THE MECHANISM OF EVOLUTION
IN LEPTINOTARSA

BY

WILLIAM LAWRENCE TOWER

PUBUSHED BY THE CaRNEGIE INSTITUTION OF WASHINGTON
WASHINGTON. 1918

CAENEGIE INSTITUTION OF WASHINGTON
Publication No. 263

BALTIMORE, MD., U. 8. ▲.

PREFACE

The data and conclusions presented in part in this volume are the product
of a project in which it was attempted to attack the " evolution problem " in
one set of organisms from a number of different aspects. Evolution in organ-
isms must be conceived of as the product of the interaction of internal and
external factors, operating in strict mechanistic manner, as in non-vital phe-
nomena, so that the central problem was the determination and proof of the
mechanism of action of these two groups of factors whose operations are pro-
ductive of evolutionary changes. Experimental analysis of the problems, prose-
cuted with rigor and thoroughness, has been held to be the only means of
progress. Before this ideal could be applied much preliminary clearing of the
ground had to be done, and to this I gave the necessary attention in a report
published under the title " An Investigation of Evolution in Chrysomelid
Beetles of the Genus Leptinotarsa." *

The results of the project have been the development of a mechanistic concep-
tion of evolution based upon the interplay of genetic and environic factors and
the demonstration that the methods of evolution are heterogeneous, even in
one group of organisms. The basis of all methods of change is found to be
directly the product of the nature of the genetic factors of composition and
their capacity for diverse modes of reaction, especially with factors of the
environic complex. Purpose, utility, and kindred concepts have found no sup-
port, every change appearing as the chance mechanistic product of the reacting
agents ; while the product of the reaction either was able or not able to operate
under the conditions of origination, so that survival is decided at once and not
after long and faltering trials.

The project as organized consists of three parts, each necessary to the success
of the others and to the hope of arriving at a knowledge of the mechanism of
evolution that is not based upon plausibility and the arrangement of isolated
facts upon the fabric of preconceived theory.

The first stage (reported upon in the publication referred to) must deal with
the discovery and testing of some limited set of organisms in nature that are
possible subjects for study and especially for the experimental portions of the
investigation. Further, it is necessary to become fully acquainted with the
organisms in nature and to make preliminary tests to discover whether it is
possible to produce permanent changes in the material and the probable success,
if any, that might be expected from an experimental attack upon this portion
of the problem.

The second portion, given a successful outcome of the first, must comprise a
rigorous experimental investigation of the factors of evolution and an attempt
to create new attributes and qualities not existing in nature in the materials,
and to discover the relation of these to old characters and their interaction when
brought into combination or into competition with existing characters. With
this vantage gained, it will then be possible to create in the laboratory new

* Carnegie Inst. Wash., Pub. 48, 330 pp., 30 pi., 31 text figs., 1906.

Ill

IV

Peeface

specific qualities and attributes, and with these and the old to create an array
of new types, new genera, or even of higher taxonomic groups of organisms,
and with this as a basis it is possible to attempt the third part of the project.

This last part involves experimenting in nature with new forms to discover
how newly arisen characters, or their combination into specific forms, behave as
they meet the conditions of the environment into which they are thrust by the
processes of their origination. It is in this way only that it may be possible to
discover methods of elimination, of preservation, of adaptation, and the study
of kindred problems, that are of vital import in the total evolutionary activities
in nature, and which are now the delight of the essayist.

Throughout this work " factor '' and " determiner " are used in a physical
and dynamic sense, and are nowhere used in the sense of a carrier, representa-
tive or otherwise, of anything pertaining to the characters of organisms. The
word " factor," an agent that makes possible a general result, has been too often
used by some Neo-Mendelians in the sense of a carrier, but I am not able to dis-
cover that this use (in the Weismannian sense) is justified, and investigations
of the Mendelian reaction do justify the use of the term in the physical sense
in which I have used it. A " determiner," on the other hand, is an agent which
by its interaction with a factor decides which one out of several possible results
shall ensue.

The presence of reactions involving these gametic agents in diverse aspects
of the same materials, the experiments showing that the factors are specific in
reaction only when present in a specific system and environment, are the reasons
for the attempt to formulate these results in a general hypothesis of evolutionary
action and causation. Unlike other evolution hypotheses any dynamic con-
ception must be heterogeneous in action and result, a complex of many factors
and types of reaction and nowhere due to the operation of a single agency. I
have found in my materials that transmutation may be by sudden changes, by
slow accumulation, by hybrid reaction, and by means of environmental forces,
and in all changes the basal operations involved are purely physical types of
reaction between the gametic agents and the condition surrounding the reaction.

I have been fortunate in the support that this project has been given. Soon
after the start it was given added impetus through aid from the Elizabeth
Thompson Science Fund; later the Carnegie Institution of Washington gave
sundry grants for some of the investigations in the tropics, and in the later
years larger and continued support at the Desert Laboratory in Tucson, Arizona,
and at the Laboratory of Marine Biology at Tortugas, Florida. Also the Uni-
versity of Chicago has liberally supported the project, and especially the experi-
mental portions of the investigation requiring controlled and exact conditions.
I am deeply indebted to President H. P. Judson for most cordial support in
the project, and to the late Professor C. 0. Whitman, and especially to my
colleague, Professor F. E. Lillie, for many kind acts and aid which in divers
ways have contributed to the forwarding of the investigation. To President
R. S. Woodward, of the Carnegie Institution of Washington, and Dr. D. T.
MacDougal, Director of the Desert Laboratory at Tucson, Arizona, is due the
credit for an opportunity to conduct upon an unusually extensive scale and,
imder favorable conditions, a series of experimental studies in the Arizona
deserts. The creation of an elaborate plant with trained assistants has given
me the best opportunity to experiment direct in nature.

Pbeface V

A diJEScult portion of my problems has received the sympathy and aid of the
Department of Marine Biology of the Carnegie Institution of Washington, and
its Director, Dr. A. G. Mayer, has spared no pains and expense in the attempts
to stock with alien races the coral islands of the Tortugas and Marquesas groups
in the effort to learn, by experiment in nature, something of the laws of evolu-
tion, by dispersion, isolation, and changed environment.

It is also a pleasure to acknowledge the helpful and loyal aid received from
many others in the progress of this investigation, and especially I am indebted
to Dr. J. K. Breitenbecher and Mr. J. G. Sinclair for enthusiastic aid in the
Tucson experiments.

That portion of the project which from 190-1 to 1910 was conducted in the
tropical regions of Mexico, and especially in the rain forests of Vera Cruz, has
been materially aided by many friends in that coimtry. Especially I am in-
debted to the late James Parkin, of the Hacienda Motzorongo, and to his admin-
istratores, Senor P. Eandolph and Senor I. P. Lorio, for valuable aid in much
of the transplantation and acclimatization experiments in the rain forests of
A'era Cruz. These friends and the patient, loyal Indian helpers and packers
made possible a series of complex experiments in the midst of a virgin tropical
rain forest.

In the preparation of the material for publication, much of the reduction of
data and compilation of the tables has been done by Miss Edna Scott. The
drawings in color, the colored photographs, and all of the prints of negatives
taken in the field, as well as the text illustrations, have been prepared by
Mr. K. Toda.

To these friends and to many others I am under obligations for many kindly
acts in aid of this investigation, which have been contributed to its good points,
but to me must be charged that which proves to be error or misdirected effort.
No finality is expected and I shall feel that my efforts have not been in vain if
either data or hypotheses serve to stimulate further advances or help to correct
present misconceptions.

William Laweence Tower.

Desebt Botanicai. Labobatoby,

November 1, 1916.

CONTENTS.

Page.

Pbeface III-V

THE PROBLEMS AND MATERIALS.
Chapteb L The Pboblems and Concepts of Evolution.

Introduction 1

Physical Conception of Life 1

Nature of the Individual 3

Basis of Characters in Non-Living and Living Substances 5

Relation of External and Internal Factors 5

Categories of Organic Characteristics 6

Evolution and Transmutation 8

Hypotheses of Species Transmutation 11

Processes of Evolution 18

Factorial Theory of Evolution 21

Chapteb II. The Matebials, Theie Taxonomy and Natubal Histoby.

Introduction 24

Methods of Obtaining Materials 24

Character and Source of Materials 25

Species and Subdivisions of the Lineata Group 31

Description of the Species 32

Evolution and Phylogeny of the Lineata Group 69

Chapteb III. The Pboblems of Gametic Constitution.

Introduction 72

The Germ-plasm Concept 76

The Gametic Agents 78

Metathesis of Gametic Agents 80

Architecture of the Germinal Material 83

Chapteb IV. Reactions and Peoducts in Intebspecific Cbosses.

Methods of Culture ^^

Leptinotarsa signaticollis X L. diversa ^^

Leptinotarsa undecimlineata X L. signaticollis 124

Leptinotarsa undecimlineata X L. diversa 142

Discussion of the Results Derived in Crossing the Species L. diversa, L. sig-
naticollis, and L. undecimlineata 143

Chapteb V. Reactions and Pboducts in Intebspecific Cbosses.

Leptinotarsa diversa X L. decemlineata 146

Leptinotarsa decemlineata X L. oblongata 152

Leptinotarsa decemlineata X L. viultitwniata 163

Leptinotarsa multitceniata X L. oblongata 168

Summary and Discussion of the Crossing of Species 169

Chapteb VI. The Pboblems of Hetebogeneity.

Introduction 184

Problems of Heterogeneity 185

Causes of Heterogeneity 190

Analysis of Heterogeneity 191

VII

VIII Contents

Chapter VII. Analysis of Hetebogeneitt in Some Simplest Chabactebs.

Page.

Analysis of Heterogeneity in Some Simplest Color-Characters 192

Analysis of Heterogeneity in Some Simplest Structural Characters 216

Chapter VIII. Analysis of Heterogeneity in Complex Chabactebs.

Composition and Modification of the Pronotal Pattern 237

Composition of the Complex Pattern 243

Chapter IX. Analysis of Heterogeneity in the Population.

In Leptinotarsa multitwniata 270

In Leptinotarsa undecimlineata 307

In Leptinotarsa decemlineata 317

Some Results of These Analyses 322

Pattern in Different Habitats 324

Effects of Transportation into a New Habitat 326

THE RELATION OF WATER TO THE BEHAVIOR OF THE POTATO
BEETLE IN A DESERT.

Introduction ^^^

Instrumentation and Conditions of Experiment 343

Materials 3"

Role of Water in the Reproductive Activity 346

Experiments with Soil Moisture 348

Experiments with Evaporation Rates 349

Role of Water in the Preservation of Life 350

Relation of Water Loss in Insects when Exposed to Changes in the Relative
Humidity of the Surrounding Medium and its Effect on the Activities

of such Organisms 354

Experiments upon Evaporation, Transpiration, and Behavior. 355

Role of Water in Hibernation 366

Entrance into Hibernation 366

Activities 'wring Hibernation 371

Water Relation of Soils and Hibernating Beetles 371

Emergence from hibernation 372

Summary and Discussion upon the Relation of Water to Hibernation 373

Effect of Changes in Water-Content upon Alterations in Tropic Activities. . . . 375

Experiments upon the Role of Water in Geotropism 376

Relation of Temperature to Outgo and Intake of Water 377

Metabolism and the Water Relation 378

General Discussion upon the Role of Water in Living Things 379

Summary and Conclusion 381

Bibliography 382

CHAPTER I.
THE PROBLEMS AND CONCEPTS OF EVOLUTION.

INTRODUCTION.

The following chapters give the results derived from an attempt to analyze
the phenomena of evolution, using chrysomelid beetles of the genus Leptino-
tarsa as materials for investigation. In this investigation I am interested only
in processes or reactions productive of evolutionary phenomena, or that are of
experimentally proven importance in evolution, because they are the modus
operandi of the evolution of living substance and are universal in action. The
chrysomelid beetles and their transmuted products and characters are of
interest only to the extent that they have been convenient reagents for use in
the investigation.

It is important to understand clearly the general philosophical conceptions
from which we interpret nature and which guide our efforts in the prosecution
of research, since this will largely determine the logical, philosophical, and
experimental methods used in investigation and the hypotheses created. It is
sufficient to state my own position ; others must decide for themselves to what
extent they can interpret the phenomena of organic life upon the same basis.

In the absence of any evidence to the contrary, the physical world, as known
to us from our sense perceptions, is all that exists, and the general philosophical
postulate from which I must approach any investigation of nature is that of
positive materialism, and all activities, from the lowest to the highest, from
this postulate are, in the end, capable of statement and explanatidi^upon purely
mechanistic physical relations and reactions.

It is our task to interpret and understand ourselves and nature from the
operation of known physical agencies. When we have entirely and honestly
exhausted all of the possibilities of physical science, when physical science shall
have reached its end, if there remain phenomena not capable of statement in
terms of the physical factors of the universe, it may then be necessary to search
for ultra-physical causes. At present we fall so far short of exhausting the
possible applications of physical knowledge to the problems of living bodies
that there is no valid excuse for even suggesting the existence of non-physical
agents or causes which find expression in living material objects and reactions.

PHYSICAL CONCEPTION OF UFE.

In the contact zones between the lithosphere, the hydrosphere, and the atmos-
phere of this planet, marvelously complex integrations of matter occur, pre-
senting activities and reactions that are conspicuously unlike, in their totality,
anything else found in nature, but upon analysis there has been found only the
action of physical principles in these living bodies, combined in an immensely
complex system of interacting factors, collectively presenting end or superficial
aspects, which to some seem sharply and finally to differentiate this product of

2 The Mechanism of Evolution in Leptinotaesa

a narrow contact zone of great stresses from all other natural productions of
the planet. This living substance, which we as part thereof seek so diligently to
understand, and the question of whose origin has been a dominating factor in
human intellectual activities through all time, presents to all — scientist, theo-
logian, or layman — questions of fundamental interest.

As human understanding of this living substance becomes clearer, and as
investigation becomes more accurate and analytical, there is increasing proof of
the sole operation of purely physical principles in the production of the char-
acters and reactions of this living stuff, and some of us, therefore, hopefully
look forward to that time in the future when all organic phenomena are capable
of statement in purely physical terms. Only some few more or less superficial
processes are capable of formulation in physical terms, while a wide array of
organic activities, of form and species, and in man the intellectual capacities —
memory, thought, and study of himself and nature — are hardly capable of
accurate formulation upon any naturalistic physical basis; that is, more than
the description of our reactions in terms of the general reactions of living
materials.

Certain properties common to all of this living substance, the evidence of
the remains of this substance upon the planet, with increasing complications
of the primitive attributes and qualities common to all, have forced upon us
belief in some natural means of production of this diversity and increasing
complexity, with the present array of living types, of which man appears to us
as the most complex.

It is profitless to debate whether there is in living things any agency or
principle above and apart from the rest of the physical imiverse, whether there
be a vitalistic content, entelechy, or soul; any and all of these terms are but
designations of a collective unknown, unanalyzed series of phenomena, whose
aid is invoked in a causative sense in the effort to present a mentally complete
picture of natural causation. The use of the unknown and unanalyzed, as a
cause, in the efforts of modern vitalists is not only unwarranted, but is distinctly
a retrogressive tendency, much of which is due to the influence of Kantian
philosophy. There is no denying the fact that the imknown with regard to the
phenomena of life exceeds the known, but at present that which is organized
knowledge is vastly increased over that of a century ago, and in this increase
understanding has at all times and in all directions been solely along the line
of the application of physical principles to the solution of the problems of living
substance, so that for the present we are justified in the belief that it is the only
safe point of departure in the effort to solve these riddles of living matter.

Further justification for this attitude comes from the fact that not a single
advance has ever come from the vitalistic point of view, and while a vitalistic
conception or principle may help in certain metaphysical ramblings, or save
some frail intellect from despair at the incompleteness of knowledge as to the
causation of the phenomena of life, it serves no other end. At no time have any
conceptions of this kind given the least aid in investigation or in the attainment
of actual results in the solution of our problems. At all points the physical
conception of life and its evolution, at present fragmentary, is nevertheless the
only conception that has been of any service as a basis for the effort to under-
stand these phenomena, and finally to comprehend what our understanding of
these means in terms of cause and effect.

Problems and Concepts of Evolution 3

NATURE OF THE INDIVIDUAL.

At present the most important question confronting us, aside from the
determination of what life is, is probably the question as to the nature of the
individual, which is in nature the unit of action in the attainment of the evolu-
tion and distribution of living species.

Two concepts seem to dominate the present situation : (1 ) That the individual
is a mosaic, composed of lesser entities, unit-characters, biophores, or similarly
conceived ultimate vital units, each the carrier of some one or more characters
of the organism; (2) that the individual is an indivisible entity, or the unit of
existence. Between these views there has been in the past no compromise;
either one or the other, in the opinion of the adherents of the special concept,
must be true, and in the last decade the data derived from the neo-Mendolian
investigations has, in the opinion of many, strengthened the mosaic conception.

If the factors and determiners discovered in these Mendelian investigations
are thought of as carriers in the Weismannian sense, the evidence obtained by
the workers with this principle can be construed to support this mosaic concep-
tion. The ideas of the nature of these gametic agents are, however, changing,
and the most active and progressive neo-Mendelians no longer regard them as
ultimate units, the carriers of characters, but as agents whose presence or
absence within or without the organic system is productive of specific end-
results or directions of reaction in the general operations of the mass. In this
sense the individual and the line of descent must be conceived of in terms of
associated gametic and environic factors of composition that must be present
and in specific relations for the purpose of maintaining the integrity of the
individual in its characters and in descent. In other words, the individual is a
compound of physical agents — factors, determiners, inhibitors, accelerators,
and so on — not one of which is anything more than some non-living mass or
some physical relation within or without the mass, which by its presence decides
the course and extent of some one or more resulting reactions and the products
thereof in the development of the individual.

In non-living materials there is present in all substances and reactions these
agents or factors of operation that are productive of the end-result or the specific
compound or substance. Zinc, sulphur, and oxygen are quite unlike when
isolated, and all are different from water; nevertheless, when combined under
the proper conditions of the medium to permit of certain reactions between
them, there results crystalline zinc sulphate, a substance with specific qualities,
attributes, and conditions of being, no one of which was brought in by any of
the components, but which are the products of the reaction of the component
constituting agents of the specific mass. The mass is a unit, an individualized
entity, whose integrity and totality of characters and capacity of reaction is
entirely dependent upon the retention in this mass of the position, proportions,
and relations of the agents that entered into its production in the first instance,
and change in any of these results is a change in the composition of the mass,
its characteristics, and in its reaction capacity.

In organisms there is no a priori reason why exactly the same conception is
not true, both with regard to constitution and also with respect to its capacities
for changed reaction. The results of the neo-Mendelian investigations have
shown an abundance of instances in which the demonstrated presence or absence

4 The Mechanism of Evolution in Leptinotaesa

of an agent was definitely productive of changed aspect of the mass, and also of
its capacity to react differently when it was combined with some unlike mate in
breeding. It is true that in the organic there is greater complexity of composi-
tion and reactions present than are found in the simple non-living example;
nevertheless, there is no evident reason to suppose that there is any difference
in principle of action in them — only in detail and complexity thereof.

There can be no doubt of the existence of these gametic agents that are pro-
ductive of the exact end-results obtained from the operations of the neo-
Mendelian investigators, and while the nature of some of the agents productive
of the characteristic in the soma in color are approaching a solution, there is at
present entire lack of information as far as the gametic agent is concerned.
These somatic agents are, however, entirely physical in their nature, and are
undoubtedly the product of antecedent physical agents in the gametes.

These gametic agents which can be so accurately manipulated in the opera-
tions of the neo-Mendelian type, giving exactness of result within expectancy of
error that is predictable, show that a real start has been made in the problem of
solving the nature of the individual and its characters, and provide a valuable
means of attack upon some of the evolution problems.

It has often been suggested that only the unimportant and superficial of the
organism's characters are found to react according to the factorial principles,
and that the " essentially specific " characters do not do so. This is not true,
and in the pages which follow I shall be able to show that in some organisms
at least many characters, important and unimportant, structural and also
physiological, react in entire accord with these factorial principles of constitu-
tion and reaction. It is probable, therefore, that final analysis of the composi-
tion of any individual species will resolve it into an array of agents that are
productive of its total sum of characters and activities, and it is not too much
to expect that in time it will be possible to dissect the composition of any race
and profoundly alter its character by the removal, introduction, or alteration
experimentally of one or more agents or groups thereof, and in several instances
in these reports it is shown how this has been actually accomplished by divers
means ; but in all instances there have been at the basis of the operation either
gametic or environic agents, specific and exact in operation.

Tlie individual is an entity to the extent that it represents in its character-
istics the summation of the total products of the activities of the genetic and
environic agents and their interaction during the development and life of the
individual, and this is in no wise different from the specific non-living entity.
Each is the product of its productive factors, and in both there are always only
physical substances, relations in the system, the product of directive conditions
in the medium at the time of reaction and production of the combination. The
agents, either internal in the mass or external in the medium, are not the
carriers of anything or of any character of the end-product. There can be,
therefore, no unit characters in the meaning of De Vries, any more than there
are those carried by the pangenes or other ultimate units of other writers;
instead, characters are entirely reaction products in the organism, precisely as
in non-living masses.

Peoblems and Concepts of Evolution 5

BASIS OF CHARACTERS IN NON-LIVING AND LIVING SUBSTANCES.
Inasmuch as the material elements comjwsing both organic and inorganic
matter are the same and exist in both in arrangements of molecules, atoms, or
lesser masses, it is the arrangements and reactions of these that must be the
basis of investigation and interpretation of organic activities.

In the physical world any mass has certain properties that are properties of
the whole, the product of the immense number of interactions between the
component molecules. These properties of the whole express themselves in
characters, weight, forms of crystallization, ductility, and so on, yet no physicist
has ever thought of removing these from the mass and setting each on the
pedestal of a representative particle, or of considering it a lesser entity which
helps like the bricks in a wall to build up the structure, but they are considered
as being what all of our experiences show them to be, namely, properties of the
whole, which may be temporarily or permanently altered, but can not be
removed as an entity from the mass. This is the condition in the inorganic,
and what possible reason is there for supposing that any different condition
exists in the organic?

I am not at all concerned with the distinctness with which characters may
or may not be present in organisms ; these exist and can be experimentally tested
and modified, and the same condition exists in the behavior of matter in the
domain of physics and chemistry, so that the logical result of the representative
particle-unit character conception is the marked admission that the known
facts of physics and chemistry are of themselves unable to serve as a basis for
the interpretation of the phenomena of living things. I can not form a con-
ception of anything existing in the universe which is not physical in first and
last analyses, and although with present knowledge there are difficulties, each
added bit of information helps us to understand nature. With this outlook,
one can not hope to attain ultimate knowledge of the ordering of living material
in nature ; there may always be an uninterpreted residue of observed conditions.
This, however, is constantly diminishing, and behind we leave the phenomena
of nature interpreted in the store of human knowledge as the results of our
collective experience. Each one may have his particular opinion, which may be
for him the correct one ; which of the numerous concepts is correct, however,
time alone will determine, and, we may rest assured, will determine correctly.

RELATION OF EXTERNAL AND INTERNAL FACTORS.

It is undeniable that in organisms there are internal factors which condition
and govern the production and manifestation of the bodily qualities, and it is
equally certain that there are factors external to the organism in the medium in
which it lives, and as far as there is any evidence, this has always been the con-
dition in living matter. It is frequently maintained that in the evolution of
organisms the factors of importance are entirely internal and all that the
external conditions do is to determine which individuals shall live. Others hold
that all of the important determining factors in evolution are external and that
the living material is a sort of plastic, homogeneous material to be molded by
the conditions of existence.

In the domain of physical science it is recognized that any state or condition
in any substance or body is the product of two groups of factors — one resident

6 The Mechanism of Evolution in Leptinotaesa

in the substance itself, the other in the medium surrounding it — and the inter-
action of these two sets of factors determines the particular qualities, conditions,,
and attributes presented. In organisms there are both internal and external
factors, and the a priori view is that they have the same relation in organic
matter as in inorganic, and if they do not have this relation it is something to
be proved and not held as an a priori dogma.

CATEGORIES OF ORGANIC CHARACTERISTICS.

Present considerations of the characteristics of organisms designating them
all " characters," without in any respect attempting to comprehend what the
nature and cause of the character is, and how modifications may be the product
of diverse causes, have lead to much confusion.

In physical terms natural specific things have three grades of characteristics :

Specific Properties or Qualities — Belonging to substances, and not capable of
change without change in the nature of the substance except by alteration of the
factors of composition; i. e., color, hardness, specific gravity, configuration of
the system (crystallization).

Attributes. — Characters distinguishing bodies of the same kind, as size,
weight, volume.

Conditions. — States of being or activity, as temperature, motion, etc., that
can be changed or removed without altering the attributes or specific properties.

In organisms there is precisely the same series of categories of characters
present, which must be considered in the investigation of the evolution phe-
nomena, and care taken that the three categories of characters are not confused
in investigation.

SPECIFIC PROPERTIES OR QUALITIES.

Specific properties or qualities are those characters which can not be changed
without altering the identity of the substance, and in the non-living this is
always accomplished in one of three ways, either by replacement within the sys-
tem of some factor that is productive of specific end-results, because of its pres-
ence, by another similar but different factor which is capable of occupying in the
system the position vacated by the displaced factor — a simple metathetic change ;
or change is produced by the loss of a factor, so that the character is no longer
produced ; and lastly, changes are produced by the production of new, or changes
in present, factors, thereby productive of new or transmuted characters in the
substance. This is precisely what happens in the non-living as in the living,
and I shall be able to show how in the materials that I have used there have been
produced in experiment permanent changes in the specific properties of these
organisms by these three main methods of change and by combinations of them.
In these modifications three general mechanisms of change can be distinguished :

(1) Combinations, or the union into one of two or more factors or factorial
groups, producing in the end a combination product of the characters present
at the beginninff.

(2) Decomposition or germinal disintegration, the breaking up of a sub-
stance or form in which there is loss of one or more of its factors and conse-
quently of one or more characters of the original, producing " varieties " of the
original.

Peoblems and Concepts of Evolution 7

(3) Complex redistributions, of which two grades have been present in my
materials: (a) Double decomposition: Metathesis (the Mendelian reaction),
in which there is in the reaction incident to the reproductive process, decom-
position of each of the gametic systems present in respect to essentially
equivalent and mutually replaceable groups, which then interchange places in
the gametic systems of the two gametically different races present in the reac-
tion, (b) Internal rearrangements, in which there is dissociation of the agents
present in the gametic system, with rearrangements of them into new relation-
ships, with the end-result that there appears in the resulting individual product
either new or changed aspects of the character or characters.

ATTRIBUTES.

Attributes, which are characters distinguishing bodies as size, stature, weight,
and the many other characters that serve to distinguish one individual mass
from another, or one individual organism from another of the same species,
are in the main those characters in the organism that have been in the past
chosen for the operations of the " selection process," and for the diverse attempts
to alter " quantitatively " the nature of the individual. The differences that
fall in the category of " fluctuating variations " belong properly to this class of
characteristics, and in the non-living as in the living are in their manifestation
much influenced by the conditions of the medium.

In organisms there seems to be a basis of permanent differences in the
capacity of these attributes to be manifested. Thus, in stature or in weight
(beans) there are values in the population that seem to be limits beyond which
change is not possible, and also in some instances values that may be maintained
in pure lines without change. What this signifies is difficult to decide, but the
investigations of Johannsen, Jennings, and others have at least made a decided
step in the solution of the problem. No doubt in final analysis it, too, is the
product of the relations of the factors that are productive of the specific
properties of the individual, and may be due to many causes in so complex a
system as organisms are. In some of the later chapters detailed experiments
in this direction show how, in some instances at least, the conditions present are
to be explained. It is not to be expected, however, that upon the basis of this
conception any alteration of the nature of the substance can be produced by the
accentuation of these attributes, because there is no point of entry for any
operation that could possibly be productive of any type of change in the specific
properties, and because throughout the factors productive of them are main-
tained in the system in their original relations, and no means is present in the
attempt to alter the attributes, whereby a change in the relations or in the
factors that are present could result. This is, in my experiments, the reason
for the failure of " fluctuations " to carry alteration beyond a certain limit by
any means. It is not due to the inefficiency of " fluctuations " and the efficiency
of " mutations," but to the fact that in the " fluctuations " only the differences
in attributes have been concerned, and in the operations based thereon there
has been no method present that was able in any way to change the nature or
relations of the specific properties present.

8 The Mechanism of Evolutiox in Leptinotaesa

conditions,

Characters that may be described as conditions in the mass are present as
completely in organisms as they are in non-living substance, and in organisms
these characters concern many of the reactions and behaviors that are present
and that are so easily modifiable. These in the non-living are the product of
the nature of the substance as constituted by its factors productive of the specific
properties and the conditions of the medium, of which many common examples
will at once occur to the reader. In organisms many of the tropic activities and
" animal-behavior " characters are of this kind. They may be held in one con-
dition by retention of the same condition in the mass and one set of conditions
in the medium, or at any time altered, without in any manner changing the
nature of the substance itself, and of these changes I have presented several
examples in later portions of these reports. The characters in organisms that
are of the nature of conditions are in many instances of great importance in the
economy of the organism, and serve immensely vital roles in the processes of
conservation and equilibration as well as in the phenomena of migration, distri-
bution, and apparent composition.

It will, I believe, be at once perceived that the three categories of characters
recognized are not only present in organisms as they are also present in the
non-living, but that this recognition of the nature of the different categories
present in organisms will aid in the further investigation of many problems. It
will be possible from this basis to clearly differentiate and formulate the prob-
lems of experimental investigation, and in some directions there appears at once
a clearer understanding of many problems and results.

The chief advantage gained is the conception of the categories of the char-
acteristics of natural things, living and non-living, from one point of view, and
in this there must be at the least an element of permanent truth, since both are
the products of the same elements and of the same general types of physical and
chemical action.

The literature upon the evolution problems has dealt and must properly deal
with the characteristics of organisms, but unfortunately there is an ever-
present " biological aspect " to these writings and failure to look at the prob-
lems of evolution from purely physical aspects in the majority of instances.
Many harmful metaphysical concepts and methods of expression have gained
entrance to the evolution literature; nevertheless I believe evolution problems
should be conceived of in a purely mechanistic physical sense, devoid of utilities,
purpose, motive, or " forces of evolution."

EVOLUTION AND TRANSMUTATION.

That there are in nature different kinds of living substance, each constant
in form and continued in endless series of generations as far as our observations
go; that there has existed in the past an innumerable array of these specific
kinds of substance and that the history of living nature on this planet, as dis-
covered, shows changes of the characters and in the historical sequence of these
forms, are undeniable facts. For the most part the facts of the history of life
upon this planet are isolated, but suggest to us nevertheless continuity of change
in the past, in conformity with some natural principle of causation, and hence
there have arisen diverse hypotheses of the causation of this diversity and the
sequence of events in the different phyla of the living world.

Pkoblems and Concepts of Evolution" 9

problems of the phylum.

Regardless of hypotheses of the causes of the conditions found in nature, two
striking facts are shown in all of the different phyla that are known, namely,
the fixity of a type of organization that is constant throughout the phylum, and
the fact that there occur changes in the different minor features of the phylum,
which are responsible for the production of the diversity of specific kinds of
living bodies, past and present. Present historical information gives little
satisfaction as to the origin of these huge lines of organic form that persist
through the history of the planet for inconceivable periods of time, and while
interesting hypothetical pictures of the relations and origin of these may be
formed, perhaps with some degree of certainty, the fact remains that there is no
actual information as to the method or point of origin of any of these major
groups of living substance.

Within any of these lines — phyla — the information increases in amount and
certainty as our point of observation approaches the present, and in the living
things of to-day we see, and in experiment produce, changes in the characteristics
of some of the members of different phyla. Methods of transmutation of the
qualities, attributes, and conditions of the phyla are thus either proven in experi-
ment or with different degrees of certainty tied to operations in nature, and the
whole is then used in the effort to project present experiences into the past to
interpret and explain the production of diversity, or the evolution within the
phylum through its history as paleontology reveals it to us.

In each of these histories of the phyla, and collectively true of them all, is the
fact that their history, as revealed in their remains, shows through time a
sequence of modifications, at the start simple organizations, little differentiated
from the phyletie type, with subsequent changes resulting in the later members
in complicated or specialized arrangements of the originally simple phyletie
characters. More rarely in restricted groups, or especially in some portions of
the phylum where it is possible to observe in some detail the history of a large
group, the history is one of progressive increase in complexity to a maximum of
diversity of species and structures, followed by an old-age simplification of the
structures and decrease of the number of different types, ending perhaps in
final extinction of the series, in which the last members approach in simplified
form somewhat the conditions of the series in its younger stages.

There can be no doubt that precise experiment is the only method of obtain-
ing exact information as to the methods and factors in evolution ; nevertheless,
it must not, in the enthusiasm of the moment, be lost sight of that the formula-
tion of hypotheses of evolution from these experimental results must keep in
mind these undeniable facts of the history of any and all lines of living sub-
stance. N"o doubt it will be a long time before experimental efforts will be able
to proceed far in the elucidation of this question, but a start has been made, and
promises to replace, with certain knowledge of the relations of cause and effect
in transmutation, the plausibilities of the past, and thus enable us to undertake
more certainly the interpretation of the causes of the production of the forms
and history of the inhabitants of the planet in the past.

Concerning the origin of the phyla and the fixity of type within each at the

present time, and the production thereof, there is little but opinion or belief as

to the origin and causes. These must be accepted as found, their remote history

and origin credited to our ignorance and not to ineffective mentality or methods

2

10 The Mechanism of Evolution in Leptinotarsa

of investigation, and hope for further historical information that shall in some
measure help to close this gap in our knowledge. Complete information as to
the nature of the form that seems so fundamentally a part of each phylum,
especially as to its method of production and change, is and must be entirely a
matter of experiment; and it is not too much to hope that within no distant time
it may be possible with increased knowledge of organic constitutions and skill in
experimental operations to attack directly and change permanently the basal
system upon which the phylum is built.

Fixity of phyletic type is axiomatic and has long been admitted, even when
accompanied by differences in theory as to origin and also in the method of
producing diversity within it. From the first to the last all have been impressed
with the fact that in the history of any phylum there has been uniformly the
progress of the type as a whole from the simple conditions when it appeared first
in historical records to later periods or to the end of its existence, and this has
produced the idea of a movement in the series based upon the phyletic type, and
has found expression in different conceptions as to its causation. From Aristotle
to the present it has been difficult to escape the cul-de-sac of an evolutionary
principle or force which in the specific phylum determines and in a way drives
it through the observed series of conditions discovered in its history. The ques-
tion why may be asked ; it can not be answered at present in any certain terms,
and belief in any agency helps only individually in forming a comprehensive
and comforting picture of nature. Any theory of evolution to be valid and
comprehensive must in some manner provide in naturalistic operations for
these phenomena in the history of all phyla of living things. There is no evi-
dence of growth force any more than there is of a principle of perfectibility,
or evolutionary force, or bathmogenesis, or orthogenesis, in the production of
the results observed.

Any naturalistic cause of the history of progressive specialization observed in
phyla must at the same time provide for the evident simplification of the mem-
bers of the series in the later stages of its history, and the return of them to
the more elementary states found at the beginning of the history of the phylum.
It may be interesting to compare these to the juvenile, adult, and senescent con-
ditions in the span of a single life, and while there may be similar processes
productive of the two, as far as present information is available, there is no
evidence that it is so, no matter how earnestly one may believe it.

Thus at the base of our conceptions of evolution in nature there lie universal
conditions, beyond doubt entirely naturalistic as to production, but at present
known to us only from the broad outlines of their results through past history
in different phyla and throughout organized nature as a whole. Most probably
the phyla are but the expression of these same underlying causes, and could we
arrive at an understanding of the nature of a phyletic form and the operations
whereby its composition could be permanently changed through experiment,
there would no doubt thus be derived the basis for understanding the funda-
mental nature of these more proximate questions of the apparent movement and
the history of any phylum, and in the rise, culmination, and decline in the
complexity of the members of groups as the special series passes through the
history of the planet.

The problems of phyla and of larger groups within the phylum are a matter
of phylogenetie speculation ; and evolution theories have, from Lamarck to the

Problems and Concepts of Evolution 11

present time, been mainly concerned with the methods of transmutation of the
units of natural species. The principal problem of the student of evolution has
been to discover the causes of the production of species, and then to be able to
experimentally produce them. This investigation in the past century has
given several hypotheses of the mechanism of transmutation.

HYPOTHESES OF SPECIES TRANSMUTATION.

Xone of the several theories propounded appears to give a satisfactory explana-
tion of the problems of origin of species. The formation of species by the
Darwin-Wallace factors has many difficulties to overcome, and its later modifi-
cations, wherein transmutation was the result of the accumulation of minute
fluctuating variations conditioned by the play of mystic entities in the germ-
plasm, involves a degree of credulity not common among scientific workers out-
side of biologists of the neo-Darwinian school. Others have tried to reestablish
the principles of Lamarck that the stress of the conditions under which the
animals live induces modifications that are transmitted to subsequent genera-
tions. Still others, through inability to substantiate the fundamental idea of
the Larmackian theory, and unable to make progress with neo-Darwinism as a
working hypothesis, have conceived of evolution as due to a jumping process,
and sought for data to prove that new characters or modifications of old ones
arose directly through sports, and that these become the progenitors of race and
species.

Xot satisfied with any of the preceding hypotheses, the attempt has been made
to find an efficient cause for the origin of species in geographical and physio-
logical isolation, but especially that geographical isolation which comes from
the segregation of the different portions of the same organized form in unlike
habitats, and hence in different en\aronmental surroundings ; while in another
line of effort, orthogenesis, one finds, in the writings of the authors supporting
the hypotheses, evidences of the old Aristotelian concept of an impelling
principle or of a goal with a fixed line of development in the evolution of
organisms. With this array of hypotheses it is no wonder that the layman is
confused and inclined to think that perhaps after all evolution may not be as
universal a truth as some of its advocates have claimed it to be, and even the
biologist often wonders what the outcome of this situation will be.

The oldest of these hypotheses of species formation — that of Lamarck — was
formulated when many things now common knowledge were unknown. Lamarck
in his time knew something of the direct effect of external conditions in inducing
changes in organisms, especially in plants when they were transplanted into the
botanical gardens of Europe from exotic locations, and these direct effects of
the transplantation were noted, and this seems to have furnished Lamarck with
a possible natural cause of the formation of species. Lamarck recognized that
unless this modification was transmitted to subsequent generations no evolution
could result — which was certainly correct — and hence the idea of the trans-
mission of these through the reproductive process. It is true that Lamarck was
never clear as to how these characters were transmitted nor how they could be
transmitted, excepting that transmission from generation to generation was in
some way associated with the reproductive process and was necessary for the
transmutation of species in evolution.

12 The Mechanism of Evolution in Leptinotarsa

neo-lamarckians.

The neo-Lamarckians, although boldly maintaining the truth of Lamarck's
fundamental proposition of the effect of the conditions of the medium, seem to
have been most uncritical in the use of the cardinal principle of their master,
and at the present time there is no evidence that the neo-Lamarckians' hypoth-
esis is true, and it is necessary to suspend judgment as to whether their principle
of the transmission of variations from the soma to the germ ever occurs.

The essential principle in Lamarck's work, however, that differences in the
medium, and especially those resulting from change in habitat, are effective
factors in evolution, certainly must receive most careful experimental investiga-
tion, and it matters little wliether the effect of the conditions of the medium are
direct upon the germ or indirectly through the soma to the germ. The principle
is now shown to be true that changing conditions in the medium are efficient
factors in the evolution process.

Although forced now to admit the truth of Lamarck's essential principle of
the effectiveness of factors in the medium, we do not commit ourselves in any
way to the hypothesis of the neo-Lamarckians, nor is it possible to do more than
recognize that if their principle is effective in evolution, further consideration
and use of it can only be the product of exact experimental investigation.

The chief principle of Lamarck, however, finds a permanent place in the
hypothesis of transmutation by means of natural selection as enunciated by
Darwin, which was a far broader conception of organic evolution, wherein he
made great use of the effect of environment so emphasized by Lamarck. Darwin
forcefully and frequently insists that the agencies most productive of the
differences in any population of organisms, which become the basis of the
selective process whereby species or changes are produced, are in considerable
part the direct product of the action of the conditions in the medium upon the
organism, and especially upon the reproductive process, and probably upon the
reproductive elements.

DARWINISM.

The hypothesis enunciated by Darwin has proven far from satisfactory when
applied generally in the attempt to solve the riddle of evolution in plants and
animals. Darwin recognized the Malthusian priiiciple that every species tends
to increase at a rapid rate and to produce a greater number of individuals than
are capable of survival, and that of these 98 per cent in the main die before
reaching maturity, only 2 per cent surviving to reproduce the next generation.
It is axiomatic that there is this excess of reproduction over survival, and the
whole of Darwin's hypothesis of transmutation hinges upon the single point of
whether those individtials which persisted and reached maturity are those which
possess variations of a nature such that they are thereby made more efficient
than their fellows, and hence better able to successfully compete in the " struggle
for existence" or whether the survivors are only those individuals luhose chance
position, when the accidents of life happen, save them from extinction, and
eliminate their less fortunately placed companions. If it is true that the sur-
viving minority are the possessors of conditions making them more fit to meet
the struggle for existence, then Darwin's hypothesis has a considerable degree
of probability of truth, but it distinctly has not been shown that the surviving

Problems and Concepts of Evolution 13

individuals are the fortunate possessors of these advantageous modifications.
Because individuals do survive, it is the custom to assume on the basis of
Darwin's hypothesis that they are better able to meet the conditions of life than
those which were eliminated, but an accurate analysis and measurement of this
problem is utterly lacking, nor can it be attacked, excepting through a long-
continued, painstaking analytical investigation upon favorable materials in the
laboratory and in nature.

If it proved to be true on adequate investigation that the elimination in
natural populations is entirely a matter, or even largely a matter, of the chance
position of the individuals when the accidents of life occur, and that the sur-
vivors are on the average no better provided with equipment for life than those
which are eliminated, then Darwin's hypothesis as a means of transmutation
and species formation in nature must be abandoned, or at the least assigned a
minor position as a productive cause of diversity in nature.

It is not worth while at this place to enter mto a lengthy discussion of Dar-
win's hypothesis. There has been too much discussion and too little investiga-
tion ; but one aspect needs attention at this place, namely, the idea advanced by
many, perhaps most vividly insisted upon by De Vries, that " natural selection "
acts solely as a sieve which allows certain individuals to persist, while others are
eliminated thereby. This " sieve-like action," however, seems to me to be one
quite different from that which Darwin conceived of in his hypothesis. There
is unquestionably in nature an action of the conditions of the environment which
in the main eliminates extremes in any population, and further, under adverse
conditions it is the mediocre in the race which has the best chance of survival.
This seems to be a fairly well substantiated generalization, as far as evidence
at the present moment goes, and in my own work I have frequently found that
this principle was actively operating when populations were subjected to hostile
conditions. This action would have a tendency of conservation, to hold the
population in a stable condition, and distinctly would not have the action which
natural selection was supposed to possess by Darwin — one of producing diver-
gence. It is possible that two operations have been confused, and that there
should be recognized the possibility of there being in nature a " natural selective
process," which might be productive of the results which Darwin supposed to
be the basis of the origin of species; on the other hand, there are clearly in
nature operations which do exactly the things which De Vries attributes to
natural selection. These latter I have often encountered in my work, and in
this report I have adopted the name " factors of conservation," meaning thereby
an association of agencies within and without the organisms, which, so long as
they remain constant, maintain the race in uniformity. These are agencies
acting for the retention of specific form, habitat, and distribution, but they are
distinctly not formative forces in the sense that they produce anything new.
Their operation is the reverse, and hence their name. In portions of these
reports I shall show how experiments with these two aspects of the problem
operate in some instances, and especially in relation to the two questions which
have been raised with regard to Darwin's hypothesis — whether it is chance
favorable constitution or chance favorable position that determines the survival
of the individual.

Through inability to make progress with the neo-Darwinian concepts, many
earnest students of the evolution problems, in the latter part of the nineteenth

14 The Mechanism of Evolution- in Leptinotaesa

century, began a new attack upon the problem of species, which was based in
the main upon the conception that if species were discontinuous when fully
formed, a 'priori there was no reason why they may not have arisen discon-
tinuously at the start. In support of this proposition there was found in the
older writings of plant and animal breeders, and especially in Darwin's works,
many records of the rise of new domesticated races of organisms based upon the
appearance of an ancestral sport. The attack upon the problem was made from
quite different points of view, but nevertheless all have arrived at a somewhat
similar result, namely, that certain discontinuous changes in the qualities of the
organisms were quite as likely to be productive of evolution as the methods of
slow accumulation so currently held by the neo-Darwinian school. Bateson's
compilation of instances of discontinuous variation and De Vries's experimental
investigation of the process which he calls " mutation " in Oenothera lamarcJc-
iana, the experimental production and observed rise of stable forms by Mac-
Dougal, Tower, Kammerer, Morgan, and others, in the main have provided the
basis for the hypothesis that effective changes in the qualities of organisms may
arise discontiuuously in many directions, producing, as De Vries says, some-
thing quite new each time.

MUTATION.

The mutation theory of De Vries has raised a number of interesting and
important points for consideration, chief of which are his efforts to distinguish
between the conditions present in any population, dividing them into sharply
separated categories of " fluctuations," which are ineffective in transmutation,
and " mutations," which bear the burden of the evolution process.

De Vries's argument is based largely upon the data of biometric study and
the more certain showing that in domesticated plants fluctuating variations are
not capable of accumulation beyond an amount which was present in the race at
the start as the limits of " variation," and further, upon the showing that all of
these variations are either plus or minus additions to the character and in no
other directions, while mutations are stable, effective, and in many directions.
There is no doubt that descriptively De Vries's account is correct, but I am
strongly of the opinion that both the account and the definitions are actually
incorrect. I have pointed out previously in this introduction that it is necessary
to recognize three general classes of characteristics in organisms, and that of
these characteristics one, especially, the attributes, which distinguish bodies
of the same kind, has been almost exclusively used in this selective accumula-
tion experimentation, and that there is not present in the process any mechan-
ism whereby the qualities of the race may be altered by accumulation of the
amount of the attribute present, and this in every instance is the effort which
the worker of cumulative selection seems to have made. With the sugar beet it
was increased in the amount of sugar. With other forms it is increase in stature
or increased or diminished amounts of one thing or the other within the race
which serves only to distinguish the individuals of the race, and not the races
from some other ; so that in all these operations there is present only the means
to attempt to accentuate the attributes present, and there is no possible mechan-
ism available for modifying the qualities of the race to which the attributes
belong. Whether these attributes vary only in one direction, plus and minus, is
largely a matter of definition and the method of investigation employed : and in

Problems and Concepts of Evolution 15

a later chapter data will be found dealing with this problem, showing that the
statement is true or not true, depending entirely upon the definitions employed
and the point of attack from which the investigation is made.

"With regard to the mutations of De Vries, I am of the opinion that De Vries
has described a specific operation, which is not the same as the formation of
sports and saltations described by other writers, and I would distinguish, there-
fore, a process of mutating as distinguished by De Vries and shown by him to be
present in Oenothera lamarcliana, and a saltation process in which there is a
single sporadic production of one or more divergent individuals as the result of
the single application of the productive force to the parental strain. Both of
these types of the production of new conditions in the qualities of organisms
are experimentally confirmed in these reports, in addition to those already
existing in the literature elsewhere, and should be recognized as distinct as to
causation. To these a third process must be added, which also produces dis-
continuous changes either repeatedly in the same strain or rarely as an apparent
sport. Morgan's experience with germinal disintegration in Drosopliila and my
experience with this same process in my materials leaves no doubt of the exist-
ence of this third category as a general type of operation, the products of which
are all now collectively characterized as " mutations."

The three processes are alike only in the sense that they produce at one opera-
tion new aspects in the organism which are discontinuous with the parent
stock. In mutation there is a process operating in which there is repeatedly
produced in succeeding generations one or more new types which appear again
and again through a series of generations. I have shown in some experiments
how such a race can be produced experimentally and give products which are in
every way in animals the duplicate of those described by De Vries in Oenothera
lamarcTciana, and hence I think it necessary to recognize a " mutating process "
as distinct from the others which may also give products of quite diverse natures.
The sporting process seems uniformly to give but a single result, following the
application of the productive cause, and there is thus far nowhere in any of my
experiments, or in the modifications produced by MacDougal, Kammerer, or
Woltereck, any indication of repeated or of subsequent production from the race
of the new types. In many of my examples the change is distinctly that of
adding a character to the organism, in others it is the displacement of the
character, and so no doubt the sporting process works both ways, either to add
or subtract characters to or from the race.

In germinal disintegration there is present an operation in which characters
are removed from the race one after the other, but which can be put back into
the race by proper crossing methods, thus restoring the original ; and differing
in many respects from the products of mutation or the products of sporting.
This germinal disintegration is possibly the process which is largely responsible
for the production of large numbers of domesticated varieties, which can be
shifted back to the original condition merely by adding the requisite character.

In these three types of change there is concerned solely the action of genetic
and environic factors, either through the combination of agents hitherto sepa-
rated, and hence with the resulting appearance of a new character, or the actual
production of new factors, and the removal of agents, all of which show that the
entire series of operations resolves itself into the study of the factorial composi-
tion and operation of the gametic substance.

16 The Mechanism of Evolution" in Leptinotaesa

These products of mutation, saltation, and germinal disintegration are largely
of laboratory origin, and at present there is entire ignorance in the main as to
how these products may behave in nature ; and it is not known whether they
could or would become the progenitors of specific groups. This is not an
impossible subject for investigation, and it should receive experimental investi-
gation by the introduction of newly arising groups into localized natural
habitats, and their behavior studied through a series of years. In this manner
it will be possible to evaluate the different agencies concerned with respect to
their efficiency in the production of diversity in nature.

ISOLATION AND SEGREGATION.

Two other hypotheses call for mention at this place, namely, the supposed
action of isolation and segregation so ardently advocated by Warming, Wagner,
Gulick, Jordan, and others. This hypothesis of species formation by isolation
is little more than the plausibilities of geographical distribution and faunistic
study and the juxtaposition of possibly unrelated facts observed in nature, with
the presentation of them interwoven into an hypothesis of natural causation,
in which the fact of their being isolated from one another is conceived of as the
efficient motive force. Within this group of writers one finds various causes
asserted to be the agency which produces the modification supposed to result
from isolation and environmental actions, the various types of isolation elabo-
rated by Gulick, and many others, wherein conceivable but unproven agencies
are called into action to explain the conditions found in nature. Habitudinal
geographic factors may well be effective agencies in the production of hetero-
geneity in nature, but these need to be investigated by experimental methods,
and I have found in some of the species that I have used that there were degrees
of geographical heterogeneity associated to a greater or less extent with different
habitat complexes surpassing in refinement that recorded by any worker. All of
these differences, however, are entirely the product of distinct gametic factors,
which can be determined and experimented with through modern methods. I
have likewise attempted preliminary experimentation in nature in the effort to
create isolated colonies of species of like gametic constitution, and then to test
repeatedly the nature of these species in the effort to determine whether isola-
tion is productive of change, and, if so, to what cause the changes are due. Thus
far the results obtained show that isolation alone is not productive of germinal
changes, provided that homogeneous materials are used for the experiment and
as long as the new conditions do not operate as effective factors in the production
of gametic changes by the direct action of the environmental conditions. Isola-
tion unaccompanied by such environmental action never shows, even with the
closest inbreeding, any change in characters, and there is no evidence that with
gametically uniform stock there is any possibility of such processes resulting in
change. When stocks are not uniform or pure, then there have resulted isolated
races in these experiments, and this experimental production of them may help
in interpreting the conditions in nature, and aid in the solution of the origin of
many geographical races.

ORTHOGENESIS.

Lastly, there is the hypothesis of evolution which is characterized " ortho-
genesis." It is notorious that, since the time of Aristotle, through the Middle
Ages, in the recent writings of Nageli, Eimer, Hyatt, Jackson, Whitman, Osborn,

Problems and Concepts of Evolution 17

and others, the fact so often recorded in nature, that modifications within the
phylum and in different minor divisions thereof, the supposed succession of
species through successive geological horizons, or over some terrestrial surface,
appear to have the relation, one to another, of a series of steps which seem to
have moved from the first observed condition to the later one of greater or less
complexity, and the fact that in ontogeny the sequence of stages during develop-
ment is always uniformly the same, and that this ontogenetic sequence to a
greater or less extent parallels that found in phylogenetic series, has led many
to postulate a causative agency which maintains organisms in these lines, giving
specific end-results. Hence the word "orthogenesis," used descriptively by
Haacke, has come to a greater or less extent to designate an unknown but active
principle, whose effect is to produce these modifications, one after the other, in
an orderly, step-like series. There is no denying the facts of our observation
as to the series both in phylogeny, ontogeny, and in the phenomena of distribu-
tion, but it is not warranted to personify the unknown agencies which were pro-
ductive of these conditions and make the unknown an effective cause. A cause
there must be, but that there is a force driving organisms through nature, or an
evolution-trend moving towards a goal, or a principle of perfectibility is some-
thing which the majority of biologists are unable to believe. As a matter of fact,
it is highly improbable that the observed conditions described by the word
" orthogenesis " are produced by uniform causes at all, and results that might
descriptively be called " orthogenetic " might well be produced by quite hetero-
geneous causes. There is no positive evidence, aside from plausible interpreta-
tions based upon the hypothesis, that the conditions, placed one after another in
paleontology or in distribution, really are in sequence with one another ; nor is
it known by exact demonstration in any instance that one arose from the other.
The data derived from ontogenesis and the frequent parallelism between onto-
genesis and phylogenesis does not signify anything more than the conclusion
that like substances react alike imder similar conditions, and that the same
reactions follow, under similar conditions, the same sequence of events. In
physical nature identical substances and identity of capacities under the same
conditions produce identical reactions, and show the same array of sequences;
and so I should regard both the ontogenetic and phylogenetic series as merely
recurrent operations present in essentially the same substance, and they are not
to be interpreted as due to a principle driving organisms through a predeter-
mined series of events. The data of orthogenesis are entirely of an observational
character, based upon studies in phylogenesis, geographical distribution, and
ontogenesis ; but there is no evidence that there are productive forces, or a force
at all, apart from the other natural operations, and it is possible to find an
explanation for these states which are observed in nature that is in harmony
with other aspects of the evolution problem.

With this array of hypotheses conflicting in principle in some respects and
overlapping in others as to the production of species in nature, one is made to
wonder whether after all there may not be a common basis upon which they can
all be tested and brought into agreement. Each one has its good points as well
as its defects, and if a common basis of operation and investigation can be found
it will be possible to test the respective propositions of each of the divergent
conceptions.

18 The Mechanism of Evolution in Leptinotaksa

PROCESSES OF EVOLUTION,

The investigations of the past century have clearly formulated certain definite
evolution processes which may be characterized as ontogeny or development,
variation or heterogeneity, heredity, conservation, and equilibration. These
comprise groups of activities which are involved in the production, in deter-
mining the survival and final location, of the products of evolution.

HETEROGENEITY.

The process which has so long been characterized " variation " is distinctly
not a uniform one, but a collective name for the results of diverse reactions
whereby there are produced in natural things different degrees of unlikeness in
many directions, or heterogeneity. Because of this absence of unity of action,
it is more correct to think of the production of heterogeneity in nature rather
than of " variation," because heterogeneity is simply descriptive of the actual
results produced by many processes, while variation implies divergence from the
average or mean condition. This conception of variation reaches its climax in
Danvin's work and in the neo-Darwinian hypothesis is unduly accentuated by
the biometricians, but it is distinctly not applicable to the problems as under-
stood at the present moment. " Variation " in the physical sense implies simply
the finding of a range of values above and below some mean, divergence of
results produced by conditions external to the reaction, which cause it to
diverge and show error or departure from the mean or true value. Such arrays,
common in all physical measurements, are adequately expressed and formulated
according to the theory of error and arrangement in various types of frequency
curves. On the other hand, it is known at the present moment that the highly
miscellaneous differences which are found in any natural population are dis-
tinctly not the product in any population of one cause, but of many, and so the
diversity existing in any population is better described as heterogeneity.

It is certain that within any natural population there may be produced
diversity not only as the result of the effect of the conditions of existence upon
the developing organism, but also as the direct result of the composition of the
organisms, and these again may interact to produce other added complications
in the array which the population presents, and when to this are added all of the
possibilities which came from the combination and recombination of the
numerous unlike factorial potentials in the gametes and the unknown series of
operations which may produce new factors in any generation, it is certain that
the problems of diversity in natural populations are among the hardest problems
to analyze at the present moment, and I have devoted in this report considerable
space to the attempt to analyze some of the problems of heterogeneity as it finds
expression in these organisms.

In this respect the conception of the natural diversity of any population again
agrees with the diversity present in any non-living group. Their multifarious
conditions are the product of numerous unlike causes and not of a single opera-
tion, and out of the array present in any population two general types of result
seem to follow in the next generation. There seem to be distinctive conditions
present in the population which are not repeated in the next, and these are the
" fluctuations," according to the Weismannian point of view, which are
" somatic " in character and not " germinal," hence their " non-inheritance."

Pkoblems and Concepts of Evolution 19

On the other hand, there are characteristics which are repeated in the next and
following generations, frequently for long periods of time ; these are, therefore,
asserted to be " germinal."

We have been too lenient with the Weismannian dogma and the criterion it
establishes, that because a character does not appear in the next generation it is
not a product of germinal origin ; but there is no longer any doubt that there are
conditions in the population that are entirely the product of gametic factors
which may not appear in successive generations, due solely to the fact that the
right combination of factors is not made. A further fault at the present moment
is the assumption that because a character is not " inherited " by the progeny
it therefore is somatic in character, and while I do not doubt the existence of
modifications which are purely somatic in character and are, on the basis of
present knowledge, hardly to be expected to be of inheritable kind, nevertheless
the backward application of this test is not justified, and I shall be able to show
later that there are characters which " do not appear to be inherited," which are
purely germinal in origin. Furthermore, it is shown in some of this work that
there are conditions appearing in populations which would be characterized by
De Vries as fluctuations, and which are not capable of fixation in permanent
form and are not inheritable, which are not somatic, but germinal, due to the
presence in the gametic system of gametic factors occupying positions or rela-
tions such that their action is to produce this irregularity. In other words, they
act as impurities in the gametic system in the same way that impurities may act
in non-living physical systems.

INHERITANCE.

Inheritance is also simply a collective term descriptive of the reaction of
gametic agents and their behavior through a series of generations, and the
results which may follow from the combination of specific gametic factors. It
is hardly possible at the present moment to distinguish between the phenomena
called " variation " and " heredity " ; and operations often characterized
heredity are distinctly important in the production of heterogeneity in natural
populations.

These two groups of phenomena, heterogeneity and heredity, are the basis, of
course, upon which evolution works, and present problems are, therefore, of two
general kinds : First, by means of the principles which have been developed in the
last fifteen years, there must be undertaken an extensive and accurate analysis
of the gametic constitution of organisms in terms of their component factors
and determining agencies; secondly, with this knowledge gained and with
materials of known gametic constitution available, to analyze the phenomena of
heterogeneity, undei? the controlled conditions of laboratory experimentation
and also in organisms in nature. In combining these two I have attempted to
trace in some degree the significance of heterogeneity in several species of these
organisms, and this effort, I believe, gives a clear insight into the real signifi-
cance of natural diversity and the potentialities present in natural species for
the production of divergent groups, and through this means perhaps there is a
new method of attack upon the problem of origin of species. Actual evolu-
tion operations of course center in and around the processes concerned in the
production of heterogeneity and the operations which are termed collectively
" heredity." Everything that ever has been or ever shall be in the evolution of

20 The Mechanism of Evolution in Leptinotaksa

organisms is the product of operations within this domain. They supply to the
race the qualities, attributes, and conditions, the modified old, the new char-
acteristics, either in combinations or separate, arranged to produce the specific
living kinds or species which must after their formation by the operations
mentioned be subjected to the tests of the factors of conservation, and lastly to
the processes of equilibration.

CONSERVATION.

The factors of conservation are those natural relations of the internal and
external factors of every organic kind whose relations determine at once and for
all whether or not the organism can persist. This is perhaps a synonym
of natural selection, but natural selection is employed in so many ways to
mean an agency productive of diversity, or as an agent which is productive only
of elimination, that I prefer the term " conservation " as indicating those
operations in which the newly arisen mechanism is tested out and it is proven
by test whether it is able or not to survive under the conditions into which it is
thrust by the process of origination. This is a complex test and it can only be
investigated by exact experimental analysis. No information can come from
the routine collecting of statistics and their treatment in biometric fashion,
and thus far the only information that I have been able to obtain concerning the
operation of the processes comes from experimental test in which it must be
proven what the specific eliminating factors are and how they work in the
system. I have experimentally tested a number of examples in this investiga-
tion, and in every instance it has been found that elimination or failure to pass
the test of conservation is entirely due to the factorial composition of the
organism, and that a factor of one kind or another by its very presence forces
upon the organism either in mode of action, or in some quality, attribute, or
condition, relations or methods of reaction such that in some portion of its life
the relations between external and internal can no longer be maintained, and
the organism is either eliminated by its own death or the race is eliminated by
the inability of the organism to reproduce itself under the conditions.

I have thus far failed to find any influence of those selection effects, and in
every instance elimination, as far as I have observed it and tested it in these
and other organisms, involves little of the " biological content," and is largely, if
not entirely, a matter of physical relationship between the factors of composition
within the organism and the physical factors of existence without, in the-
medium. This aspect of the problems of evolution at the present moment needs
extensive experimental testing, and must be carried out, in order to obtain the
best results, in combination investigations in the laboratory and in nature, and
in this investigation I have been fortunate in the nature of my material and
with the facilities which the American continents have afforded for various
types of experimentation in nature.

EQUILIBRATION.

When an organism has passed, if by its original constitution it succeeds in
passing, through the test of conservation, it must then meet an added series of
problems in the activities which are characterized equilibration, which are those-
activities whereby the organism, without changing its qualities, so adjusts its
attributes and conditions to the composition of its medium that there is the least

Problems and Concepts of Evolution 21

possible resistance between it and the conditions under which it must live.
This is a specific set of reactions which every species must undergo, and I have
seen many interesting examples of it in the introduction of one race from one
habitat to another, in which the habitat was not sufficiently different to serve
as an inhibition to its existence there, but in which there were sufficient differ-
ences in the new habitat to bring about in the introduced race distinct adjust-
ments of the organism, not in its qualities, but in its attributes and conditions,
which may become definitely altered and thereby produce a new localized habi-
tudinal race. When an organism has passed through the processes of origina-
tion, has been tested as to its ability to survive, and has adjusted itself to the
location into which it is placed, it takes its place as a member of the communit}%
and, in as far as anything which it exhibits is concerned, it may have been in
that community for hundreds of thousands of generations, when, as a matter of
fact, it may have been there less than a half dozen.

This brings us to an important point in all of these operations in evolution,
namely, that when there is change it is rapid ; that the processes of origin pro-
duce transmuted factors in the organism within one or two or at the most a few
generations, and that the product appears finished and complete in all its
characteristics in a single relatively short series of processes. This may not be
universally true, but in any event the changes which I have observed have
reached completion in a short time, and the questions of conservation are
decided at once. The organism with its system either can or can not survive
under the conditions in wdiich it arose, and this in every instance is decided in
one or in the most two or three generations, and the operations of equilibration
are again rapidly brought to a stable condition, so that in a half dozen or a dozen
generations from the beginning of the process of change the organism, so far
as appearances are concerned, in its stability and in appearance, might have
existed in its habitat for ten thousands of years.

Many of the previous conceptions, especially those of Darwin and neo-
Darwinian schools, demand long-continued, faltering operations with irregular,
haphazard reactions, when as a matter of fact in observed instances the opera-
tions and reactions are as rapid, as specific, and as complete within a short time
from their start as are the operations in physical phenomena. This must of
necessity be so, because in all these operations there are involved only the
physical component agents within and without the organism; its gametic
factors and the processes which we analyze and describe, which are nothing but
the interaction of these physical substances and relations in terms of physical
process ; and this is the reason, apparently, why the reactions which have been
witnessed in experimental evolution have given so radically different a view-
point with regard to evolution problems. This brings us to the last point of con-
sideration—the point towards which we have been moving for more than three
centuries — namely, the question of an adequate formulation and experimental
investigation of a factorial theory of organic evolution.

FACTORIAL THEORY OF EVOLUTION.

It is difficult to decide where the first ideas of a factorial theory of evolution
arose, but certainly we can follow the development of it for a long time. This
is common knowledge, and everyone knows how Mallet, and Maupertius more
than a centurv ago, formulated factorial concepts of organic constitution and of

22 The Mechanism of Evolution in Leptinotaesa

evolution. We all know to what use Spencer, Darwin, Weismann, and De Vries
have put this conception in the production of their theories, but Darwin,
Weismann, and De Vries figured their factorial elements in the gamete as single
bodies, the carriers of the characters of the organisms. Each of these bodies, in
the terms of these writers, was potentially a small organism. Any such con-
ception eliminates the possiljility of investigation from an experimental point
of view, and is certainly of an order which can not be formulated in any physical
terms. There is no known instance in which a substance is the carrier of a
character, and all characters which appear in non-living substances are the
reaction products of the combining factors of composition and the conditions
of the medium at the time of combination.

Out of the neo-Mendelian investigations there has come the most valuable
contribution to biological science in a long time, namely, the exact demonstra-
tion that the characteristics of organisms are the product of the action of two
or more productive agents, and the Mendelian operations have clearly shown
that these factors may be removed one from the other, and that one may be
removed and the other left, thereby producing the non-appearance of the char-
acter with which the original pair is associated, and that the character may be
brought into existence again when the missing one is replaced by the action of
crossing. This gives an opportunity to apply this principle further to the
analysis of organic constitution and to evolution problems. In this report the-
principle is applied broadly and in divergent lines, in every respect confirming
thus far the broad fundamental principles which are the outgrowth of a long
series of antecedent studies. And thus we may reformulate our conceptions of
organic constitution and evolution, that the organism itself in its gametic
make-up is but the associate combination of an unknown number of non-living
factors the interaction of which in many associated relations is necessary for the
production of the living substance and its characteristics. Any change in char-
acters which the organisms undergo are of the same order and due to the same
causes as are characteristic of changes in non-living substances. The method
of change of each of these characteristics is entirely the product of the factors,
their presence or absence within the system, and their interaction with the
factors within and without; likewise the facts of survival or conservation, of
equilibration, and of all the processes present in evolution are directly and
solely to be investigated and solved from the standpoint of the genetic factors
within the non-genetic factors without the organisms.

I realize that it will be contended that while this may apply perhaps with
some truth to the superficial characteristics, it can not apply to those long-time
trends of evolution and to the problems of fixity of type within phyla. As a
matter of fact the conception does give a naturalistic conception of these por-
tions of our problems. In the non-living world are found combinations of
various factors of composition which are most marvelous and tenacious in their
association. Silica and oxygen form an association — quartz — which is dis-
sociated with difficulty and one which persists for a long period of time. This
substance becomes the basis of many minor operations in producing the various
kinds of quartz — silicates and their products — which are produced by minor
additions or combinations of the basic combination of SiOg, and this might
perhaps be the equivalent in a rough sense of the general phyletic type of
organisms which when analyzed show certain securely combined gametic factors,

Pkoblems and Concepts of Evolution 23

and it seems to me that that " phyletic type " is nothing more than a chance
association of gametic factors, such that it is disrupted with extreme difficulty,
but which has also great capacity for adjustment to meet widely varying con-
ditions of existence, capacity for slight added changes, and nothing more. If
this is true, then it is to be expected that at some future time it will be possible
to dissociate the various factors productive of a phyletic type and perhaps
change the phyletic type experimentally. This may be something which will
not be accomplished for a long time, but it is not unthinkable that it may be
accomplished at any moment with the methods now available. Likewise from
the same basis of factorial composition long trends of evolution which one finds
are but the potentialities present in these types for the continued successful
addition, reconstruction, and rearrangement within localized or trivial portions
of the original factorial composition of the race. This view finds more or less
of confirmation in the expressed opinion frequently given by paleontologists
that evolution after all consists merely in the specialization or in the reduction
of the characters of the original progenitors of the group, and this conception
does account for and provide us with an experimental basis from which to
investigate the condition characterized as orthogenetic.

In physical operations, given a specific composition and conditions in the
medium, a given cause will produce a reaction only in one direction, and this
reaction will follow through a definite predetermined series of steps which are
entirely due to the relations existing between the factors of composition within
the mass and the process of decomposition, rearrangement, and loss of factors,
and it is probable that the orthogenetic sequences where they exist at all are due
entirely to this cause, which is purely naturalistic and is capable of exact
experimental production and investigation.

The factorial conception is certain to prove the most usable working hypoth-
esis available at the present time. jSTo doubt its formulation at the present
moment, and our description of the factors and the designation of them, are in
crude terms a pragmatic personification, but it is recognized that these are at
present only symbolizations ; nevertheless there can be no reason to doubt the
existence of these factors and the type of their reactions in the gamete, which
are characterized as germinal factors, any more than to doubt that there exists
in the medium various physical agents which are external factors in evolution
operations.

CHAPTER II.
THE MATERIALS. THEIR TAXONOMY AND NATURAL HISTORY.

INTRODUCTION.

The purpose of this chapter is to give as clear and complete an account of
the materials and their origin as is possible, so that no matter how taxonomic
groupings may change, there may be no uncertainty as to the characters, adult
and juvenile, and the actual locations from which my materials came. All
figures and descriptions are from the living animals, and are the species with
which I worked. The findings in experiment through this report are those for
a specific form, from a precise location in nature, and no more. I do not know
that the same " species " from another location would act the same in duplicate
experiments ; in fact, many of my experiences show that they do not, due to
diversity of the gametic composition of a phenotypical constant species that is
uniform in taxonomic aspect over its area of distribution, but is gametically
dissimilar. Until we know more of the geographical distribution of this gametic
diversity, records must be for the reactions of specific materials from specific
locations, and not be applied broadly to phenotypically uniform species.
Gametic uniformity must be proven and not assumed.

METHODS OF OBTAINING MATERIALS.

My materials without exception have been derived directly from nature, from
definitely located stations which have been under as constant observation as was
possible during the period in which the material was being used in the investi-
gation. •

In every instance where any material has been used in these experiments the
following routine has been followed : Having determined by preliminary tests
that the species was of interest in connection with this investigation, I have
then, first, gone personally over the entire known geographical range of the
species in question, with the dual object of discovering its actual habitat and
the geographic distribution thereof and the total range of variability of the
form both as regards its common fluctuations and accidental somatic aberra-
tions, and also to discover if, in any restricted habitats, it had developed isolated
local races of any description. Further, I found it helpful to become acquainted
with all of the conditions under which any species was living and did live in
nature. Two sets of information concerning the species were thus obtained —
an extensive knowledge of the range of " variability " of the organism and at
the same time data of its conditions of existence. In itself this is no small task ;
it was further complicated and made difficult by the conditions prevailing in
Mexico and Central America, where much of this portion of the work had to be
done.

At the same time that these field observations were in progress materials were
sent in from the field to the laboratories at Tucson and Chicago, and there
24

Materials, Their Taxonomy and Natural History 25

subjected to tests to determine their value for experimental work. Their
adaptability to cultural conditions and the rapidity of reproduction were of
course vital points to be determined, but the gametic composition of the species
was of far more importance. Adaptability to cultural conditions, to a very
considerable extent, can be attained with most of the organisms that I have
attempted to use, and is thus far, in all instances in my experience, entirely a
matter of external conditions.

The question of composition, however, is much more serious, and some other-
wise admirable materials have had to be discarded on account of the practical
difficulties encountered in the reduction of the material to a gametically homo-
geneous state. The chief difficulty has been the existence in the materials of
diverse minor strains which could be isolated by one process or another and then
maintained as " pure cultures." Species from nature that had no habitudinal
races or modifications of any kind, that were of low somatic variability in
response to the changing conditions of the medium, nutrition, and kindred
environmental factors, and free from minor strains, biotypes, or other sub-
divisions, have been the type of materials which I have sought for use.

With materials of this character, and with a knowledge of the total range of
the " variation " over its area of distribution, and of the conditions under
which it lives in this range, I believe that I have a proper basis for an investiga-
tion of evolution, experimentally. The actual materials used in the investiga-
tion have in all instances been taken from some accessible location, not likely
to be disturbed by agricultural or other economic operations, and during the
course of the experiments, or at least during the crucial portion thereof, the
exact locality from which the material came has been visited at least twice in
each year at a time when the population could be seen in numbers, and at that
time a census was made to determine the presence in the population of any
divergent or modified traits or characters that were not found in the first
general census of the species. This hnowledge of the status of the materials in
nature at the point of origin of the experimental material is of the greatest
importance, because if any, modifications are produced in experiment, and if
these are new or striking it is then necessary to know positively whether the new
character ivas produced in experiment alone.

I have been exacting upon this requirement in the character of my materials
for the reason that the bane of experimental evolution at the present time lies
in the unfortunate fact that all too often, as in De Vries's Oenotheras they were
" mutating " when he found them, and everyone is now at liberty to " interpret "
the results of De Vries as his own biological orthodoxy dictates. The rigid con-
ditions demanded in physical science should be for the biologist the model of
operation, a most rigorous study of the materials in nature, purification and
standardization, and then experimental operations with the proper setting to
give exact answers to specific problems.

CHARACTER AND SOURCE OF MATERIALS.
THE LINEATA GROUP OF LEPTINOTARSAS.

The animals which have furnished the materials upon which this report is
based are a homogeneous group of species — the lineata group — of the genus
Leptinotarsa. These organisms are confined almost entirely to North America,
3

26 The Mechanism of Evolution in Leptinotarsa

do not occur in the Antilles, and, as far as is known, are distributed from the
northeast corner of South America northward through Central x\merica and
into North America as far as southern Canada. It has been difficult to deter-
mine what names should be applied to many of the forms found in nature, due
to the fact that for most of the species the material existing in museums is
meager, often inferior in quality and data, and, moreover, the taxonomic
descriptions are based upon dead and often upon only a few badly preserved
specimens. Because of post-mortem changes in color, systematic descriptions,
especially of the colors, are in the main incorrect for the living animal, and
since students of the physiology of evolution are only interested in living
organisms, I have given for all species used in this report diagnoses from the
living material.

These species are specific kinds of living substances, each with its specific
qualities, which, in each, are genetically reproduced true to type, generation
after generation, in experimental cultures and in nature. To prevent confusion
and to make clear the exact nature and source of my materials, I have presented
in this report as complete a taxonomic description as is at present possible of
each of the different species in the lineata group, their juvenile stages, distribu-
tion, and ecology, and such discussion of their life-histories, distribution, and
other activities as is necessary to an understanding of the work described herein.

The descriptions are, as far as possible, compiled from the original sources,
and I have given the original descriptions as printed and supplement each
with a description of the living material that has passed through my hands.
Many questions concerning the nomenclature have presented difficulties in the
effort to arrive at a just settlement of the complicated taxonomic situation.
For example, four species (L. panamensis n. sp., L. guaiemalensis n. sp., L.
diversa n. sp., and L. undecimlineata Stal) are distinguished with difficulty
as far as museum specimens are concerned, but they are definite, clearly dis-
tinguishable organisms when alive, especially when followed through their life-
histories, and when crossed their differentiating characters are sharply alter-
native in behavior. The most conspicuous differences between the four species
are found in the larval stages, and in their habits, distribution, and ecological
relationships. Stal (1858) and in 1862 in his Monographic des Chrysomelides
de L'Amerique, described L. undecimlineata, and as far as his description is con-
cerned it would apply fairly well to the imagines of either of the forms, and as
far as the distribution given by him is concerned it would also apply to any one
of the four found, but the one which I have designated L. undecimlineata is
more probably the one which Stal had, and it is certainly the one which has in
later years most often been so designated. Much later, Duges described the
larvae and life-history of a form from Guanajuato, Mexico, which he believed to
be L. undecimlineata Stal, but his description is of the juvenile stages of the
form that is here recorded as L. diversa. Of the two forms, that which has the
wider distribution as given by Stal I have called L. undecinnlineata, and this is
in accord with the identifications given to many museum specimens. In the
Baily Collection in the British Museum there is a specimen labeled, " Coll.
Baily," named by Stal " L. 11 lineata," "Type Stal 194"; "Mexico," that is
the same form that I have called L. 11 lineata so that there can be little doubt
but that the determination is correct. The other I have called L. diversa,
although many specimens of this species exist in collections and are labeled

Materials, Their Taxonomy and Natural History 27

L. undecimlineaia Stal. L. panamensis nov. sp. from Panama, Costa Rica, and
the Isthmus of Darien in Colombia, and L. guatemalensis n. sp. are also quite
as distinct as the other two. The four arc closely related and probably represent
species that have been differentiated from the same original stock, and which
are now limited to separate geographic areas. In that I do not know of inter-
grades between them, and that their chief differentials are sharply alternative in
crosses, I have retained them as species and have not classed them as geo-
graphical varieties.

The relations existing between the species L. muUitceniata Stal, L. oblongata
nov. sp., L. melanothorax Stal, L. miiltilineata Stal, and L. decemlineata Say
are confusing. As far as I can discover, L. melanothorax does not, at any
locality in nature, sustain itself as an independent type, but always appears
periodically, sometimes rather irregularly, as a recurrent sport from L. muUi-
tceniata Stal and its geographic varieties. Upon its appearance as a sport in the
population, it crosses back with the parent species, within which it is at once
submerged. However, when isolated in a culture, it breeds true indefinitely as
a specific kind of living substance, and behaves in the same way that organisms
which are called elementary species and mutations do. The question as to what
L. melanothorax Stal is depends upon one's point of view and one's definition of
a " species." I have classed it as a " recurrent mutant."

Stal's account of L. muUitceniata and L. muUilineata leaves one in doubt,
as far as the descriptions are concerned, as to what use should be made of the
two names in describing the series of forms which live on the southern half of
the high plateau of Mexico at the present time. Throughout the entire southern
end of the plateau, southward into the Rio Balsas Valley as far as Matamoras de
Izuca, eastward to the Plains of Apam, and northward into the upper tribu-
taries of the Rio Amacusac, Rio Coetzala, and Rio Atoyac, is distributed a
variable organism, which is unquestionably L. muUitceniuta Stal. Throughout
a considerable portion of the Oaxaca-Guerrero highlands, the Balsas Valley,
and up the slope of the Mexican Plateau as far as the foot of the escarpment, is
another species closely related to the former, but different in form, distribution,
in juvenile characters, as well as in life-history. In some respects this species
agrees with L. muUilineata Stal ; but Stal considers his L. muUilineata and
L. decemlineata Say to be one and the same species, and L. decemlineata Say is
not known to me in Mexico. Stal's species, muUilineata, is, I believe, simply a
" form biotype " of L. multitcBniata Stal that is fairly common in some locali-
ties ; at least, a biotype of L. muUitceniata Stal is found which in all respects is
like L. muUilineata Stal and therefore I have used his name for it. The form is
radically different from L. decemlineata Say and is distinguished by certain
gametic factors for body proportions from L. muUitceniata Stal and its other
varieties. L. decemlineata Say, as recognized by Rogers and all the later Ameri-
can writers, is clearly distinct from the Mexican types, although in museum
specimens the several sjjecies might easily be confused, especially when badly
preserved. The living animals, however, when followed through the life-history,
leave not the slightest doubt as to the existence of the specific entities.

Thorough searching of the European museums, especially the British Museum,
has shown interesting points that have materially helped in the solution of this
confused taxonomy. In the Baily collection in the British Museum there is a

28 The Mechanism of Evolution in Leptinotarsa

specimen labeled, " muUilineata Stal," with the pin labels, " named by Stal,"
"Type Stal 176," which is evidently an immature specimen, the elytral stripes
being brown instead of black. It has, however, the characteristic dark legs and
ventral surface and the narrowed pronotum, with the same type of pattern, and
in all respects it agrees with the form that I have called " biotype " ^nultilineata
Stal, and I think that there is little doubt that muUilineata Stal is a biotypic
form of multitceniata Stal, and that oblongata is a distinct species.

There are in the British Museum five specimens, all alike, that are clearly
the same as the oblongata recognized here, and that are labeled midtilineata.
Two are from " Oajaca, Mexico," one from the " Baily coll." " Mex.," one with
no locality, and one that is marked " muUilineata, agrees well with descrip-
tion." All of these are, however, clearly oblongata and not the biotypic form of
multita;niata Stal, here designated as muUilineata.

The species decemlineata Say and multitceniata Stal, are in all respects so
distinct that there is no need for confusing them, excepting in very badly pre-
served museum materials. There is not the least doubt in my opinion that Stal's
species muUilineata is a biotype of his distinct, widely distributed species
multitceniata; that oblongata is a distinct species, as is decemlineata Say,
although the three are badly confused in all collections and in the taxonomic
literature of this genus, and this is especially true of Jacoby's determinations of
these species, as shown by an examination of his materials deposited in the
British Museum.

