeds of both species during collecting trips between 1998 and

2001. Infested host plant seeds were collected in the field and transported to an environmental chamber at the Museum of Comparative Zoology, Harvard University.

They were then transferred into cloth-covered jars and adult specimens were removed as they emerged from seeds and were immediately frozen and stored in a -80°C freezer.

Emerging adults were not allowed to begin a second generation; therefore all specimens included in this analysis were field collected.

Frozen whole individuals were flash-frozen in liquid Nitrogen and immediately ground to a fine powder with a melted pipette tip in an Eppendorf tube. Total genomic

DNA was then isolated using the ‘salting out’ protocol of Sunnucks and Hales (1996). I amplified a ~700 base pair fragment of 3’ end of the mtDNA gene Cytochrome Oxidase I

(COI) using the polymerase chain reaction (PCR). Amplifications were performed in

50µl reactions to produce double-stranded DNA product under the following conditions:

0.2µM each primer, 0.15mM each dNTP, 2.5µM MgCl2, 1X buffer supplied by the

manufacturer (Qiagen, Valencia, CA), and one unit of Taq polymerase (Qiagen).

Reactions were brought up to 50µl using water and 1.5µl DNA genomic template.

Typical temperature profiles for amplification of fragments consisted of 40 cycles of 30s at 95°C, 30s at 50°C, and 1.5 min at 72°C, followed by a 5 min extension at 72°C.

Primer sequences were s1859: (5’-GGAACIGGATGAACWGTTTAYCCICC-3’) and a2590: (5’-GCTCCTATTGATARWACATARTGRAAATG-3’). Numbers refer to the nucleotide positions in the complete mtDNA sequence of Drosophila yakuba (Clary and

Wolstenholme, 1985; Simon et al., 1994). Non-standard nucleotide designations are

IUPAC ambiguity codes (Cornish-Brown, 1985) and refer to equal mixtures of bases. ‘I’

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refers to inosine. PCR products were checked on 1.5% agarose gels and products were

purified directly or after gel extraction using QIAquick columns (Qiagen).

The amount of DNA from the purified PCR product was estimated by comparison

to a Low Mass Ladder (Gibco/Invitrogen, Carlsbad, CA) and 70-90ng (ABI373) or 20-

50ng (ABI3100) was used for sequencing reactions. Sequencing primers were identical

to those used in PCR reactions. Sequencing of this double-stranded product was carried

out using 25 PCR cycles of 96°C for 30s, 50°C for 15s and 60°C for 4 min with a 2°C increase per second in a 10µl reaction. This reaction used dye terminator cycle sequencing chemistry with the specified amount of template, 2.0µl of Dye Deoxy FS

Terminator or BigDye premix (PE Biosystems, Inc., Foster City, CA), 0.16µM primer,

and water to the final volume. Finished reactions were precipitated in 0.5mM MgCl2 in

70% ethanol, centrifuged, and dried under a vacuum. The majority of sequencing was done using an ABI 373 automated DNA sequencer, with some sequencing done using an

ABI 3100 automated sequencer (PE Biosystems, Inc.). Compiled segments resulted in

665 base pairs of CO1 for all 240 specimens included in this analysis. Both directions of

the PCR product were sequenced and contigs were assembled, edited, and aligned with

Sequencher ver. 4.1 (GeneCodes, Ann Arbor, MI). Because the fragment consists of

protein-coding sequences with no insertions or deletions, alignment was unambiguous.

Sequences will be submitted to GENBANK prior to journal submission.

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DATA ANALYSIS

Genealogy Reconstruction

All specimens from all populations were included in the analyses of the genetic and demographic history of divergence between S. limbatus and S. beali in the Mexican xerophytic province. Genealogical relationships were estimated using maximum- likelihood, although the results presented here are identical to those presented in Chapter

3—apparently including or excluding the large number of specimens of S. limbatus from

Mesoamerica and South America does not affect genealogy reconstruction. The program

ModelTest 3.0 (Posada and Crandall, 1998) was used to determine the appropriate model of molecular evolution for the maximum likelihood analysis. The model selected was a variant of the HKY85 model (Hasegawa et al., 1985) with rates estimated based on a gamma distribution (α-parameter estimated from the data) with an estimated proportion of sites assumed to be invariable (HKY85 + I + G). The parameters estimated using

ModelTest were incorporated into the searches in order to decrease the search to a reasonable time limit. The maximum likelihood analysis was implemented using

PAUP*, and consisted of 50 heuristic searches based on a starting tree derived from the random addition of taxa, with each search allowed to examine 1,000,000 trees. The close relationships of many haplotypes in this intraspecific analysis meant that more thorough tree-searching algorithms would have been prohibitively long and the results should be interpreted with this caveat in mind.

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Population Genetic Structure

Assessments of genetic variation are highly dependent upon the geographic scale at which estimates are inferred (Peterson and Denno, 1998). Therefore in order to make meaningful comparisons between the population structures of S. beali and S. limbatus all comparisons were limited to populations within the Tamaulipan biogeographic province in the plain along the coast of the Gulf of Mexico (Figure 4.1). Patterns of genetic variation and levels of genetic diversity within and among populations of both S. limbatus and S. beali were analyzed with ARLEQUIN v2.000 (Schneider et al., 2000). Population genetic structure was estimated using the analysis of molecular variance approach

(AMOVA) of Excoffier et al. (1992). This approach estimates the fixation indices (in this case FST) as originally defined by Wright (Wright, 1951; Wright, 1965) and subsequently applied to genetic sequence data (Excoffier et al., 1992; Weir and

Cockerham, 1984) and coalescent times (Slatkin, 1991) by taking into account the number of mutations between molecular haplotypes. Genetic distances were corrected using Kimura’s 2-parameter (K2P) model of DNA substitution to account for different rates of substitutions between transitions and transversions (Kimura, 1980). Statistical significance of FST values was determined based on 10,000 permutations of haplotypes among populations (Excoffier et al., 1992). In addition, geographic subdivision was examined using more stringent exact tests for haplotype × population associations

(Raymond and Rousset, 1995). This implementation of the exact test makes use of a

Markov chain in order to explore all potential states of the contingency table, and the P- value is estimated based on the proportion of values observed in the chain that are more likely than the observed value. While these frequency-based approaches do not

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Figure 4.1. Sampling localities of S. beali (black) and S. limbatus (gray) for assessment of population structure within the Tamaulipan biogeographic province. Codes correspond to localities in Table 4.1.

distinguish between genetic differentiation due to different levels of migration or different times since isolation (Nielsen and Wakeley, 2001), they do provide a heuristic to examine the genetic distinctness of populations.

Mantel tests (Sokal and Rohlf, 1995) were used to test for an association between

FST and the geographic distance separating populations among all pairs of populations in order to examine the possibility of isolation by distance (Smouse et al., 1986). Straight line geographic distances were calculated from GPS coordinates using the direct method of Vincenty (1975); this method therefore does not take into account the possibility of

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corridors or barriers to gene flow between particular populations. The significance of the

partial correlation coefficients obtained from the pairwise correlations (the r-value) was

tested by comparison to a distribution of 10,000 random permutations of the geographic

distance matrix (Smouse et al., 1986).