Similar difficulties are encountered in the case of L. dejecta Stal. The t}'pe
of organism which has been designated L. dejecta Stal by most of the American
writers, and so labeled in collections, is a close relative of L. juncta Guer., has
been described as L. texana by Schaffner, and apparently represents a modifica-
tion of L. juncta Guer., which has arisen on the semiarid plains of the Rio
Grande Basin and nearby areas. Of L. dejecta Stal I have had live materials
from Brownsville, Texas, and it is clearly closely allied to L. juncta Guer., and
its geographic variety texana Sch. is apparently rare and may be only a rare
sport from texana Sch. At least, I do not know that it anywhere exists as a
constant, genetically perpetuated member of the fauna of any locality. Stal's
original statement that its habitat is " Mexico, Texas, and Yucatan " gives
nothing upon which one could base an opinion, and furthermore, his description
and Jacoby's figures are not very illuminating.

The " dejecta " which I have used in my experiments is not the L. dejecta Stal,
although so labeled in most collections, but is L. texana Sch., which is not a
variety of L. decemlineata as Schaffner supposes, but is related to L. juncta
Guer. as shown by the character of the elytral punctation.

The lineata group represents a homogeneous assemblage of species centering
about the lower end of the Mexican Plateau and presents three main trends of
evolution, and from present knowledge may be grouped into three divisions as
follows :

Tlie species L. panamensis n. sp., L. guatemalensis n. sp., L. undecimlineata
Stal, L. diversa n. sp. (habitudinal variant rugosa n. var.), L. angustovittata
Jacoby, and L. si,gnaticollis (biotype nigropunctata Sturm) represent a group
entirely confined to the grasslands below frost-line, as shown in plate 3, a
tropical savannah group, and all feed upon perennial solanums of the species
8. lanceolatum, S. liegerii, S. chrisotrichum, and allied species.

Materials, Theie Taxonomy and Natural History 29

The species L. multitceniata Stal (habitudinal variant multitceniata Stal,
recurrent mutant melanothorax Stal; saltation tacubayensi?; biotype multi-
tcEtiiata Stal; habitudinal variant variabilis nov. var. ; habitudinal variant inter-
media n. var., recurrent mutant melanothorax Stal), L. oblongata n. sp. (bio-
types, (a) red, (&) orange, (c) yellow), L. rubicunda n. sp., L. decemlineata
Say (saltiitions pallida n. var., dejecto punctata n. var., minuta n. var., tortuosa
n. var., melanicmn n. var., rubrivittata n. var.) represent a group entirely con-
fined to the temperate and cold uplands of Mexico and the plains and prairies
of the United States and Canada, as shown in plate 5 — typically temperate or
upland savannah inhabitants, and all feed upon solanums related to S. ros-
tratuin, or S. elceagnijolium or 8. tuberosum.

The species L. juncta Guer. (habitudinal variant texana Sch.), L. dejecta
Stal, and L. tumamoca n. sp. represent a group divergent from the two above
groups and inhabiting portions of the Atlantic and Mexican Gulf Coastal Plain
and Pacific coast desert, as shown in plate 3, feeding upon annual and perennial
solanums of diverse kinds.

That I may clearly establish the nature, quality, and source of my material,
I have gone fully into the taxonomic study of it, especially in the living animals,
their distribution, habits, and ecology, and in this paper I have presented the
essential facts known bearing upon these points. It will be necessary to repeat
some facts and data already in print, but completeness in the treatment of this
portion of the subject is essential at the present time. The diverse series of
experiments, to which various members of this group have been, are now being,
and will in the future probably be subjected, demands that no effort be spared in
ascertaining the uttermost information concerning the condition and relations
of the materials in nature.

TAXONOMIC AND GENETIC DIVISIONS.

At the outset of the study of this aspect of the problem I am confronted by
the question of what it is that constitutes a species; further, there are the
problems of the lesser units or divisions present in some of the species, and of the
orientation and terminology to be applied.

In nature there exist only individuals, each with its genetic line of descent,
and the aggregation of tliese genetic lines into groups, and it is this latter aspect
of the problem that presents the greatest difficulties. The mere fact that a form
breeds true to itself is no criterion and no reason for considering it a species,
and were this basis adopted an almost infinite number of species are possible
even in one narrow group, depending upon the skill of manipulation and the
minuteness of the differentiating characters employed by the observer.

In this investigation I am of necessity forced to consider and use species, and
I have from experience come to think of them as organic systems distinguished
by a limited array of qualities, which perpetuate themselves in nature, and are
separated by a distinct complex of qualities from other related species. Within
any such group there are potentialities present in the diverse qualities and their
permutation for the isolation, by genetic methods, of a varying number of pure-
breeding lines. Also, in some specific groups in one or more areas, the accentua-
tion or suppression of one or more of the qualities marks off the members in-
habiting that area into a geographic variety. So, too, there are occasional sports,
appearing one at a time and at irregular periods, and also regularly recurring

30 The Mechanism of Evolution in Leptinotaksa

variations of the same order may appear. Sudden variation in single characters
may also give rise to minor groups, or may change in nature the aspect of the
whole lineal group.

In this material I have recognized and determined the extent to which any of
the following grades or minor groups are present in any specific form which is
to be used for experimental investigation before it is utilized in experimental
study. This analysis of the composition of the materials is absolutely necessary
and it is only by careful analysis of the composition that is is possible to avoid
frequent error and misinterpretation in the progress of investigation. In
practice I have found the following scheme of description and analysis to be
effective :

Primary Grade — Spkcies:

A. Gametic in character, or due to actual factorial composition of the organisms.

Changes therein by loss, addition, recombination or transmutation.
1. Species: A group of organisms separable from all other groups by a
constant array of qualities, and genetically perpetuated in nature
throughout their range of habitats.
Secondary Grade:

a. HaMtudinal varieties: A secondary and geographically localized
portion of the primary group which is distinguished from the
primary group by the possession of difference in, or accentuation
of the characters of the primary group in the geographic area
inhabited by the differentiated portion of the species, and geneti-
cally constant in nature.
6. Recurrent ''mutations'' : Periodic, or recurring discontinuous varia-
tions in the group, which appear as sudden deviations, independent,
genetically perpetuated races.

c. Saltations (sports): Occasional variation of greater or less magni-

tude appearing without visible cause and often becoming the basis
of races of permanency and of permanent modifications in the
specific group. Usually involve sudden change in several associated
characteristics. Rare and irregular in occurrence.
Tertiary Grade:

d. Biotypes: Lines of descent in which one or more attributes or

qualities are accentuated or suppressed and capable of isolation
and propagation by breeding. Such biotype groups present inter-
grading variations with the remainder of the population and the
" purification " results in reducing the fluctuation of the type.
Equivalent to the Biotype of Johannsen, Elementary Species of
DeVries, and many of the " varieties " of domesticated plants and
animals.

B. Somatic in character: Not due to factorial change but to developmental

alteration of the somatic expression of gametic factors.

a. HaMtudinal somatotypes: Somatic alterations recurrently produced in

any form in successive generations by influences present in the
habitat without producing gametic change. Not inheritable and
always lost on change of habitat. Variable. Character only
decided by breeding in changed environment.

b. Environmental (aberrations) : Irregular discontinuous somatic changes

produced by sudden irregularities in the medium or other excessive
conditions. Not inheritable. Character of change only proven
after breeding tests.

This arrangement of the divisions is applicable to species in a state of nature,
where genetic continuity and stability are demanded. Often there are found
year after year, associated with some species, certain types, and these are
described as varieties or even as species — e. g., L. melanothorax Stal — a pro-
cedure that is entirely justifiable on the basis of the information ordinarily
available ; but not infrequently I have found that these types are not genetically
continuous, but are produced anew as sports recurring in each generation, or

Mateeials, Their Taxoxomy and Natueal History 31

even as somatic aberrations. It would be interesting to know how many of the
rare species varieties and instances of groups of closely allied species described
as inhabiting the same area are of this character.

Study of actual material in the field, in life, is absolutely essential for all
genetic studies, and this must be done in many localities through a series of
years or generations. From different localities samples of the population must
be taken, tested by breeding under some standard set of conditions, and these
two sets of observations will eventually give the only true basis for the correct
determination of the nature and quality of the materials used for experiment.
This has been the general and inflexible plan in all of my work.

In its application I have tried to go, and in the main have personally gone,
several times over the entire range of each species, collecting data and materials
in many localities, and each location was visited again in subsequent years. This
has given extensive data of distribution, ecology, and the differences in nature.
From many locations materials were tested under standard conditions, and the
gametic constitution of the species in the locality determined. This data, plus
that from observation in nature, gave the basis for thorough knowledge of the
nature of the materials used in this experimental investigation of evolution.

SPECIES AND SUBDIVISIONS OF THE LINEATA GROUP.

In nature I have found and tested the following species and their minor
divisions, which I have arranged in two divisions, as follows :

A. Elytral markings edged with an irregular double row of impressed punc-
tations.
Undecimlineata division:

(1) L. panaynensis n. sp.
L. guatemalensis n. sp.
L. undecimlineata St3.1.

Saltation: abrupta nov. var.
L. diversa n. sp.

Habitudinal variety: rugosa nov. var.
L. angustovittata Jacoby.
L. signaticollis Stai.

Biotype: nigropunctata Sturm.
Multitaeniata division:

(2) L. multitivniata St§.l.

(1) Habitudinal variation: micltitccniata Stkl.
Recurrent mutation: melanothorax Stil.
Saltation: tacuhaytrnsis nov. var.
Biotype: obscura nov. var. multilineata StM.

(2) Habitudinal variation: variabilis nov. v&t.
Recurrent mutation: melanothorax St^l.

(3) Habitudinal variation: intermedia nov. var.
Recurrent mutation: melanothorax Stai.

L. oblongata n. sp.

Biotypes: (a) red, (&) orange, (c) yellow.
L. rubicunda n. sp.
L. decemlineata Say.

Saltations: pallida nov. var., defectopunctata, minuta, tortuosa.
melanicum, rubrivittata.
B. Elytral stripes edged with single even regularly placed row of impressed punc-
tations.
Juncta division:

L. juncta Guer.

Habitudinal variation: texana Sch.
L. defecta Stai.
L. tumamoca n. sp.

32 The Mechanism of Evolution in Leptinotarsa

These species are unmistakably distinct, easily separable in life, and while
some of them might be further divided to increase the number of species, I
can not at present find adequate basis therefor.

In the working over of the literature of the taxonomy only important
references and synonyms are retained. Many references are vague, and so
many purposeless, incorrect citations and determinations appear that I have
omitted all but those necessary to trace clearly the correct name and synonymy
of each species.

In the description of these species use must be made of a more precise
nomenclature than has hitherto been employed, especially of the elements of
the color-pattern which are constant and useful as differentials. In my 1906
paper (p. 60, figs. 1 and 2; p. 143, fig. 7) I give the arrangement and nomen-
clature of the centers of pattern-formation, which are designated in the main
from the sclerite or portion thereof occupied, with the exception of the pronotum,
where an empirical lettering is used.

DESCRIPTION OF THE SPECIES.

UNDECIMLINEATA DIVISION.

Elytral markings edged with an irregularly placed double row of impressed
punctations.

LEPTINOTARSA UNDECIMLINEATA StIl.

Myocoryna 11-lineata Stai. Ofv. af. K. Vet. Ak. Forh., 1858, p. 316.2.

Chrysomela undecimlineata Stai. Monog. Chrys. d. Am., 1862, p. 163;
Chevolat, Dej. Cat, 3d ed. p. 421; Krantz, Berl. Zeit., 1874, t. I, f. 5.

Leptinotarsa undecimlineata Stai in Gemminger et Herold, 1874, Catalog.
Coleop. t. XI, p. 3441; Jacoby, 1883, Biol. Centr. Am., vol. VI, pt. 1,
p. 234; Tower, 1906, Inv. Evol. Chrys. Beetles Gen. Lept, pp. 5-14.

I have retained the name undecimlineata for the species inhabiting the
savannahs and foot-hills around the Gulf of Campeche in Mexico. This is the
foi-m originally described by Stal. The habitat Mexico and the flavo-testaceous
basal joints of the antennas possessed by this form indicate clearly that Stal's
name should be retained for this species and that L. diversa, L. guatemalen^is
n. sp., and L. panamensis should be given equal systematic rank. Stal's
description is as follows (Monog. Chrys. d. l.'Am., p. 163) :

" Nigra, supra pallide flavescens ; basi, apice maculaque media triangulari
capitis, maculis liturisque prothoracis nigris; margine inflexo, sutura vittisque
quinque elytrorum aeneo-nigris. Long. 8| — 12, Lat. 5| — 7 millim.
" Patria: Mexico, Costa Rica, Bogota, Bolivia. (Mus. Holm., etc.)
" Statura praecedentis. Ovalis, sat convexa, nigra, nitida, supra pallide
flavescens vel straminea. Caput parce punctulatum, disco laevisculum, basi,
apice maculaque magna media triangulari nigris. Antennae apicem versis
sensim nonnihil incrassatae, articulis quinque ultimis subquadratis, clavam
vix formantibus. Prothorax elytris nonnihil angustior, antrorsum sensim
leviter angustatus, pone medium interdum parallelus, disco parce, subtiliter,
utrimque nonnihil densius et distinctius punctatus, litura media ut littera V
formata maculisque pluribus lateralibus parvis nigris, angulis anticis mucro-
natis. Scutellum laeve, seneo-nigrum. Elytra lateribus parallelis, subacervato-
seriatim distincte pimctata, serie punctorum prima paullo ultra tertiam partem
elytrorum extensa, margine inflexo, sutura, vitta suturali anteriore nee non

K. TODA DEL.

JL-LIL'S BIEN,

Colored PHoxodHAPHS of Living Materlvl ok some Species t^sed in Different

Experiments.

1. Leptinotarsa undecirnUneata. Male. X 2.

2. Egg cluster of L. undccimlineata. X 1.5.

3. Full-grown larvae, L. undecirnUneata.

X 1.5.

4. L. panamensis n. sp. 4a, male; 4b.

female.

5. L. guatemalensis n. sp. Female. X 1.5.

6. Egg cluster of L. panamensis. X 4.5.

7, 8. Larvae of L. panamensis. 8a. full-
grown, 3d stage; 8b. 2d stage. X 2.

9. L. diiH'rsa n. sp. Male and female
copulating. X 2.

10. L. diver.sa. Egg cluster and first-stage

larvs. X 1.25.

11. L. diversa. Larvae; (a) 2d stage; (b)

3d stage, fully grown. X 1.5.

Materials, Their Taxonomy and Natural History 33

utriusque vittis quinque interstitia alterua occupantibus, tertia et interdum
secunda basin attingentibus, intermediis tribus prope apicem abbreviatis, sub-
suturali et marginal! apice conjunctis, nigro-snesis.
" J* Segmento ventrali ultimo apice truncate."

Description of the Living Animals.

Imago (plate 1, fig. 1; plate 2, fig. 8). — Above: Hypodermal color greenish
pearly-white in the fully developed imago, with epicranium and pronotum often
tinged with pale lemon-yellow; with age color becomes dimmed and browned.
Head with sides, mouthparts, and ventral side black; eyes black. Antennae,
basal joint black, second to sixth brown or light yellow brown, with fifth and
sixth often with brown color deep or black; seventh to eleventh joints black
thickened, longer than broad, terminal joint conical and elongated. Epicranium
with anterior lateral epicranial spots united mediad to form heart-shaped spot
in center of epicranium, which frequently is prolonged caudad to meet fused
posterior lateral epicranial spots in median line, which are always fused mediad.
Posterior border and lateral portions behind eyes irregularly punctate with
well-marked pits. Pronotum: Constant central V-shaped spot composed of
h' + a' + am + p-m + a+b; d and c always free; often reduced; d' + e' + f and
d + e + f commonly fused, rarely united to central V-shaped spot. Posterior
border and lateral portion rather thickly and coarsely punctate; anterior
and central portion freely punctate. Elytra with 5 longitudinal black stripes
with greenish metallic luster, edged with irregular double row of large puncta-
tions. Stripes on anal edge unite to form median black stripe where elytra
are closed. Epipleurae inflexed, black, punctate. Below: Legs, mouthparts,
thoracic and abdominal surfaces dense shiny black, except that edge of pro-
notum is of same color as dorsal surface and frequently shows ventral pleural
spot, variable.

Size: Not variable; male 7 to 11 mm. long, 5 to 6 mm. broad; female 8 to
14 mm. long, 5 to 7 mm. broad.

Sexes: Female, ventral sclerite of the terminal abdominal segment rounded
smooth; male with same sclerite truncate and median groove well defined and
reaching well towards middle of plate, variable. In general the male is smaller
than the female.

Food: Preserved specimens of the lood-plants and live material grown from
the seeds were submitted to Dr. Greenman, of the Field Columbian Museum, for
identification. Knabe, in 1907, gives the food as 8. torvum, which, according
to Greenman, is confined to the Antilles. It feeds equally well upon Solarium
hertwigi Bereth (Camaron, Tierra Blanca, San Marcos), S. diversifolium,
Schlecht (San Marcos, Vera Cruz, Coatzocoalcos), and in captivity upon
*S'. lanceolatum Cuv., and *S'. clirysotnclium Schl.

Juvenile Stages.

Eggs: Laid in bunches upon the lower surface of leaves (plate 1, fig. 2), from
10 to 175 in a bunch; pale yellow in color, with stalk of coagulated gelatinous
cement; 1.25 to 2 mm. long, 0.75 to 1.00 mm. broad; flattened and concave on
ventral side. Incubation period from 7 to 18 days; average 9 or 10 days.

First larval stages: Head, pronotum, and legs, black; body-spots each with
one or more spines. Average length at end of stage 2 to 3.25 mm.

Second larval stage: Head, pronotum, and legs, black; body white or trans-
parent, without spots or spines, except spiracula and wing-spots ; white color
increasing in intensity with growth, especially in the sternal and pleural por-
tions. Average length at end of stage 5.5 to 6.5 mm.

34 The Mechanism of Evolution in Leptinotabsa

Third larval stage: In color and markings exactly like second stage, but with
body-color ivory white, becoming opaque or yellowed at pupation. Length at
maturity, 8 to 14 mm. (plate 1, fig. 3).

Pupa: Pupate in ground, 0.5 to 6 inches below surface. Pupa, pale yellow
white, with spiracula spots very small. Duration of pupal stage, 10 to 20 days,
average 12 days.

Length of larval period: Eange 20 to 42 days, average 27.

Length of ontogeny: On average 49 days, range 30 to 80 days. The larval
stages of L. undecimlineata Stal described by Duges are not of this form, but of
L. diversa, which occurs (rarely) at the location where Duges obtained his
material near Guanajuato.

Geographical Distribution.

In its distribution this species as now limited is confined largely to the
savannahs and lower foot-hills and valleys about the Gulf of Campeche (plate
3). I have included here only citation of localities where there is absolute cer-
tainty of the correctness of the determination. Many older records, even when
the material has been examined, are of no value, owing to poor preservation,
and can not be corrected. The records of localities submitted apply only to the
form as here limited, and while further information would no doubt change the
range and perhaps the relation of these closely placed species, nevertheless the
data given here is for a single form, irrespective of its systematic value. I have
found it at Campeche (near the city) at Esperanza, Tenabo, state of Campeche;
Frontera, Chilapa, San Juan Bautista, Embarcadero, Huimanguillo, state of
Tabasco; Coatzacoalcos, Minatilan, Hidalgotilan, Tortugas, Santa. Lucretia,
Medias Aguas, San Juan Evangelista, in the valley of Eio Coatzacoalcos, and in
many other unnamed localities in the same valley ; Tlacotalpam, Achotal, San
Marcos, Teschoacan, Los Narancos, Chirpo, Perez, El Hule, Tuxtepec, Tierra
Blanca, Tepextempa, Motzorongo, Vista Hermosa, Agua Fria, and many other
localities in the valley of the Eio Papalaopan and its tributaries; Vera Cruz
(near the city), Paso del Macho, Camaron, Misantla — all in the state of Vera
Cruz. At all of these localities I have seen this species in all of its stages, and
at all of the localities the characters were uniform and constant.

Habits and Ecology.

An adequate general account of the habits and ecology of this species is given
in my 1906 report (Carnegie Inst. Wash. Pub. 48, pages 14 to 16).

Source of Materlax.

The living material for the various cultures and experiments described herein
came in the main from Tierra Blanca, San Marcos, and Coatzacoalcos.

{a) The location at Tierra Blanca, Vera Cruz, Mexico, 3 kilometers SW. by
W. of the railroad station is on the Coastal Plain at an altitude of 200 feet upon
a flat and typical tropical savannah, with an open, park-like, thorn-forest, and
poorly developed fringing forest along the water-courses. Well-developed wet
(June to January) and dry (January to May) seasons occur here, and the
activities of the beetles follow closely the climatic cycle. All material for experi-
mental purposes has been gathered from a restricted locality on the edge of a
small stream. Stocks were obtained as follows: No. 700 in August 1903;
No. 701 in May 1904; No. 702 in July 1906 ; No. 703 in June 1908.

Materials, Their Taxonomy and Natural History 35

(b) The location at San Marcos (Vera Cruz, Mexico) is also on the Coastal
Plain, at an altitude of 600 feet, in a wide, poorly drained plain, with a short,
compact growth of grasses and a well-developed tree-island forest. The
climatology is about the same as at Tierra Blanca. All material has been
gathered on the edge of a tree island 0.5 kilometer SSW. of the railroad station.
Stocks were obtained as follows: No. 710 in May 1904; Xo. 711 in July 1905;
No. 714 in June 1907; No. 715 in June 1908; No. 716 in June 1909.

(c) The Coatzacoalcos (Vera Cruz, Mexico) location is 2 kilometers north-
east of the town, at the edge of the sand-dunes. It is also a savannah, but modi-
fied by the approaching dunes and high humidity brought in from the Gulf of
Mexico by the onshore winds. The climatology is modeled on the same plan as
at the other two sources of material. Stocks were obtained as follows : No. 720
in May 1904; No. 721 in August 1904; No. 722 in January 1908; No. 723 in
August 1908.

Materials from all three localities were, as far as analyses could discover,
identical, and not one has thus far shown any gametic characteristics that were
strictly limited to any one locality.

Saltation: L. abrupta nov. var.

Leptinotarsa angustovitta Jacoby. Tower, 1906, p. 6, pi. 16, fig. 2, where It
is recorded as angustovittata Jacoby.

Imago : Exactly like undecimlineata in all respects except that elytral stripes
are suppressed or wanting and greenish metallic iridescence gives elytra greenish
tinge, or greenish white. In breeding it proves to be a recessive— ^i. e., absence
of stripes is recessive — and breeds true in cultures as an extracted recessive
when crossed. It is a rare sport accompanying certain strong climatic
influences.

Occurrence.

In nature, Tierra Blanca, 1904, 1907, 1910. Produced in experiment,
Chicago, 1905.

Juvenile Stages.

Like L. undecimlineata; food and habits also identical.

Jacoby's doubt as to the standing and relationship of L. angustovittata led
me to believe in 1906 that ahrupta was the same or essentially the same form.
I have since 1906 found L. angustovittata in nature, reared it, and its affinities
are clearly with diversa and not with imdecimlineata Stal.

LEPTINOTARSA DIVERSA N. SP.

Leptinotarsa undecimlineata Stkl. Material from Orizaba, Oaxaca (Jacoby,
1883); Orizaba, Jalapa, Oaxaca, Mitla, Tlacolula, Tomellin (Tower,
1906), are references to L. diversa n. sp. and not to L. undecimlineata
Stai. Duges, 1883, Ann. de la Soc. Ent. de Belg., T. 28, pp. 1-6, pi. 1,
life-history from Guanajuato, are of forms here designated diversa.

Descriptiox.

Imago (plate 1, fig. 9 ; plate 2, fig. 4) . — Above : Head, pronotum, ivory white,
more or less tinged with yellowish : pattern of black spots. Elytra, ivory white,
often greenish, with 5 dense black longitudinal stripes ; costal edge inflexed,
black, punctate. Below: Mouthparts and eyes, dense shiny black, x^ntennae:

36 The Mechanism of Evolution in Leptinotaksa

Black, joints 7 to 11, broad as long, distinct, terminal joint reduced, conical,
posterior portions of epicranium punctate, with large pits. Pronotal color-
pattern uniform, spots and central V-shaped spot constant, composed of

am
a'-f- -|- +fl and a large spot in each outer posterior angle composed of

pm

d' + e' + f and d -\-e + f. Lateral third of pronotum coarsely punctate, middle
third polished, few minute pits, scattered larger punetations.

Size: Larger than L. undecimlineata Stal and somewhat smaller than L. sig-
naticolUs Stal. More robust than former, less so than latter. Male 8 to 14.5
mm. long, 5 to 7.5 mm. broad ; female 8 to 17 mm. long, 5 to 9.5 mm. broad.

Sexes: Female, sternal sclerite of last abdominal segment rounded, not
truncate ; male, same sclerite truncate, with well-marked median groove from
posterior border to about the middle of the plate. In general the male is smaller
than the female and differs in being narrowed posteriorly, while the female is
broadened.

Food: At Orizaba, Solanum chrysotrichum Schl. ; Duges (1883) gives
8. torvum; Dr. Greenman states that S. torvum is not found in Mexico; at
Guanajuato, on ^S'. hertivigi Bereth ; on west coast, on S. diversifoUum Schl. and
S. hertivigi Bereth.

Juvenile Stages.

Eggs (plate 1, tig. 10) : Laid on lower surface of leaves in bunches; 20 to
200; yellow, oval, ventral surface slightly flattened and concave; attached by
stalk of varying length composed of coagulated gelatinous cement. Long, 1.25
to 2.5 mm,, broad, 0.75 to 1.25 mm. Development requires an average of 8 days
under normal conditions ; range 6 to 12 days. Duges records eggs of two sorts —
first fixed by foot and second stalked. This difference, which exists in nearly
all the species of this group, is, as far as I have observed, due entirely to the
manner of laying and the drawing out of the material used to cement eggs to
leaf into a longer or shorter attaching stalk. It is not a part of the egg.

First larval stage (plate 1, fig. 10) : Head, legs, and pronotum, black; body
yellow, with full system of spots. Length at hatching averages at end of stage
3 to 4.25 mm.

Second larval stage (plate 1, fig. 11a) : Head, pronotum, and legs, black;
body yellow, due to the fact that body has no hyperdermal pigment; spiracular
and wing spots only present. Mid-dorsal line and middle of each segment
laterally grayish, owing to lack of fat-body in under-lying structures, giving
regular pattern of yellow and grayish on back. Length, 4 to 7.5 mm.

Third larval stage (plate 1, fig. 11&) : Head, pronotum, and legs, black;
terminal part of legs brown ; body yellow or chrome yellow, due to increased
density of pigment in fat-body. Second to sixth abdominal segments with
broad, tergal black band reaching and often fused with spiracular spots. On
first abdominal segment, inner and middle tergals only present, rarely united;
seventh segment, all spots present, variable. Length at maturity, 9 to 21 mm.
Coloration of this stage somewhat variable, due to the fusions of the different
tergal centers.

Length of larval life: Varies with condition, but averages 20 days under
normal conditions ; range, from 12 to 50 days.

Pupa: Pupates in ground from 0.5 to 4 inches in depth, at foot of food-plant.
Pupa pale yellow, with small spiracula and tergal markings. Pupal stage lasts
on an average of 12 days ; range, 8 to 21 days.

Length of ontogeny: Average 60 days in nature; range, 35 to 70 days.

Materials, Their Taxonomy and Natural History 37

Geographical Distribution.

Mexico (plate 3), on slope of eastern and western escarpment, at altitude of
3000 to 6000 feet; from the state of Vera Cruz, Mexico, at Cerro El Borrego,
Cerro Escamela ; Sumidero near Orizaba, Jalapa, Cocomatepec, Huatusco ;
Mitla, Tlacolula, Ejutla, Oaxaca, Villa Alta, state of Oaxaca ; Guanajuato
(Duges), canyon of Eio Grande de Santiago, at many points below Cascada de
Janacatlan in Jalisco; Tepee, El Cora, territory of Tepee; and it is probably
distributed along the Pacific coast to the Gulf of Lower California and south
into Oaxaca-Guerrero highlands. On the east it is probably distril)uted through
the foot-hills and north to the Rio Panuco Valley.

Habitat.

The general distribution of this species is shown in plate 3. It is not a gen-
erally distributed form, but, as far as I have observed it, is always local, limited
to narrow habitats, either on hillsides, the edges of the steep-walled barrancas
that cut the edge of the Mexican Plateau, or in the immediate vicinity thereof.
It apparently is distributed along the eastern and western edges of the plateau,
on the east as far north as the valley of the Eio Panuco, and on the west it
apparently reaches nearly to the head of the Gulf of California ; at least, good
museum material from the Pacific coast of Mexico is of this type. On the south,
in the deserts of the Tehuacan district, it is modified into the geographical
variety rugosa, and on the Oaxaca-Guerrero highlands it again assumes its
typical form. South in Chiapas it occurs typical in form, and apparently it
extends southward into Guatemala ; at least, museum material from the plateau
region of Guatemala is apparently of this same type.

SouBCE OF Material.

Living material for experiment has come from (a) the east side of Cerro el
Borrego and from the southern end of Cerro Escamela near Orizaba, Vera
Cruz. Stocks were obtained as follows : No. 815 in August 1903; No. 816 in
May 1904; No. 817 in June 1905; No. 818 in August 1907; No. 819 in May
1909. (b) From the canyon of the Eio Grande de Santiago at point due north-
west of Guadalajura ; in Jalisco known as " la l)arranca." Stocks were obtained
as follows: No. 800 in September 1903; No. 801 in June 1905; No. 802 in
May 1907. (c) Two kilometers north from Tlacolula in Oaxaca. Stocks were
obtained as follows: No. 810 in June 1904; No. 811 in August 1909.

I have used the material from Cerro el Borrego in the larger part of my
experiments. The materials are gametically alike at all three locations.

HABITUDINAL variety rugosa NOV. VAR.
(Plate 2, figs. 5, 6, and 7.)

Like diversa in form, color, and markings, but larger. Distinguished from
divcrsa by coarse punctations of head and pronotum and the very large irregular
punctures along the edges of elytral stripes, which are two to five times as large
as in diversa. Elytral stripes vary from a complete shiny black stripe to almost
complete absence thereof. Some examples show only faint brownish tinge in

38 The Mechanism of Evolution in Leptinotaksa

the stripe, with dense black spots on the edge, and more rarely the stripe breaks
longitudinally into two, not unlike L. an gusto vittata Jacoby. All stages occur
in the same restricted localities and frequently have been reared in the progeny
obtained from a single pair of parents. Sexes distinguished as in diversa.

Distribution: Mexico: Desert areas in headwaters of Eio Balsas Valley,
Recorded: Teuhuacan, El Eiego, Pantzingo, Venta Salada, Mihuatlan (Estado
de Puebla), in Guerrero.

Food: S. lanceolatum.

Juvenile stages: Like those of L. diversa.

Habitat: A strictly desert form inhabiting some of the most extreme desert
areas in America. It is always limited in its habitat to edge of water-courses,
either natural or artificial, and its distribution is, therefore, extremely local in
character. It has the capacity for surviving through long periods almost free
from desiccation by hibernating in earthen cells, which condition, while it
apparently allows air to enter, prevents evaporation from the cell, thus retaining
the necessary water to enable the animal to pass the critical season.

Source of Materials: All materials in experiment have come from a point
1 kilometer south of El Eiego near Teuhuacan in Puebla. Stocks were obtained
as follows: No. 900 in May 1907; No. 901 in June 1909.

LEPTINOTARSA PANAMENSIS N. SP.

Leptinotarsa undecimlineata Stkl, 1862, Monog. Chrys. d. Am., p. 163. St§,l
gives Costa Rica, Bogota, and Bolivia as localities for undecimlineata.
In Panama I have not found undecimlineata, but an allied form here
described, and in the lower portion of Costa Rica panamensis is also
the only species present. Dr. Calvert, from Cantago, Costa Rica, sent
me all stages of panamensis. All references to undecimlineata from
these regions are therefore probably to panamensis as here described.
Also quoted by Jacoby, 1882 (Tower, 1906), and many museum speci-
mens in Europe and America from Panama and Costa Rica are so
labeled.

Imago (plate 1, fig. 4; plate 2, fig. 9). — Above: Head and pronotum bright
yellow, with black markings ; mouthparts black with yellowish hairs ; elytra
ivory white, often with greenish tinge marked with black longitudinal unbroken
stripes, 5 or 6 on each elytra, with costal border strongly inflexed, punctate,
black. Below : All parts deep, shiny black ; hind wings well developed, polished,
blackish, deepest on costa and toward base ; antenna?, basal four or five joints
brownish to yellow, variable, terminal joint short, conical. Seventh to eleventh
broad as long and minutely pubescent. Palpi, basal and first joint brownish to
yellow, variable; third joint enlarged distally to breadth equal to its length,
terminal joint short, conical, black. Head: Anterior lateral epicranial spots
fused into heart-shaped spots and prolonged caudad to meet fused portion of
lateral epicranial spots. Posterior epicranial border and lateral border to eyes,
punctate. Pronotum with all elements present, small, variable, with little or no
fusions.

Size: On average smaller than L. undecimlineata Stal. Male 6.5 to 11 mm.
long, 4 to 6 mm. broad ; female 7 to 12 mm. long, 4.5 to 7 mm. broad.

Sexes: Female with sternal sclerite of last abdominal segment rounded,
slightly truncate ; male with same sclerite sharply truncate, with median groove,
variable in length reaching anteriorly. In general the males smaller than the
females, and less broadened posteriorly.

Food : Solanum diversifolium.

K. TODA DEL.

JULIl'S BIKX,

Adult Forms of the Uxdecimli.xeata divisiox. ix their Livixg Colors.

BUT IX THE Posas COMMOX IX TAXOXOMIC LITERATURE.

1. L. signaticollis. X 3.

2. L. signaticollis, biotype nigropuyivtata. X 3.

3. L. angustovittata. X 3.

4. L. (liversa n. sp. X 3.

5. 6, and 7. L. diversa geog. var. nigosa nov. var. X 3.

8. L. undecimlineata. X 3.5.

9. L. panamensis n. sp. X 3.

Materials, Their Taxoxomy and Natural History 39

Juvenile Stages.

Eggs (plate 1, fig. G) : Laid on lower surface of leaves in bunches, from 10
to 250; pale, yellowish white, oval, ventral surface slightly concave, attached
by stalk of gelatinous cement. 1.5 to 2.25 mm. long, 0.60 to 1.20 mm. broad.
No sculpture. Development requires from 7 to 15 days, depending upon
temperature and rate of evaporation.

First larval stage: Head, legs, and pronotum shiny black; body pale trans-
parent yellow, with fully developed system of color markings, each spot provided
with one or more spines. Length at end of stage 2 to 3.5 mm. Closely resembles
the same stage in L. undecimJineata St<il, L. signaticollis Stal, and L. diversa
n. sp.

Second larval stage (plate 1, fig. 86) : Head, legs, and pronotum shiny black ;
body, well-developed row of spiracula spots and with tergal plates of last two
abdominal segments black below and pleurae yellow, due to yellow color of fat-
body below the transparent integument ; tergal portions of thorax and abdomen
grayish, due to transparent integument and absence of fat-body in median line.
Length at end of stage 4 to 6.5 mm.

Third larval stage (plate 1, figs. 7 and 8a) : Markings and color precisely
same as in second stage, yellow color (due to fat-body) increasing throughout
this stage until at the end of the tergal gray area is entirely or nearly replaced
by yellow, often becoming a deep lemon yellow. Length at end of stage 10 to
18 mm.

Length of larval stage: Varies with the conditions of growth, food, tempera-
ture, etc.; averages 25 days, range from 15 to 35.

Pupa : Pupates in ground at foot of plant, 1 to 6 inches from surface. Pupa
pale yellow with yellow white spiracula spots. Pupal stage lasts from 8 to 14
days ; average 10 days.

Length of ontogeny: Average 56 days; range 36 to 64 days.

Geogbaphical Distbibutiox.

Panama, Costa Pica, south to Isthmus of Darien. I have collected and tested
material of this species and find it to be genetically constant from Corozal,
Pedro IMiguel, Tabernilla, Gatun, Colon, Balboa, Cerro Ancon, in the Canal
Zone, Panama; Bocas del Toro, Changuinola Junction, and numerous points
in Sexola Valley in Costa Rica. I also have dead material that was not tested
by breeding from the lower portion of Panama, valley of the Rio Succio, from
Cartago in Costa Rica (coll. by Dr. P. P. Calvert), and other dead material
from Costa Rica which is identical. I have, therefore, on plate 3 given in solid
color the distribution of the tested materials and the probable distribution in
dotted color, as far as I can, of this species over lower Central America.

Habitat and Ecology.

Little comment concerning the ecology of this form is needed, and while
more general in habitat distribution than the two preceding species, it is, never-
theless, limited in its distribution and ecology by the same factors and in the
same manner as is L. undecimlineata Sttil. Its more widespread habitudinal
occurrence within its range is due entirely to the topographical and climatic
character of the area of distribution, rather than to differences of habit or of
ecological complex demanded.

40 The Mechanism of Evolution in Leptinotarsa

Source of Material.

Materials utilized for experimentation were obtained at Pedro Miguel (stock
No. 1705, obtained August 1913), about 1 kilometer north of village ; at Corozal
(stock No. 1700, obtained August 1913), near railroad station; at Gatmi
(stock No. 1710, obtained August 1913; No. 1711, obtained September 1913),
and 3 kilometers south of the railroad station, and at Culebra (stock No. 1715,
obtained August 1913; No. 1716, obtained September 1913). Habitats fairly
uniform for all, but at the Gatun location, in the hills, it rains almost the
entire year, while at Corozal a fairly well-marked dry season occurs between
January and May.

LEPTINOTARSA ANGUSTOVITTATA Jacoby.

Leptinotarsa undecimlineata Stk\ var. Jacoby, 1883, Biol. Centr. Am.,

vol. VI, pt. 1, p. 234. Described as a variety.
Leptinotarsa angustovittata Jacoby, 1891. Biol. Centr. Am., vol. VI, pt. 1,

suppl., p. 254, pi. XLI, fig. 15.

This species, in its distribution, habitat, as well as its structural and color
characters, and in its juvenile stages, is clearly more closely related to L. diversa
than any other species in the group. Its occurrence in semidesert habitats, and
the fact that it is often closely approximated by the geographical variety of
L. diversa (rugosa nov. var.) lead me to suspect that it is either a desert habi-
tudinal variety of L. diversa or a modification thereof that has become perma-
nent in semidesert locations. The data is not available to reach a decision of the
relationship, and I have therefore allowed it to stand provisionally as an inde-
pendent species. It is possible also that this form may have arisen and is now
arising through hybridization, at any rate I have produced in experiment by
hybrid reactions a form that I am not able to differentiate from Jacoby's
L. augustoviUaia. Stock from nature at Guanojuato breeds genetically true,
even under changed environment. Jacoby's description (Biol. Centr. Am.,
vol. VI, pt. 1) is as follows :
" Leptinotarsa undecimlineata, Huj. op. (partini).

" Black : the head with two flavous spots ; the thorax and elytra flavous, the
former spotted with black, the latter with eight narrow black, strongly punc-
tured, longitudinal stripes. Length, 5-6 lines. Habitat : Mexico, Guanajuato
(Salle), Morelia, and Tacambaro in Michoacan (Hoge), Xucumanatlan in
Guerrero (H. H. Smith).

" This insect was previously treated by me as a variety of L. undecimlineata,
but as we now have received twenty additional specimens, all alike, I am com-
pelled to treat it as a distinct species. It agrees in everything with L. undecim-
lineata, except that the elytra have eight very narrow black stripes, each of
which is abbreviated at a short distance before the apex ; these stripes are deeply
punctured in single, sometimes in double and very closely contiguous rows, this
character separating the species from L. undecimlineata, in which each elytron
has four much broader stripes, and the stripes singly or doubly punctured along
their margins."

Description of Living Animals.

Imago (plate 3, fig. 3).— Head: Pronotum, gTeenish ivory white, often
yellowish, marked with black spots ; eyes and mouthparts shiny black ; elytra
grayish white or grayish-gray white, with 4 pairs of closely placed parallel
black lines, strongly punctured. These are equivalent to the elytral stripes of

Tropical and Sub- tropical America, to illustrate Known Range of Species
AfJD Varieties of the undecimUneata and juncta Divisions.

Materials, Their Taxonomy and Natural History 41

other species, except that in this species the central portion of each stripe is
wanting and only the punctate margins thereof are pigmented. Even in this
there is considerable variability; examples are not uncommon in which the
pigment is almost entirely lacking, giving an appearance much like L. signa-
ticoUis Stal var. nigropunctata Sturm. Color-patterns of head and pronotum
precisely like L. diversa in pattern, fusions, and variations. Antennae black;
last five joints as broad as long, terminal joint conical. Below: Shiny black;
under wings, pale blackish brown, densest at base and along the costal edge. The
full extent of coloration is not attained until 3 to 5 days after emergence.

Size: Male 7 to 14 mm. long, 5 to 7.5 mm. broad; female 7 to 18 mm. long,
5 to 8.25 mm. broad.

Sexes: Female with central sclerite of last abdominal segment rounded
smooth ; male with the same sclerite truncate and grooved to about the middle
of the plate.

Food: Solanum hertivigi Bereth., ;S^. diversifoUum Schl., and ^S^. lanceolatum
Cuv.

Juvenile Stages.

In all live material that I have seen these stages are indistinguishable from
the corresponding stages of L. diversa. Length of larval periods and habits
identical. Pupates deeper than L. diversa — i. e., 3 to 8 inches.

Geogbaphical Distbibution.
(Plate 3.)

Mexico, Guanajuato (Salle), State of Guanajuato; Morelio and Tacambaro,
State of Michoacan (Hoge) ; Xucumanatlan, State of Guerrero (Smith),
Jacoby, 1891. I have found it in the State of Guanajuato at Guanajuato,
Irapuato, Dolores Hidalgo, and at Morelia, Zitacuaro, Uruapan, Tinguindin,
State of Michoacan,

Habitat.

Ecologically this species presents little of interest. Its habitat for breeding
and hibernation is almost identical in kind and conditions with that of
L. diversa, excepting that it is uniformly more arid, less water present, with
a longer dry season, and higher soil temperature at the end of the dry seasons.

SOTJECE OF MaTEBIAL.

This species has not been used to any extent in experiments thus far, but
material has been reared and tested in the laboratory from Dolores Hidalgo in
Guanajuato (stock No. 1416 obtained in June 1908), Morelia (stock No. 1400
obtained in August 1905; No. 1401 in July 1907), and Uruapan in Michoacan
(stock No. 1416 obtained in June 1907; No. 1417 in June 1908), Mexico.

LEPTINOTARSA SIGNATICOLLIS SxAi.

Myocoryna signaticollis Stai. Of. "Vet. Ak. Forh., 1859, p. 317.5.

Chrysomela signaticollis St§.l. Monog. Chrys. Am., 1862, p. 163.

Leptinotarsa signaticollis St§,l. Gemminger et Harold, 1874, Catalog.
Coleop., t. XI, pt. 1, p. 3441; Jacoby, Biol. Centr. Am., Jan., 1883, vol. VI,
pt. 1, p. 232, pi. XIII, fig. 20, suppl.. May 1891, p. 253; Tower, Inv. Evol.
Chrys. Beetles Gen. Leptinotarsa, 1906, p. 6. Var. nigropunctata Sturm,
in literature; Gemminger et Harold, 1874, Catalog. Coleop. t. XI, pt. 1,
p. 3441, given as syn.; Jacoby, Biol. Centr. Am., 1883, vol. VI, pt. 1.

4

42 The Mechanism of Evolution in Leptinotaesa

There is no difficulty in the recognition of this species, even in badly pre-
served museum material, but the variety nigropunctata Sturm, is a biotype,
breeding true in its distinctive character, and behaves as an alternative domi-
nant character in heredity. Stal's description of L. signaticollis (Monog.
Chrys. Am., p. 163), is as follows:

" Nigra, supra sordide fiavescens ; maculis prothoracis capiteque nigris, hujus
vittis duabus flavis; elytris subvage nigro-punctatis, vitta angusta suturali
margineque inflexo aeneo-nigris. Long. 12, Lat. 7 millim.

"Patria: Mexico. (Mus. Holm., Coll. Dohrn). Ovalis, sat convexa, nigra,
nitida. Caput parce punctulatum, vittis duabus obliquis maculisque duabus
minutis ante medium sordide flavis. Antennae articulis quinque ultimus leviter
transversis, clavam minus distinctam formantibus. Prothorax sordide fiaves-
cens, elytris nonnihil, angustior, lateribus parallelis, antice angustatus, disco
parce, subtiliter, utrimque paullo densius et distincte punctatus, litura media
ut littera V formata maculisque lateralibus pluribus, plus minus confluentibus,
nigris. Scutellum Iseve, nigrum. Elytra lateribus parallelis, dilute sordide
fiavescentia, vage distincte nigro-punctulata, punctis hie illic in series dispositis,
vitta angusta suturali margineque inflexo aeneo-nigris."

This description applies very well to the dead dried specimens of collections.
The following description is from living material in all stages :

Imago (plate 2, fig. 1). — Above: Hypodermal color of head and pronotum
pale grayish white, often pale yellowish ivory-white, becoming darkened with
age; elytra, pale gray or pale gray-green. Head, mouthparts black; antennae
black, terminal joint truncate, eighth to eleventh thickened, broader than long.
Epicranium with anterior lateral epicranial spots fused medianward to form
heart-shaped spot, and prolonged caudad to fuse with posterior lateral epicranial
spots, which are united mediad. Posterior border and lateral portions of epi-
cranium behind eyes irregularly punctate with fairly well marked pits. Eyes

am
black. Pronotum with variable pattern of black spots. h' + a'+ -f +a+b

pm

are always fused to form widely open V-shaped median spot, c' and c always
free, and d, e, f groups somewhat variable. Margins, especially anterior lateral
portions, punctate, with well-defined pits ; central portion with few minute pits
irregularly distributed. Elytra with black anal edge giving median black stripe
when closed. Each elytron with ten irregularly placed double rows of pimcta-
tions, marking position of chitinous columns between elytral lamellae, and with
slightly variable amount of black pigment in bottom of each pit. These rows
are arranged in a definite pattern shown in plate 2, figs. 1 and 2. Under wings,
pale yellowish brown, transparent, darker towards the base. The adult colora-
tion is not attained until about the third day after emergence.

Size: Male 7 to 13 mm. long, 6 to 7.5 mm. broad; female 7.5 to 14.5 mm.
long, 7 to 9 mm. broad.

Sexes: Female with sternal sclerite of last abdominal segment roimded ; male,
same sclerite sharply punctate, with median groove reaching nearly to anterior
border.

Food: At Cuernavaca, Solarium hertwigi Bereth., S. diversifolium Schlecht.
At Atlixco and Matamoros, S. diversifolium and S. lanceolatum Cuv. Will also
feed upon S. chrysotrichum Schl., but will not eat S. torvum from the Antilles.
Identification of food-plants by Dr. Greenman.

Materials, Their Taxonomy and Natural History 43

Juvenile Stages.

Eggs: Yellow, rarely lemon-yellow or yellowish white, laid on lower surface
of leaves, in bunches of 5 to 200. Length, 1.75 to 2.5 mm. ; breadth, 0.75 to
1,35 mm. Concave on ventral side and attached to leaf by a stalk of greater or
less length composed of a coagulated gelatinous material which is extruded
ahead of eggs at laying. Development requires from 5 to 12 days, depending
upon external conditions.

First larval stage: Head, pronotum, and legs black; body yellow, with color-
pattern shown in plate 7, figure 10a. Tergal spots all with one or more spines.
Length at hatching, 2.25 mm.

Second larval stage: Head, pronotum, and legs black; body yellow, with
single row of black spiracula spots (plate 7, fig. 10 6). Length, 4 to 7.5 mm.

Third larval stage: Head, pronotum, and legs black; body yellow, with two
rows of spots on pleurum, spiracula, and baso-pleural ; inner, middle, and outer
tergal spots present; more or less fused lateralward; frequently fused with
spiracula spots ; inner tergal always fused antero-posteriorly on each segment
(plate 7, fig, 10c). Length, 10 to 19 mm. Length of larval period varies with
meterological conditions, food, and the local race ; may be passed over in 15
days or prolonged to 40 or 50. In nature and in cultures it averages about 25
days.

Pupa: Pupates in ground at foot of plant, from 0.5 to 5 inches deep, depend-
ing upon soil hardness, moisture, and air-supply. Pupal stage lasts from 10 to
30 days ; average 15 days. Of this period 25 per cent is passed in prepupal stage,
60 per cent as pupa, and 15 per cent as imago. Length of ontogeny from egg
laying to adult averages 40 days ; range 28 to 70 days.

Geogbaphical Distribution,
(Plate 3.)

Mexico, Stal, 1859, 1862 ; Gemminger et Harold, 1874 ; Jacoby, 1883 ; Tower,
1906. Eecorded from Izuca, Puebla, State of Puebla (Salle; Jacoby, 1883) ;
Cuernavaca, State of Morelos; Aurula, Xucamanatlan, State of Guerrero (H. H.
Smith; Jacoby, 1883); Alarcon, Cuanutla, Jojutla, State of Morelos; Mata-
moros de Izucas, Atlixco, Tatetla, State of Puebla (Tower, 1896).

The habitat of this species is confined to the upper portion of the eastern
and northern branches of the Eio Balsas, and in the main lives close to the foot
of the great Mexican escarpment (plate 23). I have not found it to extend
northwestward beyond the Rio Balsas Valley, eastward beyond the Rio Coetzala
Valley; neither does it reach an altitude of over 6000 feet, rarely above 5500
or below 4000, It is narrowly limited in range, and is also still more restricted
in its ecological relations.

Source of Material,

Of this species my chief source has been from localities near Cuernavaca,
Morelos, Mexico: (a) Rancho Basoco colony: Stock No. 418, obtained July
1903; No. 419, May 1904. (6) Quauhtemotzin colony: Stock No. 417,
obtained July 1903, (c) San Antonio colony: Stock No. 420, obtained May
1904; No. 421, July 1905 ; No. 422, June 1906. Other material has been used
at different times from near Atlixco and Matamoros in Puebla. The chief stock
location has been that at Rancho Basoco.

44 The Mechanism of EvoLUTioisr in Leptinotaesa

BlOTYPE NIGROPUNCTATA StURM.

Similar to signaticolUs (plate 2, fig. 2), but with elytra having numerous
large black punctations, which may or may not show arrangement in pattern of
ten irregular double rows ; punctures often confluent, giving the appearance of
longitudinal vittae. Appears also in experiment, most often as a dominant, and
when isolated breeds true; female sex-linked, but not uniformly so. (See also
Jacoby, 1883, p. 232, who had Sturm's specimens for study.) Recorded from
Cuernavaca, Cuautla, Estado de Morelos, Atlixco, Estado de Puebla.

MULTIT/ENIATA DIVISION.
LEPTINOTARSA MULTIT^NIATA Stax.

Myocoryna multitcrniata Stai, 1859. Ofv. af K. Vet. Ak. Forh., p. 317.4.

Chrysomela multitwniata Stkl, 1862. Monog. Chrys. Am., p. 164.

Leptinotarsa multitcrniata St§.l. Gemminger et Harold, 1874, Catalog.
Coleop., t. IX, pt. 1, p. 3441; Jacoby, 1883, Biol. Centr. Am., vol. VI, pt. 1,
p. 253; Tower, 1906, Inves. Evol. Chrys. Beetles, Gen. Lept., Carnegie
Inst. Wash. Pub. No. 48, pp. 7, 18.

This species has given me more trouble in the effort to untangle its taxonomic

relations than all of the others combined. It is a plastic, variable, polymorphic

form, showing curious phenomena of heterogeneity which I shall discuss later.

From present data I have arranged the species and its minor forms as follows :

L. multita^niata St§,l.

Habitudinal variation: multitcrniata Stkl.
Recurrent mutation: melanothorax St§-1.
Saltation: tacubayaensis nov. var.
Biotypes:

multilineata StM.
obscura nov. var.
Habitudinal variation: intermedia nov. var.

Saltation, melanothorax StS,l.
Habitudinal variation: variabilis nov. var.
Recurrent mutation: melanothorax StS,l.
Biotypes:

Red hypodermal color.
Orange hypodermal color.
Yellow hypodermal color.
White hypodermal color.

Habitudinal variett L. MUi.TiT^NiATA-MxrLTiTyENiATA Stal.

Stal's description of the typical muUitceniata form, taken from his monograph
(1862), is as follows :

" Ovalis, supra flavescens, subtus cum antennis, pedibus, maculis prothoracis,
limbo scutelli, sutura vittisque quinque, exteriore intramarginali, elytrorum,
nigra ; maculis pectoris limboque apicali segmentorum ventris flavis ; elytris
minus regulariter seriatim pxmctatis. Long. 10, Lat. 6f millim.

" Patria: Mexico (Mus. Berol., Coll. Dohrn).

" C. multilineata simillima, prothorace latiore, elytris regularius seriatim
punctatis differt. Ovalis, sat convexa, nigra, nitida. Caput sat dense, distincte
punctatum, vittis duabus obliquis, basi conjunctis, flavis. Antennse articulis
quinque apicalibus transversis, clavam sat distinctam formantibus. Prothorax
elytris nonnihil angustior, sat convexus, antrorsum sensim, angustatus, disco
parce, subtiliter, utrimque densius, subfortiter punctatus, flavescens, maculis
compluribus lateralibus lituraque media ut littera V formata, plus minus con-
fluentibus, nigris ornatus. Scutellum flavescens, plus minus late nigro-limba-

X«.7

K. TODA DEL.

JTrlilUS BIKIf,

1. L. multitwniata. Typical form. X 2.

2. L. multitamiata, biotype multilineata. X 2.

3. L. muUitaniata. saltation tacuhayirnsis nov. var. X 2.

4. 5, and 6. L. multita'niata recurrent mutant melanothorax. shown in three

biotypical forms, yellow, orange, and red, in which it may occur. X 2.

7. L. rubicunda nov. var. X 2.

8. L. multitamiata. biotype obsoleta nov. var. X 2.

9. L. multita-niata geog. var. interyntdia. X 2.

10, 11. L. multitipniata geog. var. variabilis nov. var. X 2.

Materials, Theie Taxonomy and Natural History 45

tiim, la2ve. Elytra flavescentia, lateribus parallelis, minus regulariter seriatim
vel subacervato-seriatim distincte punctata, linea suturali basin versus latiore
nee non vittis interstitia alterna occupantibus, nigris, vitta interstitii decimi
medio et ante medium a margine paullo remota, interstitio none reliquis fere
duplo latiore, medio vage, remote et distincte punctato; margine infiexo pos-
terius nigro. Pectus maculis nonnullis plus minus distinctis flavo-testaeeis.
Segmenta ventris apice plus minus late flavo-limbata."

Habitudinal vabiety multit^niata-multit^niata Stal.
(Plate 4, fig. 1.)

I have reserved this name for that portion of the entire species which is
limited to the southern end of the Mexican Plateau and which agrees with Stal's
descriptions, and is also the form described and recognized by Jacoby, 1883-1891,
in the Biologisches Centrallblatt under this name.

Imago: Oval, convex, robust in form. Above: Head and pronotum light
tan, often with tinge of red marked with black color-pattern. Epicranium and
pronotum punctate, especially on lateral and posterior portions, with irregularly
arranged variable punctures. Elytra pale yellow white or yellowish, rarely
reddish yellow, marked with five longitudinal black stripes, which are edged by
an irregular double row of impressed punctations, variable in size. Costal edge
inflexed, dark brown or black, slightly grooved and finely punctate, sparingly
or smooth. Second and third elytral stripes often united posteriorly, and may
also be united to fourth stripe. Eyes black, epicranium with anterior lateral
epicranial spots fused and reaching to anterior border and to fused posterior

am
lateral epicranial spots posteriorly. On pronotum a' + 1)' -\- -\- +a^-h usually

pm
fused to form broadly open V-shaped spot, c usually free, and d' e' f and d e f
group variable, usually united, and often forming extensive fusions with other
parts of pattern. Below : Uniform light tan-color or brownish yellow marked
with black, variable from all color-centers distinct to completely black. Legs :
Femora dark brown or black, tibia, ends always black, central portions brown
or black, sometimes reddish brown, variable. Entire ventral surface and legs
punctate, irregular, variable, slightly pubescent.

Size: Variable, depending upon food and climatic conditions. Females
always larger than males. Female, 7 to 13 mm. long, 6 to 9 mm. broad ; male,
6 to 12 mm. long, 5 to 8 mm. broad.

Sexes: Female with ventral sclerite of last abdominal segment rounded flat;
male with same sclerite truncate, slightly grooved; female also more robust and
broadened posteriorly than male.

Food: Feeds exclusively in nature upon Solarium rostratum Dun., or close
Mexican allies thereof, on S. elceagnifolium, and more rarely upon S. tubero-
sum, and very rarely upon other SolanaceiB. S. rostratum is its food. In
captivity, when deprived of other food, will eat S. diversifolium, lanceolatum,
and Jiertwigi.

Juvenile Stages.

(Tower, 1906, plate 17, figs. 10, 11, 12.)

Eggs: Bright yellow, sometimes pale yellow, laid upon the lower surface of
the leaves, in bunches of from 5 or 6 to 200, and cemented to the leaf by a
coagulated gelatinous cement; oval, flattened and concave ventrally, smooth,
polished. Length, 2.5 to 3.25 mm.; breadth, 1.3 to 1.9 mm.; incubation, 5 to
10 days, depending upon temperature and moisture conditions.

46 The Mechanism of Evolution in Leptinotarsa

First larval stage : Head, pronotum, and legs, black ; body yellow, with full
system of body color-markings, each with one or more spines. Length at end
of stage, 3.5 to 4.5 mm.

Second larval stage: Head and legs black, pronotum with posterior edge
black, anterior border and sides to middle, yellow or brown ; body bright yellow,
ornamented with spiracula, wing and basi-pleural spots, and a variable number
of the anterior members of the inner tergal spots. Length at end of the stage,
6.5 to 8.25 mm.

Third larval stage : In color and pattern identical with the second. Length
at end of the stage, 10 to 18 mm. Length of larval period varies from 13 to 25
days, average 17 days; but much depends upon surrounding conditions of
growth.

Pupa: Pupates in ground, from 2 to 6 inches in depth ; pupa yellow. Period
of pupation, 6 to 15 days, but average about 9 days.

Length of ontogeny: From 19 to 40 days; usual average is between 25 and
30 days. The larval stages are very constant in this geographical variety and
all of its minor divisions.

Habitat and Ecology.

This form passes the winter — i. c., the dry season, from the middle of
September to May — in hibernation in the ground as an imago. It emerges
from hibernation in May, usually about the 15tli, in the region of Mexico
City, and begins breeding about June 1 to 15, depending upon the advent of
the rainy season. It produces a brood that becomes adult about July 20,
which in turn breed at once and give a second brood, which emerges about
the end of August. These latter feed for a few days, and with the cessation
of the rains and cooler nights of September, enter into hibernation, and
by September 15 or October 1 they have all gone into hibernation, with the
exception of a few that never do enter the ground. These latter are curious
hybrid forms produced in the following manner : Some members of the over-
wintering hibernating population may remain alive and breed with their
descendants, giving a cross of summer fornix winter form, thus producing
hybrids which are variable in their breeding activities and some of which are
extracted summer forms in Fo, and do not hibernate, and are, therefore, elimi-
nated by starvation and desiccation during the dry season.

Geographical Distribution.

The range of this habitudinal variety and its minor divisions is, as indicated
on plate 5, limited to the southern end of the Mexican Plateau, between the
altitudes of 5000 and 8000 feet. It is especially characteristic of and common
in the valley of Mexico (7400 to 8000 feet), the plains of Apam, and eastward
to the edge of the plateau (6000 to 8000 feet), the plateau portion of the State
of Puebla, especially the headwaters of Eio Atoyac, and less common on the
high plateau of the portion of the valley of the Eio Lerma and westward to edge
of plateau; northward it passes over into northern habitudinal variety inter-
media, in the region of the headwaters of the Rio Panuco.

Stal gives Mexico as its habitat; Jacoby, 1883, 1891, gives Toluca, La Parada
(Salle), Cerro de Plumas (Hoge) ; Tower, 1916, Z. c, records it from Puebla,
Atlixo, Matamoros de Izecas, San Marcos, in the State of Puebla ; Apizaco and

Materials, Theik Taxonomy and Natural History 47

Tlascalo in the State of Tlascala ; Mexico City, Guadalupe, Tacubaya, Tacuba,
Santa Fe, San Angel, Tlalpam, Tlalnepantla, Texcoco, and Mixcoco in the
Valley of Mexico.

SouKCE OF Material.

The stocks for different experimental purposes have been derived as follows,
and in the body of the paper will be referred to according to the name of the
location from which they were derived.

(a) Guadalupe stock: From a location on the shore of Lake Texcoco, on an
old shore-line east of the Shrine of Guadalupe. Location described, Tower
(1906), plates 5 and 6, pages 18 to 20. Improvement and cultivation destroyed
tliis location in 1907-8. Stocks were obtained as follows: No. 500 in July
1903; No. 501 in May 1904; No. 503 in August 1904; No. 503 in July 1905;
No. 504 in August 1908.

(b) Chapultepcc stock: From the plain to north of the Cerro Chapultepec,
in waste places and pastures; is fast being built up and the location is now
nearly obliterated. Stocks were obtained as follows : No. 540 in August 1903 ;
No. 541 in May 1904; No. 542 in July 1905; No. 543 in August 1906; No. 544
in June 1909 ; No. 545 in August 1908.

(c) Tlalnepantla stock: From west shore of Lake Xico, near Tlalnepantla.
Stocks were obtained as follows: No. 510 in August 1903; No. 511 in May
1904; No. 512 in June 1907.

(d) Puehla stock: From open plain to northeast of the city, about one-half
mile north of Cerro de Guadalupe. An open rolling plain, more or less culti-
vated; well drained. Stocks were obtained as follows: No. 515 in August
1903; No. 516 in May 1904; No. 517 in June 1905; No. 518 in August 1906;
No. 519 in June 1909 ; No. 520 in June 1910.

(e) Apam stock: From the open plain of Apam, on the edge of a wet-season
lake about a mile northeast of the town. Stocks were obtained as follows : No.
525 in August 1904; No. 526 in August 1905; No. 527 in June 1909.

(/) Chalcicomula stock: On the road from Chalcicomula to San Andres
Chalcicomula, a mile from the plaza ; location is a cold, dry upland savannah.
Stocks were obtained as follows: No. 530 in July 1904; No. 531 in June
1905 ; No. 532 in August 1906 ; No. 533 in June 1908 ; No. 534 in June 1910.

These stocks and several others have been tested, analyzed, and made the
basis of experiments in many lines, and the data of these analyses have entered
largely into the understanding of the real nature of this species as it is found
in nature, and without the analysis of the laboratory a correct understanding
would be impossible.

Recurrent Mutation L. multit^niata-melanothorax Stal.

This form has hitherto been recorded as a species and is so included in all
lists and collections. In 1906 I was noncommittal as to its status, but further
study shows in the clearest possible manner that it is not a species, but a
recurrent mutant, as here recorded.

Myocoryna mulanothorax StS,!, 1859. Ofv. af. k. "Vet. Forh., p. 317.7.

Chrysomela melanotJiorax St§,l, 1862. Monog. Chrys. Am., p. 166.

Leptinotarsa melanotJiorax StS,l in Gemminger et Harold, 1874. Catalog.
Coleop., t. IX, pt. 1, p. 3441; Jacoby, 1883, Biol. Centr. Am., vol. VI, pt. 1,
p. 234, suppl., p. 254; Tower, 1906, Inves. Evol. Chrys. Beetles, Genus
Lept., Carnegie Inst. Wash., Pub. No. 48, p. 720.

48 The Mechanism of Evolution in Leptinotaksa

Stal's description is as follows :

" Ovalis, nigra ; prothorace lasviusculo ; elytris testaceo-flavis, minus regu-
lariter geminato-seriatini distincte punctatis, margine inilexo, vitta angusta
suturali interstitiisque alternis nigris. Long. 9, Lat. 6^ millim.

"Patria: Mexico (Mus. Holm., Coll. Dohrn).

" Ovalis, sat convexa, nigra, nitida. Caput parce, subtiliter punctulatmn.
Antennae graciles, articulis quinque ultimis siibtransversis, clavam sat dis-
tinctam formantibus. Prothorax elytris nonnihil angustior, antrorsum sensim
leviter angustatus, laevis, utrimque punctulis raris adspersus, angulis anticis
subacutis. Scutellum laeve. Elytra lateribus parallelis, minus regulariter
geminato-seriatim distincte punctulata, testaceo-flava, margine inflexo, vitta
angustissima suturali nee non vittis quinque, interstitia alterna occupantibus,
nigris, vittis basin haud attingentibus, tribus interioribus prope apicem abbre-
viatis et conjunctis.

" ^ Segmento ventrali ultimo apice truncato."

Easily distinguished by black head and pronotum and black ventral surface.
Jacoby, 1883, noted that third and fourth elytral black stripes are sometimes
united. This is not common and is due to crossings with another form,
tacuhayaensis nov. var., and its presence in the strain. The elytral ground-color
is variable, dark ocher-yellow, yellow, or red. These differences can be separated
in breeding, giving the melanothorax type in four minor biotypes : L. multi-
toBTiiata-melanothorax &i, red elytral ground (plate 4, fig. 6) ; L. multitcEniata-
melanothorax h^, orange elytral ground (plate 4, fig. h) ; L. muUitceniata-
melanothorax b^, yellow elytral ground (plate 4, fig. 4) ; L. muUitceniata-
melanothorax h^, white elytral ground. All of these breed true genetically, and
are alternative in their behavior in crossing. (Plate 4, figs. 4, 5, and 6.)

Food, juvenile stages, distribution, habits, and ecology as in L. muUit(£niata
Stal.

Saltation L. multit/Eniata-tacubayaensis nov. vab.
(Plate 4, fig. 3.)

Like L. melanothorax Stal in size, form, head, and pronotum; elytra orange
or red, with longitudinal stripes, broad brownish black, the first confluent with
anal edge, forming large wedge-shaped median stripe, the second broad, united
to fused third and fourth stripes posteriorly. Costal edge black, inflexed portion
yellow brown and sharply grooved. Impressed punctations, large, 2 to 3 rows,
interspaces smooth, arched, conspicuous. Below, all parts shiny black, except
posterior and lateral portions of sternal sclerite of abdominal segments.
Antennae second and third joints brown or yellowish, remainder black.

This rare sport breeds genetically true and is in cultures different from
L. melanothorax. In nature examples of this form are frequently contaminated
by attributes from other portions of the species, but no difficulty is encountered
in purifying it and establishing it as a stable, uniform race.

Food, juvenile stages, habits, and ecology: The same as in general species.

Geographical distribution: Valley of Mexico; I have found it at Tacubaya,
Tacuba, Chapultepec, and Texcoco.

Further complications are introduced by the presence of a biotype form that
occurs in the species and with regularity in some locations, to which I have given
Stal's name, L. multilineata, in that Stal's description fits perfectly the biotype
in question. In museum material it is frequently not unlike some specimens
of L. decemlineata Say and L. oblongata, but it has no relations to these two

Materials, Their Taxonomy and Natural History 49

species, and it further occurs in the region from which Stal's material probably
came. I have considered it best to retain his name for this division of the
species,

BlOTYPE L. MrXTIT^ENIATA-MUI/TrLINEATA STAL.

(Plate 4, fig. 2.)

Myocoryna multitwniata Stai, 1859. Ofv. af. K. Vet. Ak. Forh., p. 316.3.

Chrysomela multitwniata StS.1, 1862. Monog. Chrys. Am., p. 165; considered
as synonym of L. decemlineata Say, and apparently confused with it;
Harold, 1874, Ber. Ent. Zelt., p. 444, synonym with decemlineata : Jacoby,
1883, Biol. Centr. Am., also synonym with decemlineata, following St&l,
gives Mexico and Costa Rica as habitat of decemlineata incorrectly.

Stal's description, which follows, taken from his monograph (Chrys. d.
I'Am.), fits perfectly the portion of muUitceniata designated here as a biotype:

" Ovalis, pallide flava, subtus cum capite prothoraceque nigro-maculata, hoc
sat convexo, minus dense, sat distincte, utrimque fortius punctato; elytris
subacervato-seriatim punctatis, sutura, margine inflexo posterius interstitiisque
alternis subaeneo-nigris ; antennis fere totis, apice femorum tarsisque nigris.
Long. 8 — 10, Lat. 5| — 6 millim.

" Patria: Mexico (Mus. Holm., etc.) ; Nebraska et Texas (see Eogers, 1. c.)
Praecedentibus affinis. Ovalis, sat convexa, pallide sordide flava vel straminea,
nitida. Caput subremote, distincte punctatum, lateribus baseos maculaque
media triangulari, interdimi etiam ore, nigris. Antennae basi flavo-testaceae,
articulis quinque apicalibus clavam indistinctam formantibus, subtransversis.
Prothorax elytris tertia parte angustior, lateribus parallelis, anterius angusta-
tus, disco minus dense, sat distincte, utrimque dense et fortius punctatus, sat
convexus, litura media ut littera V formata nee non maculis nonnullis laterali-
bus, plus minus confluentibus, nigris, ornatus. Scutellum laeve, nigro-limba-
tum. Elytra lateribus parallelis, subacervato-seriatim distincte pimctata, serie
punctorum prima paullo ultra quartam partem elytrorum extensa, margine
inflexo posterius, sutura, vitta suturali anteriore angusta nee non utriusque
vittis quinque interstitia alterna occupantibus, omnibus prope basin, intermediis
tribus etiam prope apiceni abbreviatis, subsuturali et marginali apice conjmictis,
subaeneo-nigris. Pectus nigro-maculatum. Ventris segmenta macula utrim-
que laterali fasciaque abbreviata basali, interdimi interrupta, nigris ornata.
Pedes flavo-testacei, apice femorum tarsisque, interdum etiam macula femorum,
tibiarumque basi et apice aeneo-nigris.

" (^ Segmento ventrali ultimo apice truncato."

Food, geographical distribution, habits, ecology, and juvenile stages exactly
like the remainder of the species.

In life the elytral color is pale yellow-white, or rarely yellow in young, imma-
ture specimens. Its more narrow, anteriorly constricted pronotum, elongate
form, and general color in live specimens give no suggestion of decemlineata.
The reduced amount of fusion in the pronotal pattern, however, gives a con-
dition closely similar to the northern species; it possesses, however, entirely
different juvenile stages which are like L. muUitceniata Stal, In collections,
especially where data of localities is poor, it might easily be confused with the
northern type. This " biotype " is identical in all respects, as here distinguished,
with a specimen in the " Baily Coll." in the British Museum labeled " multi-
lineata Stal," " Named by Stal," " Type Stal 176."

50 The Mechanism of Evolution in Leptinotaesa

BlOTYPE L. MULTIT^NIATA-OBSCURA NOV. VAR.

This form occurs in nature, especially in the populations of muUitwniata, in
the valley of Mexico, and in the region of Puebla, often with considerable fre-
quency, and is an extracted homozygous recessive of F2 or F^ that comes out in
the crossing of the typical muUitceniata and melanothorax. It has been the
experience that the majority of the specimens found in nature were homozygous
when isolated in cultures, and continued to maintain their type, but not infre-
quently individuals are found that are heterozygous, often of great complexity.
The typical aspect of the pure-breeding recessive from nature is shown in
figure 8, plate 14. I have seen it in nature only in that form, where it has the
form and aspect of the modal L. muUitceniata Stal, but with the head and pro-
notum entirely black, excepting for the edges of the pronotum, which are
brownish or yellow, but with the pronotal markings entirely obscure. The
ventral side and the legs are entirely black.

This form does not, as far as I am aware, maintain itself in continued genetic
lines anywhere in nature, but is constantly breeding back into the general
population. Its habits, juvenile stages, and ecological relations are in every
respect like those of L. multitcmiata Stal.

This form has been produced many times in crossing experiments between
muUitceniata and melanothorax, and as far as experience goes is always a
recessive.

Habitudinax, Variety Intermedia nov. var.

Leptinotarsa multitceniata-intermedia nov. var. nov. sp., Tower, 1906, I. c.

This form, a variety of L. muUitceniata Stal, is found in the main in the north
plateau of Mexico, and is distinguished from the southern variety by its smaller,
less robust form, lower variability, conspicuous absence of minor divisions,
uniform pale yellow-white color, and its freedom from extensive fusions of the
elements of the color-pattern. It differs also in the larval stages, which are
yellow, yellow-red, or pale red in color, with the same system of ornamentation,
but more pronounced. As known from museum material, it might well be
confused with the varieties of muUitceniata or decemUneata, but the fact that it
is a stable, genetically perpetuated type, distributed over a wide geographical
range, and that it is alternative in its attributes to corresponding attributes of
muUitceniata in crosses, and its distribution leaves no choice but to class it as a
habitudinal variety of the same species, or perhaps to raise it to specific rank.
(Plate 4, fig. 9.)

Description of Living Animals.

Imago. — Above : Oval, convex, head and pronotum light yellow-brown, orna-
mented with black spots. Elytra pale yellow-white, with five longitudinal black
stripes, second, third, and fourth often fused ; edged with irregular double row
of punctations, interspaces arched, polished; costal edge inflexed, grooved,
smooth brownish or yellow-white. Head : Eyes black, mouthparts dark brown,
anterior basal parts brownish, last six joints black, faintly pubescent, eleventh
joint broader than long, twelfth conical, truncate, last six joints forming an
elongated cone-shaped club. Epicranium faintly irregularly punctate, spots all
present, variable. Pronotum smooth, polished, few faint punctures in posterior
outer angle. Spots all present and not extensively fused, central V-spot

a' — -f — a is a common condition, often also include h' — h; spots d', e', f, and

pm

Materials, Their Taxonomy and Natural History 51

d, e, f, variable, but never fused to central system and never reaching to pro-
notum border ; scutellum brownish black, polished. Below : Light yellow-brown,
thoracic and abdominal segments with color-centers all distinct, rarely fused ;
legs: femora, ends black, often extending to near middle, which is brownish
or reddish ; tibia and femora yellow brown or yellow, ends black ; tarsus brownish,
legs smooth, polished, few minute punctures, and rarely minute spines.

Size: Male smaller than female, and both smaller than L. multitceniata Stal
■or L. decemlineata. Male, 7 to 11.5 mm. long, 5 to 6.5 mm. broad ; female, 7 to
13 mm. long, 5 to 7.25 mm. broad.

Sexes: Female with sternal sclerite of last abdominal segment rounded
smooth, and in male truncate and faintly grooved.

Food: Solanum rostratum or its allies, and Solarium elceagnifolium in the
northern part of its range.

Juvenile Stages.

Eggs: As in L. multitceniata, but smaller; length 1.5 to 2.25 mm., 1 to 1.4
mm. broad. Incubation 6 to 10 days.

First larval stage: Head, pronotum, and legs black; body dull yellow color
with sternal and pleural spots present, minute; tergal spots, especially outer
and posterior members, reduced or wanting, variable. Length, 2.25 to 3 mm. at
end of stage.

Second larval stage: Head, legs, and pronotum yellow-brown, latter with the
posterior border black. Body dull yellow-red or ocher, spiracula spots strong ;
baso-pleural small, posterior members usually w^anting or reduced to mere
trace ; tergal spots present only on thoracic and two front abdominal segments.
Length, 8 to 12 mm. at end of stage.

Third larval stage: Like the preceding, but baso-pleural and tergal spots
frequently almost gone. Body-color variable, dull yellow to dull yellow-red,
never clear yellow of L. mutitceniata or clear wine-red of L. decemlineata.
Length, 8 to 12 mm. at end of stage.

Pupa: Pupates in ground at depth 2 to 6 inches. Pupa like multitceniata.
Pupal stage lasts 5 to 12 days, on average 9 or 10 days.

Length of ontogeny: Twenty to thirty-five days.

Geographical Distribution.

In 1906 I gave localities of occurrence. Further investigations have given a
range indicated on plate 5, where it is seen to extend northward over the Mexi-
can Plateau, up the Eio Grande Valley into New Mexico, and into Arizona
towards the Colorado Eiver. I have observed it in all its phases at the follow-
ing locations : In the Rio Grande y Lerma Valley at Ocatlan, La Barca, Guada-
lajara, Zopopam, Encarnacion, State of Jalisco ; in Eio Panuco Valley at San
Bartolo, Eio Verde, Cardenas, San Luis Potosi, Espirito Santo, Matehuala,
Vinegas, Catorce, State of San Luis Potosi ; Monterey, Montemorelos, State of
Nuevo Leon; Matamoros, Forlon, State of Tamaulipas; Monclova, San Pedro,
Jimulco, State of Coahuila ; Gomez Palacio, Durango, State of Durango ; Aguas
Calientes, State of Aguas Calientes; Eincon de Eomos, Zacatecas, Fresnillo,
State of Zacatecas; Ojito, Santa Barbara, Parral, Escalon, Jimenes, Sta.
Eosalia, Chihuahua, Montezuma, State of Chihuahua; Tucson, Benson, Bowie
in Arizona ; Lordsburg, Eincon in New Mexico.

52 The Mechanism of Evolutiox in Leptinotarsa

Recltbrent Mutant L. multit^niata-intebmedia-melanothoeax.

This recurrent mutant has only been observed at Octlan in Jalisco, San
Bartolo in San Luis Potosi, and Sta. Kosalia in Chihuahua. It differs from the
same mutant form in the southern portion of the range of L. muUitceniata, by its
smaller size and duller color of the head and puncture, Avhich have little or no
luster. I have not been able to find this mutant in New Mexico and have no
knowledge of the relations of the " melanothorax " species found by Snow at
Las Vegas. These, while undoubtedly this same mutant type, do not seem to be
regular in occurrence in New Mexico, and in fact the mutant is rare over the
entire range of the habitudinal variety intermedia. (Plate 12, fig. 7.)

Habitat, Ecology, and Life History.

Like the preceding, this form passes the long, dry season (winter) in hiber-
nation as an adult in the ground. It emerges over most of its geographic
range when the summer rains begin, which is a variable climatic event. It
breeds at once, producing progeny that grow rapidly and give adults in 3 to
5 weeks ; these, over most of the range, after feeding for a few days may hiber-
nate or breed again, giving a second generation which hibernates soon after
emerging. Whether there be one or two generations per season is entirely
dependent upon the climatic conditions of the locality. In localities like Tuc-
son, where the summer rainy season may be short and not begin until August,
one generation seems to be the rule, although I foimd two in 1909. On the
plateau of Mexico, at Guadalupe, Zacatecas, {wo generations are the rule,
although here there are exceptions. When brought to the laboratory two gen-
erations are the normal cycle, but the second one of these is easily suppressed
by the use of desert complexes. Materials from nature are usually heterozygous
with respect to the reproductive rhythm and must be purified before they are
used in experiment.

Ecologically this form presents nothing different in principle or in the
factors involved from L. multitcenvita Stal, but differs considerably in minor
details and relationships, especially in the strictly desert areas.

Source of Material.

Stock used for experimentation has been obtained at La Barca in Jalisco;
San Luis Potosi, Catorce, Monclova in Coahuila ; Jumilco, Zacatecas in Zacate-
cas; Chihuahua in Chihuahua, Mexico; Tucson in Arizona, and Deming in
New Mexico, and has been fairly uniform in composition from all locations.

Habitudinal variety multit^niata-variabilis nov. vab.
(Plate 4, figs. 10, 11.)

In the upper portion of the Eio Lerma Valley, especially on the plains at the
foot of the Nevada de Toluca and eastward to the Sierra de las Cruces, occurs a
geographically isolated portion of L. muUitceniata, which differs therefrom in
constant genetically reproduced characteristics, and even though no fast line of
demarcation can be drawn, the gametic distinctness and the behavior of the
restricted group leaves one with little choice as to its taxonomic place. I have

Materials, Their Taxonomy and Natural History 53

classed it as an habitudinal variety on account of its separation in space and its
known differences of gametic constitution.

Desceiption of Living Animals.

Imago: Broadly oval, convex, robust; in general, shorter and broader than
multitceniala, color variable, orange-red or reddish yellow, pattern dense black;
punctations weaker and surface more polished than L. multitceniata. Above:
Head and pronotum reddish yellow, often orange or red, marked with dense,
extensively fused pattern not variable, few scattered weak punctations on epi-
cranium behind eyes and on lateral portion of pronotum; polished; on epi-
cranium pattern center fused into general one which nearly covers entire part.
Pronotum, central triangular spot reaching to anterior margin, h' — a' — am —
a— I with central lighter area; d' , e! , f and d, e, f always fused and usually
reaching posterior border in outer angle. Not infrequently fused with central
area ; c' and c always free. Mouthparts black or deep brown ; antennae black ;
basal joints sometimes brownish, terminal joint very short, conical ; scutellum
black, polished ; elytra orange, yellow, red, variable, with 5 dense black longi-
tudinal stripes, broader than in any other member of group, and edged with
an irregular double row of feeble pmictations. Second and third always fused
partially. First and darkened anal margin form wide median band. Costal
edge inflexed, smooth, flat. Below: Legs almost always black, tibia rarely
bro\vnish in central portion. Legs, polished, few punctures and minute
scattered hairs. Thoracic and abdominal surface yellow-brown, with variable
amount of fusion between color elements.