Demographic History

Genetic diversities within S. limbatus and S. beali were estimated based on π, the

average number of pairwise differences between sequences (Tajima, 1983), and θW, based

on the number of segregating sites using ARLEQUIN (Schneider et al., 2000). In

addition, a Metropolis-Hastings sampling routine of the sequence data was used to

estimate species-wide θ’s (Kuhner et al., 1995). This method incorporates genealogical

structure into the estimation of θ by using Kingman’s (1982) model of the coalescent process to calculate the prior probability of sampled genealogies given an estimate of θ:

P(G| θ). Genealogies are sampled according to a decision-making algorithm in a manner that creates a Markov chain of genealogies that if run long enough will travel through genealogies in proportion to their posterior probabilities, providing a maximum- likelihood estimate of the parameter θ as well as confidence intervals. This estimation was implemented using the program FLUCTUATE v1.5 (Kuhner et al., 2002) which implements the algorithm described in Kuhner et al. (1995) when g is set to zero. In order to estimate starting parameters for the long chains, I ran 20 short chains of 200 steps sampling every 20 genealogies. Four long chains were then run for 40,000 generations while sampling every 40th genealogy. This is well above the number of steps recommended by Kuhner et al. (1995) to obtain reliable estimates. Multiple runs

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using this strategy converged on the same estimate of θ. Starting parameters for the analysis were based on the observed transition/transversion ratio as estimated using the program SITES (Wakeley and Hey, 1997), the estimate of θW, and a starting genealogy

based on a UPGMA tree estimated using PAUP* (Swofford, 2000). Because this study is

based on the haploid, maternally transmitted mitochondrial DNA, the three estimates

listed above are estimates of 2Nfµ, the effective population size of females multiplied by

the mutation rate per generation, estimated per base pair. When reference is made in the

remainder of the paper to effective population size, it is implied that this is the female

effective population size. Throughout all analyses µ was assumed to be 8.5 × 10-9 per

base pair, an estimate derived from an analysis of mitochondrial divergences in numerous

arthropod groups (Brower, 1994). Because of this, all values that are scaled by the

mutation rate (θ, π, τ) are expressed per base pair instead of per sequence. It should be kept in mind that this estimate of mutation rate likely has high between-taxon variation

and should be taken as a heuristic only.

The demographic history of each species was assessed in terms of expectations

and models that examine changes in population size based on coalescent theory. While

many of these estimates are indistinguishable from the effects of selection when using a

single locus, the analyses are done under the working assumption that the mitochondrial

genome has been evolving neutrally since the divergence of the two species.

I used Tajima’s D (Tajima, 1989c) to examine whether there is evidence for either

population expansion or population subdivision in both species. This metric is based on

the fact that while θW and π are equivalent estimators in a population that has been at

demographic equilibrium and selective neutrality, θW is more sensitive to low-frequency

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polymorphisms than is π. Tajima (1989c) initially developed this statistic to examine the

effects of selection, as a population that is returning to equilibrium after a selective sweep

is expected to contain a large number of rare mutations in loci affected by the selective

sweep, while a gene that is under balancing selection will be deficient in these

polymorphisms. As a result, θW is expected to be higher than π in directional selection,

resulting in a negative D, while the opposite is true under balancing selection. However,

a rapid population expansion will have a similar effect on low-frequency polymorphisms

as a selective sweep because the probability of coalescence is directly proportional to

effective population size (Tajima, 1989b). Population subdivision will have the opposite

effect, preserving mid-frequency polymorphisms through longer time intervals and

causing an elevation in π relative to θW, resulting in a positive D (Tajima, 1989a). I

estimated this statistic using ARLEQUIN (Schneider et al., 2000). Statistical significance was determined by generating 1000 random samples under demographic equilibrium and selective neutrality using a coalescent simulation algorithm adapted from Hudson (1990).

The P-value was also assessed by reference to Tajima’s Table 2 (p. 592, 1989c) which estimates confidence intervals of D by assuming the beta-distribution.

I assessed the number of pairwise differences between pairs of haplotypes within both species in order to examine the ‘mismatch distribution’ (Rogers and Harpending,

1992). Because this will reflect the highly stochastic nature of the coalescent process, this distribution is expected to be multimodal in a population at demographic equilibrium.

However, it is expected to be unimodal in a population that has recently experienced a demographic expansion (Rogers and Harpending, 1992; Slatkin and Hudson, 1991). I examined this distribution via the expansion model of Schneider and Excoffier (1999),

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who implement a parametric estimator of this distribution based on a generalized non-

linear least-square approach using the coalescent algorithm of Hudson (1990) mentioned before. Using parameters estimated from the data, they assume an immediate expansion from an ancestral θA at some time τ ago to the present day θ1 and then generate a

mismatch distribution based on these simulations. If the observed data do not reflect this

demographic history then there is a significant deviation from this model. A similar

approach is implemented by Wakeley and Hey (1997) in their model of population

expansion following speciation. This method generates the expectation of the frequency

of different categories of site frequencies under the same model of population expansion

described above. I assessed the fit of the distribution of these site frequencies in both S.

limbatus and S. beali using the program SITES (Wakeley and Hey, 1997). Due to

software limitations, the maximum size of the dataset for this analysis is constrained to

200 taxa. These analyses are therefore done based on 100 randomly chosen individuals

from each species.

Both of the methods described above assume an immediate expansion from an

ancestral population size to the present-day population size, and neither is based on

explicit sampling of genealogical information. A more realistic model (although still

rather simplified) of population growth might incorporate, for example, a model of

exponential growth (Slatkin and Hudson, 1991); and a more efficient estimator should

incorporate genealogical information directly into the assessment of the probability of the

data (Felsenstein, 1992). I estimated the likelihood of a demographic history of

exponential growth in both S. limbatus and S. beali using a similar maximum likelihood

estimation based on the coalescent as described for the estimation of θ previously.

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Kuhner et al. (Kuhner et al., 1998) jointly estimate θ and the exponential growth rate, g, using the same Metropolis-Hastings sampling of Monte Carlo chains described above.

Starting parameters and Metropolis-Hastings sampling regime are identical to those

described previously. The likelihood of the hypothesis that the species have experienced

recent exponential growth was evaluated against the null hypothesis of no exponential

growth. This was done by comparing the likelihoods when g and θ are jointly estimated

versus when g is held at zero and only θ is estimated using the Akaike information

criterion (Akaike, 1985). Using this criterion, the model that minimizes -2 × [Ln(L) – di]

is chosen as being most likely, where di is the number of parameters of model i (i = 1

under the null hypothesis and 2 under the alternate hypothesis).

RESULTS

Genealogical relationships

Because of the non-recombining nature of mtDNA haplotypes, they will

essentially have a history of splitting. In this case genealogical and phylogenetic

relationships are equivalent terms. Among the 132 individual COI sequences determined

from S. limbatus, 57 display different nucleotides at one or more sites (Table 4.2a).

Among the 108 individual COI sequences collected from S. beali, 39 have different

nucleotides at one or more sites (Table 4.2b). In Chapter 3, maximum-likelihood,

Bayesian analysis, and parsimony give similar results for the genealogical relationships.

Unlike interspecific phylogenetic analysis, under the coalescent ancestral haplotypes are expected to coexist with derived haplotypes. Because of this, the maximum-likelihood trees presented here are shown as networks and include sampled haplotypes that are

308 reconstructed to reside at nodes within the phylogenetic tree. The frequency of individuals with a particular haplotype is directly proportional to the area of the circle

representing them on the genealogical tree. The tree for S. beali (Figure 4.2) displays a

Table 4.2. Number of singletons and frequency of haplotypes from the sampling localities where more than one individual was sampled. (a) S. limbatus. AZ2, AZ3, SO, TX2, and JA are not included in table because only one individual was sampled. However, the haplotype from AZ2 was identical to a haplotype from CH (*), and AZ3 is haplotype B (†). All haplotypes are included in the ‘Total’ row at the bottom. (b) S. beali.