Size: Constant; male 10 to 13 mm. long, 7 to 9.5 mm. broad; female 10 to
14 mm. long, 7 to 10 mm. broad.

Sexes: Female with sternal sclerite of last abdominal segment rounded
smooth and in male truncate, deeply grooved; female larger than male, but
difference in size is less than in multitcenmta.

Food: Solarium rostratum or allied species.

Juvenile Stages.

Eggs: Dark yellow or reddish yellow, laid on lower surface of leaves in
bunches of 10 to 150, as in other forms; not often stalked, oval, ventrally
concave, or flattened, smooth, polished. Length, 2 to 2.75 mm., 1.25 to 1.75 mm.
broad. Incubation 6 to 12 days; average 8 days.

First larval stage: Head, pronotum, and legs deep shiny black; body reddish
yellow or deep yellow ; full complement of body-spots, each with one or more
spines. Length at end of stage, 2.5 to 3.75 mm.

Second larval stage: Head, pronotum, and eyes shiny black; pronotum with
anterior border deep reddish brown, brown, or rarely red ; body-color variable,
deep chrome-yellow, yellow-red, ocher ; spiracula and basopleural spots present,
strongly developed ; few pairs of the inner tergals present at anterior end of the
series. Length at end of stage, 4.5 to 6.5 mm.

Third larval stage: In color and pattern identical with second stage. Length
at end of stage, 8.75 to 14 mm. Length of larval period varies from 15 to 25
days ; averages about 18 days.

Pupa: Pupates in ground from 2 to 5 inches below surface; pupa yellow,
with spiracula and variable tergal spots. Pupation lasts from 6 to 12 days;
on the average 10 days.

54 The Mechanism of Evolution in Leptinotaksa

Length of ontogeny: Twenty-one to thirty-seven days ; average 25 to 30 days.
The juvenile stages are variable in this geographical variety and some of the
larval colors may be isolated by proper breeding into line-cultures, breeding true.

The range of this form has already been indicated and is shown on plate 5.
It has been observed in all stages at the following points : Toluca, Lerma, Sala-
zar, Ixtlahuaca, Malacatepec, Vale, Zitacuaro, Angangueo, Maravatio, in the
States of Mexico and Michoacan.

Recitbbent Mutation, multit.«niata-melanothobax Stal.

Identical with same mutant of L. multitceniata, except that stripes are often
broader upon the elytra.

Color biotypes are present and easily isolated by selective purification in
cultures. These relate entirely to hypodermal color, and the following can be
easily established : L. variabilis h^, red elytral ground ; L. variabilis 6^ orange
elytral ground ; L. variabilis ¥, yellow elytral ground ; L. variabilis b*, white
elytral ground.

The life-history is like L. multitceniata, excepting that the growing-season is
somewhat shorter and less favorable. Two generations per year are the general
rule. The last generation may be seriously retarded or nearly annihilated by
early autumn frosts, cold spells, or early advent of the dry season.

Ecology of this variety differs only in trivial details from the ecological
relationship of multitceniata, excepting that it is in general compelled to meet
more rigorous conditions than L. multitceniata.

Stocks for analysis and experiment have been obtained at the following
points: (a) Toluca, from the open plain west of Toluca, especially on the
northern side of Cerro de Zopilacalco and Cerro de Huitzila and from the banks
of a small rainy-season pool east of El Temple de El Eanchito ; ( & ) Lerma, on
open plain to south of town on shore of small lake; (c) Malacatepec, 3 miles
south of plaza, near an old ruined rancho; {d) Zitacuaro, near town, in waste
places, especially in corrals or pastures.

LePTINOTABSA oblongata NOV. SP.
(Plate 4, fig. 12.)

Leptinotarsa oblongata Tower, 1906. Inv. Evol. Chrys. Beetles genus Lept.,
Carnegie Inst. Wash. Pub. No. 48, pp. 6, 20; Jacoby, as L. decemlineata
Say, etc., Biol. Centr. Am., vol. VI, pt. 1, p. 233, and suppl., p. 255.

Jacoby refers specimens of this species from many Mexican localities to
L. decemlineata Say, on account of " fulvous legs, more or less marked or
spotted with black." The "fulvous" color of dead material is meaningless
and useless as a diagnostic character, and the highly variable black markings
are of service only when taken in conjunction with other characters. Some of
the localities recorded by Jacoby refer undoubtedly to this form (Cuernavaca
in Morelos) ; other localities are L. multitceniata Stal or its different geo-
graphical races. Jacoby's specimens now in the British Museum show clearly
much confusion with respect to this species and L. decemlineata Say. I have
been over this material and have given in tabular form below the determinations
reached.

Materials, Their Taxonomy and Natural History

55

5°-.

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56 The Mechanism of Evolution- in Leptinotarsa

Five specimens in the Jacoby collection in the British Museum, collected by
Hoge at Ventanas in Durango, Mexico, and recorded as L. decemlineata Say,
are in all respects like L. oblongata, but of nearly double the size, with the same
form and color-pattern system. I have never seen this type in nature, and do
not know that oblongata is found that far north. They may represent another
localized species of this group.

Descbiption of Living Animals.

Imago: Elongate, elliptical in outline, convex, distinctly and constantly
longer and narrower in proportions than any other species in the group. Above :
Epicranium and pronotum yellow, rarely tan or yellow-brown, marked with
black pattern. Epicranium rather regularly and uniformly punctate with
medium-sized punctations. Pronotum evenly punctate on lateral portion and
posterior border ; less punctate in median anterior portion. Mouthparts dark
yellow or yellow-brown, never black, eyes black; antennae, basal joints yellow
or yellow-brown, last six to eight black, faintly pubescent, sixth to eleventh
broad as long, twelfth truncate, conical; epicranium pattern, anterior lateral
epicranial spots fused, forming heart-shaped spot, lateral posterior epicranials
free, distinct, reaching to posterior border of eyes, rarely fused in median line.

Pronotum, color-pattern, spots a' and a parallel, united a'— —a, forming

U-shaped spot; b' and b sometimes fused entirely to a' and a. d', e', f and
d, e, f groups often completely united, forming dense black spot in the posterior
outer angle, and frequently fused anteriorly to b' and b. The modal condition
of the pattern is expressed by the formula :

c' b' ■}-am+ b c

e' + d' a' a d + e

+ b +pm-i- f +

The pattern is constant and typical of this species; scutellum brownish,
polished ; elytra pale yellow-white typically, rarely yellow or red, with anal
border black and five longitudinal black stripes ; second and third always united
posteriorly, sometimes also fourth ; all edged with irregular row of punctations.
Costal edge inflexed, flat or slightly concave, smooth, yellow, shoulder reduced.
Below: Yellow or yellow-brown, spots all present, variable amount of fusion;
legs, yellow or yellow brown, rarely red, femora mostly black or deep brown,
tibia ends black, tarsus black, femora sparingly punctate.

Size: Smaller than muUitceniata or decemlineata, constant. Female larger
than male.

Sexes: Female with sternal sclerite of last abdominal segment rounded com-
plete, smooth, and in male truncate, grooved, extending to near middle of plate ;
variable in male ; female also more robust than male and broader posteriorly.

Food: Solanum rostratum or near relations, but will eat S. tuberosum,
8. el (Bagino folium, and S. hertwigi when other food is denied it, but they do not
thrive thereon and soon die out.

Juvenile Stages.
(Tower, 1906, pi. 19, figs. 13, 14, 15.)

Eggs: Pale yellow, oval, smooth, polished, laid in small bunches of 5 to 50,
on lower surface of leaves, rarely stalked; ventral side flattened and slightly

Materials, Theie Taxonomy and Natural History 57

concave. Length, 2 to 2.75 mm.; breadth, 1 to 1.35 mm. Incubation lasts 5 to
10 days ; average 7 days.

First larval stage : Head, pronotum, and legs, black ; body bright yellow,
spiracula spots only present. Length at end of stage, 2.5 to 3.25 mm.

Second larval stage: Exactly like first stage in all respects, except that
anterior edge of the pronotum may be brownish or yellow in variable degrees.
Length at end of stage, 3 to 5 mm.

Third larval stage: Like second, except that pronotum always has anterior
portion yellow. Body color deep yellow. Length at end of stage, 9 to 14 mm.
Length of larval life from 12 to 25 days; average 15 days.

Pupa: Pupates in the ground at a depth of from 1 to 5 inches. Pupa yellow,
spiracula spots present. Pupation lasts from 8 to 16 days; average 10 days.

Length of ontogeny: 25 to 46 days ; average 32 to 35 days.

Geoghaphical Distbibution.

As far as information is available, this form is strictly limited to the Rio
Balsas Valley below the Mexican escarpment on the west, the Oaxaca-Guerrero
Highlands, and southward to the Isthmus of Tehauntepee (plate 5). I have
seen it in all its stages many times at the following locations: Cuernavaca,
Yautepec, Jojutla, Triente, Puente de Ixtla in the State of Morelos; Iguala,
Naranjo, Los Amates, Balsas, State of Guerrero; Cholula, Atlixco, Tatetla,
Matamoros de Izucas, Tlancualpican, State of Puebla; Oaxaca, Mitla. Tlaco-
lula, Tula, San Juan, El Parian, Tecomavaca, Venta Salada, State of Oaxaca
(Tower, 1906, p. 7) . The records of Guadalupe and Texcoco, valley of Mexico,
are of L. multitceniata, biotype of L. multitcBniata Stal. Added localities:
Mescala, Venta, Zopilote, Chilpancingo, Chilapa, Tetela de Eio, State of
Guerrero ; Ocotlan, Ejutla, State of Oaxaca, extend its known range well over
the Oaxaca-Guerrero Highlands, but I have no records from the Pacific slope,
and I have not found it as far south as the Isthmus of Tehauntepee, nor north
beyond the Eio Tepalcatepec Valley.

Habitat, Ecology, Life Histoby.

Living everywhere in regions having a long, hard, dry season and a short,
sharp, rainy season, two general periods occur — one of quiescence passed as an
adult in hibernation in cemented cells in the earth and the other in active growth
and reproduction. The beetles emerge from hibernation at the end of May or
early in June, begin breeding by the middle of June, and produce a generation
which emerges by the latter part of July, which again breeds at once and gives a
second generation, which emerges at the end of August or early in September.
The generation soon enters hibernation and remains dormant until the follow-
ing May. At Cuernavaca and Oaxaca this cycle is very regular, although there
is frequent overlapping and intercrossing of broods at all locations. Materials
from nature are, therefore, very frequently impure in respect to the reproductive
rhythm, and must be purified before utilization in experiments.

SOUBCE OF MaTEBIAL.

The materials for experimentation in this species have come from the follow-
ing localities, with the exception of a few unimportant strains : {a) Cuerna-
vaca: Stock No. 617, from Rancho de Basoco, to east of town, on a loma sepa-
5

58 The Mechanism of Evolution in Leptinotaksa

rated by two deep barrancas from the rest of the valley; stock No. 619, from
sides of deep barranca between Cuernavaca and San Anton ; stock No. 621,
from the top of the loma between the barrancas north of trail from El Hacienda
to Tetlama. (&) Atlixco: Stock No. 615, from cultivated fields 2 kilometers
to south of town, (c) Oaxaca: Stock No. 607, from foot of Maote Albans, on
rolling, well-drained area ; stock No. 605, from north and west sides of Cerro
del Fortin, to northwest of Oaxaca City.

BlOTYPES.

Four biotypes are capable of being developed from some strains of this species,
especially those from the northern and eastern edge of its range : L. oMongata
y, red elytral ground-color; L. oblongata &-, orange elytral ground-color; L.
oblongata b^, yellow elytral ground-color; L. oblongata &*, white or pale yellow-
white elytral ground-color. These are especially prone to develop in material
from that portion of the habitat along the south flank of the Nevada de Toluca,
but I have not been able to develop them from materials obtained in the Oaxaca-
Guerrero Highlands.

LEPTINOTARSA RUBICtJNDA NOV. SP.

(Plate 4, fig. 7.)

Leptinotarsa ruMcunda, Tower, 1906. Inv. Evol. Chrys. Beetles, genus
Lept.; Carnegie Inst. Wash. Pub. No. 48, pp. 7, 21.

The history of this form as I have found it leaves me greatly in doubt as to
what status it should have in this taxonomic orientation. When first found in
1903 it occupied a considerable range on the plain between Toluca and the
Nevada de Toluca, and the Sierra de Las Cruces, covering an area estimated at
10 to 12 miles square. It differed constantly in all stages from L. multitceniata-
variabilis, and was not observed inter-breeding with it at any time. In cultures
it is genetically constant and true to type indefinitely, and it genetically perpetu-
ates itself in nature in the same constant manner. In 1906 I was able to find it
only in a limited area of about half an acre in extent near Mexicalicingo, and
in 1908 only 30 specimens could be found in a week's search at the height of the
season, and in 1909 and 1910 no trace of the form could be found. The last
members of the race were as constant and were in all respects like the first
found, and while never abundant at any time, it was fairly easy to discover 20 to
50 in a day's search, and it was obtained in the earlier years in considerable num-
bers. Its rapid decline and apparent extinction raise some interesting questions.

Because of its genetic distinctness and constant differences from the obviously
near relatives I have retained it here as a species. Future information may
change this orientation.

Descbiption of LrviNG Animals.

Imago: Broad, rounded, convex, dark red or crimson, with heavy, shiny
black color pattern. Above : Epicranium, pronotum, and elytra uniform deep
red or crimson, never orange or yellow; epicranium evenly punctate with dis-
tinct pits, anterior lateral epicranial spots fused and reaching anterior border,
rarely prolonged caudad; lateral posterior epicranial spots forming band on
posterior portion and anteriorly to eyes ; eyes black ; mouthparts black, polished,

Materials, Their Taxonomy and ISTATrRAL History 59

pubescent; antennge, basal joints reddish, remainder black, pubescent, sixth to
eleventh broad as long, twelfth truncate, conical, broader than long. Pronotum
minutely, evenly, sparingly punctate; few larger scattered punctations near
lateral margin, polished. Pattern constant, central widely open V composed of

c' b'+ +b + c

a +07)1 +a
+
e' + d'+ pm +d + e

+ h'+ +h +

in which elements are entirely and constantly obscured in all examples. Spots
d', e', f and d, e, f always fused and reaching posterior border, and usually
fused to central area, all by J'-anterior end of a' and {^-anterior end of a. Cen-
tral area frequently reaching border of pronotum. Elytra : Anal border shiny
black with 5 broad, shiny, black longitudinal stripes ; third and fourth usually
fused posteriorly, all edged with irregular row of rather small punctations,
arrangement variable, in some specimens only single row, in others some rows
are partly double; in none are they developed to the extent found in niultitce-
niata or its varieties. Elytra polished, costal edge inflexed, red, smooth, flat,
polished, never punctate. Below: Eed body-color, nearly covered by color-
pattern composed of fused elements to the extent that usually only edges of
sclerites are red; legs, all shiny black, slightly pubescent femora and tibia
punctate, often strongly.

Size: About same as multitceniata but less variable, and sexes of nearly equal
size. Male, 9 to 12.25 mm. long, 7.5 to 9 mm. broad; female, 9 to 13.5 mm.
long, 7.5 to 10 mm. broad.

Sexes: Female with sternal sclerite of last abdominal segment complete,
rounded, smooth ; male truncate, grooved ; female also broader posteriorly than
male.

Food: Solatium rostratum, S. tuberosum (wild stock) on Nevada de Toluca.

Juvenile Stages.
(Tower, 1906, plate 17, figs. 16, 17, 18.)

Eggs: Bright red, large oval, polished; 3 to 3.5 mm. long, 1.25 to 1.75 mm.
broad ; ventrally flattened and concave, laid in bunches on lower side of leaves,
10 to 50 in a bunch, never stalked. Incubation lasts 8 to 12 days ; averages 10
days.

First larval stage: Head, pronotum, and legs shiny black; body bright red
with full set of color markings. Length at end of stage, 3 to 4.5 mm.

Second larval stage: Head, pronotum, and legs, shiny black ; body bright red,
spiracula, baso-pleural, and the anterior two or three pairs of inner tergal spots
present. Length at end of stage, 5 to 7.5 mm.

Third larval stage: Exactly like the second stage, excepting that pronotum
has the anterior edge red and the anterior tergals wanting. Length at end of
stage, 10 to 17 mm. Length of larval period, 12 to 20 days; average 15 days.

Pupa: Pupates in ground, 2 to 4 inches below surface. Pupa red, spiracula
and tergal spots present, variable. Pupation lasts from 8 to 17 days; averages
10 to 11 days.

Length of ontogeny: 28 to 50 days; averages about 36 days.

Geographical Distbibution.

Northern slope of Nevada de Toluca below oak zone, 8,500 to 9,500 feet; in
open grasslands. Recorded from following locations in all its stages. (Plate 5.)

60 The Mechanism of Evolution in Leptinotarsa

Habitat, Ecology, and Life History.

As far as known, the life-history of this species is like that of L. muUitceniata
Stal. It passes the long, dry, cold winter in the imago stage, hibernating in the
ground, emerges at beginning of rainy season in June, breeds by the middle of
June, and produces a first summer generation which emerges about the end of
July. Then these breed again and give a second generation, which emerges
late in August or early in September. This second generation was always
observed to be small and was several times apparently absent. In cultures a
normal rhythm of two generations in each reproductive cycle was found.

SoxmcE OF Material.

The stock for breeding and testing came from the following locations : (a)
Toluca, on open plain to south of city, about 1 kilometer south of Capilla de
San Sebastian; (&) Mexicalicingo, on open plain near town.

LEPTINOTARSA DECEMLINEATA Say.
(Plate 6, fig. 5; plate 7, fig. 2.)

Doryphora 10-lineata Say, 1824. Journ. Acad. Nat. Sci. Phila., Ill, pt. 2,
p. 453; Rogers, 1857, Proc. Acad. Nat. Sci. Phila., VIII, p. 302; Suffr.,
1858, Ent. Zool., XIX, p. 244.2; Walsh, 1865, Pract. Ent, I, No. 1;
Harold, 1874, Berlin Zeit., p. 444.

Leptinotarsa decemlineata Gemminger et Harold, 1874. Catalog. Coleop.,
t. IX, p. 1, p. 3440; Jacoby, 1883. Biol., Centr. Am., vol. VI, pt. 1, p. 233;
pi. XIII, fig. 24, suppl., p. 253.

And many other authors. No doubt is attached to the determination of this
form.

Say's original description is as follows:

" Yellow ; thorax litterate, with black ; elytra each with five black lines.
Inhabits Missouri and Kansas. Body yellow ; head with a triangular, black,
frontal spot; thorax two abbreviated black lines, divergent before; about six
black dots on each side; elytra, sutures, and five lines on each, black; the
interior line is confluent with the suture behind ; exterior line marginal ; three
intermediate ones joined or approximated at tip; beneath, incisures and three
or four series of ventral spots black.

" Length two-fifths of an inch.

" Var. a elytra white ; the two outer, intermediate lines are united at base
and tip.

" This species seems to be not uncommon on the upper Missouri where it was
obtained by Mr. Nuttall and myself. The variety I found on the Arkansas."

Description of Living Animals.

Imago. — Above: Oval, convex, robust, variable in form, size, and color,
depending upon age, geographic location, and conditions of growth during
ontogeny; head, epicranium, and pronotum yellowish brown, rarely reddish, in
sexually mature specimens, often reddish in freshly emerged specimens, with
variable pattern of black spots; epicranium rather uniformly punctate with
moderately developed pits, most numerous behind eyes, eyes black, niouthparts
yellow brown, rarely black; mandibles black, tips of palpi black, basal joints
brownish ; antennse, basal 5 or 6 joints brownish, last 6 broadened, broad as long,
black, slightly pubescent, last joint short, conical, often nearly oliscured.

Materials, Their Taxonomy and Natural History 61

Pattern elements all present but variable. Pronotnni rather coarsely punctate,
often strongly so on lateral and posterior portions, center more freely punctate,
polished. Pattern: All elements present but highly variable, from mere trace
to extensive fusions, a' and a always divergent, forming V more or less open.
Scutellum yellow-brown, smooth, polished, edges darkened. Elytra, in sexu-
ally mature specimens, pale yellow or straw-color, often more yellow or even
yellow-red, variable with locality, food and age; anal border black, 5 longi-
tudinal stripes, edges with irregular double row of moderate punctations,
second stripe often united to third and these in turn to fourth stripe posteriorly ;
interspaces not raised, polished, costal edge inflexed, fiat, or slightly concave,
smooth, polished, yellow or brownish ; hind wings strong, well developed, trans-
parent, reddish or bright red in center, darker yellow or brown on costa and at
base, variable. Below : Yellow-brown with pattern elements all present, variable,
often much fusion; thoracic and abdominal surfaces smooth, polished, rarely,
faintly punctate ; legs, femora and tibia yellow brown or reddish, rarely black,
joints darker brown or black, femora smooth, polished, rarely and freely
punctate ; tibia, few irregular, variable, well-marked punctures and spines.

Size : Female variable ; male, 7 to 16.5 mm. long, 4.5 to 9 mm. broad ; female,
8 to 12.5 mm. long, 5.5 to 10.25 mm. broad.

Sexes: Female with sternal sclerite of last abdominal segment rounded com-
plete ; male with same sclerite truncate, faintly notched, rarely grooved. Female
larger than male, more rounded and broader behind; male more elongate in
form and often very small.

Food: S. tuberosum^ S. rostratum, and more rarely many species of Solanum.

JxrvENiLE Stages.

(Tower, 1906, plate 17, figs. 19, 20, 21.)

Eggs: Laid in bunches, 5 to 200, not often stalked, oval, polished, smooth,
concave on ventral side; length 1.T5 to 2.50 mm., breadth 0.75 to 1.1 mm.
Incubation, 5 to 15 days ; average 6 or 7 days.

First larval stage : Head, pronotum, and legs, black ; body red with full set of
color markings, often spined. Length at end of stage, 3 to 4.5 mm.

Second larval stage: Head and legs, black; pronotum, posterior edge black,
anterior portion red, reddish yellow, or brown ; body red, varying in tint with
the condition of the animal, food, climatic conditions, etc., baso-pleural and
few anterior inner tergals present. Length at end of stage, 5 to 6.75 mm.

Third larval stage: Like second stage, color variable; length at end of stage,
11 to 17 mm. (Plate 12, fig. 2.)

Length of larval life, from 10 to 28 days ; average between 13 and 15 days,
but much depends upon food and climatic conditions.

Pupa : Pupates in ground, from 1 to 5 inches below surface ; pupa reddish
yelloAv. Pupation lasts from 6 to 15 days, but averages 8 to 10 days.

Length of ontogeny: 21 to 58 days; averages between 28 and 31 days.

Geographical Distribution.

(Plate 5.)

North America, eastward from the foot of the Rocky Mountains to the
Atlantic coast, northward into Canada, and northeast throughout the St. Law-
rence Valley, southward to the Gulf coast and to lower Texas. Common in most
localities; is everywhere a grassland form and becomes a serious agricultural
pest in many areas, especially in the north and east. (See Tower, 1906, for
further account of distribution and migration.)

62 The Mechanism of EvoLUTioisr in Leptinotaesa

Habitat, Ecologt, Life Histoby.

Like the other species in this division L. decemlineata passes the winter as an
imago, hibernating, but there is a great variation over the area inhabited in the
dates of entrance into and emergence from hibernation, due almost entirely to
varying climatological conditions. On emergence it breeds, giving the first
summer generation, which emerges about the middle of July over most of its
range, and these breed again, giving the second summer generation, which
emerges in August or early September and soon hibernates. It is not imcom-
mon to find a third small brood, especially in the south, and in favorable seasons
in other places, and this due to the fact that in nature the overwintering popu-
lation may live and breed with the second summer generation, producing hybrids
between generations of different potentialities. The progeny of these matings
often numerous and strong, behave in a very curious and variable manner, so
that in materials in nature, two, three, or even four broods are found at times.
When, however, stocks from many different portions of the country are fully
analyzed, it is found that all, both north and south, are two-brooded like the rest
of the species in this division.

Minor Divisions.

Many years of breeding and experimentation have shown that an endless
number of true-breeding races of low variability can be created in experiment.
None of these are well defined, or of any independence in group-cultures or in
nature, and none has any homologue in nature. All are due to purely artificial
isolations and fixings of trivial variations of attributes present, and while an
imposing array of pure biotypic lines could be created, it would be the acme of
folly to do so, because they have no discoverable permanence in the species in
nature.

Sports are known sparingly in nature. In 1906 I recorded the occurrence of
several which were then regarded as such, some of which proved on breeding to
be gametic variations, and it is only by breeding that the real nature of these
variations can be determined. It is not uncommon to find somatogenic varia-
tions, some of which closely resemble the germinal variations. Breeding tests
alone can determine the true nature of these extreme variations.

Sports Observed in Nature and Tested by Breeding.

LePTINOTABSA PAI>LIDA NOV. var.
(Plate 6, fig. 2.)

Imago : Oval, convex, more elongate than decemlineata Say, white or faintly
yellow- white ; smaller than L. decemlineata Say, with sides more nearly parallel.
Above: Epicranium sparingly, irregularly, and not deeply punctate, densest
behind eyes, center polished, few punctations, color pale white with tinge of
yellow, pattern extremely reduced, but center usually variable; eyes black,
mouthparts pale; mandibles, tips brown or black; antennje, four basal joints
white, remainder very light brown, faintly pubescent, seventh sharply broadened,
broad as long, as are also eighth to eleventh, twelfth conical, rounded. Pro-
notum, few small scattered punctations on lateral and posterior margin, whole
part polished; pattern reduced, elements all present, but fusions are rare.
Pronotum, distinctly longer and narrower than in L. decemlineata Say; scu-
tellum smooth, polished, brown, edges darkened ; elytra white or pale yellow-

.V. , ., J.

K. TOD A DEL.

JtTLinS BIEN,

Types of Modifications produced ix Earlier Experiments from L. decemlineata.

1. Form rubrivittata. X 2.5.

2. Form pallida. X 3.

3. Form iortuosa. X 2.5.

4. Form melanicum. X 3.

5. L. decemlineata. Normal stock form

used. X 3.
fi Form albida. X 2.5.

7. Form found by Snow in upper part of

Rio Grande Valley, near Las Vegas,
and called L. melanothorax. I have
found it once at Las Vegas on S.
eleac/nifoliiim — not the same as L.
melanothorax. Figured from spe-
cimen in Kansas Museum. X 3.

8. Form minuta. X 3.5.

9. Form defectopunctata. X 3.

Materials, Their Taxokomy and Natural History 63

white, anal edge brown, never black, 5 longitudinal dark bands, fifth always
brown, remainder always dark brown, never black, all edged with irregular
double row of punctations. Interspaces flat, while elytra have frequently granu-
lated appearance due to numerous fine furrows which often radiate from the
punctations. Shoulder more depressed than in L. decemlineata, costal edge
inflexed, smooth, flat, white ; hind wings always white or transparent, no color
present. Below : White or whitish yellow, color-centers often wanting entirely
upon thorax and abdomen, which are smooth and polished. Legs : Light joints
browned, tarsus brown, femora sparingly punctate, first pair more so than other,
tibia faintly punctate, small spines.

Size: Eather constant, body index, male 7 to 12.5 mm. long, 4.5 to 8 mm.
broad; female, 7 to 13 mm. long, 5 to 8.5 mm. broad.

Sexes: As in L. decemlineata Say.

Occurrence: In nature near Cold Spring Harbor, Long Island, New York,
1899, tested, breed true ; Cabin John Bridge, Maryland, 1900, not tested fully,
some breed true ; in F-, Clifton, Ohio, 1901, 6 tested, breed true ; Chicago, 1902,
2 tested, breed true. (Tower, 1906.)

Juvenile stages: As in L. decemlineata Say, but uniformly lighter in color
and lower development of color elements.

LePTINOTAESA MELANICUM NOV. VAB.
(Plate 6, fig. 4.)

Imago: Bounded, convex, robust, shoulder prominent, raised, dark ocher-
yellow with dense black, much-fused color-pattern. Above: Epicranium,
mouthparts, antennae, eyes, dense shiny black; antennae, seventh to eleventh
joints nearly as broad as long, pubescent, terminal joint conical, longer than
broad; epicranium strongly punctate; pronotum rather regularly and densely
punctate, least so in center ; ocher-yellow with pattern having all elements fused
and much enlarged ; scutellum black, polished ; elytra, dark ocher-yellow, anal
border black, 5 longitudinal black stripes, second, third, and fourth always
united posteriorly, costal edge inflexed, smooth, flat, black or brownish an-
teriorly, shoulder prominent, all elytral punctations strong. Below: Head
black, thoracic and abdominal segments quite or nearly so; all color-pattern
elements fused, body-color ocher-yellow; legs entirely black, femora and tibia
strongly punctate.

Size: Larger and more robust than the average L. decemlineata Say and
much broader in proportion to length; not variable. Male, 12 to 16 mm. long,
7 to 10 mm. broad; female, 12 to 16.5 mm. long, 8 to 10.25 mm. broad.

Sexes: As in L. decemlineata Say, but male is more strongly marked.

Occurrence: In nature: West Bridgewater, Massachusetts, 1895, 2 tested,
breed true; Cold Spring Harbor, Long Island, New York, 1899, 1, not com-
pletely tested, indications were that it would breed true; Cabin John Bridge,
Maryland, 1900, 29, not tested.

Food: As in L. decemlineata Say.

Juvenile stages: As in L. decemlineata except that body-color of larvae is
always dense red, verging towards a brownish tinge,

LiEPTINOTABSA BUBBIVITTATA NOV. VAB.
(Plate 6, fig. 1.)

Imago: In all respects like L. decemlineata Say, but characterized by the
bright-red hypodermal color of the head, pronotum, elytra, and ventral surface ;

64 The Mechanism of Evolution in Leptinotaesa

breeds true ; color is often almost a scarlet in freshly emerged specimens, but
changes to full red at maturity.

Size : As in L. decemlmeata.

Sexes: As in L. decemlineata.

Occurrence: In nature: Stock from McPherson, Kansas, 1903, gave in
1904 at Chicago 1 male able to transmit character. Liberal, Kansas, 1905,
1 male, 2 females, breed true.

Food: As in L. decemlineata.

Juvenile stages: As in L. decemlineata.

LePTINOTARSA TOBTUOSA NOV. VAB.
(Plate 6, fig. 3.)

Imago: In form, color, sculpture, and pattern, except elytral pattern, like
L. decemlineata; elytral pattern, anal edge black, five longitudinal black stripes
fused into a variable network of many transverse fusions. Median stripe
shortened, much so posteriorly, not reaching more than two-thirds length of
elytron, bent anteriorly at region of middle band and unites with ramous stripe
which is bent posteriorly and fuses with inner subcostal stripe distally; sub-
costal approximated and much anteriorly, and other subcostal bent at middle
base sharply forward to meet costal stripe which is shortened and often not
well developed ; punctations much reduced and consist of a single irregular
row along edge of each stripe ; interspaces flat, polished, smooth.

Size: As in L. decemlineata.

Sexes: As in L. decemlineata.

Food: As in L. decemlineata.

Juvenile stages: As in L. decemlineata.

LePTINOTARSA ALBIDA NOV. V.VR.
(Plate 6, fig. 6.)

Imago: Elliptical, elongate, convex, smaller than L. pallida, white with
light-brown pattern. Above: head, pronotum, and elytra white, with color-
center present, greatly reduced, and all light brown in color; elytral stripes
shortened and narrowed, punctations on head and pronotum about like those of
L. pallida; antennae, sixth to eleventh joint broader than long, terminal joint
short, rounded, almost hidden ; elytral pmictations, single irregular row. Below,
like pallida.

Size: Smaller than decemlineata. Male, 7 to 10.5 mm. long, 4 to 6.5 mm.
broad; female, 7 to 11.25 mm. long, 4 to 7 mm. broad.

Sexes: As in L. decemlineata.

Occurrence: San Antonio, Texas, 1904, 1, tested, breeds true — i. e., was able
to transmit its characters in toto.

Food: As in L. decemlineata.

Juvenile stages: As in L. decemlineata, only smaller larvte and body-color
pale red or pink.

Leptinotaesa minuta nov. var.

Imago: Body-form, proportion, color-pattern, and sculpture like L. decem-
lineata, excepting that L. minuta is always less than half the average size and
frequently only one-third that of L. decemlineata. (Plate 6, fig. 8.)

Size: Body index, male, 5 to 9.5 mm. long, 4 to 5.25 mm. broad ; female, 5 to
10.25 mm. long, 4.5 to 6 mm. broad.

Sexes: As in L. decemlineata.

K. TODA DEL.

JVLIUS BIEN. N. Y.

1. L. jiincta. Larvae, 3d stage. X 1.

2. L. decemlincata. Larvae, 3d stage. X 1.

3. L. juncta. Prom life. X 1.5.

4. 5. L. decemlineata. From life. X 1.5.

6. L. texana. Larva, 3d stage. X 2.

7. L. Uimamoca n. sp. Larva, 3d stage.

X 2.

8. L. tumamoca n. sp. From life. X 2.

9. L. signaticollis. Larvae, from life: (a)

1st stage; (b) 2d stage; Cc) 3d
stage. X 1.

10. L. signaticollis. Larvae, from life: (a)
1st stage; (b) 2d stage; (c) 3d stage.

Materials, Their Taxonomy and Natural History 6o

Occurrence : In nature : Cabin John Bridge, Maryland, 1, not tested ; Yellow
Springs, Ohio, 1901, 1, tested, bred true; Chicago, Illinois, 1902, 1, tested, bred
true; San Antonio, Texas, 1904, not tested (Tower, 1906).

Food: As in L. decemlineata.

Juvenile stages: As in decemlineata, excepting that larvse in second and
third larval stages are always very small in size.

Ecology: Elytral stripes edged with single regular rows of impressed puncta-
tions. Often depressed and placed at the bottom of a groove.

JUNCTA DIVISION.
LEPTINOTARSA JUNCTA Gieb.

(Plate 7, fig. 3.)

Chrysomela juncta Guer., 1824. Ins. spec, nov., pp. 590, 825. Stai, 1862,

Monog. Chrys. d. I'Am., pt. 1, p. 165; Kraatz, 1874, Berl. Zeit., p. 442, 1,

fig. 6.
Doryphora juncta Rogers, 1857, Proc. Acad. Nat. Sci. Phila., VIII, p. 30, 1;

Suffrian, 1858, Ent. Zeit., XIX, p. 243, 1.
Leptinotarsa juncta Guer. Gemminger et Harold, 1874, Catalog. Coleop.,

t. XI, pt. 1, p. 3440.

This distinct and easily recognizable species is clearly separated from all of
the foregoing forms by the character of the elytral punctations first noted, I
believe, by Walsh (Am. Ent., vol. I). The original type I have not seen, but
Rogers's and Stal's determinations leave no doubt of the correctness of the
identification. Stal's description, taken from his monograph, is quoted merely
for completeness in taxonomy :

" Ovalis, flavo-testacea, antennis apiceni versus nigro-f useis ; maculis capitis,
prothoracis ventrisque nee non scutello nigris; elytris stramineis, striato-
punctatis, interstitiis alternis nigris. Long. 9^11, Lat. 6-7^ millim.

" Patria: America borealis, Georgia. (Mus. Holm., etc.)

" Ovalis vel subovata, flavo-testacea, nitida. Caput apicem versus distincte
punctatum, maculis tribus vel quinque parvis, una basali, duabus mediis, duabus
prope apicem, nigris. Antenna? apiceni versus sensim nonnihil' incrassata?,
articulis apicalibus infuseatis, latitudine paullo longioribus. Prothorax elytris
nonnihil angustior. antrorsum sensim subangustatus, anterius utrimque leviter
rotundatus, obsolete parce punctulatus, utrimque punctis raris distinctis in-
structus, angulis anticis acutiusculis, vittulis duabus discoidalibus abbreviatis
nee non maculis pluribus parvis nigris ornatus. Scutellum l»ve. Elytra
lateribus parallelis vel retrorsum leviter ampliata, distincte striato-punctato,
seriebus punctorum basin baud attingentibus, quarta et quinta, septima et
octavo longe ab apice, sexta et nona prope apicem coeuntibus, interstitiis
secundo, quarto, sexto et octavo nee non dimidio interiore interstitii decimi
nigris, interstitio septimo reliquis angustiore, interdum fusco-testaceo vel nigri-
cante. Venter maculis, in series transversas dispositis, nigris ornatus. Femora
subtus interdum macula nigra ornata.

" ^ Segmento ventrali ultimo apice truncato."

Description of Living Animals.

Imago: Stal's description of this distinct form leaves only comments as to
color necessary. In life the elytral ground-color is ivory-white and the head,
pronotum, and ventral side is light yellow-brown, making it one of the most
beautiful members of the genus. Much variation exists in the color-pattern.

66 The Mechanism of Evolution in Leptinotarsa

and between the type and variety (a) as described by Stal all kinds of inter-
grades occur. (Plate 7, fig. 3.)

Size: Larger and more robust than L. decemlineata; sexes of nearly equal
size. Male, 11 to 16 mm. long, 8 to 11 mm. broad; female, 11 to 17.5 mm. long,
8 to 11.75 mm. broad.

Sexes: Female with sternal sclerite of last abdominal segment rounded;
male with the same sclerite truncate and faintly grooved.

Food: Mainly Solanum carolinensis. More rarely egg-plant or other succu-
lent or herbaceous Solanacese. Experience in cultures shows that S. carolinensis
is the only food upon which it thrives, and while it will eat other plants (S. escu-
lentum, 8. tuberosum), it in my experience does not thrive upon them, and can
not be reared for more than one or two generations thereon.

Juvenile Stages.

Eggs: Large, oval, rarely stalked, polished, smooth, pale yellow-pink in
color; 2 to 3 mm. long, 1 to 1.5 mm. broad; ventral flattening very slight.
Incubation, 7 to 12 days.

First larval stage: Head, pronotum, and legs, black or dark brown; body,
pale bluish-pink, variable, spots variable, spiracula always present. Length at
end of stage, 3 to 4.5 mm. (Plate 7, fig. 9.)

Second larval stage: Head, pronotum, legs, as above; but the dark color on
the pronotum is limited to the posterior edge and is variable ; body, pale bluish-
pink, spiracula spots only present. Length at end of stage, 5 to 7 mm.

Third larval stage: Exactly like second (see Walsh, Am. Ent., I). Length
at end of stage, 10 to 16 mm. (plate 7, fig. 1). Length of larval life, 12 to 30
days ; average, 20 days.

Pupa: Pupates in ground at depth of 1 to 5 inches. Pupa yellowish. Dura-
tion of pupation 10 to 25 days ; average, 14 days.

Length of ontogeny, 30 to 60 days ; average, about 40 days.

GEOQBAPHKJAIi DiSTBIBtJTION.
(Plate 3.)

Irregularly and sparingly distributed over Atlantic Coastal Plain and nearby
areas. Not often common, and considerably restricted and reduced in numbers
and range since the advent of L. decemlineata. Does not extend southward into
Mexico, as far as has been discovered.

Material for experimentation has come from the following locations : Atlanta,
Georgia; Mobile, Alabama; Lexington, Kentucky, and New Orleans, Louisiana.

Geographical variety texana Sch.
(Plate 7, fig. 5.)

Much confusion exists concerning this not uncommon form from Texas and
the Eio Grande Valley. It has been variously classed as L. dejecta Stal, as Say's
variety of decemlineata, while Townsend has described its larvae as those of
L. undecimlineata.

I have reared it for many generations and find that it is genetically true and
constant to its type when grown side by side with L. juncta under the same
conditions and on the same food-plant — i. e., S. carolinensis. It differs from
L. juncta Guer. in a few minor attributes and these only in degree, not in kind.
I have, therefore, classed it as a geographic variety, it being comparable to the
other similar varieties here recognized. In that I have no means of knowing

Materials, Their Taxonomy and Natural History 67

whether Say found this form on the Arkansas or not, I have only included his
possible notation because it is often so included in taxonomy.

Doryphora decemlineata Say, 1824. Journ. Acad. Nat. Sci. Phila., Ill, pt. 2,

p. 453.
Doryphora dejecta StS.1. Specimens identified by Horn and others are found

in many museums and collections, and while closely like St^l's dejecta,

both are distinct, as I can testify from having both alive at the same

time to compare.
Leptinotarsa decemlineata Say var. texana Schaeffer, 1906. Bull. Brooklyn

Inst. Arts, Sci.. I. No. p. p. 239.

Schaeffer's description is as follows :

"Leptinotarsa decemlineata var. texana, new variety. Form and size of
decemlineata but differs in having the punctures of the striae regularly placed,
not confused. The first black elytral vitta abbreviated at about the middle, the
submarginal entirely absent and the epipleurae entirely pale, from base to apex.

" Brownsville, Texas.

" I have given this form a name, which seems to be quite constant in southern
and southwestern Texas, as it stands in several collections as dejecta Stal, from
which it differs in several points. In dejecta the suture is always black, the
third and fifth intervals are only slightly wider than others, very distinctly
and much more in var. texana, the inner margin of elytral epipleurae and apex
is always black and all the markings on elytra have a very distinct aeneous tint,
which is never the case in decemlineata and var. texana, and is twice mentioned
in Stal's description. The specimens of dejecta, which I have taken from
Brownsville, Texas, have the suture, the sixth elytral interval, from apex to not
quite to base and the eighth from base to not quite to middle blackish-feneous ;
the eighth interval is in some specimens at apex also darker, as well as the
fourth, which is then more or less connected with the vitta on the sixth interval,
the sutural interval being counted as the first."

Schaeffer is entirely correct in distinguishing the form from Stal's L. dejecta
and correct in his notation of the elytral punctation, a character that at once
places it as allied to L. juncta and not L. decemlineata. When reared, the
juvenile stages are unmistakably like juncta and not like decemlineata.

Description of Living Animals.
(Plate 7, fig. 5.)

Imago: I would add to Schaeffer's description as follows: Color in life,
head, pronotum, and elytral ground-color pale yellow, often almost white.
Pronotal color-pattern variable in size of spots, but fusions between them are
rare.

Size: Constant.

Sexes: Female with sternal sclerite of last abdominal segment rounded flat;
male with same sclerite truncate, grooved.

Food: Solanum carolinensis, S. elceagnijolium.

JirvENiLE Stages.

Eggs: Exactly like L. juncta, only paler; 2.5 to 3 mm. long, 1 to 1.5 mm.
broad. Incubation lasts 5 to 12 days ; average 7 or 8 days.

First larval stage: Exactly like L. juncta, only paler. Length at end of
stage, 3 to 4 mm.

Second larval stage : In all respects like L. juncta, but color is paler. Length
at end of stage, 5 to 7 mm.

68 The Mechanism of Evolution in Leptinotarsa

Third larval stage: Like L. juncta, smaller, paler (see Townsend, Trans. Tex.
Acad. Soe., vol. 5, pp. 51-101). Length at end of stage, 8 to 14 mm. (Plate 7,
fig. 6.)

Length of larval life: 15 to 30 days; average 22 days.

Pupa: Pupates 1 to 3 inches below surface of ground. Pupa pale yellow.
Pupation lasts 10 to 20 days ; average 1-4 days.

Length of ontogeny: 30 to 60 days; average 50 days.

Geogkaphicax Distribution.

Eio Grande Valley in Texas and northern Mexico; possibly northward into
the valleys of Eed and Arkansas Rivers. I have one specimen labeled Pueblo,
and to this has been added Colorado, but I have no confidence in the correctness
of the locality. Accurate records : Dallas, Brownsville ( Sch. ) , Laredo, Corpus
Christi, Fort Worth, Santa Tomas, San Antonio, Texas; Matamoros, Rosita,
Ciudad, Porfirio Diaz, Mexico.

Habitat, ecology, life-history: In cultures this form has always given two
generations per year — one early in the season, then a long wait and one late in
the season, then hibernation through the winter. In nature I suspect that this
is also its behavior, but I have not had opportunity to fully verify this point.
It is also probably variable in behavior in nature.

Leptinotarsa tumamoca nov. sf.

Description of Living Animals.

(Plate 7, fig. 8.)

Imago: In shape, general color, and pattern much like L. texana, but only
half the size. Above: Oval, convex, epicranium and pronotum light yellow-
brown marked with black; epicranium strongly and densely punctate on sides
and posterior portions, center polished, few small or no punctations present.
Pronotum densely punctate on lateral portions; center and posterior margins
smooth, often polished; eyes black, pattern on epicranium lacking, or anterior
lateral epicranial spots showing only a trace thereof ; pronotal pattern, /' and /
almost always absent, as are am and pm, a' and a never fused, but &' and h almost
always fused with a' and a; scutellum dark brown, polished ; elytra, ivory-white
or yellowish ivory-white; anal edge yellowed or brown (cubital and anal stripes
lacking), medials present, well developed ; ramous and anterior subcostal always
united posteriorly and posterior subcostal often united to anterior subcostal at
or anterior to its junction with the ramous; costal stripe bent, shortened pos-
teriorly, all edged with regular single row of punctations. Costal edge inflexed,
smooth, flat, yellow. Below : All parts uniform pale yellow, edges of sclerites
and joints sometimes brownish, anteunEe and terminal joints slightly darker,
no pattern on thorax or abdominal segments.

Size: Smallest member of group and possibly of the genus; is not larger and
probably averages smaller than L. lineolata.

Sexes: Female, sternal sclerite of last abdominal segment rounded, flat,
smooth; male with same sclerite truncate, slightly grooved. Male slightly
broader and larger than female.

Juvenile Stages.

Eggs: Small, oval, ends squared, not stalked, laid on lower surface of leaves
singly or in small bunches of 10 to 20; pale bluish-yellow or yellow; 1.25 to
2 mm. long, 0.6 to 1 mm. broad. Incubation lasts from 5 to 10 days; average
6 or 7 days.

Materials, Their Taxonomy and jSTatural History 69

First larval stage: Head, pronotum, and legs dark brown, or rarely black;
body pale lavender or yellowish, spiracula spots alone present, small. Length
at end of stage, 2.5 to 3.5 mm.

Second larval stage: Exactly like first. Length at end of stage, 3 to 4.5 mm.

Third larval stage (plate 7, fig. 7) : Strongly resemble small larvae of L.
texana, color variable, pale dilute lavender to pale bluish-yellow ; pronotum and
head often brown or yellow-brown ; legs variable, brown to black. Length at
end of stage, 5 to 8.5 mm. Length of larval life, 10 to 18 days; average al)out
14 days.

Pupa: Pupates in ground from 0.5 to 2 inches below the surface. Pupa
yellow-white ; pupation lasts 6 to 12 days ; average 8 days.

Length of ontogeny: 25 to 40 days; average about 30 days.

Geographical Distribution : Limited to desert areas of Arizona and Sonora.
Recorded from Hermosville, Guaymas, and Xogales, State of Sonora, Mexico ;
Benson, Tucson, and Maricopa, Arizona.

Habitat, ecology, life-history : Apparently there is but one generation per
year of this form. This has been our experience at Tucson and in cultures.
Hibernates as an imago.

Source of material: Bottoms of Santa Cruz River in moist midsummer at
foot of Tumamoc Hill, Tucson, Arizona.

EVOLUTION AND PHYLOGENY OF THE UNEATA GROUP.

The present conditions in any group or organisms in nature afford little basis
for creating schemes of descent, and where fossil evidence is lacking the usual
substitute of ontogenetic stages, similarities, distribution phenomena, and the
like usually result in expressing the bias of the student rather than relation-
ships. To many this procedure is proper and correct, and many believe it is
still able to give accurate pictures of descent. When applied to large groups
and longer trends of evolutionary development this may in a very crude manner
be partly true, but for minor groups and for the building of schemes of relation-
ships within genera the method can hardly be considered as safe. Former
methods and consideration in phylogeny were based upon the unproven assump-
tion that descent had always been by dichotomy, either by slow divergent
variations and extermination of intermediates or by a saltation, both a dichot-
omy of existing materials, and so grew up the familiar tree-like schemes of
descent, but to what extent these represent the truth no one at present can
honestly decide.

The accumulated experience of the neo-Mendelian investigations shows be-
yond doubt that attributes and qualities can be and are shifted about, replaced,
and recombined, giving quite different expressions to the new combinations.
As a result nothing new appears; only an organic metathesis occurs, which,
nevertheless, produces distinct, divergent, individual entities. To what extent
are the grouped lines of descent which we call species the product on a larger
scale of metathesis in nature ? Who can answer ? The Linnsean view that cross-
ing is indicative of and s}Tiom-mous with degeneration in standard and stamina
in the species is hardly tenable at this time, and in the attempt to arrive at some
comprehension of the probable past history one can not forget the part that
crossing may have played in conjunction with other processes in the formation
of the evolved group.

70 The Mechanism of Evolution in Leptinotaksa

The inertia of taxonomy and orthodox biology seems to be yielding to the
opinion that species in nature are not all due to dichotomy and extermination,
but that intermingling or crossing is a potent factor in species formation, and
increasingly frequent are the expressions of opinion that species may after all
be hybrids (Morgan) and that frequent intercrossing has occurred (Cook).

In the Uneata group of the genus Leptinotarsa, fossil evidence is lacking, and
I have no confidence in ontogentic sequence of characters as a basis for deter-
mining relationship.

The data of distribution also are subject to valid criticism as a basis for
phylogeny, less valid than ontogeny; but too much reliance can not be placed
upon distribution data. Nor can habitat and adaptability be of any consider-
able value, because the physiological capacities which make for habitat selection
and ecological restrictions are as fully capable, and are transferred by meta-
theses as often as are purely morphological characters.

The Uneata group is limited to North America ; beyond doubt the undecvm-
lineata division is strictly tropical grassland in distribution and ecological
relations. The muUitceniata division, on the other hand, is always a temperate
grassland form, either on the high plateaus of the tropics which have a temperate
climate or on the plains farther north ; at any rate, it in the main is absent from
the areas occupied by the first division. The juncta division, in its range, gives
one the impression of being decadent and limited to areas of peculiar complex
like the coastal plains and subtropical deserts — areas of high temperature and
considerable humidity for a short growing season, then a long period of drought
and quiescence. These are observed conditions in nature where the divisions
present more than distributional and ecological distinctions, each having
marked morphological characters, and between them intercrossing is not only
not common, but is difficult in most instances to obtain.

In each of the three divisions of the group there is an undefinable impression
of a fundamental basis, a form nucleus in each upon which the array of shifting
characteristics is placed ; whether this is a reality or only a product of the com-
bined characteristics present opinions would differ, and all would be merely
opinion, not demonstration. Within each division, however, the arrangement
of the characteristics gives species, which cross and show metatheses of their
attributes in the products of crossing.

I could, of course, create a phylogeny for these materials, but it would be
necessary to assume certain premises as true which could not be tested, and too
much of this sort of biology has already been created. In their earlier history
this general group, the Chrysomelinse, reaches far back towards the base of the
Mesozoic era, and there the genera show species distinguished by much the
same characteristics as distinguish species and genera to-day. Since the rise
of the Chrysomelinge no one has any conception of the myriad of species and
lesser divisions that have come and gone in their history, and yet there persists
through all a basis of form, and the array of attributes and qualities capable of
metathetic shifting and recombination, giving species and varieties. The time
has passed when schemes of phylogeny can be of interest and current assump-
tions of monophylitic origin and dichotomy, either slow or rapid, are vanishing
into meaningless metaphysics.

That there are long-continued lines of descent no one doubts ; and perhaps
all of our trends of evolution and also phyla are but syntheses of factors from

Mateeials, Their Taxonomy and Natural History 71

diverse sources, producing stable and pliable aggregations of qualities which are
incessantly worked over, modified, recombined, and adjusted to newer con-
ditions from time to time, giving a trend of evolution and a multitude of species.
For us nature contains only other organic forms diverse in kind, intricate in
action, and all of most vital interest to us, since we are one of them and have
come into being through the same processes that gave rise to them. Phylogeny
and its schemes all too often lead to a static condition of mind and to orthodoxy
in biology. There exists only living matter with its qualities manifest in
diverse combinations. These species, genera, etc., of our scheme of cataloging,
lasting though they may be, are but expressions of the operation of the general
processes of germinal distintegration, synthetic recombination, and the rise of
new factors in the germinal complex, all interacting in an amazingly complex
series of operations. These are the centers of present inquiry because they are
the modus operandi of evolution. As for schemes of past evolution in these
materials, I have none and have no basis for building one. I know which species
are most alike and which are most different, but likeness does not always mean
nearness of kin, nor unlikeness remoteness of genetic relationship. They are
to me only so many kinds of living substance, each with its proper and restricted
qualities, attributes, and conditions, which I am able to use, like reagents in a
test-tube, for many operations of analysis and synthesis, and thereby help, I
hope, to add to our knowledge of the fundamental processes that are productive
of heterogeneity and evolution in organisms.

CHAPTER III.
THE PROBLEMS OF GAMETIC CONSTITUTION.

INTRODUCTION.

Knowledge of the gametic constitution of organisms has been one of the
desert spots in biology, because there has been, up to recent times, no adequate
working hypothesis for use in investigation, with the result that there has been
endless interpretation and profitless speculation that has served more to con-
fuse than to advance the analysis of the gametic constitution and the behavior
of characters in genetic lines of descent. Darwin's provisional hypothesis of
pangenesis, and the subsequent conceptions that were the natural outgrowth of
it, lead only to controversy, interpretation, and plausibilities, and were not
capable of use as working hypotheses in experimentation. The Galtonian
hypothesis stated only the results of a statistical blending of the data and
arrived at no analytical results, nor can the hypothesis be utilized in the
orientation of any critical experimental analysis. The rediscovered work of
Mendel and the neo-Mendelian developments that have taken place in the past
decade, especially the advances made by Cuerrot, have produced, at the least,
a useful working hypothesis.

In the course of this investigation I have had abundant opportunity to test
the two current hypotheses, without prejudice to either. The inherent defects
in the Galtonian hypothesis were early apparent ; the early dogmatic assertions
with regard to the Mendelian principles, the rabid partisanship of many of its
adherents, unfortunately gave it the setting of a cult, and led, naturally, to
suspicion of its principles. Both of the hypotheses were put to severe test
through the course of this investigation.

The net result of the severe tests was the complete failure of the Galtonian
hypothesis in all respects and at every point and the demonstration that the
principles of Mendel were true in their broader aspects when stripped from the
encumbering conceptions of " fixed units " and Weismannism. The conceptions
of the separateness of manifestation of "characters" as the result of the inter-
action of specific agents in the germinal material, the capacity of these for
transfer or metathesis, and the random metathetic distribution of characters
into different classes of gametes, were put to every sort of test that I could
devise, but in no instance, thus far, have I been able to satisfy myself that tests
made have in any manner been at variance with the real principles, and, in short,
have in every respect throughout confirmed them and in some directions carried
the investigation farther than friendly investigations in its support would have
done. In the effort to find characters that " did not Mendelize," meaning the
fact that they were not definite in manifestation and were not able to " segre-
gate " but blended in crossing, I have had no success. In these cases on further
testing I have always found either conditions in the medium, in the organism,
or associations of the character with others that, when worked out, showed
entire agreement with the principles of factorial constitution and operation.
72

Problems of Gametic Constitution 73

In this second section of this report I shall present such facts concerning the
behavior of different characters, and of the gametic agents productive thereof
as have a bearing upon the other portions of the investigation, and also some of
the data of the attempts to analyze the gametic constitution of my materials.
In this I have used simple characters of structure, color, and complex associa-
tions thereof, and simple reaction characters and complex associations thereof.

The problems of heredity for the present, in experimental investigations,
center entirely about the methods of attack that arose from the rediscovery of
Mendel's work and as formulated in the present neo-Mendelian conceptions.
At the basis lies the idea of the individuality of manifestation and production
of " characters," and out of this has grown the misconception that the " char-
acters " are separate entities or individualized in the organism. It is distinctly
not true that the characters are so individualized, excepting in our recognition
of them as end-results or products of the operations of interacting antecedent
agents, and even in the observed product our methods of perception and
description tend to an excessive symbolic delimitation of them that is not
justified or representative of the truth. If it can be kept clearly in mind that
the characters in organisms are the same as in non-organized substances, the
resultant of the combining, interacting materials or factors of composition
and the conditions of the medium at the time of combination, no difficulty is
introduced into the problems of gametic constitution by the pragmatic indi-
vidualization of characters in plants and animals.

With this conception of characters in organisms is associated the idea of the
antecedent gametic factors of composition whose interaction and presence in the
germinal material, the interactions and products of which must be provided for
by proper conditions in the medium. This idea of the factor or agent that
makes possible, and the determiner, the agent that of several possibilities
because of its presence decided the direction of the resultant reaction, are most
decidedly not " biological " conceptions, but are in all respects a purely physical
conception of the problem, and precisely the same as exist in non-organized sub-
stances. Neither factor or determiner nor any other agent is the carrier of
anything, any more than water of crystallization is the carrier of the crystalline
form or color or hardness. Determiners and factors need not even be sub-
stances, but may well be, in many instances, physical positions and relations in
the parts of the gametic substances. Many instances in non-organized sub-
stances could be cited where this single fact of configuration of the system was a
determiner of the action of the substance, and produced differences in the
reaction and in the products because of this initial difference of physical position
and arrangement of the parts of the system, and there is no a prion reason why
the same may not be true of organisms.

The fact of the segregation or distribution during gametic genesis of these
agents into different classes of gametes does most to disrupt cherished traditions,
and some of the conceptions and discussions that have been put forward by
neo-Mendelians, attempting to give a particulate shifting and sorting in the
gametes, is on the face of it absurd, and no better than Weismannism. In every
mating there are numerous contrasting pairs of equivalent characters, but why
some cross from one gametic system into the other, and others do not, and what
determines which shall undergo metathesis, is entirely unknown. In one series
6

74 The Mechanism of Evolution in Leptinotaesa

of crosses that I had there were 41 contrasting characters that were known to
be alternative and that had been proven to undergo metathesis in experiment.
This number of characters would, on the pure particulate basis, demand the
formation in F^ of thousands of classes of gametes and an impossible number
of combinations thereof. No such extensive metatheses were found, nor have
been found, in any organism that I know of, but there is a firm association of
these agents in the gametes, and only a few show metatheses in any one combina-
tion, and this is most important. The association of the agents, and the deter-
mination of their position and relation in the system of the gametic mass, receive
much-needed impetus from the recent developments in the study of sex-limited
and other characters in Drosophila by Morgan, and gives the first published,
clearly investigated data, upon this most important aspect of the problem.

No portion of the entire subject is of so great import as is this point concern-
ing the position and relation of these agents in the system of the organic mass,
and the relation of these to the possible dissociations and recombinations that
may be produced during gametogenesis, and the number of kinds of gametes
that are produced. This is the basic problem in the investigation of the gametic
constitution of organisms in the future, and has to do with the analysis of the
composition of the system of the organism, the position and relations of the
agents, and the capacities for the dissociation, recombination, and replacement
under standard conditions, and also under divergent constellations of environic
factors. There is no reasonable doubt but that there are dissociation, recombina-
tion, replacement, and that in gametogenesis there are different factorial com-
binations produced in the gametes, such that when in combination in the
zygotes they produce in one or more " characters " unlike results. That there
is the random sorting of all the agents in gametogenesis, giving chance distribu-
tions of all of the agents and equal numbers of all possible kinds of gametes, is
so highly improbable that it is not worth any consideration at all.

It is true that there is something acting in the production of gametes of
different kinds that gives wonderfully consistent and precisely repeated results,
and no doubt there is at the basis of the operations a series of phenomena with
which we are dealing in accurate operations, but of which our commonly
expressed conceptions are, no doul)t, far from the truth. The mere fact that the
verbal description of the reactions and results has been crude, particulate,
vitalistic, or otherwise objectionable lies entirely in the wording and formula-
tion of the operations as we have tried to think of the mechanism " in biological
terms," and not at all in the operations or results — a condition that is not
limited to this field alone. One needs but recall the first formulations of the
agents operative in the field of immunity for an example of a professedly untrue
picturing of the mechanism and of the agents, but with no question of the
existence of the agents and the certainty and positiveness of the resultant
reactions.

One of the most influential conceptions introduced by Mendel was the demon-
stration that the union of the gametes in fertilization was purely a chance one,
and that the resulting combinations or classes of zygotes produced followed
closely the expected array that results from the random union of the number
of classes of gametes present in equal numbers. It gave little chance for
" selective fertilization " and for " selection " or " choice " of any sort, pur-

Peoblems of Gametic Constitution 76

poseful or otherwise. As far as investigation has gone all instances show that
the combinations are purely chance, and although the use of " selective fertiliza-
tion '"' has recently been utilized in " explaining" some results of crossing, it is
not as a demonstration but as interpretation. It is safe to utilize for working
the hypothesis that all fertilization is chance and await the demonstration of the
instances of selective fertilization and the discovery of the actual mechanism of
the process.

One aspect of the fertilization problem that has not received the attention
that it deserves is the role of environic conditions to which the uniting gametes
are subjected in the production of changes in the operations of the zygote, and
even in the non-operation of some combinations. In this there is no selection,
only the physical inhibiting of action or development by conditions in the
medium, which, if the conditions persist, result in the elimination of the com-
bination, solely because it can not operate, and so ceases to exist. This is a vital
portion of the problem that has as yet been too little studied and may have much
to do in determining dominance and in other ways modify the action of the
combinations produced by the union of unlike gametes. The work of Tennant
on echinoderm fertilizations and my modifications of the behavior by means
of external conditions indicate some of the possibilities in this field. Further
data upon this same topic is presented in the following pages. It should be
considered as a possible agent in the investigation of " irregular " results in the
products of crossing, and it is not at all improbable that the condition of the
somatic tissues and fluids surrounding the gametes of internally fertilized
organisms might well play an important role in the decision of the direction and
character of the ensuing processes.

It is not unlikely that the further knowledge penetrates into these processes
the more frequently will the play of the conditions of the medium about the
point of reaction become of recognized importance, and this may involve the
entire organism in its medium, or the portion, or character in the system of the
organism, in which the surrounding portions and the external medium become,
in combination, the important and efficient agents in determinations of the
resultant reaction and its products. In evolution there seems, in the organisms
I have used, the best of reasons for utilizing this idea as one of the means of
attack upon otherwise difficult problems, and one that promises some measure
of profit.

While one can not overestimate nor fail to admire the work of Mendel and
its influence upon the developments of the past decade, all too often its broader
implications have been lost in the desire that has apparently actuated many to
make of it a cult, " Mendelism," as something apart from the rest of related
phenomena, and attempts are made to attribute to it many things not possible
from the very nature of its fundamental conceptions and means of operation.
Some adherents have, seemingly, at least tried to make of it a highly special
and permanent " force " in nature. This is most mifortimate. Mendel developed
a viewpoint, a concept of the nature of characters, a method of analysis of the
problems of constitution of the organism, and of the phenomena of heredity,
and out of these the neo-Mendelians, especially Cuerrot, have developed exten-
sions of the ideas of Mendel. Our understanding of the nature of the agents
will change from time to time; their terminology may alter, and the discovery

76 The Mechanism of Evolution in Leptinotarsa

and isolation of actual agents well extend our knowledge and conceptions far
beyond that of the present, but at the base there is a method of approach to the
problems that has permanence of usefulness and philosophical value which will
insure its retention in the methods of investigation. With this some may not
agree, but the past decade has moved far beyond the knowledge and insight of
the problems that Mendel had; but in all, the method of investigation, the
determination of the relations of the factors of composition to the products, or
characters, and the dynamic, mechanistic conception of these most fundamental
operations of organisms, in terms of the interaction of the combining materials,
are in essence the same as the formulation of the same problems of constitution
in the non-living world.

The problems of heredity as investigated by Mendelian methods lead directly
into some of the most fundamental portions of the general problems of the
constitution of living bodies, the system of organization, their development, and
transmutation in evolution.

THE GERM-PLASM CONCEPT.

The collective experiences of the last quarter century, and especially the
concepts that have been the natural outgrowth of the writings of Weismann,
have forced upon us concepts from which there is no present escape, namely, of
the existence of gametic material that bridges the gap between generations that
are constant in composition and organization, highly conservative with regard
to changes, which persist without alteration throughout long-continued genetic
lines of descent. Moreover, in allied lines of descent there are basal similarities,
with alteration of portions of the system, that give species, genera, and all of
the groupings of natural living things into the taxonomic divisions, into which
we are by custom prone to classify the objects of our study.

From many fields of investigation — phylogeny, evolution, experimental
embryology, genetics, and experimental evolution — consistent and mutually
confirmatory evidence supports the concept that this genetic material, the germ-
plasm, is not only remarkably stable in its composition and in its arrangement
into a physical system of action, but also that all changes of an evolutionary
character first occur in this material, by some alteration in its composition,
whereby the action thereof is altered in respect to some character or characters.
If therefore, any conceptions of the mechanism of evolution are to be had from
experimental investigations, it is necessary, first of all, that some exact knowl-
edge of the gametic system itself be had before much can be done in the attempt
to modify it experimentally. In other words, it is necessary to understand
something of the nature and composition of the essential materials with which
we are working.

In the attempts to determine this we are unfortimately compelled to deal
entirely with indirect analysis, in which the composition and structure of the
genetic substance is described in terms of its results, as observed in the soma,
and from these results project experiences backward into the germ-plasm, form-
ing such idealized concepts of its factors of composition and of its structure as
our perceptual experiences with the soma seem to justify. Indirect and unsatis-
factory as this method is, the methods now in use enable one to derive lines of
descent with stability of organization, and to repeat series of events, or to

Problems of Gametic Constitution" 77

produce desired combinations and results that are well within the expectancy
of error, thus showing that the method has at least some present value in
determining the nature of this substance, its factors of composition, its struc-
ture, and exact mode of action in specific combinations.

The conception of exact conditioning agents in the germ-plasm, so logically
and philosophically developed in Weismann's writings, and the proof of these
tliat came through the advances made after the recover)' of the investigations
of Mendel by the neo-Mendelian investigators, does not leave any doubt of the
existence in the germ-plasm of agents of diverse potentialities, whose presence
is necessary for the production of specific end-results in the soma.

The investigations of the neo-Mendelians show in an incontrovertible manner
that there are gametic agents whose action can be predicted with entire mathe-
matical accuracy ; but it has been asserted that these agents concern only super-
ficial and unimportant characteristics in the organisms investigated, and that
there is a basis that is not at all dissociated to which these agents are attached
as more or less unimportant additions.

It is undeniably true that the great majority of the characters of any one
parent entering into a mating not only retain their identity, but also their asso-
ciation with the total complex of characteristics that came from that parent, and
that only one or at most only a few are lost or transferred or replaced as the
result of the crossing. There is a retention of the specific parental gametic
complex of the species. Does this indicate a different material basis, or only
the retention of the integrity of composition and structure of the system, with
capacity to act for a time in union with some other system, but no ability to enter
into a permanent union therewith ?

In the crossing of natural species it has been my experience that the species
base retains its integrity with exactness, but with varying degrees from complete
integrity, giving only exact and typical monohybrid reactions and the extraction
of pure parent types in F^ through series in which one or two or a few char-
acteristics were interchanged, giving new synthetic extracted types in subse-
quent generations to complex cases in which there was much interchange, and
lastly to most intricate examples in which as far as could be determined new
and permanent combination had been made between the two uniting gametes,
with loss of many portions of the two parental systems, giving rise to new com-
binations that were, as far as could be determined, permanent in character and
action. In these series of experiences, details of which are given in the chapters
that follow, I have not been able to draw any line of demarcation, on one side
of which is the species base and on the other the superficial gametic agents that
are capable of interchange or loss. As far as my experiences go, it seems the
most logical concept that if the factorial working of these basic phenomena of
organisms are true in part they are also true in all characteristics of the organ-
ism, and that the germ-plasm must be thought of as a physical system with
exact composition and structure, precisely as in the non-living materials of the
planet, with differing degrees of capacity for dissociation and metathesis of
factors. This capacity for dissociation and metathesis is not alone due to the
nature of the specific form, but also to the reactions and the conditions of
reaction to which the complex is subjected. That is, in one set of reactions
under one set of conditions, there may be complete retention of integrity, as in

78 The Mechanism of Evolution in Leptinotaesa

the cross of L. signaticollis and L. diversa, giving a simple monohybrid reaction,
with no interchange of characters, while both species in other combinations
show interchange of characteristics, and L. diversa and L. decemlineata form
at the other extreme a fixed combination, composed of elements from both
parents which it has not been possible to dissociate.

It therefore appears to me that capacity for dissociation is not necessarily a
property of the basal species complex, but rather due to the reactions to which
the parental gametic masses are subjected, both in the way of the combination
in which they are united, and also depending upon the conditions of the medium
under which the union or reaction is carried out. In this there is no difference
in principle from the union of non-living materials Avhere the nature of the
reaction is entirely a product of the combination made, and the conditions under
which the reaction is set to operate.

This capacity of the two gametic systems to unite for the production of a
heterozygote, acting in harmony for the production of a soma, with two parental
groups of potentialities which attain a balance for the space of a single genera-
tion, but with no permanent capacity for union, with the total separation of the
parental systems in gametogenesis, presents no end of fascinating problems for
the embryologist and geneticist, but concerning which there is at present entire
lack of knowledge.

THE GAMETIC AGENTS.

Since we must admit the existence of specific gametic agents, or factors of
composition, and since there is entire probability that the total sum of char-
acteristics of the organisms are conditioned by the factors of composition
arranged in a system, what about the agents themselves? Are they discrete,
invariable units, present or absent in their totality, or do these agents represent
centers of activity, foci, of greater or less magnitude ? The Weismannian con-
cept of fixed units was adopted bodily by the neo-Mendelians, and much the
same idea is retained by De Vries in his mutation theory ; but the fixity of these
units seems to be a constant cause of trouble to Mendelian workers ; hence the
concept of multiple allelomorphs and kindred concepts developed by Bateson
and others, all with the purpose of retaining the concept of the fixed, quantita-
tive unit factor as a static morphological element of structure.

All of our ideas upon this point are based upon our perceptual experiences
with the soma ; certain body-characters appear or are absent in their totality in
a given cross ; it follows, therefore, that each is due to a factor which we sym-
bolize as A or a, and unless further investigated each is asserted to be a unit
factor, an entity. In some other combination, A is shown to break up into two
or more portions, and it is therefore a compound allelomorph ; but where is the
limit of this compounding? In the first experience the action was a unit or
center of activity, while in the second experience a hitherto single center of
action, divided into two, each acting independently as far as the end-results are
concerned; but what evidence is there from these experiences that we have
reached the limit of divisibility ? Further, what logical basis is there in this
perceptual experience for the assertion that individualized entities have been
discovered or isolated in the operations, or shifted therein ? Conclusions as to
the unit nature of the agents in the gametic material productive of these
observed results in the soma are based upon the a priori assumption that organ-

Problems of Gametic Constitution 79

isms are composed of ultimate " biological units of structure " which must be
thought of in the same manner as Weismann conceived of his biophores. Any
such idea is essentially vitalistic, for the reason that the unit is in all respects
a living individual, and between it and the non-living there must be a sharp
division, or else we must think of more remote agents of composition, and so on
without limit.

On the other hand, if we think of the observed results produced in the soma
merely as products of gametic reaction centers of a purely physical nature, no
difficulty is introduced into the interpretation of the observations, because there
are in physical phenomena ample evidences of the fragmentation of reaction foci
into two or more discontinuous and independently acting centers, which may
be permanent or transient, depending entirely upon the surrounding materials
and conditions in the medium.

The basis of the ideas with regard to the fixity of these ultimate units of
structure and activity was derived first from the interpretation of nature and
evolution phenomena, and later by the neo-Mendelian experiences in crossing,
in which morphological considerations dominated the logic and philosophy of
the writers, developing dogmatic definitions and concepts of the discrete, dis-
continuous nature of both agents and of somatic characters. The neo-Mendelian
observations showing the alternative transfer of characteristics is no criterion
for the deduction that the agents are discrete and exist as units. It is con-
venient to symbolize them for purposes of description, but it must not be for-
gotten that it is only a symbolization and nothing more. In organisms we are
not dealing with static masses, but with highly complicated integrations of
material, in which endless physical and chemical reactions are in ceaseless
operation. In this mass there are innumerable centers of activity at all times,
in the germ as in the soma, and as the result of experiences with the materials
that I have had to study, I can only regard the gametic agents as expressions of
centers of activity of greater or less magnitude, bound into a system of opera-
tion w^hich in the main retains its totality of manifestation throughout successive
generations ; but this retention of uniform aspect is not so much the product
of its structure and composition as it is that it has in its successive generation
cycles the same kind of materials to react with, and also essentially the same
conditions in the medium in which to carry on these reactions ; therefore repeti-
tion of reactions and results must naturally follow.

The fact of the perceptual experience of the metathesis of characters in cross-
ing seems to me no evidence of the transfer of fixed units of any kind. The
same gametic system, L. signaticollis, in one combination acts as a unit, in
another combination it acts as two, or three, or more, depending upon the losses
or replacements in its minor portions. Moreover, in my materials I have not
been able to decide where the lower limit of unit manifestation was to be drawn
in any instance. For example, in the complex pronotal pattern it may behave
in crossing as an entity and may be symbolized as a unit of activity, or specific
and trivial elements of it may show this same capacity for metathetic action,
giving typical ]\Iendelian reactions, and discontinuity of the somatic manifesta-
tions, and stable extracted products, in F,, and in no instance have I been able to
satisfy myself from experimental evidence that I had attained the limit, present
in the materials for the production of this alternative, discontinuous type of

80 The Mechanism of Evolution in Leptinotaesa

activity in process and products. I see no escape from the conclusion that there
are not fixed and discontinuous units in the gametes, but rather that the results
that we observe in the soma, and with which we are able to deal in accurate
analytical and synthetic operations, are after all nothing more than reaction
systems of greater or less magnitude, capable of endless fragmentation, depend-
ing upon the nature of the materials and the conditions of the medium at the
time of combination. Each of these portions of the system as we observe its
somatic manifestations is symbolized for purposes of description, but is no
warrant for the concept of the existence of final indivisible units of any sort,
nor for discontinuity between them. In the material composition of the gametes
there can be no other discontinuity or isolation of the factors of composition
than that which exists in any physical substance or in a complex mixture of
substances.

At present there is no evidence as to the nature of any gametic agent, and it
is quite possible that factorial differences may be due not only to actual differ-
ences in component materials, but also to differences in molecular arrangement
or to changes in reaction directions or in velocity. In fact, there are so many
possible modes of the production of factorial differences in materials that are as
complex as the living substance of the gametes that it is hardly worth considera-
tion at the present.

The location of the different agents in the gamete is, and is likely to remain, a
question of debate for a long time. Cytological considerations and argument
have for many years placed all of the " bearers of the heredity characters " in
the chromatin, or at least in the nucleus, but there seems to be increasing evi-
dence, especially in the field of experimental embryology, that the cytoplasmic
portion of the gamete, especially of the egg, is also the bearer of hereditary
characters. These relate especially to form and symmetry, or to kindred deep-
seated phyletic qualities of the species.

Depending upon the nature of one's perceptual experiences with these prob-
lems of organic phenomena and the initial philosophical viewpoint hinges the
concept of the structure of the germinal material that we create.

METATHESIS OF GAMETIC AGENTS.

Certain facts with regard to the behavior of characteristics of the parent
lines in crossing are becoming increasingly evident and certain. (1) Somatic
differences result from the combination of gametes of unlike potentiality in the
same character manifestation, and that out of many such combinations there is
interchange of the characters as far as the somatic manifestation is concerned,
and, therefore, we conclude interchange in the gametic masses also, and evi-
dence, from the derivation of pure stable lines in Eg showing the interchanged
characteristics, is from many observations in plants and animals, regarded as
proof that some gametic change has taken place. This is best described and
interpreted as interchange of gametic agents, (2) In all observed instances of
metathesis the interchange is always between equivalent characters of the same
kind, and occupying in the organism the identical location and manifestation ;
that is, flower characters of form or color or pattern interchange, leaf form or
plant habit interchange in plants, while in animals it is coat color, pattern,
activities, and so on of the same position and time of expression in the life of

Problems of Gametic Constitution" 81

the individual that cross over from one to the other of the two parental gametic
systems, while the major proportion of the features of each parent retain their
integrity and come out in gametogenesis, either pure or with interchanged
characteristics.

This regular interchange of characteristics, the typical Mendelian reaction,
is now so well established in many plants and animals from the observations of
so many independent observers that there is no question but that it represents a
general type of activity in the reactions of the germ-plasm in all living forms.
It, moreover, is the mechanism whereby much heterogeneity is produced in any
population of organisms.

The essential problem is, however, what is the cause of the interchange of the
gametic factors, and why is it so regularly between equivalent productive centers
of action in the germinal material ? In my materials, why does L. signaticollis
show no interchange of characteristics when crossed with L. diversa, and inter-
changes when combined with L. undecimlineata? In the first combination the
entire gametic mass acts as a unit, and in all cases retains its integrity and
identity through all operations, in all its qualities and attributes, while in the
second there is interchange of larval pattern and larval body-color, each as
independent units, as the regular behavior, and, under certain conditions of
combinations, also show interchange of characteristics of the antennae, pronotal
pattern, sculpture and so on.

That the observed conditions are due to any chromosome behavior, at least
any that is now known, seems to me highly improbable. That the interchange
is due to difference in gradient or in potentiality there is no evidence. More-
over, if it is some sort of chromosome reaction, produced by the breaking of the
rods in synopsis or at some other time in gametogenesis, what is the mechanism
that produces the regular random interchange and the mathematically regular
classes of gametes ?

It seems to me, from the many instances that have passed through my hands,
that the operation is not that of any morphological action, but of some chemical
transposition of materials between the two gametic masses and to be a reaction
rather typical of ordinary metathesis of the type common in chemical com-
binations, i. e., 2HCl-l-CaC02 = CaCl2-f HjCOg. In other words, the gametic
materials of one parent, in certain combinations, through some purely chemical
affinity, have stronger attraction for the position held by an equivalent com-
ponent than for the original position, pass over to the new, displace the second,
and occupy its position, while the second passes back to the position occupied by
the first. Or it may be that the conditions in the united gametic masses, before
their final separation, set free all of the agents of this one sign which are then
free in the germinal material, and later attain to a, purely chance distribution in
the gametic compositions, giving essentially equal numbers of the different com-
binations of the two gametic bases and the transposed or one-time dissociated
agents.

However it is attempted to symbolize the nature of this reaction, the fact of
there being some reaction that is productive of the differences in perceptual
experience with the different somas produced is beyond doubt. The most logical
interpretation of the gametic portion of the phenomena, upon the basis of known
chemical or physical bases, is that there has been actual passage of centers of

83 The Mechanism of Evolution in Leptinotarsa

activities of greater or less magnitude between the parental gametic systems,
and represented by integrated substance of specific composition, with specific
capacity of reaction when combined with some one or more complementary
agents in its new position.

The most striking fact in this metathetic reaction is that the agent always
goes over to a precisely similar position, mode, and time of somatic expression,
or, in other words, it goes to a basal point of attachment in the new gamete,
where it finds or establishes physical relationships that hold it firmly in position
and in whole or in part govern its reactions in the development of the zygote
which it may help to form.

Another striking fact of these reactions is the uniform finding that the
metathetic reaction is always between qualities having the same general type of
manifestation, as interchange of color, of form, of pattern or symmetry, and so
on, and never is there interchange of a color character with one of form, food-
relations, or reproductive activity, so that we are coming to see that there are
groups of these agents and of their associations, and that the Mendelian reac-
tion is between differences in these groups, and not between different groups.

A second point that has been forced upon my attention is that it is the deter-
mining agents, the determiners, that are chiefly, if not exclusively, concerned
in this metathetic reaction, while the compliment or factor in my materials
never shows this type of reaction. Further, the factor is in my experience
always associated with the basal species complex, firmly bound into one associa-
tion, productive of the basal species complex. What these are in the composi-
tion of the gametic material no one knows, but their reactions, as I have
observed them, suggest that they are possibly different colloids in the general
colloidal mixture that forms the ground-substance of the living mass, and of
this interpretation support is found in the fact that in my materials these factors
are properties of the whole mass, and so strongly suggest a general distribution
throughout the entire germinal material.

The determiners, on the other hand, are numerous, and are the agents that
show not only metathesis, but also capacity for fragmentation, groups of them
being dissociated in one case or lesser groups in another, and in this class of
agents many interchanges, from extensive on the one hand to increasingly
minute on the other, have been possible in my materials.

These agents do not behave as properties of the whole mass, but in relation
to specific portions, locations, in time, in ontogeny, and in relation to specific
activities, and appear to be associated with basal material points of attachment
that are specific for the source from which the gametic material came. These
latter seem not to be dissociated from the original mass in the usual metathetic
reactions, but to maintain their association with the species complex, inter-
changing only the specific portions that are productive of localized determinative
actions.

These experiences with my materials, and the data that has come from the
investigations of the neo-Mendelians, lead directly into the problems of the
architecture of the germinal material.

Problems of Gametic Constitution" 83

ARCHITECTURE OF THE GERMINAL MATERIAL.

The concept one forms of the germinal material depends to no small degree
upon the nature of one's underlying philosophical viewpoint, and to a lesser
extent upon the nature of one's perceptual experiences with organisms, whether
descriptive and morphological, genetic, developmental, physiological, vitalistic,
or mechanistic, with plants or animals.

With those who look upon living substance as something different in kind
from the other integrations of the planet I have nothing in common. From
what we know of this substance upon this planet I see no reason for thinking
that the same order of cosmic phenomena are to be found upon any other, nor
any reason for supposing that the living materials are anything other than the
natural geological consequences of a zone of great and constantly changing
stresses between the atmosphere and lithosphere and living objects, but the
complex integrations of the materials and forces acting in ceaseless integrations
of matter and changes in energy in this contact zone of intense metamorphic
activity.

I must look upon organisms merely as a geological-stratum complex, ever
changing in thickness and intensity of manifestation, presenting rhythmic alter-
ations with the astronomical rhythm of the seasons, and in longer periods as
earth forms change in geologic activities, and I must conceive of " life," its
origin, activities, and evolution, as due to the same physical and mechanistic
forces of action and result as are found in other earth phenomena, only more
complicated and labile.

Through all the history of the planet there has been, as far as there is
evidence, since the beginning of living things, genetic continuity of a germinal
basis, relatively minute in quantity, wonderfully complex in its potentialities
for producing almost endless secondary manifestations under appropriate con-
ditions of the medium. Of this genetic continuity, first clearly put forth by
Darwin, and of the existence of a genetically continuous germinal material basis
so clearly presented by Weismann, there is no doubt. In this aspect, often
termed self-perpetuating, this product of the planet differs from all others, but
is not of itself self -perpetuating solely — only to the extent that it has presented
to it in the zone of its action to requisite conditions of atmosphere and litho-
sphere, plus the larger astronomical relations of the planet, to produce the
necessary constellation of conditions for the phenomena we call development
and growth.

There is no escape from the concept that evolution and all organic activities
are conditioned and governed by the relations of this germ-plasm to its environ-
ment, and that understanding of the many phenomena that we and all other
organisms possess and show depend upon the discovery of the nature of the
germ-plasm and of its relations to the different environmental factors that
provide for its secondary or somatic modifications and for its metamorphosis
or evolution.

Many concepts of the architecture of this material have been formed; no
doubt many more must receive consideration before its nature is comprehended.
Moreover, the problem is not the same throughout all living things, having one
aspect in plani^, another in animals, and different aspects within both of these

84 The Mechanism of Evolution- in Leptinotaesa

two main divisions of organisms. The remarkable specialization and localiza-
tion of this material in higher animals has, no doubt, tended to restrict and
codify our conception of this material, to limit it unduly ; but in plants many
portions of the plant tissues, serving a purely somatic role, may on the advent of
a proper environmental reaction regulate, and from a purely somatic aspect
develop, divergently into the buds and the production of gametic masses or
germ-cells. In the development of the plant there had been specialization along
somatic lines, perhaps development of special features of structure and function,
but not complete loss of the capacity to recede from this position, to regulate,
return to an initial position, and produce buds with the proper specialized germ-
cells characteristic of the species. With higher animals this regulatory capacity
seems to have been lost and only that portion of the zygote that enters into the
formation of the gonads goes on into the next generation, while opinions differ
as to what really happens in lower animals. In all, however, there is the pro-
duction, by one means or another, of the necessary germinal masses or germinal
materials to carry on the specific combination of qualities and attributes of the
genetic lines of descent.

The development by Weismann, Nageli, De Vries, and others of the concept of
a germ-plasm, idioplasm, and so on within the mass of the germ-cell, resident
perhaps within the chromatin of the nucleus, is entirely an anticipation of
nature and not even an interpretation.

The independent position given to this germ-plasm in Weismann's hypothesis,
a microcosm within the germ-cell, the biophores being essentially living indi-
viduals, is a vitalistic conception not open to investigation, and, therefore, hardly
worth consideration until there is some exact experimental demonstration of its
reality. Whether the germ-plasm is a special portion of the reproductive cell
or whether the whole germ-cell is germ-plasm, is at present a matter of a priori
opinion, but it seems most naturalistic and reasonable to think of the whole
germ-cell as having germinal potentialities.

The primordial germ-cells, through the division periods, seem to me to repre-
sent the real germ-plasm in its simplest condition, and are usually assumed to
have the same potential composition as the zygote in which they are located, and
no doubt are so, at least at the start. The organization and development of the
growth period are, however, it seems to me, the initiation of ontogeny, which
progresses for a certain time, but as a rule goes no further without fertilization
or the influence of some external agent, in order to set up the reactions neces-
sary to the series of ontogenetic reactions. Loeb's discoveries and those of his
followers with artificial parthenogenesis show most clearly that external physical
agents can provide the necessary impetus for the egg to develop without union
with some other germinal mass. These results that have come from the investi-
gations of artificial parthenogenesis show that development is not independent
of external motive forces, but is entirely dependent thereon, either through
normal fertilization or the action of purely physical forces, or both.

Since the discovery of the crude aspects of the mechanism of fertilization, the
union of the pronuclei, we have assumed that the gametes were the same in their
hereditary potentialities ; but the facts of artificial parthenogenesis show clearly
that at least they are not the same in their development and it is not impossible
that they may also be different in their hereditary or germ-plasm potentialities.

Peoblems of Gametic Constitution 85

No sperm is able to develop at all parthenogenetically ; many, possibly all, ova
are so able to develop. What is the significance of this from the standpoint of
germ-plasm constitution ?

In the terminology of hereditary reactions the two germ-cells are asserted to
be equivalent and to carry essentially the same potentialities or agents. Is this
really so ? What evidence is there for this assumption, aside from the pairing
of relatively trivial characters ?

In the crossing of widely divergent types it is, as far as I know, entirely the
maternal parent that finds expression in form, symmetry, rates of development,
and all general properties ; and only in the special determinative type of action
in localized portions that the paternal type shows its influence. In my materials,
where I have had, in the crossing of species, many examples of this dominance
or prepotency of the female type in widely separated species, the segregations
in F2 have shown, not the presence of the paternal factor, but of the maternal
factors combined with the paternal determiners, so that the segregated line, like
the female parent, was the same as the original stock ; but the segregated male
type was quite uniformly not the same as the original type, but differed in con-
crete respects. In other words, experience with the materials I have used gives
no indication that the male gamete carries any of the general factors of its
species, but rather the determiners — that is, the female gamete carries both the
essential groups of agents, its specific factors and determiners, while the male
gamete carries mainly, if not exclusively, the determiner complex. An egg can
develop without fertilization ; a sperm can not ; and may this not be the reason
for the decided difference of the two as regards their capacity for development
parthenogenetically ? If it be assumed that the two are equivalent in constitu-
tion as regards their gametic composition, then why wall eggs develop par-
thenogenetically and sperms will not? Further, if both are the same in com-
position— that is, carry all the gametic agents proper to the species — why in
widely separated materials is it always the maternal parent that is dominant in
form, rates of development, and symmetries, the male having influence only in
the determination of special characteristics that may be common to both species ?
It seems to me that the question of the equivalent composition of the two
gametes has been too commonly assumed to be so, following hypotheses now
more or less outgrown.

Following Weismann and others, we have been prone to think of the germ-
plasm as a specific single kind of material with organization into some fixed
system, with the different " determiners " occupying in it a specific position,
like the side-chains in some complex molecule and its symmetries compared to
crystalline organization. My experiences have led to the view that it is not a
single substance, but a mixture, or solution of substances which, like many other
mixtures of material, shows specific symmetries and special organization char-
acteristics. The evidence for this conception comes from experiences with the
crossing of different species and the showing that there are different groups of
activities, the agents in each group interchanging with similar agents in
equivalent groups and with no other.

The following scheme of composition and classification of the agents in the
germ-plasm seems to hold throughout my materials and presents, as far as sym-
bolization will permit, the ideas that have been developed with regard to its

86

The Mechanism of Evolution in Leptinotarsa

architecture. I distinguish two major types of agents : basal agents, which in-
all experiences are properties of the whole type and do not segregate in any of
the normal reactions incident to development or in crossing, thus retaining their
integrity ; and definite agents, or those that are specifically concerned in the
determination of the specific aspect that some quality presents. Of the basal
agents there are clearly two groups, those that I have called the basal factors,
few in number, and always properties of the whole, never altered or inter-
changed, and which may be the possible mixture of ground-substance colloids
that seems to be the basis of the cell organization, and chromatic receptors, the
material substratum upon which the great majority of the determining agents
seem to find their distinctive seat of localization in the gamete. Of the definitive

Table 2.

1..
2..
3..

4..

9..

ES

Vo.
Ac.

Mm.
Lm.

Basal gametic agents.

Basal factors.

PFF (phyletic form factor)
POF (ontogenetic factor)
PMF (metabolic factor) . .
PNF (neural factor)

PSF (sex factor)

PPF (pattern factor)

MCF (melanoid color fac-
tor)

LCF (liquid color factor)

SCF (surface color factor)

Chromatic
receptors.

rCFR'

\CFR"

r COR'

\COR" (?)

rCMR'

\CMR" (?)

rCNR'

\CNR" ....
"CSR'(?) ..

CSR"

rcpR'.....

\CPR"

rCMR'

\CMR" (?)

f CLR'

ICLR" (?).

rCSR'

\CSR"(?) .

Definitive gametic agents.

Chromatic determiners.

Fd.

An., At., Aa., Elt.

Od.

None found.

Fo.

None found.

Ct., Tr., Hr.
None found.

Op., Rr ^Hi.

Sx.

JS JS JS JS Ad.

Hd., Pr., Th., Ab., Hw., Elp.

Bm., Brm., Ylm.
None found.
Wl., Yl., 01., Rl.
None found.
Gr., Bl., Vi.
None found.

agents there are two groups — a small one, always associated with the basal
factors and having a distinct action as determiners of action, the cytoplasmic
determiners ; and the chromatic determiners, which are by far the most numer-
ous and the agents that commonly interchange in crossing. Of these four
classes I am of the opinion that the basal factors and the cytoplasmic deter-
miners are located in the colloidal matrix of the gamete and the chromatic
receptors and determiners in the nucleus.

Of the basal factors I have found nine such cases of activity, each concerned
with certain general types of activity in the organisms, and with which specific
determiners act to produce any given result. I have no evidence of their segre-
gation or fragmentation, nor any evidence of their having been modified in any
experimental operations to which I have subjected my materials. The combina-
tion of these nine basal factors constitutes the phyletic base — that which in the
constitution has been likened to a nucleus, and in a certain sense it is a nucleus
of potential activities, needing only the special definitive agent go give expres-
sion to some concrete manifestation in the organism.

Peoblems of Gametic Constitution

87

Table 3.

Chromatic receptore,
corresponding to so-
matic receptors.

Chromatic determiners and arrangement in groups as discovered in experiment.

Istgr. CPBS* ...

2d gr. CPBS' ...

3d gr. CPOR» ...
4th gr. CPOR'.
5th gr. CPMR^ ..
6th gr. CPMR^

7th gr. CPNR» ..

8th gr. CPNA^

17th gr. CPSRi ...

18th gr. CPSR» ...

9th gr. PPRi ....

10th gr. PPR» ....

Fd Body form index determiner.

An Antenna form determiner.

At Antenna Tolteca type determiner.

Aa Antenna Azteca type determiner.

EIT Elytral tracheal pattern determiner.

EIP Elytral pattern determiner (sculpture).

Od.

Fo Food determiner.

Ct Chemotaxis (sex) determiner.

Tr Temperature reaction determiner.

Hr Humidity reaction determiner.

Oh Ovoposition habit determiner.

/ B 1
Rr 1 I Reproductive rhythm determiner \ hibernate

Hi Hibernation determiner.

Sx Sex determiner.

J» First larval pattern determiner.

J p Third larval pattern determiner.

Ad Adult pattern determiner.

Hd Head pattern determiner.

Pr Pronotal pattern determiner.

Th Thorax pattern determiner.

Ab Abdominal pattern determiner.

■ Elp Elytral pattern determiner.

Hw Hind wing pattern determiner.

Pu Elytral punctation determiner.

V Shape determiner.

Factor. 1 Determiner.

MCR — Melanin cc

)lor receptor

f B. = Black determiner.

- Br. = Brown determiner.

( Ylm = Yellow melanin determiner.

f Wl = White lipoid color.

J Yl = Yellow lipoid color.

1 01 = Orange lipoid color.

[ Rl = Red lipoid color.

f Gs = Green surface color.

\ Bs = Blue surface color.

[ Vs = Violet surface color.

Hd = Pattern amboceptor.

Pdd.

Thd.

Abds, abdap, abdp, abdt.

Abds, abdap.

LCR = Lipoid cole

SCR = Surface col
PPS = Phyletic pa

Pl = Lar
Pc = Pui

Pa = Adi

POS = Phyletic on
PRS = Phyletic re
PMS = Phvletic m
PSS = Phyletic se

>r receptor

or receptor

ttern system:

fHd...

Pr
val pattern factor -l -pi^' ' '

1 Ab!!.'

Hd...

Pr
)al pattern factor >, „j-' ' '

1 Ab!!!
fHd...

lit pattern factor - ^h

Ab!!!

togenetic system,
action system,
etabolic system.
X system.

88 The Mechanism of Evolution in Leptinotaesa

Of the somatic determiners I have found only three thus far, although further
study may no doubt show more. Corresponding to each of the basal factors
there are in some instances two chromatic receptors, each with its determiners.
For three of these basal factors two chromatic receptors have been identified,
each with one or more determining agents, and for the remaining six, one
chromatic receptor with its determiners. The chromatic determiners are
numerous, about 50 such major agents having been found, many of which are
obviously compounded of many minor agents, or at least are capable of frag-
mentation and extensive modification through different processes.

It is perhaps suggestive, certainly not conclusive, that the number of basal
factors, 9, corresponds with the reduced number of chromosomes in the gamete,
and that for some of these there are two groups of chromatic agents, which, if it
is true for all the basal factors, would give 18 such groups, which is the %X
number of chromosomes in the zygote, so that there is possibly some real rela-
tion between these findings ; in fact, I am at present inclined to the hypothesis
that there is a rather specific relation.

It seems to me that the best interpretation of the findings is that the com-
monly dissociated and interchanged agents are, as Morgan shows in Drosophila,
probably located upon the chromosomes. The apparent number of basal factors
in Leptinotarsa, 9 (with the two pairs of interacting groups of agents for three
of them) suggests, but by no means proves, rather important relations that
further investigation may make certain. In tables 2 and 3 I have given a list
of the different agents and the symbols used in their description in the follow-
ing pages :

I picture the gametic material, then, as a mixture of substances, each more
or less independent and concerned in special manifestations of the whole, result-
ing through the numberless interactions in the correlation of form and activi-
ties, qualities, and attributes in local and general characters, producing the
total array that we know. In many aspects it appears to me not unlike many
another terrestrial stratum — a mixture of different materials, yet presenting
generic characteristics and endless minor specific differences. Granites, com-
pounded of certain substances, all have generic likenesses of form and structure ;
but with the differences in proportion of the factors of composition, with the
varying action of the conditions of the mediurn at the time of combination, or
by the press of conditions after the original production, produces granites of
many kinds. May it not be profitable for a time at least to view organisms in
much the same manner as a product integrated in the contact zone between the
atmosphere and lithosphere as purely physical and mechanistic productions of
the geological and cosmic forces incident upon the planet's surface? At the
least I have tried to show the standpoint from which I must approach the prob-
lems and the concept held of the nature of the gametic material or germ-plasm,
which is the basis of organic evolution and activity. I can not but feel that in
the intensity of modern life and its necessary specialization we have become
too intensely biologists, too little natural philosophers; that in the specializa-
tion, so characteristic of our period, there is unfortunate liability to lose sight
of broader relations and points of view, and in the restriction of activities to
narrower and narrower fields unintentional error and misconception unwit-
tingly creeps in. The naturalistic point of view is true in part, it must be in all.

Problems of Gametic Constitution 89

and there must be common concepts, underlying agents and processes, methods
of production and evolution or transmutation, that should be conceived of and
pondered much ; otherwise, individually or collectively we are soon lost in some
intellectual cul-de-sac, from which escape is difficult.

In the presentation of the data of this attempt to determine the composition
of the gametic material,* I shall present first the results of interspecific crosses,
first those of the simplest reactions, later those of more complicated characters ;
second, the findings in intraspecific crosses, especially the experiments that have
showTi the nature of the chromatic determiners and their various capacities for
fragmentation ; and at the end attempt a further analysis of the general aspects
of the findings and their bearing upon the problems of germ-plasm constitution
and architecture.

* The complete account of the gametic analyses made with these materials is in
process of preparation, to be presented in a separate report.

CHAPTER IV.
REACTIONS AND PRODUCTS IN INTERSPECIFIC CROSSES.

I have had to deal with many interspecific crosses, and these I have uniformly
treated as follows: Species freshly introduced into the laboratory were first
acclimatized to the conditions of the breeding-quarters, which were the average
conditions of the breeding-season of their native habitat. It was found that
3 to 6 generations will suffice to fully adjust any wild stock to the conditions of
the breeding and experimental rooms. In this way I eliminate in crosses
between species many of the disturbing effects of conditions in the material,
" adaptation " to one especial complex, and thus make the crosses the product
of the constitution of the organisms as completely as is possible at present. I
was forced to do this, because I found that species direct from diverse habitats
in nature were giving most irregular results that were difficult of repetition and
analysis.

It may be objected that this treatment is abnormal, and that it is productive
of change in the species ; but none has been found, and only those species are
used for these interspecific crosses that are able to completely adjust themselves
to one environmental complex. The mere fact that they are not able to do so is
all the indication needed to exclude them from crossing experiments where
effects of environment are to be eliminated. It has been my experience that
species so brought into the same set of conditions do not change ; do not lose
any capacities or characteristics, are not weakened, and when returned to the
original specific native environmental complex are able to take up at once the
original routine without effort or waste of individuals or energy.

METHODS OF CULTURE.

The most essential factor in the successful breeding of animals of the kinds
that I have used — phytophagous Coleoptera, Lepidoptera, and Hemiptera — is
the constant presence of a plentiful supply of perfectly fresh and normal food,
under conditions such that the normal transpiration of both plant and animal
can go on, and with adequate air-movement to remove from the vicinity of the
organisms the air contaminated by the products of respiration and transpira-
tion. It has been my experience that unless this is attained in a complete and
constant manner trouble will inevitably follow in the cultures — disease,
weakened vitality, with the probable loss of the culture and termination of the
work.

In all experiments it has been my first care to have the organism, regardless
of the kind upon the specific living plant, which is the normal food for the
species in the location from which the material originally came. The plant
must not only he alive, but in the usual actively growing condition, as in nature,
so that the organisms may have an adequate, normal, and hygienic food supply.

90

Fig. 1. — Photograph of breeding-cages used for pedigree ciilturcs. showini: arrangement
of the cage, the large earthen pot. and the plant used as food, the use and relations of
which are described in the text.

Keactions and Pkoducts in Inteespecific Crosses 91

Cut food wilts in a short time and with especial rapidity under high tempera-
tures or under arid conditions, and attempts to keep the food fresh by placing
it in water end in failure or in indifferent results at the best. When a healthy
growing plant is used as the food, the animals always breed and lay their eggs
exactly as in nature, and in my experiments over 90 per cent of the fertilized
eggs laid (by actual count) hatch, and the freshly emerged, delicate larvae can
at once begin feeding in a perfectly normal manner without in any way being
molested or injured.

The most satisfactory type of cages thus far devised for this work consists of
a light, strong drum of fine wire-netting (pearl wire, No. 23 mesh), of which
the sides and top are of wire and the bottom of galvanized iron, with a sleeve
that projects down into the earthen pot that serves as the base as 8ho"\vn in
figure 1. The pots that I have used are 12 inches in diameter and hold a con-
siderable volume of earth, in the center of which is placed the food-plant in a
smaller pot. The breeding-cages are uniformly 18 inches in diameter and are
either ] 8 or 24 inches in height. A cage of this character is used for each mated
pair and for their eggs and young progeny. With this arrangement the breeding
pair has normal, healthy, and fresh food, proper conditions for egg-laying, and
imcrowded space about them. They have for all practical purposes natural
conditions for their breeding activities.

I allow one pair to breed in such a cage, and when the progeny are half grown
they are put into sorting-cages in which the same construction is employed,
excepting that they are only 12 inches in diameter (fig. Ic) ; though if the
progeny are especially abundant or valuable the parents are removed to a fresh
cage to breed further and the first 100 or 200 are allowed to complete their
ontogeny in the original cage. As soon as the food-plant is defoliated a new one
is easily put in its place.

Entrance to the cages is had by means of a sliding door in the side, large
enough to permit the passage of an 8-incli pot and food-plant. This type of
breeding-cage has the advantage that it is self-sustaining, and needs but little
daily attention aside from the routine spraying of the plant and the adding of a
regular amount of water to the soil in the pot. The mechanical labor of the
project is thus reduced to a minimum, with constant efficiency of operation.
With these arrangements I have been remarkably free from epidemics of disease.
The only epidemic that I have had to combat was not a very serious one. It was
introduced during my absence and was not recognized by the assistant in charge,
because of his never having encountered anything of the kind before.

The larval stages are thus passed under optimum and normal conditions and
they pupate in the soil of the same cages in which they pass the latter portion of
the larval period. As a rule it has been found advantageous to keep the later
stages in the sorting-cages, which occupy less room and leave a larger number
of breeding-cages free for the mated pairs. After pupation the cage is watched
with care to keep the soil at the optimum condition as regards moisture, and
also for the adults to emerge. These are isolated at once, the males from the
females, in separate sterile cages, and given living food as in the breeding-cages.
This separation is made two or three times daily during the period of emer-
gence, within 1 to 4 hours after emergence, and before the post-pupal develop-
ment has been completed. The emerged adults are without exception allowed
to remain in these cages, with the sexes separated from one another, from 5 day&

93 The Mechanism of Evolution in Leptinotarsa

to 4 or 5 weeks before they are mated for the next generation. This period of
feeding and inhibition from breeding gives the stock an opportunity to become
fully nourished before they begin the breeding activities, and also serves to
eliminate the weaker members of the population, which will uniformly die
within a few days of emergence. This procedure insures an almost certain and
uniform breeding from strong and well-nourished individuals, with the result
that inbreeding can be practiced to a far larger extent than if the more usual
custom were followed of mating the individuals as soon as they were ready to
breed. Another advantage of this method with the sexes isolated and upon
normal fresh growing food is that the stock can often be held for months with-
out serious consequence ensuing. It is thus possible to breed part of the popu-
lation, hold the remainder in reserve for 3, 6, or even 10 months, and thus be
able to check the first matings or to make other combinations not made at the
start. This is of immense advantage in the working out of the results of
hybridization.

All of my pedigree work with tropical species has been done in large glass
houses, with high walls, giving a large volume of air, in which the temperature
was controlled automatically, and arranged to correspond with the average
night minimum of the growing season in the natural habitat of the species
undergoing investigation at the time. The maximum at mid-day is of less
importance and in the summer months was variable, in the sun often from
29° C. to 35° C. and in the shade from 26° C. to 30° C, which is comparable with
the natural conditions found in nature in the tropical regions from which the
material came. The humidity was controlled either by the amount of water
available for evaporation and by the water of transpiration set free in the air
by the plants in the room, whose evaporating surface amounted on the average
to about 25,000 square feet of leaf -surface, or by special humidity control. The
actual water-content of the air remained constant or nearly so, but with the
range in the daily temperature there was of course a change in the relative
humidity of the air of the room and, therefore, of the evaporation from the
surfaces of the organisms contained in the cages.

The food-supply is an important and vital portion of the project and must
be met by growing the food appropriate for the species and maintaining a con-
stant supply of it, so that at any time matings can be made as the animals are
ready therefor, or when any portion of an experimental series needs to be
analyzed. These are best grown as potted plants, after the usual routine of
florists, and must be kept free from infestation by pests or the presence of eggs,
larvae, or the adults of any of the different species that are under investigation.
I have grown my food-supply in an ordinary glass house that was separated
from the room where the animals were kept. The food was kept sterile by
frequent fumigations with HCN" gas and by sprayings with solutions of nicotine.
This has given complete protection against the possible introduction of any
stray individuals into the cultures.

Many of the food plants that I have used will last for a long time, a year or
more, and will give several crops of foliage in the course of their life, thus
materially reducing the number of plants that must be grown in the course of a
year to maintain the project. My experiments at Chicago have required in the
neighborhood of 10,000 plants per year, and a constant supply of from 2000 to
3000 plants must constantly be maintained.

Eeactions and Products in Interspecific Crosses 93

Frequent sterilization of the pots, soil, and cages by live steam under a
pressure of 10 to 20 pounds for periods of 4 to 10 hours for each sterilization
has materially contributed to freedom from disease, parasites, and kindred
troubles in both the animals and in their food plants. For this purpose steel
drums have been used. These are about 3 feet in diameter and 4 to 6 feet long,
and are connected to the steam mains that heat the quarters used for the experi-
ments. Live steam enters at a pressure of 00 pounds and with a temperature
that ranges from 163° to 170° C, and then dropping to a temperature of 108°
to 113° C. for the temperature of the sterilizer.

With properly sterilized cages, good food, and healthy stock, the chances of
numerous and vigorous progeny are as good as in nature, and I have often sus-
pected that in my cultures the progeny really surpass in number and in vigor
their less favored brethren in nature. Once a culture is set up, the maintenance
thereof is not difficult nor time-consuming. Only a moment is required daily
in which to thoroughly spray the cage and the food plant with the hose and to
add to the soil a fairly regular amount of water in order to maintain optimum
conditions of growth. This in general is the method of handling pedigree
cultures that I have used, and it has uniformly given satisfactory results.

I have been especially interested in the relation of the Mendelian reaction to
the problems of species and groups in nature, and the question whether in the
crossing of species a special order of reactions is shown. All of the older workers
seem to be of this opinion. De Vries clearly thinks of the Mendelian reaction
as present only in those forms standing in the relation of species and variety,
while elementary species or specific groups present a different type of reactions.
The same conclusion is expressed by many observers as the result of diverse
experiences.

The materials that I have used all come direct from nature, have never been
under domestication, nor in any way subjected to the operations of man. They
are natural species, and some of them have been known for more than a half
century. With this material I have made sundry studies to determine whether
in the crossing of species the reactions and laws of action were different from
those found in the lesser groups in intraspecific crosses. I could discover no
a priori reason why this should be so ; but the common expression of opinion that
it was so made the test necessary for the investigations that were in progress.

Species differ in diverse, concrete qualities ; some that are not easily expressed
in terms of " character," as for example, the " sense of difference," one soon
learns to recognize, even though one is not able to state it in accurate terms.
There is the total type aspect, with which are associated certain more or less
easily described characteristics. I have been so fortimate as to find in the
material that I have used species that could live with success under the com-
plexes provided in my laboratory, and that showed in these interspecific crosses
an array of characters such that it was possible to get a picture of the opera-
tions in the crossing of species. The general outcome of the entire series is
that in this set of organisms there are no differences in the crosses of species
that differentiate the reactions between species from intraspecific crosses. In
all respects the principles of factorial composition and reaction are fully con-
firmed, and many instances hopelessly confusing at first, and contrary to the
findings in intraspecific crosses, have been solved, in no instance with any dis-
cord in principle.

94 The Mechanism of Evolution in Leptinotaksa

It is not of any interest to present or even record the " dominant " and
" recessive " " characters " and the arrangement of them in schemes of their
" epistatic " or " hypostatic " relations, but my object has been the development
of an understanding of the structure of the gametic system, the nature of the
zygotic reaction complex (soma), and the basis of the operation of the different
agents involved and their relation to internal and external conditions, as an aid
in the further investigation of development, transmutation, and evolution in
these organisms when used as test objects. In the investigation, characters
that are "morphological," those that are "physiological," have been investi-
gated, although I can not decide where one " class " ends and the other begins ;
both in their productive agents and in operation are the same in all respects,
save in the resultant manifestation in the living mass.

LEPTINOTARSA SIGNATICOLLIS X LEPTINOTARSA DIVERSA.

Crossing of these two species has given the simplest condition that I have
found in the crossing of natural species. As shown in Chapter II, the two
species are similar in many of their characters, especially in the juvenile stages ;
the most conspicuous visible difference is the presence in L. signaticoUis of the
elytral stripes and their absence in L. diversa. To this must be added the
specific difference in general appearance, shape, and size, which is best deter-
mined and expressed in biometric determinations of the form index. iSTowhere,
as far as is known, do the two species live in the same habitat or in the same
environmental complex, L. signaticoUis living in the Rio Balsas Valley on its
eastern side, L. diversa along the edge of the Mexican Plateau on both the east
and west sides. My materials for experiment with L. signaticoUis Stal all came
from the Eancho Basoco location near Cuernavaca, State of Morelos, L. diversa
n. sp. from the Cerro Borrega colony at Orizaba, State of Vera Cruz, the two
separated by the high, dry, plateau of southern Mexico, where life is impossible
to both species.

Stocks obtained from these locations, taken to the laboratory, were reared
under the same conditions for 4 to 6 generations, and then utilized in the
experiments first described. No effect resulted from the change from nature to
the laboratory conditions except altered rates of growth, both living in perfect
health, giving vigorous offspring in large numbers, in long, continued series,
with the same growth-rates after 4 to 6 generations.

THE NORMAL REACTION IN CROSSING.

When these two species are crossed under the conditions of the breeding-
quarters, between stocks that have become adjusted to like conditions with the
same rates of development, a most regular and stereotyped series of results are
obtained, irrespective of the direction of the cross, age of the parents, and
strength or vigor of the mated pair. Crosses of old and young are completely
without effect upon the progeny, as far as any discoverable effect upon either
characters or reactions is concerned, nor is there any indication that the gametes
show changes in constitution or reaction with age, or changes corresponding to
or correlated with ontogenetic changes in the parent of any sort. I made
especial effort to check this aspect of the crossing, that I might eliminate the
" interpreting " of irregularities that might be found as due to ontogenetic or

K. TODA DEL.

JXJr.ILrS BIEK, N. Y.

1. L. juncta. Showing elytral piinctation.

2. L. decemlineaia.

3. L. texana. Showing elytral punctation.

4. L. dejecta. Brownsville, Texas.

5. F, heterozygote of L. signaticoUis X L. dwersa.

6. L. tumamoca.

7. SignaticoUis type extracted from marked heterozygous race.

8. L. decemlineata.

9. Modified signaticoUis type from marked heterozygous race.

Eeactioxs and Products in Interspecific Crosses 95

orthogenetic modifications in the constitution of the gametes during the life of
the individual. In these forms there is no rhythm in the constitution of gametes
produced, and the germ-cells are identical in constitution and array at all por-
tions of the reproductive period, as far as I can determine.

When crosses are made between the two species, in either direction, with
pedigreed, guarded parents, under conditions common to the two stocks, the
result in F^ is the imiform production of an intermediate type that is truly
intermediate in all respects, as shown in figure 7, plate S."" When these
are inbred they uniformly produce Fj fraternities that in all instances show
complete segregation in the proportions of : diversa 1 : mid-type 2 : signati-
collis 1, in remarkably close approximations to the expected arrays, not infre-
quently in perfect proportions of 1:2:1. Both of the extracted forms are
distinctly cut off from the heterozygous type and are the entire type extracted,
and are not the separation of the elytral or other characters alone. The extracted
types when crossed back upon the parent stock show no indication of difference
in composition or in any other respect as the result of its passing through the
cross. The total type has remained as a unit in the gametogenesis of the F^
heterozygotes, no interchange of characters taking place between the two sys-
tems, although there are many that might have done so, so that the F, array
shows the two parent forms as unaltered extracted Fg types, and the mid-type,
which is heterozygous.

The results obtained from the crossing of these two species under neutral
conditions, and also a complete testing of the crossing in all combinations,
everywhere and at all points in the series, show that the two types come out of
the cross with perfect regularity, unchanged, showing most constant and
regular behavior and retention of the integrity of the gametic systems through-
out. Each acts as a " single character " in the cross, and the entire series pre-
sents the aspect in operation, as in results, of a typical monohybrid reaction,
in which the contrasting elements are the two gametic systems present, one
from each species. In table 4 are given some of the results of the breeding of
these two species in this cross, and the working-out of the relations in Fj and F3,
showing the uniformity in close approximation of the arrays to the theoretical
proportions of each type in the population. I have bred the heterozygous types
through to F12 without any change in the array presented or deformity in the
proportions.

The extracted types in F, and following generations were always true to type
without exception. When crosses were made between the F^ heterozygotes and
either of the parent species — and the same is true of a heterozygote at any point
of any origin in the series — they give in F^ a 1 : 1 ratio of heterozygotes and
extractives of the same kind as the parent stock used. These, when bred out in
Fj, showed that the heterozygotes always gave a typical and close approximation
to the 1:2:1 Fg array, while the extracted type came true in all. Identical
results came from the crossing of the extracted types and any of the heterozygous
individuals. Crosses between any of the extracted types and the natural species
gave, if of like types, uniform lines; if of unlike types, the characteristic F^
heterozygotes and the Fg array seen in other crosses ; and likewise crosses between

* Compare plate 8, figure 7 with parent species, L. signaticollis, plate 8, figure 9;
plate 1, figure 9.

96

The Mechanism op Evolution in Leptinotaesa

Reactioxs and Products in Inteespecific Crosses

97

the extracted types gave, no matter what the point of origin nor what the
history, the same uniform results obtained in the crossing of the original stocks.

That there might be no personal equation involved in the series presented
after the initial combinations were made, the censuses of the fraternities were
made by a technical assistant, who was not instructed what to look for, but to
separate the fraternities into the different groups that he could recognize, and
to isolate any that were questionable as to where they belonged. After this was
done I took the materials and checked the counts and determinations of the
array; any that we did not agree upon where used in breeding to test their
character. Likewise, matings of the different portions of the fraternities were
random, the separated classes of a fraternity being put into glass jars and the
pairs draT\Ti at random for the matings. The results presented are as free from
the errors of personal equation as is possible.

From the inspection of the F^ and F, fraternities that came from the crossing
of these two species, as well as the extracted types in Fg and in later generations,
the impression was forced upon the observer that the total type had remained
as a unit, segregating out in the gametogenesis of the heterozygous forms. There

Fig. 2.

was no evidence of the interchange of any characters between the species dis-
coverable by inspection. To test this point, several of the fraternities were
investigated, as also the parent species of the stocks used in experiment and
from nature.

For this purpose I studied the form-index and its behavior in the materials,
in that it is a good indicator of the persistence and integrity of the gametic
type. With each type are associated many minor characteristics, and these,
together with the form differences, make for a distinct difference between the
two species in general aspect as well as in special characteristics.

This form-index is determined in these materials from measurements of cer-
tain parts, not liable to change in drying in preserved specimens or by changes
incidental to reproduction, or other ontogenetic processes. In figure 2 I have
shown the measurements that were made of the adults, and the index as used
was determined by the summation of the values 1, 3, and 3, divided by the
value 4. These readings were made with special vernier calipers reading to
tenths of a millimeter. Experience has shown that this index is the most
reliable and least variable, as well as the least liable to distortion by ontogenetic
or post-mortem changes in the specimens examined.

98

The Mechanism of Evolution in Leptinotaesa

The form-index of the two species was first determined from a series of
random samples, and the results are shown graphically in figure 3, showing the
low range of variability of the value, as well as the distinct grouping of the two
species about separate modes. The overlapping of the two polygons does not
seem to have more than the significance of ordinary fluctuating variations of
integral variates, and although the attempt has been made to breed pure lines

170
160
150
140
130
120

110

100
1 90

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from the extremes of these polygons, no results have come therefrom, so that as
far as experience goes the value is a single one, with the fluctuation common in
graduated varieties.

In that this first determination was made upon materials from many localities
and in different years, confessedly heterogeneous materials as regards genetic
relationships, a second study was made upon materials from the two locations
most used as the source of materials for this work.

Eeactioxs and Peoducts in Inteespecific Ceosses

99

Frequencies

ineachyeari {Frequencies In

census. 1 t„tal r(.n-,,i=

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Fig. 4. — Showing results obtained in the determination of the variability in
form-indpx iu the pure p.m'nt stiuks of /^. diversa and L. signaticoUis and by the
similar treatment of the peculiar pure-breeding extracted race that was super-
ficially L. sigrmticollis, but which showed itself to be made up of three modal
groups as far as form-index was concerned. (See text for explanation.)

100

The Mechanism of Evolution in Leptinotaesa

In figure 4 I have shown in condensed schematic form the results of this
study of L. diversa at the source of material at Orizaba and of L. signaticoUis
at Cuernavaca, through the years 1904 to 1909. This study of the form-index
in each of the two sources of materials shows clearly that the condition is not
only stable for the species in the location, but also that, taking the location as a

Form Index modes of the Parent Specioi

, *

L. diversa.

L. s^gniticoliiS;

1

—

—

F3

AAA)

J

\

7^1 AAA) U
■Extracted 1

Extract

ed-

-'-

/

\

M:

=.

^X

/

i

N

=

^

=

^

,

/

KM

—

^.

V

^

/ _

^El

=^

i^.

1=

^

^

fe

'^,-"=1

1=^

ti-

L

Div.

Type

Mid.

1 Sig. Tyua

»

' '

1

J

/ k

t

/\

/ \

' \

F,(A

^)i

-/

\,

^^

}\

4

M

s

^^ r=^

--\l..

/

1 ^

s\:i

--

hF,(C)

1

F

=p

^

^

^^^^

^

M

—Ti

-^

^::i^

,

1

1

\=t::^

L

"

^

r

/

5^

—

=

a--

-•

-

--

-■

-

-F,(C)

>

. 1

=

==

^

1

Fi(

A)-

--

">

y

\fim

^

.V-

_.

._

..

-F,(B)

'/

A

=^^

m.

ii

=.^

^

^

^^EJ

w

^

--

=

S3

^^

^

\

t

^

1

f-'

c

;

■-"?/

B?^

A

[<

A5-^

sV~<.

?

ii

^^

\^

','-

1 .

-'^^

.>

^

fe?

c5^

^l\

#'

i-

EH

e4

n

h

^

^^4\

A

si

%

A

==

— j —

^

■ISli

im

-^

-;^

-—

=

r^^

^

^•^li^

^!tl

ii

1

Pig. 5. — Diagram of apparatus for control of relative humidity in the hreeding
room. The figure shows the complete apparatus. (For explanation of Its parts and
working see description in the text.)

whole through the period of observation, it has the same index as determined
from the random samples shown in figure 3. Some divergence of the polygons
of distribution in each of the species is shown for the species mode in different
years, but no divergence of this sort has been found to have any permanency,

Keactions and Products in Inteespecific Ceosses

101

either in nature or in experiment, and may be, therefore, regarded as the results
of aberrations induced by incident conditions during ontogeny or as the result
of small numbers observed, the modal divergence in different years being purely
a chance one.

The inheritance of the form-index and its behavior in crossing is shown
graphically in figure 5, where the products of three crosses are shown between
these two species, as well as the fraternities from which the parents of each
cross came. Modal individuals were used for the matings in each of the three
shown. From figure 5 and table 5 it is seen that the F^ heterozygotes are in
modal position between the modes of the parent species, are intermediate in this
respect, although the exact position of the mode upon the scale of values differs
in different fraternities, and the range of the polygon in all instances overlaps
that of the parents to a varying extent.

Table 5.

No.

Mode 0

f L.

diversa.

Mode of L. signa

tieollis.

Remarks.

CO

3"

o

CO

05

CO

i

o

lO

CO

i

o

o

a
1"

00

ei'
1

o
o

00

05

00

00

i

o
o

OJ

05
©1

eo
1

o
o
o
CO

C5
Ci

o

i

o

05

1

o
o
■— (

C5
Ci

i

o

CO

o

CO

1....
2

1

1

2
1
5

1

i

1

2

3
1

4
7
1
3
1
1
1
2

10
4
10
11
1
8
5
2
4
3

16
9

14

28
4

17
5
2
3

13

28

5

40

35

16

40

12

7

7

30

46
3
61
26
31
71
19
3
15
46

31

3

42

13

14

42

11

2

8

31

17
1

12
9
5

20
4
1
5

15

6

"i

1
11

1

2

1
4

2

1
2
1
1
5
2

signaticollis P X ^ diversa.

Do.

Do.

Do.

Do.
signaticollis cf X ^ diversa.

Do.

Do.

Do.

Do.

Total, 1,090.

1

2

3

4....
5

1

6....

1

1
1

8 .. .

9....

10....

1

2

1

1

1

4

14

24

58

105'220321

197

89

34

17

6

1

Matings of the heterozygotes show uniformly in Fj that the visibly segre-
gated products are also segregated with respect to the form-index, and that
the form-index of L. diversa is in all instances associated solely with the more
easily visible characters of that species, and that none of the individuals with
this index carries signaticollis characteristics; the same is also true of the
extracted signaticollis type, in that it is entirely associated with signaticollis
characters. The mid-types, or heterozygotes, in their mode and distribution,
are always in an intermediate position, and individuals from this polygon always
give in Fg the same behavior as is found in the progeny from the F^ hetero-
zygotes. In other words, there is complete segregation of the form-index as
well as of all of the characteristics that accompany it in the parent species, and
no evidence has thus far been discovered in these nonnal crosses of the inter-
change of any characteristics between the two gametic systems as the result of
passing through this normal crossing reaction.

The point of special interest is the behavior of the gametic system species as
a unit in crossing under the conditions of the experiment, and the non-dissocia-
tion of it in any portion of the series. There are present the two basic species

103 The Mechanism of Evolution in Leptinotaesa

gametic complexes, between which characters might be shifted, producing in the
end a considerable array of types in Fo and F3 differing in small but none the
less distinctive and permanent aspects. No such metathesis is observed in this
series, and I believe that this type of reaction represents the simplest that can
exist in the crossing of species.

It may be objected that the materials are not species because they cross and
give fertile F^ and F2 progeny, and the fact that they cross at all would indicate
to many a lack of specific distinctness. Whether the above objection is tenable
or not need not be discussed, as it is simply a matter of anticipatory definition.
When the species come into the laboratory from nature they are less fertile and
often cross with difficulty, producing quite different results, as will be shown
presently. They are reared and crossed under the conditions of experiment
given, that is, imder the mean average of each chief environmental factor of their
breeding-period in nature, so that the conditions of experiment may not intro-
duce any incidental agents that might change them ; only the rhythm of native
habitats is changed, and instead of the pressure of the brief season in the habitat
of L. signaticoUis, both are given continuous mean breeding-season conditions,
to which the species adjust themselves without discoverable change except in the
rates of reaction in development. As the two become attuned to the same con-
ditions in the medium, their interfertility increases until in the end the fertility
is nearly as high in interspecific crosses as it is in pure-line matings. When this
state is reached the reactions shown in the crossing of these two species are
uniform and represent the true basic reaction of the two gametic systems when
combined in crossing under neutral conditions of the medium, and it is the
reaction of the systems in simplest terms under conditions that permit of the
manifestation being entirely the product of the composition and structure of the
interacting masses.

Crosses of the kind indicated between these two species have been repeated
yearly since 1907 as part of the annual routine to test this point and also the
condition of the stocks, with no indication of any differences whatsoever. These
yearly tests are random matings, with random testing into F2, and sometimes
into F3 or F^, depending upon the space and help available.

MODIFICATIONS OF THE NORMAL REACTION.

If the crossing of these two species presented only the behavior shown, it
would be of itself of interest because of its simplicity and the integrity of the
gametic systems shown, but this is not the reaction shown when the materials
are fresh from nature and not adjusted in their reactions to the same set of
external conditions. I found in 1904 that these two species, when fresh from
nature, in guarded parentages, gave in F^ a behavior that suggested that one of
the parents was heterozygous, the other homozygous with respect to the elytral
striping, with the result that in F^ two classes of about equal numbers appeared.
Thus when the cross was made between L. signaticoUis female and male
L. diversa, the uniform result in F^ was about 50 per cent of mid-types and
50 per cent of the female signaticoUis types, and these when inbred in each class
gave in F^ and in subsequent generations the extracted female signaticoUis type,
''breeding true" and the mid-type splitting in F, into signaticoUis, mid-, and
diversa types in the usual ratios. This result I have shown in plate 9. It is in
every way typical and suggestive of the crossing of a DD x DE or DR x RE.

Eeactioxs and Peoducts in Interspecific Ckosses

103

This condition was uniformly present in the crosses of the first and second
generations in the laboratory, but in the following generations this behavior
changed progressively with decreasing percentages present of the female sig-
naticollis type, until at the fifth or sixth generation none was present, nor did
they appear again in the crossing of the two species as long as the stocks were
maintained under like conditions. There is shown in this series of observations
a condition that might be of much importance that would produce unlike results
in different locations in nature from the crossing of the two species, permanently
altering the history of some individuals. I undertook, therefore, a careful
examination of the stocks and attempted to experimentally produce the modifi-
cation in behavior that was found.

Careful examination by measurements, by crossing back upon the fresh stocks
from nature and upon older strains that had been longer in the laboratory, failed
to show any heterozygous condition present in the gametic constitution of the
stock species that could be detected.

Table 6.

stock No. 419 (L. signaticollis) .

Stock No. 817 {L. diversa).

Nature.

Chicago.

Chicago.

Nature.

Period.

Days.

Period.

Days.

Period.

Days.

Period.

Days.

June 2 to July 17.
Mav 26 to July 5.
Aug. 2 to Sept. 5.
June 30 to July 10.
July 25 to Sept. 2.
June 4 to July 10.
June 7 to July 17.
Aug. 5 to Sept. 10.
Aug. 1 to Sept. 4.
May 24 to July 31.

Average

45
39
34
40
39
36
40
36
35
38

April 18 to June 10.
Nov. 2 to Jan. 4.
Sept. 20 to Dec. 15.
Nov. 2 to Jan. 6.
Mar. 11 to May 11 .
April 13 to June 4.
April 13 to June 8.
Oct. 7 to Dec. 6.
Sept. 1 to D(;c. 2.
Nov. 2 to Jan. 4.

Average

53
63
55
65
61
62
66
60
61
63

Nov. 10 to Jan. 6.
Sept. 30 to Jan. 1.
Nov. 2 to Jan. 6.
Mar. 11 to May 11.
April 6 to June 5.
Mar. 11 to May 11.
April > to Jan. 5.
April 1 to June 1.
May 11 to June 9.
April 5 to June 1.

Average

57
62
65
60
60
61
58
61
59
57

May 4 to July 5.
June 8 to Julv 8.
July 4 to Sept. 4.
April 29 to June 30.
April 24 to June 26.
May 14 to July 20.
June 4 to July 12.
May 27 to July 31.
June 1 to Aug. 5.
May 31 to Aug. 2.

Average

63
61
62
62
60
68
69
65
65
63

64.1

38.2

60.9

60.2

Records made at Cuer-
navaca of stock from
Rancho Basoco Col-
ony.

Records made, taken from records at random,
between 1907 and 1912 and are of the behavior
after four or more generations in the conditions
of the breeding quarters used.

Records made at Ori-
zaba of stock from
Cerro Borrego Col-
ony.

In nature the average length of ontogeny in L. signaticoUis is about 40 days
and in L. diversa it is about 60 days. In the records of my cultures I found
that the stocks fresh from the natural habitat retained the same rates under the
conditions of the quarters at Chicago in the first generation there, but that they
progressively changed in this respect, so that at the sixth generation at the
latest, often earlier, they had come to have the same rate of development and
the same length of ontogenetic period from egg to the adult. In table 6 are
given data taken at random from the records showing the rates of development
at Cuernavaca, State of Morelos, Mexico, for L. signaticoUis Stal, at Orizaba,
Vera Cruz, for L. diversa n. sp., and the rates in two stocks at Chicago that
were obtained at these locations after the period of probation had passed, when
the crossing of the two gave the monohybrid results described. At this time
both had the rate of 60 days each, a rate retained indefinitely under laboratory
conditions as far as known.

104 The Mechanism of Evolution" in Leptinotaesa

In the natural habitat at Cuernavaca, the growing-season, from mid-June to
mid-September, 90 days, allowed only the requisite time in the year for the
species to complete its two annual generations and prepare for the oncoming
of the dry season in September. In the habitat of L. diversa at Orizaba the
growing-season was longer, from early June to the late autumn or even into
January, giving the species ample time to undergo its development before the
onset of the dry season. In the laboratory the average growing-season con-
ditions of the habitats were maintained throughout, so that there was no pressure
to hasten ontogeny in the case of either. The uniform result under laboratory
conditions is to slow down the rate of development of L. signaticolUs and to
slightly hasten that of L. diversa, both coming to have the same rate of onto-
genetic progression.

It would seem, therefore, that this condition in the stocks on introduction
from nature might be productive of the difference found in the crossing on
introduction and after establishment. If this were true, it then ought to be
possible to produce in experiment, from races that were giving the monohybrid
behavior, the conditions shown in plate 9. It ought also to be possible to dis-
cover something of the mechanism of the production of the difference, especially
in the production in F^ of the " pure-breeding " types.

To eliminate the possibility that the condition was the product of a local
difference, in the spring of 1907 I brought to the laboratory from Atlixco, State
of Puebla, Mexico, fresh materials of L. signaticolUs Stal and of L. diversa n. sp.
from Guadalajara, State of Jalisco. These I obtained in late June in the larval
condition, and they were taken to the laboratory as pupae and emerged, the
sexes being immediately separated. A generation fresh from the field, repre-
senting random materials, but in which the virginity of the mates was absolutely
certain, was available and at once crossed, with the result that the different
crosses of the signaticolUs X diversa gave uniformly the same result as had been
obtained many times then and since from stocks from the two other locations.
These I continued breeding and crossing at random through the seasons of
1907, 1908, 1909, and 1910, with the results sho^vn in table 8, showing in all
respects the same behavior that was found in all of the other stocks. This result
made it certain that the condition was a general one and not due to special
local conditions.

In the years 1908, 1909, and 1910 the same test was made with new stocks
from the Eanclio Basoco and Cerro Borrega locations, with the same results,
showing close agreement in the progressive change in the rate of ontogeny, and
with this a similar change in the P^ array presented, which was as far as the
tests were carried. These tests were made at the time that otlier portions of this
investigation were in progress, as checks to make certain that the conditions and
the results that were found and being obtained in the two main sources of
material were not vitiated by the use of only one source of supply, in which
something unique might be present and productive of the complications found.

Analysis of the conditions in the habitat of L. diversa at Orizaba and L. sig-
naticolUs at Cuernavaca, as far as data were available, showed during the grow-
ing season that essentially the same temperature conditions were found in both
habitats, with differences in ranges that were not great. Eelative humidity as
ordinarily determined showed nothing of interest, while the rainfall at Orizaba

Reactions and Peoducts in Interspecific Crosses

105

•s=a

d a

<U (V
bo b£

(1> HH l-C

^ . .

S M bO

c<3 C3

a a

<U tt>

bJO bD

•2 «

-5 a

D. ^

O) O) aj S ^

S S O ^ c3
o o 5 (D _ iZ

a

Cj

(Tl

u

U

A

>>

4.^

a a

TS

<p a>

n

crt

be bo

d

bO

u u

o o

a

^

<t-l <»-l

ii

'd

f1

Clj

o

73

fe

>>

d d

(u <i> .;.

be U) p^

o o .S

o t>

o o

CO CO

"3 d

aa

bX)

a

s 2 =i

o «

o o

■(-> ->J
in u

d d

aa

o o
a a bD

■^ '-«J _, ra

iH T-t rj

aaa'a'l asg^aa

t- 00 •^ t-l
rH iH •<*< U5

tH O

TJ< Oi t- IM

rH 1-1 IM ?0

d

+1+1

^+1
+1

"to

'm a

Ol

t-(00

■ r-i

CO i-i ;

M XI

+1 +1 :

+

CO t-;

e4 M bo >
+1+1 «^

to t>;

T-i CO

+ 1+1

+1+1 +1 +1+1 +1

o ?

o o

bo bo

Ol Pi

O O

u u
bO bo

.£liS

"O HH,

a

X

5 a

a

J 1— 1 >— c

d o

d *>-!

3

0<!

>_bo
13 'w

O d ^

ii a

=1-1 tS •"

> bi _bo

'd 'm w

CI CI

bo bo

O O

J J J J J

fl CI
<U (D
bO bO

aa

o o
.«- f-

> _bO

h4 h4 j hJ j

h3

a

X

a

Ol Ol

o p
bo bo

>>

a a
bo bo

o o

> bo

>;_bO

a

X

w a spa

,H «i-t <i-i s-i «i-i

O 01

bo bo

aa

> Ml bo

"O OQ 'to

1-3

o

' . CO

I o .

Ǥ

•-5

. 6

Ol*-'

bo • •
.9 w

QJ ^Q

Eh GS

CO CO

l-HM S

cr* I— 1 1— (

I— I HH W

106

The Mechanism of Evolution in Leptinotarsa

was greater than at Cuernavaca. The greatest difference in the locations is in
the water-relation of the organisms. At Orizaba the relation is one of high

Table 8.

Moath.

April
May-
June
July
Aug.
Sept.
Oct.
Nov.

CUERNAVACA, 1905.

Period.

20 to 30
1 to 10

11 to 20

21 to 31
1 to 10

11 to 20
21 to 30

1 to 10
11 to 20
21 to 31

1 to 10
11 to 20
21 to 31

1 to 10
11 to 20
21 to 30

1 to 10
11 to 20
21 to 31

1 to 10
11 to 20

Atmometer.

(Fig. 5),
av. daily loss

in c. c*

107.4
119.8
112.2
98.7
92.5
66.4
64.3
61.7
54.5
51.3
54.1
64.5

Free wateri
surface,

av. daily loss
in c. c.f

93.2
108.7
100.4
91.0
84.1
55.1
54.9
53.4
47.6
40.1
41.1
58.7

Max. loss
in c. c.

Free
H,0.

Atm.

Min. loss
in c. c.

Free
H,0

76.5

70.1

79.4

74.1

84.2

81.3

89.8

85.4

87.4

80.1

117.4

148.1

141.0

117.1

108.1

80.0

80.0

81.0

73.0

77.0

81.0

96.4

124 is
121.1
127.1
129.4

141 ! 4

164.3

78.4

186.4

92.2

204.3

86.1

149.5

78.1

146.4

70.4

93.4

7.1

90.1

24.1

85.1

40.0

76.4

21.4

84.4

29.1

96.1

31.4

114.1

34.1

177! 7

54!4

179.1

61.1

186.4

65.4

193.5

64.0

261.8

68.1

87.2
101.4
98.4
84.5
77.0
19.7
30.4
42.0
29.3
35.8
37.7
39.9

6i!2
50.1
60.3
69.9

74.1

ORIZABA, 1906.

April

May

June

July

Aug

Sept. 1908..

Nov. 1905..
Jan. 1910..

20 to 30
1 to 10

11 to 20

21 to 31
1 to 10

11 to 20
21 to 30

1 to 10
11 to 20
21 to 31

1 to 10
11 to 20
21 to 31

1 to 6
10 to 18

1 to 5

7 to 14

77.0
79.4
87.4
60.1
43.1
31.1
31.7
33.5
21.4
20.4
23.6
31.2
33.4
34.5
33.5
37.7
45.7

65.2
74.3
76.1
44.2
32.1
20.9
24.3
29.2
17.3
14.1
26.1
26.4
27.7
30.1
31.1
34.6
39.4

109.0

141.0

56.2

105.0

117.0

54.4

80.0

101.1

63.2

61.0

75.4

7.3

56.0

61.0

0.0

41.0

48.0

0.0

41.4

44.1

0.0

42.0

45.9

0.0

40.4

43.7

0.0

33.1

40.1

0.0

41.1

56.4

0.4

54.1

61.0

0.0

54.7

60.7

0.0

56.7

50.4

0.0

54.7

61.1

0.0

84.1

97.4

0.0

81.1

87.7

0.0

61.0
66.1
71.4
11.9
1.7
0.4
0.0
0.0
0.0
0.7
0.9
0.0
0.0
0.0
4.1
0.0
0.0

* Evaporating surface = 800 sq. cm. heavy filter paper hung vertically in open.
t Evaporating surface = 800 sq. cm. in dish, 40 by 20 cm., in open in sun.

water-content in the medium, with relatively low evaporation-rates, hence the
prevailing type of plants and animals characteristic of moist habitats in the

Eeactions and Products in Interspecific Crosses

107

tropics, essentially a montane rain-forest, while at Cuernavaca the conditions
are those of an upland savannah complex with intense desiccation during nine
months of the year, and daily desiccations of varying degrees during the rainy
or growing season, from June to September. Records of the evaporation-rates
at the two locations have been taken at different times during the progress of
this work as opportunity permitted, and some of these are given in table 8.
These are taken with the filter-paper atmometer and the free water-surface ;
the result recorded is the loss of water in cubic centimeters of water evaporated.
These and other determinations are shown in the series of curves in figure 6,
which show graphically the progress of the season in this respect and the rela-
tion of the breeding-periods to this environmental relation. It is at once clear
that the complexes are very different in the two locations in this respect, and the
rate of evaporation has been found in experiment to be one of the most important
agents in the environmental complex; that is, this rate of water-loss from the

'-g 140 — T
"5 130 '
I- '20

~"

—

~

—

—

~~

~^

/

\

''

\

f

\

/

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/

\

^

r

t

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/

\

ifc

,'

/

\

■^

>

^

<'

/

,

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\

/

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k

\

^'

^

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,

'L'

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,,

/

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s,

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V

s

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-

-.

.

^

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-

--h'

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y

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,

,

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\^

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-Average lengin
-of breeding

\

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location.

u

—

—

—

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(at Orizaba fo

,

L.

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sa

Maximum atmo-

eter record
Cuernavaca.

Average daily at-
mometer record
Orizaba

Average daily at-

r record
Cuernavaca .
Maximum atmo-

eter record
Orizaba.

oooooooooo

Fig. 6. — Showing differences in evaporation rates in the parental habitat of
li. siffnaticollis at Cuernavaca and of L. diversa at Orizaba, as determined from
various atmometer readings through several years.

surface of the body, which is in general terms comparable to that shown in the
atmometer, alters most profoundly the action and rates of reaction in the
organism. It will induce changes in the trophisms of the organism most readily,
and reversal of the normal activities, as well as serve as an accelerator in
ontogeny. Thus the conditions at Cuernavaca are such as to produce rapid onto-
genetic development, and the species from that location have high rates of
reaction and short ontogenetic cycles, in signaticollis about 40 days, while at
Orizaba the conditions are such that there is little or no pressure of the environ-
ment, and as a result the ontogeny is longer in each of the two annual genera-
tions, and there is much overlapping and interbreeding of them.

In the laboratory at Chicago the conditions were a close approach to those
found in the Orizaba location, and under these the two species come to have the
same rates of development. Each has, therefore, a specific rate of ontogenetic
progression that is characteristic of each in its original habitat, but which is

108 The Mechanism of Evolutiox in Leptinotaesa

easily changed on introduction into a new habitat, without any change in the
characteristics of the organism. It is, as far as I am able to decide, a condition
in the organism, a state of being, that exists wdthin the mass and is the product
in its values of the conditions in the medium in which the organism is living,
acting upon and directly modifying the reaction values made possible by the
gametic factors of composition, and is in this respect comparable to the con-
ditions of the mass that one finds in non-living materials.

The ontogenetic group of developmental reactions are immensely complex,
but they have clearly the relation and values in these species, which I have
designated the ontogenetic rate determiner (Ac), and changes in the reaction
value of this agent, induced through changes in the medium, produce in the
organic mass changes in the rate of ontogenetic progression, which are measured
by the length of the time taken for the completion of growth, from fertilization
to the adult condition. The quantitative value of Ac in any species I have
expressed in terms of elapsed time demanded by the ontogeny of the species, as
expressed in days. Thus Ac*^ or Ac^°, simply signifies that in the stock from
which the individuals came under the conditions of their culture, this factor had
the value of reaction, such that the total series of ontogenetic reactions would
be completed, on the average, in 40 or 60 days.

This agent serves as a most important factor in disturbances of the Mendelian
reaction in species crosses, and changes the character, as far as visibility is con-
cerned, of many individuals in the population for indefinite periods of time. I
have shown that the two species, L. diversa and L. signaticollis, when crossed
under common conditions, and when the values of ^c are the same or essentially
the same Ac®°, give perfect monohybrid reactions in all instances, and without
exception showing the production of gametes that are pure for the total system
of each of the two species, giving true signaticollis and diversa as extracted
products. On the other hand, it is shown in plate 9 that when the two are
crossed where the Ac values are different there ensues in F^ and in following
generations a different series of events, in which the fate of certain individuals
is permanently altered, and the F^ reaction suggests the interpretation that one
of the parents is heterozygous, the other homozygous.

Thus far there has been no exception to the two types of results when the
conditions in the medium and the values oi Ac are those stated, and this repre-
sents the condition of the crossing of the two species fresh from nature, or of
fresh L. signaticollis crossed with the L. diversa that has been in the laboratory
for any length of time. Also, the same results are produced if L. signaticollis is
in experiment made to attain the value of Ac*^ or thereabouts, regardless of
what its former rate of ontogeny has been.

In the earlier experiments from 1904 to 1907, the series described were dupli-
cated over and over again, and many tests were made that gave profitable
indications for further work. From 1907 to 1914 the reactions found, and
represented in plate 9, have been extensively tested and an analysis made of the
products. Starting with a series that is easily obtained by the crossing of
L. signaticollis from Cuernavaca, when freshly introduced into the laboratory,
and L. diversa, either fresh or in stock, the cross made under the standard con-
ditions of the testing-room gave uniformly 50 per cent of the progeny that were
of the typical mid-type, and 50 per cent that were to all appearances signaticollis

Eeactions and Peoducts in Inteespecific Ceosses 109

in type. In Fj the signaticollis type bred true in all instances, the mid-type
gave the customary F, array in nearly perfect proportions. This obviously
heterozygous line shows nothing of any interest or irregularity, no matter how
far it is carried and regardless of the conditions of the medium within limits.

In the signaticollis line, however, I learned to recognize differences in the
population, delicate and not capable of determination except by measurement.
In general appearances the population was uniformly signaticollis type, bred
true, crossed with pure signaticollis stock, gave only signaticollis types in the
progeny, so that one might easily pass the entire population as extracted signa-
ticollis. I found that with close attention I could separate three form-types
from this F2 signaticollis population. These I first recognized as present in an
F2 population that was signaticollis to all outward appearance, of form-types
that had the suggestion of diversa, of signaticollis, and of an intermediate type
with intermediate conditions in body, form, and aspect.

In 1907 I had measured three random fraternities of this F2 signaticollis
type to obtain the form-index. The result of the determinations showed in the
Fa signaticollis type population a trimodal polygon, one mode of which was
upon that of the diversa parent, the other that of signaticollis, the third variable
and intermediate, that of typical heterozygotes. There was not the least doubt
of the presence of the three modes in the F2 fraternities measured, which were
supposedly homogeneous. In figure 7 I have shown the results of this determi-
nation for the fraternities and the determinations for the two parent species.
I also made like determinations of the form-index in a normal heterozygous
line and which showed that the conditions in the intermediate groups and in
the recognized mid-type was essentially the same. The three fraternities, each
the progeny of a single pair of parents, were large — 171, 180, and 204 — and
each showed the same arrangement into a trimodal curve as is shown in the
summation of them shown in figure 7. I was thus able also to detect these
modal differences of the form-index and so was able to select mates from the
two extreme modes that corresponded with the two parent species modes for
breeding, and found that the response was immediate to produce in the next
generation complete isolation of the two groups, with no more intergrades in
form-index than are present in the normal species. These lines were kept
breeding on through 1908 and 1909 without change or transgression of the
limits of the condition first determined in 1907.

In the latter part of 1908 it was observed that in fraternities belonging to the
lower end of the range there was not uncommonly a development of faint,
diffuse, irregular pigmentation along the rows of punctation on the elytra
(plate 8, figure 11), in position and relations in which pigment is never found
in the pure signaticollis stock, and has not been made to develop by the extension
of the pigment in the pits. It was further found that these conditions in the
race were permanent and could by mating likes be intensified to the extent that
the elytron presented often a faint brownish tinge extending out on all sides
from the system of pits in the surface of the elytron.

These two experiences lead to the hypothesis that the jP^ signaticollis race
was not signaticollis at all, but a masked heterozygous race that had the diversa
gametic system present but in combination, such that it was not able to produce
the normal reaction in the complex to give the usual heterozygote form. This

110

The Mechanism of Evolution in Leptinotaksa

idea was supported by the ability to separate the F^ signaticollis race into the
three groups in Fj, having the modal conditions of form-index respectively of
the two parent species, and the known heterozygote, and the presence in the lower
portion of this trimodal polygon of the development of dark pigment in an
association not known in the original signaticollis materials. If the V deter-
miner * was present, but in new combinations in the gametic complex, it was at
least worth the effort to try to recover V from this race F^ of signaticollis in
appearance, but trimodal in form-indexes in Fj, and derive the diversa form
from this race as an extracted product by bringing about in some manner the
recombination in the gametes of the proper arrangement of gametic agents,
which were possibly present but in new arrangements.

'\

I V

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(

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;

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A

1^ -

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+1 C L

lu

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rt (^ ^ ^_ IT) lO vD lO l<_ r\ 03 CO <T>_ 05 O o_ -_ — tv; i\ o ro V.
w ru" (\r oi oi" N~ (\j fvj" c\i" f\j" cj" cy oj cm' CO ro co oi ro oo" co fO en

oooooooooooooooooooo OOO

m o ^ ■* iS ui ID CD r^. !<■. CO oq <J) <n o q -. - fy t^_ to n ■^^
pj" (vT cu <\J' tu" rj rvj cj" (y oj (\l cm' oj tu (^ n' n co ro c'i' « co co

Pure §tockof
L. diversa.

Pure stock of
L. signaticollis

Fig. 7.

To test this point I used only strains of signaticollis, so that there might be
introduced no traces of the V determiner other than that already there. In
1908 and 1909 I made many crosses of this peculiar derivative from the tri-
modal signaticollis race (index mode 2.600-2.649), in which both had the Ac
determiner with a value of Ac'", with the uniform result that in no fraternity
did any trace of the V determiner producing the diversa striped elytra appear.
All of the progeny were signaticollis in aspect, but two series of crosses that
I had measured showed F^ heterozygous for form-index, monomodal, and as of
the usual F^ type, and trimodal conditions in Fg. In all 22 crosses were made,
with the constant results described, and from these it is certain that the S'igna-
ticollis pure species does not have any agent that matches the V, or otherwise

^ Determiner for elypitcal stripe see tables 2 and 3, pp. 86, 87.

Ee ACTIONS AND PeODUCTS IN INTERSPECIFIC CROSSES 111

enables it to be recovered from the signaticolUs-like races that arise in F^ as an
extracted stock.

Another test was made with a signaticollis stock that had the Ac determiner
with a value of Ac^^. This was a pure native stock that had been in the labora-
tory for 15 generations and had from the tenth generation onward been made,
by external conditions, to attain the value of Ac^^. This was done by placing it
for breeding under conditions in which the temperature ranges were those of
the native habitat at Cuernavaca, but in which the water-relation was much
more rigorous, especially in the evaporation in the daylight hours. Under these
conditions two or at the most three generations are all that is needed to attain
the full development of the faster value of Ac. It was found that when a race
of pure signaticollis, with the value Ac^^, is crossed with the suspected race of
signaticollis, having the diversa form-index, and having the value Ac^°, under
the conditions used to develop the Ac^^ value in the pure signaticollis, the result
is to produce an F^ array that is signaticollis in general aspect, but whose index
is intermediate between the two, on the heterozygous mode, and these when
inbred showed in F, a variable number of diversa types, with several others,
showing that V, the elytral stripe determiner, has been returned to its normal
relations, resulting in the production of gametes that are pure diversa in all
respects, and these when combined with likes produce pure diversa types.
The^e were found to breed true in subsequent generations, to be pure diversa
stock in every way. This reaction became the test upon which this complex
and apparently unconformable series was finally elucidated, and is referred to
in the remainder of this account of the crosses between the species signaticollis
and diversa as the test reaction.

This reaction shows that in the gametes of the extracted signaticollis appear-
ing line with diversa index there is still present the proper agent, V, which, in
certain relations of interaction, is able to produce the typical diversa elytral
pattern; but that in other relations it is not, but produces the condition of a
diversa form, with the signaticollis appearance, and the possible development of
pigmentation in irregular spreading areas in connection with the system of
elytral punctations. There is present in the elytron the agents needed to pro-
duce pigment, and it is constantly active in the production of pigment in the
pits that form the elytral punctation series. The T^ determiner is an agent that
acts, as far as known, as a pattern-determiner in conjunction with the general
pattern-factor of the elytron. For the purposes of the series of crosses between
signaticollis and diversa, only the Ac determiner, Math its values represented by
the exponent accompanying it, the elytral pattern-determiner Elp, the deter-
miner for the punctation system {Pu) upon the elytron, and the pattern-
determiner for elytral longitudinal stripes (F). These are agents of specificity
in the gamete as is shown by the ability to replace the Pu determiner of signa-
ticollis by that from some other source, as also the F determiner of diversa can
be replaced by another agent occupying precisely the same relations in this
system and productive of its specific type of resultant product. Or, in other
operations, the entire group may be dissociated from the gametic complex and
transferred entire to a new association. In the figures and plates I have shown
these agents with bonds between them, which represent no more than the direc-
tions of interaction, or the combinations of action that experience with these

113 The Mechanism of Evolution in Leptinotaesa

agents shows them to have. At present they are but graphic representations of
the associations and interaction of these gametic agents in the production of
specific end-results.

The results shown in plate 9, while suggestive of the crossing of one hetero-
zygous and one homozygous parent, are in reality not that at all, but are dif-
ferent, as shown by the test reaction applied to the F^ fraternities of any of
these arrays, which uniformly produces in the following F, fraternity, after
the test, pure-breeding diversa types along with several others, showing that
the pure-breeding F^ signaticollis race has in reality in it the V determiner,
which could not possibly have come from any other source, and that the F^
signaticollis type, while it bred true in aspect, is in reality a masked hetero-
zygote with the V determiner inactive, as far as the production of stripes is
concerned. This is further made certain if an F2 fraternity of the questionable
signaticollis type be separated into three groups by measurements of the form-
index and then applying the test to the modal classes of each group.

The test, when it is applied to the portion of the trimodal Fg polygon having
the highest or signaticollis index, gives in all following fraternities nothing but
signaticollis, regardless of what is done to it. The lowest mode, having the
diversa index, always gives in Fg pure-breeding extractive types that are diversa,
while the middle mode also gives in F, following the test diversa types and
others of complex nature. Everything shows clearly that the race of F^ ex-
tracted signaticollis is a masked heterozygous strain, a " fixed hybrid," in which
the agents that are commonly employed to differentiate the species are inactive.
However, biometric testing imiformly shows that F2 in any such race is tri-
modal, the extreme modes being that of the parent species form, proving clearly
that the F^ is really heterozygous. The further fact that the race from the
lower mode, pure-breeding, and in appearance signaticollis, will give the diversa
type as pure diversa following the test reaction; that the other race derived
from the higher mode can not be made to do so, shows that V is constantly in
association with the complex that segregates to produce the lower mode, and is
not present in the upper modal group of the F2 array.

The further observation that in the race derived from the lower mode of the
F2 array, while it is constantly signaticollis in its aspect, shows the development
of pattern in connection with the punctation system in a way not known any-
where else in all of my materials or in nature, led to the hypothesis that Y had
changed in its relations in the gametic system, such that it was entirely asso-
ciated with the punctation-determiner, the two acting as a single group. This
was shown by the ability to develop, out of the race (plate 8, fig. 11) derived
from the lower mode, a derivitive race that showed in all of the members of the
fraternities irregular pigment-pattern developing in association with the punc-
tation system where none was known before, and the ability to transfer this
condition entire to other series of combinations as in crossing with L. unde-
cimlineata or L. panamensis. From any such race, either originally or sec-
ondarily placed by crossing, the test reaction uniformly gave in the Fg following
the test the pure-breeding extracted diversa type.

All of these results show that the real explanation of the apparent inhar-
monious array shown in plate 9 is in reality due to the forming of new arrange-
ments in the gametic agents. This change consists, as far as determined, in

Extracted diversa

Ls,gnat,col/.

Reaction in cross of L. signaticollis and L. diversa when values of the Ac determiner are different,

BUT conditions OP MEDIUM ARE NEUTRAL. FIGURES REPRESENT THE FRATERNITIES OBTAINED IN A SINGLE
LINE OF CULTURES.

a;

tl

S}
Z""

f(
tl
tl
tl

si

C(

si
ir

tl
si
d',
w
o1
ti
tl
H

IE

tl

lo

fi

as

F
oi
w
cl
ci
w
fi
fi
ta
cc
ci

01

th

m
m

I

J^

Products of the Masked Heterozygo

of the Normal Hete

This array in fz and through subsequent generations
continues to breed true to type" appearing uniformiy as
an extracted 'sig type". Is not the same as extracted sig-
type of other class of Ff heterozygotes. True condftion
of the popu/ation is shown in this figure.

Ex tra c tec/ fz Majked fz

sig. type Heterozygote

Pu'£lp-Ac-Ac£lp-f^ Pu-Elp-AcAc'flp'f^

Masked Fz
Extracted di^ type

Pu-flp Ac-Ac Elp-Pu

Extractsd Fz sig'type.
Breeds true and is
pure for characters
shoivn and like
parent sig. stock.

Extracted F2

sig type
PuEip'AcAcflpFu

Fz heterozygous type Extracted div. type .
gives tn /5 duplicate Breeds true and is
of the Fz array and pure for characters
so on shown and is like

paren t div. stock .

Fz heterozygote

orm.d.type
PuElpAcAc'Elp-Pu

appea

Gametes.

F,
Zygotes.

•ion of these gametes produces a masked /^ array that
oear as all ofthe sig- type. True compositions shown above.

Extracted Fz

div. type

Pu-Elp-Ac'Ac-E/p-^

Union of these gametes produces a typical Fz array of these type.

h-Elp-Ac Ac'Elp^Pu AcElp-Po AcElp

@ -

Pu^Elp-Ac AcElp:Fh Pu-Elp-Ac Ac-flp-Pu
divXv fsigS (di^V

These gametes when crossed under conditions of experiment
gives in F/ two types of heterozygotes in about eij/ual numbers

Zygote

Schematic representation of results obtained in crossing i. signaticollis and L. diversa \

THE SAME conditions AS SHOWN IN PLATE 9, BUT REPRESENTING BEHAVIOR OF GAMETES AND CHARACT
COMBINATIONS WHICH HAVE BEEN SHOWN TO RESULT.

Products of the Normal Heten

fenerations
m/form/y as
'tracted s/'g-
•e cond/tion

3sked Fz

ted d/'v type

-Ac-AcElp-Pu
1/
V

He terozy^ ote

Extracted Fz sig.type.
Breeds true and is
pure for characters
s/iown and li/ce
parent si^. stock.

Extracted Fz
sig. type
PuE/p-/lcAc-Op-Pu \^^

Fz heterozygous ty/
gives in F3 duph'cat.
of the Fz array an>
^o on.

Fz heterozygote ;
or mid. type

PuE/p-AcAcE/p-Pu
' /

y

ray that
own above.

fie terozygo us
'g appearing type

Union of these gametes produces a typical]

Pu-Elp-Ac AcElp-Pu Pu'Elp-Ac
sig. \ I diu:\V ( ■sig-

)F RESULTS OBTAINED IN CROSSING L. signaticolUs AND L. diversa UNDER
•>? PLATE 9, BUT REPRESENTING BEHAVIOR OF GAMETES AND CHARACTER OF
SHOWN TO RESULT.

Eeactions and Peoducts in Intekspecific Ckosses 113

the formation of the determiners for striping and punetation into a closely
bound association, which thenceforward acts homogeneously to produce no trace
of stripes in subsequent generations, owing to the lack of interaction of V with
Elp. What it is that changes this arrangement in the system I do not know,
except that it occurs only when Ac has the relative values of 60 and 40, and
imder conditions of the medium as given. It would at present be useless to
speculate upon what the actual change is in the material constitution of the
gamete. The entire complex series of crosses between these two species thus
becomes intelligible and in accord with the principles of factorial constitution
and operation, capable of elucidation without making assumptions of agents
either internal or external, or relations, associations, or results that can not be
put to test in one way or another. The entire series shown in plate 9 and all
of its consequences works out as is shown in the following analysis of the opera-
tions as outlined above.

In plate 10 is shown the parental conditions and the events in the F^ and Fj
of an array like that shown in plate 9. The complex under which this cross has
in general been made is that of the breeding quarters, as follows :

Table 8a.

Temperature.

Relative humidity.

Daily average

80° -*- 5°
75° ± 8°

75° ± 5°
75° ± 5°

Night average

Wind movement continuous and causing a complete change in the medium
at least once in 30 seconds, giving an evaporation-rate higher than that in the
habitat of diversa in the growing-season, lower than that of signaticollis.

The two parent stocks with the Ac values of diversa {Ac^°) and signaticollis
(Ac*°), when crossed under the conditions given, produce uniformly the stated
results in about equal ratio of 1:1. This ratio, however, is only a product of
the special complex, both within and without.

The Fi types both produce the same array of gametes, except that in the
mashed heterozygote, the V determiner in the diversa gametes has in this series
formed in the interactions in F^ the new association shown in the graphic rep-
resentation in plate 10. From this point onwards, Pu-V exists as a group,
acting as a single agent in the gametic operations. Each of the F^ heterozygotes
forms two classes of gametes, giving in Fj the typical array of three classes in
the ratio of 1 : 2 : 1, in one set readily visible, in the other only detectable by
biometric measurement and the test reaction. In both the extracted types
breed true. In plate 11 is shown the continuation of these true-breeding lines,
from F„ onward through F^, Fj, and Fg, the mid-type or heterozygous mode not
being continued, as it duplicates repeatedly the Fj behavior. In Fj is shown,
in graphic form, the application of the test reaction to the two lines. When
crossed with the line derived from the F, higher or signaticollis mode, nothing
happens, and the reason is here shown in the gametic compositions that enter
into this combination, no elements but pure signaticollis entering; consequently
none other is brought out. In the other line, the cross of an F^ extractive from

114 The Mechanism of Evolution in Leptinotarsa

the lower or diversa mode produces an F5 zygote that is heterozygous, but
appears as signaticollis in type.

The Ac values of all the lines, and indeed after F^ of the original cross, are
Ac^°, all trace of the value Ac'^'^ having gone from the lines. When the cross is
made between the test signaticollis at the value of Ac^^ or Ac*", under the gen-
eral conditions of the experiment as far as temperature and relative humidity
are concerned, there always appear, following the test, diversa types in the Fg
fraternities. The numbers present are not constant, and it has been found that
the highest number appears when there is little or no wind-movement, hence
low evaporation ; that the least frequent comes with wind-movement producing
rapid evaporation. Plate 11 shows only the fact that the diversa type comes out
as the result of the test reaction.

In plate 12 is shown the full array that comes in the test reaction when made
between pure signaticollis with Ac*° and the tested form from the lower mode
with Ac"", under the conditions given. The full Fj array shows the following
types :

(a) diversa, a type in all respects diversa that breeds true.

(&) mid-type a, that is, in all respects a heterozygote in appearance and in
reaction between the two species.

(c) mid-type h, of lighter appearance than the preceding, heterozygous, and
giving in F2 : 1 diversa, 3 si^rna^icoZZis-appearing types within close
limits to the expected ratios.

{d) mid-type c, different from the other two, and in Fj giving 1 diversa, 2 mid-
type c, 1 new signaticollis type, also in close realizations to the ex-
pected frequency.

(e) signaticollis type, that is, the extracted lower mode race, breeding true.

(/) signaticollis type, in appearance pure signaticollis, in reality heterozygous,
giving in F2 all signaticollis, but arrayed in trimodal polygons, the
modes being on the index-modes of the parent species.

(g) signaticollis type, that is, pure, true-breeding signaticollis.

(h) signaticollis type, that is, heterozygous, for a new arrangement that comes
out in the test.

(i) signaticollis type, that appears as signaticollis, but is really heterozygous
for the two arrangements Pu-V and Elp^V.

(j) signaticollis type, that breeds true, is homozygous, for the association of the
V determiner with the Elp factor producing general pigmentation in
the elytron, and by inbreeding almost black individuals can be
obtained.

As far as experience goes, the F^ test hybrid produces two main classes of
gametes, 50 per cent that are signaticollis and 50 per cent diversa. The signa-
ticollis gametes in all tests are apparently pure and unaltered, but the diversa
gametes show with changing conditions recombinations between the three
essential agents involved. The condition shown in plate 12 is that realized most
frequently under the conditions of experiment, with three classes of diversa
gametes present. The relative number of these is apparently entirely the
combined product of the external conditions, the Ac values in the two stocks
interacting in the gametogenesis or at some other time in the life of F^.

This in general is the result of the test reaction, revealing as it does the
existence of the V determiner and its restoration in some of the gametes to its
normal relations, resulting in the recovery of the lost diversa type. Of interest

epresentation of some res
This plate shows the continued breeding of certain lines sta
AS regards gametes and placing of certain factors. In Fa IN •;

■ described in the text to suspected stock
come from the application of this test to these suppressed heterozy

Pu-Elp-Ai° Ai%£lp'Pu Pu-Efp-Af ApEl^^ Pu-Efp-Af A^/p-PO Pu-E/p-Af° Af^Elp-Pu

IZ.5%±

Pure slgnatlcoliis stock
is shown when crossed with
one of the extracted F^
lines that appear also
as a sig type coming from
the true breeding F, sig. type

To SHOW IN DETAIL THE COMPLICATION

DESCRIBED IN TEXT.