(a) S. limbatus Localities # of # shared Shared haplotypes Total # # of singletons local haplotypes individuals haplotypes A B C D E F G AZ1 5 0 5 5 CH 9 0 1* 10 10 TX1 4 0 1 1 5 6 NL1 5 1 (3) 2 7 10 NL2 3 3 (3,2,2) 6 10 NL3 2 1 (3) 4 1 5 10 TS1 1 1 (2) 3 2 1 5 9 TS2 0 3 (3,3,2) 2 4 10 TS3 2 0 2 1 3 1 6 9 TS4 1 1 (2) 3 3 1 5 10 TS5 0 0 8 1 2 9 TS6 0 0 4 4 1 3 9 VC1 2 2 (3, 2) 3 5 10 VC2 1 0 8 1 3 10 Total 35 12 2* 38† 11 2 5 2 4 57 132

(b) S. beali Localities # of # shared Shared haplotypes Total # # of singletons local haplotypes individuals haplotypes H I J K L M N TX3 6 0 1 1 8 8 TX4 1 1 (2) 1 3 3 5 10 NL4 1 1 (8) 1 3 10 TS7 3 0 3 4 3 10 TS8 1 0 7 2 3 10 TS9 0 1 (2) 3 5 3 10 TS10 3 1 (2) 2 2 1 7 10 TS11 3 2 (2,2) 1 1 1 8 10 SL 3 1 (2) 1 4 6 10 VC3 0 2 (4,4) 1 1 4 10 VC4 1 1 (2) 5 1 1 5 10 Total 22 10 11 10 5 4 18 6 2 39 108

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Figure 4.2. Genealogical relationships among Stator beali haplotypes based on the HKY85 + G + I model and rooted with S. limbatus from the Mexican xerophytic province. Branch lengths are proportional to the expected number of substitutions. Closed circles represent unsampled haplotypes. The area of open circles is directly proportional to the number of haplotypes sampled.

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Figure 4.3. Genealogical relationships among Stator limbatus haplotypes in the Mexican xerophytic province based on the HKY85 + G + I model and rooted with S. beali. Branch lengths are proportional to the expected number of substitutions, although they are collapsed and represented by slashes for distances ≥ 5. Closed circles and slashes represent unsampled haplotypes. The area of open circles is directly proportional to the number of haplotypes sampled.

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very star-like topology with 23 lineages radiating from the second most-common

haplotype. The tree for S. limbatus (Figure 4.3), on the other hand, shows considerably

more 2-dimensional structure. There are three clades separated by numerous mutational

steps, and there is considerable genealogical structure within each of these clades.

In addition, the range of sequence divergence (corrected using the K2P model)

between populations of S. limbatus within the Tamaulipan province is 0.42%-2.9% and

extends well beyond the range found between populations of S. beali, which is 0.24%-

0.83% (Table 4.3). The average sequence divergence between individuals in S. limbatus

is 1.33% with a maximum divergence of 4.06% (1.23% and 3.75%, respectively, within

the Tamaulipan Province). The average sequence divergence between individuals in S.

beali is 0.48% with a maximum pairwise sequence divergence of 1.52%. Comparing

these to the average (2.59%) and maximum (4.08%) sequence divergence between S.

limbatus and S. beali suggests that diversity within S. limbatus is similar to diversity

between the two species.

Population Genetic Structure

Given the current spatial scale and density of sampling, genetic diversity within

many populations of both species is quite high, occasionally higher than between

populations (diagonal vs. above diagonal, Table 4.3a and 4.3b). This varies considerably

between population comparisons for both species, however. As a result, FST-values among pairwise populations of S. limbatus are significant, following a sequential

Bonferroni correction (Sokal and Rohlf, 1995), in 15 of 66 comparisons (below diagonal,

Table 4.3a, significant comparisons in boldface). The southernmost population (VC1)

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accounts for 11 of these significant comparisons, suggesting that it is rather isolated from

the remaining populations. The overall FST for the Tamaulipan populations of S.

limbatus is significantly non-zero (FST = 0.19, P < 0.0001), including when population

VC1 was excluded (FST = 0.13, P < 0.0005). However, the exact assignment of

haplotypes to geographic locations does not differ from random (P = 1.00), as is apparent

from the examination of the geographic distribution of individual haplotypes (Figure 4.4).

The phylogenetic reconstruction of S. limbatus haplotypes also indicates a lack of association between haplotype relationships and geographic region, both within the

Tamaulipan biogeographic province and when haplotypes from the Chihuahuan and

Sonoran biogeographic provinces are included (Figure 4.3). Only one of the three major haplotype clades of the Mexican xerophytic clade of S. limbatus is limited to one of the above biogeographic provinces, although a second that is found in all three includes specimens that are limited to the very northwestern portion of the Tamaulipan province.

Of those haplotypes within the Tamaulipan province, most populations contain haplotypes that are distributed throughout the area.

The situation is similar for S. beali, both in among-population comparisons and species-wide metrics. Pairwise FST-values are significant in 14 of 55 pairwise comparisons (below diagonal, Table 4.3b, significant comparisons in boldface). As in S. limbatus, one peripheral population from the northwestern extent of S. beali’s range

(NL4) accounts for 8 of the total significant comparisons. The estimated species-wide

FST is very similar to that estimated for S. limbatus and also indicates significant

population structure (FST = 0.20, P < 0.0001), which is significant when the peripheral

population NL4 is excluded (FST = 0.15, P < 0.0001). Again, while there appears to be

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Table 4.3. CO1 sequence diversity within and between populations of Stator limbatus (a) and Stator beali (b). Above the diagonal are percent average pairwise sequence diversities between populations, corrected using Kimura’s (1980) 2-parameter model. On the diagonal are percent average pairwise sequence diversities within populations (in italics). Below the diagonal are estimates of FST from pairwise population comparisons. Significant values, P < 0.05 after adjusting for multiple comparisons using a sequential Bonferroni correction, are in boldface. Numbers of specimens for each population are in Table 4.1.

(a) S. limbatus

TX1 NL1 NL2 NL3 TS1 TS2 TS3 TS4 TS5 TS6 VC1 VC2 TX1 1.92 2.65 2.42 2.49 2.79 2.67 2.66 2.72 2.60 2.74 2.97 2.61 NL1 0.09 1.59 1.55 1.61 1.51 1.46 1.43 1.38 1.40 1.44 1.56 1.41 NL2 0.13 0 1.15 1.32 1.74 1.05 1.19 1.45 0.99 1.76 1.14 1.14 NL3 0.11 0.01 0.07 1.31 1.67 1.00 1.11 1.40 0.89 1.71 1.28 1.02 TS1 0.07 0.15 0.2 0.12 1.66 1.51 1.42 1.42 1.38 1.45 1.76 1.36 TS2 0.30 0.11 0.17 0.04 0.26 0.60 0.74 1.17 0.49 1.61 0.90 0.67 TS3 0.17 0.1 0.16 0.03 0.11 0.02 0.85 1.13 0.56 1.50 1.11 0.68 TS4 0.06 0.07 0.13 0.04 0.07 0.16 0.02 1.37 1.03 1.41 1.39 1.08 TS5 0.41 0.23 0.29 0.12 0.31 0.14 0.02 0.21 0.24 1.50 0.79 0.42 TS6 0.09 0.19 0.24 0.17 0.1 0.35 0.2 0.03 0.41 1.54 1.80 1.46 VC1 0.48 0.31 0.38 0.38 0.47 0.52 0.5 0.41 0.68 0.51 0.62 1.07 VC2 0.26 0.17 0.23 0.06 0.17 0.09 0.07 0.08 0.03 0.27 0.58 0.27

(b) S. beali

TX3 TX4 NL4 TS7 TS8 TS9 TS10 TS11 SL VC3 VC4 TX3 0.58 0.48 0.56 0.63 0.43 0.39 0.57 0.63 0.54 0.83 0.47 TX4 0.08 0.31 0.47 0.39 0.39 0.24 0.44 0.44 0.38 0.64 0.32 NL4 0.33 0.47 0.19 0.58 0.35 0.34 0.50 0.54 0.52 0.80 0.48 TS7 0.24 0.10 0.49 0.39 0.54 0.36 0.53 0.48 0.45 0.67 0.43 TS8 0.04 0.28 0.35 0.39 0.26 0.28 0.44 0.50 0.46 0.77 0.38 TS9 0.10 0.12 0.54 0.29 0.32 0.12 0.34 0.36 0.30 0.59 0.24 TS10 0.01 0.03 0.27 0.12 0.09 0.04 0.54 0.54 0.49 0.72 0.44 TS11 0.11 0.05 0.33 0.04 0.21 0.10 0.01 0.53 0.50 0.75 0.46 SL 0.07 0.03 0.40 0.09 0.25 0.07 0.01 0.04 0.43 0.66 0.40