I

i

Reactions and Products in Interspecific Crosses 115

in this test series is the formation of a new association of the V determiner,
giving a new race in the series that breeds true.

In the further breeding the new race (J) that comes out in the test and
its testing with the same reaction show that it is in no wise an exception to the
entire series, but that the test will, under the conditions of experiment, produce
the dissociation of the agents, restoring them in some of the gametes to their
original relations, and giving diversa as the result in lines in which it had not
been present for generations. This dissociation, in this instance, is easy to
accomplish if the temperature and humidity are increased above that previously
used and if the evaporation rate is low.

There results from this testing and other operations two races that in appear-
ance are signaticollis, in reality are diversa, with the Y determiner in different
positions in the gametic system, and that breed true without limit.

This series of observations and experiments with crosses between two species
under conditions that give the reaction shown in plate 9 are interesting and
suggestive of many important considerations. In that portion of the F^ popu-
lation that was signaticoTlis-\\ke in aspect there were possibilities for misinter-
pretation and wrong conclusions and operations. The fact that it bred true, so
that in ordinary operations it would have passed as a pure extracted race of
signaticollis, that had come out as the result of one or the other of the parents
being " heterozygous " for some complement or factor present in homozygous
condition in the other, might easily lead to confusion, and the further test cross-
ings would have been interpreted as mutations in the extracted line. If the
operation had taken place in nature and had been unknown, and the stock had
come into our hands in the laboratory, the conclusion womld be drawn that it
had " mutated," giving new " elementary species " or races.

Aside from the potentialities for mistakes in the laboratory, the series is
suggestive of important roles in nature, both in the matter of rendering species
in nature complex and also in the role of serving as an actual motive force in
evolution. It is not diflBcult to picture a series of events in nature, such that
introduced forms in crossing would, in the manner shown here, gametically
change the nature of the resident form without in any way altering its pheno-
typical aspect, so that it would pass as one form, " variable " perhaps, which at
some future time might mutate, producing quite new characteristics, which were
in reality only the reconstitution of an arrangement that had previously formed
a portion of the attributes of some intercrossed species. An apparently pure
signaticollis race, when crossed with another of known purity under the con-
ditions internal and external that have been stated, showed in the test in Fj an
array of 10 types in differing proportions. Some of these 10 are pure-breeding
when mated with likes and remain constant, even though they may be hetero-
zygous. Others are pure, as far as tests can determine, and others are hetero-
zygotes in the ordinary sense. The array might in nature well be the basis for
the origin of new groups or species.

In many respects the action of the signaticollis race that comes out in F^
reminds one of the fixed hybrids that are often described. I have had a number
of instances in my cultures of fixed pure-breeding heterozygous races, and all
appear as fixed hybrids in the ordinary tests that are applied. The fact that the
form comes true in breeding may be of importance, but it is not a reliable

116 The Mechanism of Evolution in Leptinotarsa

criterion that it is either homozygous in action or in composition, and its
apparent homozygous action may well be the product of inability to make mani-
fest the conspicuous differentiating characters, and so the series is passed on as
pure-breeding extractives, when in reality the thorough testing of such races
is necessary to determine their true characters. In the great majority of the
instances I have had to deal with, the usual tests of present neo-Mendelian
operations are not sufficient.

The most important internal agent is the Ac determiner and its values, and
the most important external one is the water-relation, as measured in terms of
evaporation from the body. These animals obtain water only from their food
and as hygroscopic water that is absorbed from the air through respiration.
They are extremely sensitive to changes in the water-relation, and it is sug-
gestive that a relation has been found between the water-loss, the determiner Ac,
and the recombination of the V determiner in the operations of F^. The
modifiability of the internal mechanism represented by the Ac determiner, its
rapid change to a balance with the conditions of the medium, and its readiness
to shift its values within limits, suggests most strongly not any substantative
thing in the organism, but rather a general state of the entire mass of the system
in the relations of its component atomic or molecular elements; that is, pro-
ductive of rapidity of reactions in one instance or their prolongation in others.
Within any species or group of species this agent is capable of change only
within limits, and I have not in any instance been able to modify it beyond those.
Thus, in L. signaticolKs, 29 days is the absolutely low limit, and one that can
not be maintained, while 37 to 40 days is the lowest that can be maintained with
any success. The upper limit is 63 days as the condition that can be held in the
strain for purposes of work. In L. diversa, the lower limit is about 50 days, the
upper 70 days or thereabouts. Even though plastic in its nature, this general
condition in the mass of the system is specific within limits for that mass, and
the specificity of the condition segregates with the mass of each gamete with
certainty and distinctness. None of the extracted diversa gametes or zygotes
in these experiments take on the rate found in signaticollis at its more rapid
limit, nor is signaticollis made to react as slowly as can diversa, but in the cul-
tures both may and do react perfectly within the mid-limits of their ranges, and
when the two are reacting in these values, crossing of the two species is always
accompanied with the most regular and stereotyped monohybrid Mendelian
reaction. Divergence in the rates of action of this agent in the gametes is
productive of the changed arrangement in the agents that are productive
of elytral characters, although why it should be these and not others
it is not possible to state. It may well be true that there are others that are
not observed that are changed, and that added investigation will disclose them.

Precisely the same series of events occur if the cross is made in the other
direction, diversa female and signaticollis male, so there is no discovered rela-
tion of the action to sex, or sex-linking.

Decidedly interesting relations of this reaction to the external conditions in
the medium have been developed as the outcome of this series of experiments
and its analysis thus far. These experiments show a relation of the operation
of the internal agents to the conditions in the medium that is most suggestive.

Eeactions and Products in Interspecific Crosses

117

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118

The Mechanism of Evolution in Leptinotarsa

In figure 8 I have put in condensed form a schematic presentation of the
general results that have been obtained in the study of the role of external con-
ditions in modifying the reactions in crossing. In these experiments there are
two important agents — the value oi Ac and the composition of the climatic
conditions in the medium ; but since the value of J. c is the direct product of the
conditions and duration of the race in the medium, the external complex plays
the major role in the production of changes in the Mendelian reaction, I have
used two sets of conditions, as follows:

Table 8b.

Climatic complex X.

Climatic complex Y.

Temperature.

Relative
humidity.

Temperature.

Relative
humidity.

Day

' F.
75 ±5
50 ±5

Per cent.
50 ±10
80 ±15

Day

° F.
90 ±5
75 ±8

Per cent.
75 ±5
75 ±5

Night

Night

Air-movement, 425 to 450 ft. per min.;

av., 440.
Evaporation, loss in cm., av. 84.6 cm. per

24 hrs.

Air-movement, 200 to 230 ft. per min.;

av., 220.
Evaporation, loss in cm., av. 41.7 cm. per

24 hrs.

With stocks of known standard reactions, when crossed, under the conditions
of climatic complex X, with the Ac values 40 and 60 respectively, the uniform
result is the production in F^ of all signaticoUis type, and these in F, breed true ;
but the polygon of all fraternities that have been tested are trimodal, whereas
the line continues to appear to breed true as a ^i^rnaiicoZHs-appearing type, as
shown in case 1, figure 8. Under these conditions the desiccation is intense and
the result is to accelerate ontogeny, so that the reaction in F^ is to alter the
arrangement in all of the gametes, and even in the F^ heterozygotes, so that they
are in aspect signaticoUis. This is a striking result, and although the result
breeds true in ordinary terms, it is truly in all instances thus far seen hetero-
zygous, as may be demonstrated by biometric analysis of the individual fra-
ternities, or by application of the test reaction. Both tests are equally good, but
the biometric test must not be applied to more than the members of a single
fraternity if the results are to be clear and useful. In table 9 I have given the

Table

9.

Date.

P.

All signaticoUis.

1

Fi

F2

Fs

F4

Fe

F,

M.

F.

M.

F.

M.

F.

M.

F.

M.

F.

M.

F.

Apr. 1904
July 1904

Mar. 1907
Apr. 1907
Apr. 1907
Aug. 1907
June 1909

Aug. 1910

Sig. m. A38 X div. fem. A^i

F'em. sig. A*" x m. div. A"

M. sig. A*" X fem. div. Asi

Fem. sig. A*i x m. div. A^**

Fem. sig. A*o x m. div. A"

M. sig. A*2 X fem. div. A^^

Fem. sig. A*" x m. div. A""

Fem. sig. A" x m. div. A«»

M. sig. A*i X fem. div. A^o

Fem. sig. A^' x m. div. A«2

M. sig. .\" X fem. div. A«2

76

49
55
14
27
74
38
12
44
77
73

95
46
61
17
24
86
39
9

151
214
314
56
156
46
86

150
226
327
51
154
54
92
197
156
31
41

199
196

46
121

44

204
199

54
127

51

42

44

74

77

151

150

1393
930
857
658
453
260
718
404
387
938
1315

46

50

92

84

146

137

87

92

42 145
74 27
61 29

214

276

226
281

56
171

54
192

92
85

87
106

Total obs.

8318

i

Keactions and Products in Interspecific Crosses

119

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120

The Mechanism of Evolution in Leptinotarsa

data that has been derived from experiments of this character extending from
1904 to 1910 and carried in all far enough to make certain of the result. In
the 11 tests of this sort that are given, the uniform result is the entire failure
of these heterozygous types to split up in any of the 8313 individuals in the
record. All of the lines have been tested at one time or another, either bio-
metrically or by the test reaction, and all of them showed unmistakable hetero-
zygous conditions in the fraternities, either in the trimodal polygon, with the
modes on the modes of the parent species, or the recovery of the diversa form
from the signaticollis strain in the manner described in the preceding pages.

When the same materials are crossed under the conditions of the climatic
complex F, the stereotyped result is the production in Fj of the signaticollis type
and mid-type in about equal numbers, as is shown in plate 10, and in diagram
in figure 8, case 2. In table 10 is given the data from 5 experiments, showing
the uniformity of behavior in case 2, and aside from the change in the reactions
of the masked heterozygotes, the remainder of the population reacts in perfectly
regular manner.

The behavior and modifications of reactions shown in figure 8, cases 1 and 2,
only follow when the values of Ac are at about 40 in signaticollis and 60 in
diversa. As the two values approach, the reactions change, and many transi-
tion stages have been found between the two types represented, so that in the
end the reaction is that shown in case 3, in figure 8, in which the reactions are
normal monohybrid in all respects. This type of reaction, shown in case 3,
IS of interest in that it is not disturbed by the conditions of the medium, even
when more extreme than those in complexes Z and Y. For example, stocks of
these two species have been sent from the laboratory at Chicago to the plant
at the Desert Laboratory at Tucson, where the conditions given them were
desert surroundings ; nevertheless, there has not been any change in the pro-
duction of the Fi of the usual mid-type array, with Fj showing the three
usual types. In the same manner, in the experimental conditions in the
laboratory, the conditions of the medium do not, within limits, change the re-
action of the crossing of the two species in any respect when the Ac values are
the same. In table 11 are given some arrays of the crossing of these two when
the Ac values are the same.

Table 11.

Date.

p.

Fi

Fs

Fs

F«

Fj

■ 2.56 sig. . .

181 sig...
'177 sig...

93 sig...

46 sig...

r 122 sig...

107 sig.
64 sig.

May, 1907.

Sig.fem.A"" X
div. m. A°°.

93 mid. ...

. 516 mid. .
260 div.. .

- 469 mid

182 div. •
— div..-

\ 252 mid..
1 124 div...

— div...

— div...

131 mid.
63 div.

■ 576 sig...

72 sig...
■ 90 sig...

46 sig...
121 sig...

[201 sig...

93 sig.
73 sig.

Do. ...

Sig. m. A"° X
div. fern. A""".

107 mid. ...

- 1158 mid..

■ 181 sig. .
91 div. .

\ 404 mid.

[198 div...

310 div...

214 div.

581 div.. .

291 div...

443 div...

106 div.

Reactions axd Products in Inteespecific Ceosses 121

An interesting complication is introduced into the series by the reactions
shown in figure 8, case -i, in which the output from a single pair of parents may
be altered, if the parents have the proper Ac values, by the application of the
climatic complexes X and Y to the parents at the time of fertilization of the
gametes and in the early stages of development. In the figure is shown the
process of production in the same pair of parents of the two discordant re-
actions of cases 1 and 2 in the progeny, merely by the use of the proper climatic
complexes at the appropriate moment. In this there is nothing that is not in
harmony with the other findings in this series of experiments, but it checks
some of the results of the other cases, showing that the differences produced
are really the product of the differing Ac values, the conditions of the medium,
and the nature of the two gametic systems. In all, it is purely a dynamic pro-
duct of the interaction of agents, within and without the organic mass, and in
no wise anything but purely physical in conception and operation. In the
laboratory the order in this type 4 can be varied so that the role of any possible
age affects strength of parents, " orthogenetic," "ontogenetic progressive,^'
gametic change, and kindred "interpretations" may be checked and effectually
eliminated. No trace of the operation of any of these supposed agencies has
been found. In table 12 is given data from four experiments of this sort,
showing the average results that are obtained from type 4 reactions.

I have presented in this digest of the crossings of these two species the re-
sults of much more work than appears upon its surface. From 1907 to 1914
the series has been in continuous operations, with a minimum of 4 generations
per year, and more than 100 matings per year that were productive of progeny.
Throughout, the fraternities have been strong and often have been purposely
limited in size to save labor and food, so that the total numbers might easily have
been far larger than they have been, but no purpose would probably have been
served by this increase in mere numbers. I have never had the total numbers
compiled that have passed through our hands in this study, but breeding not
less than 100 fraternities per year, with on the average of 50 progeny per
pair, gives an estimated number of about 25,000 that have in one way or
another been consumed in this investigation. Many of these are either routine
tests of the material or tests of the different products of experiment, and
have been used merely as indicators of the composition in the manner shown.
The counts of a considerable part of these fraternities have been made by
Mr. Kuehnel or others, and I have in many and in all critical instances checked
the determinations, suspicious cases being bred out and otherwise tested to
decide their nature. The earlier series from 1904 to 1907 are regarded as
preliminary tests and do not enter into this account to any extent.

The role of a reaction of this kind in a state of nature is obviously important.
It is not difficult to discover in nature, or to conceive of conditions and acci-
dents producing exactly the array that is shown in case 1, where the result
would be, while in all aspects and classification a signaticollis race in the
location, one with potentiality for change, per saltum, on the realization of
the proper agent entering into the history of the race, producing diversa or
some or all of the other types described. In the laboratory, if perchance the
reaction seen in case 1 were all that had been seen, the immediate conclusion
would be that the production of a fixed hybrid that breeds true for generations
had been accomplished. Case 2 in nature might also be a potent agent in evo-
9

123

The Mechanism of Evolution in Leptinotaesa

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Reactioxs and Products in Ixteespecific Crosses 123

lutiou; and in the laboratory, if it alone had been seen, an easy interpretation
would have been that one parent was homozygous, the other heterozygous, for
some character or agent. Case 3 and case 4 show the remainder of the relations
and the complexity of the actual series.

The essential agent in the entire series beyond question is the Ac determiner,
and its changes with the change of environment of the stocks, its capacity
to be altered rapidly in experiment, and the limits of change that it shows in
both of the species. It is clearly, in its operation to the conditions in the
medium, closely and accurately balanced, and especially to the relation of
water-loss in the organism and the consequent change in the relations and
associations of the agent productive of elytral color marks and pattern. That
the Ac determiner is a specific agent in the gametic complex is shown by its
limits in any one species that are specific for that, and by the fact that it is not
capable of replacement in the complex by the corresponding agent from another
source, thus giving in materials that are visibly the same, different Ac value
ranges and different lengths of ontogeny, the present measure of the Ac deter-
miners reaction value. Every indication regarding the nature of the Ac agent
tends to show that it is a property of the whole, a cytoplasmic determiner, and is
a condition of the material rather than any specific substance or body, but its
action is important in the life of the species and active in the production of dis-
turbances in the gametic system, when present in differing rates or reaction,
in any zygote. Another peculiarity is its tendency to change rapidly in cross-
ing within its limits to a common value of operation in F2. As a gametic agent
it is a very delicate one, easily neglected, but able to produce wide differences
in the results of crossing, so that one is made to ask to what extent an agent of
this character may have played the role of confusing us in some of the Mende-
lian reactions, leading to the assuming of conditions in the materials that were
not really there at all. Throughout this series it is certain that when the Ac
values are like, the conditions of the medium do not within limits have any
action upon the reactions in crossing these two organisms, the resultant arrays
being in all portions of the series entirely the product of the gametic constitu-
tions present.

Eather interesting in the series is the tenacity with which each of the gametic
systems retains its entirety. There are in this series no instances of the crossing
of agents from one gametic system to the other, although there are plenty of
opportunities for this to happen, so that the entire reaction is a most typical
monohybi^id one. At no point is there indication of difference in this cross in its
reactions from those found and described in other monohybrid crosses. How-
ever, "monohybrid crosses" represent to me more a type of reaction than an indi-
cation or statement of the differences present in the crossed lines. In this
instance there are plenty of differences that in all stages might have become disso-
ciated from the original association and formed attachments to other systems
present in the germinal material during gametogenesis. In some respects this
series of experiments is different from the crossing of many domesticated races, in
that in many of these there is only one species base to which are attached the
agent or agents that are capable of displacement and rearrangement. In the
signaticoUis X diversa series, the total gametic complex acts as a unit in the oper-
ations of gametogenesis. Both are the same in action, differing only in the
character and magnitude of the unit systems that are engaged in the operations.

124 The Mechanism of Evolution' in Leptinotaesa

This instance is, I believe, the simplest thus far described in the study of mono-
hybrid crosses and represents the simplest type of operation that can at present
be thought of in the intercrossing of two natural species. The only complexity
that enters into the series is that due to the differing values oi Ac and the role
of conditions in the climatic complex in inducing changes in the gametic
system through the activity of Ac.

The nature of fixed hybrids is suggestively indicated, as are some of the
alleged "permanent blends" between species. So, too, it is strongly suggestive
of the manner of production of many of the sports or mutants that occur,
especially in the crossing of domesticated races, and it may well be true for the
species in nature. Whether this is in any way related to the mutation behavior
found by De Vries in Oenothera will receive attention in its proper place.

LEPTINOTARSA UNDECIMLINEATA X LEPTINOTARSA SIGNATICOLLIS.

Crossing of these two species in its simplest conditions always shows reactions
of a trihybrid nature, and is, therefore, more complex in its relations and reac-
tions than the crosses of signaticollisxdiversa. These two species differ in
many respects in the adult, and in the juvenile stages inhabit entirely unlike
environmental complexes, with consequent differences in many of their
reactions.

In the adult condition there is difference in the form index, the elytra have
the same difference as in the diversa X signaticolUs cross, undecimlineata
being striped, signaticollis not, due to the same set of agents present in the
gametes. These agents, while similar, are not in diversa and undecimlineata
the same, and do not produce the same end-result ; that is, the elytral complex
from diversa is alternative to that from undecimlineata when the two are
crossed, the differences showing in the results of the 'Pu determiner and in the
character of the stripes produced by the V determiner.

In the juvenile stages there are differences of lipoid color and in the pattern
of the second and third larval stages. In undecimlineata the body-color is
white, with only the spiracular spots present in all of the stages ; in signaticollis
it is yellow, with only the spiracular spots present in the second and the full
system of spots in the third stage. Three alternative pairs of characters are
formed, no one of which is a simple one, namely, the unresolved species residue
characterized by the elytral-pattern group, the larval-pattern group, and the
lipoid-color group. There are others present, but they do not in this series
become dissociated from the general species complex, which, as far as discovered,
retains its integrity in the same manner that was present in the cross between
signaticollis and diversa. These three groups of agents are "dissociated" in the
ordinary operations of crossing. In addition to the agents mentioned, there are
also present in this cross the differences in the Ac determiner found in the pre-
vious instance, only in this the average value of ^Ic in L. undecimlineata is, in my
cultures, 64 days, same as in nature, within small range. In table 13 are
given figures, from cultures of this species, in the laboratory at Chicago and in
nature, showing the length of ontogeny, that is, the value of the Ac determiner,
which is not changed in this species by introduction into the laboratory.

The two species live in quite different conditions; undecimlineata living in
regions of high humidity and temperature, low evaporation rates, and long

Shows results obtained in crossing L. signaticollis and L. undecinilineata when the Ac values are

ESSENTIALLY THE SAME AS DESCRIBED IN THE TEXT, AND CONDITIONS UNDER WHICH THE CROSSES ARE MADE
ARE NEUTRAL. ThE ARRAY SHOWN ON THIS PLATE IS A TYPICAL TRI-HYBRID MENDELIAN REACTION.

Reactions and Products in Interspecific Crosses

125

seasons for development, longer even than in the habitat of L. diversa, in sharp
contrast with the conditions in the habitat complex of L. signaticollis. The
first results obtained in the crossing of these two species in 1904 and 1905 were
most confusing.

When these two species are crossed under common conditions, with the values
of ^c in each about 60, the uniform result is the production, regardless of the
direction of the cross in F^, of a uniform progeny that are intermediate in the
adults, with the undecimlineata larval groups dominant when the female parent
is undecimlineata, and with the signaticollis larval series dominant when
signaticollis is the female parent. The real dominant, if such there be, is
undecimlineata, as shown in the working out of the F2 array. In Fg there is
produced the typical array of a trihybrid. In plate 13 is shown the results pro-
duced when these two species are crossed, when the Ac values are the same and
the conditions in the medium neutral, and the same result is produced when the
cross is made in the opposite direction, the only difference being the dominance
in the juvenile stages of the female parent over the male in the juvenile charac-
ter, so that the only difference between the reciprocal crosses is the dominance
in Fj of the larval complexes characteristic of the female parent.

Table 13.

stock No. 722, from Coatzacoalcos, Mexico.

Stock No. 714, from San Marcos,

Vera Cruz

Mexico.

Chicago.

Nature.

Chicago.

Nature.

Period. Daj's.

Period.

Days.

Period.

Days.

Period.

Days.

Jan.

6 to Mar. 11.

64

Dec.

27 to Mar. 2.

65

June

5 to Aug. 4.

65

June

1 to July

29.

59

May

11 to July 14.

64

nee.

29 to Mar. 1.

62

June

Ito Aug. 1.

62

Aug.

10 to Oct.

14.

65

Mar.

9 to May 6.

58

Jan.

4 to Mar. 10.

65

Mav

30 to July 29.

60

Julv

7 to Sept.

11.

66

Mar.

5 to May 1.

57

May

11 to Julv 12.

62

Mav

30 to Aug. 5.

67

July

5 to Sept.

7.

64

Mar.

20 to May 23.

64

Mav

8 to July 13.

66

Apri

4 to June 2.

59

Aug.

1 to Oct.

4.

65

May

30 to Ju!v 31.

61

Aug.

4 to Oct. 1.

58

Jan.

5 to Mar. 12.

66

Oct.

29 to Jan. 6.

67

June

7 to Aug. 1.

55

Jan.

7 to Mar. 6.

58

Nov.

2 to Jan. 5.

64

June

5 to July 31.

57

Aug.

5 to Oct. 10.

66

July

25 to Sept. 29.

66

Jan.

1 to Mar. 4.

63

Aug.

2 to Oct. 1.

60

July

20 to Sept. 27.
Average

69

Jan. Ito Mar. 8.
Average

68

Aug. 2 to Sept. 28.
Average

57

i

63.4

62.1

62.0

63.8

Note. — The Chicago records are random samples from the breeding stocks at Chicago, taken from F] to Fio.
The records from nature are from tests of breeding in the native habitat made mostly in 1905 and 1906.

If in the crossing of these two species the conditions of the medium be those
used in the normal cross of signaticollis and diversa, the dominance in F^ is
always of the female parent in larval characters, but in the adult the result is
always uniformly mid-types strictly intermediate between the parents. The
Fj dominance of the female larval characters may be changed partly or com-
pletely by the conditions present in the medium, or incident upon the progeny
during ontogeny, so that the aspect of the F^ larvse is variable, depending
upon the conditions of life. Under neutral conditions F^, while variable in the
manifestation of the larval condition, is constant in the adult array and
remarkably uniform in the Fo products that come out.

While none of the groups of agents or characters that are alternative are
simple, but are in instances compound associations of agents whose collective
interaction is necessary to produce the character, the reaction in this cross is

126 The Mechanism of EvolutiojST in Leptinotaesa

that of the group collectively and not as so many individual agents, and as a
result there are three groups that need be considered, namely, the mass of the
gametic system, symbolized by the elytral pattern group, which for brevity may
be represented by A for undecimlineata, a for signaticoUis, B for the pattern
condition in the larval stage of undecimlineata, b for the same in sig7iaticollis,
and C for the white lipoid-color group of undecimlineata, c for the yellow of
signaticoUis. In this cross other agents are neutral and do not act in the opera-
tion, so that the composition of signaticoUis is abc, undecimlineata ABC. In
all respects this series can be and is, in reality, as far as this cross is concerned,
a typical trihybrid reaction, as typical and constant in its reaction as the classical
case of Mendel's trihybrid peas, with this important exception. Mendel's peas
had the three characters present in the same stage of the individual; in this
series the three contrasting characters are not present at the same time in the
ontogeny of the individuals, but at successive periods in the life of the indi-
vidual, and none is in any way dependent upon those antecedent for its mani-
festation,- so that this series is of interest as showing that the condition of
trihybrid or any other is not limited to the manifestation at one stage, but that
the condition governs the entire life-cycle.

In plate 13 the results shown are of the cross of these two species when the Ac
determiners are the same in value and th-e conditions of the medium are uniform
and neutral. The result in Fg is that, in the larvae in the second stage when the
first contrasting characters come in, there is a sharp separation of the population
into two classes, white and yellow, in the proportions of 3 white, 1 yellow, the
array appearing in all instances that of a monohybrid type. In the third larval
stage each of the two classes again separate, upon the basis of the pattern system
present in this state, into two groups that have the general relations of 3 without
pattern, 1 with pattern ; but in the entire array in the third larval stage there
are present four classes of larvae: 9 white without spots, 3 white with spots,
1 yellow with spots, 3 yellow without spots, showing the " dominance " of the
white over yellow and of no spots over spots, or undecimlineata pattern over
signaticoUis. In the adult each of the four larval groups gives three kinds of
adults : extracted signaticoUis type, undecimlineata type, and mid-types. These
adults are always pure for the adult characters in the extracted types, but may
be hybrid for one or two of the larval characters, or pure in all. The mid-types
are never pure for the adult characters, but may be pure for the larval, or hybrid
in 1 or 2.

That there might be no mistake, I have tested out the different products in Fj
as a routine laboratory operation, and have found that all of the expected com-
binations were present, giving the actual kinds of individuals that Mendel's
hypothesis demands. I have found 27 different gametic conditions in the Fg
array and no more, and these were in entire agreement with Mendel's hypothesis
as to their composition and their products in Eg. Th-e combinations have been
found and tested to the extent as shown in table 14.

This series has been a fortunate one, in that the condition of the larval char-
acters is shown in the ontogeny, so that one is able with fewer matings to make
an analysis of the Fg array than if the three characters were all present in the
same stage, and the fact that when A and a are present in the gamete the product
is always intermediate, separates at once, in adult characters, homozygous from
those that are not. With experience I learned to separate in Fj nearly all of

Eeactions and Products in Inteespecific Ceosses

127

the types with a high percentage of certainty, so that only a few of the matings
made were other than the intended combination.

This is the typical reaction given by inbreeding F^ mid-types with equivalent
Ac values and standard conditions, and no exception or variation thereto has
thus far been observed in F,, and it is everywhere consistent in ratios, products,
and the dominance in the larval characters of those from undecimlineata,
regardless of whether the undecimlineata parent was male or female. Unlike

Table 14.

Adult.

11-lineata . .
11-lineata . .
11-lineata . .
11-lineata . .

Mid

Mid

Mid

Mid

Signaticollis
Signaticollis
Signaticollis
Signaticollis
11-lineata . .
11-lineata . .

Mid

Mid

Signaticollis
Signaticollis
11-lineata . .
11-lineata . .

Mid

Mid

Signaticollis
Signaticollis
11 lineata . .

Mid

Signaticollis

Appearance.

Larvae.

Color.

Wh.

Wh.

Wh.

Wh.

Wh.

Wh.

Wh.

^Ti.

Wh.

Wh.

Wh.

Wh.

Wh.

Wh.

Wh.

Wh.

Wh.

Wh.

Yl.

Yl.

Yl.

Yl.

Yl.

Yl.

Yl.

Yl.

Yl.

Pattern.

In testing the composition
proved to be —

ABC = ABC

ABCc = ABCc

ABbC = ABbC ....
ABbCc = ABbCc ..

AaBC = AaBC

AaBCc = AaBCc . .
AaBbC = AaBbC .
AaBbCc = AaBbCc

aBC = aBC

aBCc = aBCc

aBbC = aBbC

aBbCc = aBbCc ...

AbC = AbC

AbCc = AbCc

AabC = AabC

AabCc = AabCc .. .

abC = abC

abCc = abCc

ABc = ABc

ABbC = ABbC

AaBc = AaBc

AaBbc =:AaBbc . . .

aBc = aBc

aBbc = aBbc

Abe = Abe

Aabc = Aabc

abc = abc

No. of pairs

tested of

each.

9

2
1

14
2
3
2
7
5
2
1
2
3
1
5
2
4
1
2
4
2
1

11
4
5
2
8

Total tested between 1907 and 1910 (pairs)

105

Note. — The number of pairs tested in the above table has no relation to the
frequency, owing to my being able to rather accurately determine by inspection the
composition and many matings made for purposes other than this test.

the c^o^5s of diversaxsignuticolUs, the array in F, shows nine different kinds of
mid-types, only one class of which is comparable to the F^ condition and occurs
closely in tests and recognized counts on the average of eight times in the total
frequency of combinations. All other mid-types are different in their constitu-
tion and reaction from the F^ and the one class of F2 mid-types. I have no
doubt that this represents a typical and perfect instance of the behavior present

128 The Mechanism of Evolution" in Leptinotaesa

in trihybrids, entirely comparable with those investigated by others, and in all
respects a duplicate of the classic trihybrid cross of Mendel with peas. The
contrasted characters represent groups of agents associated in reaction that are
transferred entire in the rearrangements of the qualities in Fg. Two points are
of especial interest in this series, namely, that the characters are successive in
ontogenetic sequence, a condition quite different from that present in most other
trihybrid crosses examined, and the presence in the series of a meristic series
of color-marks that is inherited as a unit system.

I have repeatedly made this cross in both directions, testing to Fg or beyond,
as far as space and aid permitted, and in all there has been not one indication
that the behavior in any respect was other than the most orderly and typical.
This statement applies only to the cross when made under conditions that are
neutral to the two, and when the Ac determiner has the value of about Ac^'^ in
both, so that the reaction represented with this complex, and conditions in the
medium, is the basal reaction between the two species complexes. The same
cross has given most perplexing arrays at different times, the production of
which are now to some degree understood. It is important to make certain that
the reaction between the gametic complexes is typical, or else to determine the
agents that produce the discordant result. In table 15 I have given examples of
data derived in the testing of this cross in Fj, where the Ac values were about
60 and the conditions in the medium were neutral.

One peculiarity present in this series is the dominance in F^ of the larval
color-characters of the female parent in the cross, unless the conditions are in
one way or another inhibitory to this, a change which is easy to produce experi-
mentally. There is no indication that the dominance is in any manner asso-
ciated with sex, or any other sex-relation. The corresponding groups of deter-
miners are introduced by the male gamete and are present in full intensity in Fj,
so that there is no suggestion of the contrasted characters being absent, and the
fact that the same result occurs in the reciprocal at once throws out any idea of
the strength of one parent species being superior to the other. The fact that
conditions in the medium were able to easily change the relations of the four
groups of agents in the larval stages, as far as their manifestation in F^ is con-
cerned, but not in any way in the Fj array, leads to the conclusion that the con-
dition of apparent female dominance in the F^ juvenile stages represents a
condition in the mass of the female gamete, already has an initial velocity along
the line from which it came, that is imparted to it by the developmental activi-
ties which produced the matured and which egg determines F^ juvenile domi-
nance. Therefore, the initial developmental momentum in the egg continues
in the same direction these groups of agents as uppermost in visibility, until
they are in one way or another, by internal or external conditions, retarded until
the two sets of agents present are on a level, and then the visible dominance in
the Fj heterozygotes depends entirely upon the role of conditions within the
organism and without it, a relation that is shown by simple experiments, so that
the reciprocal F^ juvenile arrays can, within the limits of the time and change
possible with the different molts, be altered at will. This is especially true with
regard to the lipoid body-colors, which can be changed about at will in the larvae
merely by the alteration of conditions. I am of the opinion that this is the
cause of the conditions found in the F^ crosses of Fundulus, described by New-

Eeactioxs and Products in Inteespecific Ceosses

129

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130 The Mechanism of EvoLUTioisr in Leptinotaksa

man and others. In this case there is no indication that there is any agent or
relation of the juvenile characters in the female stock that are productive of this
result, and the conception advanced here seems entirely in harmony with the
facts observed. I think no one will deny that the gamete, especially the female,
has at the time of fertilization considerable developmental momentum, as
shown by the fact that unfertilized eggs may go on developing for a time without
any inciting cause, either fertilization or artificial parthenogenesis, due to the
developmental momentum acquired in the growth-period, and that in this period
certain directions of reaction and rates of reaction become established and con-
tinue on in the zygote, unimpeded by the cross when made under neutral con-
ditions, so that this dominance has only a temporary existence and no genetic
significance.

Out of this cross there result 8 pure breeding races that are homozygous in all
l-espects and that continue homozygous without limit either pedigreed cultures
or in mass cultures. These appear as shown in table 14, a result different from
that present in the cross of signaticollis X diversa.

This series shows that in the crossing of species there is no detectable differ-
ence in the reaction from that present in crossing intraspecific conditions ; in
both the reaction is the product of the number of individual agents or the groups
of agents that are capable of being dissociated and metathetically redistributed
between the gametic systems present. I have made every effort of which I am
capable to discover if this instance was not nonconformable to the results of
Mendel, or that the interpretation was not correct. There have been abundant
complications which will be described presently, but in all the net result has
been complete confirmation of the reaction as described by Mendel in his tri-
hybrid cross of peas. It may seem a waste to attack this instance so extensively,
but the materials have served as the basis of some other investigations, and this
served to test fully whether in this respect they were in accord or discord with
materials used by other observers. The series has an interesting outcome in the
production in F, of 8 arrays that are different in their life-cycle, so that there
are 8 potential species which would have been described as species by taxonomists
had they been found in nature and their pure breeding and constant characters
been determined, and in fact such Juvenile differences have lately been made the
basis of separation of species of Culicidoe by different workers, no doubt with
entire correctness, but is it not possible that many of them have arisen or been
distributed in the series by processes of this character ? The taxonomist knows
nothing of the genetic sequence or constitution in his species, only the fact that
the same form occurs repeatedly in the same locations, and genetic continuity
is assumed, in most cases with correctness ; but one is made to suspect that the
idea has been too liberally applied.

The reaction that has been described may be called the hasic reaction between
these two gametic systems, and shows the extent of the dissociation of the sys-
tems that takes place under neutral conditions of reaction, and not in any way
influenced by external conditions. When, however, differences in the value of
the Ac determiner in the medium and in other agents that are present are
brought into the complex, then the reactions become much complicated, pro-
ducing results that, at first sight, appear entirely at variance with the principles
of factorial composition and reaction, and " gloriously non-Mendelian."

Breeds true in tOO^

Reactions and Products in Interspecific Crosses

131

The most common departure from the routine condition in the crossing of
these two species is shown in plate 14, in which the results are graphically given
when the cross is signaticollis with the Ac*^ and undecimlineata A&^. The con-
ditions of the cross external to the organisms have been on the average as
follows :

Day .

Night

Temperature
(average).

° F.
101 ±4
85 ± 3.5

Relative humidity
(average).

Per cent.
77 ±6
97 ± 3

Wind movement, av., 440 ft. per min. ; range, 425 to 455.
Evaporation (av., 52.3 c. c. per 24 hrs.) :

Day (7 h. 30 m. a. m. to 5 h. 30 m. p. m.), av., 43.1 c. c.

Night (5 h. 30 m. p. m. to 7 h. 30 m. a. m.), av., 9.2 c. c.

These conditions presented evaporation-rates in excess over that found in
the normal habitat of undecimlineata and about that present in the habitat of
signaticollis. It has made no difference that can be detected whether the stocks
came direct from nature or whether the rate of action of ^c had been produced
in the laboratory, the result is the same.

The products of the cross are in F^ that the larvae divide into two groups most
clearly determined in the second larval stage, yellow and white larvae, and this
division is present in the third stage, with both having the pattern-system of
undecimlineata dominant; the larvae are either yellow or white without spots.
The numbers are highly variable but on the average are 3 white to 2.7 yellow,
essentially a 1 : 1 ratio. The white larvae give only adults that are undecim-
lineata in type; the yellow larvse give only mid-types. The two classes of
adults are also present in essentially equal numbers. The F^ undecimlineata
types when inbred " breed true " without limits, as pure-breeding races, in
aspect showing only the presence of the undecimlineata adult and larval char-
acters as long as the breeding is continued. Inbreeding of the mid-types
derived from the yellow larvae shows in Fg only the normal and entirely typical
array produced by the mating of typical F^ heterozygotes between these two
species, and many dozens of tests of these were made between 1906 and 1914, so
that we need not concern ourselves further with this portion of the series. The
undecimlineata types that are derived in F^ are the portion of the experiment
that is of interest.

In superficial respects in general behavior this experiment is much like the
reactions shown in plate 9, in which it was seen that the two F^ classes were
different conditions of heterozygosis, and it is on the surface probably the
same in this cross. There is one conspicuous difference in the apparently
extracted type that comes out in F^ in this cross that is not found in the first
case, namely the decreased rate of reaction present in this undecimlineata type.
Unlike the normal form which has diO. Ac value of about 60 to 64 days, or the
Fi heterozygote, which in this cross has the Ac value between 60 and 65 days,
the Fi undecimlineata types have the Ac value from 70 to 98 days. The actual
length of time between generations is 290 to 335 days, or about a generation in 10

132

The Mechanism of Evolution in Leptinotaesa

to 11 months, showing a considerable lengthening of the developmental period.
This result is attained regardless of the direction of the cross, and uniformly so
long as the conditions in the materials combined and in the medium are those
given.

This cross and the results that come out are shown in table 16, in which are
given the parental conditions, the F^ results, and the breeding of undecimlineata
types in Fj or beyond. No purpose is served by presenting the breeding-test of
the mid-types ; enough has already been given to show the characteristic reac-
tions they present.

The Fi undecimlineata types have been kept in the laboratory for gener-
ations ; they have been sent to Tuscon ; placed in isolated locations in Mexico,
and planted upon some of the Florida Keys. Nowhere have they shown any
change in the length of life-cycle or in the character of the form, or any

Table 16.

Parents of the cross.

F, adult a
types.

rray

Fn arrays and
reaction rates of Ac.

F3 arrays and reaction
rates.

11-lin.

Mid.

Min.

Av.

Max.

No.

Min.

Av.

Max.

No.

Sig. m. Ac3» X 11-lin. fem. Ac«=

Sigr. fem. Ac*o x 11-lin. m. Ac«*

Totals in 10 tests:

Sig., av. Ac, 38.3 )

11-lin. av. Ac, 62.2 (

46

, ' V

Pair A . . .

52

78
74

75
84
87

81
79
78
74

89
90

90
91
90

89
90
90
88

95
98

93
95
93

94

97
98
92

131

146

149
73
171

71
46
137
46

fAA 73
J AB 70
1 AC 79

Lad 83

fEA77
J EB72
1 EC 87

Led 80

rCA78
CB 79
J CO 83
1 CD 71
CE 78
LCF84

83
90
89.5
89
89
90
91
91

90
91

89
88
90
90

97
98
93
92
93
97
95
97

95

97
91
93
98
96

46
69
171
141
24
146
73
92

46
59

193
71
44

139

Pair B . . .

Pair C . . .

Pair D ...

Pair E ...

31

' 7^ >

37

Pair B . . .

Pair C ...

Pair D ...

292

1

301

70

89.7

98

2,214

70

90

98

3,144

The values given are for the length of ontogeny, egg to adult, and the number observed.
Average length of ontogeny has been 90 days = A^.
Average elapsed time between generations, 316 days.

other discovered change or return to the parental rates of development; nor at
any point in the series did there appear any trace of the signaticolUs type
coming out of the line. In Mexico, where they were introduced into a location
north of Jalapa in 1908, they persisted for at least two generations without any
change that I could discover; and material taken from the location in 1910 and
tested showed that the same condition was present as at the start. The type
appears highly resistant to differing external conditions and is in reality well
organized to persist in many different conditions of nature.

In the laboratory this type has been subjected to most varied tests in the
effort to prove it to be in composition a masked heterozygote. When crossed
with signaticollis the reaction is, when it is obtained at all, a duplicate of the
reaction from which the type arose, with about equal numbers of the two types
present in F^, and the undecimlineata type that comes out in this test
breeds true.

Eeactions and Peoducts in Interspecific Ceosses

133

Thus far only one test agent has been found that serves to even partly show
the composition and nature of this stable heterozygote, namely, the pure
■undecimlineata. The suspected race appears as undecimlineata in all respects,
and the test of its heterozygous nature would be to recover from it in some
manner the signaticollis character, either in whole or in part. When pure
undecimlineata and the suspected type are crossed under the ordinary breeding
conditions of the laboratory nothing results, the two breeding with perfect
freedom and no separation of the types appearing ; and perhaps the only reason
for so persistant pursuit of the race is the constant presence in the suspected
type of the trimodal condition in an otherwise perfectly uniform fraternity and
series of fraternities.

By applying the same test to the fraternities that I used in the cross of diversa
X signaticollis it was found that the Fo fraternities were trimodal, and that the
modes of the compound curve were essentially the same as the parental modes,
with the intermediate mode that of the heterozygous type. It was further
found that breeding from the modal conditions of the extreme modes at once
gave constant lines so far as the form-index was concerned, and that this was
in value the same as the parental modal values in pedigreed lines. Only meas-
urements of fraternities were of any use in this analysis, as fraternities are the
only homogeneous materials that we have to work upon. In figure 9 I have
shown the results of a biometric analysis of the suspected race and the products
of breeding in line from the modal conditions.

In many respects this would by many be considered competent proof of the
heterozygous nature of the suspected line, but the final proof is to recover the
suspected contaminating agents from the line. In this I have, to the present
time, been only partly successful, due in the main to the lack of the requisite
facilities for control and production of the necessary external conditions.

I have found that when the suspected race is crossed with a pure line of
undecimlineata under conditions to be shortly described, there resulted in the
progeny of such a cross the recovery of signaticollis larval characters — t. e.,
the yellow larval color and the meristic pattern system of the third stage.
These could only have come from the suspected race, as they are kno-mi not to
be present in the test materials used — i. e., pure L. undecimlineata.

When the test race is crossed with the race to be tested under the following
conditions, the Fg fraternities show signaticollis larval characters in widely
varying proportions in about 50 per cent of the trials. The combination of
conditions that I have employed are those of a desert complex, with high daily
temperature and intense desiccation, and low night temperatures, with satur-
ation, or nearly so. The average conditions were, in the tests that have been
made:

Table 16 a.

Day .,
Night

Temperature (average).

• C.
42 -f 2.3
21 -f 1.2

Humidity (average).

Per cent.
34 -f- 6.2
96 -f 4.5

Wind movement by day, av., 412 ft. per min.; night, av., 10 ft. per min.
Evaporation by day, av., 94 c. c; range 81 to 107; night, av., 2.3 c. c.

134

The Mechanism of Evolution in Leptinotaksa

This set of conditions is rigorous and serves to stimulate or to accelerate dif-
ferent ao-ents and operations in the two lines. When the cross is made between
the pure and the suspected materials, F^ shows no trace of the signaticollis
conditions at any point, nor does it appear in F, unless the same set of condi-

L. undecimlineata. L. signaticollis^
Fig. 9.

tions are applied to the breeding pairs of the two at the time of breeding ; and
moreover, the result is not produced unless the same complex is applied to the
Fi. The mated pairs ready for breeding are introduced into the apparatus
under the conditions and allowed to remain there until the eggs are deposited,.

Eeactions and Products in Intekspecific Ckosses 135,

when they may be removed to the usual conditions of the laboratory for the
remainder of the cycle, and then again treated as before in the mating of Fj.

The test has usually been applied to F., and F3 fraternities of the suspected
strains and to lines whose form-index was that of the parental signaticollis
species. The results show that the recovery of the signaticollis larval characters
from the different tests is by no means found in every test, nor in any regular
proportions, and sometimes it is only yellow color, at others it is the meristic
spot system, in others botli that are recovered, showing that the operation needs
further investigation.

After the recovery of the larval signaticollis characters from the race, they
continue to breed as true and to be as accurately alternative in crossing as if'
they came from pure signaticollis, and the end-result is that from one of these
tests it is possible to get certain lines with these constantly present as the larval
characters in materials otherwise undecimlineata. No indication of the posi-
tion and relations of these hidden groups of larval characters in the suspected
race that I care to discuss has been determined. However, the test, in com-
mon with the trimodal condition in the Fo fraternities in the suspected line,_
hhows conclusively that the line is in reality heterozygous and is in all respects a,
suppressed heterozygous series, excepting that in this series the combination
that the signaticollis groups of agents have made are binding and broken with
great difficulty, so that only certain most precise conditions and interactions re-
sult in the recovery of the agents from the suspected complex. It would be of
interest to know how long this condition would remain in the line. I have
found them present in F5 in one series and in Fg in another, but I do not know
how much longer they may remain. I do not know whether they play any other
role in their new situation, as there is no evidence of new characters having
come out in the line as a result of the contamination present.

One other suggestive test has shown that the adult elytral-pattern system is.
also present in the suspected race and has been recovered in the two tests that
have been made of this kind. It was found that when the test was made with
tlie same day conditions as in the first test series, but with the night conditions
having a much lower temperature, as low as 10° C, with saturated atmosphere
and no wind-movement and no evaporation, the number of individuals showing
the larval characters increased and that a few individuals arose showing the
signaticollis adult character.

These adult signaticollis characters thus recovered do not change, but are per--
manent in the race from that time on, and in one test produced in F4 a pure--
breeding homozygous race that was signaticollis in its adult characters. I have
not carried this testing further, as the requisite apparatus has not been available,
and an ice-box will not serve for the nocturnal low-temperature portion of the
test conditions; its conditions are too unsanitary and are productive only of-
failure.

Collectively the results of the testing show that the undecimlineata type that
appears in F^ is in reality a fixed or masked heterozygote in which all of the con-
trasting characters are present, but, in some way not discovered, are bound in
associations that are not broken excepting by certain specific and energetic
treatments. There is no doubt but that in the cross between signaticollis x
diversa and signaticollis X undecimlineata, when the Ac values are different,.

136 The Mecha^shsm of EvoLUTioisr in Leptinotaksa

there results, in conjunction with conditions of the medium, changes in the
association of agents or groups of agents in the gametes, such that in a portion
of the population the F^ heterozygous condition in appearance resemhles one of
the parent species and continues to breed true until the gametic system is again
changed and the agents freed from their position of nonvisible manifestation.
In both there is the same separation of the fraternities in Fj of the suspected
race into trimodal polygons, showing that the species form-base as measured by
the form-index is not affected by this firm association of the several agents in
the heterozygous individuals. It is true that the series in both species is quite
unusual — call it a non-Mendelian reaction if need be — but there is at no point
that I have been able to demonstrate with certainty anything either in action or
in result that is in any manner at variance with the principles of factorial com-
position and reaction, and further in the series the reactions, as far as they have
been analyzed, are most clearly in entire accord with the principles of the
Mendelian reaction. When it is possible to carry this further I have no doubt
that it will be capable of as complete analysis as was the simple case in the cross-
ing of signaticollis and diversa.

A feature of interest that has come out in these crosses is the slow breeding
of the undecimlineata race that appears in F^, giving about one generation in
10 or 11 months. As far as I know this slow breeding persists under ordinary
conditions indefinitely and remains a permanent character in this race. Some
indications of its origin have been obtained that are of interest. In Chapter II
it is shown that in signaticollis the normal cycle is two generations in rapid
succession, then a period of rest for about 9 months, passed either in the ground
in aestivation or in part on the plants in practical inactivity. Under the con-
ditions of the laboratory the same rhythm is maintained, but the length of
ontogeny spread out and the period of quiet between successive cycles is
shortened. In experiment, when the rate of reproduction is shortened by con-
ditions in the medium, the stock always shows longer periods of rest and more
uniformly enters into hibernation for this period than when the Ac values are
larger, at about 60. On the other hand, undecimlineata, while it has the same
rhythm of reproduction, is much less sharply delimited and the period of repose
between successive cycles is shorter and to a large degree a product of the con-
ditions of the medium.

I may later show something of the nature of this rhythm and its relations to
agents within and without the organism and its general methods of inheritance.
In general in these forms in pure reacting lines, in which the rhythm is homo-
zygous, the sequence is followed with precision, of an overwintering or resting
generation that emerges from the period of inactivity, especially on the advent
of favorable conditions in the medium, reproduces at once, giving a summer
generation that reproduces without rest, giving a second generation in the cycle,
which, however, hibernates or rests in one way or another for a varying period
of time. There are thus in the cycle two conditions in reproduction that follow
in rhythmic manifestation — winter generation, which after resting produces
the summer generation, which in turn produces a winter generation without
resting, which winter generation rests from reproductive activities in signati-
collis as much as 9 months in nature and in the laboratory strains with Ac values
of 40 or thereabouts.

Eeactioxs and Products in Interspecific Crosses 137

In crossing of these species it has been the uniform custom to mate in the
original crosses only individuals of like condition Avith regard to this repro-
ductive cycle; otherwise troublesome complications in the breeding-out of the
series is sure to be encountered. Thus in practice it has been most common to
mate the materials on emergence from the resting-period, as one is thus sure to
get the first two generations of the series within a half year or less, and thus
facilitate the operations. More rarely are matings made between the- summer
generations which give only F^ before a period of rest intervenes, and only for
special purposes are the two aspects of the cycle mated, when the result is
complications in abundance.

These crosses between L. signaticoUis and L. undecimlineata have been, as far
as this portion of the investigation goes, made entirely between the stocks on
emergence from hibernation, and with respect to the character of the reactions
the two types of reaction described present quite different results. In the
normal cross, when the Ac values are about the same and the conditions of the
medium are neutral, the result is the regular cycle in the reproduction of the
series, the second generation following at once after the production of F^, giving
Fo within the usual period of 4 months from the initial cross, and then F„ is
followed by a period of rest in the F, fraternities, with a duration on the average
of from 6 to 10 weeks, depending entirely upon the conditions in the medium.
In the second type of result in the crossing of these two species, as shown in
plate 14, the result is different. The suspected undecimlineata race, now known
to be heterozygous, acts at once as a winter generation, commonly enters within
a few days into typical hibernation, and is not to be forced into reproduction,
although it may be forced from hibernation. The F^ heterozygous types that
are normal give the characteristic response by breeding at once, giving the F2
by the end of 4 months as in the normal cross. Further breeding of the two F^
types show that in the manifestly heterozygous series the reactions in the cycle
are normal and can be carried through to at least Fg within 12 or 13 months,
while the undecimlineata series in this same time produces only one generation.

It appears that in this suppressed heterozygous race there are some important
rearrangements produced in many of the properties present, and that there have
arisen associations of the agents in the gametes, such that the arrangement
present allows of only certain visible end-results. There is no doubt that the
race is heterozv'gous, as is demonstrated by the fact that the F, fraternities show
accurate separation of the series into a trimodal polygon, whose significance
with regard to the form-index is known ; that certain methods permit of the
recovery from the race of the nonvisible signaficollis larval and adult elytral
characters ; and further, the race shows a complete suppression of the summer-
brood production, so that the race appears in a long monobrood cycle that in
general aspect is undecimlineata, the total complex being retained without
change as far as has been carried and without regard to external conditions as
far as is shown in the observations. Only special operations, under precise
complexes of the medium, produce dissociation of this complex that arises so
uniformly in F^.

One series of this strain I carried from 1906 to the end of 1914, in which I

had 10 generations after the initial one, but at the end it was the same in

behavior as in composition as at the start. One point that came out in this series

was of much interest. It has been shown that the Fo fraternities are trimodal

10

138 The Mechanism of EvoLUTioisr in Leptinotaesa

in the form-index ; that from these can be reared pure lines of the form-index ;
but this is possibly only by isolation and inbreeding. During the same time
that these were in progress certain mass-culture lines were carried, with the
result that a test of the form-index that I had made in the fourth generation
showed only a monomodal curve, and the same was true in the sixth, which I
also had tested. From the fourth I was able, by mating likes, to produce by P^
much-restricted lines in the modes, so that there had taken place in the mass-
breeding a mixing with the result of obscuring the real conditions present, but
out of which I could isolate pure lines with respect to the form-index.

The production of this undecimlvneata line is essentially the production of a
good species in every sense. It is strong, well able to survive widely ranging con-
ditions ; the fact of its slow breeding at once isolates it from crossing back with
the rest of its relatives in the production of Fg ; its low power of dissociability
with respect to its contained potentialities, the fact that it is produced in num-
bers of both sexes in the one operation, shows how in nature a new group might
easily arise that would thereafter become a permanent element in the fauna of
the location. Any of the usual tests of breeding would in natural materials
have shown only uniformity, when in reality they were heterozygous in origin
and in character, and by appropriate means the fact of this can be shown. It is
not known how long the signaticollis groups of agents would remain in the race
unseen, nor is it known what part they may be playing in the complex as it is
seen, but as far as one is entitled to an opinion it seems to me that they are most
probably present in some association that ties them with other agents, the
totality acting as a unit system in reaction, but in which a portion of the group
is relatively unimportant and may be recovered without altering the aspect of
the series.

That the reproductive cycle is also in reality heterozygous is shown by the
experiences in two series — one in F3, the other in F^ in another line entirely
independent of the first, in which, after subjecting the lines to severe conditions,
there came out of each a line that showed the normal rhythm with some changes.
I was not able to duplicate these experiences in later tests, and, therefore, am
not disposed to base more than the suggestion upon them that the experience
shows that in this character of the reproductive cycle the usual double brooded
condition could be recovered if we knew how to dissociate or analyze the present
combinations. To all intents and purposes the line is a stable typical species,,
and the series shows how such might easily arise in nature. In ordinary terms
this is a fixed hybrid, and while it is fixed in one sense, it is by no means
irretrievably so altered, nor is the condition present in any manner at variance
with the principles of factorial composition and evolution, but as far as I am
able to discover are entirely a confirmation of them and an extension of their
operation into a type of operation that may well have played a frequent and
important role in the production of new specific groups in nature. But any
such group, pure as it might come in the usual breeding operations, has present
within it the associated agents ready, on the realization of the right conditions,
within and without, for the production of new characters or races by sudden
changes.

Three other main types of reaction have been found in this series, which,
while in first appearance entirely at discord with any of the usual considerations
in crossing, are nevertheless entirely in harmony, as far as tested with the

Masked Hstarozygote.
Breeding true 'n aspec

•zygote, g^^-es only i-yp.osl Fz array

Masked Heterozygote.
Breeding trc/e in aspect

Eeactions and Products in Inteespecific Crosses

139

principles of the Mendelian reaction and the general evolutionary conceptions
of the factorial theory. These in most respects are duplicates in a broad way of
the differences in reaction found in the cross of L. diversa and L. signaticollis,
the differences in both series being the product of the conditions of the medium
in which the cross was made.

I first found, in 1907, that when L. signaticollis with the Ac value at 38 to 40
was crossed with uiidecimlineaia with the usual Ac value of about 62 under the
following complex, that the results in F^ were further upset and productive of
much confusion.

Table 16b.

Temperature.

Humidity.

Maximum

98
59

80.5

Per cent.

95
40

68

Minimum

Average

Wind-movement, 200 to 418 ft. per mi
Evaporation, from 1 to 12 c. c. per hi

n.; av., about 356.

This arrangement of the complex and its rapid extreme changes in the experi-
ment, when brought to bear upon the crossing at the time of mating, maturing
of the gametes, and during fertilization and early development produced a most
distressing array in F^, especially so when the behavior of other crosses of the
same materials is known not to give anything but the most orderly and typical
reactions. The actual result of this cross under the conditions is shown in
plate 15 which shows that in F^ there appear the three types of adults — visible
undecimlineata, mid-type, and signaticollis types — in about a monohybrid ratio
of 1:2:1.

The F^ larvae split in the second stage into two groups of about equal numbers
of yellow and white, and in the third stage are still, in equal numbers, yellow and
white, both without the meristic-spot series, so that the L. undecimlineata was
the dominant one in this respect. The white larvse always gave types that were
undecimlineata, that in all respects are duplicates of the F^ appearing undecim-
lineata tv^es of other series, slow breeding, with difficulty dissociated, in fact
masked heterozygotes. The yellow larvae gave two types of adults, on the aver-
age of two mid-types to one signaticollis type. The mid-type gave only the
normal reaction, and were in no observed respect different from the other F^
heterozygotes that have been seen and tested.

The signaticollis type that appeared in F^, however, was pure-breeding, at
least to the extent that they came true to type for an undetermined number of
generations. They were a fixed type arising in F^ that bred true to type and
had all of the characters of the signaticollis normal type. They bred at once,
had about the same length and type of rhythm in reproduction as the mid-types,
but were true to the Fj condition in subsequent generations. These were
another type of heterozygote, as could be shown quite readily by applying the
test reaction used in the cross of diversa and signaticollis, which showed the
recovery in Fg following the test of the undecimlineata type. Biometric tests
of the pure breed Fj also showed in two fraternities that I had tested the typical

140

The Mechanism of Evolution in Leptinotaksa

trimodal condition. The numerical results attained in this cross are shown in
table 17, which shows a close approximation of the array in the F^ to the mono-
hybrid ratio of 1 : 2 : 1.

Table 17.

Parent,

Fi larvae,
third stage.

Fi adult arra}'.

White.

Yellow.

White
larvae,
11-lin.

Yellow larvae.

Mid.

Sig.

Sig. m. A^ X 11-lin. fem. A" . . . .
Sig. fem. A« X 11-lin. m. A*^ . . . .
Sig. fem. A'» X 11-lin. m. A«' . . . .
Sig. m. A" X 11-lin. fem. A''^ . . . .
Sig. fem. A=" X 11-lin. m. A" . . . .
Sig. fem. A« X 11-lin. m. A^" . . . .

Sig. fem. A" X 11-lin. m. A'"

Sig. m. A" X 11-lin. fem. A"' . . . .
Sig, m. A^ X 11-lin. fem. A«» . . . ,

Totals

121
54
28
11
39
11
12
71
93

246
96
21
36
11
84
23
14

201

41

21

4

2
27

4
11
63
74

89
44

7
31

4
46
11

2
136

33
11

5
1

0*
27
10

2
62

440

732

247

370

152

* 1 dead.

Continued breeding and testing of the P^ heterozygous signaticollis strain
gave many indications of the reactions and associations that had been formed in
the production of it.

This line, when treated with the test reaction used in the cross of diversa X
signaticollis, showed in F„, following the test, the uniform appearance of the
type in which the V determiner in the elytral-pattern complex had been brought
into relation with the Elp determiner, the two producing a general color distri-
bution over the elytron. Also, there were produced types in which the V de-
determiner manifestly had relations, productive of the strain where the pigment
was linked with the Pu determiner. These two conditions have been tested out
and their composition determined by the test reaction used in the signati-
collis X diversa cross, and from each of them the undecimlineata type can be
recovered by the same methods in the same ratios that were found in the first
example. In fact, this signaticollis race that comes out in F^ of the cross is in
its adult characters the duplicate of that found in signaticollis X diversa in F^,
in so far as reactions and principles are concerned.

In the larval characters the stock breeds true to the yellow with the meristic-
spot system in the line as it arises, but it can be broken and is broken up by the
test reaction, giving resolution into the component character agents that went
into it, showing in all respects its complete heterozygous character.

The point of essential interest in this cross under the stated conditions is the
production of three sorts of heterozygotes, differing in their aspect, not in com-
position, but in arrangement of the agents in the gametes and in the reactions
of them in development. Two are of especial interest in that they breed true,
are stable associations in reactions and in gametic associations, which can, how-
ever, be broken up into the orginial agent groups of which they are composed.
The Fi production of the three types, while in the summation is an approxima-

Eeactions and Products in Interspecific Crosses 141

tion to the ratio of 1 : 2 : 1, in individual experiments shows wide ranges there-
from, and these are in close correlation with the differences present in the dur-
ation of the ranging conditions used in the medium. The range shows oscilla-
tions towards both of the extreme or pure-breeding conditions, and in nature
the same cross might well be productive of different results and sequences of
consequences in the local fauna as the product of the conditions surrounding
the initial cross.

The last complication in this series of experiments that need be mentioned
here is the crossing of these two species, under conditions of high temperature
and moisture with little evaporation and air-movement. The following com-
plex of conditions with these materials has given the result that the progeny in
Fi are all alike and of the undecimlineata type, breed true, and appear as if the
other parent had gone from sight. This result I have thus far obtained only
when the female parent was undecimlineata, in 12 trials out of 19, giving in F^
217 adults. The converse of this has not been produced when signaticoUis is
the female parent. These crosses have been tested in three series, and by
the tests that were applied to the other F^ pure-breeding types are shown to
be in all respects heterozygous and of the same composition and apparent ease
of dissociation; likewise they have the same slow-breeding reactions that are
present in the other strains of the same origin. Aside from this aspect these
last experiments are without much interest.

The intercrossing of these two species under different conditions shows the
production in experiment, and the same no doubt is true in nature of 10 pure-
breeding strains, or races that are different, so that in nature they might easily
be described as species. By pure-breeding I mean only the usual application of
the term, the production of progeny that are the duplicate of the parents, a
term and description that are not of much significance at present. The 10 pure-
breeding lines show two groups: (1) a group of 8 that are homozygous in
action and in constitution, and (2) a group of two that are homozygous in
action, hut heterozygous in constitution. In appearance these are as follows :

(a) Homozygous in constitution and in action, always derived as Fo extractives.

L. signaticoUis (a) signaticoUis in all respects, with yellow-spotted

larvge.
L. signaticoUis (h) signaticoUis with white larvae, spotted with black in

third stage.
L. signaticoUis (c) signaticoUis with yellow larvae, not spotted in the

third stage.
L. signaticoUis (d) signaticoUis with white larvae, not spotted in the

third stage.
L. undecimlineata (a) undecimlineata with white larvae not spotted.
L. undecimlineata (h) undecimlineata with larvae white spotted with

black.
L. undecimlineata (c) undecimlineata with larvae yellow spotted with

black.
L. undecimlineata (d) undecimlineata with larvos yellow spotted with

black.

(b) Homozygous in action, heterozygous in constitution and always derived

in Fi.

L. undecimlineata (e) undecimlineata with white larvae not spotted.

L. signaticoUis (e) st^naiico/fe with yellow larvae spotted.

14:2 The Mechaj^ism of Evolution in Leptinotaksa

These two groups are alike in reaction under ordinary conditions and unlike
in constitution, actually and in principle, but ordinarily would be rated as
homozygous, and so used in the further crossing of them. The first group ofPers
nothing of interest or difficulty and is the product of the usual metathesis of
groups of agents that takes place in crosses of the type where there are three
agents or groups of agents capable of dissociation and rearrangement. The lat-
ter group would in usual operations be interpreted by the assumption of some
agent in one parent not present in the other, one being heterozygous, the other
homozygous, therefore with the result of the production in F^ of the extracted
t}'pe. That this is not the case in these experiments is shown by the fact that
from all of these in F^ races it has been possible in varying degrees to recover
from them the other characters, which would not be possible were they true
homozygous extractives. There are thus available 10 homozygous-acting kinds
of substance, 8 of which are homogeneous in composition and action, 2 in action
only, and in addition there are the diverse kinds of heterozygous combinations.

LEPTINOTARSA UNDECIMLINEATA X LEPTINOTARSA DIVERSA.

Crossing of these two species is simple, and also serves to show that the reac-
tions are normal metatheses of the character groups involved. Between the
two there are the larval character groups of lipoid color, yellow or white, and the
spot system or its absence, while in the adult the elytral-pattern system of
agents, while not precisely alike, is with difficulty separated, so that it is not so
clearly a diagnostic character in the adult as they were in the former crosses.
However, the antennse in their coloration serve as a certain mark of recognition
for separating the two adult conditions. This is not in this type of cross dis-
sociated from the general species complex, as far as known, always appearing
in correlation with the form-index of the proper species. The basal joints in
L. undecimlineata are light in color, the distal are black; and in L. diversa
the entire antennse are black. This in common with the index serves well to
separate the adult conditions. In both the Ac determiner values are alike, so
that no derangement of the crossing reaction occurs, and the conditions
employed, unless in extreme ranges, do not change the array. Eeciprocal crosses
show identity of F^ results, giving yellow larvge with the spot system minutely
present or not visible, so variable in this manifestation that precise statement is
possible only for individual instances, due to the fact that slight variations in
the conditions of the medium produce oscillations of the manifestation of this
character. The adults are also an intermediate in all of the characters, as
antennse, index, pattern, and so on. The production of Fg from these shows
separation of the larvas in the second stage into white and yellow ; into an array
in the third stage that consists of 9 white without spots, 3 white with spots, 3
yellow without spots, 1 yellow with spots, and each of these give three classes of
adults that appear as one, but that can be separated on the basis of the antennse,
the form-index, and to some extent by the elytral pattern. There is no indica-
tion that the reaction is in this instance anything but of the most orderly tri-
hybrid type in all respects.

With this combination I have not attempted many crosses when the Ac
determiner had divergent values experimentally produced, because of the
technical difficiilty that the separations of the adult types were slow and must be

Reactions and Peoducts in Inteespecific Ceosses 143

done by either biometric testing, which is time-consuming and expensive for
extensive operations, or by minute examination of antennal characters or the
elytral punctations. Further, there were not found results or reactions differ-
ing in principles. The crossing of the two has shown only the fact that nothing
present appeared or was brought out that was hidden, and might have been
productive of the complications found in the previous series.

DISCUSSION OF RESULTS DERIVED IN CROSSING THE SPECIES
L. DIVERSA, L. SIGNATICOLLIS. AND L. UNDECIMLINEATA.

I have presented at some length and in detail the crossing of these species,
and out of these experiments, which were begun in 190-i and are by no means
terminated, certain positive advances have, I believe, been obtained, in addition
to many aids in the analysis of the composition of the constitution of the
germinal material. Begun at a time when the furor of enthusiasm resulting
from the recovery of Mendel's work was at its height, I purposely took a most
uncompromising position, and in every possible manner made effort in this and
other materials that I was using to break down the principles. The reason for
this was that I believed that if the principles were true, and not another variety
of "Weismannism, no amount of adverse investigation would alter the findings
and might open the way for extensions of the principles in diverse directions.
I have no hesitation in saying that I made every effort of which I was capable,
aside from biometric messing-up of heterogeneous things and assuming homo-
geneous arrays, and disregard for the accuracy of technical operation, to break
down in practice the Mendelian reaction in these materials. To this end I
made especial efforts after it became increasingly certain that the reactions were
in accord with the principles of reaction described by Mendel.

In all of this I have most positively failed, and not one certain instance have
I been able to find that will stand the tests that it has had to undergo in my
examination of it. Mendelian enthusiasts will say that the effort was a waste
of time and energy, and had far better been employed in adding to the array of
cases. I have no interest in added cases, only in principles, their validity and
extension. It was further necessary to test fully the role in these forms of this
reaction, in which other operations directed at the problems of evolution were
being carried on.

Current neo-Mendelian conception and terminology characterized the opera-
tions in the gametes productive of the differences and arrays that result as due
to the " segregation " of factors and determiners, that all too often are conceived
of as " carriers of characters," or as " specific nuclear substances," the char-
acters being isolated units, compared to blocks in a wall, the entire terminology
and conceptions in general being modified Weismannism. The experiences
with these crosses of natural species shows that there is no general shifting of
characters or units of any sort, either as agents or as conditioning entities. In
all it is seen clearly that there is a gametic system characteristic of each species,
and two systems when combined in crossing are not able to form permanent
combinations, but separate at once in F,, and this regardless of the visible
differentiation. For proof of this, the uniform experience with the fixed hetero-
zygous races, which were in appearance alike and uniform, were shown by
accurate measurement to be not like, but different, and to produce groups whose

144 The Mechanism of Evolution in Leptinotaesa

modes were the modes of the parent forms from which pure lines could be
grown, pure for the parent form-index, showing that the two parental basic
gametic systems did not intermingle ; but while they might react in common in
the heterozygote, as they must or perish, at the first opportunity they separated,
each into gametes that were pure for the species gametic system in question.

In the cross between diversa and signaticollis this is all that took place, the
two systems maintaining their integrity throughout the series, and no indica-
tion of metathesis being found in the series. This to me seems the simplest
reaction that one can find in the crossing of two forms of organisms and is the
simple interaction of two systems that are sufficiently like in substance, struc-
ture, and reactions so that in combination they are able to react as one, pro-
ducing the compromise end-results of the heterozygote, which in most respects
are intermediate between the two parent forms, but not able to form any
permanent association as far as experience has shown, and between which no
interchanges of agents or groups of agents take place in the interactions that
go on during ontogeny and in the gametogenesis of F^.

Quite different are the reactions and results in the series shown in the crossing
of signaticollis and undecimlineata, in which there is the same retention of the
integrity of the gametic system ; but in the reactions incident to gametogenesis
in Fi certain interchanges of groups of agents occur. The array has the appear-
ance of a trihybrid, owing to the fact that the three pairs of masses are present
and interacting: (1) The non-dissociated gametic mass, characterized by
form-index, elytral pattern, etc.; (2) larval pattern; and (3) larval color.
These present the array of three independently interacting characters. In
reality the elytral system is not in any of these dissociated from the basal
gametic complex, and is the indicator of its presence, a fact that can and has
been tested by breeding and by biometric measurements in the diverse lines
with full confirmation in all respects. That which happens in this instance is
the separation of the basal systems in gametogenesis into two great groups,
distinguished by the elytral-pattern sign, and then the interchange of the two
other groups of agents in metathesis, such as would in all reactions take place
between unlike complex substances when in mixtures and under conditions
that permit of interactions and interchanges between them.

With considerable possibility of truth one can symbolize these substances and
reactions in one's imagination as two collodial mixtures, each a highly organized
system in itself, with structures and reactions such that they may for a time
interact in common to produce the soma of F^, but are not able to form per-
manent combinations. We think of this simple relation in terms of a single
pair of reacting groups of agents, and all of the evidence from Fa separations,
measurements, and breeding of extracted lines gives support to the symbolic
conception of the two gametic systems retaining their identity and integrity
throughout the operation. There is no evidence that the gametic systems in
this cross are impure or contaminated in any way by the passage through the
cross, nor are extracted stocks and parent species different in any point that
has been detected.

The cross of signaticollis X undecimlineata shows in essence the same general
operation as in the simpler cross, with the exception that in gametogenesis the
two gametic systems which retain their individuality in the divisions of the
germinal materials into the two main types of the parental stocks, with respect

Eeactions and Peoducts in Interspecific Crosses 145

to the larval color and the meristic pattern system of the larvae, permit of inter-
change or metathesis of these groups between the separating systems of the
groups of agents that are productive of the characters in question, and these
interchanges seem to be equal in both directions. How this is produced or
what it is that passes between the two systems is unknown, but it is clear that
something does pass from one to the other system ; that there is true metathetic
change between the two interaction masses that represent the germinal sub-
stance derived from each parent and which are separating from the combina-
tion of them into masses pure for the substance contained therein, with the
exception of such additions or removals as may have been produced by the
metathesis resulting from the interaction of the two gametic masses. On the
surface it appears much like some type of mass-reaction, accompanied by inter-
change between the two interacting masses. These interspecific crosses show
that the mass of the gametic system, acting as a whole, as an indivisible unit
of reaction in one combination, in another allows of interchanges in parts of
its system, resulting in new pure breeding combinations.

These results raise the question whether the changes found are the extent of
possible change, or whether there is a specific residue which may or may not be
incapable of dissociation and recombination. Bateson and others have in some
measure pictured the conditions as the combination of a basal portion upon
which is placed or attached the agents capable of being changed in position and
recombined. This basal portion is simply the unknown unanalyzed residue of
the gametic complex, and there is no reason to expect a specific indivisible base
upon which to build, and if the principle of factorial constitution and operation
is true in part it seems most probable that the same condition holds throughout ;
at least that is the condition that one finds in unorganized substance, and is, no
doubt, true for all substance.

CHAPTER V.
REACTIONS AND PRODUCTS IN INTERSPECIFIC CROSSES.

In Chapter IV, data and the analysis of the crossing of species from nature
were given, showing interesting possibilities for the production of irregularities
in the laboratory, as well as potentialities for the production of natural groups
capable of continuation and independent existence in nature.

Clear indication was presented that the gametic complexes behaved as units
in the production of the gametes in F2 and of the purity of the types found in Fj
as determined by genetic testing and biometric measurements. Between these
some interchange of factorial groups was observed, as well as the formation of
combinations that were permanent and stable. These latter were, however,
capable of dissociation, showing the manner of their production and the nature
of their composition. In this chapter is presented further data derived from
the crossing of species from nature.

LEPTINOTARSA DIVERSA X LEPTINOTARSA DECEMLINEATA.

The cross between these two species is difficult to make, and out of many trials
I have thus far obtained two series that gave progeny that would breed on into
F2 and beyond. Many of the trials have given juvenile stages, fewer adults,
only two lines that were able to breed through several generations.

The species differ in almost all of their attributes, not only in the color and
pattern characters presented in all of the stages of each form, but also in the
form-index, the rates of ontogentic development, in the food that each is able
to utilize, as well as in many other characters. Perhaps the chief difference
that is of interest in this cross is the specificity of the food-relation presented
and which enters into the results of the cross. L. decemlineata normally feeds
upon Solarium rostratum, S. tuberosum, S. nigrum, etc., but will eat some few
of the woody types, but none of them with success. L. diversa will not eat the
first-named types ; as far as experience goes it feeds only upon the woody types
that are its food in nature (-S^. liegerii, S. chrisotrichum, 8. lanceolatum, etc.).
So specific is this relation that L. diversa will starve before it will eat potato or
S. rostratum, while L. decemlineata will eat the food of L. diversa when forced,
but never thrives thereon, and in all instances perishes by continued use of the
L. diversa food.

In the crosses between these two species I have used only materials of L. decem-
lineata that were pedigreed and came from my oldest line, and of L. diversa
also only pedigreed stock. Between these two species I have made over 200
trials, always in reciprocal pairs, and thus far only two matings of L. decem-
lineata female X male L. diversa have given lines capable of any analysis, and
these were both in all respects precisely alike. The crosses were made under the
conditions of the breeding-quarters, to which both species had been fully

146

Reactions and Products in Inteespecific Crosses 147

accustomed for generations, and their progeny and testing thereof was all done
under the same conditions, so that the conditions of the medium play little or
no part in the history of this combination.

On July 18, 1907, a pedigreed L. decemlineata (Fjo female) was mated with
a pedigreed Fc, male L. diversa. Both were young, freshly emerged, and typical
of their respective lines. These gave progeny that in the larval stages were in
all respects precisely that of the L. decemlineata type ; from these there emerged
on September 18, 11 male and 8 female adults that were all of the decemlineata
type. On July 12, 1908, a precisely similar mating was made that gave progeny
from which were obtained on September 16, 22 males and 25 females. In this
series all the juvenile stages in all respects were indistinguishable from decem-
lineata, and the adults were also fully of the female parent type. In both of the
series they were the only pairs out of 20 in the July 1907 mating, and out of
32 in the July 1908 series that gave progeny from which adults were obtained.

In making these crosses it is necessary to have in the cage two different food-
plants — that of each parent ; otherwise the chances are entirely against obtain-
ing any reaction from the mating. Under these conditions the female decem-
lineata will deposit the eggs irregularly on both of the plants, with an average
larger number on the normal food than on the other. These eggs, when they
hatch, give larvje that do not feed on the succulent type with any degree of
success, and in those larvae that are on the potato the mortality is, if left thereon,
always 100 per cent. If on the woody type, or transferred thereto, they thrive
well and in the two series there were 75 larvaj in the 1907 set and 82 in the 1908
set, all healthy, well nourished, and the progeny that came from them also were
fine, large specimens, and like the larvae would only feed upon the woody type
of food-plant. In the length of ontogeny F^ showed the rate characteristic of
the diversa parent, which was about 60 days, whereas the same value in the
decemlineata parent in the strain used was 38 to 40 days.

These F^ hybrids were clearly composites, showing in gross bodily qualities
entire dominance of the female parent's characteristics ; in rates of growth, and
in food preferences the strict limitations of the male parent. In other attributes
difficult of statement, these individuals showed the type of reaction now of the
male, at other times of the female parent, but in all respects uniformly so, the
entire population composed of the two fraternities being strikingly uniform.
In both cultures the F^ fraternity entered into hibernation by the end of Sep-
tember, remaining until the following March, when they emerged, and were
tried first upon potato, which was not touched, then put upon Solanum chriso-
trichum, which was at once eaten, and by April 1 reproductive activities had
begim. In April 1908, I mated 7 pairs of the first series that arose in 1907,
and these bred at once, all 7 pairs giving progeny, which emerged between
June 5 and 15, having an average ontogeny of 61 days. The record from the
pairs is shown in table 18.

The larvae were without exception all pure decemlineata tv'pe, like the
female-parent stock, and the adults were all like the F^ fraternity, with no trace
of separation into types. Biometric tests, made of fraternities from matings
A and D, gave no indication of multimodal conditions, the findings in the
measurement of the form-index showing a monomodal polygon that was in all
respects precisely that of the pure decemlineata stock in position and distribu-
tion of frequencies.

148

The Mechanism of Evolution in Leptinotarsa

From pairs A and D matings were made and began breeding early in July,
giving adult progeny that had all emerged by September 12, and then hiber-
nated. These F3 fraternities gave 416 larvae and 318 adults in all respects like
the populations in F^ and F,, and one line was continued to Fg without any
indication of change either in the characters or reactions of the line.

Efforts were made to cross this line with L. diversa and with L. signaticoUis
as well as L. undecimlineata without success, but it crossed feebly with L. decem-
lineata. This cross, made in the effort to break up the combination, showed
not the breaking up of the combination but its dominance in F^ and the separa-
tion in F2 of pure decemlineata, and the pure combination, which could be
easily distinguished and tested by their rate of development and the limitation
of food. In no test did I succeed in breaking up the combination that had been
formed by the cross.

Table 18.

Pair.

Larvae.

Adults.

Males. Females.

A

42

92

126

154

27

89

177

707

8
31
56
61
12
33

16
26
27
49
11
31

B

c

D

E

F

G

68 54

269 244

Total

Total adults. .

513

1

Both of the series that have been observed behaved alike, were in all discovered
respects the same, and neither yielded to any of the efforts to disrupt the com-
bination. That it was a combination there is no doubt. The virginity of the
females was certain, the fact of the cross equally sure and proven by the
presence in the combination of characteristics that could only have come, under
the conditions of experiment, from the male line, and in all respects the product
of the cross was distinctly a combination of the lines, even though the form and
usual taxonomic characters were of the female type entirely. The length of
ontogeny, the food limitation, and the many peculiarities of action that came in
with the male line showed fully the existence of a combination of the parental
lines into a complex that did not dissociate in the production of F, or subse-
quent generations. A new and stable type had arisen by this combination that
was stable from the start, existed as a group from the start, that with its slow
progression in ontogeny and limitation in food would have, in nature, effectually
isolated it from possible intermingling with the female-parent species, while it
was unable to cross with the male-parent stock.

It is not difficult to imagine the advent of a female L. decemlineata into
the habitat of L. diversa, and perchance if the crossing took place there would
be produced in the location a new species, if its origin were not known, that
would feed with diversa upon the same food plant, but not cross with it, at least
with any ease ; that would be by food-relations, growth-rates, and other agents

Eeactions and Products in Interspecific Crosses 149

isolated from crossing with decemlineata, or if it did cross would retain the
purity of its type ; so that the conditions are not unfavorable for the origin of a
group in nature, the " origin of a species."

I made the effort to put this hypothetical case to a test by the introduction
of L. decemlineata females into some of the habitats of L. diversa. I have not
thus far been successful, owing to practical difficulties in the control of the test
and the fact that L. decemlineata does not thrive in the habitats of L. diversa
in nature. In 1908 introductions were made at five places in the forested
valleys to the north of Cocomatepec, Vera Cruz, Mexico, where the food and
L. diversa were found in isolated locations. At these I saw the copulation of the
L. diversa males with the L. decemlineata females that were fresh from my
laboratory stock, I could not remain to observe the outcome, but when the
colonies were visited in August 1908 and again in 1909 no trace of the intro-
duced stocks was found. I saw the locations again in 1910, but found no indi-
cation of the success of the test. I hope at some future time to repeat on larger
scale and with better means this or similar lines of experimentation.