VC3 0.24 0.23 0.46 0.20 0.39 0.32 0.16 0.19 0.16 0.68 0.68 VC4 0.04 0.00 0.46 0.15 0.22 0.07 0.01 0.06 0.05 0.26 0.33

some localization of haplotypes this subdivision is not strong, as evidenced by the fact that the exact assignment of haplotypes to geographic locations does not differ from random (P = 1.00). Swift perusal of the geographic distribution of haplotypes (Figure

4.5) shows why this is the case, as numerous haplotypes are shared across the distribution

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Figure 4.4. Frequency and distribution of haplotypes sampled for S. limbatus within the Tamaulipan province of the Mexican xerophytic region. Haplotypes are significantly localized within the area (FST = 0.19; Prandom>observed = 0.00000), although there is no significant association between haplotypes and populations based on an exact test of geographic subdivision (P = 1.00), nor any correlation between FST and geographic distance (Mantel test; Prandom>observed = 0.10) suggesting that there is no isolation by distance within this area.

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Figure 4.5. Frequency and distribution of haplotypes sampled for S. beali. Haplotypes are significantly localized within the species (FST = 0.20; Prandom>observed = 0.00000), although there is there is no significant association between haplotypes and populations based on an exact test of geographic subdivision (P = 1.00), nor any correlation between FST and geographic distance (Mantel test; Prandom>observed = 0.54) suggesting that there is no isolation by distance.

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Figure 4.6. Plot of FST from pairwise population comparisons versus geographic distance within the Tamaulipan geographic province for (a) Stator limbatus and (b) S. beali. There is no significant correlation in either analysis (Mantel test; S. limbatus: Prandom>observed = 0.10; S. beali: Prandom>observed = 0.54). However, when populations of S. limbatus from the Chihuahuan and Sonoran deserts are included, there is a significant association between FST and geographic distance (Prandom>observed = 0.04).

of the populations. There is no phylogeographic structure to haplotypes (Figure 4.2),

perhaps because of the distinct star-like topology of haplotype diversification.

The situation is slightly different between the two species in terms of isolation-by- distance, although this is not significant. S. beali shows no correlation between pairwise

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FST-values and the geographic distance separating those populations (r = 0.00, P > 0.50), as is readily apparent from a plot of pairwise FST as a function of geographic distance

(Figure 4.6). However, there is a non-significant trend to an association between FST and

geographic distance in S. limbatus (r = 0.20, P < 0.10; Figure 4.6a). While this trend

becomes significant as populations from the Chihuahuan desert and Sonoran desert are

included (r = 0.46, P < 0.05), it decreases the utility of the comparisons between species.

Demographic History

Estimated effective population sizes of females (Nf) based on θW or π, µ = 8.5 ×

-9 10 (Brower, 1994), and the relationship θW or π = 2Nfµ are shown in Table 4.4a for both

species and for the different populations of both species. Overall diversity estimates

6 indicate that population sizes are rather large for both species: 1.2 × 10 (θW) and 7.8 ×

5 5 5 10 (π) for S. limbatus and 7.9 × 10 (θW) and 2.8 × 10 (π) for S. beali, with local

samples of both species also having large population sizes. The estimated species-wide

effective population size is higher for S. limbatus than for S. beali, although the large

variance in the estimate does not support statistical significance (ts = 0.78, P > 0.05).

Similarly, individual population estimates are considerably higher for S. limbatus than for

S. beali, and using these point estimates alone indicates that S. limbatus populations have

higher θW (ts = 5.40, P < 0.001) and π (ts = 4.83, P < 0.001) than S. beali.

These estimates of effective population size are based strictly on either pairwise

differences or the number of segregating sites and do not explicitly take into account

genealogical information. Felsenstein (1992) showed that incorporating phylogenetic

information allows more efficient and effective estimation of the effective population

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size. Implementing genealogical information using the method of Kuhner et al. (1995)

results in considerably higher estimates of effective population sizes of females for both

species (Table 4.4b: 3.22 × 106 for S. limbatus; 1.90 × 106 for S. beali) as well as non- overlapping 95% confidence intervals, supporting significantly higher long-term effective population sizes for the generalist S. limbatus than the specialist S. beali. This difference is likely due to increased efficiency and accuracy through the incorporation of

genealogical information into the estimate of θ when using a single locus of fairly short

length (Felsenstein, 1992).

Estimates of Tajima’s D for S. limbatus and S. beali are -1.143 and -2.018,

respectively. These negative values support the conclusion from the analyses of genetic

structure (Figures 4.4 and 4.5) and genealogical relationships (Figures 4.2 and 4.3) that

there is no evidence for long-term genetic structure in S. limbatus or S. beali. Rather,

these negative values are consistent with a model of rapid population expansion for both

species. For S. limbatus this negative value is not distinguishable from a population at

selective neutrality and demographic equilibrium when referenced to the Beta-

distribution (Prandom

However, the specialist species S. beali does show significant evidence of a rapid

population expansion, with a D-value of -2.0181 being highly unlikely given expectations from the standard coalescent (reference to Beta-distribution: Prandom

compared to simulations: Psimulated

While the multi-modal frequency of pairwise differences between pairs of

haplotypes in S. limbatus (Figure 4.7a) suggests a population not significantly departing

from demographic equilibrium, the unimodal mismatch distribution of S. beali is in

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Table 4.4. (a) Diversity estimates and effective population sizes for Stator limbatus and S. beali and each population of both. Standard deviations of estimates are in parentheses. -9 Estimates of Nf based on relation of θ or π = 2Nfµ, µ = 8.5 × 10 (Brower, 1994). (b) Maximum-likelihood estimates and confidence intervals of species-wide θ based on Metropolis-Hastings Markov Chain Monte Carlo genealogy sampling (Kuhner et al., 1995).