The series of experiences with this cross and its products is highly suggestive.
The crossing of widely divergent types in the genus is of interest; one a tem-
perate savannah, the otlw^r a tropical montane rain-forest type with different
food-relations, the two differing in nearly all characters throughout; and only
in the general type of structure and in the large relations of parts and in general
pattern plans do the species compare, as is shown by the comparison of the
figures given of these types in Chapter II. The finding that less than 1 per
cent of the matings give progeny that are able to mature and continue the line,
and then only from one side of the cross, shows plainly that the problems pre-
sented in this combination are by no means solved. The lack of separation of
the Fj population into types of any sort shows that there is no gametic segrega-
tion or production of gametes of unlike kinds present. All indications show
that the gametes are like and permanent as far as experience goes in their com-
bination of the characters. The character of the strains that have arisen indi-
cates that the two parent types have each given to the new complex certain of
their determiner groups and that others have been lost or else are present in
inactive state or serving other purposes.

All examinations and tests that have been made show that the type in body-
index is that of the decemlineata parent, and with this are the many character-
istics of structure and color-pattern, all those of the decemlineata type in pure
form. This, perhaps, indicates that the basis of the new combination has been
the gametic system of the female from which have been lost certain groups of
agents that have been replaced by other gametic agents from the male line. As
far as I have been able to discover, the limitation of food is complete, and
associated with this are a considerable series of reactions that involve the entire
body. The diverse catalitic agents that are engaged in the metabolic activities
and many sensory reactions are also distinctly present in the new type that came
from the male. The rate of development and many developmental details are
also those of the male line, so that the type is most complex.

It is idle to speculate upon what has taken place in this combination, and only
further investigation can help in the solution thereof. It is clearly not like
any of the fixed heterozygous conditions thus far encountered. These have all

150 The Mechanism of Evolution in Leptinotaesa

yielded to test and have been capable of some dissociation, showing the presence
of the lost characters in the erratic type. In this no indication of dissociation or
disintegration of any sort has been produced, and the type is as stable and fixed
in all tests thus far made as are any of the types direct from nature which we
call species. I can only suspend Judgment as to its production. It would be of
interest to know the reason for the limitation of the production of these lines to
one side of the cross and its realization in less than 1 per cent of the total
crosses made. These are problems for the future, and as time and opportunity
permit I hope to be able to penetrate farther into the mechanism and nature of
this method of the production of stable, physiologically isolated groups. This
represents perhaps the most complex type of interspecific cross, with com-
plete permanent combination and production of new groups, that of diversax
signaticollis the simplest, with no dissociation and the non-contamination of the
gametic system by their entering and passing through the P^ hybrid combina-
tion. Between these two extremes are all sorts of intermediate conditions, some
of which have been given in the preceding chapter, and others are to be presented
in the remainder of this.

The point of most interest shown in the series is the reaction of the nutritive
activities as a unit group being retained in one totality in the combination in
dominant positions, so far as the feeding and foods that are capable of use are
concerned. Interesting new relations must, however, have been established in
the new combination, so that the products of this new nutritive arrangement
and limitation may be used by the agents productive of the characteristics of the
female line. The adjustments that have been made in the type to bring this
about would be of much interest if known, and might give important informa-
tion regarding the inheritance of metabolic relations and capacities, as well as
of the gametic agents present that are responsible therefor. It is certain that
the agents in the gamete, productive of restricted nutritive relations, are as
specific and capable of dissociation and metathesis and of entering into new
associations as are any of the other gametic agents experimented with. Further,
they have the ability to produce in the new position some of the same end-
results that were shown in the original association, and this in many respects is
suggestive of the transfer only of determining agents, rather than the transfer
of the total nutritive complex. At any rate, it is certain that agents of the most
fundamental nature are able to act as units in the interchange, and recombina-
tions have taken place in the production of this race. Other experiments in
which the food-relations are subject to analysis have shown the presence in the
gamete of a general basic growth and metabolism factor, with which are asso-
ciated several determining agents or determiners that may be dissociated and
replaced in the combination. In this series the probability is that the food
determining agent, determiner Fq from diversa, has combined with the chro-
matic receptor of the female parent, CMR^, the combination being productive
of the limitation as to food. What became of the determiner Fq from the female
line remains to be discovered.

The importance of reactions of the kind here described in evolution in nature,
I believe, merits attention. By this method of reaction there arises at once a
precise, isolated group with capacities and characters derived from its pro-
genitors fitting it to be successful from the start as an element in the fauna of

Reactioxs and Pkoducts in Interspecific Crosses 151

the location. The agony of origin, of initial stages, and struggle for place is not
seen ; the type is produced or not as the result of a specific reaction, and at the
close of the reaction is completed as far as that reaction is concerned. In setting,
operation, and result the series is purely mechanistic and devoid of the uncer-
tainties of time-elements and time for fixation needed to produce stability of
type.

In the instance here presented a precise reaction is produced that in about
1 per cent of observed instances results in a specific stable combination — that
from the start is physiologically isolated, and in all observed respects the
equivalent of a natural species and fully able to survive in nature. It arose only
in experiment. Thus far I have not been able to obtain it in experiment in
nature, but my attempts are not thorough nor extensive in this effort. I am
convinced, however, that the type of reaction seen shows clearly that the
realization of an operation of this sort in nature would at once produce a com-
bination which, if found in any habitat and of unknown origin, would pose in
faunistic lists as a species or other taxonomic element. There is no reason why
operations of this sort should not be of frequent occurrence in nature, and I have
no doubt that if we really knew what goes on we would be astounded at the
diversity of the trials of this sort that are by chance made in each season of
developmental activity.

I know that the type that arose was stable from the experiences in the labora-
tory and inability to break it up, and its ability to survive was tested at a loca-
tion created for it at Tepextempa, Vera Cruz, Mexico, where it was placed in
tropical savannah conditions in 1908 and without effort passed through four
generations in 1908 and 1909, being exterminated in the winter of 1910 by the
clearing of the fields for the planting of cane. It was introduced into the deserts
at Tucson in 1909 and thrived through two generations in that year, but was
killed in the winter of 1909-10 by defects in cage construction which prevented
them from penetrating deep enough into the earth to escape the desiccation of
the winter months. The entire population was found dead in the spring of 1910
along the wire bottom of the cage. The type could not be tested in nature in
any of the northern regions and could not be turned loose in nature, because
the food it could eat could not grow and survive the northern winter. I tried
it on potato at Chicago in inclosed spaces, and it was completely extermi-
nated, never even making a start at reproduction. It has the capacities, limita-
tion, and characters of natural species — and the experience is suggestive of a
method of origin of groups in nature that may prove to be of wide occurrence
and productive of much diversity in natural forms.

It is suggestive that the observations show the origin of a group and not of an
ancestor, and perhaps for this one reason such origins would on the whole be
uniformly more certain of successful competition in the habitat into which it
was thrust by the method of its production. This series, it is true, showed only
recombinations, nothing in the way of new characters; but other reactions of
somewhat similar nature have shown new conditions in the substance, and there
is no reason why in similar instances new attributes may not well be the product
of reactions like the one observed in the combination of these two species in
my experiments.

152 The Mechanism of Evolution in Leptinotaesa

LEPTINOTARSA DECEMLINEATA X LEPTINOTARSA OBLONGATA.

The crossing of these two species has given further data concerning the
gametic comiDosition and reactions different from that derived in the crossing
of the other species described in Chapter IV. In Chapter II, I have described
these two species, and the chief points of difference, between them at all stages
in their ontogeny are shown. There is difference in the form and general type
that is measured by the form-index ; there are differences of color and pattern
in the larvae, in pattern and color of the adult, in reactions to different environ-
mental agents; but both have about the same rates of development {Ac values)
and feed upon species of succulent solanums ; L. oblongata upon S. rostratum
and its allies; L. decemlineata upon either the rostratum or tuberosum groups.
L. oblongata, however, does not thrive as well upon 8. tuberosum as it does upon
the 8. rostratum, and although it can be made to eat tuberosum, it always shows
a decided preference for its native food. Both are easily grown side by side in
the laboratory with perfect success, showing no changes as the result of the
enforced captivity, so that crosses can be made under the conditions of the
breeding-quarters, and thus to a large extent eliminate the possible complica-
tions that external conditions might introduce into the experiments.

The crossing of the species is not a perfectly free one, although it is obtained
with relative ease in all of the stocks of these species that I have seen. Some
cross more freely than others, and all with greater certainty of success after they
have been under the same conditions for several generations and have been
subjected to the same routine breeding treatments. There is no entire sterility
between them, and infertility seems on the whole to be failure to complete
development rather than failure of the two to breed. All of the F^ hybrids are
fertile and have given fertile progeny, so that the operations are not compli-
cated by the problems of sterility or lack of interfertility of F^. It is true, how-
ever, that they are less certain to produce progeny when the stocks are fresh from
the original habitat than when both the lines have been long grown under the
same conditions.

Under the conditions given as to source of stocks and conditions of experi-
ment, the crossing of these two species is always complicated by the extent to
which the cross shows dissociation of the two gametic systems and the number
of interchanges of elements that occur. This is evident even in F^, and in Fa
the complexity is realized more fully when one attempts to work out any of these
series of crosses. All of the crosses recorded here are between stocks of L. oblon-
gata that originated from the " Temisco colony" near Cuernavaca, Mexico
(No. 607 and No. 619), and my stock of L. decemlineata at Chicago (No. 99).
When crosses are made between these two species, in the F^ fraternities the
larvas show uniform dominance in color and pattern of the decemlineata parent,
but in the adults the fraternity shows several well-marked combinations of the
parental characters in widely varying ratios. The types commonly found in F^
are as follows :

{a) decemlineata in form, with pale yellow-white hypodermal color.

(&) decemlineata in form, with pronotal color of decemlineata and yellow ely-

tral color of oblongata.
(c) decemlineata in form, with yellow-white elytral color of decemlineata, and

yellow pronotal color of oblongata.

Eeactions and Products in Interspecific Crosses

153

(d) decemlineata in form, no lipoid color in any part, appearing translucent.

(e) decemlineata in form, but in all other characters oblongata.

If) intermediate in form, and showing variable combinations of the characters

of the two parent lines.
(g) oblongata in form, and in all other characters decemlineata in aspect.
(h) oblongata in form, with pronotal color of decemlineata.
(i) oblongata in form, without lipoid color, appearing translucent in color.

There are three chief types that appear, decemlineata, intermediate, and
oblongata in form, all more or less complicated by the appearance of the char-
acters from both parents, so that the F^ array is most diverse in appearance.
This diversity is not limited to appearance, but is shown fully in the breeding
of these F^ types for Fj, which shows that the F^ fraternity is not a uniform

Table

19.

First group.

Second
group.

Third group.

Total

a
6

6
14

c

7

d
1

e

t

9
13

h
9

t

Fern. obi. X 10-lin. m

29

79

Fern. obi. X 10-lin. m

46

4

11

21

6

71

43

11

4

217

M. obi. X 10-lin. fern

1

68

27

92

41

4

233

Fern. obi. X 10-lin. m

92

14

1

4

21

11

74

9

226

M. obi. X 10-lin. fern

i

6

141

1

7

156

M. obi. X 10-lin. fern

11

17

29

31

4

107

92

31

4

326

M. obi. X 10-lin. fern

19

11

21

1

26

4

4

39

125

Fern. obi. X 10-lin. m

11

14

4

41

4

56

28

43

71

268

Fern. obi. X 10-lin. m

9

4

12

6

31

M. obi. X 10-lin. fern

. . .

i

5

86

i

93

Fern. obi. X 10-lin. m

147

. • •

147

Fern. obi. X 10-lin. m

9

5

• • .

11

81

3

175

M. obi. X 10-lin. fern

27

46

7

.5

93

181

359

Fern. obi. X 10-lin. m

11

4

49

17

92

12

18

9

212

M. obi. X 10-lin. fern

Totals •

241

1
131

77

21
240

7
82

171

5
251

9
384

3
329

217

1133

771

1133

964

2864

heterozygous population at all. It is all heterozygous, but not all in the same
manner, so that out of an F^ array diiferent mated pairs show many differences
in the production of F, and in the progeny produced. The character of the F^
arrays is shown in table 19, which is the summation of 15 crosses between these
two species under the conditions of experiment, as far as the F^ arrays are con-
cerned.

The breeding of these F^ types of heterozygotes shows that there are three
main types of action which are in the main coupled with the type that one starts
with in Fj. Those in which the decemlineata type is dominant in F^ shows
uniformly one type of reaction in this series ; those intermediate another, and
the set with oblongata form dominant still a third set of reactions in the produc-
tion of Fg and subsequent generations, which are in the main centered about the
reactions of the form-producing agents, and little or not at all concerned with
the color and pattern agents that may be present.
11

154

The Mechanism of EvoLUTioisr in Leptinotaksa

BREEDING OF Fi HETEROZYGOTES.

The chief point of interest in this series is the extent to which there has
been dissociation of the gametic systems with interchange of agents or groups
thereof. All of these F^ fraternities are so complex that in no single instance
have I had the aid and space for a complete analysis of the entire condition pre-
sented in one fraternity which would involve in F^ and F3 hundreds of simul-
taneous matings to adequately test the total complexity of the interchange of
agents. I have, in the main, limited my efforts to the breeding and testing of
types and lines that showed points of interest with regard to certain gametic
agents and their capacity for change in position and their action in the new
positions. The complication, or diversity, is due to the numerous color and
pattern determiners that suffer interchange in crossing and the numerous
possible combinations thereof. Eight pairs of character groups capable of
metathesis are concerned.

WITH THE DECEMUNEATA FORM DOMINANT.

As shown in table 19 diverse types come out in F^ with decemlineata dominant
in form, and of these, (a) , that is decemlineata in appearance, has given some

180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10

~

~

~~

^

■^

■~

~~

~~

-*

~"

/

\

/

\

f

\

/

I

\

/

\

^

\

\

\

\

\

1

1

\—

/

\

1

f

\

1

/

/

/

<«

S

J

\

/

N

S

f^

r

s,

r-"

A

s

%

J

t

?

J

\

•

;

0
0

%

■

•^

?

T

J

[

J

0

L. decemlineata

L. oblongata

Fig. 10.

interesting results. The frequency of this type in F^ is shown in table 19, in
which it appears that the occurrence is highly variable, may even be absent or
present in the population in considerable numbers, and none that has been
tested breed true. In figure 10 I have shown the determinations of the form-
index in the two parental lines. In L. decemlineata the mode and mean of
the index falls upon the class value 2.600-2.649, with a normal distribution on
either side. In L. oblongata the mode and mean fall in or close to the class value
3.250-3.299, also with even distribution on both sides. There is little or no over-
lapping of the index-curves in these two stocks; the form-index values are
constant, and little trouble is encountered in separating them by inspection in

PLATE 16

Homozygous
.Tf'ting

Heterozygous

Homozygous
afting

- F„

- F,

- P„

Pair A — 22

•• B— 12 U59obs

C — 75 I 155.5 exp.

•• D— 50

Fj (a) type, dominant decemline-
ata Index mode— 2.600 2.649

Pure L. oblongata stock of
No. 617 Temisco, Mor. Mex.
of Blotype 10. Index
Mode 3.250 3.299

Shows in Graphic Form Results obtained in some Test Reactions
IN crossing L. decemlineata and L. oblongata.

K. XODA. DEI..

jur-rcs BrETN trra.. N. Y.

Eeactions and Products in Inteespecific Crosses 155

a fraternity of hybrids, doubtful cases being measured and tested by breeding;
the two methods combined give certainty of operation.

When the F^ apparently entirely dominant decemlineata is inbred the larvae
produced from such matings show pure reds, reddish yellow, and yellow in
proportion based on the scheme of 1 red : 2 red yellow : 1 yellow, although the
individual fraternities are highly variable. The adult progeny produced from
these groups is complex, and only that from the red line of larvae is of interest
here, the others not being presented. The red larvae produce adults that are in
pattern and coloration pure decemlineata; but show in form-index wide varia-
tion and a bimodal polygon with two modes, one on 2.600, the other on 2.950.
Matings from these modal classes develop from the 2.950 form a pure line that
is stable at once for the character of form-index and uniform for other decem-
lineata characters. The group that centers about the value 2.600 is variable,
some giving pure-breeding lines, others showing wide deviation or bimodal
conditions, but in all the numbers are not constant and the ratios are not in
harmony with present neo-Mendelian ratio systems.

When a race is isolated that has the form-index 2.950 as its constant modal
value and is crossed with pure decemlineata from the parent strain, the product
of the cross in Fg is the production of an array all decemlineata in character
that shows a bimodal condition, with the exception that the form-index values
of the modal classes of each modal group are different ; namely, 2.600, 2.600-
2.800, 3.150. The appearance of these types is erratic and the fraternities show
diversity in the numbers presented, but from the modes 2.600 and 3.150 pure
races are derived. Two of the modal groups are apparently homozygous, but
the other contains individuals which when mated give pure-breeding lines with
the form index 2.600, while others are heterozygous. I have presented this
series of reactions in plate 16, and also data of some fraternities showing the
irregularity of the array with respect to the numbers present.

The two lines that originate from this F, array give two different form-types
of an organism that is decemlineata in all other respects, and I have not been
able to derive from either of these lines any characters that indicate the presence
of the oblongata parent. The race developed with the modal value of 3.150 is
perhaps the most interesting to us in that in all respects save form it is typical
decemlineata, even to minor habits and reactions, as well as in color and pattern
characters. The odor of its secretions are characteristically decemlineata and
not those of oblongata, which, even to my senses are recognizably different.

It is probable that we are dealing in this race with one of two possible con-
ditions ; either that the line is the result of the transfer of all the commoner
decemlineata qualities onto an oblongata form-base, or that the form-determiner
of oblongata has been dissociated from its complex and replaced in the pure
decemlineata complex, the normal agent for form determination of that species.
The latter seems the more probable, and indication of its probability is found in
the change produced in the pronotal pattern of the race, which gives an appear-
ance much like that found in pure oblongata.

Testing of this race by crossing with both of the parent species has given
suggestive confirmation regarding its constitution. When crossed with oblon-
gata, the product in F^ is intermediate between the two in all respects. For this

156

The Mechanism of Evolution in Leptinotaesa

test I have used only pure white, biotype 10 lines of oblongata. F^ in the test
shows an array that is suggestive only of a monohybrid reaction, the oblongata,
the tested type, and an intermediate coming out in nearly perfect ratios (table
20) showing that the types in the test have acted as units, neither gametic
system having been broken or interchanged agents. This test is of interest in
that it shows that the race does not have in it the characters of oblongata in at
least easily available form, else there would have appeared in F^ of this com-
bination, or at least in Pg, some indication of them. As a test reaction this is
only suggestive, not in any way final.

When crossed with the decemlineata parent stock, the product in F^ is an
intermediate array, and in Fj there appear the three types of form-index values,
as shown in figure 11. The reaction seems to have been of the simplest type
possible, in that the two gametic systems acted as units and suffered no change
in the passage through the P^ hybrids.

Table 20.

Pair A, F„

Pair B, F.,

Pair C, F„

Pair D, F....

Pair E, F,

Pair F, F„

Observed

Expected on basis of monohy
brid reaction.

Decemlineata
type.

20
37
12
13
20
16

118
117

Intermediate
type.

47
74
3
15
Gl
33

233
234

Oblongata
type.

19
36
9
11
27
15

117
117

Total.

86
147

24

39
108

64

468
468

A more conclusive test was made by crossing the type with a line of L. multi-
tceniata biotype melanothorax, that was known to be pure for form-determiner
of a value of 1.1950, and with only the pronotal pattern of melanothorax present.
This combination showed dominance of the melanothorax type in F^, but in Fj
there appeared a complicated array that gives indication of the nature of the
special race investigated. This I have shown in schematic form in plate 17.

The array given in plate 17 does not show the entire complexity of the Fg
product, which is further complicated by juvenile stages and groupings of
adult characters, but shows the essential adult conditions that are present in
Fj, indicating what the suspected race was composed of. An interchange of the
characters takes place, such that the Fo shows new combinations and types not
present in the original lines. Most striking is the occurrence in Fn of a biotype
7 multitceniata type that is the product of a synthetic combination between the
pattern system of the pronotum of the tested type and the melanothorax stock, in
which the pronotal pattern systems have been interchanged, giving as a result the
type shown in F2 of plate 17 {A). The pattern type of the tested line is that of
oblongata, in which there are present all of the elements that occur in multi-
tcBniata, but in different relation. There also is obtained the decemlineata type
(B), the tested type (C), melanothorax type (F), tested type with melano-

Tested 7 pairs iu F,,

3 pr. Honioz.vfcous

4 pr. Heterozygous

Tested 4 pr. in P^
All Homozygous

- F,

- P„

^ SI

O «

Synthetic homozygous acting
race with form index 2.950
tested by crossing with L. mul-
titrpniata,l)iotj-pe melanothorax

Graphic Presentation of Test Reactions in L. muUitceniata, showing the Action
OF certain Form Determiners.

K. TOnA DEL.

Ji.n:.iTrs bien i-nn.. n.y.

Eeactions and Pkoducts in Inteespecific Ceosses

157

thorax coloration {D), and others that breed true, and also combinations that
are hybrid and not shown. The point of interest is the showing that a form-
determining agent is present, acting, and capable of change, and the interchange
of the pattern systems of the pronotima.

70

—T"

—

—

—

"■

■'

1

-

-

~

~

1

\

—

^

—

"~

~

1

1

/

\

\

V

/

\

\

■ ■

•V,

1

,M

^

1

1

~"

I

,<?

/

1

1

1

r

■k

/

1

1

1

^-

r

/

^

1

_

i^i

/

1

/

1

o7

1

\

f

I

~"

1

\

\

1

1

1

y

i

I

/

f

\

/

v

r

/

/

\

,

\—

,

1

_

_..

/

/

^-

hv

\

_

/

f

[\.

A

_

F2 AA

T

3

5

14

7

1

z

5

13

29

II

i>

I

3

2

5

13

8

2

Fj AB

1

3

W

9

6

3

2

2

7

23

8

2

1

1

1

5

10

7

2

"■

Fj AD

2

w

8

7

1

1

1

1

2

8

20

'*

1

1

2

3

5

4

1

^ F2 CC

1

5

II

3

3

6

9

22

7

3

2

2

1

2

5

14

7

1

1

' F, A

1

1

2

1

4

8

II

7

3

S

1

1

.

F, B

1

4

3

5

14

le

10

8

4-

1

2

1

. fl ^

1

1

5

8

IZ

\k

19

11

7

5

3

1

1

1

J;

Class J ~
Values.' i

0

J-

0

0

0

S

0

-4-

0

0
J-

0

0
0

0

J-

0

0

5
0

0

0
0

7
0

0

0

0

0

0
0
0

c

0
0

0
0

0
0

3

0

r>

3
0

0

0
0

J-

0

0
0

0

5
0

<->

5

0
0

0

0

Fig. 11.

The existence of the form-determiner is shown by the derivation from the Fj
array of decemlineata forms that are approximately the result of the combina-
tion of the basal form-factor of the tested race and the form-determiner from
the melanothorax type, which result could only be produced in this test by the
transfer of the form-determiner from the melanothorax type to the tested type,
and the result is precisely that which is expected.

This test is in all respects a routine Mendelian reaction, uninteresting, except-
ing in the ability which it gives to test the nature of the agents that are being

158 The Mechanism of Evolution in Leptinotaesa

dissociated and recombined in the crossing of these two species. In this race,
the subject of the test, there is clearly present in the interactions two groups of
agents, or two agents, a form-factor group that is not dissociated and a form-
determiner, the interaction of the two being to produce the characteristic form
or type as measured by the form-index, or recognized by inspection in many
instances in its pattern modifications. These two groups of agents are purely
symbolizations at present and no indication is to be had that shows what they are.

The race tested is in all respects decemlineata, with the exception of form,
which is that of oblongata, while its breeding true shows that there has been
formed a permanent combination, that is, a harmoniously acting one, so that it
gives uniform fraternal arrays, generation after generation, the usual test of
purity and stability. That the race is due to the combination of the decem-
lineata complex with the form determiner from oblongata is shown in the test
cross with melanothorax in which, in the complex interchange that takes place,
there comes out a decemlineata form as the result of the combination of the
decemlineata form-factor complex with the determiner from the melanothorax
race. This result could only have come about in two ways ; either it is the prod-
uct of the process stated, or the production of the decemlineata form in Fg is
due to the presence in the tested line of the decemlineata determiner being pres-
ent in hidden form in the tested race and not acting. However, tests in cross-
ing with the pure decemlineata and oblongata have failed to show the existence
of it in reactions where it would have been revealed if present. It is hardly to
be expected that the race that is being tested is due to the transfer of its very
numerous decemlineata characters to an oblongata base. There are too many
of these and too decided an absence of oblongata attributes in the combination,
nor is the combination precisely the oblongata parent condition, but below it,
towards decemlineata, showing that the combination does not react in the same
manner as in either of the parental stocks. The fact that the line is in color,
reaction, odor, habits, development, and in all respects decemlineata, save in the
element of form which is nearly that of oblongata, the experiences in the tests
of the line that have been made force one to the conclusion that there has been
produced a combination of agents from oblongata with the decemlineata com-
plex, producing a new, homozygously acting genetic line. The series shows, I
believe, the dissociation and recombination of the form-producing agents in the
gamete and the existence of two groups, the interaction of which is necessary to
tlie production of the specific end-result, a form-factor group, and a determiner
group, and the evidence shows that it is the determiner group that is transferred
and recombined and not the factor series. It is further shown in the test made
with melanothorax that the pronotal-pattern series are capable of interchange
as a whole, another example of the action as a whole in crossing of meristic series
of characters, other examples being the behavior of the larval patterns in this as
in other series of crosses already described.

Three matings of the (a) type in Fj have given different results from those
presented, and have not shown the presence in Fo and in later generations or in
tests of the breaking up of the line into the different form-types shown in the
series just described. These three lines have been uniformly decemlineata in
form-index and showed only the production of diverse F, groupings dependent
upon the metathesis of different agents that came from the parent stocks, espe-

Eeactions and Products in Interspecific Crosses 159

cially color-characters. Each of the three lines has been bred out to at least Fg or
beyond, and no breeding or testing has been able to break them up into different
form-index groupings, the decemlineata form remaining stable and constant.

At no point in the crosses of these two species has there been any linking or
correlation of the different color and pattern characters with these form-produc-
ing agents, and so in the three pairs that breed true for the decemlineata form
there was abundance of combinations of the color and pattern conditions. The
findings suggest the existence either of another instance of a masked heterozy-
gote for form with the decemlineata dominant, or the obliteration of the oblon-
gata form and other agents in the F^ combination. In crossing natural species
it has often seemed that the results of the interactions in F^ have been to produce
a total obliteration of some of the agents of one or the other parent, as far as
further ability to get them out of the combination is concerned. Whether they
have actually ceased to exist as agents, having been thrown out of the complex
produced in F^, or are held in fixed form not manifested or manifest in new
actions, is a problem for further investigation.

About 60 per cent of the F^ (a) type decemlineata matings are heterozygous
in the ordinary sense and show in Fg only routine combinations of the different
characters and groups thereof that are dissociated from each of the two parental
complexes. From these, extracted types of each of the parental species as well
as many stable combinations are commonly derived, while other rarer combin-
ations and associations are erratic in their appearance.

WITH THE FORM INTERMEDIATE.

In every cross between these two species there are certain intermediate forms
as regards form-index, with variable associations of visible parental characters.
Breeding of these shows in all that have been tested that they are uniformly
typical Fi heterozygotes, and the F, arrays that are produced from them show
only routine separations into the parental form-types with differing combin-
ations of the color, pattern, and juvenile characters, so that out of the series one
can obtain a very considerable number of stable combinations of the characters
shovm in the two species. The F^ arrays are in all instances irregular and the
ratios are not in accord with the expected arrays, owing to the chance non-dis-
sociation of two or more characters in one or both parents. At no point in the
series is there any indication of blending of these characters, and the reactions
are always clean-cut and precise, the uncertainty being entirely inability to
devote enough space and labor to any one series to fully analyze its composition.

These F^ heterozygotes are interesting because of the irregular metathesis of
one or several characters which at other times or even in the same lines are dis-
sociated, and suggests agents present either within or without that act to
influence this association of factors and the subsequent behavior of them as a
unit. The direct result of this condition is that one can derive a large number
of races from the cross, which breeds true— i'. e., is homozygous acting in genetic
lines — and these when crossed give simple Mendelian arrays, usually, those char-
acteristic of monohybrid or dihybrid reactions. Some notion of the extent of
the array that can be obtained is shown by the series of pairs of characters that

160

The Mechanism of Evolution in Leptinotaesa

have been found to interchange in the crossing of these two species. The com-
moner are shown in table 21.

Tabu: 21.

Character.

Decemlineata.

OlDlongata.

Form determiner

Larval body-color

Larval pattern-system..
Elytral color

Fd,

Fd,.

Y.

[(Pd. + Pd. + Pds) (ob)].

Y.

Y.

oblongata.

R

[(Pd, + Pd, + Pd3) (10 L)]
Yw

Pronotal color

Species base

YR

decemlineata

These six pairs of agents, which are recognizable as being shifted in crossing,
give, if they act independently, a possible hypothetical Fg array as follows :

(1) Number of individual combinations (4°) or 4' = 4096.

(2) Number of terms in the series (3°) or 3* = 729.

(3) Number of stable combinations (2") or 2' = 64.

To this series could be added the combinations of some of these into associ-
ations that act singly, and also the dissociation of some of the groups into com-
ponent simpler ones. Thus, in color of the body there has been obtained no
hypodermal color, white, pale yellow, yellow, chrome yellow, orange, red, and
Vermillion in pure-breeding strains out of this cross, and localized sometimes in
the pronotum, sometimes in the elytra, or at others uniform over the body as a
whole; these, of course, in homozygous-acting races that behave when crossed
with other races as pure Mendelian pairs. I have not the least doubt that if
time, space, and labor Avere available one could easily obtain from this cross
several hundred of pure-breeding lines, each distinctive in one or more char-
acters, alternative to one another throughout.

Two points of general interest come out in this complicated reaction : (1) the
dissociation of each of the gametic systems and the numerous interchanges that
are shown as the result of this cross; (2) the action of agents and groups of
agents as units, irrespective of the number of contained agents present. This
second point is of especial importance as showing that the type of reaction is not
one that involves the ultimate nature of the interchanging agents. The entire
series of reactions, the numerous stable end-products, presents a condition that is
strikingly in contrast with that shown in the crosses of species described in the
previous chapter. In plate 18 I have shown some of the homozygous biotypic
lines that have been derived as the products of the intercrossing of these two
species. Those shown are all derived from the middle group of P^, as far as
the complications presented in the adult condition are concerned, and consist
only of a few combinations of form and color. When these are added characters
in the juvenile stages, very many lines, each with its own sequence of characters,
are possible of derivation from this cross. The details of the production are not
worth presenting, involving only routine operations, with interplay of external
agents for the attainment of certain end-results. Each one of the 20 conditions
shown in plate 18 is biotypic for its adult characters and with ease can be made
biotypic for its entire constitution. The 20 shown can be made 40 by change
in larval color alone, yellow and red larval races being possible of each one of
these, and so on the biotypic lines could be multiplied.

12

"b^r

K. TODA DEL. •"^''"^^ "'"''• "• "'■

This plate shows graphically some possible combinations that can be made
in L 7nultit(rniata after pure biotypic lines have been established for these
characteristics. In this instance body-color of red, yellow, and light yellow and
body-form are concerned, giving the array of types shown in the figure, all of
which have been actuallv produced and found to breed true for at least two
generations as e.xti acted types. Many of these have been bred true for G to 8, and
even 10, generations in biotypic pure lines.

I

Eeactions and Products in Interspecific Crosses 161

with oblongata dominant.

The third Fj group in the primary cross of these species shows dominance of
oblongata, with relatively few complications. None of them is pure, all that
have been tested being the heterozygous, but in varying degrees and directions.
From the third F^ group many lines of homozygous-acting combinations have
been derived, of which the most interesting are those that involve the form deter-
miners, and a duplicate series shown for the first group is found, showing that
the form-determiner is dissociated and interchanged in all F^ interactions. The
series, however, gives nothing in action or principle that differs from that seen
in other aspects of this species cross.

One of the chief points of interest in the crossing of these species is in the rela-
tion of the two gametic systems in F^. In F^ there appears an equivalence and
blending of the two when the cross is not complicated by certain agents within
and without, whose action has been analyzed. As shown in table 17, the F^ prod-
uct under the conditions of the laboratory quarters is highly diverse, even when
the parent lines have been fully accustomed to these conditions for generations.
There is, of course, not constancy in this environic complex, but rhythmic regu-
larity of conditions which diurnal changes produce in the organisms — possible
differences in physical condition that may be in some measure productive of the
complex Fj array and the amount of the dissociation of the gametic systems
shown. That the conditions of the medium do have something to do with the
complexity of F^ is shown by the crossing of these species under the mean condi-
tions of the breeding-quarters, in special apparatus, but constant, when there is
produced in F^ only the blended intermediate type with uniformity in the entire
array in all characters ; but in breeding these are all strictly heterozygous, and in
Fj give heterogeneous arrays, depending upon the conditions present in the
medium.

The complete analysis of this cross would require many times the facilities
that I have had for controlled conditions, but enough is given concerning the
cross of these two species to show that the complexity of the products of the
cross are dependent upon the condition of the materials and the oscillation of
factors present in the medium. Tlie crossing of these two should first be
worked out under constant conditions, and then the role of departures and
changes in the medium could be determined. The cross shows how, in the ever-
changing conditions in any natural habitat there would be produced many pos-
sible combinations and rearrangements, giving many distinct lines of descent.
It is this operation in nature that has much of interest, because of its possible
bearing upon the production of heterogeneity in nature, and also because the
same two species have in nature been utilized in experiments in the production
of mutating stem-forms.

Crossing of these species under the usual conditions of life produces what
seems to be an extensive disruption of both of the gametic systems, which is in
experience even more complicated than here indicated. Not only are the unim-
portant characters of color and pattern dissociated and interchanged, in differ-
ing aggregations, but habits, food-relations and growth are involved. As a
result, hardly any two cultures are alike, and derivation of homozygous-acting
lines is often attended with endless difficulties; nevertheless, at no point has
there been anything which on test proved not to be regular in its action and con-
stitution. I wish that it were possible to present at this time even an approxi-

163 The Mechanism of Evolution" in Leptinotaesa

mately complete account of the analysis of the intercrossing of these two species,
but the complexity of the dissociation has made it largely a practical matter of
inadequate facilities.

Both of the species are stable and constant and even under extreme changes of
the environment they retain their stability, altering only characters if at all,
but in combination in crossing the lines that have been experimented upon show
no end of diversity. Constant conditions surrounding the cross reduce or elim-
inate the dissociation of the gametes, the reaction appearing as simple, but
with ranging conditions in the medium the disruption is extreme. It may be
objected that in reality only a few character agents are involved, and this may
be true ; but there are some agents, that are specific and localized, capable of sep-
aration and recombination through the reactions of crossing plus the conditions
of the medium, so that it makes little difference what is personified or symbol-
ized to express our experiences. The important point is that these unseen
agents react precisely and orderly. In this series there are many color-con-
ditions, especially of the hypodermal colors, that are unstable and break down
at death, but which are nevertheless distinct and precise in life, and react
as distinct contrasting units of operation. I am aware that the color-producing
molecules may well be the same in all, and that the color differences may be
only differences in the arrangement of the component atoms and atom groups
of the molecules; but there is some precise cause of this.

The result of the crossing of these two species under ordinary conditions may
be characterized as a collision between them, with disruption of each of the
gametic systems into fragments of different sizes and composition, which are in
form and position to be built into new positions and arrangements, so that the
results of a cross of this order might be the production of many different lines
of descent. It is the antithesis of the reaction of signaiicollis X divcrsa, where
the systems were not disrupted at all. The experience is suggestive of what
might conceivably happen in nature and be productive of some of the groups of
related species that occur, where lines of demarcation are wanting or difficult
to draw. It would be impossible to decide whether in any instance in nature
where there was extensive heterogeneity it is due to this or some other cause, for
the reason that the production of the condition would soon pass away, leaving
behind only the heterogeneous population, in which in nature nothing is known
as to the relations in breeding or in genetic reactions of the component elements.
I have put this to test in experiments described in a later part of this report.

Whether all cultures of decemlineata crossed with all cultures of oblongata
would give this result I do not know. I have tested only crosses of my 99
decemlineata stock and oblongata from Temisco and Cuautla. They may
well be local differences in these species, such that the reactions of other sources
of materials would not be as complex as I have found them, or might be more so.
Whatever the solution of this problem may be, the relation between the two
species will not be any simpler than I have found it, and probably is much more
complex, taking the species as a whole, so that the potentiality for heterogeneity
between these two is very great.

This cross reminds one strongly of some of the events that may have happened
in the production of the diverse strains of domesticated plants and animals.
Many garden fruits and flowers, dogs, poultry, and pigeons show endless
combinations of character that are present in the wild originals; and also

Eeactions and Peoducts in Interspecific Crosses 163

numerous accentuations and developments of these, with few apparently new
characters. There have been in domestication crossings without number, con-
trol, and perhaps all possible combinations, so that it may be probable that the
initial impetus given to primitive man to breed and utilize some of these then
wild organisms came from this same sort of initial disruption of two originals,
by chance giving combinations pleasing to the savage fancy, so that they were
perpetuated with his crude breeding operations, and come down to later periods
in culture. The initial cross would in all probability not be intentional, but the
product of breeding of the tamed with the surrounding wild forms, as is known
to be the case in savage or barbarous establishments.

One point further is of interest in this cross, namely, the fact that the initial
Fi and F^ arrays are on the whole tlie most complex, and that thereafter the
reactions of the products of the primary cross are all increasingly towards
simplicity, as the lines are reduced progressively to homozygous-acting biotypic
strains, between which crosses give only the most orderly and monotonous
Mendelian reactions and products. This experience is also in harmony with the
crossings of domesticated plants and animals, to which, thus far, the majority
of the orderly and fully analyzed Mendelian operations are confined. Thus, the
20 types shown in plate 18 are, when crossed in biotypic condition, productive
of monohybrid or dihybrid reactions, rarely of trihybrid, while the huge
majority in the tests thus far are of the simpler types. In many respects this
cross of decemlineata and oblongata shows the difference between species or
groups in nature and the purified races of cultivation in the complexity of the
natural, the homogeneity of the cultural, in reactions that test their gametic
constitution ; but in both there is no difference in the basic principle of reaction.
This is the essential point and one upon which attention is directed in the
summary at the close of this chapter.

LEPTINOTARSA DECEMLINEATA X LEPTINOTARSA MULTIT/ENIATA.

The crossing of these two species is complicated and can best be presented
under two headings — the crossing of the natural species and the crossing of
biotypic lines. These species, especially rmiUitceniata, are in natural conditions
complex in their constitution, and when they are crossed the ensuing F^ and Fj
arrays are diverse, depending upon the original composition of the stocks used.
In some of the earlier chapters it has been shown that for muUitceniata the
composition is complicated by differences in form, pronotal pattern, sports, and
other means of gametic complexity. As a result the crossing gives in each set
of matings differences and arrays depending upon the stocks used. I have for
the purposes of this analysis purified the parent stocks used, and then utilized
them in crosses in the determination of the relations between these two species
when crossed. I shall describe the results of crossing the biot}'pic lines first,
and then the general reactions when the crosses are made between the stocks
as a whole.

CROSSING OF BIOTYPIC UNES IN DECEMLINEATA AND MULTIT>tNIATA.

The simplest and standard cross between these two species is that made
between a biotypic line of pronotal pattern biotype 7, mean form-index, mean
yellow-white pronotal color, and mean pronotal color, each with the pure non-
variable larval characters proper to each species. Two biotypes of this sort are

16i The Mechanism of Evolution in Leptinotaesa

easy to derive from the parent species, are perfectly homozygous in action, but
must be tested by intraspecific crossings to determine the presence of non-
manifested gametic contaminations. These when found must be eliminated.
Two such lines reared under the same conditions of the medium for several
generations when crossed give a relatively certain test of the reaction proper to
the two gametic systems, independent of the conditions of the environment, and
not complicated by nonhomogeneity in the stocks.

In the crossing of these two species I have used only pedigreed materials that
had been reduced in cultures to a biotypic condition, and my crosses have been
between the decemlineata stock from Chicago (No. 99) and multitceniata from
Chapultepec (No. 543), Puebla (No. 541), and Texcoco (No. 547) in Mexico.
Of this species I have used the pure multitceniata biotype 7 and multilineata bio-
type 13. All of these have been of the typical yellow hypodermal color, so that
the crosses have not been complicated by the array of hypodermal colorations
found in the previous cross. The two species are also not greatly different in
form-index, except for the narrow form that is present in the biotype multi-
lineata and the broad index that is associated with tacuhaycensis.

After these two species have been reared in the same conditions in the labora-
tory at Chicago for three to five generations and then crossed, the uniform result
is the dominance of decemlineata in F^ in the juvenile stages, especially in larval
body-color, the patterns and larval-pattern sequences being the same in both.
The dominance of the decemlineata in F^ can in many respects be altered, by
the use of desiccation, high temperatures, and other agents, to diminish the
intensity of the F^ red, but in no instance has the color been modified to more
than an intermediate condition of yellowish red. The F^ adults show rather
uniformly the equal blending of the adult characters of the two parents when
the parent lines are in both biotype 7 in pronotum pattern. The pronotal
hypodermal color may be variable, swinging between the two parental types,
but in the main it is intermediate between them, while the elytral colors are not
sufficiently different to produce any change. The form-index, however, shows
complete dominance of the decemlineata type, with relatively little oscillation
and decemlineata is the dominant type.

Inbreeding these F^ for the next generation shows that on the whole the two
have been through the cross with no dissociation of the characters, there appear-
ing in the adults in F2 three classes that are by inspection clearly decemlineata,
heterozygotes, and multitceniata. Some fraternities show this separation with
remarkable distinctness, others show the presence of intergrading conditions
in the heterozygous series, so that the separation of the entire array can not be
made by either inspection or measurement, but in such arrays no difficulty is
experienced in selecting by inspection the extreme conditions that always breed
as extractives, or of the centrally placed heterozygotes. Other conditions in the
array are not so easy of calibration and often are only properly placed after test
by breeding. This difference in F2 in different fraternities is a product of the
conditions surrounding the cross, which in general seem to produce, when con-
stant and with little or no oscillation, sharply marked F, arrangements; but
when the conditions are oscillating the groups are less clearly defined or often
obscured entirely by the range in the heterozygous members of Fo.

In these reactions it is certain that the characters of the adult have not been
dissociated in passing through the F^ reactions, coming out of the operation in

Eeactions and Products in Inteespecific Crosses 165

original form and arrangement, so that the reaction, as far as the adult con-
ditions are concerned, presents only the array of a simple monohybrid type as
far as the crossing of these two biotypic lines are concerned.

In the larvae the arrays presented are complicated by the unstability of the
larval body-colors when in heterozygous combination, so that F, always presents
two classes, reds and yellows, in variable numbers.

This is entirely the product of the potentiality possessed by the heterozygous
members of the fraternity for range in the characters presented, and is in the
main, if not entirely, a product of the conditions of the medium or other con-
ditions of life and of purely transient nature. When these fraternities are
reared under constant conditions the Fo array of larvae shows yellows, yellow-
reds, and reds, in close approximations of 1 : 2 : 1.

In nature and imder the usual conditions of operation, the F, array in its
heterozygous members is able to range widely in both directions, so that the
result is to produce the curious ratio of an apparent 1 : 1. The homozygous
yellow is always more easy of separation than the red, which even in the pure
stock is variable in its intensity, depending upon conditions of nutrition and
surroundings, so that separation of F, fraternities is by no means easy and is
complicated by the action of heterozygous individuals, which tend to swing to
one or the other extreme of the possible series, giving either reds or yellows,
and few uncertain intermediates under ordinary conditions. The Fj hetero-
zygotes in all of these experiments are more sensitive to the action of the
external conditions, as shown by their ability to fluctuate between the possible
extremes. This fluctuation of the heterozygous individuals is by no means
common in described crossings, largely due to the absence of observations upon
the juvenile stages of organisms in crosses. Xewman has recorded variations
in the aspect of the embryos of Fundulus, and I have seen in many of the
materials that have passed through my hands this same fluctuation in the
juvenile characters in ontogeny, and have experimentally altered them one way
or the other by change of conditions or food. These changes are thus far in my
experience entirely without genetic significance, although they show in the
heterozygotes susceptibility to external conditions not shown in the homozygous
types, and further these are always far more strongly manifested in the juvenile
stages than in any of the adult characters where fluctuations of this sort are not
common.

The evidence from this cross of the mean biotypic lines of these two species
shows that the two gametic systems act in crossing as unit systems coming out
of the cross in totality, without interchange of the characteristic of either parent,
so that the reaction in the cross is of the monohybrid type. This result is true
only of the condition in the stocks used and under the conditions of experiment
given. Further, in experience with the cross the regularity of the reaction is
increased with the approach to constancy in the conditions of the medium, the
sharpest reactions being observed in controlled series, where the conditions
were constant and at the mean of the environmental complex present in the
breeding-quarters antecedent to the time the stocks were crossed.

These two modal biotypes represent the species complex devoid of any
accessory or complicating agents, and the reaction shown between them in this
cross is, no doubt, that which is entirely proper to them when brought into
combination. Any such reaction is in all probability entirely a laboratory event.

166 The Mechanism of Evolutiok in Leptinotaksa

and in nature it is highly improbable that anything of this sort would happen,
owing to the rarity or absence in both of the species of this modal biotype
uncomplicated by other conditions or agents productive of other characters in
the gametes. No materials direct from nature have ever given any such result
entirely, so that natural crosses are more complicated than the one described.
Another cross of interest is that between the modal biotype of L. decemlineata
that was used in the first instance and L. multitceniatd biotype melanothorax,
with other characters those of modal standard. In this combination decem-
lineata is dominant in F^ in all stages, especially in the juvenile, and F^ when
inbred for F, shows in this instance in the larvas the same array as in the first
cross, due to the same causes, and is of no further interest. In the adults,
however, there come out in F, sharp indications of the dissociation of the
gametic systems, as far as certain adult characters are concerned, showing uni-
formly the following types in variable proportions :

decemlineata in all respects.

decemlineata with melanothorax pronotum and head.

multitceniata in all respects of biotype 7.

melanothorax in all respects.

multitceniata of biotype 2 aspect.

decemlineata of biotype 2 aspect.

Of these there are some decemlineata that breed true ; the majority are hetero-
zygous as far as their reactions are known. The decemlineata with the melano-
thorax characters has always the form-index of the parent (2.699) and breeds
true in part. Multitceniata in all respects of biotype 7 has in all tests bred true ;
melanothorax in all respects also breeds true, while none of the remainder has
been found to breed true. Out of the combination I have derived thus far the
following stable combinations :

decemlineata in all respects. Eatio, homozygous 3 : heterozygous 8.

N. = 96.
decemlineata with melanothorax pattern ; 4 out of 6 pairs mated. Eatio,

homozygous 2 : heterozygous 1. N. = 141,
multitceniata in all respects of biotype 7; 14 out of 14 tests. Eatio,

homozygous 1 : heterozygous 0. ]Sr. = 73.
melanothorax in all respects ; 12 out of 12 tests. Eatio, 1 homozygous : 0

heterozygous. N. = 134.

All of the other types that come out in Fj do not breed true in any tests thus
far made.

The point of interest in this cross is the crossing of the pronotal-pattern pro-
ducing agents from one gametic complex to the other, and this is shown clearly
by the finding of them in combination with the characters and form-index of
each of the two parent types. Interesting also is the derivation in Fg of multi-
tceniata biotype 7 as a result of the cross, showing that the pattern present on
the head and pronotum of decemlineata had crossed to and replaced in melano-
thorax the corresponding agents, thus synthesizing multitceniata biotype 7 out
of the combination, and also giving decemlineata forms with head and pronotum
of the melanothorax type.

None of the juvenile characters have been observed to be dissociated from
their respective gametic systems, and in Fj all homozygous forms are red where

Keactions and Pkoducts in Interspecific Ceosses 167

the decemlineata form-index is present, yellow where that of multitceniata is
present, and complicated when the individual is heterozygous.

The reaction in this cross is plainly of a dihybrid type in which the gametic
systems represent one unit in the reaction, with all of the non-dissociated agents
and characters proper to each, and the two head-pronotal patterns the other pair
of characters. In reality these are the dissociated agents, although in this
instance each is complex, even though acting as a unit in the reaction. I have
not the requisite data to attempt a statement of the ratios present, owing to the
necessity of testing out many more pairs of the heterozygous individuals, whose
composition can not be approximated by inspection, and whose study is further
made complicated by their great sensitiveness to conditions in the medium
which act to cause extreme fluctuations that obscure their true nature. The
existence of the four pure-breeding lines are, however, all of the data needed
here, in that they show fully the interchange of the head pronotum-pattern
producing groups as units in toto, and their combination with the other parental
gametic system. I have only been interested in the fact of interchange and the
number of possible stable combinations that are derived and the type of reac-
tion. Crosses so plainly dihybrid in type are not worth the effort to work out
the actual composition of the heterozygous types present, and especially so as
there has been no indication that there was anything unusual or interesting in
them.

In this cross precisely the same species bases are used, the only difference
being the presence in multitceniata of the agents productive of the melanothorax
type of head and pronotum coloration. The type used was not the recurrent
mutant melanothorax, but only the transfer of the head pronotum group of
pattern agents into the biotype 7 complex, displacing the corresponding com-
plex, giving the type used. The cross had only the one difference present, that
of the two pattern-agent groups mentioned and the specific non-dissociated base.
In the first instance the crossing showed only uniform monohybrid reactions,
the two systems remaining intact; in the second the two remained intact, but
with the exception of the one interchange between the two groups of agents
observed. Why this should be so can not at present be decided, but it is clear
that the reaction is not the product of a chance shifting of independent agents,
but is more probably the interaction of the two gametic systems upon the basis
of their nature and conditions of reaction, these determining the extent and
character of the dissociated and interchanged agents or groups of agents.

From this point it is possible to make all sorts of complications in the cross-
ings of these biotypic lines, simply by adding to one or the other agent or groups
of agents by the appropriate methods. Thus, for example, if to the same two
stocks utilized in the preceding experiment there be added to the L. multitceniata
line the red elytral color, the complications are greatly increased in the array
of pure-breeding types found in F,.

The red may be derived from any source, but the most convenient one that I
have found is the geographic variety variabilis. This is readily reduced in cul-
ture to a homozygous-acting red race, and when crossed onto the combination of
multitceniata and melanothorax types gives a race that is pure for all of the
characters, is homozygous in action; and this, when it is crossed with the
decemlineata race used, further complicates the Fo array by the production of
yellow, orange, and red in pure-breeding races, thus adding to the possibilities

168 The Mechanism oe Evolution in Leptinotaesa

already present to make a considerable array of possible pure-breeding Fj
extractive races. The F^ in this combination shows nothing of interest with
these lines; decemlineata is dominant, and the fraternity is as in the other com-
binations of these species, except that the elytral color is variable from yellow
to red, with no observed regularity of proportions. Fj is complicated, and I
have never tried to analyze out the entire conditions present, as it required
space and labor that I could not afford to devote to this relatively unimportant
portion of the general project.

These experiments establish first, the point that decemlineata is the dominant
form in crossings of these two species, and this has an important relation to
some of the experiments to be presented later. It suggests also that in the test-
ing of species in initial crosses it is in all respects best to reduce them to a
modal biotypic condition, from which races all contaminating agents or char-
acters have been removed, or the lines otherwise reduced to basal conditions in
which the agents productive of diversity were eliminated. When this is done it
is possible to test fully the true method of reaction between the two species and
the extent to which dissociation occurs in crossing. From this as a point of
departure it is possible in any instance to test by experiment the action of added
gametic agents or the role of external conditions in the production of hetero-
geneity of action and product in the crossing of the two in experimental con-
ditions or in nature.

As far as the crosses of these two species are concerned, further description
at this point would add only uninteresting details and no points of general
interest. Added data of the crossing of them appears from time to time in other
portions of this report as it has a bearing upon the operations under discussion.
At all points, however, it should be kept in mind that these two species when in
combination are not in crosses dissociated to any considerable extent, and that
the basal form, comprising those agents that produce the form as measured by
the form-index, with which are associated many characteristic and non-dissoci-
able characters, remains intact in the intercrossings, and only some of the more
easily displaced agents are shifted. The condition in this cross is therefore
quite different from that of decemlineata and oblongata^ in which the reaction
between the two reminds one of an extensive disruption of the two systems
when they meet in the F^ heterozygotes. The main point in presenting these
details in this report is to make clear the fact that the reactions of these species
in crossing are in no respect different in principle from the reactions found in
many other organisms studied, as measured by the neo-Mendelian reaction in
the last ten years.

LEPTINOTARSA MULTIT/ENIATA X LEPTINOTARSA OBLONGATA.

The crossing of these two species presents essentially the same set of problems
that are present in multitceniata and decemlineata. It is necessary that the
materials be reduced to homogeneous conditions, to modal biotypic lines, before
the crossing is made. When this is done and the cross is made between multi-
tceniata modal biotype and oblongata modal biotype, the reaction is a perfectly
simple one, monohybrid in all respects, without any dissociation of either
gametic system. This cross is not complicated by the juvenile body-colors,
which are the same, yellow, in both, and the only juvenile difference is the

Reactions and Pkoducts in Interspecific Crosses 169

pattern, which is not in experience dissociated in this combination. The chief
distinction is the form-index, which permits of sharp differentiation even by
inspection and separation of the F, extracted groups.

SUMMARY AND DISCUSSION OF THE CROSSING OF SPECIES.

It has been shown in these two chapters that organisms from nature that have
the taxonomic value of species, some of which have been distinguished as such
for more than a half century, can be crossed and give fertile progeny and types
of reactions that are interesting. The first question that naturally arises is, are
these forms true species ? But what are true species ?

It has been shown in Chapter II that each of the forms used here have precise
habitat relations and differences, are at all stages in life distinguished by con-
stant characters of color, pattern, form, and the many specific methods of
reaction characteristic of each, between which species there are no present inter-
grades. Nevertheless, they cross and produce fertile progeny to greater or less
degrees, and in this transgress one of the oldest criteria of distinctness. It is
now certain that there is no certain line of separation in this respect, and that
from complete fertility one passes by all sorts of transitions to complete infertil-
ity. What constitutes fertility or infertility remains for the future to deter-
mine. Some data of significance is gathered from these experiments.

The problem of fertility of species hybrids presents two aspects : ( 1 ) as to the
factors productive of limitation of breeding in the initial cross; (2) of the fac-
tors productive of sterility or nonsterility in differing degrees in the F^ array
in breeding. In Darwin's time, and even before it was known that the sterility
of hybrids between species was a complicated relation and the work of Gartner,
Kolreuter, and others, as well as his own experiences, forced Darwin to admit
that " we have conclusive evidence that the sterility of species crosses must be
due to some principle, quite independent of natural selection," " and from the
laws governing the various grades of sterility being so uniform throughout the
animal and vegetable kingdoms, we may infer that the cause, whatever it may be,
is the same or nearly the same in all cases." Thus Darwin clearly recognized
that sterility, of which so much could be made in discussion of species and their
origin and its advantage to incipient species, rested upon other principles, and
in the end he concludes that " the primary cause of the sterility of crossed species
is confined to differences in their sexual elements."

Sterility, in the broad use of the term, comprises several different aspects of
the fact of nonproduction of progeny in a given mating. Commonest are the
instances of mechanical inhibition of breeding or copulation, which is usually
due to structural relations or conditions whose origin is unknown ; second, there
are the numerous series of instances where the gametes are able to unite and ini-
tiate developments, which, however, do not progress beyond a certain stage in
ontogeny, a stage which seems in many instances to be a constant point for the
particular combination; third, the array of numerous examples of the produc-
tion of an Fi hybrid, frequently with unusual personal strength and vigor, but
inability to produce gametes that are capable of either fertilization or develop-
ment or both. Between these three modal points there are many intermediate
conditions described in the voluminous literature of hybridization.
12

170 The Mechanism of Evolution in Leptinotaksa

The first group of causes of sterility is at present not open to profitable dis-
cussion, owing to the fact that in no case is it known what the agencies were
that produced the mechanical inability to copulate or inhibition of the gametes
to unite or even come into proximity. These are matters of existing structures,
wdth specific modes of function, of whose method of production entire ignorance
prevails. Apply interpretation as one will, nothing but a miserable array of
plausible arrangements of possible causes and effects is the result. It is not
even probable that these diverse inhibiting mechanical devices arose because of
their utility in keeping the species pure or aided in its origin, and so we must
pass them by until such time as there is real basis of discussion.

Of the two remaining categories of agencies productive of sterility and
associated with the union and nature of the gametes themselves, direct experi-
mental investigation is possible.

It has been recorded in many plants and animals that the fertilized zygotes
resulting from a cross begin development, progress normally up to a certain
point, then cease normal development, go on in abnormal development for a
time, and then die at approximately the same point. It is needless to cite
instances ; these are too well known to the readers of hybridology and experi-
mental embryology.

In the crossing of these natural species I have had many indications that the
problem of sterility is entirely one, as far as the gametes are concerned, of their
factorial composition and of the position and action of these agents in the
gametic complex. A priori this might be asserted, but can experimental con-
firmation of it be obtained, investigated, and the role of specific agents deter-
mined as to their relation to the production of this phenomenon ?

Uniformly in all of the species used the stocks fresh from nature do not cross
with the same ease or certainty as they do after they have been in the laboratory
under the same conditions for several generations. This has been noted in one
way or another for nearly a century, and Darwin makes much of it in his writ-
ings, but I do not know that anyone has made a serious investigation thereof.
With the species used I have had no difficulty in their breeding under the cul-
tural conditions provided for them, so that my problems are not those so often
mentioned in the breeding of wild organisms — of their refusal to breed under
domestication. I have no"t found any that would not reproduce when given the
proper food and conditions in the medium. Nevertheless, while I have had con-
stant success in the breeding of wild species in my laboratory conditions, the
crossing of these shows in all most decidedly that at the start the cross is not only
far more difficult to make, copulation is not so easily effected, and after copula-
tion, if fertilization is effected, the mortality in the developmental stages is
immensely greater than in the same type of cross in the same strain two or
three years later. Data upon this point is given in condensed form in table 23
which shows in the three species L. signaticolUs, diversa, and undecimlineata
the change in respect to the fertility of the matings in crossing of these three
during the first six generations of captivity. In the pure species cultures there
has been no appreciable change in the fertility of the species from the introduc-
tion to the end of the sixth generation, and the matings not producing adult
progeny are the same in the first as in the last generation ; nevertheless, in the
crosses there is in the matings made in the stocks fresh from nature much lower

Eeactioxs and Peoducts in Interspecific Crosses

171

fertility than is found in later matings, although at no time does the fertility
become equal to that in pure stock matings in any of the species.

In any combination between these three species there is never entire infertil-
ity in any line found thus far in nature. I have recorded in table 22 only mat-
ings in which copulation was observed or known to have taken place, but in these
matings there are instances in which there is no copulation of the pair and they
pay no attention to one another. These cases have their causes in the breeding
reactions of the parent species and are in part instinctive reactions dependent

Table 22.

■a

TS

■a

"O

£?

v

c

3

'^^

'•B

fcT

3

o

t

o

>>c

>,

S

g2

B

c

o

1

Z

a

Condition of stocks with ref-

<£

o

&

&•=

^

fa

d

erence to time under cul-

Species crossed.

Hi

o

bo

0^

a, c

o

•o

tural conditions.

as

bL-6

§ .

be

■o

o

s

'S-o

^1

^t

">

■2 v

^c

O

.2

3

'^a

o^

"o"

'5

Sf

■■S.2

Oi

. j:

. o

e£i rt

d

o

o -"

6a

d «

d

O"-

c*^

132

51

19

7

5

50

43

0

Direct from nature . .

L. signaticollis X

L. diversa.

Second to fourth gen-
erations in condi-

185

46

11

3

1

124

77

0

tions of culture.

Sixth and later gener-
ations in culture.

151

18

4

3

2

124

67

0

L. signaticollis X

Direct from nature . .

L. undecimllneata.

211

65

41

17

19

69

60

0

Second to fourth gen-
erations in condi-

179

31

23

9

4

112

97

0

tions of culture.

Sixth and later gener-
ations in culture.

243

19

5

7

7

205

174

0

Direct from nature . .

L. diversa X
L. undecimllneata.

73

19

3

4

6

41

38

0

Second to fourth gen-
erations in condi-

151

31

5

8

2

105

78

0

tions of culture.

Sixth and later gener-
ations in culture.

82

7

1

0

1

73

60

0

upon sex-attractions of one kind or another, most commonly that of odoriferous
secretions and possibly to other agencies.

I have been most successful in the crossing of these species if they are mated
at the height of the first reproductive activity, at which time the instinctive reac-
tion against an outcross seems easiest to overcome, aided perhaps by the pres-
sure of accumulated reproductive elements within. My data show that in
these species there is at the start infertility such that only a small proportion of
the matings give adult progeny and that continued existence under the same con-
ditions results in the raising of this fertility to a considerably higher percentage
in the matings, but never entirely reaches the fertility of the matings within
any of the species.

173 The Mechanism of Evolution in Leptinotaksa

In Chapter IV, I have sho^^oi that the rate of development, the determiner, Ac,
and its values had a role in the production of certain interesting results in the
crossing of these species, and examination of the records of different matings
gives data which show that the same agent is in some respects concerned in the
production of the degree of sterility in these crosses. The data given in table 22
are drawn at random from the records of the crossing of the different species
and the stock matings, under the same set of cultural conditions, and in that
time the species attain the same Ac values, about 60, I find that in strains of
signaticollis in which the Ac value is artificially kept at about 40, that the degree
of fertility in crosses is lower and approximates the initial condition on being
taken direct from nature.

Added data in the same line comes from the testing of one of the races that
arose from the crossing of signaticollis and undecimlineata described in Chapter
IV. This curious undecimlineata race arose as a pure-breeding one in F^ and
was characterized by its excessively slow ontogeny and long life-cycle, irrespec-
tive of conditions in the medium. This race I crossed repeatedly with signati-
collis having the Ac values between 40 and 60 as a test reaction to break it up
and so determine its composition, in which the crossing was successful in 11 out
of 131 trials, and then only with the Ac value at 60, never at 40, but even at
Ac 60 the cross is sterile.

From these data, gathered by the wayside as the by-products of other experi-
ments, it is shown that the activities whose presence and action is symbolized
by Ac and its value expressed in elapsed time is an agent in the gametic complex
whose action is productive of changes in the degree of fertility in the crossing
of these species. The data accumulated show that when the values of Ac are
wide apart fertility is diminished, when equal fertility is increased, but that the
attainment of the same values in the Ac determiner does not raise the fertility
to the standard found in the pure species.

This experience is in line with the collective experience of those who have
spent years in crossing organisms as was well stated by Darwin, Gartner, Kol-
reuter, and others among the older students of these problems ; but why do the
species from nature become increasingly fertile after they have lived under the
same conditions of existence for varying lengths of time, and what is it in the
species that changes ?

In the species L. signaticollis, diversa, and undecimlineata it is the Ac deter-
miner values that change, made manifest to us by the rate of ontogenetic
development under the standard conditions of the experiment, although not the
least evidence is to be had as to what changes in the physical composition of the
gametes. Numerous possible causes can be suggested, but evidence is wanting
that it is any of them. It would be interesting to know how general the relation
between this changed rate of development and the degree of fertility is, but I
have not been able to find any data in the literature that has any direct bearing
upon the problem.

Even though the rates of development be the same as can be made in experi-
ment, the fertility never reaches the point found in the normal cross, so that
there are other factors in addition to the Ac determiner that are not altered, or
not enough, by the changed conditions of life so that fertility reaches the normal
for the species. A point of interest in this connection is the interfertility of
extracted Fg types, such as are derived from the crossings of these species. In

PiEACTIOXS AND PRODUCTS IN InTEESPECIFIC CkOSSES 173

the extracted types coming from the crossing of signaticollis and diversa, the
degree of fertility is as high or higher than in either of the parent species. Com-
parison of the data shows clearly that there is relatively higher fertility in the Fg
extractives, showing that there has heen a change in the nature of the gametes
by the reaction of crossing, and inhibition of uniform fertilization and develop-
ment is removed by passage through the F^ reactions. A point of interest
arises in this connection, namely, the increased interfertility between these
extracted Fj stocks and the parent strains.

The reactions in these crosses are of monohybrid or trihybrid types, and the
extracted products show no change in any detectable attributes or qualities, so
that the reason why the extracted products of crossing have higher interfer-
tility is a problem for further study. There clearly is some change in the
aametes as a result of the passage through the F^ reactions which in some way
removes the barriers to complete fertility in the matings of these otherwise dis-
tinct types.

I was further interested to see what the data would show with respect to the
fertility of the Fg extractives coming from different crosses. I had compiled
from my mating-records data of the crossing of Fg homozygous-acting extrac-
tives, and although the crosses of this sort have not been numerous, the data
derived as the by-product of other experiments show clearly that the fertility
is strikingly lower than in the matings of Fo extractives from the same type of
species cross.

Out of these data three interesting general points come : (1) the demonstra-
tion that alternation in the degree of fertility follows directly any change in
the value of the Ac determiner; (2) the demonstration that Fg extractives have
entire interfertility between themselves and with the parent species; (3) the Fj
extractives from different species crosses, but of the same extracted type, do not
have the same interfertility as extractives from the same type of cross.

In the crossing of the species L. decemUneata, oblongata, and muUitcBniata
the Ac determiner values are nearly the same and culture of these species affects
no appreciable change in the Ac value; nevertheless, the fertility at the onset of
any stock direct from nature is decidedly lower than it is later in its history.

As in the first three species, the fertility between these three never has reached
in experience the same ratio of progeny production from the total matings as it
does in the normal stocks under cultivation, but the F, and Fg extractives show
precisely the same relation that was found in the first group of species, showing
that the principles at work are the same.

The fertility between these two groups of three species is very low, and thus
far the only series of crosses that has been at all successful are the two lines
from the crossing of decemUneata and diversa, described in the early portion of
this chapter, and which gave such unusual results. Only two have given adult
progeny out of many trials. No other successful crossing between the two
groups has been attained. This has been attempted, but there seems an aversion
of the two series to one another, so that copulation can not be obtained excepting
at the climax of reproductive activity, and then is best attained if the antennae
are cut off or covered with neutral gmm, so that the sex-reactions depending
upon the odors of the species secretions comes feebly or not at all into play. By
subterfuges of one sort or another I have been able to make all the possible com-
binations of these two groups of species, able to obtain matings and fertilized

174 The Mechanism of Evolution in Leptinotaksa

eggs in all of them, most of which developed for short periods, but few of which
liatched. Very many die before or at the period of the forming of the limb-
buds. The two groups have quite different rates of development, Ac values,
and differences in these may be primarily responsible for the death of the
embryos.

It may be possible to produce hybrids between these groups by the altering of
the Ac values through the use of external conditions, but thus far the permanent
alteration of tl^is character has not been affected by this agency. It is shown in
this and the preceding chapters that this character or the agents which it repre-
sents are specific for the species, and, while capable of change, has in all thus far
tested shown rigid upper and lower limits not transgressed by the action of the
pressure of conditions in the medium.

In one way or another the gametes of the different species in this genus are
able to unite and begin development, some to complete it. The problem of the
intercrossing of species consists, therefore, of two sets of problems : the initiation
of the mating reactions, which are independent of the second, namely, the inter-
actions of the gametic systems when placed in the zygote. Evidence is derived
from these experiments showing that in the gametes the production of F^ adults
is directly associated with rates of development in the two gametic systems, and
that likeness of rates is productive of fertility and development, unlikeness of
infertility and cessation of development; but there are other at present unde-
termined agents present contributing to the infertility and non-development.
Many interesting and suggestive openings into this most difficult and important
portion of the problems of evolution have been obtained, and I hope to be able to
follow them further.

Suggestive points with regard to the inhibition of the reproductive reactions
have also arisen in the course of this crossing, which collectively indicate — in
some instances show conclusively — that the reproductive isolation of these
species or types is independent of the factors governing the interaction of the
gametes and in most instances is an incident in the origination of the line, a by-
product of the process through which the form was produced, and I have
observed, in the course of this work, instances of this isolation which have arisen
in experiment by the reaction of origination, and the isolation has no relation to
utility, purpose, or adaptation, nor plays any role iji the origination of the line.
It is a purely physical resultant of the reactions that have taken place in the pro-
duction of the specific form. In the data of crossing species already given there
is positive evidence for this conclusion, while later portions of this report con-
tain added data in support of the same proposition. An interesting instance of
the rise of a race that is physiologically isolated in different ways is the line that
arose from the crossing of L. diversa and L. decemlineata. This race or species
crossed with L. decemlineata is slightly fertile, and entirely infertile with L.
diversa, L. signaticollis and L. decemlineata, and no method has been found that
decreased the infertility in any degree. This was true of the line so long as it
was maintained and was the direct and completed product of reaction that gave
rise to this race, so that there were produced factorial conditions that effectually
inhibited the reproductive activities with the nearest of kin, the parent species;
nor has fertility with other species changed as far as has been tested. The condi-
tion is not that of either parent, but something new, a fact that is clearly mani-
fested by the infertility with both parents. A stock of this character arises as a

Reactions and Products in Intekspecific Crosses 175

group, not as an individual, and its isolation is a process of production and not
accumulated progressively in any manner, from any process of survival, selec-
tion, or utility. It is purely a result of the reaction, and there is no reason why
it should be present other than that the gametic factors active in the production
of this line in their interaction produced a stable complex such that this was one
of the resultant products.

The stock is also of interest in that it shows that other aspects of the isolation
problem are touched upon in an interesting way. The resultant combination of
different characteristics of the parent species has given habitudinal isolations
that are barriers to the breeding of the form in one direction at least. Its food
demands are for Solanum chrisotrichum, or S. liertwigii, upon which it feeds,
and upon which the only form with which it has been made to cross does not
feed, and there were other differences of habits and developmental rates, and
on the whole it was in many ways thoroughly isolated from its progenitors. All
of its character associations are the product of the act of origination, are in no
respect due to slow development or survival, and are entirely produced without
regard to utility in the form. Conditions of isolation seem to act in this useful
way only after they are fully established in the special life-relations into which
the processes of production have by chance happened to place it. It is not im-
probable that the same product under other conditions of life might well have
the same attributes and qualities in all respects, which might not there serve at
all in the capacities to give the degree of physiological isolation that the observed
series showed.

What constitutes fertility or infertility is clearly and only the capacity for
interaction of the gametes and inhibition of the parents from breeding, both of
which are the direct product of factorial composition, method, and rate of reac-
tion of the combining gametes. Whatever there may be of inhibition in repro-
duction is, in some instances at least, solely the indifferent results of the process
of origination ; but how generally this is true of the conditions existing in nature
is and must remain entirely a matter of opinion and interpretation.

This is not the only instance in these experiments in which the problems of
fertility in species crosses has been elucidated by data derived as a by-product of
other work. In the crossing of undecimlineata and signaticollis, as described in
Chapter IV, there arose under certain conditions a race that was exceedingly
slow in its developmental reactions and equally long drawn out in its life-cycle.
This race was produced by the single series of interactions of the cross as a group,
with all of its attributes and qualities present and formed by the reactions pre-
ceding its appearance in an adult F^ array. It was, as showTi by the test, of a
heterozygous nature and could by proper means be broken up, but in the usual
conditions of life it was as stable as the majority of species in nature. It would
breed, as far as the act of copulation was concerned, with either of the parent
species, but the different rates of development, or something that was the product
or associate of this difference, produced a huge mortality in the progeny of these
crosses. In this respect it is isolated, and while it might go through the reac-
tions of breeding, the differences in rate of reaction in the gametes, or factors
associated therewith, directly eliminated the probability of more than sporadic
or small numbers of progeny.

This race had another interesting aspect of its composition which in the con-
ditions of culture was of no moment, but in nature would have been in the form

176 The Mechanism of Evolution in Leptinotarsa

of an added agent, isolating it from its relatives in breeding. It was the uni-
form experience with this stock that it came out of aestivation 1 to 3 weeks earlier
than either of the parents under the same conditions. This was uniformly true
where all had gone into aestivation in the early winter months voluntarily ; under
identity of conditions this special race was out first and well started in breeding
before the parent lines had started into action. Had this been the reaction in
nature, and I can discover no reason why it would not, the new race would then
be out and breeding in the spring before the others had emerged. Trivial con-
ditions of this sort in the composition of organisms are productive of not a little
of the separation of natural groups of organisms, and in the main it seems most
probable that characters of this sort have arisen as the result of the product of
the reactions productive of the origination of the group rather than in the indi-
rect ways of survivals and slow accentuations through long periods.

In these experiments I have in the main eliminated, by the method of work-
ing, the sterility that comes through nonbreeding as the result of new conditions
by the provision of conditions such that the stocks from nature could breed in my
laboratory, and although there is much in the eifects of these conditions of life in
their action upon the reproductive activities, I have eliminated as much of it as
possible, my operations being adequately complicated without the addition of
this group of factors. There is abundance of evidence that this relation to the
conditions of the medium are vastly important and govern the breeding activities
of all organisms directly in nature and in experiment.

Probably the most common record in the accounts of the crossing of species is
that of the sterility of the F^ progeny and the inability to obtain Fg progeny of
crossings back upon either parent form. It has been discussed adequately in the
literature; tests by breeding and examinations of the germ-glands have shown
that there are slight or extensive derangements of the gametes, and there the
entire matter rests. In my experiments I have had but few instances in which
if Fi progeny were produced they were also not able to produce Fg and subse-
quent generations. It was found in the peculiar race of undecimlineata men-
tioned in preceding pages that often, when it had given progeny in crosses, it
gave no second generation when these F^ were inbred. No reason could be dis-
covered except that often the males or less often the females would not develop
any mature germ-cells. Examinations showed that the gonads were not active
and were producing only a few or only defective germ-cells. In normal mate-
rials similar individuals are also found which have a relation to lines of sterility
in the race. In these hybrids the reactions were the same and the defective
nature of the gonads much like the conditions in sterile individuals of normal
stocks. There the matter rests for the present. I have no means of determin-
ing what the sterility is due to, whether to disease, inheritance, or incident
agents, but these cases are sterile independent of any conditions in the medium
that I have been able to discover.

It is a constant experience in the crossing of these species by close attention to
the conditions of life, as temperature and food relations, that in the production
of F2 there are effected many times in such production a distinct increase in the
fertility, and often in practice the obtaining of F^ has depended upon these nice-
ties of adjustment in the conditions surrounding the organisms. One is led to
wonder to what extent the recorded sterility of F^ hybrids among animals,
especially in the crude conditions of zoological gardens, may not in reality be

Reactions and Products in Inteespecific Crosses 177

due to this cause rather than to the defective reproductive apparatus. Nor is
the fact that the gonads on examination have not produced gametes proof of their
sterility, because I have seen examples where there was not the least activity
until I had made some trivial change in the environmental complex. The repro-
ductive process is so delicately balanced in the organism, so tied with diverse fac-
tors within and without, that the problem of sterility presents endless complica-
tions to the experimenter.

In this investigation fertility and sterility have been only incidentally studied
in the practical aspects of the breeding operations ; nevertheless, as a by-product
from these routine crossings of species, data are derived showing that the prob-
lems of this character are entirely matters of factorial causation and that the
production of conditions of fertility or intersterility are the direct result of the
reactions incident to the origination of the stable type, which, if it were found in
nature and first seen by the taxonomist, would have been a species. Isolation in
reproduction and intersterility have long been and still are by many considered
the distinguishing mark of species, but I can not see any a priori reason why this
is necessarily so. Specific distinctness in the non-living is solely the possession,
by the contrasted materials, of constant and characteristic attributes and quali-
ties, manifested to our perceptions, such that they may by us be separated one
from the other. They may be specifically distinct, yet interact with violence, ease,
or not at all. The capacity for interaction is not a criterion of specific distinct-
ness. In organisms fertility or sterility in its many manifestations is only the
capacity of the specific masses to react, and the mere fact that they act readily
and uniformly when combined, with difficulty, or not at all, has no bearing upon
their specific distinctions, which in each are proper thereto and constant at all
times, irrespective of the capacity for interactions. Enough has been presented
at this point to show the trend of ideas with reference to these problems and the
substantial support that comes to them from these experiments in the crossing of
species. Interspecific sterility in animals has been regarded as an indelible char-
acteristic of specific distinctness; but as Darwin was forced to admit, and all
since have seen, this was not universally true. Darwin, however, thought that it
was true in nature, but could be removed by domestication. Cultural conditions
can alter the factors concerned in the production of sterility, within the limits of
fluctuation proper to the species, not beyond, and further change must come as
the result of the product of the reactions of interacting gametic systems, wherein
the factorial nature of the system is itself changed. I have reared diversa and
decemlineata for many generations and seasons in the same conditions, but the
degree of fertility is no higher now than at the beginning. Others have changed
within the limits of fluctuation of some specific and efficient factor, not beyond,
and perfect fertility is not in experience produced in any instance by the condi-
tions of culture.

THE PROBLEMS OF THE NATURE OF SPECIES CROSSES.

De Yries has clearly crystallized the general opinion so long prevalent that the
crosses of species are different from the crosses of varieties in principle, in his
now familiar statement that there are two main types of crosses — the bisexual or
Mendelian and the unisexual — in which the chief criterion is that the bisexual
gives in Fg segregation into different groups with interchanges of the character-
istics of the parents, some of which breed true as varieties, while the unisexual do

178 The Mechanism of Evolution- in Leptinotaksa

not segregate, but are fixed from the start. This experience is projected back-
wards from that point to a hypothetical difference in the gametes, which is not
put to test in the effort to discover whether it is capable of dissociation and thus
determine its nature. Uniformity of action in succeeding generations has been
the test of the constitution of organisms in the past ; but, as is increasingly shown
by the work of the Mendelian hybridologists this is not true, and in this work the
crossing of species has shown us both of the main sorts of crossing reactions that
De Vries and the older authors distinguished in the work of hybridizing
organisms.

In the course of this investigation I have had under cultivation and crossed
many species from nature, and as the collective result of this experience, some
of which is given here, I must conclude that there are not two main kinds of types
of crosses, but that in the crossing of organisms there is evidence of only one
type of reaction, namely, that the union of the gametes in fertilization results in
the interaction of the two gametic systems in F^, in such fashion as their struc-
tures and compositions permit, to produce the resultant adult zygotes, and which
in gametogeuesis produce, through the action of the two intermixed gametic
systems, separations of these with or without dissociation and interchange of
larger or smaller associated groups of gametic factors.

Examination of the data presented in the description of the different crosses
shows, as for example in the simplest series, the crossing of diversa and signati-
collis, that from the same parent stocks different types of reaction were obtained ;
sometimes the reaction was such that it would be described as the bisexual, at
others it was only the imisexual, and at still other times it was intermediate.
The parents are quite distinct, do not live in like habitats, and are not completely
fertile, but their interfertility is easily altered within limits. It is shown in
these experiments to what the mechanism of this difference in the reactions of
crossing are due and hoAv they may be produced in experiment. It is further
shown that the pure-breeding lines that arise in F^ are capable of being in some
instances broken up and their composition determined, showing that they were
in actual composition heterozygous, but through the action of the gametic agents
the relation of the factors in the gametes was so altered that it persisted, giving
the appearance of a pure race, because its successive generations were alike when
bred on without test. Proper conditions readily dissociated it, showing its heter-
ozygous nature, which was also strongly indicated by the trimodal polygons of
the fraternity. In species crosses in which the two or more types of reaction are
observed, if when analyzed show that the principle of reaction is the same in all
aspects thereof, we may well speculate as to the probable nature of many untested
series in which the only test applied was that of their " breeding true." The
point has been reached where the only indication that " breeding true " gives is
that it tells little of the composition of the line that breeds true, and as a result I
have distinguished in this work very carefully between homozygous in gametic
constitution, and homozygous in actions. The two may or may not be present
in the same line.

In these experiments many lines arise that are heterozygous in composition,
but have been homozygous in action, and it has been shown that some of these
can be proven to be such by the proper tests, either complete or in part, while
a few lines have with present methods and knowledge resisted the effects to
dissociate them in tests. These lines, which have resisted the efforts to dissoci-

Keactions and Peoducts in Inteespecific Ceosses 179

ate them, are of the nature of what has been called fixed hybrids, numerous
examples of which have been described in the literature, especially that of horti-
culture and in the older writings. There would be little question as to the
stability of these types and of their position in nature, and no doubt they would
receive at the hands of taxonomists the designation of species. However, it is
sho^vn in the examples that have come up in the course of these experiments that
they are heterozygotes, of such arrangement of the factorial agents that the
separation of the two gametic systems in F^ gametogenesis is either obscured as
in the fixed heteroz}'gous lines arising from the crossing of signaticoUis, diversa,
and imdecimlineata, or suppressed, as in the products of the cross of diversa and
deceinlineata. As far as the evidence shows us this diversity is purely a result
of the factorial action, within and without, that is productive of these conditions
in the crossing of species.