5 5 (a) θ/bp Nf × 10 π/bp Nf × 10 (using θ) (using π) Stator limbatus 0.0204 (0.0052) 12.0 (3.1) 0.0133 (0.0068) 7.8 (4.0) AZ1 0.0209 (0.0109) 12.3 (6.4) 0.0192 (0.0122) 11.3 (7.2) CH 0.0181 (0.0078) 10.6 (4.6) 0.0134 (0.0077) 7.9 (4.5) NL1 0.0159 (0.0069) 9.4 (4.1) 0.0122 (0.0070) 7.2 (4.1) NL2 0.0138 (0.0061) 8.3 (3.6) 0.0115 (0.0066) 6.7 (3.9) NL3 0.0144 (0.0063) 8.4 (3.7) 0.0131 (0.0075) 7.7 (4.4) TS1 0.0111 (0.0051) 6.5 (3.0) 0.0166 (0.0094) 9.7 (5.6) TS2 0.0048 (0.0024) 2.8 (1.4) 0.0060 (0.0037) 3.5 (2.2) TS3 0.0116 (0.0053) 6.8 (3.1) 0.0085 (0.0051) 5.0 (3.0) TS4 0.0101 (0.0046) 5.9 (2.7) 0.0137 (0.0078) 8.1 (4.6) TS5 0.0039 (0.0021) 2.3 (1.2) 0.0024 (0.0018) 1.4 (1.0) TS6 0.0100 (0.0046) 5.9 (2.7) 0.0154 (0.0088) 9.1 (5.2) TX1 0.0138 (0.0070) 8.1 (4.1) 0.0158 (0.0097) 9.3 (5.7) VC1 0.0027 (0.0015) 1.6 (0.9) 0.0027 (0.0019) 1.6 (1.1) VC2 0.0106 (0.0048) 6.3 (2.8) 0.0062 (0.0038) 3.6 (2.2) Stator beali 0.0135 (0.0037) 7.9 (2.2) 0.0048 (0.0028) 2.8 (1.6) TX3 0.0081 (0.0040) 4.8 (2.4) 0.0058 (0.0037) 4.8 (2.3) TX4 0.0027 (0.0015) 1.6 (0.9) 0.0031 (0.0021) 1.6 (0.9) NL4 0.0021 (0.0013) 1.3 (0.8) 0.0019 (0.0015) 1.3 (0.8) TS7 0.0053 (0.0026) 3.1 (1.6) 0.0039 (0.0026) 3.1 (1.5) TS8 0.0032 (0.0018) 1.9 (1.0) 0.0026 (0.0019) 1.9 (1.0) TS9 0.0011 (0.0008) 0.6 (0.5) 0.0012 (0.0011) 0.6 (0.5) TS10 0.0069 (0.0033) 4.1 (1.9) 0.0054 (0.0038) 4.1 (1.9) TS11 0.0064 (0.0031) 3.8 (1.8) 0.0053 (0.0033) 3.8 (1.8) SL 0.0053 (0.0026) 3.1 (1.6) 0.0043 (0.0028) 3.1 (1.5) VC3 0.0048 (0.0024) 2.8 (1.4) 0.0068 (0.0041) 2.8 (1.4) VC4 0.0032 (0.0018) 1.9 (1.0) 0.0033 (0.0023) 1.9 (1.0) (b) Maximum-Likelihood Estimation 5 θ/bp 95% confidence interval Nf × 10 Stator limbatus 0.0547 0.0435-0.0733 32.2 (25.6-43.1) Stator beali 0.0324 0.0202-0.0428 19.0 (11.9-25.2)

strong agreement with a distribution predicted from a population experiencing a rapid population expansion (Figure 4.7b). Table 4.5 shows the estimated values of θA, θ1, and

τ (twice the time since the population expansion, measured in mutation units) based on

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this analysis (Table 4.5a) and the corresponding values of the effective population size of

females and the number of generations using Brower’s (1994) estimate of the per base

pair mutation rate (Table 4.5b).

The population size change model of Wakeley and Hey (1997) produces very

similar results: while the pattern of site frequency polymorphisms for S. limbatus do not

fit a model of rapid population expansion, the data of S. beali fit the model well (Figure

4.8). Table 4.5 shows the same values as described above for this estimation. There is

general agreement between the models, as the estimates from Wakeley and Hey’s (1997)

model fall within the 95% confidence intervals of the estimated parameters of the

mismatch distribution under the same model of size change. This is at least in part due to

the very large confidence intervals of θ1 for the mismatch distribution, which are known

to be inflated using this estimate (Schneider and Excoffier, 1999). Regardless, there is still support for a population expansion, as evidenced by the non-overlapping confidence intervals between θA and θ1. The estimate of confidence intervals for τ is considerably more robust and the overlap in the estimation of this parameter between the two methods is encouraging.

The maximum-likelihood estimate of θ in conjunction with the growth parameter g provides further evidence that S. beali has undergone a recent population expansion, while S. limbatus has not. The likelihood of the data is not significantly increased by

allowing the added parameter g to vary along with θ in S. limbatus (LnL(θ) = 0.168;

LnL(θ, g) = 0.307; AICθ - AICθ,g = 1.72). Examining the likelihood surface of the joint

estimation and the estimation of θ alone suggests why there is no significant increase due

to g (Figure 4.9). The wave-like form and horizontal ridge of the 3-dimensional graph of

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θ and g versus the likelihood suggests that at the maximum estimate of θ, g can take a

wide range of values without significantly altering the overall likelihood surface. On the

other hand, adding the exponential growth parameter g to the estimation of the likelihood

for S. beali significantly increases the likelihood of the data (LnL(θ) = 0.051; LnL(θ, g) =

1.27; AICθ - AICθ,g = -0.443). The more peak-like form of the likelihood surface (Figure

4.10) supports this conclusion. The fact that g and θ will covary is also seen by the

somewhat diagonal ridge that proceeds from θ = 0.03 at g = 0 up to θ = 0.09 at g = 1243.

A deterministic backwards-approximation of population sizes of S. beali and S. limbatus given their maximum-likelihood values of g and θ heuristically demonstrates the importance of recent rapid population growth in S. beali and the relative lack thereof in S. limbatus (Figure 4.11): when S. beali is predicted to have a single individual (~1.46 million years ago), S. limbatus still has 3.3 × 105 individuals. In order to make this result

comparable to the estimates of τ from the previous analyses I calculated the number of generations backward that it would take for S. beali to decrease to the estimates of the ancestral population sizes Nf(ancestral) from both the model of rapid expansion using the

distribution of pairwise differences (Rogers and Harpending, 1992) analysis and using the

distribution of site frequencies (Wakeley and Hey, 1997) using the relationship Nf(ancestral)

-gtµ = Nf(current)e . The results are presented in Table 4.5c. While the results indicate slightly

deeper times, they are certainly not drastically different, and this result could be due to

the tendency of the estimation of g to have an upward bias (Kuhner et al., 1998).

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Table 4.5. Estimates from models of size change in S. beali. (a) Estimated values of θA, θ1, and τ based on the estimates of the mismatch distribution (Rogers and Harpending, 1992) and the population size change model of Wakeley and Hey (1997). (b) Estimated values of the effective population size of females (Nf) and number of generations (t) -9 based on µ = 8.5 × 10 (Brower, 1994) and the relationships θ = 2Nfµ and τ = 2tµ. (c) Estimated values of t to arrive at ancestral population sizes of the above estimates based on the maximum-likelihood estimate of g (-1243, s.d. = 90.16) and θ1 (0.0901) using the -gtµ relationship θA = θ1 × e . All estimates are per base pair. Standard deviations are in parentheses. Standard deviations in (c) are based on g only.

(a) θA θ1 τ Mismatch 0.0014 0.2542 0.0035 (0.000 – 0.0042) (0.0232 – 12.29) (0.0023 – 0.0068) Size change 0.0007 0.0236 0.0045 5 5 5 (b) Nf(ancestral) × 10 Nf(current) × 10 t (generations) × 10 Mismatch 0.80 150.0 2.08 (0.00 – 2.5) (13.7 – 7230) 1.36 – 4.00 Size change 0.43 13.9 2.61 (c) Estimate of number of generations to reach Nf(ancestral) from column 1 above based on maximum-likelihood estimation of g and θ1. Mismatch 0.80 53.0 3.97 (estimate from above) (3.70 – 4.28)

Size change 0.43 53.0 4.55 (estimate from above) (4.24 – 4.91)

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Figure 4.7. Mismatch distributions for (a) Stator limbatus, top; and (b) S. beali, bottom. Expected values were obtained by simulating 10,000 datasets under a coalescent algorithm modified from Hudson (1990) by implementing parameter estimates based on a sudden demographic expansion (Rogers and Harpending, 1992; Schneider and Excoffier, 1999). S. limbatus departs significantly from the unimodal expectation of a population expansion, while S. beali fits the predictions very well.

Figure 4.8. Observed and expected site frequency distributions for S. beali given a coalescent model of rapid population expansion (Wakeley and Hey, 1997). The full site frequency distribution has been reduced to the expected value of these three classes for convenience in estimating the three parameters of the model (θA, θ1, τ). The model could not be fit for S. limbatus.

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Figure 4.9. Maximum likelihood estimation of θ and population growth rates (g) for S. limbatus based on a Metropolis-Hastings Monte Carlo algorithm to sample genealogies from the Coalescent (Kuhner et al., 1998). (a) Joint estimate of θ and g, with maximum- likelihood estimates of 0.084 (SD 0.007) and 220 (SD 29), respectively. The wave-like form of the likelihood distribution is indicative of the result that adding a growth parameter did not significantly increase the likelihood of the data. (b) Coalescent estimation of θ without including a growth parameter. Estimate of 0.055, with 95% confidence interval of 0.043-0.073.

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Figure 4.10. Maximum likelihood estimation of θ and population growth rates (g) for S. beali based on a Metropolis-Hastings Monte Carlo algorithm to sample genealogies from the Coalescent (Kuhner et al., 1998). (a) Joint estimate of θ and g, with maximum- likelihood estimates of 0.090 (SD 0.006) and 1243 (SD 90), respectively. The more peak-like form of the likelihood distribution is indicative of the result that adding a growth parameter significantly increases the likelihood of the data. (b) Coalescent estimation of θ without including a growth parameter. Estimate of 0.032, with 95% confidence interval of 0.020-0.043.

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Figure 4.11. Deterministic backward extrapolation of female effective population sizes of S. limbatus and S. beali given the maximum-likelihood estimations of θ and g using Kuhner et al.'s (1998) coalescent model of population growth. A history of exponential population growth is significantly more likely for S. beali, whereas adding this does not significantly increase the likelihood of the assessment of historical population demography for S. limbatus. S. limbatus: θ = 0.08 (S.D. = 0.007), g = 220 (S.D. = 29); S. beali θ = 0.09 (S.D. = 0.008), g = 1243 (S.D. = 90).

DISCUSSION

This analysis allows me to compare the genetic differentiation and historical demography of a specialist seed predator, S. beali, with its sister-lineage of the generalist seed predator species S. limbatus. This approach is intended to gain insight into the process of speciation and divergence between these two lineages; as well as to contrast the demographic history and population genetic structure of a specialist and generalist lineage while controlling for the confounding influences of time and geography. Insights gained from genealogical analyses, assessments of population genetic differentiation, and coalescent analyses of demographic history provide substantial insight into the highly divergent history of these two lineages.

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Divergence in Population Genetic Structure. S. limbatus and S. beali have very

similar FST-values within the Tamaulipan region of the Mexican xerophytic province

(0.19 and 0.20, respectively); this similarity is remarkable given the extreme divergence in the breadth of their ecological associations. Furthermore the distribution of significant pairwise FST-values is essentially the same (Table 4.3), with a peripheral population in each accounting for the majority of significant pairwise differentiations (73% in S. limbatus and 57% in S. beali). These species-wide FST-values are significantly non-zero

and indicate that there is similar geographic-based genetic structure in both species. This is perhaps surprising given the assumption that extreme specialists are expected to (1) experience more patchy resources and therefore experience greater barriers to gene flow

and/or (2) use hosts with limited geographic ranges and therefore experience selection

against long-distance dispersal ability (Barton and Charlesworth, 1984; Futuyma and

Moreno, 1988; Peterson and Denno, 1998; Price, 1980). It is, however, in line with other

recent analyses suggesting that there is no correlation between diet breadth in

phytophagous insects and population differentiation at demographic equilibrium (Kelley

et al., 2000; Peterson and Denno, 1998).

However, these two populations appear to have arrived at very similar levels of

population differentiation via different processes. FST-values reflect levels of gene flow

only in a population at demographic equilibrium (Nielsen and Wakeley, 2001; Slatkin,

1993; Wright, 1943). Under non-equilibrium conditions FST-values can reflect historical

demographic factors such as bottlenecks or range expansions. The similar FST-values of

S. limbatus and S. beali appear to reflect reciprocally varying historical factors and

current restrictions on gene flow. One way to address this is to examine the influence of

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isolation-by-distance. Under the hypothesis of demographic equilibrium, FST-values are expected to be positively correlated with geographic distance (Slatkin, 1993). While neither lineage shows significant isolation-by-distance within the area of sympatry

(Figure 4.6; S. limbatus, P = 0.10; S. beali, P = 0.54), this phylogroup of S. limbatus shows a trend in that direction that becomes significant when populations are included from farther afield. The distribution of pairwise FST-values with respect to geographic

distance is essentially random for S. beali. The distinctly star-like topology of S. beali

(Figure 4.2) is also suggestive of recent population growth and range expansion from a

population bottleneck. These combined results indicate that pairwise FST-values for S. beali are not good surrogates for assessing levels of migration between populations; and

the species-wide significant FST-value is more likely to reflect a history of rapid

population expansion than to reflect low levels of gene flow. This conclusion is

supported by the demographic analyses discussed below.

S. limbatus, on the other hand, appears to deviate only slightly from an isolation- by-distance model, and does not when the entire population is examined. The genealogical reconstruction of this lineage (Figure 4.3) is also considerably less star-like, with the co-occurrence of at least three fairly old clades each with moderate levels of genealogical structure. While these results are currently inconclusive, they do suggest that the pattern of genetic differentiation within this haplotypic lineage of S. limbatus is due at least in part to limits on gene flow between populations separated by large distances. The non-significant trend in the predictions of isolation-by-distance, a similar result for Tajima’s D (discussed below), and the absence of derived shared haplotypes with respect to internal shared haplotypes (Crandall and Templeton, 1993; Templeton et

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al., 1995) indicate that some of this could also be explained by a modest recent

population expansion combined with populations within biogeographic regions

historically connected by moderate levels of gene flow. Regardless, it is clear that these

nearly equivalent FST-values do not indicate a similarly equivalent evolutionary history in this specialist and generalist, and at a more general level these results show that FST- values in the absence of geographic or demographic data do not provide an explanatory metric for the process of population differentiation.

Divergence in Demographic History. Long-term effective population sizes of the

Mexican xerophytic phyloclade of the generalist S. limbatus are estimated as being larger than S. beali based on either θW or µ, although the large variances in these estimates do

not exclude the possibility that these are simply due to sampling error (Table 4.4a). On

the other hand, the more efficient estimator that takes into account genealogical structure

does indicate that the long-term effective population sizes are significantly larger in the

generalist (Table 4.4b). Additionally, local population sizes of the generalist tend to be

considerably larger in the generalist than the specialist (Table 4.4a). This result indicates

that a generalist habit in this biogeographic region supports higher long-term effective

population sizes than a specialist habit. Regardless, even the smallest populations of S. beali are estimated to have effective population sizes in the tens of thousands and

estimates are in the same order of magnitude as for the generalists. Whether this

indicates that being a generalist is a less risky strategy than being a specialist depends on

the relevance of these differences to the population biology of the beetles.

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The long-term effective population size is based on estimates assuming the standard coalescent—selective neutrality and demographic equilibrium. It is clear from the data that the demographic histories of S. limbatus and S. beali diverge notably in this respect. Based on four separate metrics—Tajima’s D (Tajima, 1989b), the fit of the distribution of pairwise distances to a model of population expansion ('the mismatch distribution', Rogers and Harpending, 1992), the fit of polymorphic site frequency classes to a model of population expansion (Wakeley and Hey, 1997), and the joint likelihood estimation of θ and g, the exponential population growth rate (Kuhner et al., 1998)—the demographic history of S. limbatus does not significantly depart from that expected by the standard coalescent. This does not apply to any hypotheses concerning the initial colonization of the Mexican xerophytic province from the south and subsequent range expansion throughout the region by S. limbatus, as the monophyly of the haplotypes indicates that there has been enough time since this event for the haplotypes to coalesce even given demographic equilibrium. But it does suggest that this generalist has not experienced a recent population bottleneck and that population sizes have been relatively constant at least since the diversification of the current Mexican xerophytic haplotype group. Using the estimate of 2.3% sequence divergence per year (Brower, 1994) and the average corrected (K2P) pairwise distance between haplotypes in the clades separated by the most basal split within this phyloclade of S. limbatus, this divergence began about 1.1

(s.d. 0.3) million years ago.