An interesting series of data are derived from these species crosses, showing
that the ]\Iendelian reaction, which is so universally present in them all, is a type
of reaction that is entirely independent of the size of the factors that are trans-
posed between the two crossed species. It is interesting to find, as in the cross
of diversa and signaticollis, when the Ac values are similar, that the reaction
in crossing is in all respects most accurately and typically Mendelian in its
showing of F^ and Fo arrays ; but more of interest is the action of the entire
gametic complex of each of the parent species as a unit in the operations, giving
the most perfect monohybrid displays. There is no dissociation from the
masses ; each in all experiments retains its integrity ; and the resultant reaction
is of the purest monohybrid type possible, and complications come in only when
the parent stocks differ in the Ac values, which differences produce not dissoci-
ations but derangements in the nature of the F^ heterozygotes, giving arrays that
might easily lead to false conclusions, as the nature of the gametic constitution
of the parent stocks.

In more complicated crossings, as in that between signaticollis^ and undecim-
lineata, the results show uniformly the retention of the integrity in the main of
each of the gametic systems, but with some dissociation of certain juvenile char-
acters of color and pattern that are transposed in F^ gametogenesis, giving the
expected chance array of the F, combinations of different degrees of stability,
precisely following theoretical expectation on the basis of the trihybrid Men-
delian reaction. Also, there arise the same general type of complications from
differences in the Ac values of this cross. Between this cross and the preceding
certain important differences can not fail to be noted.

These two crosses slioiv that the Mendelian reaction in type and in operation
is independent of the character of the substances involved, and this is shown
clearly by the action in the first cross of the total gametic system as a unit, and
in the second by the action of the larger portion of each of the two systems as
units and the simultaneous action of two portions of them also as units. These
two dissociated portions — the color-determiners and the group of agents produc-
tive of the larval pattern — are not of the same nature or extent. One is probably
a single a2:ent. not capable of present change or di>^sociation into smaller agents,
and this is the condition of the yellow-color-determiner that is dissociated and
recombined. The larval pattern is known to be a system of minor agents that
collectively act to produce the pattern, a conclusion that is warranted by the
capacity shown for alteration of this pattern and the removal of specific portions

180 The Mechanism of Evolution in Leptinotaesa

thereof, the presence of which is dominant to its absence in typical Mendelian
reactions. In this species cross the typically Mendelian reaction, trihybrid, is
entirely a product of the number of the dissociated groups, and independent of
the character or size thereof.

In the crosses between the species decemlineata, oblongata, and multitcjeniata,
another aspect of the same problem appears, namely, the complexity or simplicity
of the reaction depending upon the complexity of the gametic constitution, as in
the cross of decemlineata xmuUitceniaf a, where factors added to the complex
also added complexity to the type of reaction resulting. The cross of decem-
lineata and oblongata, however, showed still other conditions in the extensive
dissociation that followed the combination of these two in a hybrid reaction. In
decemlineata xmultitceniata the modal biotypic conditions in homozygous lines
reacted in strict monohybrid reactions ; added factors to either disrupted this and
also induced dissociation in the gametic complexes ; and so through the entire
series it is uniformly the result that the reaction which follows the combination
of these species gametic systems is the direct product of their differences, and the
dissociation resulting depends upon the number of equivalent factorial groups
capable of metathesis. This sequence of events following the cross we may call
the Mendelian reaction and not Mendelian heredity.

The chief point that I desire to make in this connection is that in the crossing
of species direct from nature there is but one fundamental type of action,
which depends entirely on the factorial constitution of the gametes combined,
multiplied by the conditions of the medium at time of combination, and that
the resultant reaction is the direct product of their differences and the capacity
for metatheses between equivalent factorial groups. If the reaction proceeds
under conditions in the medium that are common to and act in like manner upon
the two systems, the reaction and products will appear in proportions and com-
binations that are the product of chance, but if the conditions of the medium
act to accelerate action in one gametic system or to retard it more or less than in
the other, then the dissociation and combinations are disturbed and the result-
ant array of products is entirely dependent upon the nature and action of this
active agent in the medium. This Mendelian reaction is our first and at present
most important means of investigating the gametic constitution and action of
organisms. It is not a law of heredity; it tells usnothing of the way characters
are maintained through countless generations of similar organisms nor why
characters exist at all ; but it shows a first method of investigating the factorial
agents in the gametes that are, in ways at present imknown, concerned in the
production of the characters in endless repetitions.

But once for all let us discard the notion of characters and of kindred concep-
tions, and recognize that the presence of a character in an organism is there as in
the inorganic, the end-result of the interactions of the component non-living sub-
stances, and so the physical basis of these characters that are used here as mere
indicators of the presence and type of reaction present may be only HO groups
in the material substance that bridges the gap between generations. We do not
know the exact agents that produce these end-results, but their precision of
action, and the certainty of their duplication, times without number, in the same
line of living beings can admit of no other viewpoint than that the production of
a character is, in organisms, the same resultant of the physical interactions of
the materials of composition, and due to the same precision of process that pro-

Eeactions and Products in Inteespecific Ceosses 181

duces blue rhomboidal crystals when CuSO^ and SHgO are combined. Not that
it is the same, but that it has the same physical principles of operation and causes
of the resultant characters, which are after all only temporary states of stability
in the ceaseless integrations and dissipations of matter that characterize the
progression of organisms in nature.

In this crossing of species from nature I have presented for tlie most part only
data that tested to the limit of present operations the truth and application of
the Mendelian reaction. I have found no blends, no instances, totally at vari-
ance with the principles of action of this test of gametic composition, and while
it is true that there remain in this account lines that have arisen whose entire
composition has not been entirely solved, or whose production is not entirely
clear, still no instance has been found that has not at least given exact evidence
that the Mendelian reaction and the principle of gametic factorial agents are not
present and operating, even though at present obscured by unsolved portions of
the problem. This latter is, however, open to continued test of experiment, and
empirically it is possible to progress with certainty in the solution of these com-
plications.

The literature is abundantly supplied with instances of the rise of pure-breed-
ing and stable races as the product of an initial-species cross, and in them the
fact of their pure breeding in following generations has been the criterion of
purity, blending, and kindred assertions concerning them. Many Mendelian
workers, Bateson especially, has expressed doubt of this being true, and while
it may be true in some instances, I am at present not informed of any instances
where the testing of them has been even a tenth part as rigorous as that given
in similar lines in my experiment; and so, on the basis of experience, in this
series we may well ask that there be other tests applied than those of " breeding
true " and of the simpler routine Mendelian reactions.

In my experience this Mendelian reaction has thus far stood the test of the
most hostile treatment I could give it, and everywhere it has proved to be certain
and precise, correct, and when one dissociates it from misconceptions and the
drag of biological units it takes on a new significance and in investigation
becomes one of the most important tools of progress that we possess to-day. The
probable nature of this reaction is discussed in Chapter IV.

THE PROBLEMS OF THE CROSSING OF SPECIES AND SPECIES PRODUCTION.

In spite of assertion that the problems of evolution are those of character
origin, it must still be admitted that the problem of the origin of the groups in
nature that are called species is a real problem for investigation. The older
writers upon this problem have interpreted and argued the situation over and
over again, with the well-known results that the problem of the origin of these
groups is by no means solved, nor is the probable mechanism of production much
more than a matter of opinion.

Considerations of origins have largely centered about single individuals as
the progenitor of the group, or at least a pair, in the thoughts of those who con-
ceived of species rise through saltations, while the separation or differentiation
of a group by slow changes remains largely a pure speculation. It is not at
present possible to decide how any given species in nature arose, nor how long
it has existed, aside from the evidences derived from fossil remains, which are
on the whole fragmentary and insecure. All that is possible is to discover

183 The Mechanism of Evolution" in Leptinotaesa

methods by which groups might originate in nature, and if possible detect or
produce such origins in experiment in nature.

The idea of the role of crossing in the production of natural groups in a state
of nature as an agency of any importance is of recent origin. The older writers
admitted the possibility of crossing in nature, but, following Darwin, thought of
it as incidental to other operations, often as the product of a destruction of the
barriers of interspecific sterility, while in the minds of all the main type of
production has been that which was the product of dichotomy of some ancestral
line, either by slow or rapid processes of separation. This conception is retained
in De Vries's theory of species production, the separation being one of rapidity
instead of by slow transformation. Since the rediscovery of the Mendelian reac-
tion and its verification in numerous instances, the experiences and expressions
of opinion have been increasingly towards the idea that it may be possible that
species in nature are of hybrid origin. One of the first of the modern writers to
express this opinion was Morgan, and it has been raised also by others, while the
findings of many observers have shown that crossing is going on in nature
between natural groups, but to what extent no one has any information.

The great problem in all species origins has been that of the rise of the initial
group, and the discussions have to considerable extent centered around the fate
of the first ancestor and what was necessary for this lone individual or pair to
encounter or overcome in order to survive and produce progeny in the location.
It seems to me that some of the results of these experiments in crossing species
give what may be a method of the origin of groups in nature which under the
conditions of existence would breed true without limit and which might rarely
throw a sport as the result of its hybrid nature and be in all respects a true-
breeding species. Any one of a number of the pure-breeding fixed heterozygous
lines that have appeared in my cultures would have survived in nature and gone
on as effective members of the fauna of the location in which they arose. This
appears as a possible method of origin ; I do not have any data as to how probable
it is, nor do I know of any method of deciding whether species in nature have or
have not arisen by this or any other method. This type of origination seems logi-
cal and suggests that it is of common occurrence in the production of natural
specific groups.

Precisely the same general type of sequence of events is productive of much of
the specific diversity in the nonliving series in nature ; the specific thing, com-
pound, mineral, or complex rock is the immediate product of the reactions of the
interacting combining materials or factors of composition, and the product
appears fully developed and in amount corresponding to magnitude of the oper-
ations. If organisms are complex compounds whose operations are purely phy-
sical in nature, I see no a priori reason why in the origination of specific organ-
isms the process should not be in the main of the same order, and its rise follow,
as a group of varying size, entirely dependent upon the magnitude of the origina-
tion process. In this way it is possible to conceive of and possibly produce
experimentally the rise of groups in nature by processes akin to those I have wit-
nessed in the laboratory, giving pure-breeding, distinctive, physiologically iso-
lated groups. The conception and experience is interesting and worthy of
extended test in nature and in laboratory experiment. That it is not an idle one
is fully shown by the products derived in the experiments described in the preced-
ing chapters.

Eeactions and Products in Inteespecific Crosses 183

I have given data iu the preceding pages to show that these species, of which I
have made use in my experiments, are in no respects special cases in any way, but
that in the principles of reaction in crossing they show the same phenomena that
De Vries so clearly puts into two categories, which without extensive testing
appear to justify this distinction ; but in the examples that I have had to test
there appears no true distinction. Breeding true is not a test, and instances that
have been classed as unisexual must, I think, be adequately tested before it be
admitted that they show a special type of reaction. Throughout, the crossing of
species is fully in accord with the principles of factorial composition and action,
and in this the Mendelian reaction appears most prominent as a method of
analysis.

CHAPTER VI.
THE PROBLEMS OF HETEROGENEITY.

INTRODUCTION.

In Chapter I it was shown that the characters of organisms are grouped under
three chief categories: the specific properties belonging to the specific living
kind, which can not be altered without change in the identity of the kind;
attributes, belonging to and distinguishing bodies of the same kind from one
another; and conditions, or states of being or activity which can be changed
or removed without altering the identity of the body or its kind in any way.

Since organisms have specific properties, attributes, and conditions, and
because evolution is entirely dependent upon change in the specific properties
through transmutation and secondary adjustments in the attributes and condi-
tions, it is important to understand what transmutation signifies and to dis-
cover as many of its modes of manifestation as possible before attempting to
produce new states or to recombine existing states into new kinds of living
substance.

It is essential that by transmutation should be understood only those differ-
ences giving permanent change in the specific properties. Many of the differ-
ences found in any population — " variations " in the older terminology, and
which may give permanent changes which may be factors in evolution, are pro-
duced by metathesis through the rearrangements of the factors productive of
organic characteristics by the interbreeding of two more or less unlike types.
In this reaction, while differences in bodies and in substance may result, it is not
transmutation but heterogenetic rearrangement, which may or may not be
accompanied by transmutation, " Variation " in the older usage is synonymous
with the two divisions recognized here — i. e., the transmutation of qualities and
recombinations of existing characters into new complexes. The first is transmu-
tative heter agenesis, or the origin of new factors, the latter metathetic hetero-
genesis or the rearrangement of existing factors.

In nature, these two phenomena are continuously associated and active in the
production of the diversity of specific natural forms. It is necessary to untangle
the relations and interactions of these two methods of producing diversity before
proceeding with the problems of experimental evolution. All too often " vari-
ation " has been thought of as a process ; it is in reality devoid of unity of cause
and presents unity only in the diversity of end-results, a fact most clearly stated
by De Vries.

If further progress is to be made in evolution investigations, it must be known
what the conditions of organisms in nature are, and these must, as far as possible,
be analyzed and determined by the methods of modern experimental
analysis in the laboratory. The latter will give us a glimpse of what may happen
in the population observed, and will give accurate knowledge of the way recom-

184

Problems of Heterogeneity 185

binations are made and how transmutations may arise. The census-taker and
field naturalist will see one side of the problem, the laboratory student another ;
both are right in their frequently hostile utterances, but both partly wrong.

In the making of a census in any population these main facts are desired : the
number showing a particular character; the amount and permanency of the
characteristic shown ; the minimum, maximum and mean deviation presented ;
the direction and, if possible, the cause of the condition.

An entirely false conception of the nature and role of small fluctuations has
arisen through the uncritical use of biometric methods, and while it is undoubt-
edly true that many characters, as for example the variation in any meristic
series, do follow the law of probable error in their measured dimensions, is it
certain that the differences in number or dimension are the essential features of
the heterogeneity ? These numerical differences, after all, are but the result of
operations not discoverable or measured by the methods of the census-taker.
Moreover, in color-patterns in animals or plants the amount of the color present
may be measured in terms of the area exposed, and from this data the biometri-
cian then proceeds to an exhaustive mathematical analysis in which the result, if
any at all, is mathematical and not biological.

The biological facts of the arrangement of the character in the organism, the
relation of this to surrounding conditions, and its effects as it varies upon the
welfare of the individual, are points which may be of paramount importance;
but with these the statistical method does not and can not deal.

PROBLEMS OF HETEROGENEITY.

In the literature on " variation " confusion reigns supreme — dogmatic defini-
tions, anticipatory assertions, arguments without point ; it is small fluctuations
with preservations per utility and described a la biometrics, or it is disconti-
nuity per saltum preserved by Mendelian recessiveness or dominance, or it is
determinate variation ever in line conditioned by organic growth or by ortho-
genesis repeating ancestral states and adding new ones, which are held in line by
internal automatic regulating and determining tendencies; or again, it is an
inherent tendency to vary determinately, indeterminately, or per saltum, and so
on ad infinitum. In reality there are many processes that are involved in this
confusing mass of phenomena which are called variation.

In any population two general sets of operations are interacting. There is the
action of the total population as it encounters the conditions of existence, the
way in which it responds to its environment and in divers ways meets incident
physical conditions and reacts to social relations of the society of which it is a
member by population responses. Opposed to this is individual action tending
to diversity, frequently to extremes. Both are but parts of the same process;
only in the mass action of the population there is obtained a view of what the
final effect of the innumerable processes which went on among the individuals in
the production of the generation is upon the population in each generation.
From the individual is obtained an idea of the methods of interaction, but this
could never give complete knowledge, even in a limited and simple population.
So, therefore, for the present at least, the general action of the population must
be considered until it is known more completely what the mass effects may or
may not accomplish in organic evolution.
13

186 The Mechanism of Evolution- in Leptinotaesa

Darwin, in the Origin of Species, conceived of heterogeneity in a far less
refined manner than that current now. Eecognizing that individual differences
were numerous and in many instances showed intergradations, but that spontan-
eous departure or sports were also found, he expressed his inability to draw a
hard-and-fast line between them. He further saw clearly that the nature of the
organism and the nature of the conditions were always concerned in the produc-
tion and determination of the directions of variation, and the effects of these
essential factors in the cause of variation were clearly to produce two kinds of
" variation " response, definite and indefinite. " Definite variation " is variation
in which all, or nearly all, of the individuals in a generation living under the
same conditions of existence varied in the same way or direction, and " indefinite
variation " was the condition where the individuals did not vary in the same,
but in many directions. Definite and indefinite variations are clearly always
mass action, and continued definite variation may result in decided change of
the population in one direction, while indefinite variation might equally well
result in change, but there would be diversity in the population. Darwin applied
this conception of the response in heterogeneity to the departure of a single char-
acter or a group of characters; to the individuals of a local group or to a
species as a whole. I believe that this recognition of the method of response in
a population is too mtich neglected and that it is of vital importance in evolution
in nature. Darwin's exposition of it leaves neither possibility for misunder-
standing its character nor reason for not testing its importance as a transmuta-
tive factor in nature.

Since Darwin, the same two words are often used to designate two entirely
different conceptions with respect to variation. Many of the neo-Darwinians,
the Lamarckians, the followers of Eimer, and especially workers in the lines of
paleontology, comparative anatomy, and descriptive ontogeny, have used definite
and determinate variation as interchangeable terms to express the idea that
" variations " are limited to narrow lines in the individual and in the phylogeny
of the race. This use of the term is either for a particular character or for the
entire individual. The converse of this concept is expressed by indefinite or
indeterminate variation.

These involve two fundamentally different concepts, intended to describe, first,
the response to the cause of diversity in the species as a whole or in some isolated
portion thereof; second, the limitation of the direction of the response in the
individual or in the whole population, in particular qualities, attributes, or con-
ditions. To anyone who is at all acquainted with the conditions of organisms in
nature the proposition of Darwin is evident and needs consideration and investi-
gation. Moreover, it is not to be ignored in the consideration and construction
of evolution theories, although far too much neglected of late years. On the
other hand, the concept that is expressed by definite or determinate as meaning
the limitation of departures in the individual or in the race, or as designating
trends of evolution in larger or smaller systematic groups, is a no less real and
certainly known condition, regardless of what its explanation may be. In these
reports I shall use the terms definite and indefi,nite variation to designate the
method of response as described and used by Darwin, and I propose and shall
use in this work the term delimited to designate those instances where the modi-
fications resulting are limited to certain restricted paths of change ; that is, they
are departures whose direction is marked out by physical limitations, impossible

Problems of Heterogeneity 187

combinations, in the physical material itself. It must be understood that these
terms are purely descriptive, indicating only an observed condition in the hetero-
geneity present in species.

Any particular instance of determinate change may be so because it is also
delimited, but not necessarily so ; because if determinate, the environment may
determine the response, if delimited, the organism. The same kind of organisms
might, if undelimited, exhibit determinate variation in different directions in
different portions of its natural habitat in a given character, while a delimited
character would show determinate variation always the same under different
environments. The effect which these relations produce in the population
responses of a species is truly important when one considers organisms in
nature, and is beautifully demonstrated in some of my experiments in trans-
planting populations into new environmental complexes. The earlier writings
of the Darwinian period much emphasized these variation phenomena, but made
no experimental test of their effects.

With the development of modern methods of genetic analysis, attention of
necessity was more and more focused upon the individual and upon the char-
acters thereof, so that now our problems are almost entirely those of the individ-
ual and its characters and not so much the mass action of the population.

In the development of the mutation theory, De Vries created added compli-
cations in the problems of heterogeneity. The familiar assertion that there are
really two distinct kinds of variation — mutations (sports) and fluctuations —
seems an attempt to separate by definition the two extremes of " variation "
which Darwin recognized but was unable to separate. If mutations are the salta-
tion-like changes by which a new unit-character is added or an old one taken
away or made latent or recessive, and these steps are multifarious in direction,
and if the ordinary " variations '' are quantitative, adding to or subtracting
from the character in question, then all such variations are always in line, like
the swing to and fro of a pendulum, and this is of necessity made so by the
definition and the method of description which is mathematical in terms of
measure. Associated with this idea is the further assumed difference between
the two kinds of variations — that mutations are qualitative and fluctuations are
quantitative.

The sharp distinctions made by De Vries have seemed to many a questionable
subterfuge by which the unit-character concept becomes more clearly differen-
tiated, and of the two kinds of heterogeneity one, fluctuation, is rendered inoper-
ative in evolution, while mutation bears the entire burden of evolution change
and is due to transmutation from present to subsequent unit-characters. Even
though this proposition could be shown to be true, it does not follow that fluctua-
tions in a unit-character are always plus and minus, and that the mutations of a
unit-character are always multifarious. If there are unit-characters, as con-
ceived by De Vries, each must have relations in space and in interactions, and it
is hardly to be supposed that a unit-character is so fixed, so stable and invariable
that no matter where placed it is only a little smaller or larger, depending upon
the conditions of growth present during its development.

According to Weismann, all variation is ultimately quantitative, due to an
increase in the number and vigor of particular determinants, which increase is
due to quantitative increase in the determinants. If this be true, it follows
that the difference between species, or between their characters, are quantitative.

188 The Mechanism of Evolution in Leptinotaesa

whereas it has generally been the opinion of biologists that the essential differ-
ences between specific living substances are of a qualitative nature. This is
important because, if true, quantitative accumulations ought in the end to pro-
duce diversity of the kind spoken of as qualitative differences. It raises the
question, can quantitative accumulations in the parts produce resultant qualita-
tive differences of the whole ? This is a problem well adapted to exact experi-
mental test ; that is, can the accentuation of an attribute produce a change in
the quality of which the attribute is a mass manifestation ?

The quantitative concept implies change in the amount of something, with the
retention of the same spatial relations ; the qualitative implies the establishment
of different relations from those hitherto existing. This concept can not be ap-
plied at present to the conditioning factors in the germinal material, but it is
possible to determine whether the variations are mere plus or minus differences
in quantity or material or activities, without reference to spatial relations, or
whether the common fluctuations are small differences in the spatial and other
relations of the character in question.

The discontinuity which species in nature show is, by this conception, ex-
tended to the characters of which organisms are supposed to be composed;
whether there is discontinuity between the unit-characters became, therefore, a
problem for solution. Because many species show in nature a certain discon-
tinuity in that there are no intergrades, it seems more reasonable to some
(Bateson, De Vries) that they arose in that way and that there have never been
intergrades, while the fact that there are in nature species groups with inter-
grades seems to others evidence to maintain that all species groups have arisen
through the gradual separation of groups and the extinction of intergrades. In
the same way the variations are far too often classed as continuous whenever
intergrades can be foimd and as discontinuous where such are wanting. As a
matter of fact, the existence or nonexistence of visible intergrades is of no
importance; only intergrading gametic conditions as determined by breeding-
tests are of interest. However, in nature the variation conditions in a popula-
tion are always confused, and mere somatic aberrations, rare chance reconstitu-
tions, and true transmutative variations may easily be confounded and classed
as mutations and as discontinuous in character.

Another point upon which there is difference of opinion is raised by the asser-
tion that the only safe basis of study in variation is the " unit-character," mean-
ing thereby some attributes which, as far as is known is not divisible in the
organisms, but is present or absent in its entirety. To some these attributes
represent the initial unit of structure and activity in the organism, and are,
therefore, the only basis from which to proceed in the analysis of organic phe-
nomena, while others hold that the problems must be considered from the point of
view of the whole. Naturally these two bodies of workers will arrive at quite
different end-results, and not infrequently will fail to appreciate the results
obtained by each other. This situation results, naturally, from the difference in
the initial philosophical starting-point, and if one's conception of organisms is
of the unit-character mosaic variety, unit-characters of necessity become
supremely important in the constitution, evolution, and activities of organisms,
while if the individual is considered as entity, then all too often that point of
view considers only the sum total effect produced on his senses by the particular
organism, and his statement of the conditions becomes a sort of impressionistic

Peoblems of Heterogeneity 189

one, in which the values and behaviors of the individual qualities and attributes
are lost sight of or ignored.

In heterogenetic phenomena, as elsewhere, the unit-character conception seenis
unnecessary and a cumbersome method of reasoning, and it is essentially vitalistic
in its conception. In physical phenomena specific properties are present or
absent, are changed with sharp alternativeness, and no need is felt of either such
concepts or for inventing mythical pangenes or biophores. If such concepts are
not needed in physical science, why introduce them into biology ?

For the purpose of this paper I shall consider first those attributes which, as
far as known, are not capable of being divided into lesser ones, or simplest char-
acters, and from these shall proceed to the consideration of characters of increas-
ing complexity. It must be understood that this method of attack does not
imply in any degree a preference for any current hypothesis. I shall begin with
the simplest characters that can be certainly and generally recognized. The
criterion for the recognition of this condition has already been stated, and while
the criterion will always remain the same, the condition it defines will change
from time to time. A name is needed for the conditions from which I start,
and throughout this paper I shall call them the " simplest characters," implying
no more than the idea already expressed, that they are the simplest conditions
that can now be certainly determined and profitably studied among the multi-
tude of specific properties, attributes, and conditions presented by organized
bodies.

The development of the "pure-line" hypothesis of Johannsen introduces
further complications into the problem. A basal unit or " gene " comparable
with " unit-characters," " unit-factor," " element," " allelomorph," and so on,
and mainly an expression of the results of the experience of neo-Mendelian
hybridology, is conceived of as having a fixed value or manifestation which
behaves as a constant factor in organic evolution. The combination of genes in
the organic form into a harmoniously acting state of stability, a genotype, is
essentially of the same order as De Vries's conception of the constitution of
species by combined elementary species. The isolation of genotypes by Johann-
sen and others, of clones by Jennings, leaves no doubt that there are in
the population states of stability which can be separated and maintained as pure
homozygous-acting strains. These are always to be distinguished by pedigree
breeding methods and are different from " phenotypes," which are separated by
purely descriptive methods and may or may not represent " real things."

The genotype conception, while it expresses many experiences in language that
is not liable to be misconstrued, nevertheless introduces complications. Opinion,
supported by practice in line-breeding, has rapidly established the view that
genotypical constitution changes by jumps alone, without intergrades, and that
the obvious and constantly present overlapping of genotypes when isolated is
only the " fiuctuations " of the genodifferences present. The added current
belief that no selective accumulation of the fluctuations of the differentiating
genes can alter the mean of the " gene," results in much of the work in selection
being interpreted as due to the isolation, by " selection," of " genotypes."

With respect to the " gene," as with the " unit-character " or the " simplest
character," the essential present need is more real knowledge of these characters.
It is necessary to determine whether their commoner differences are mere quanti-
tative oscillations about a mean, whether their rarer jumps to new states are

190 The Mechanism of Evolution in Leptinotaksa

jumps or not, and whether they are qualitative or quantitative in character. If
the gene be so independent and have a mean about which common differences
fluctuate, a different conception than is current of those of inorganic bodies
mu^t be formed of organic attributes.

Heterogeneity is, of course, the sole basis of evolution, is evolution at work,
and the processes which follow are entirely of conservation to preserve, rearrange,
and distribute among organic bodies the changed and new attributes presented.

CAUSES OF HETEROGENEITY.

The causes of heterogeneity are certainly diverse, but appear in two main
groups of causation: First, transmutation in the qualities with subsequent
adjustments in the attributes and conditions of organisms; and second, diversity
resulting from recombinations (metathesis).

The opinions concerning the causes of transmutation current with Darwin and
his co-workers placed much weight upon the conditions of life as efficient forces
for initiating change. This was, however, not much more than opinion and
not at all the product of exact investigations. The Weismannian hypothesis
also considered the conditions of existence as the essential force in the produc-
tion of change, but placed the process in a different setting ; but the principle was
the same — the stress of conditions produced either increase or decrease in the
qualities and attributes of the living substance. Some difference of opinion as
to how this came about existed, all of hypothetical character and not of any mo-
ment here. All saw that in some way there must come to exist in the germ
something to give expression to the changed character in subsequent generations.
Arguments concerning this method have in the main revolved around the
origin peripherally, and the aggregation in the germ, or of the origin directly in
the germ and subsequent manifestation in ontogeny. Opinions have differed and
still differ with respect to these problems, but in the last decade researches show
concretely that transmutations may arise through the direct action of incident
forces upon the germ, and that action upon the soma has not produced inherit-
able changes. These investigations serve in some measure to clarify a much-
befogged situation. Clear, consistent proof is provided of the alteration in the
germ of that something which conditions a character in the soma, and the evi-
dence available strongly indicates that the changes found are not the product of
a struggle between determinants, but changes which appear suddenly, are fixed
from the start, and are physico-chemical in character, and not tlie outcome of a
struggle for place and food between lesser organic units.

It is certain that the action of incident conditions is a potent force in trans-
mutation phenomena, but whether it is the only productive force remains to be
proved. Many are of the opinion that purely internal operations are in con-
siderable measure responsible for evolution, if not entirely. These automatic
internal conditioning and determining forces are dangerous concepts for the
biologist. If organisms are conceived of as automatic, internally regulated and
propelled mechanisms, they are the only self-contained mechanism in existence,
all others depending upon the interplay between the mechanism and external
forces for their operation. Even if it be admitted that organisms are automatic,
they would, unless possessed of an " inherent tendency to vary," go on in per-
petuity in the same form and condition. Inherent tendencies of any kind are

Problems of Heterogeneity 191

not at present worth consideration, and the attributing of changes to the play
between internal parts after all only changes the scenery and not the nature of
the play. That is, it is interaction between the something which conditions the
character and the materials or forces, or both, external to itself at the point and
moment of reaction.

It matters little at present whether I speak of the germ as a whole or of the
hypothetical lesser parts; whichever is chosen, the fimdamental operation in
transmutation is the interaction of the constitution of the gametic substance with
the conditions under which its activities go on. Two series of events are ever
passing — the antecedent genetic sequence of events in the material composition
of the gametes and the sequence of events in its medium ; at any moment the
constitution of the germinal material and its resultant soma are the product of
the dynamic interactions of the two. The two series of events in all instances
must be the prime factors in all transmutation phenomena, and they alone are
responsible for all transmutative changes of specific properties in organism. If
the two series of events are constant, there will be constancy of the resulting
organisms ; if the organism be plastic in its mechanism and adjusts itself easily
to divergent conditions, constancy with considerable adjustive oscillation will
result ; but if the organic mechanism be little or not at all plastic, divergence in
the conditions of its medium will result in adjustments between the mechanism
and its medium with change in the mechanism — a transmutative change.

Eegardless of what future advances may bring us, the dynamic conception of
transmutation provides an easily applied working hypothesis of the cause of
change and one that is best expressed at present, though imperfectly, in terms
of physical science.

ANALYSIS OF HETEROGENEITY.

One of the most decided advances of recent years is the unquestioned demon-
stration that any real analysis of the heterogeneity and any expectation of under-
standing these phenomena must rest upon exact experimental methods of investi-
gation. Biometric analyses, descriptions of phenotypes, have their place as
methods of preliminary calibration of materials, but further knowledge depends
exclusively upon experimental analysis. Experimental analysis, however, is apt
to be pointless unless the results can be directly applied to the understanding
and investigation of conditions in nature. Obviously it is neither possible nor
profitable to consider all the characters presented, and the results obtained from
a few studies will be presented, as illustrating principles common to all specific
properties and attributes of organisms.

In the study of heterogeneity of these qualities or specific characters I shall
begin with the simplest that I have been able to discover. ' These, while always
parts of a system, are specific in position, form, relations, etc. The differences
are often minute and would not ordinarily be used as " specific differences " by
taxonomists; however, in all respects they are distinct in character and alter-
native in behavior in crosses.

CHAPTER VII.

ANALYSIS OF HETEROGENEITY IN SOME SIMPLEST
CHARACTERS.

ANALYSIS OF HETEROGENEITY IN SOME SIMPLEST COLOR-CHARACTERS.

It is known beyond doubt that color characters are as fully " law-conforming "
in their origin, development, and behavior as structural or physiological
features.

In Leptinotarsa the color-patterns in the species utilized consist of two super-
imposed sets of pigments, one in the outer layer of the cuticula, the cuticula
pigments, and the other in the tissues of the hypodermis or immediately below it,
the hypodermal colors. The relations of these I have described in previous
papers. In the formation of color-patterns these two sources of color behave
quite independently of each other, the lipoid or hypodermal colors forming the
usual color-ground while the cuticular pigments may be either of general distri-
bution over the surface, in which case it may hide completely the lipoid color,
or in areas arranged in a precise pattern. The latter colors are always dark
browns or blacks, rarely light browns or dark yellow, and may belong, though
it is by no means certain, to the melanin series of pigments (Gortner), and
arise as the result of the action of an enzyme upon the soft primary cuticula, pro-
ducing the pigment and the hard outer surface of the integument. The color
areas are always developed in precise positions or centers of expression, which,
as I have shown, are as fixed in the constitution of the organism as are the rela-
tions and positions of nerves or muscles. Many of these areas are simplest
characters in that they are indivisible, are present or absent in toto, and invari-
able in position.

I have endeavored to discover what these characters can reveal in the way
of concrete information concerning the following problems :

In simplest characters are the differences :

( 1 ) Quantitative or qualitative in character ?

(2) Continuous or discontinuous, or both, and what is the relation of the

two?

( 3 ) Delimited or undelimited ?

( 4 ) Determinate or indeterminate ? In restricted localities and groups ?

In groups and species of wider range ?

a, in the adult. — The members of the lineata group, save one, all have a spotted
pronotum in which the colored areas are developed around a series of centers
shown in figure 12. I have elsewhere shown that these centers are invariable in
position, but that two or more of them often unite to form a larger color area.
Three spots, c, h, d, meet all the requirements of simplest characters in that each
is always present or absent in its entirety ; they are never known to break up into

192

Analysis of Heterogeneity in Some Simplest Chabacters 193

minor areas or component centers ; are common to the species in the group. To
varying degrees these areas enter into combination with other areas in the for-
mation of the general color-pattern, especially b and d, and these combinations I
shall consider later.

(1) Are the differences found in the simplest characters, c, d, and h, qiuintita-
tive or qualitative ? — The spots meet in a fair way the idea of unit-characters in
that they behave quite independently in many processes, and are either present
or absent in their entirety; as far as known are indivisible, and in heredity
behave as allelomorphs ; are present or absent, and the presence of one of these
spots in crossing is dominant to its absence, satisfying fully the conception of an
allelomorphic pair, but of the simplest kind. The characters meet all of the
concepts as to the nature of the most elemental manifestations of gametic consti-
tution as held by the neo-Darwinians. Their invariable position in these ani-
mals shows them to be characters whose positions and relations are, and have
been for a long time, conditioned in the germinal material. In every way they
are comparable to warts, wens, or other extremely localized attributes so often

k; k;k;a'" b

Fig. 12. — Showing the pronotum of the
litieata group in Leptinotarsa and location of
the different centers of color formation. The
designations are purely empirical, but are
those used throughout the text to denote ele-
ments of this complex system.

discussed by the Weismannians as certain proof of the existence of conditioning
entities in the " germ-plasm," and whose variations, which must form the basis
of their transmutations, are " ultimately quantitative."

The question whether the small variations of these characters are quantitative
or qualitative is far reaching and of fundamental importance in the analysis of
transmutation phenomena, and in heredity and evolution. Putting aside opin-
ions as to the manner in which the various attributes are conditioned in the
germinal material, only admitting that they are in some manner so conditioned,
the conception of Weismann that all variation is ultimately quantitative is
clearly shown in his statement in The Evolution Theory, vol. II, pp. 151-2.

The standpoint that there are two distinct kinds of variation in organisms
quite independent of one another, quantitative and qualitative, has been clearly
stated by De Vries.

The clearness of De Vries's statements and argument leaves not the slightest
room for doubt as to his position and his belief in the existence of two entirely
distinct kinds of variation, whose behavior centers around the unit-character.
The fluctuations of one of these characters must, by De Vries's proposition, be
plus and minus, always in line, the mutations in many directions or multifarious.

194 The Mechanism of Evolution in Leptinotaksa

The first De Vries considers as due in the main to the conditions of nutrition
or an amphimixis-like process, the second to processes at present unknown but
having no relation to the first. If the proposition of De Vries is true, then the
ordinary or the common intergrading differences of our " simplest characters "
ought to be all in line, and this would also be true if Weismann's proposition is
true, the chief point of difference being the part these might play in the evolu-
tion of organisms.

The first three " simplest characters " for examination, c, l, and d, are deposits
of pigments at invariable positions on this portion of the body; they are end-
products and as such are the visible manifestation of a specific property of the
entire mass in the same way as the angles in the faces of a crystal are specific
properties. Spots c, b, and d have position, dimensions, area, volume, and as a
consequence of position sustain exact relations to adjacent parts and characters
and present within themselves something of internal organization.

Fig. 13. — Showing variation in shape of spot c as it occurs in L. undecimlineata.
The figures represent examples chosen from many thousands of individuals taken
In successive years over an entire known range of species, but show diversity in
shape as well as in size that this area may present in this species.

In L. undecimlineata, spot c is sometimes absent, as in figure 13a, but in the
great majority of individuals it is present, ranging from the conditions of a min-
ute spot (figure 136) to even larger areas than are shown in figure 13d. In
any and all individuals the spot is present or absent as a unit, and never breaks
up into lesser centers of color deposition. A careful examination of the spots
found on the pronota of this species at the center (c) shows that they are not
all of the same shape, any more than they are of the same size. The difference
in size, as far as it is expressed in area exposed, may be a purely quantitative
matter, but the shape of the spot is quite another matter. Is this difference in
shape quantitative or qualitative ?

The simplest condition in which this spot appears is in the form of a small
round area of pigment (fig. 13&,) and from this as a center the spot pushes out
first in one direction, then in another, sometimes in two opposite directions
(figures 13/ and 13h), to produce an oblong spot; at other times it pushes for-
ward and outward (fig. 13^) and medianward at the same time, or it may push
out in four directions, as in figure 13y. It is not uncommon for the spot to grow
larger without development in any one direction, simply adding pigment equally
on all sides, as in figure 13d. It must be fully comprehended that these direc-
tions in which the spot extends are not in the least purely chance directions, but
are firmly fixed in position, delimited in some manner in the physical constitu-
tion of the matter involved.

Analysis of Heterogeneity in Some Simplest Chabacteks 195

The quantitative aspect of this spot is presented in figure 14, where is given in
the form of a curve the distribution of the areas found in the left spot (c) in 500
individuals. This statistical data presents simply the fact that in 500 individ-
uals from Tierra Blanca, State of Vera Cruz, Mexico, the area covered ranged
from 0 to 3.7 sq. mm., with the modal condition ^^^
at 1.5 sq. mm. From these data I could, if 95

90

desired, calculate many mathematical values of
no significance. From the statistical aspect the
variations are in line mere plus and minus addi-
tions, made so by the method of study; but ex-
amination of the areas shows that there are fea-
tures that are not and can not be stated statis-
tically; the character has spatial relations, and
these can not be analyzed by biometric methods.
In figure 15 is given the condition of the spot
c found in L. multitceniata Stal, geographic
variety multitceniata Stal, at Puebla, Mexico.
In this location the spot is nearly always present
and usually of considerable area, but shows the
same direction of variation as in L. undedm-
lineata, pushing out from the center along the
same lines and in the same relative position on the pronotum. Statistically
it shows nothing more than in the first case, a distribution of the area of

Fig. 14. — Statistical examina-
tion of area of 500 left c spots in
L. utidecimlineata from Tierra
Blanca, Vera Cruz, Mexico.

A
A

/V /%/^

m

Fig. 15. — Showing difference in size, shape, and directions of extension of spot
c. in L. multitwniata from Puebla.

exposed pigment in a curve extending between different extremes with a different
mode, and hence with the possibility of deriving different mathematical coeffi-
cients if desired (fig. 16).

The same character in the same species from Guadalupe, Mexico, when exam-
ined shows only differences in the end-results, but everywhere the same direc-
tions of variation movement. Biometrically considered, the data from 500 indi-
viduals show a different mode and distribution of areas (fig. 17). One might
go on in this manner without limit and in all it would be found that when the
areas were measured the variations would all fall into line, plus and minus.

196

The Mechanism of Evolution in Leptinotaksa

It is only in one very trivial aspect, however, that the clifEerences in spot c con-
form to "^this condition, as has been shown in the three sets of examples given.
Area in this character is not necessarily quantity of pigment, but area of surface
pigmented, and can not show truthfully either the quantity, process, product, or
distribution in either materials, duration of processes of pigment development,
or relations to adjacent parts.

00

1

i

TV

/ ^

L

_4

\

1 ~

L

I

\

7~

\

t "

\

I "

\

r '

~\

\_

i "

\

rft "

\

t

~ J L

-A -

tt

^

/\ '<

1

/

yS

1

lu

/

\

7^

\_

1

110

1

100

/

/'

f

,

j

1

1

j

,

I

/

/

1

/

l/

\

-

\

1

i

/

\

f

/

\

1

1

\

1 I

1 1

r-

1

o — —

o — —

Fig. 16. — Area of 500 left c spots in
L. multitwniata from Puebla.

Fig. 17. — Area of 500 left c spots in
L. muUitwniata from Guadalupe.

If the distribution of the pigment in the spot c is considered, and not the
amount of pigment, it appears that there are several directions of change in the
distribution of pigment and in relation of these to the other centers of coloration.
Is it the amount of the pigment, the area colored, that is important, or is it the
arrangement and changes in arrangement and relation to adjacent parts that
are important? Spot c presents two distinct sets of "variation" data, of

Fig. 18. — Showing difference in size, shape, and direction of
variation in spot b in L. utidecimlineata.

which the relative importance is entirely dependent upon one's initial philo-
sophical viewpoint, upon definition. Other similar areas of pigmentation
on the animal show identical conditions in principle and confirm in every
way the statement made. Different manifestations of spot h on the prono-
tum of L. undecimlineata are represented in figure 18, and show that the

Analysis of Heterogeneity in Some Simplest Chakactees 197

40

spot may be entirely absent or present as a simple round area and that
it may retain its round form and increase greatly in size. As in the spot
c it also changes now in one direction, now in another, or in two or more
at the same time, but these directions are never chance or haphazard, but
always are along certain invariable lines. Sta- ^^^
tistically, a lot of 500 h spots shows a typical
curve of distribution, plus and minus (fig. 19). '°°
In L. multitceniata spot h shows the same con- 9c
dition present or absent in its entirety, present gg
in the simplest condition as a simple round spot,
often of considerable size as a round spot; at
other times pushing out in different directions, co
but never in chance or irregular directions. The 50
statistical statement from a lot of 500 left spots
offers nothing of interest; it is simply the plot-
ting of a lot of areas having upper and lower ^°
limits — a mode, a mean, and coefficients not 20
worth calculating. ,0

It would be easy to go on endlessly accumu-
lating data of this kind, and I could fill many
pages with the mere statement of the facts
gathered concerning spots occupying the posi-
tion 16c in chrysomelidae, and also in other
families of Coleoptera. Naturally, not all show the same conditions, but all
give the same results in principle, namely, that the common variations of these
simple indivisible color-characters present two superficially distinct variation
phenomena — diversity with respect to area in units of some measure, and in the
arrangement and relation of the character, both external and internal.

It is obvious from the data given that the common differences of simplest char-
acters are not all in line, plus and minus, but that there are changes first in one
direction, then in another, or in one or more directions at the same time. If the
biometric definition of fluctuations be true, then all changes found that are not
plus and minus in terms of quantity, but are multifarious in direction and rela-

n L 1 1

1^ ' i

\ 1

\

i x- -^

i: A

\

i L.

t t \

t >».^

t - i

r

, t

4 \

t H

t i^

-i dtt

^ ^lt

t A

r -^

7

Fig. 19. — Statistical treatment
of a uniform lot of 500 spot &
areas in L. undecimlineata from
Tierra Blanca.

Fig. 20. — Spots having the same area, but not the
same shape or direction of variation ; statistically
unlike things are often confused on the basis of mea-
surement and treated as like.

A ^ ^ » •

tion, are mutations, and only those measurements of the area of pigmented sur-
face are fluctuations and are of necessity in line, because forced there by the
method of description. Area, moreover, is not an accurate method of describing
the condition of these spots, and this I have shown in figure 20, where there are
shown spots all of the same measured area, but their shape is quite diverse and
might be productive of quite diverse results. Statistical methods force these
spots into the same class and count them in statistical analysis as all alike. They
are decidedly unlike.

198 The Mechanism of Evolution in Leptinotaesa

If the changes in different directions producing unlike shapes of spots are
mutations, there ought to be visible or demonstrable discontinuity, but there is
nowhere the slightest trace of breaks to be discovered. The " variations " which
these spots show are not of the same kind, or at least have different directions and
hence differing consequences in the composition of the color-pattern of the part.
Movement medianward is different and is productive of different pattern-results
from one that moves caudalward. The departures shown by these spots in sev-
eral directions can be found in many if not in all simplest characters, showing
first one, then another, or one or more of its potential possibilities at the same
time. I see no escape from the conclusion that the biometric definition of
fluctuations is not only not true as a general proposition concerning variation in
simplest characters, but must be regarded as an anticipatory definition framed
in the interest of a particular hypothesis, and in no wise a carefully derived con-
clusion directly deduced from data carefully considered.

It is possible that supporters of the biometric proposition may maintain that
the different directions of variation found are in reality the basal " unit-char-
acters," and that variations along the different lines are the variations to be con-
sidered, and statistically analyzed. Experience has not shown these spots ever
to break up into lesser units in nature, and I have not been able to accomplish
it by experimental means. Further than this I can not go at present ; as it repre-
sents the limit of experience and the conclusions to be logically derived there-
from. Any further statement would, therefore, be anticipatory, dogmatic, to
support some one or other theory, but any other similar statement has just the
same chances of being true or false. It must be shown that the simplest char-
acters in nature and in experiment can be broken up into lesser units, and until
this is done it is fairest to consider that spots c, h, and d really represent simplest
characters, indivisible, and that they clearly do not conform to the requirements
of the De Vriesian hypothesis as regards their common or fluctuating variations.

The question whether the pigment as a substance which should be measured in
terms of quantity or area colored and the pattern or distribution of it considered
as another character, and that the two are necessary for visibility can hardly be
answered with any exactness. Neo-Mendelian hybridology has shown clearly
that pigment and pattern can be shifted independently of one another in crossing.
If this proposition be admitted as true and as applicable to these simplest char-
acters, the problem is not solved. Pigment then becomes a character that fluc-
tuates in quantity as statistically measured ; pattern, its companion, shows noth-
ing that can be called fluctuation, nor that can be called mutation. The decision
would largely depend upon definition in the absence of experimental evidence.

Each of these characters presents, therefore, heterogeneity; and depending
upon what is considered the correct point of view hangs the kind of statements
that will be made. If the character is calibrated only in units of area, diameters,
length of perimeter, and angles between the directions of variation, the state-
ments will all of necessity be in terms of more or less ; the determinations are
forced to become quantitative. What is actually present are differences existing
between the same character in different individuals, and these are neither entirely
quantitative nor entireh/ qualitative, hut are differences in the quantity of mate-
rial, in composition, and in the physical position, arrangement, and relations to
surrounding parts. From a descriptive standpoint it is impossible to go further. .

Analysis of Heterogeneity in Some Simplest Chaeacteks 199

It would be rash, with present knowledge, to attempt further separation which
would be purely anticipatory as to the nature and constitution of organisms. I
believe the conclusion valid that there is no sharp demarcation in nature.
Aspects of any character that are measured in units of some measure will be
arrayed in line and expressed in terms of measure ; those that are not or can not
be measured must be expressed in terms of position and relationship ; and there
the matter rests for the present. It is not possible to express in quantitative
terms, plus and minus, the facts of the fluctuations of these single characters.

(2) Are the differences of the simplest characters, spots c, h, and d, continuous
or discontinuous, or both, and what is the relation between the two? In recent
years many authors have drawn sharp distinction between continuous and dis-
continuous " variations " and their supposed role in evolution. That variations
do arise suddenly is undeniable, at a single step, and stand much apart from
their fellows without intergrading conditions; and from changes of this kind
stable races unquestionably have arisen. As a protest against the complacent
assertions regarding the omnipotent efficiency of minute, constantly accumulat-
ing, utilitarian variations as the sole cause of evolution, and in the hope that a
new viewpoint and mode of investigation might be the outcome, many have
enthusiastically advocated the idea that sudden transformations in the attributes
of animals and plants accounts better for the rise of stable races and species than
slow accumulation of utilitarian variations. Discontinuities, or sudden jumps
with gaps wherein there are no intergrades, must of necessity follow in De Yries's
system, from the very nature of the initial concept as to the nature of organic
bodies. Eegardless of the assumed underlying entities, actual investigations of
the origin and behavior of these sudden variations have been organized and com-
pleted with marked success, in striking contrast to the masterly inactivity of the
neo-Darwinians. These variations in most instances are the same as the
" sports " or " spontaneous variations " of Darwin and Wallace, the " saltations "
of other writers ; but in De Vries's Oenotheras a different phenomenon is clearly
present.

It is necessary to keep constantly in mind the possible differences in these
changes ; one arising as " variations " in the sense of a departure ; the other as
due to rearrangements of characters following crossings. De Vries's Oenotheras,
in their recurrent production of the same types, clearly suggest this origin for
their " mutations," and the recent studies of Davis, Herbert-Nilsson, Gates on
Oenothera, and mine on Leptinotarsa, show clearly the fact and a method of
producing these mutating races.

It is shown that the fluctuations of simple characters were not linear, but in
several directions, and that there were also variations in amount, extent of area,
and other measurable features ; in other words, that there was a complex varia-
tion of the simplest character.

In the study of the variations of the simplest characters many conditions have
been found which might be described as discontinuous, where in the population
there were no intergrades. One of the most common discontinuities is that
found in statistical study, of which one illustration is presented in figure 31,
where in 1000 left c spots of L. undecimlineata Stal, from Tierra Blanca,
Mexico, there were a few individuals that stood some distance from the rest of
the variates of the lot and without intergrades. These latter were all collected

200

The Mechanism of Evolution in Leptinotaesa

from one small plant, in a very moist situation, and were noted at the time of
collection and on measuring the lot these individuals stood out from the rest as a
group by themselves. Two months later, in the same location, I collected a sec-
ond lot of 1000 (the next generation) and examined the left c spots statistically.
The results of this examination are presented in figure 21, where the isolated
group found in the first curve of distribution is also present, but in this case it is
included in the curve of continuous variations. Another set of observations
made at a point near San Marcos, in Vera Cruz, Mexico, gave different data,
but results that were identical in principle in that the data of the variability of

90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40

s 1

v lu

J t

i A

4l^J

it^

it H

t t

-t

1 1

11

' tj

"7 V

-[-■ X

"t "

I" "-\

-t-

j

r

T t- E 4l

j-

l

X

I

t 4 ^-^

l^V ^

r ±1 I .^ Jv

U t A 7 '^ ^tx

I 5.^11 \t s^ tv

^ --) ^L ^^

S S 3

■n

Fig. 21. — Measurements of left spot c in
L. undccimlineata at Tierra Blanca to show
differences in the array in two successive
generations.

the left c spot in 500 individuals gave a polygon of distribution with a small
group on one side, but connected with it by intergrades. A collection in exactly
the same locality in the next generation gave a different condition in the polygon
of distribution, with the group standing below the general population entirely cut
off, and intergrades were wanting. This extreme group came entirely from an
area of coarse gravel with little loam, and dry at all times. The first collection
was made in the early part of the rainy season, when it had been rather dry over
the plain as a whole, so that there was a rather continuous series of stages of dry-
ness from the extreme state of the gravel bed to that of the rich soil of the

Analysis of Heterogeneity in Some Simplest Chaeactees 201

savannah ; and the second lot was collected later in the season at a time when it
was raining daily, the only dry place was the gravel-bed, and here the individuals
were found that were discontinuous from the general population. These statis-
tical discontinuities can be multiplied without number, and are sharp changes,
often only produced in a few individuals by chance conditions of location, but
the majority of the population was not so influenced. It was not observed that
the influence had produced any change in the shape of the spots or in the different
directions in which this spot varied ; it was simply in one case an accentuation
and in the other a diminution in the amount of the pigments formed as measured
by the area darkened by them. Both were simple climatic aberrations and on
testing failed to appear in the next generation so that statistical discontinuity or
aberration means little or nothing.

Is there any discontinuity in the presence and absence of these spots ? Care-
ful examination shows the gradual diminution of any or all of these spots until
the traces are no longer visible to the unaided eye, but proper preparation shows
on microscopic examination a long scale of decreasing color values. In their
lowest terms these simplest characters may be invisible to the eye, only micro-
scopic traces persisting, often a faint light yellow-brown color in the lower por-
tion of the cuticula which gradually vanishes completely. In these characters
there is no visible discontinuity in the present and absent conditions ; neverthe-
less the two extreme conditions are capable of fixation in " lines," and when
crossed these show clear dominance of the presence over the absence and segre-
gation in Fj.

There is divergence between the different directions of variation of these spots.
Spot b varies in an antero-posterior and in a medianward direction, rarely in
others. Between these directions of variation no intermediate directions have
been found. Occasionally a direction not hitherto observed arises suddenly in
some individual or individuals. The new direction of variation starts from the
center and goes in its own direction with remarkable precision. There is diver-
gence in the directions of change in these characters but whether there is discon-
tinuity depends upon definition.

In this section the answer to the question turns in a way not unlike that of
the first section ; there is diversity, divergence, with discontinuity of end-results
of diverse sorts, but the proposition of the unity of direction in " continuous
variations " seems entirely unfounded. There are statistical discontinuities, but
from the method of examination these can exist only in plus and minus aberra-
tions ; and further, this method of statement " lumps " so many different condi-
tions as one homogeneous class that it is extremely doubtful if the results of sta-
tistical study are in reality biological at all. There remains the undoubted fact
that in these simplest characters there are differences that are continuous, in that
there are plenty of intergrades, and those that are divergent, giving discontinu-
ous end-results between which present knowledge has found no intermediates.
Actually, the " variations " are diverse relations in constitution, amount, space
relations, and interactions with the rest of the mechanism, which result in differ-
ing arrangements in the visible product deposited as the outcome of these pro-
cesses. That intergrades are present in some aspects of the complex, lacking in
others, is not a basis for the creation of sharply marked categories. That inter-
grades are present or absent is no criterion of the nature of the processes, and it
14

203 The Mechanism of Evolution in Leptinotaksa

has not yet been shown that in any of these simplest characters, or any of the so-
called unit-characters, there is a different composition where there is statistical
discontinuity.

(3) Are the vaj-iations of the simplest characters delimited or undelimited in
the spots c, h, and d? The answer to this question has been to a considerable
extent given in the preceding sections. All of the variations thus far seen are
delimited in that they are all confined to certain well-marked directions of varia-
tion. These spots do not vary at random, but in conformity to certain rules ;
what these are need not now be stated, as it does not affect the situation.

The delimited nature of the variations of these simplest characters shows cer-
tainly that they are not homogeneous or undifferentiated things, but each has
structure and relations that are its own, and entirely characteristic of it. That
is, in the areas called spot c or & or tZ there are forces and physical relations exist-
ing which center in the location c, h, d, and the same is true of all other color
areas in these organisms, which control the distribution of the pigment and limit
it to certain positions, which means that the underlying processes producing the
pigment are themselves thus localized and vary. For convenience of expression
it can be said that each of these spots has an organization peculiar to itself,
which delimits the variations which it shows, but it must be clearly understood
that this does not mean that the spot exists as an entity, independent of the rest
of the pronotum. Experience gives no indication of any such condition. All
that is known is the fact of the localization of the results of certain processes at
a fixed point on the surface of the pronotum, which there carry out certain reac-
tions from which, as a product, pigment is produced and distributed in the same
position as the processes themselves are localized. These localizing conditions,
as far as it is possible to analyze them, seem to be properties of the whole organ-
ism presenting most exact and complex arrangements.

It is important to recognize that in these minute and unimportant simple char-
acters the variations, to use Elmer's phrase, are " law conforming " ; that is, are
orderly and not disorderly. This signifies of course a complete control of the
conditions in the smallest of the attributes that it is profitable at present to study.
These characters in organisms stand in the same relation to the organic body as
do angles, hardness, color, etc., in inorganic bodies. They are not entities in a
mosaic, but manifestations of physical and chemical interactions giving as an
end-result the things seen and used as a biological alphabet.

(4) In local areas and in species as a whole, are the variations of the simplest
characters definite or indefinite f Whether variations are definite or indefinite
in Darwin's meaning of the term is a question of prime importance in the solu-
tion of many problems in organic evolution, especially in the formation of groups
and species in nature, and has been altogether neglected in recent years.

In Darwin's conception of this response in " variation " there is a broader rec-
ognition of the general relationships of " variation " phenomena in organisms
that is found in the writings of his critics, or in any of his professed followers.
This aspect of the study of variation has been tested in spots c, h, and d in L.
undecinilineata Stal, L. multitceniata Stal, L. oblongata n. sp., L. decemlineata
Say, L. signaticollis Stal, and L. diversa n. sp., and in all the results have been
the same in principle, different only in statistical details. I shall present the
data of a few of the studies for consideration.

Analysis of Heterogeneity in Some Simplest Chakactees 203

Statistically, the problem must be answered from four aspects: (1) in the
species as a whole, regardless of where or when the specimens lived, it is
necessary to determine the entire range presented; (2) comparisons of one local-
ity with another are necessary to discover possible local influence ; (3) successive
generations in the same location must be compared to determine the constancy of
relation between local causes and effects; (4) comparison of series of genera-
tions in the same and different localities to determine the existence of continued
or permanent local influences. These four examinations will give all the infor-
mation possible from this source, and when combined, will give some basis for the
determination of the nature of diversity in these simplest characters, and provide
the proper data for initiating experimental analysis of problems not touched by
the methods used. In this study I have examined these characters in many thou-
sands of specimens, and in living material and museum specimens have looked
constantly for new or unusual " variations." In figure 22 I have shown outline
drawings of the main types of variations found in L. undecimlineaia.

Fig. 22. — Diagrammatic representation showing common types of variation in
spot c found in L. undecimlineata.

Fig. 23. — Diagrammatic representation of outer portion of pronotum in L.
undecimlineata, showing relation of different directions of variation in spots c, e,
and relations of these to the element x.

Examination of the variations shows clearly that the character in its variations
is delimited to a few directions of change. These are diagrammatically shown in
figure 23, which is a composite of the spot in outline to show the direction of
departure. As compared with other species, it is quite distinct in its variations,
which are few and rarely of any magnitude. Medianward fusions with 6
have not been found, although c may extend towards b, but h does not move
towards c; hence there is no fusion. Caudalward unions with e are rare, and
result from combined action of c and e ant. towards each other ; and still more
rare are fusions with d, which depend upon the presence of a rare element h.
Its absence is not infrequent and its presence never reaches an area of over 5
sq. mm., 4.89 sq. mm. being the largest seen, and that a product of selection plus
environmental stimulus.

Parallel observations in nature were made near Tierra Blanca, 57 miles south
of the City of Vera Cruz, at El Hule, 39 miles south of Tierra Blanca, and at San
Marcos, 68 miles south of El Hule, all upon the savannahs of Vera Cruz, Mexico.

The location at Tierra Blanca was on tlie edge of a small stream 3 miles from
the town on the trail of Estanzeula. The immediate location was typical trop-
ical savannah with thorn forest, well watered, good drainage, and about 225 feet
above sea-level. The El Hule location was on the southern edge of the flood-
plain of the Eio Papalaopan, west of the station buildings about 1 mile, on the

204 The Mechanism of Evolution in Leptinotaksa

trail of Tuxtepec; also typical savannah but more open and park -like, fewer
trees and many annual and shrubby perennials, and about 300 feet above sea-
level. At San Marcos, 600 feet above sea-level, the location was about 1.5 miles
west of the station on the eastern edge of a rainy season lake. The area was
savannah, but with tree islands, poorly developed drainage, abundantly watered
and covered with short grass and low herbaceous plants.

At all three places the annual cycle of seasons is the same in type but differs in
details. Eecording thermometers that were secreted in the open and read on
returning gave the highest and lowest temperature that had elapsed since the
last observation. Tierra Blanca gave the highest temperature, 107° F. in the
shade. May 1906, and the lowest record obtained there was 54° F. in December
1905, during a " norther." San Marcos was always cooler, the extreme records
being 97°F. in May 1906, and 45°F. in February 1906. El Hule was more
moist and always had fairly high humidities at all seasons, while Tierra Blanca
had the least rain and the lowest humidities.

The plan adopted was to go to each location at proper seasons and there make
determinations of the value of at least 500 individuals. Owing to the peculiar
localized character of these colonies, none, or at least only a few, were removed
because it early developed that artificial removal soon altered the tone of the local
group under observation. To have removed 500 would often have taken nearly
the entire population of the place. Usually one or two natives were employed
to gather the specimens, while I determined and recorded the values. To facil-
itate this, scales of areas were made as a basis of comparison, and the material
seriated quantitatively, and also camera outlines were made and measured by
planimeter later. Either method gave the same results, the latter more detailed,
but no more accurate or significant. Variations in shape were recorded in
terms of type of variation. Two sets of observations were obtained, therefore,
at each location, and for each period of observation a quantitative measure and
an expression of the frequency of different types of distribution of the pigment
and the variation directions of the spot. As a rule, two to four days at each
place were necessary to obtain the requisite number of records. No specimens
were returned to nature until after the records were completed.

The results obtained in the years 1905, 1906,. 1907, 1908, and 1909 are pre-
sented in the condensed form of tables.

Quantitatively, the San Marcos gi'oup showed (table 23) constantly in all
of the ten determinations the least amount of pigment, but a considerable varia-
tion range, while the Tierra Blanca group gave constantly the largest amounts
of pigment, and also considerable range of variation ; the El Hule determinations
gave the greatest variability and the least constancy in values. In all it is
clear that the members of the population responded as a mass to the conditions
of life, in that the polygon moves as a whole plus and minus in different deter-
minations. From quantitative determination the conclusion might be drawn
that quantity varied determinately in this character, but it does not give any
basis for a discussion of its action in evolution until it is shown whether the
variation is permanent or can be made so. Statistics can not determine this,
but they might give the basis upon which to base experimental operations. The
variation differences found in these locations may be due to different external
conditions, to localized racial characters resulting from partial isolation, and

Analysis of Heterogeneity in Some Simplest Characters 205

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206

The Mechanism of Evolution in Leptinotaesa

so on. They can be either somatic or germinal, but no trace of knowledge of
this either one way or the other is found from the biometric analysis. From any
such body of data any one of many " conclusions " can be drawn, but in all the
conclusion is an assumption made to interpret the array of mathematical values
which the biometric method has obtained. The method shows a condition only,

Fig. 24. — Showing directions of variation found in spots c, x, d,
and e, in L. panamensis.

and none too accurately, because it can not distinguish germinal and ontogenetic
differences, or any other kind of heterogeneity present. It is, however, a fairly
good crude method in obtaining preliminary data of conditions in a population.
Examination of the directions of diversity found in L. panamensis (fig. 24)
shows differences in the areas, but the general result is clearly a delimited vari-
ation. In restricted localities the population shows remarkable delimited move-
ment of the population as a whole in certain directions. To what agencies these
movements and responses are due is not determined by the methods of the census ;
an endless array of possible causes for the observed effects could be suggested and
plausibilities put together to no purpose whatever.

Fig. 25. — Sliowing directions of varia-
tion, especially in spots c, h, and e, in
L. signaticollis, and some results which
these directions of variation may produce
in the production of pattern differences.

In L. signaticollis the same spot (c) occurs and shows some of the same direc-
tions of variation as in L. undedmlineata, but in area it much exceeds the first
species. In figure 25 are shown the variations thus far discovered, together with
the results produced in the pattern. In this species spot c is most commonly
present as a round area of variable size, is never absent, with the observed

Analysis of Heterogeneity in Some Simplest Charactees 207

extremes as represented in figure 38, a and h. It may extend medianward
towards h, but this is always accompanied by a lateralward movement from h
towards c. These two always move toward each other at the same time, produc-
ing a mutual movement to unite. Likewise c and e ant. may simultaneously
move towards each other and unite. The movements of c and h towards each
other, of e and f, and the movements of c and e ant. are shown in figure 25.
These states are inheritable in pure homozygous biotypic lines and are alterna-
tive to their absence in crosses, No other variations in direction are known in
spot c in L. signatxcoUis, either in nature or in cultures, and none has thus far
been induced by experimental means.

The biometric study of this area shows little of interest. In figure 26 are
given the statistical determinations for the populations in restricted colonies at

1

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li.

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Fig. 26. — Biometric treatment of the left spot c in
L. signaticollis at Cuernavaca and at Atlixco.

Atlixco, in the State of Puebla, and Cuernavaca, in the State of Morelos,
Mexico. In the Cuernavaca colony determinations of left c spots were made in
1904, 1905, and 1906, and show a mode and a wude range, but the spot is never
absent. In the Atlixco population for the same time is shown a lower mode.
Both modes are higher than the modes for this character in L. undecimlineata,
and the mode for Cuernavaca is the highest known and occurs only in one
restricted area. Other colonies in the Cuernavaca Valley have much lower
modes.

Biometric observations upon the state of the population at these colonies in
the Cuernavaca area were made in 1904 to 1908, a total of ten observed gener-
ations. The three colonies were different in conditions, especially in the water-
relation. The Bosoco colony was located in the shallow, steep-sided barranca
(50 to 100 feet deep) to the east of the town, and so located that the deep, rich
soil was moist, due to drainage conditions. The Quauhtemotzin colony was on

208

The Mechanism of EvoLUTioisr in Leptinotaesa

Table 24.

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

3
2

1

3
4
6

5
11

178

12
31
?11

21
89
183

77

165

qo

98
92

141
26
6

67
5

1

17

1

3
2

2

1

Basoco Colony

San Antonio Colony

Table 25. — Observed frequency of occurrence of the different types of variation in
spot c in each of the colonies during ten successive " generations."

Basoco Colony:

June, 1904

August, 1904

June, 1905

August, 1905

July, 1906

September, 1906

July, 1907

August, 1907

June, 1908

August, 1908

Quauhtemotzin Colony

June, 1904

August, 1904

June, 1905

August, 1905

July, 1906

September, 1906

July, 1907

August, 1907

June, 1908

August, 1908

San Antonio Colony:

June, 1904

August, 1904

June, 1905

August, 1905

July, 1906

September, 1906 . . .

July, 1907

August, 1907

June, 1908

August, 1908

Observed shape and direction of spot c.

Absent . Small. Large

c-^d.

c + d.

c->-e.

If

17
24
51
106
77
15
49
74
10
92

142

171
86

105
88
21
55
71
45

109

71
121
59
50
92
46
31
100
18
77

:ii

171

39

31 1

186

51

15 5

77

5

3

118

29

2

71

14

5 4

37

10

2 1

95

34

15 6

83

41

4

21

15

2

109

4

214

12

1 4

186

19

3

92

4

77

10

i

31

25

2

36

15

3 1

67

10

4

175

36

2 19

56

12

5

211

71

6

281

14

145

19

314

71

49

3

86

14

92

i

19

55

4

210

6

37

4

88

1

10

4

7

3

14

2
17
4
7
9
17

2
3

2

Analysis of Heterogeneity in Some Simplest Chaeacteks 209

the eastern slope of a large barranca and always moist, owing to constant seepage
of ground-water. The San Antonio colony was located on the west side of the
river above the Falls of San Antonio, and was the best drained and driest of the
three. All were about the same altitude and received the same rainfall. Evapora-
tion, however, was most intense at the San Antonio, least at the Quauhtemotzin
colony. Statistical determinations made by measurement and seriation were
used, frequently together, and the results show constant differences between the
three locations, between which there was little chance of interchange. They
have not been found to cross the elevated ridges from colony to colony, but work
up and down the barrancas, so there was little or no intercommunication prob-
able and none was discovered between them. In table 24 are given the results
from these determinations. It is evident that the Quauhtemotzin colony has
always the highest mode, that is, c is larger here than in either the Basoco colony
or the San Antonio colony, where it is smallest. As far as the biometrics are
concerned, here the story ends. No more information of any importance is
forthcoming as the result of the application of this method of study. The con-
ditions and any mass actions of spot c in the population are known ; the reasons
for these conditions can only be analyzed through experiment. At best, only a
plausible interpretation of the observed condition based upon a favored assump-
tion is possible, which is neither proven nor investigated ; and to assume that
large water-content in the medium means much pigmentation and lesser
amounts mean less pigment is nonsense. This relation of water-content may be
an entirely correct conclusion, but by no means do the biometric data in this or
similar instances prove the conclusion at all.

Table 26. — Percentages of fusion or movements to produce fusion in the total popu-
lation of the Cuernavaca district that were observed in the three colonies at
Rancho Basoco, San Antonio, and Quauhtemotzin colonies with respect to
directions of fusion.

Source of the stock.

Round, either
smaH or large.

c'^b or c+b.

c"^e or c+e.

Basoco colony

Per cent.
23

32 —
31 +

Per cent.

5 —
2.9 —

Per cent.

0.5

1.—

3.—

Quauhtemotzin colony

San Antonio colony

Much more interesting and indicative of more significant conditions in the
population are the results from a census of the directions of variation of spot c
in the three locations. This I have shown in table 25, where the census is
recorded for each of the three stations at Cuernavaca for 10 consecutive periods
of observation. In this census a decided difference is evident in the San Antonio
colony, in that c X & is not found, and its presence is only indicated in 2 females,
neither of which was able to transmit the observed variation. Both were tested
by breeding. At the Basoco and Quauhtemotzin colonies all the variations were
found, but in different populations. In all, 6377 records were made at the
three colonies in the 10 censuses. Eegarding this as the population of the
Cuernavaca area, the three local populations show their differences in percent-
ages of the whole as shown in table 26.

210

The Mechanism of Evolution in Leptinotarsa

Table 26 shows local diversity in the directions of variation, and they may be,
as far as biometrics go, either germinal or somatic. Experimental testing, aided
by adequate field observations alone, can decide, and this I have done in this
instance by bringing stocks from all of the three local populations to Chicago,
where under favorable conditions of culture they thrived and gave a combined
result shown in the table 27.

Table 27. — Results found in stocks from three Cuernavaca District colonies, when
reared under like conditions at Chicago.

Source of the stock.

Small
or large.

c'\"d or c+d.

c"^e or c+e.

No. of
generations.

Basoco colony

Per cent.
25 +

29 +

30 +

Per cent.
7 +
2 +

Per cent.

1 +

2 +
3.5 +

8
6

5

Quauhtemotzin colony

San Antonio colony

It is certain from this test that the three stocks are different in gametic con-
stitution ; otherwise why should they retain, under like conditions, when grown
side by side, the same differences observed in nature and tested in this simple
experiment? The result shows the fixity and specificity of these minute and
trivial characters in the organisms, and also the delimited, determinated
response in the directions of heterogeneity in the different locations, but to what
agency this determinate response is to be attributed is not indicated. In the
amount of pigmented area perhaps more certain determinate response is
observed, but this is of no significance.

In L. diversa and its geographic variety rugosa, little need be said. No
directions of variation are found, and it has not been observed to be absent in
any individual that was able to transmit the character as absent. Spot c (fig.
27) is always a round area of variable size and it is always free, never fusing

Fig. 27. — Showing absence of variation in directions and
difference in size found in spot c in L. diversa.

with any other center ; even in area it is fully as conservative as it is in shape.
In the former its greatest change is a condition having an irregular outline.
Quantitative determinations made of the area at El Borrego and Sierra de
Escamela, near Orizaba, Mexico, and at El Riego, near Tehuacan, Puebla,
Mexico, for its variety rugosa, show the constant differences and shape of
polygon, represented in figure 28. That their value is of no moment is shown
by their lack of permanency ; when stocks are taken to the uniform condition,
of the laboratory at Chicago at once all assume the same condition. In this
example the differences are, as far as determined, merely unfixed habitudinal
somatic fluctuations in quantity of pigment due to external conditions acting
upon growth conditions.

Analysis of Heterogeneity in Some Simplest Chaeacteks 211

Examinations of the other species that have formed the material basis of this
portion of the investigation with reference to these simplest characters show
the same kind of data and the same general results as those set forth. All of the
species in the group have been gone over in this way and none have given results
in any wise different from those stated. Only in detail, which is of no general
interest, do the sets of observations differ. Spot c has been most frequently
presented in this account, because of all the pronotal characters it is least liable
to fusions and least variable. In all instances this " simplest character " shows
heterogeneity which, by definition, may be made fluctuating or mutative, quanti-
tative or qualitative, as one may wish. It is shown
that the simplest characters differ in amounts when
measured, change in different directions, and enter leo
into diverse combinations. They are always de- ,50
limited, are apparently never undelimited, and may
or may not respond to heterogenetic causes in
definite or indefinite reactions, so that these trivial ""^o
characters react individually in much the same way uo
that individuals and species do. To some this may ^^^
mean that the individual as a whole so responds,
and the character reacts in the same way because '°°
correlated with the whole. To some extent this is 90
true, but not entirely so, because these areas have a go
specificity of behavior and an individuality of re- ^^
action and stability that is astounding.

In L. undecimlineata, in the same locations, c is *°
often wanting in a low percentage of the popula- 50
tion, and from these individuals a race in which c 40
never appears can be easily created. Of interest,
therefore, is the experience of crossing one of these
races (biotype) based upon a minute and trivial ^°
character with L. diversa or L. signaticollis, where to
c is never absent, and then to discover that Fj
extractives of signaticollis with c absent are ob- ^
tained, and from them arise pure biotype lines
differentiated by this character. The presence and
absence relation is to be conceived of in this in-
stance as appljdng only to the one area c, but
equally precise behaviors have been observed in h, d, and in the absence of

am
the a' + &'+ + +a+b group. The contrasting characters, minute as they are,

pm
behave in the same precise manner as do larger and more generally distributed
characters. This can only mean that somehow in the gametes there are exact
and detailed conditionings of this character (spot c), and that in crossing there
is a precise recombination of the germinal agencies, such as to introduce, where
it was not before, a condition of the character found in another species. WHiile it
may well be true that whatever does produce the new condition in the c-less
extracted signaticollis of F, may be tied to or be a property of the whole, there
still remains the grave difficulty of accounting for so exact and trivial a mani-

1

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Fig. 28. — Statistical treat-
ment of values of left spot c
in L. diversa and its variety
rugosa at El Borrego near
Orizaba, Sierra de Escamela,
and El Riego.

213 The Mechanism of Evolution in Leptinotaesa

festation of the " general property of the whole " and always in the same place.
Moreover, c does not come back into such a strain except as it is put back by the
reactions of interchange of gametic factors between a c-less race and a race
that has c, which may be either another homobiotypic strain or any heterobio-
type that has c present.

Not for a moment h cot I ox d or any other area to be thought of as a single
thing or entity. It is absolutely not a unit-character in any sense. The pig-
ment of the spot is the product of a black determiner and a color-factor; the
pattern rests upon two distinct sets of agents — a general pattern-factor of the
pronotum and the pattern determiner of the particular area, and this pattern
determiner is always, except in pathological examples, precisely localized in the
pronotum, but from this fixed position in space it is able to, and does, form
different combinations by movements mutual between it and nearby areas.
These combinations are always specific for the species, can be fixed and isolated
in biotypes, and are alternative in crosses of such biotypes. For example,
L. diversa, angustovittata, and rugosa have no directions of fusion; signati-
collis forms c + h and c+e ant., but never both at the same time in the same
individual. L. undecimlineata and panamensis may have c wanting or form
c + e ant., c + d,OT c + t combinations. L. multitceniata shows the greatest num-
ber of combinations, oblongata only c-\-e ant., decemlineata c + e ant. and no
other, while juncta, texana, tumamoca, and dejecta have not thus far shown in
nature or in cultures any fusions or heritable directions of variation. This
behavior has a strong analogy to the condition of many molecules or radicles
where there are one or more free bonds at times, at others with no unsatisfied
bonds. It is not for a moment to be supposed that the reactions shown are
such ; only the type of action seems to have something in common in the effort
of the localized mass of matter to place itself in the system with all bonds
satisfied and no free bonds unsatisfied, which at any moment may become a
possible entrance-point for incident dissociative forces.

These simplest characters, while not capable of disruption into lesser areas
or characters, are each the visible product of the interaction of the factors and
determiners above described. These are the common normal agents most con-
cerned in such areas. There are others present, active, and important, which
need not be considered here. All react to produce the character " simplest "
observed, and the observed product varies in space, volume, direction of com-
bination, quantity of pigment produced, and area occupied. Variation in these
characters is not entirely of any one aspect, but is quantitative or qualitative,
continuous or discontinuous, depending upon the aspect, method and point of
view, and responds in delimited changes which may be definite or indefinite in
the action of the observed population.

There are very many simplest color-characters in the adult that are precisely
like those discussed. The head, abdominal segments, legs, and wings present
color-marks and arrangements of coloration that would be serviceable in the
investigation of this problem of the nature and diversity of simplest characters.
I have in the course of this work gone into the study of more than a dozen of
these with greater or less completeness, but always with the end-result found in
those presented, and no purpose would be served by presenting a larger array at
this place. It might be asserted that the instances chosen and presented are

Analysis of Heteeogeneity in Some Simplest Chakactees 313

special cases, of a highly specialized pattern, and are through " organic correla-
tion " bound together, so that all show essentially the same action, even though
they are diverse ; and in order to meet this situation as well as one can, I have
investigated simplest characters in the young, also structural and physiological
characters.

IN THE YOUNG.

The young stages of these animals possess many characteristics that might
be made the basis of studies of simplest characters. A good example of a
simplest character in the larva are the spiracula spots arranged on the pleurae in
a single longitudinal row sujrounding the openings of the spiracles. In all the
chr}'somelid larvas that I have seen this row of spots is always, if present, found
as single indivisible pigmented areas about the spiracular openings. They are
never, as far as I have been able to observe in nature or produce in experiment,
broken into two or more elemental areas or components of any sort, and as far as
evidence is available they are simplest characters arranged in segmental series
upon the abdominal segments. They may be absent or they may be removed by
experimental means, especially by combined selection and environmental influ-
ences ; and in this instance the presence of spots is dominant over the absence of
them in races created by this means. Never is there any trace of a division into
elements, so that they are to be regarded as simplest characters. In the majority
of the species in this genus the spots are small, rounded, rather uniform in size,
presenting little in the way of variability in exposed area on corresponding seg-
ments in a series of several hundred individuals. Reference to the figures given
in this work at a different place for divers purposes will show that the character
in question is extremely uniform for all, and that there is little range in size or
shape in the segmental series or in the genus as a whole. Long ago, in a fit of
biometric enthusiasm, I measured the spots from camera outlines made from 100
larvas of L. decemlineata. Those from corresponding segments were seriated,
and the whole series showed only the expected monotonous array of uniform
steep-sided curves, all much alike in limits, identical in form, and only signifi-
cant of uselessly expended time and energy.

When one searches for directions of combination with other spots, there too
the quest is not productive of any certain results in the larger part of the forms
examined. In L. diversa and L. signaticollis, however, there exists precise and
distinct lines of fusion, and once one knows the position of these, it is recognized
that the rather squared form of the dorsal edge of the spots that is often present
has a meaning. In the two species mentioned, the top of the spot is squared, and
from the anterior and posterior sides thereof there are locations and directions
of pigment production medianward and that may unite with corresponding lat-
eralward developments of the lateral tergal spots, fusing to give a U-shaped spot
with a heavy base and the arms reaching dorsalward towards the middle tergals,
with which fusion is not infrequently effected. In figure 29 I have shown some of
the conditions in this spot that are found in L. signaticollis. In other species
there is shown only an aborted attempt to develop pigment in these locations and
directions.

In L. undecimlineata even this aborted tendency is absent; the spot is nearly
round, and when crosses are made of these two species some interesting results

214

The Mechanism of Evolution in Leptinotarsa

are produced in the larvae. In the Fg extracted dgnaticollis types there is a
regular proportion of the fraternity in which the normal type of spots has been
replaced by that from undecimlineata, giving a yellow larva with the usual num-
ber of black tergal markings, but without the characteristic fusions. An
extracted race of this sort does not have the tendency to produce the direc-
tions of fusions that the normal species does; the typical spiracular spot of

Fig. 29. — Diagram showing centers of coloration
on the abdominal segments in the larval stages of
L. diversa and L. signattcollia, and directions in
which fusion between these elements may take
place.

signaticollis has been replaced by an equally specific condition from another
species by the reaction in crossing. In figure 30 I have shown these two types
of larvae, which are different enough in pattern so that anyone can at once
distinguish the two. When two such biotypic races of signaticollis are crossed
typical Mendelian arrays are always produced in Fg, showing the remarkably
exact and specific nature of the productive factors and determiners of these

Fig. 30. — Showing pattern In third larval stage of L. undecimlineata and in
L. signaticolUs and effects produced In the larvae by the crossing of these two, which
results in the production of extracted races of signaticolUs in F2 having larvae
of two distinct types In the third stage; those like the original parent stock (C),
and those Intermediate between the two parental stock (B), In that in the latter
extracted race the different elements never fuse, hut remain separated as a stable
characteristic, so that the amount of pigment presenting this stable race (B) Is
Intermediate between the conditions represented In the two parents.

trivial, nonutile characters. There are clearly present and operative in this
instance the general color-factor for black pigment (Cm), the black pigment
producer (Bm), the general body-pattern factor (PIII), and a specific
spiracular pattern-determiner (Sp) that are intimately concerned in the pro-
duction of these spots, which are absent if one of these essential elements in
its array of producers is absent. If either Cm or Bm is absent, no color is pro-

Analysis of Heterogeneity in Some Simplest Chabacteks 215

duced in the location; likewise if {PHI) is absent no general localization
of color occurs, a diffuse distribution with no specific spots resulting, while Sp
is necessary to the series to give specific form to the area in question.