In contrast, all of the above metrics provide significant evidence for rapid recent population growth in the specialist species S. beali. While determining whether this recent population growth is due to a recovery from a genetic bottleneck or rapid

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population growth from already large populations requires information from multiple

unlinked loci (Fay and Wu, 1999; Hey and Harris, 1999), estimates of ancestral

population sizes include very small ones (Table 4.5). Using the sudden size change

models, information regarding the demography of speciation is lost in this mitochondrial

locus, as the timing of this change in population size is estimated to have occurred

approximately 200,000-500,000 years ago (Table 4.5, assuming one generation per year),

well after the proposed divergence of these species around 1.4 million years ago.

However, if one is willing to make the assumption of a constant estimate of g, the

exponential population growth rate, extrapolation backwards indicates a population size

of less than 2 at 1.4 million years ago. Given that this population is not at demographic

equilibrium, the current effective population sizes are estimated to be considerably larger

than the long-term effective population size. They range from a low of 1.39 million

females (estimate based on the model of Wakeley and Hey, 1997) to 5.3 million females

(estimate based on the maximum-likelihood estimation of Kuhner et al., 1998) to 15.0

million females (estimates based on the model of Rogers and Harpending, 1992—

confidence intervals for this measure are significantly biased upward).

Implications for Speciation. Stator beali appears to have diverged from S. limbatus about 1.4 million years ago within a fairly localized geographic context, the

Tamaulipan biogeographic province of the Mexican xerophytic biogeographic region.

This speciation event was accompanied by an intense specialization in host plant use, behavioral adaptations to use the larger seeds of the novel host plant more effectively,

and a decrease in the armature of the apical sclerite of the male genitalia. All of these

332 imply a role for selection, either natural or sexual, in the speciation of S. beali. Whether or not these caused the initial divergence and reproduction of the two lineages, or were permitted by the elimination of the homogenizing effects of gene flow from the generalist ancestral populations that allowed reproductive isolation when the populations came into secondary contact has implications for our understanding of how adaptive radiations in phytophagous insects proceeds. The reciprocal monophyly of the local phylogroup of S. limbatus and S. beali precludes direct assessment of the demographic circumstances at the time of speciation by reference to mitochondrial haplotypes. While this means that more direct examination of different modes of speciation will require a combination of increased sampling of nuclear loci and tests of biogeographic patterns of reproductive isolation (Kliman et al., 2000; Noor, 1999; Turelli et al., 2001), we can indirectly examine the likelihood of alternative models of speciation given the biology of the organisms and the divergent demographic histories that have occurred since the time of speciation.

The phylogeography of S. limbatus as a whole, and evidence of isolation-by- distance across long distances within the phylogroup addressed in this study both suggest that gene flow in this species is structured to some extent by long-term vicariant divergence between broadly separated populations. However, within the area in which S. beali is suspected to have originated, there is no evidence that the ancestral species S. limbatus forms localized populations. A micro-allopatric mode of speciation (Mayr,

1947; Mayr, 1963) does not invoke the need for long-term and profound barriers to gene flow for the evolution of reproductive isolation. Instead, intrinsic properties of the organisms, most notably low vagility or habitat fidelity, cause seemingly minor features

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of the environment to become significant barriers to dispersal; or temporal barriers to

gene flow such as those caused by periodic glaciations permit the initial species

divergence. In these cases, initial divergence is not driven by natural or sexual selection,

although both can contribute to the rapid evolution of reproductive isolation. Seven

observations suggest that if the reproductive isolation of S. beali were strictly allopatric

or microallopatric it must have been acquired rapidly (Knowles, 2001a; Knowles et al.,

1999; Schluter, 2001): (1) the observation of modest levels of gene flow between

populations of S. limbatus in the Tamaulipan region; (2) the complete lack of observed or hypothesized borders to gene flow within the low floodplain of the coast of the Gulf of

Mexico; (3) the apparent lack of host races and locally specialized populations in S. limbatus (Fox, 1997; Fox, 1998; Fox et al., 1997; Fox and Savalli, 2000), indicating that habitat fidelity alone is not likely to promote micro-allopatric speciation; (4) the rapid colonization of introduced hosts by S. limbatus (Fox and Savalli, 2000; Johnson and

Kingsolver, 1976; Johnson et al., 1989) suggesting that they are not strictly limited to the

hosts that their parents used (the Hopkins' host-selection principle, Hopkins, 1917); (5)

the implication from palynological data that the ancestral host plants of S. limbatus were

unlikely to have been absent for any extended period from the geographic area in which

the shift and specialization on Ebenopsis ebano is likely to have occurred (Graham, 1976;

Graham, 1987; Graham, 1988; Magallón and Cevallos F., 1994); (6) the dampened

climatic oscillations of the Pleistocene as one moves toward the equator (Graham, 1997);

and (7) the rapid periodicity of these oscillations (Graham, 1999; Roy et al., 1996).

Otherwise, the homogenizing effects of gene flow between the larger source population

of S. limbatus and the sink population of S. beali would have saturated the localized

334 adaptations of populations using E. ebano (Futuyma, 1987; Mopper et al., 1995).

Therefore, if microallopatric separation allowed the speciation of S. beali, rapid reproductive isolation was almost certainly caused by intense natural selection (Schluter,

2001; Via, 2001), divergent sexual conflict (Gavrilets, 2000; Knowles, 2000), or both.

Paraphyletic relationships and asymmetric range sizes of the type displayed by S. limbatus and S. beali have been taken to imply distinct differences in effective population size and local speciation; thereby supporting an hypothesis of peripatric speciation (Avise et al., 1983; Harrison, 1998; Hey and Kliman, 1993; Knowles et al., 1999; Patton and

Smith, 1994). However, paraphyly in general is an almost inevitable consequence of the temporal procession from polyphyly to reciprocal monophyly during any mode of speciation (Harrison, 1998; Neigel and Avise, 1986; Tajima, 1983) and only an extended history of paraphyly provides direct evidence for peripatric speciation (Hey, 1994). Due to the stochastic nature of the coalescent, and of the mitochondrial genome in particular

(Hudson and Turelli, 2003), assessment of multiple loci is necessary in order to determine the duration of different genealogical patterns (Edwards and Beerli, 2000; Hey,

1994). The phylogeographic distribution of lineages of S. limbatus and S. beali does not immediately recommend peripatric speciation even taking these caveats into consideration. The reciprocal monophyly of sympatric lineages of the two species does not necessarily imply differences in effective population size and the geographic range of

S. beali does not lie on or outside the periphery of the range of S. limbatus. But current geographic sympatry does not necessarily imply historical geographic sympatry, particularly given the northward movement of S. limbatus (Chapter 3) and the climatic fluctuations of the Pliocene and Pleistocene; and the founder effects associated with

335 peripatric speciation could explain the rapid speciation apparently necessary under a selectively-neutral model of initial divergence. Therefore one must explicitly examine whether there is any evidence of a population bottleneck in the history of S. beali that may have facilitated speciation. As discussed above, the monophyly of the Mexican xerophytic clade of S. limbatus, while not significantly departing from demographic equilibrium, implies that we can no longer address the demographic circumstances at the time of speciation. On the other hand it is clear that S. beali has experienced significant and rapid population growth at some time since speciation. Estimates based on models of a sudden change in population size followed by demographic equilibrium do not allow one to distinguish between a population bottleneck at the time of speciation and a population bottleneck within S. beali well after the speciation event. But the star-like topology of S. beali (Figure 4.2), low sequence diversity (Table 4.3), and low estimates of ancestral population size (Table 4.5) are suggestive of a bottleneck at least for the mitochondrial genome. The somewhat more realistic model of exponential growth is perhaps more in line with a population bottleneck at the time of speciation, with very small population sizes estimated at 1.4 million years if one is willing to extrapolate backward long enough. However, the estimate of g is maximized only over the time since the most recent common ancestor of the current haplotypes of S. beali and does not pertain to demographic histories earlier than that. Therefore, while this evidence of a population bottleneck does not directly support a peripatric model of speciation, it is certainly in line with a prediction of an association between reproductive isolation and a reduction in effective population size. Notably, S. limbatus is able to use E. ebano quite well when not in the competitive presence of S. beali (Fox and Savalli, 2000), and genetic

336 variation for ovipositing in clutches is present in S. limbatus, albeit at low levels (Charles

Fox, personal communication). In addition, a single small population of E. ebano can almost certainly support very large population sizes of Stator. These factors indicate that the adaptations that have accompanied specialization on E. ebano were likely segregating at low levels in the ancestral population of S. limbatus. Whether or not the ‘genetic revolution’ envisioned for peripatric speciation (Carson and Templeton, 1984; Mayr,

1963) is a necessary or sufficient condition to allow these preadaptations to go to fixation while at the same time causing reproductive isolation is a matter of debate (Barton and

Charlesworth, 1984; Slatkin, 1996; Turelli et al., 2001). It does seem, however, that if this were to occur, then the initial founder population could have rapidly reproduced and expanded onto the abundant resource that E. ebano provides.

The most difficult model to examine using a single non-recombining locus is that of sympatric speciation. Certainly the current biogeographic distribution of S. beali with respect to its most closely related lineages of S. limbatus combined with the evidence of adaptive divergence and divergence in sexually-selected characters are in line with the predictions of sympatric speciation (Barraclough and Vogler, 2000; Berlocher, 1998;

Bush, 1994; Gavrilets and Waxman, 2002; Schluter, 2001; Via, 2001). However, the reliance on a single, non-recombining locus makes it difficult to examine the genealogical predictions based on the initial divergence of populations due primarily to the action of divergent natural selection. For example, allopatric, selectively-neutral speciation will have genome-wide effects and the variance in genetic diversity between loci will be low (Kliman et al., 2000; Wakeley and Hey, 1998), while ecologically driven speciation makes no such prediction because speciation is occurring against a background

337

of gene flow. Because the duration of speciation should be shorter in sympatric

speciation (Avise and Walker, 1998; Bush, 1975; Bush, 1993; Kondrashov et al., 1998;

McCune and Lovejoy, 1998), evidence from multiple loci is necessary to distinguish

these modes on this basis, as stochasticity in single loci, particularly in mtDNA, can be

quite large (Edwards and Beerli, 2000; Hudson and Turelli, 2003).

These results may seem unsatisfactory due to their inability to distinguish

between different modes of speciation. They are particularly noteworthy in numerous

respects, however. First of all, they indicate that broad allopatry is the least likely scenario of speciation in this sister-species pair. This mode of speciation is generally thought of as the most likely mechanism of diversification and is often taken as the null model to which other modes are compared (Bush, 1975; Bush and Smith, 1997; Futuyma and Mayer, 1980; Turelli et al., 2001). Second, there is substantial evidence for a population bottleneck in the demographic history of the specialist, indicating that peripatric speciation is a distinct possibility. While the analysis of Groman and Pellmyr

(2000) also provides evidence to this effect, other recent analyses from phytophagous insects are unable to distinguish between a population bottleneck and demographic equilibrium, leaving evidence for or against lacking (Knowles, 2001b; Knowles et al.,

1999). Finally, and perhaps most importantly, this analysis indicates a very important role for selection in speciation. While it cannot be determined whether this played a role in initiating the divergence of the species, the apparent necessity of either rapid reproductive isolation for the maintenance of these divergent phenotypes in sympatry or strong selection for the creation of these divergent phenotypes in sympatry illustrates the importance of ecological divergence in generating evolutionary diversity. Indeed, the

338 observations of distinct ecological divergence in hosts used and the adaptive divergence in behaviors related to host use support this hypothesis (Fox et al., 1996b; Fox and

Mousseau, 1995b). Additionally, these very attributes may be functionally correlated with diverging levels of sexual conflict as evidenced by the decreased competition between offspring in conjunction with the loss of genital armature in S. beali

(Crudgington and Siva Jothy, 2000; Haig, 1999; Johnson and Kingsolver, 1976). These two aspects of divergent ecology and divergent sexual selection are expected individually to rapidly cause reproductive isolation (Gavrilets, 2000; Gavrilets and Waxman, 2002;

Hawthorne and Via, 2001; Kondrashov et al., 1998; Schluter, 2001) and could potentially interact to emergently accelerate the process.

Implications for the Maintenance of Specialization. At the macroevolutionary level, specialization is often considered to be a mixed blessing. On the positive side are the selective advantages associated with specialization including an increased competitive ability, more efficient neural-based host-searching capabilities, and avoidance of generalist predators (Bernays and Graham, 1988; Bernays and Funk, 1999;

Denno et al., 1995; Futuyma and Moreno, 1988; Holt and Lawton, 1993; Jaenike, 1990;

Thompson, 1994). On the flip side are the long-term extinction risks associated with specializing in an ecologically fluctuating world (Cope, 1896; Simpson, 1953;

Thompson, 1994). The results of this study illustrate both the negative and positive aspects of specialization. The generalist S. limbatus appears not to have experienced extreme fluctuations in population size and has long-term effective population sizes roughly twice that of the specialist S. beali. This can be seen as a more conservative

339 strategy in which the beetle is riding neither the booms nor busts of climatic oscillations to any significant degree.

On the other hand, the specialist S. beali shows signs of a potentially severe bottleneck at some point in its history. If this bottleneck was not associated with speciation but is instead the result of climatic fluctuations during the glaciations over the last half-million years, then this illustrates the potential risks associated with specialization. As a result, its long-term population sizes are roughly half that of the generalist. The massive population growth since this proposed bottleneck and large estimated current effective population sizes illustrate the advantages to a specialist when its host plant becomes a dominant member of a community. While S. limbatus has the ability to use the Texas ebony tree quite well either in the laboratory or where it has been planted as an ornamental outside the range of S. beali (Fox and Savalli, 2000), it has never been reared from this host plant within the area of sympatry, indicating a distinct competitive advantage of the specialist on this host plant.

Conclusions. The results presented in this chapter illustrate the utility of a combined genealogical and demographic approach to the study of the connection between ecology and diversification. While the conclusions are based on a single non- recombining locus of fairly short length and are therefore conditional upon this caveat, they indicate that the study of phytophagous insects is a fertile field for addressing fundamental questions of evolutionary biology including the link between ecology and diversification, the mode of speciation, and the evolutionary consequences of ecological specialization.

340

The goal of this study was to address whether ecological specialization drove the

speciation of S. beali from its parent generalist species S. limbatus. While it seems clear

that selection played a key role in the rapid differentiation of these two lineages,

determining whether it played a role in the initial divergence is beyond the scope of this

study. Research into this species pair is illuminating as to the specific circumstances

surrounding their split, particularly with respect to the biogeographic context and the

subsequent divergence in demographic and population genetic histories. However, in order to determine whether this example is representative of more general phenomena in seed beetles or other phytophagous insects will require replicated research. To this point, there is not a discernable trend in the mode of speciation in phytophagous insects, perhaps because many results, like this one, are inconclusive on this subject (Brown et al., 1996; Feder and Filchak, 1999; Feder et al., 1999; Funk and Bernays, 2001; Groman and Pellmyr, 2000; Knowles, 2001a; Knowles et al., 1999). However, it is becoming clear from all of these studies that selection is highly important in the rapid acquisition of reproductive isolation whether or not it plays a role in the initial divergence.

341

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