It is easy to show by present methods that there are four necessary active agen-
cies at work in the production of this series of spots, and while it is not known
what the nature of the agent is that produces the action in any instance, the
accuracy of operation and the certainty of the result leaves not the slightest
doubt that these agents are at work in the production of the spots in question.

I have tried repeatedly to obtain, from the study of the spots in the larvae of
these organisms, evidence that there were various categories of " variation " in
the differences shown, but I find myself unable to decide which differences are
quantitative or qualitative, which in line or multifarious, unless I ignore obvious
mistreatment of the data and use unproven assumptions as true. The larvae
afford numerous simplest color characters, and can with adequate tests show in
L. decemlineata or any other species that the character presents differences in
quantity, arrangement, relations in space, quality and specificity of manifesta-
tion ; in short, it presents the array of attributes, qualities, and conditions that
any resultant of interacting substances and forces do, and these are heterogene-
ous in their manifestations.

In the effort to understand the meaning of heterogeneity in the characters of
organisms, using these beetles as materials for study, I have made such use of
these simplest color-characters in the effort to understand the significance of the
characters themselves and the nature of their production and significance of their
differences. There has been the problem to satisfy myself concerning the two
categories of " variation," as defined by De Vries, to discover if the distinctions
made are valid, because if so they are fundamental for further investigations of
evolution problems. Thanks to neo-Mendelism, the nature of characters has
received a searching study and analysis as never before, and while incompleteness
is apparent in neo-Mendelian work, it has shown clearly that there are in the
production of the characters of organisms agents, carriers of nothing in partic-
ular but the properties of their own substance and capacity of action, but which,
when present in the mechanical system of the organism, produce the concrete
product and interactions in exact and predictable results.

I have tried to give the De Yriesian criterions with regard to the diversity in
organisms an adequate and fair test in this material in its color, as well as in
other characters, as illustrative of a great series of " organic characters." If it
be assumed that pigment is the character, which is contrary to fact, and if it be
further assumed that the amount of it present represented the homogeneous
result in the population of uniform causes, which is also contrarj^ to fact and if
it be further assumed that the measurement of the area of pigmented surface is
an accurate measurement of the amount present, which is also contrary to fact,
one is ready to begin the routine statistical study of the character and arrive at
" exact mathematical proofs " and demonstrate some " fundamental facts of
biology." De Vries took at their advertised face value the supposed accurate
results of the biometricians, and therein lies a serious defect in his treatment
of the problems of the origin and meaning of " variation " in organisms. No
statistician has taken care to determine that his measured material is homo-
geneous, or that his measured " characters " are single characters or dimensions

216 The Mechanism of Evolution- in Leptinotaesa

of single characters, and the neglect of these alone is sufficient to invalidate their
findings. The added diflSculty that these fluctuations were not capable of unlim-
ited accumulation through quantitative selection seemed an insuperable obstacle
to De Vries, and while there is no doubt of the general correctness of this limi-
tation and the frequent regressions to the mean, both limitations and regressions
are due to other agencies than those considered by De Vries.

ANALYSIS OF HETEROGENEITY OF SOME SIMPLEST STRUCTURAL

CHARACTERS.

With structural characters it is necessary to apply the same criteria for theii
determination as in the color-characters, in that they must be the indivisible
resultant of the interaction of a single constellation of interacting agencies, the
absence of any one of which results in the non-production of the character, in
precisely the same manner that the absence of an agent in a complex physical
system results in the non-production of the proper product of the operation of the
mechanism.

IN THE ADULT.

Prime favorites with the statistician and the taxonomist are such " structural "
features of the organism as the dimensions of parts, length and breadth or
indices, form, i. e., elongate, robust, depressed, general aspect of the surface,
rugose, glabrous, punctate, and so on without limit. These characterizations of
the appearance and form of the object serve well in the operations of the taxono-
mist to distinguish the different species, but finer analysis is needed for the
investigation of the problems of the origin and meaning of diversity in organ-
isms, and especially in the effort to untangle some of the conflicting statements
regarding the problems of heterogeneity.

In the case of structural characters, the same set of questions need to be asked
and investigated with care as in the case of color-characters, and the same exact
requirement needs to be followed so that homogeneous states shall be investigated.
Both must be the indivisible product of a group of interacting agencies, in which
the absence of any member is followed by the non-production of the character in
question. It is necessary to deal with simplest structural characters, as the basis
of the analysis.

In the organism with which I have been working there is an unusual dearth of
spines, scales, or other ornamental characters of a " structural " type, only the
integumentary punctations being present. It has, therefore, been necessary to
use more important portions in the study of the characters of this class, although
it is by no means as easy to obtain simplest conditions as in color-characters.

Body-weight. — One of the first characters investigated was the variability in
the body-weight of the population, which, obviously, is the product of a large
number of contributing factors in the population and in the individual. A deal
of time was spent in 1899, 1900, 1901, and 1902 in the statistical study of body-
weight in L. decemlineata Say. Both the live weights and the dry weights were
obtained, but the outcome of the study was the demonstration that the method
and the material could give nothing of value. I was using materials from
nature, and these were obviously of unlike composition genetically, of dissimilar

Analysis of Heterogeneity in Some Simplest Charactebs 217

experiences in their ontogeny, and came into my hands for study from the field
in quite heterogeneous states of nourishment, age, and reproductive activity.

It was thought that the " character " might be of some interest in the study
of " variation " phenomena, and in that similar weight determinations had been
used in investigations, there was no a priori reason why the same might not be of
interest in this material. This first study of the weights as a structural char-
acter was so unsatisfactory that I did not follow it further at that time. There
were too many obvious sources of error in the set of observations, too little homo-
geneity of materials or determinations, even though the statistical method did
blend them beautifully in the end ; so that the attempt was abandoned from the
point of view of materials taken in nature, even with the greatest care as to
location, generation, and conditions of life and stage of development.

In 1901, 1902, and 1903 I made a different plan of attack upon the problem
of weight as a " unit-character," this time breeding from single pairs, obtaining
the weight of the progeny at the onset of the breeding-period, and in the next
generation breeding only from the individuals that had the same weight as the
parents, and so on. Effort was made to have the material as uniformly nour-
ished at all times as possible, to give it all the food it would eat, and to weigh at a
fully nourished state at the onset of reproduction activities. Other conditions
were not controlled or measured and no attention was paid to them, excepting
to make them as nearly normal as possible, so as to give the best growing-condi-
tions for each group and produce homogeneous materials. These measurements
were made in a series of experiments to modify the size of this species by selec-
tion, and the measurements here recorded were a part of the measurements
made in the course of that study, and serve well in this place to illustrate what
one really finds in the variation of characters of this class.

In the population which I was using for this work, which came from eastern
Massachusetts, I weighed both males and females, and the means of the weight
at breeding were males 7.5 to females 10. In figure 31 is given the results of the
weighings of the males in four generations. At the start three sets of weights
were mated — light, average, and heavy — the male being used as the basis of the
determinations. They were mated with females whose weight was to the males'
weight as 10 : 7.5., thus preserving " statistical equality " in the matings. From
the lightest condition mated (a) a line of descent was carried out as shown in
figure 31. In the first generation are shown the matings from the different
light, average, and heavy pairs, with the male weights alone given, showing that
the three conditions of weight gave about the same conditions in the array of the
weights in the progeny. In figure 31 are given the results in the line of descent
from the light condition (.-1) for four generations. The materials subtended
certain weight values, in terms of more and less, and in consequence were at once
arranged in a line distribution of plus and minus ; but the array of values showed
nothing of any interest, save the shortcomings of statistics and their inability
to analyze the condition in the material under investigation. I was interested in
this set of determinations for the reason that so many similar determinations
had been made with far less care than I had employed, and not infrequently far-
reaching conclusions had been drawn, the reason for which I could not discover.
The general result is simply that statistically determined weight is not a simple
character of the organism, although not infrequently so considered.
15

218

The Mechanism of Evolution in Leptinotaksa

Corresponding lines from the average and the large weights were also carried,
but are not shown in the figure because they would only complicate it with the
larger number of curves and add nothing to the results in that they were precisely
similar to those shown in the A series.

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weight as a single character, showing distinctly that it was not possible to purify
this material with respect to weight and reduce it to uniform pure lines.

In the years 1907, 1908, and 1909 I made a similar set of determinations upon
races of L. signaticollis from the Eancho Basosco location, these being bred out
in line cultures and selected on the basis of Aveight. The general result was the
same as in the two preceding instances, and there were no results but a lot of
values subtended by the animals in question and having no certain relation to the
organism.

Analysis of Heterogeneity in Some Simplest Characters 219

Much use has been made of the instances of improvement made in the domesti-
cated organisms by selection of " quantitative variations," and all are thoroughly
familiar by this time with the extensive use that has been made of these instances
by De Vries in the formulation of the mutation theory, and by Johannsen and
his followers in the development of the pure-line hypothesis. In all of these
instances it is assumed that there is " quantitative variation " ; but is it proven
that there is such ? Has it not been assumed that there are quantitative vari-
ations solely on the basis of the inaccurate findings of the statisticians ? In what
instance has a biometric worker really determined the nature and homogeneity
of his material before he began to investigate it through statistical methods?
Eecall, for example, the many determinations made by Pearson, Galton, and
others of the same school in the human species, with entire lack of homogeneity
of parentage, race, conditions of growth, present conditions of life, and other
factors that may enter into the production of the individual. It is results from
examinations of this sort that have led to the existence of " quantitative fluctuat-
ing variations " in the literature of the last two decades, and lead primarily to
the creation of the situation in this field that arose with the mutation theory.

The history of the sugar beet has been again and again used as an example
of the existence of " quantitative " or " fluctuating variations " ; but is the deter-
mination of the saccharine content of the beet the character, and does it repre-
sent this group of substances as they exist in the beet ? In this instance a heter-
ogeneous group of compounds, the saccharine content, has been removed from
the organism, is determined in percentages of total weight of the body from
which it came, but the extracted product may have many mono- and di-saccha-
rides in it, but is rated as " one character," and no attention is paid to its diver-
sity in composition and the fact that it is an extracted product and not a charac-
ter in the organism. As to the materials in the organism, there is relatively little
information, but that available indicates complex relations, the product of
divers processes at work. For the practical purposes of the sugar industry the
amount of " sucrose " that can be extracted is important, and the development of
strains that produce large extractable quantities is commercially desirable, but
extractable amount measured quantitatively is hardly a determination of the
character as it exists in the organism. There is nothing in the work to indicate
whether the differences in the saccharine content in the sugar beets are fluctua-
tions in quantity or not. In the mono-saccharides and di-saccharides present
there is remarkable specificity of the different substances in composition, action^
and relations in the organism, and differences of most numerous sorts may well
exist without being discovered or even considered by the statistical treatment of
the " character."

In this instance saccharine content is not the only " character " that has been
considered; form of the root, woody content, leaf character, and others have
come under the observation and manipulation of the beet grower, have been puri-
fied, and races derived having the desired commercially valuable characteristics.
In this practical work the operators, misled by the selection idea and the com-
placent uncritical methods of the statisticians, applied " measures " to the char-
acters, which although fairly useful to a limited extent for the practical purposes
of industry, did not and can not express or indicate the real relations of these
" characters " in the plants. These are most complex, and differences of all

230 The Mechanism of Evolution in Leptinotaksa

sorts are presented which can not be separated into rigorously limited classes of
quantitative and qualitative " variations." In no instance in operations of this
kind have the w^orkers been dealing with simplest characters, or even with unit-
characters in the sense of De Vries, but usually with an undetermined complex,
capable of subtending some mathematical value, and the mathematical value has
been regarded as a true representation of the character in question. It is largely
this sort of thing that has led to the establishment of the idea of quantitative
variations by De Vries as distinct from qualitative.

The fact that the continued " selection " of these characters does not result
in the production of unlimited divergence of the character, which is a diflficulty
in the minds of many, is not really an element in the present problem. It is
quite distinct, and it may well be asked whether any kind of character is
capable of unlimited " improvement " by any method of transmutation whatso-
ever. It seems that the very nature of natural substance would indicate that
there are limits to combinations, to arrangements, and to operations, and the
limitation of " selective action " by bounds that are not passable is not surprising.

The " difficulty " that has entered so largely into the discussion of the demerits
of fluctuations as an effective element in the modification of organisms, " that the
products of fluctuations can not be fixed in position," does not seem at present to
offer any difficulty. Fixity in position is simply stability of composition,
method of reaction, or both, and homogeneity of organization ; and it is precisely
this that the selectionists have not taken into their considerations and operations.
No effort has been made to obtain materials or characters that were single and
pure, and a heterogeneous mess extractive like saccharine content, that can be
measured in mathematical values, is the limit of their ability to analyze either
method or product or character. I shall have occasion to deal with this question
further when I come to consider some of the experiments in " selective improve-
ment," but the main point that I wish to make at this place is the uncritical use
of the biometrical efforts in the last two decades, as an example of an inefficient
type of investigating the origin of heterogeneity, in which the differences were
assumed to be mere differences in the amount of something present. Without
reference to the correctness of some of the conclusions regarding the lack of effec-
tive transmutation produced by these differences, and all of the consequences
that follow therefrom, it is becoming increasingly clear that diversity is not one
of amount present, but that the origin and nature of diversity is best stated as
integrated products of associations of productive agencies whose presence is nec-
essary for the production of the result and in whose array any break results in
nonproduction of the usual manifestation. In some instances it might be pos-
sible to express this integration in single terms of measure, but these can
never express the really complex nature of the result, in which there are
always present relations of position in space, interaction with adjacent materials,
the intricate series of antecedent series of events that preceded the end-result,
with the constant play of many incident agencies from within and without the
mass of the system, and to regard this complex series as a mere quantitative
swing of the amount present and as expressing in measure the amount of units
subtended on the biometricians' meter-stick, is to blind ourselves to some of the
most obvious facts of nature, and to indulge in the entirely useless and false cre-
ation of categories and definitions that are not in any way useful in investigation
or expressive of certainly determined facts of nature.

Analysis of Heterogeneity in Some Simplest Chaeacters 221

In 1906 I gave the results obtained in the study of the size of L. decemlineata
as determined statistically in two locations. In these determinations more than
the usual care was taken to have the materials of uniform composition, as regards
location, generation, and the possible presence of material of other generations.
The result expressed at that time in quantity showed only the customary array
in a polygon of distribution without significance. This I followed in the popula-
tion at West Bridgewater, Massachusetts, through 16 generations, and at Chicago
I followed the same investigation through 8 generations, with the same results at
both places. I was interested in doing this because of its bearing upon many
problems of evolution and transmutation. The net result of this and similar
examinations made in the same way was to show that the biometric method was
seriously invalidated by the failure to consider the action of place variation, and
the further false assumption that the materials collected in nature are homoge-
neous, even when collected in one location of the same generation and at the same
time. It was early evident in my determinations that the material was heteroge-
neous, and hopelessly so, as far as any effort to simplify it by biometric means
was concerned. I kept up the effort, using all of the precautions possible with
the collections made in nature, both with our native species and those of the
tropics, with the idea of giving this " quantitative variation idea " the fullest and
fairest test that I could, and for this express purpose I kept in the background
other ideas and methods of investigation.

" Graduated variates," as determined by measurement of stature or other
dimensions, have been the basis of much " quantitative variation " literature
and arguments, and the stature, or longest diameter of the mass of the system,
is often assumed to be a unit. Neo-Mendelian investigations have shown beyond
doubt that in many instances " stature " is a specific property of a mass and does
not blend into intermediate masses in fi:xed combinations, but segregates out of
any mixtures into which it enters. The experiences with plants of different
statures, of different sizes of animal forms that segregate out, and of which an
ever-increasing array of instances is being discovered by enthusiastic workers,
leads one to be highly doubtful of the " blended " nature of these characteristics
in any organism and of the existence of a long series of " graduated variates "
therein.

Without any exception, I believe, everyone has accepted the idea that at
some point in the series there was a certainty of there being " graduated vari-
ates " that were " quantitative fluctuations " in the " amount " of the character
present. The biometricians, starting with heterogeneous unknown material,
measured anything that was convenient and further complicated it with the
application of their treatment of the data, while the De Vriesians, starting with
refined material in pedigree cultures, attained an exactness of analysis and refine-
ment of results quite beyond the ken of the former school. The unit-character,
however, was not destined to long survive the searching analysis given " charac-
ters,^' and gave place to the conceptions of Johannsen and his followers. All
have retained the conception of the existence of quantitative fluctuations. In
reality the problem has not been analyzed ; rather with the recognition of more
and more refined units of operation in the investigation, and the establishment of
the reality of these, the fundamental propositions as regards the nature of the
diversity presented by the unit has been assumed and not investigated.

233 The Mechanism of Evolution in Leptinotaksa

Significant as are the investigations of Johannsen with the genotypes of
plants and of Jennings in the clones found in Paramcecium with respect to these
problems, there still remains in both the idea of quantitative as opposed to quali-
tative " variations " bearing much the same relation to one another that they do
in the original De Vriesian statement.

Since most of us can not admit the possibility of any " specific biological prin-
ciple," or vitalistic content in the phenomena of our science, it follows that the
essential processes in one of the most evident phenomena of organism, the role
of heterogeneity in transmutation and evolution must be capable of expression
in terms and in principles that were equally applicable to both organized and
non-organized bodies ; and the same principles of investigation should, with dif-
ferences in the means employed, be applicable to and give consistent results in
both sets of material aggregations. This working hypothesis may be formulated
as follows :

(1) The recognition that only physical principles are operative in the phe-
nomena classed as organic — a principle that is in entire accord with all of the
investigated and analyzed organic phenomena.

(3) The recognition that the characteristics of organisms are of the same
origin and kind as in non-organized bodies and are specific properties, attributes,
and conditions, and have in organisms precisely the same origin and relation to
the whole that they do in non-organized masses.

(3) The hypothesis that heterogeneity is the product of the composition of
the material by the conditions of the medium during the production of the com-
bination and influenced by the degree of purity of the combining materials and
presence of uncombined substances, which may influence the end-result in the
manifestation of specific properties and attributes, and be the active causes of
heterogeneity. These operations and results, when fully analyzed, will be
found to be but expressions of established principles of physical science.

An analysis of heterogeneity must, therefore, be made on the following
scheme :

(1) An analysis of the constitu,tion of the substance under standard con-
stant conditions of observation.

(3) The determination of changes in the medium upon constancy of the
substance.

(3) The determination of loosely combined or uncombined "impurities"
in the organic system, and the role which they play in the production of fluc-
tuations in the characters of the material.

(4) The isolation of non-fluctuating lines and study of the action of changed
conditions and introduced factors in the system, to determine the nature of the
so-called " fluctuations."

The defect in biology is that homogeneity has been assumed without being
tested at all until recent years, and then inadequately.

I began in 1904 an inquiry into this aspect of the " variation " problem, and
for this purpose I took the most variable and complex member of the group,
L. multitcBniata Stal, which I was certain would give me no end of trouble and
present about all the complications that one would expect to find in any organ-
ized body. In this I was not deceived.

It was recognized at the start that it was possible to obtain with the methods
in use an abundance of data, which could, with ingenuity, be arranged into

Analysis of Heterogeneity in Some Simplest Chaeactees 223

curves of " quantitative variations " and those that stood apart and were to be
characterized as " qualitative." These same data would, with proper treatment,
give an imposing array of constants of no value or meaning, as far as the analysis
of the origin and meaning of the very evident heterogeneity was concerned, and
having already obtained an abundance of this sort of information regarding
these materials, at the onset of this study I discarded entirely the method and
attacked the problem from the point of view of genetic composition and the
effects of conditions in the medium.

I have sho^^^l in Chapter II something of the natural complexity of this
material, of the forms that exist in nature and of the wide range in many of its
attributes. I was forced into this study by the attitude of De Vries in the
mutation theory and the position of Johannsen in his 1903 paper, and by all of
the current writers at that time, who as a body held to the two categories of
" variation." Since that time the progress of neo-Mendelism has produced
many changes in the viewpoint, with closer and closer approach to the concep-
tion of heterogeneity conceived of in purely physical terms. It is clear that
De Vries thought of mutations and fluctuations as distinct, and that the latter
were either of germinal or somatic origin, distinguished by the fact of their
inheritance, and if so, capable of accumulation up to certain limits. East in
1911 has in common with many others limited the term "fluctuation" and
placed the germinal variations as follows : " A fluctuation is a non-inherited
variation produced upon the soma by environmental conditions, while the in-
herited variation, the mutation if you will, is any variation quantitative
or qualitative, that is germinal in character." It follows from this that any
diversity in the observed series that is not inherited is then the product of the
environment upon the organism during its ontogeny. Since it was non-visible
in the following generations, it follows that it was absent as a part of the
germinal complex, which may or may not be true, and the further assumption
that since it was not inherited as per test it was the product of incident external
conditions upon the organisms during its growth. This is characteristic reason-
ing from effect to cause, and then broadly applying the result as a criterion to
separate the kinds of heterogeneity present.

FORM-INDEX.

For the study of form-index I took a body-index as representing a measur-
able character of the entire mass, and comparable to stature, tall and short
in some of the neo-Mendelian operations, and roughly equivalent to the same
index that might be derived from inorganic masses. These animals, like other
organisms, are during ontogeny changeable in some of the measurements of the
body that shift with the state of nutrition, reproduction, and age, so that I used
only measurements that were free from these sources of obvious error. In
figure 2 I have shown the measurements taken, the distance between parallels
passing through the anal apex and the anterior margin of the elytron (1), the
same measurement between the parallels passing through the anterior and pos-
terior margins of the pronotum (2), and the corresponding measurement of the
epicranium (3) . These are not influenced by the conditions of nutrition or the
changes in form incident to breeding. They were added to give the length or
longest measure of the mass ; this was divided by the greatest breadth (4), taken
as the greatest distance between the lateral margins of the pronotum, and this

224 The Mechanism of Evolution in Leptinotaesa

gave body-index. The breadth of the elytra would have been useful, but too
much difficulty was encountered in eliminating obvious sources of error in the
measurement. This index is a good indicator of the general " body-forms " so
ofton useful in taxonomic work and a real characteristic of the mass. On
plate 26 are shown some of the different forms of body which this species
exhibits, and in the main can be grouped into three assemblages, of which the
multilineata form represents one extreme, some of the melanothorax types the
other, and the typical multitceniata the intermediate. Any one of these types is
" variable " in its form as found in nature and even in cultures that have been
purified with reference to the color and pattern differentials. My problem was
to purify one of these forms if possible, and then analyze or produce in it
" variations," in the hope of being able to gain some clue to their causation and
significance. I took the multilineata type as the start in the problem as being
the simplest, and the problem was to obtain a constant and pure multilineata
type.

In the years 1904, 1905, and 1906 many preliminary tests were made, so that
at the end of 1906 I had 6 lines of this material that were thought usable as a
basis for the study. In the course of breeding experiments with this species I
had found and identified three sets of factors that were productive of form and
form changes in the species L. multitceniata, and I had been able to prove the
reality of these by the repeated transference of them to the species L. decem-
lineata and L. oblongata, replacing in the normal form the " body-form factors "
from the strains of L. multitceniata. The materials which I had at the end of
1906 were 6 races with the form-factors so purified by being repeatedly extracted
and purified in genetic operations that the strain was highly constant in both
size and shape, ranging well within the index limits of 2.500 and 3.000, and
there also had been an effort to reduce the complications in color and other
characters as far as was possible, so as to give a homogeneous-acting biotypic
form. The strains were now in the condition that would have admitted being
characterized as highly refined " biotypes " showing only the usual expected
" fluctuation " about an average value.

In 1907 I carried 5 of these through 2 breeding-periods, or 4 generations in
all, and of the 5 all showed the same results in that they were constant to the
type of the line, with the usual range of divergences in the character chosen for
the examination. All would have been characterized as constant stocks, and
the problem became, what was the cause of the range presented by the index ?

The sixth line of the original series at the beginning of 1907 was composed
of 62 males and 66 females that were the progeny of a single pair of parents
and emerged from the hibernation in the winter of 1906-07, and were an ex-
tremely uniform lot of individuals. These were divided at random into 4 lots
of 15 each, and within each of these lots 5 pairs were mated at random, and these
bred out through 2 generations in March, April, and May 1907, and entered
hibernation in the early days of June. The progeny from these matings were
imiform and like those from the 5 main stocks reared during the same time and
under the same conditions; 17 of the pairs gave progeny and the range in the
index of these, as well as the same character in one of the 5 " stock " lines, is
shown in table 28 for the first generation of 1907 and in table 29 for the second
generation of 1907.

Analysis of Hetekogeneity in Some Simplest Characters 225

All that the tables show is that the 5 stocks and the 17 pairs were like in their
index and with ranges of the individual fraternities that in the main were
similar. In all respects a " pure " biotypic strain had been isolated in this
complex species, showing the " customary fluctuations." What was the nature
of the fluctuations ?

Table 28.

No. of mating.

1. gP

2. gP

3. g P

4.gP

5.gP

6. g P

7. gP

8. g P

9. g P

10. gP

11. g P

12. g P

13. gP

14. gP

15. g P

16. g P

17.gP

Stock 2 Mass. . .
Stock 2 Pt. A . .
Stock 2 Pt. B...
Stock 2 Pt. C...

Parents,
index.

2.757

2.741

2.569

2.701

2.564

2.778

2.731

2.521

2.631

2.700

2.754

2

2.761

2.965

2.581

2.913

2.600

r2.924

2.531

L2.754

2.634
2.595
2.781

2.558
2.962
2.754
2.754
2.776
2.983
2.728
2.725
2.940
2.799
2.961
2.892
2.760
2.769
2.794
2.622

2.810

2.728
2.835
2.744

2.911
2.701
2.811

Fraternity No.

1-gI

2 gl

3.gl

4 gl

5. g I

6.gl

7 gl

8. g I

9.gl

10. g I

11. g I

12. g I

13. g I

14. g I

15 g I

IC. g I

17. gl

M. 2. g I . . .
M. 2. g 1(A)
M. 2. g 1(B)
M. 2. g 1(C)

Progeny.

Range of inde.x.

{I

(M.

If.

{f.

{?:
{I
{?:

{?:
{f.

(M.

If.

JM

IF.

{f.
{I

!?.
U':
\f.

1

2

4

7

3

2

9
12

4
11

19
21 18

1 .

1'
7i
8| 5

2 1
3l 1

Totals.

431
44)
64)
49)
27)
34)
23)
30)
S3)
36)
42)
39)
38)
46)
16)
21)
27)
34;
49)
60 i
21)
11)

J?!

24)
31)
17)
171

87

53

84

37

109

32

19

55

34

118

551
631

163 ) 097

164 J '*'"

37

14)
23)
35)
37)

23)

72

Total.

.1817

Three possibilities existed with regard to these ranges in the index: (a)
they may be entirely environmental in origin and show simply the plasticity
of the material in its response to the conditions of the medium; or (6) the
range may be due to " impurities " in the organism, which are present in a more
or less loosely combined state, but produce divergence and a range in some of

226

The Mechanism of Evolution" in Leptinotaesa

the characteristics, as do similar conditions produce like ranges in characters
of non-organized matter; and (c) it is possible that the range is due to many
small actual differences in the constitution — " germinal variations " — which
the methods used or the criteria of the index used are not able to separate out
into the component groups.

Table 29.

No. of mating.

Parents,
index.

Progeny.

Fraternity No.

to

Range of index.

Totala.

gl

gl

gl

gll

gl

gl

gl

gl

gl

gl

gl

gl

gl

gl

gl

gl

gl

2.gl

2. gI(A) ...
2. g KB)...
2. gI(C) ....

2.761
2.749
2.560
2.739
2.570
2.790
2.738
2.529
2.619
2.700
2.752
2.689
2.766
2.969
2.580
2.900

2.600

r2.951

2.600

L2.754

2.630
2.594

2.785

2.599
2.931
2.751
2.760
2.771
2.870
2.731
2.700
2.901
2.791
2.898
2.894
2.715
2.769
2.763
2.715

2.825

2.700
2.815
2.749

2.911
2.715
2.826

1. gll ....

2.gn ....

S. gll ....

4. g n ....

5. gll ....

6. gn ....
7.gn ....

8. g II ....

9. gn ....

10. g u ....

11. g n ....

12. g II . . . .

13. g II ....

14. g n ....

15. gll ....

16. g II

17. gn

M. 2. gn ...

M. 2. g 11 (A)
M. 2. g n(B)
M. 2. g 11(C)

{?;

JM.
IF.

!^;
!^:

fM.

If.

(M.

If.

fM.

If.

(M.

If.

!^;

JM.
IF-

{?;

(M.

If

(M.

If.

fM.

If.

f M.

If.

fM.

If.

f M.

If.

JM.
IF.

If:

11

10
46
4:
20
17
46
13
13
14
13
17

9
24

9
11
17
21

9
15
16
16
16
19

4

7
48
47
61
47

3

4
14
11

122
151

7

11
24
25
11
12

417
46 J

87

154)
159 j

114 1
79 j

l'^2l 275
103 J ^'^

651
81]
431
59]
531
61 ;
52]
491

313

193

146

102

;jii4

101

157

95

111

461
49 j

571
54 j

tl] ^^3

38

14)
24)

21)
31 J
57)
611

52

118

6501
562]

50)
76)
71)
73)
36)
42f

1212

78

Total 4113

The first attack was made to determine whether the " environment " was
either primarily or even partly responsible for the condition foimd in the range
exhibited in the index. For this, therefore, I set up 12 breeding-tanks of the
construction and arrangement of control shown in figure 32 (Chap. I), and
arranged to give constant conditions of temperature and moisture, air-move-

Analysis of Heterogeneity in Some Simplest Chaeacteks 227

ment, evaporation, and soil-moisture. The temperature was set at 21° C, the
relative humidity at 75 per cent, and the air was changed once each minute,
giving uniform evaporation rates.

In September 1907, I mated, under the controlled conditions, 3 pairs from
pair 1 of stock 6, 2 from pair 8, 4 from pair 10, all of these taken at random in
the population of each parent fraternity. The method of doing this was to
separate the sexes, place each in a dish under a dark or opaque cloth, and then
draw them out singly as needed for the matings, thus preventing any possible
selection in the matings of the pairs within the population. These 9 pairs of
random matings in different lines were placed in the tanks, and there 8 of them
gave progeny. At the same time 3 mass cultures were mated at random, and
consisted of 3 of each sex taken from the progeny of pairs 2, 14, and 16, and
these were allowed to breed under the conditions of the experiment as were the

Fig. 32. Diagrams showing different measurements talcen to determine the body

index in these animals. (See text.)

pairs, all giving progeny. The small size of the breeding-chambers made it
necessary to grow only small populations, but each pair was allowed to breed
until death ; the elimination was made in the eggs and was, of course, purely a
random one, and consisted of destroying about half of every batch of eggs laid.
Mated pairs and mass cultures gave normal, strong progeny in the middle of
October, and presented no difference in the indices or in the range thereof in
any of the populations resulting. At the end of October each line was continued
by another random mating, and in the same tanks as before, and these all gave
progeny, reduced in number as before, in the first half of December 1907.
The resulting progeny, however, showed exactly the same ranges in the index
as did the previous generation and as did the other portions of the series, namely,
the progeny of the other stocks that were reared at the same time, but not under
accurately controlled conditions. I was greatly surprised at this result, having
expected that there would be an alteration of the range in the index in at least

228

The Mechanism of Evolution in Leptinotarsa

some of the lines from the mere fact of growing them under uniform and con-
stant conditions ; but no such effect was found.

All of the strains hibernated from late December 1907 to the first of March
1908, and were then mated within the same lines at random and in the same
set of conditions, giving a generation in April and a second one in late May and

Table 30,

No. of mating.

1. gn(A) .,

l.g 11(B) .,

l.g 11(C) .

8. gn(A) .,

8. g 11(B) .,

10. g 11(A) .

10. g 11(B) . ,

10. g n(C) . ,

10. g n(D) .

1. g m(AA)
1. g in(BA)
1. g in(CA)

8. g III(AA)
8. g m(BA)
IC. g ni(AA)
10. g III(BA)

10. g m(CA)

10. g III(DA)

Parents,
index.

M.

F.

2.715

2.841

2.619

2.737

2.771

2.742

2.779

2.601

2.658

2.711

2.811

2.614

2.719

2.732

2.748

2.755

2.644

2.791

Progeny.

Fraternity No.

1. g ra(A)
1. g ni(B)
1. g ni(C)
8. g ni(A)
8. g in(B)
10. g m(A)
10. g m(B)
10. g m(C)
10. g in(D)

Range of index.

Totals.

341
35)
23)
29)
331
40)
26)
23)
20)
19)
14)
16)
16)
17)
28)
23)
16)
24 f

Total 436

2.779

2.651

2.741

2.618

2.771

2.754

2.786

2.870

2.593

2.911

2.701

2.891

2.658

2.840

2.631

2.843

2.712

2.935

1. g IV(AA)
1. g IV (BA)
1. g IV(CA)

8. g rv(AA)

8. g IV(BA)
10. g IV(AA)
10. g IV(BA)
10. g IV (CA)
10. g IV(DA)

1^; :::

1

2

4
1

5
4

3
6

1
5

1
1

2

2

1

JM. 1...

If. ...

1

"i

2

2

3

7

9
11

4
5

1
3

1
1

(M. ...
(F. ...

1
1

7
5

11
10

4
5

2
1

1

\?: :::

2

1
. 1

4
1

1
2

1
6

"3

1
1

"i

\f :::

"i

1

1

3
1

5

7

2
3

1
1

"2

1
1

|M. ...
IF. ...

1

2

4

5
7

8
10

4
5

1
3

"i

\f. :::

1

1

1

2
3

9
4

4

2

2
3

"i

1
1

IS :::

"i

"2

1
1

2
6

4

1
2

1

1

"i

!?; :::

1

1

2
2

3
2

6
4

3
3

1
2

1

40

52

48

I 25

i-

!'■

I 35

I 23

I 30

Total 336

early June of 1908. Identical methods were followed in both of the 1908 gen-
erations, with no change in the result, as far as the range in the index was
concerned.

It was most surprising to find that in the different lines all carried under
controlled conditions, with random matings, that the range in the index was not

Analysis of Heteeogeneity in Some Simplest Characters 229

in any way changed or even deformed by the removal from the organisms of the
supposed forces productive of the range in the character. These results I have
shown in concentrated form in tables 30 and 31.

Table 31.

No. of mating.

Parents,
index.

Prog'eny.

Fraternity No.

M. 2. g II

r2.76l
-^2.894
L2.668

2.750"!
2.692 )■
2.6S1J

M. 14. g II....

r2.708
^2.692
L2.923

2.750-)
2.774 y
2.661J

M. 16. g II ....

f2.778
■i 2.810
L2.746

2.7641
2.689 y
2. 891 J

M. 2. g III....

r2.751
^2.863
12.641

2.811"1

2.751 y

2.554J

M. 14. g III...

r2.776
-^2.781
12.894

2.846"!

2.651 y

2. 758 J

M. 14. g III...

f2.777
-^2.731
L2.6I8

2.58n
2.774 y
2. 841 J

M. 2. g III..
M. 14. g III..
M. 16. g III.
M. 2. g IV...
M. 14. g IV..
M. 16. g IV..

fM.

If.

{f.

f M.

If.

(M.
5F.

Range of index.

Totals.

177

40?
44)

261 fi7
42 1 ^^

Total.

All of the stocks went into hibernation in June and remained quiet until
October of the same year. I then decided that the series was not worth the space
and attention that it required, and therefore drew lots for three to be continued
for two more generations in the autumn of 1908. The choice fell to pairs
8 g IV (AA), 10 g IV (BA), and mass culture 2 g IV, and these were continued
for two generations more, completing the series early in January 1909. The
result was in no wise changed, as is shown in table 32.

In the literature, ranges in characters of the kind used are commonly
attributed to the action of incident environmental conditions of temperature,
moisture and the like, and to conditions of nutrition, all of which were con-
trolled in the series, and were uniform and optimum, and should have given
uniformity and decreased ranges in the attribute under consideration if the
differences were due to environment as is commonly asserted. The entire series
in this direction was killed off at this point, nothing of any value being expected
from its continuation,

I do not know of any other tests of this question that have been made with
the same refinement, or even any tests of any sort. If I am not entirely mis-
taken, the instances of range in fluctuations of characters have been of reason-
ing from effect to cause, and instances where observed ranges were not other-
wise accounted for have been attributed to this " environmental action," which

230

The Mechanism of EvoLUTioisr in Leptinotaesa

has become a dumping-ground for differences of this general class. My first
proposition that " environment " was responsible for the range seems to have
been answered negatively in a certain and emphatic manner.

Two possibilities remain : either the range is the product of " impurities "
in the substances or due to minute differences in the constitution of the material
which the methods used did not entirely isolate. I sought to test the question
of the possible existence of minute " germinal variations '' which might be
small differences in the composition of the substance itself, through mating
individuals of identical index, to determine if it were possible to obtain within
the group minutely characterized races.

Table 32.

No. of mating.

Parents,
index.

M.

Progeny.

Fraternity No.

Range of index.

Totals.

10. gVI(BA)A

10. g VI(BA)B

10. gVI(BA)0

10. gVI(BA)D

10. g VI(BA)E

10. g VI(BA)F

10. gVn(BAA)A ....
10. g VII(BAB)A ....
10. gVn(BAC)A ....
10. gVr[(BAD)A ....
10. gVn(BAE)A ....
10. g Vn(BAF)A ....

2.554

2.557

2.679

2.681

2.753

2.758

2.991

2.980

2.904

2.911

2.893

2.891

2.556

2.553

2.677

2.678

2.753

2.756

2.989

2.993

2.901

2.912

2.895

2.890

gVn(BAA) .,
gVn(BAB) .,
gVnCBAO .
g Vn(BAD) . ,
gVn(BAE) ..
gVII(BAF) ..
g Vni BAAA
g Vni(BABA)
g Vin(BACA)
g Vni(BADA)
g Vm(BAEA)
g Vm(BAFA)

(M.

If

\%:

(M.
IF.
f M.

If.

(M.

If.

(M.

If.
5 M.
If.

If.

f M.

If.

(M.

If.

22} 59

341

43]

321

35)

40

45j

^!-
il 5^

Total 934

30 I
29'

i!^06

401
431

59

83

77

85

In October 1907, I mated from stock 6, line lOgVI (BA) (table 32), 6
pairs, when both parents had the same index, but reared under the conditions
of the general culture quarters. The progeny obtained from these pairs matured
at the end of November and showed some indication of the influence of the
method that had been practiced. Early in December I again mated from the
same lines pairs having the same indices as in the first instance, and obtained
a second generation early in January 1908, in which there were unmistakable
indications of the action of the operations in some of the lines, but by no means
in all, a condition that might mean one of several possibilities. All hibernated
from the middle of January to the first of March 1908, when they were forced
from hibernation and mated. In table 33 I have shown the conditions of the
series in the two generations of 1907.

Analysis of Heterogeneity in Some Simplest Charactees 231

Three lines shown in table 33 showed distinct tendencies to become restricted
in their ranges, and these three, 10 g VIII (BAAA), 10 g VIII (BACA), and
10 g VIII (BADA), and were preserved for further analysis to see if continued
operations of the same kind would further restrict the range in the fluctuations
of the index. The remainder of the lines were killed off or used for other work,
having given no indications of curtailment of the range as a result of the opera-
tions. The three lines reproduced between the middle of March and the middle
of June, gave two generations in which the same standard of parental indices
were maintained in both generations, with the result that at the end of the tenth
generation in June 1908, three clearly defined groups had resulted, and which

Table 33.

No. of mating.

Parents,
index.

M.

Progeny.

Fraternity No.

Range of index.

Totals.

8. g IV(AA) .
10. g IV (BA) .

8. gV(AA) ..
10. g V(BA) .

M. 2. grv ...

M. 2. g V

2.625

2.770

2.753

2.701

2.650
2.761

2.883

2.681
2.891
2.714

2.851

2.741

2.761

2.626

2.741
2.789
2.675

2.741
2.814
2.762

8. g V(AA)
10. g V(BA)
10. g VI(BA)
10. g VI (BA)
M. 2. gV...

M. 3. gVI..

JM.

If.

fM.
IF.

fM.
IF.

371

64

75

1S3

Total 515

could no doubt have been maintained in the same condition and range of index
as long as required. The differences between the three strains were so small that
it was not possible to certainly distinguish them in the cultures by inspection,
and only measurement could reveal the true state of affairs.

The condition in these three strains continued through the four generations
that they were carried, showing that there were minor differences in the original
" biotypic group " which could be isolated by more minute methods of analysis
and perpetuated by common methods of isolation, proves that the original
" fluctuations " are not the simple set of " environmental " disturbances they
are sometimes asserted to be.

In the autumn of 1908, after hibernation since June, the three cultures were
again put to breeding, and two more generations of precisely the same character
were produced, indicating further the constant nature of the small differences
that were being dealt with. In the last three generations it was found that
there was a tendency for the groups with the higher and lower indices to
transgress the bounds of the variability of the original " biotype " stock, espe-

232 The Mechanism of Evolution in Leptinotaesa

cially in the direction of a higher index, which was away from the condition in
the species as a whole. It would have been of interest to have followed out this
suggested tendency in the upper group, but space in my laboratory was at a
premium, so that the entire culture at the beginning of 1909 became the victim
of a struggle for space. In any event the culture had shown clearly the main
point at issue, namely, the presence in the population of minute differences,
fixed in the germinal constitution of the stock and capable of being isolated, and
held as distinguishable groups when the proper refinement in method was
employed, showing clearly that the " fluctuations " are not all uninheritable
in these " biotypic " lines.

It must be remembered that these lines were not "genotypes," and as
" biotypic " lines are not fully comparable with the " genotypical lines " with
which Johannsen, Shull, East, and others have worked, and concerning which
so many of the recent qualifications have dealt. However, as most narrowly
delimited lines of biotypical breeding, they are good examples and do show, in
these lines, that the problem of the " fluctuations " is by no means as simple as
it might seem from some rather emphatic statements as to the limitation and
nature thereof.

The issue might be raised that the original group was not a true biotype at
all, but a complex assemblage, and this may be true ; but where is the limit to
be drawn? With the ordinary methods in use the first group satisfied the
requirements fully, and remained as a constant assemblage with the customary
range in the index after once it was established without further attention or
selection on the part of the breeder. If I had gone further no one would for
a moment have even thought of the group being anything but a simple one.
Nor do I know how complex it was in any instance, nor how many of these
minor strains I could have isolated from the mass of a single population, with
time and refinement of method. In all of the pure-line work the same seems to
be true, that with refinement of method the lines may well become finer and
finer, less and less remotely separated, and lines only in the sense that our
methods of analysis and pure breeding hold them as such, and in a most
unnatural condition.

The third possibility remains, that some of the " fluctuations " may have been
due to loosely combined " impurities " in the constitution of the lines, which
would in this instance be in the nature of substances present as "factors,"
which, not being in the proper place and relation in the system, or lacking the
proper complement, gave no direct resultant of their own in the reactions of the
other " factors " of the mass, but by their presence served to disturb the normal
workings of the system and thereby produce distortion of the action and deforma-
tion of the resultant " character." It is well known that in the non-organized
bodies, this type of deformation is common ; indeed is the most common one in
nature, and there is no a priori reason why the same may not well be true in
organized matter, although not so easy to test or to certainly determine.

In the culture 10 g VII (BAF), and again in the next generation of the same
line, as shown in table 32, a wide range in the index is shown, and the same
method that produced the restriction of the range in the other cultures had
failed in this one, so I tested it for two possible causes of the condition found.

First, four pairs were put into controlled conditions and carried through four
generations with the results shown in table 33, and the graphic representation

Analysis of Heteeogeneity in Some Simplest Chaeactees 233

in figure 33A. No decided influence of the uniform conditions in restricting
the range of the index was found, the range at the end being about the same as
at the start.

Second, there was the possibility of " impurities " in the constitution of the
strain. I went over the living stock carefully and over such of the preserved

60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10
60
50
40
30
20
10

I 1

l.c"'A-c'

^^r£

/j Ll\

^--^212^=-

-■ i i

c" c'"| Ic'

P A , /[\

f ^ -r^ "^ °~t ^ S~ ■ '"^

1 V 1

"•" i^ i yV A

K^\ IV- C, ^ \ '"

/A^V/ ^ / \/ ^ ' \

-'2Lr^3^t:_\^ iZZ^t ^5T^

it IT ^ it m

1 V-C"; 1 1

1 ^ C 1 IOBAC,\ 1, lOBAB

C; ---kl/ I08A^ /^. A

y-r.^^ } i'v_ /T\ / X '\

^z^'" ^•-'tHs •■-. •^- - / ^^^-^ \', \

' / ! c - - . ^

^c", ^^

C" /S^' i \ yv.

■^V^-\ i ■-. -^ .'vIOBA

1 ;

1 1

1 1

/k. --"'A 8AA^..-|-

/..-^.J^- ..V<•■^^IOBA

k. 1 1

^i 1 1

Zlt-^Ip -i-

^ 3 It^ '

/ /I V_| \ BAA^.I- ^10 BA

^^-■'' j^-^'"^-_ ^-7-^ —1^ "N :^ >v

' • I

IT 13: i

i ^ ^^ t

aZTSd-

,-^ ;^ \ ! V 10 B . . 8A

^^ .,''i -— (__ ^--^ ^<^'^' ^

' 1

a f .prJ-b

^^ IT . __ c

z:z^iji:!l!!^^--d

t ^Jnix^^S

Xl!^^

a, ", l'/ .c

^"0SC ^-:^ ^„v-

b-Z^E^^^ ^^K^^ it

^^5-4^^, _,|;?^ \

Fig. 33. — Diagrammatic representation of results obtained in the attempt to
breed pure lines as regards form-index in L. multitwniata. (See text.)

material as I had, and found in this " line " a suggestion upon which I went to
work. In all of the biotypical lines of this muUilineata series the spots a and a'
are parallel with the median axis of the body and are also moved close thereto.
I found in this strain that these two spots were in many of the specimens some-
what divergent at the anterior end and were, on the whole, more widely separated
16

334 The Mechanism of Evolution in Leptinotaesa

than in the other lines. I knew from other portions of this investigation that
the position and relation of these spots is directly the product of a complex set of
body form-factors and that this complex set was capable of dissociation, and that
in some instances the factors went far astray as loosely attached members in
other portions of the system. There was no possible method of even guessing at
what the specific cause of the disturbance might be in this instance, if it were
that at all, and there were so many possibilities that any attempt to test out its
nature within the species L. multitceniata was hopeless or at least not hopeful. I
tried the tests with another species, L. oblongata, in which there is not present
the factors productive of the wide separation and divergence of the spots a and a'.
In this form they are always parallel and closely set to the median line. I had
some pure-line cultures of this species at that time and crossed the suspected
material with the L. oblongata lines, and the F-^ results showed at once that the
suspicion was well founded, as the four fraternities reared showed strong indi-
cations of the multilineata type of pattern ; and in the Fg results some forms
came out in the progeny that were in pronotal pattern multitceniata in every
respect, with the widely divergent a and a', as well as in other characters. The
result was clearly an indication that the " biotype strain " had something in it
that gave, in the new opportunity which cross-breeding gave it, the capacity to
form new associations and initiate diversity of characters, while in the first posi-
tion its location in the system, or lack of proper complements allowed only mani-
festation by the lack of stability found in the series of narrowly restricted strains.

Thus far I had found only a possible source of contamination, but did not
know what the contaminator was, excepting that it visibly deranged the two
spots mentioned, a rather trivial indicator. It was hopeless to try to purify the
strain through the use of neo-Mendelian methods, as the characters were too
fine for certain recognition, and the fact of crossing would only introduce addi-
tional complications. The only resort left was the effort to purify the race
through the elimination in the breeding members of any indication of the con-
tamination as revealed in the spots a and a'. This I began early in 1909, paying
especial attention to the character of the spot system, with the result that at the
end of 1909, having put the stock through four generations, that the range in
the index had fallen to less than half of its former range, even though the indices
of the mated pairs had not been so rigorously coincident as in other matings.
The figures from these generations are shown in the table 34.

After hibernation from the end of December 1909 to the first of April 1910,
the stock was put to breeding again, this time without any attention to either
pattern or index, mated at random for the first mating. The progeny showed
no change; and again mating as a group culture, the last generation reared
showed only the same narrow range in the index. This rather rough treatment
of the line gave some indication of its purity and fixity, as well as the freedom
from the formerly present factor or factors that had been productive of the wide
range in the index in the earlier generations, and there is no doubt that the proc-
ess of elimination carried on as I had begun it would have resulted in the for-
mation of a race that was free from this one impurity and would have been nar-
row-ranging in the index, as were some of the others.

Not all of the lines gave results as easily as this one, or even were capable of
solution at all. Thus, the line shown in 10 g VII (BAB), table 33, was of the
same wide range in the index, but the same source of impurity was not present,

Analysis of Heterogeneity in Some Simplest Charactebs 235

or at least was not to be detected by the same methods. It was tested with con-
trolled conditions to no purpose, and no clue was found to the range in the index
present, so that selection through 1909 and 1910 failed to change the array in
any respect. This may be interpreted in different ways, but the range was surely
not environmental, else it would have shown at least an indication to respond
to regulated conditions, which it did not at any time; or if smaller germinal
differences were present it must surely have been possible to have done at some
time the same here as in other lines, but more probable seems the hypothesis
that there was some " impurity " present, which my methods of detection did
not reach, and which by its presence in the system continued in spite of rigorous
methods to produce wide ranges in the index of this biot3rpic line.

The results in this series were a decided surprise to me, and indicate clearly
the difficulties of the problem and the immense complexity thereof. As far as
the cause of the " fluctuations " was concerned, the outcome was not expected or
anticipated on the basis of the assertions made regarding these differences in
organisms, and the quite general independence of the index from environmental
influences is striking, as is the possibility of finer divisions and the presence of
impurities. It would be of interest to completely analyze the nature of the
entire fraternity resulting from any pair of parents, and I have often thought
that it would be worth while to attempt it, but I have never been able to obtain
the space or time free from other work to allow of the attempt being made.

At the same time that the experiments with the muUilineata biotype series
were in progress, another part of the same species gave interesting confirmation
of the same principles, but, as might be expected, with diversity in the details.
In the course of the preliminary testing of this species I had separated out a
form " biotype " in which the index was low, on the average about 1.750, with a
range upwards to about 2.000 and on the lower side to about 1.550. The same
elimination of color and pattern differences were practiced, so that at the end of
1906 I had a weU-defined set of four races of this material, which were utilized
in some tests, that are companion to those already presented.

The original stocks were uniform in aspect and in behavior in crosses and
in every respect appeared as homogeneous racial line cultures, and all showed
essentially the same " amount " of " fluctuations " in each fraternity, as shown
in figure 33B, in the pairs a, h, c, and d; and in the following generation progeny
of matings from these same fraternities showed the expected range in the " fluc-
tuations." These were random matings within the population, reared under the
conditions of the general breeding quarters, without any control of the condi-
tions. I then reared them through four generations under the same conditions
as in the muUilineaia series, with the same general result in the two lines carried
from pairs a and c. This difference was found in that in the line c the modal
condition remained at the same point on the scale, while the mode of the a
series was constantly shifting its position. Neither of the lines showed the
slightest indication of restriction in its range as a result of the controlled and
more uniform conditions of development, so that the environmental influences
were apparently not operative in this set of " fluctuations " more than in the
first one.

The next step in the testing was the attempt to breed minor races from the
lines, as was done in the first series. With the line c, three sets of matings were
made from three points in the array of the fraternity, as shown in figure 33B.

236 The Mechanism of Evolution in Leptinotaesa

Two of these gave at once the results that had already been obtained in the other
series, giving in the sublines c' and c" fraternities of decreasing range in the
index and increasing isolation from one another. The third mating, c'" , did
not respond to this treatment, and throughout the test retained its range in the
fluctuations and showed considerable shifting of the mode during the course of
the four generations. The c subseries shows, at any rate, that in the population
there were present minor conditions that could become fixed as minor races by
the use of appropriate and delicate methods of analysis and isolation. There
were clearly present in the same fraternity those that did not respond to the same
treatment, and which retained the range in the index and showed a shifting
mode. It is clear that the condition in the original c line was not a simple one,
and this may be entirely responsible for the failure to respond by decreased fluc-
tuations to the conditions of the medium when held uniform.

From the a line four matings were made, only one of which was found to
respond to the conditions of the effort and become restricted in the range of the
fluctuations as in other series, while the other lines preserved their original
range of index and the shifting of the mode, so that the result, which is not repre-
sented in the figure, indicated only the complexity of the assemblage, the absence
of environmental influences, the presence of minute fixable differences, and sug-
gested the existence in the strain of agencies invisible in manifestation as to
characters in the usual sense, but productive of unstable conditions, admitting of
such diversity in the range of the indices under investigation.

The entire series is suggestive of the conditions that may be expected in a
large number of characters, and the findings are more in line with the conditions
found in the simplest color-characters than might at first have been expected.
From time to time other series of determinations have been made in the effort to
analyze these problems of " variation," and thus far with identical results. In
the juvenile stages similar dimensions, especially the head-breadth, have been the
basis of some similar examinations, with entirely similar results. It is not nec-
essary to add to the already rather long mass of data presented, in that nothing
new in principle is gained and added cases are not of interest at this place. The
analysis of the meaning of heterogeneity in these simplest characters has shown
interesting developments in this material, but further discussion and analysis of
the situation is best deferred until after some data of heterogeneity in systems of
pattern and structure have been described and analyzed ; then it will be profitable
to return to an analysis of the entire problem of heterogeneity in the individual
and the population.

CHAPTER VIII.
ANALYSIS OF HETEROGENEITY IN COMPLEX CHARACTERS.

COMPOSITION AND MODIFICATION OF THE PRONOTAL PATTERN.

The pronotal color-pattern of this group, the genus leptinotarsa, affords an
opportunity for the investigation of heterogeneity in a highly " variable "
organic " character." The species as a rule are narrowly limited in distri-
bution, and some show minor habitudinal divisions. Within their habitats they
live in restricted colonies, as described in Chapter II, and are, therefore, localized
in differing habitats between which there is little interchange of populations.
These conditions produce isolated populations in which it is possible to test
some of the relations between the " variation " phenomena and the conditions of
existence. The pronotal color-pattern is chosen as a subject of observation
because of its composition of exactly localized elements and the ease with which
differences may be estimated, seriated, and recorded. It is a complex of simplest
characters, some of whose departures have already been analyzed. The pattern
of this part may be considered as a system largely independent of any of the
other parts.

IN LEPTINOTARSA MULTlTitNIATA.

In species like L. multitceniata, examination of a collection of a thousand or
more individuals, taken at random over its geographical range, shows a wide
range of pronotal pattern, as shown in figure 34. Conditions from a totally

Fig. 34. — Showing great diversity in color-pattern in Tj. multitwniata througli-
out its range. The first appearance of these patterns on inspection would seem
to warrant the view that their aspects were entirely without order or uniformity.

black surface to one with only the simple spots present are found, and between
these are all sorts of patterns, of differing amounts of pigment, and differing
combinations between the elements. In a given generation in some locations in
a large sample it is possible to find all or nearly all of the variations shown, while
in other locations and generations only a few are present. The treatment of a
population showing a character of this diversity depends largely upon the point
of view and the philosophical background applied to the problem.

237

238 The Mechanism of Evolution in Leptinotaesa

The simplest and easiest method is to assume that this condition represents
wide fluctuation in the quantity of pigment and that it spreads in all directions,
producing the fusions and different patterns found. Upon this basis the essen-
tial phenomenon is the amount of pigment and the variation is one of quantity.
In my 1906 paper, page 99, the place variation of L. muUitceniata at Guadalupe,
Federal District, Mexico, is given for six generations, i. e., 1903, 1904, and 1905,
from the biometric standpoint, and is expressed in terms of the area of the prono-
tum covered by the dark pigment, no pigment being 0 per cent and entirely black
100 per cent. When seriated the successive generations show only that the
amount of surface colored black varies from generation to generation ; that the
polygon of distribution of these quantities shifts plus and minus with different
means and modes and unlike curves. These determinations I might have plot-
ted in curves of distribution, but from them no further information can be
obtained, and no amount of mathematical treatment can extract a single addi-
tional fact of interest. All the essential facts of the different patterns pre-
sented in these years, the way in which these patterns reacted in the reproduc-
tion of the population, and the real relations of the elements composing the
pattern are lost in the statistical methods employed.

In the years 1903 to 1910 observations upon the heterogeneity of several trop-
ical species were made, together with the attempt to analyze the findings. In the
northern form, L. decemlineata, similar analyses of the variation at Chicago
from 1902 to 1907 were made. It was the experience with L. decemlineata from
Massachusetts and Chicago that gave the original clue to undertake a more exten-
sive analysis with the more favorable materials of the Mexican tropics.

L. muUitceniata, as shown in Chapter II, is variable in composition, is limited
in distribution to the Mexican Plateau, where its actual habitat is localized, so
that little interchange between different colonies takes place. From 1903 to the
close of 1910 continuous observations were made upon this species in several
locations. By continuous observations I mean study and actual contact with this
species in nature at stated periods in each of its two annual generations through-
out the entire period. This necessitated either actual residence in the country,
as in 1903, 1905, and 1906, or two or more visits to Mexico per year for purposes
of observation. A series of from 10 to 16 consecutive generations were thus
tested and analyzed in this investigation.

LOCATIONS FOR OBSERVATION.

In 1903 a series of locations were selected after a preliminary series of obser-
vations had been made, and actual work was begun in 1904. These locations
were chosen along the southern edge of the Mexican Plateau, and presented a
diversity of conditions. At these locations two sets of problems were studied :
(1) the analysis of heterogeneity in the individual in different locations; (2)
the analysis of heterogeneity in the population through a period of years. The
two are interdependent, and especially is the second dependent upon the first.
The locations selected were as follows :

A. Valley of Mexico : Chapultepec colony, Tlalnepantla colony, Tex-

coco colony.

B. Eio Atoyac Valley : Puebla.

C. Slopes of El Volcan de Citlaltepetl : San Andres Chalcicomula.

Analysis of Heterogeneity in Complex Characters 239

These locations are shown in plate 19, and while close together and easy of
access by transportation, are diverse in their conditions of life, so that as far as
possible any influence of localized conditions or other environmental factors
could be detected and made the basis of experimental analyses. The three gen-
eral regions were the Valley of Mexico, Eio Atoyac Valley, and the western
slopes of Volcano of Arizaba.

The ChapuUepec colony — A location 1 kilometer southwest of the Palate of
Chapultepec (near Mexico City), in the open plain just above an old shore-line,
of Lake Texcoco, was chosen in 1903, and regular observations were made begin-
ning with the first generation of 1904 and continued without interruption until
the middle of 1910. Thirteen generations were thus observed. This location is
an open savannah, sloping from the hills to the west of the valley towards Lake
Texcoco. It is well watered by rain and less influenced by drainage operations
than the location about the Sierra Guadalupe. Further, the observations of the
meteorological conditions obtained at Tacubaya were fairly applicable to the
location.

The Texcoco colony. — Another location was chosen in 1903 on the east shore
of Lake Texcoco near the town of Texcoco, Federal District, Mexico. Two rea-
sons determined this location ; first, its being an old lake-bed and still at about
the lake level, so that it differed strikingly in soil-water supply from the Chapul-
tepec Station, and second, the fact that fairly systematic meteorological observa-
tions were made in the town. These latter, although not exactly what I would
like to have had, were still a fair measure of the difference between the two
localities, one to the west and the other to the east of Lake Texcoco. Observa-
tions were begun in 1903 and continued until the close of 1909 or 13 generations.

The Tlalnepantla colony — In the north-central portion of the basin near
Tlalnepantla, Federal District, Mexico, where meteorological observations were
also to be had, a location was chosen near the Eio Tlalnepantla, along the border
of some cultivated fields. Observations were begun here in 1903 and continued
until 1907, 10 generations being under observation. In addition to these loca-
tions records were also continued near the foot of the Sierra Guadalupe until
the end of 1907, or for 8 generations. Other records were also made for loca-
tions near San Angel, Santa Fe, Tlalpam, and Huehuetoca, also in the valley of
Mexico ; at all, results the same in principle but not in detail were obtained, and
these, together with the fact that meteorological observations fairly applicable
to Chapultepec, Texcoco, and Tlalnepantla were available, led to the concentra-
tion of effort upon the colonies at these points in the valley of Mexico.

RIO ATOYAC VALLEY.

Only one location near the city of Puebla, State of Puebla, Mexico, was estab-
lished. A number of locations were examined, but the absence of climatic rec-
ords of any value and the difficulty of having them made decided the location of
the Puebla colony. On the open plain about 4 kilometers north of the city,
in uncultivated land used for pasture, a thriving colony was found, and to the
climatic conditions of this location the meteorological records of two stations in
the city of Puebla were fairly applicable. Observations were begun in 1903 and
continued until 1908, or 13 generations in all.

240 The Mechanism of Evolution in Leptinotaesa

slopes of el volcan de citlaltepetl.

On the cold, dry, grassy slopes on the western side of this huge volcanic cone a
small colony was found near the station of San Andres Chalcicomula, State of
Puebla, Mexico. This location, although without meteorological records, is of
interest because of the cold, dry, tundra-like area in which it was located, the
short growing-season, and the generally rigorous conditions of life. Observa-
tions were begun in 1904 and continued until 1909, 12 generations being
observed.

Observations were made at a number of other points : At Ometusco, Federal
District, Mexico, 1903-1905; Apizaco, Tlaxcala, Mexico, 1903-1907; Esperanza,
State of Puebla, Mexico, 1904-1907; all on the open high plateau; at San
Martin, State of Puebla, Mexico, 1904-1906; and Tlaxcala, State of Tlax-
cala, Mexico, 1904-1906. The purpose of these was to discover if there were
different processes at work in locations other than the five chief points of obser-
vation. None was found, and it was not thought wise to expend time and
energy upon more than the five locations. At each of these colonies exactly the
same method of observation was applied, and as far as possible observations were
made at all of the colonies, especially the five important ones, at as near the
same time as possible. This was made fairly easy by the rather good railroads
operating in that portion of the country, and without these the project would
have been impracticable, if not impossible.

METHODS OF OBSERVATIONS.

It has been the universal custom in studies of this character to make collections
in the locations chosen and preserve them for study later. In this way a consid-
erable proportion of the population of the colony is removed and an unknown
population of undetermined character left to continue the species at that point.
In organisms as localized as these are, and often inhibited from extensive dis-
semination by unfriendly environs, it is conceivable and highly probable that the
removal of large numbers from a restricted area would introduce complications
not easily determined or compensated for. It was decided at the beginning to
eliminate any error of this nature by not removing any of the population, or at
least in any numbers, and only those needed for experimental testing. This plan
has been rigorously followed and made necessary the determination of the condi-
tion of the population in the field. This was easily accomplished. Two, three, or
more natives were instructed to collect all of the kind of beetles they could find
in a given area, and those alive and uninjured were recorded by me, and the sex
and type of variations presented by the part were examined. These after exami-
nation and recording were kept caged until the census of the colony was complete,
and all were then returned to the colony to breed and do whatever other things
they might. I am therefore certain that the conditions described are those
resulting from causes proper and natural to the location, and that no error has
entered into the observations by elimination on my part from making collections.

At Irolo, State of Hidalgo, Mexico, I made a test in 1904, 1905, and 1906,
removing a large part of the population from the colony, and by the end of 1906
it had become very weak in numbers and its character otherwise much altered.
It seemed on the whole best not to remove from any colony under observation
many individuals in any given generation.

Analysis of Heterogeneity in Complex Chakactees 241

At the start two methods of recording for this part were used : (1) an exact
record of the type of pattern found and the extent and direction of the unions
between the component elements; (2) a record of the proportion of surface pig-
mented to that unpigmented, a purely quantitative statement of the " vari-
ation " in the amount of pigment present. This latter determination, esti-
mated on the scale of 0 to 20, was abandoned at the close of 1906, the biometric
results being utterly meaningless and of no significance whatever.

Each year, during the term of observation, the colony was visited at or near
the time when the first and second summer generations were at their maximum
in number. The life-history, Chapter II, shows two annual generations of this
form; the second, passing the dry season in hibernation and emerging in late
May or early June, become the parents of the first summer generation, which in
turn breed at once, giving the second summer or hibernating generation. In
nature it is not always possible to be entirely sure that the population examined
is entirely of a given generation, because of the fact that a few of the overwin-
tering generation may live on and mix, and even breed with the first summer
generation. These can commonly be separated by the distinctly duller color and
generally aged appearance. Som£, however, may have escaped detection, and
have been recorded as first-summer generation. So, too, the first generation
may mix with the second. As a rule these mixings can be detected easily in the
living specimens, but a few possibly have passed into the wrong records.

Having established the general plan of investigation, the next step was to
determine what variations were present in the pronotum of this species over its
entire range. Accordingly, in 1903 a collection of over 8,000 specimens of this
species was made from all portions of its geographical range and these carefully
analyzed, and from this collection it was found that several distinct pattern-plans
were present, and upon these pattern-plans much, if not all, of the variations
found were based. In figure 34 is shown an array of these variations. In L.
decemlineata I had previously found that the variations could be arranged
around several central plans, which in turn could be arranged in a large
scheme covering the entire range of variations of the species. In L. multitoeniata
12 such pattern-plans exist, as shown in figure 35, and these are in turn capable
of being arranged in a scheme of " variation " of the whole species as regards
the pronotal color-pattern, as shown in figure 36. It was further found that,
using figure 36 as a basis of plotting, a far more accurate expression of the char-
acter of the population as regards this feature was possible than by any
other means. It expressed well the direction, kind, and extent of the variations
presented by this part. In figure 36 are given all the variations found in the
species, and only rarely and in large populations are all of these ever present at
one time. It was found that the variations of this species could be accurately
separated by this method of plotting, precisely as had been previously used in
L. decemlineata. Blanks of this form (fig. 36) were prepared and taken into
the field, each individual being entered on the proper part of the blank and also
on the second record, indicating whether in the given pattern, the amount of
pigment was increased or decreased below the average for that pattern. In this
manner there was obtained by direct seriation a statement of the number of
individuals showing a given pattern and the variations of that pattern towards
others, and also in the quantity of pigment present.

242

The Mechanism of Evolution in Leptinotarsa

@S^ <S§) (^ @>

12

11

Fio. 35. — Showing the 12 biotypic groups Into which it has been possible to
separate the pronotum pattern of L. multitmniata. These groups are based upon
the fact that each has been isolated from the general population and reared in pure
homozygous acting pure lines through several generations in each, and represent
with present methods all the stable groups of the pattern as a whole that I have
been able to isolate.

Fig. 36. — Showing arrangement of
the 12 biotypic groups into a general
scheme for the population as a whole
and different directions of combina-
tion and variation within the pattern
as presented by the population. This
schematic base showing the position
of the 12 biotypes within the popula-
tion has been used as the basis for
recording the heterogeneous condi-
tions found within the population of
this species in nature.

I

Analysis of Heterogeneity in Complex Chaeactees 243

From time to time materials were taken from each of the colonies to Chicago,
and there tested experimentally to determine the heritability of the pattern
and the gametic constitution of individuals showing it. Pure races of different
patterns were obtained, and these, after testing and analysis, gave a good basis
for the interpretation of conditions found in nature.

COMPOSITION OF THE COMPLEX PATTERN.

An abundance of instances of complex patterns or parts have been recorded
and arranged in a system, a common practice in studies where phylogeny is the
basis of arrangement and perspective is limited to some system of phylogeny and
hypothesis of evolution. It is here attempted to unravel the intricacies of the
complex pattern of the pronotum of these beetles in the effort to discover the
laws governing the obvious heterogeneity. It has been shown (fig. 34) that in
L. multitceniata there are many patterns giving seeming diversity, and that these
can be arranged in 13 series (fig. 35) ; and further, that these 12 biotype groups
of true-breeding types can be further combined into a pattern or scheme express-
ing the whole range of diversity in the entire species (fig. 36), and upon such a
scheme coujd be graphically represented the variations of the species in any
particular location and generation.

One conspicuous fact of this arrangement is the divergence from biotype tj^pe
7, through types 4, 3, 2, and 1 to a totally black surface, through types 9, 10, 11,
and 12 towards a different extreme, and through 5, 6, and 8 towards still other
divergent types. In any population there is evident a complete gradation along
any of these lines towards the extreme type, and perfectly continuous series in
some of them are at all times evident. The first question to be decided was, is
this population a unit with a complete gradation towards some race extreme, or
is the population a composite of lesser types which, assembled, produce the
scheme of the whole and its apparent " continuous variations " ?

I had found much earlier that the simpler population of L. decemlineata could
be broken up into several lesser groups, depending upon the characters used and
the degree of rigorous inbreeding practice. Were these groups natural or arti-
ficial ? The work of Johannsen and De Vries raises this question in more urgent
form. De Vries postulates " elementary species," and such divisions are found
in this species, while Johannsen postulates lesser lines, biotypes, genotypes, and
clones, of which biotypes only are found in sexually reproducing species. This
material has afforded an opportunity to put to a test some of the recent develop-
ments in genetics.

A postulate common to the conceptions of De Vries, Johannsen, and all of the
neo-Mendelian workers is belief in the inability of a minor strain, when pure, to
be modified by continued selection of the extremes of its fluctuations. The
problems are: (1) can races breeding true without constant selection be
obtained ; (2) when obtained, can these be modified by the selection of extremes ?

One of the most striking characteristics of this material is that when obtained
in nature it rarely breeds true with respect to a specific type of pronotal pattern,
and usually from a pair of wild parents different types appear in the progeny. A
priori, this may be due to wide " variation," or hybrid constitution of the par-
ents respecting the types of pattern represented. Breeding will show accurately
which one of the two is correct.

244

The Mechanism of Evolution in Leptinotaksa

Figure 37 shows in schematic form the results from the progeny of a single
pair, both of which were in aspect type 1 pronotum. A pair of wild beetles with
black pronota from Chapultepec ' were bred in 1905-06, and gave in F^ the array
sho-wTi in the diagram, with some fluctuations of each of the five types which
are represented. Of the five types found, examples of each were mated with
likes. A pair of black type 1 was mated (A), and gave in F2 equal nimibers of
black A' and a type 2 form A". Each of these when inbred gave in F3 : from
the black of A' came 100 per cent black, and this continued to breed true, while
from the type 2 A" came black A" and type 2 again A" a, in about equal numbers.
The blacks in Fg A" bred true for three generations, while the type 2 A" a split

F7.

F6

F5

F4

Fs

Tz

Fi

LA'

I A'

^m

^ a^

««l«l«ll»«i

A'

A":

I I

wwum^^

A'

A'

A"

^"1 I A"ab I A"ac

A'-'J — , — I A"a

A' I — I A" 1 I I

m m.

J I - J. ^^

D"b D"a. D"c E'ca E"c E"cb

^ ^ ^ E"c E"b E"a |

Fig. 37. — Schematic results showing complex nature of a pair of L. multitwniata,
having a pronotum of biotype 1 and heterozygous in nature.

into blacks A" a and type 2 again, some of which A"ac continued to breed true,
and some A" ah split again. In Fj of type 2 two pairs were mated and only one-
gave progeny, and in Fg two pairs were again mated and only one gave progeny,
but in F4 six pairs out of eight gave progeny from type 2 matings and two gave
the behavior shown in A"ac, and four that of A" ah. Two races, stable, without
selection were obtained from A.

From B no pairs were mated, but from C in F^ out of three such matings one
bred true and gave a stable race. From D, of which I bred only one pair, two
types appeared in Fg, a low extreme of biotype 4 (D') and a biotype 3 {D").

^ In all tests of materials from nature the larvee were taken and reared, and the
sexes isolated at emergence, so that the females were in all cases certainly virgin.

Analysis of Heterogeneity in Complex Chaeacters

245

The biotype 4 {D') was not mated and one pair of biotype {D"), which gave
progeny, gave in F3 biotype 2 {D"a), biotype 3 {D"b), and an extreme of biotype
8 {D"c) . One pair of Y^ of the type 4 {D"a) which gave progeny was the basis
of a line breeding true and constant. The other pairs gave biotype 4 and biotype
3 (not shown). Three matings of biotype 4 (DEa) gave results like similar
matings in F3. The biotype 8 extreme {D"c), of which there were only two
pairs that gave progeny, gave in one case all biotype 8 extreme {D"ca), and in
the other pair {D"ah) two biotypes, 8 and 4, appeared, which, however, were not
fully tested.

From the mating E in F^ the results from two pairs are shown, one (£")
breeding true in F2 and subsequently ; the other giving in Fg a population of a
biotype 12 {E"a), a biotype 4 {E"h), and an extreme biotype 2 {E"h) . Of these
only the extreme biotype of 2 {E"c) were mated and one pair gave in F3 biotype
2 again {E"c), a biotype 3 {E"ca) and the other extreme of type 4 {E"ch).
Only the biotype 2{E"c) was mated in F3, and this pair gave in F^ biotype 2
{E"c) and biotype 3. In F^ five pairs of biotype 2 were mated, one of which
gave biotype 2 {E"c) and types 3 and 4, and of this F5 population matings of the
biotype 2 {E"c) showed one that was constant in F^ and F^.

F4

Fig. 38. — Showing results in a second pair of
L. multitwniata from the Chapultepec colony,
having a pronotum of biotype 1 ; although ob-
viously heterozygous in composition they were
apparently much less complex than the similar
pair shown in figure 37.

F3

F2

Fl

It is evident that the original pair ' were not pure black type 1, but complex
hybrid combinations in which different potentialities were combined in such a
series of complexes that the chance union of these two gave rise to a striking
array of types, out of which several distinct races were extracted and maintained
as pure-breeding strains. It is also shown that although the original stocks
were exceedingly heterozygous in character, their progeny were, nevertheless,
quite fixed in type and the array found was entirely due to diverse germinal
constitution.

Although the pair and their progeny show extremely complicated germinal
constitution, not all tested pairs were so complex. Thus in figure 38 is shown
a pair obtained at the Chapultepec colony in 1908, which gave a simple behavior,

^ Both of these were typical " L. melanothorax Stai " as found in all museum col-
lections and would unless tested by breeding have passed as pure typical specimens
of that " species."

246

The Mechanism of Evolution in Leptinotaksa

showing simple gametic composition. In this pair of biotype 1 blacks from
nature were mated and gave in F^ biotype 1, biotype 2, and biotype 6. The F^
blacks when inbred gave, out of 7 pairs, 4 that came true and 3 that gave blacks
and type 2. In F3 and F^ the pnre-breeding blacks continued to breed true and
the type 2 was heterozygous, giving type 2 and blacks of biotype 1, the latter
breeding true. Of the biotype 6, which appeared in F^, 2 pairs out of 8 gave
progeny, and these came true to type in Fo, F3, and F4. The biotype 2 in F^
inbred gave biotype 1 and biotype 2 in Fj, but these were not mated for further
testing. The simple behavior of this pair shows a relatively simple gametic com-
position and the capacity for extracting pure-breeding biotypic races by the
mating of likes.

F8

Ft

Fa

F5

F4

•Fs

•Pz

Fi

^

m

^ mm ^

lE"b

^ m

"C'a

lC1_-C"

Fig. 39. — Showing results obtained from a single breeding pair of L. multitCBiiiata
from the Chapultepec colony of biotype 4.

Even more simple cases have been found where blacks of biotype 1 were pure
homozygotes, but in nature these are not common, but may be found in almost
any population of emerging stock by testing enough samples. It is certain,
therefore, that as regards this biotype that it exists in nature in all degrees of
gametic constitution from pure homozygous to highly complex and intricately
involved heterozygous constitutions, where there are correlations with other char-
acters, giving extreme diversity in the progeny of a single pair. These correla-
tions, while of importance in the production of diversity in the pronotal char-
acter in the population, can not be discussed in this part of the paper.

The conditions found in the biotype 1 pattern are equally true of any of the
others found in the entire population. In figure 39 are shown the results

Analysis of Heterogeneity in Complex Chaeacters 247

obtained from a pair of biotype 4, also from the Cliapultepec colony, as wild
stock. In Fi this pair gave biotype 3 (A), biotype 4 (5), and biotype 5 (C).
In Fj two pairs of biotype 3 (A') were found that gave 100 per cent of this same
type and continued to do so through F^, F^, Fg, F^, Fg, and Fg, while 5 pairs
A" gave biotypes 3 and 4. Four pairs of biotype 3 {A"a) were found that came
true in Fg and subsequent generations when inbred, while the biotype 4 of Fg,
only 1 pair gave progeny, and these were of biotype 4 and biotype 2. In F3 the
biotype 4 showed 3 pairs out of 11 breeding true in F^ and F^, but in F5 one pair
again gave biotype 2 in small numbers. In F3 1 pair out of 12 of biotype 2 gave
all biotype 2.

From the mating B in Fj, 1 pair, all that gave progeny, continued to breed
true through F3, F4, F-,, and Fg. Mating C of biotype 5 was heterozygous, 5
pairs giving in Fg two classes : biotype 5 (C") and biotype 9 (C) . Matings in
Fg gave of biotype 9 only one pair with progeny, all of biotype 9, which continued
to breed true in F4, F^, Fg, and F^. Three matings of biotype 5 were found to
breed true C"a, and came true in F^, Fg, Fg, F^, Fg, and Fg, while 10 pairs did
not breed true in Fg, but were not analyzed further {C"h).

One might continue this type of analysis without limit. From the natural
population of L. mul titceniata at any point in its geographic range, materials
taken from nature show this diversity of gametic constitution as regards this or
any other character that might be considered. It follows that each individual
may be different from every other in the population, even in a single character,
and any individual from nature may produce many geno-different gametes. It
has been clearly shown by Johannsen, Jennings, and others that in self -fertilized
or sexually reproduced forms any genotypic constitution that may be selected by
the observer can be made to continue in straight lines, but in sexually reproduc-
ing types it is these " biotypes," or groups of like gametic make-up, that are to
be looked for, isolated, and studied.

A considerable number of writers has isolated genotypes and biotypes that
breed true and are asserted, especially by Jennings, to be incapable of modifica-
tion by cumulative selection. Jennings, in Paramcecium, worked only with
clones, which are not exactly comparable to genotypes of self-fertilized organ-
isms, and not at all to biotypes of cross-fertilizing forms.

In almost all organisms genotypes and clones will be absent and the biotype
will be the group mostly studied and employed. Johannsen's clear definition
that a biotype is a group of individuals possessing the same germinal constitu-
tion (identical genotypically) leaves no opportunity for misconception regard-
ing the nature of biotypes. A current idea among the supporters of the pure-
line hypothesis is that genotypically different gametes have no intergrades
between them, but are discontinuous. Woltereck, working with Daphnia, has
denied this, and others from less secure data have also criticised the view.

Two questions seem of fundamental importance in this connection and
involve the validity of the entire conception of minor groups within the
" species."

(1) Where in any instance does the limit lie with respect to the determi-
nation of a genotypical constitution ? I can with my materials, as shown in the
first part of this section, easily isolate a race that is hornozygous in behavior,
absolutely so, which breeds true without limit to the one type, and is in every

248 The Mechanism of Evolution in Leptinotarsa

way a typical group of individuals of like gametic constitution — a biotype.
This character of the pronotum is composed of many elements, each capable of
independent expression, and each to a greater or less extent independently capa-
ble of modification. In figure 41 are shown certain true-breeding races, homo-
zygous in action, with patterns that are capable of having their elements modified
greatly, without changing the homozygous nature of the pattern as a whole. Are
there biotypes within biotypes? If so, where is the limit? Would it not be
possible to go on indefinitely, as skill and methods become acute and refined,
more and more to get ad infinitum smaller and smaller genes and geno-differ-
ences, until a stage is reached in the distant future where the only difference was
one of presence or absence of one electron or other minute material unit ?

(2) Do these groups — biotypes or aggregations of genotypically like individ-
uals— exist in nature as isolated realities in the population, or are they merely
individual momentary conditions present in members of the population which
can be separated and when isolated breed true as long as the isolation is main-
tained? What is the position and meaning of these experimentally isolated
groups in populations from nature.

There is no doubt that from beans, grains, etc., it is possible to isolate geno-
types as Johannsen and others have done, or to isolate clones from organisms
like Paramcecium, as has been accomplished by Jennings; but were all of the
genotypes isolated, and how many other genotypes based upon different char-
acters could have been secured ? Are we not still thinking and talking of these
matters in terms of the morphologist and anatomist ? Are not genes and geno-
typical constitutions at base purely morphological concepts in that they involve
identity of parts or constitution ? The key to the whole situation lies, I believe,
in the determination of what homozygous and heterozygous conditions of the
germinal material really signify,

Neo-Mendelians, descriptively at least if not in fact, adhere to the anatomical
picture, and homozygous gametes are those of like factorial constitution, and
heterozygous gametes those of unlike composition. This is not true. Homozy-
gous as well as heterozygous are descriptive of uniformity of reaction in the ger-
minal material and not of the conditions of constitution per se. That is, homo-
zygous gametes are those in which a state of physico-chemical adjustment has
been attained, such that, regardless of the active potentialities present, the action
of the gamete is always the same in normal development and in combinations.
Genotypical gametes are the same in character and action, in that a genotypical
gamete or group may be different in their potentiality constitution, but they
react alike, behave alike in ordinary breeding, and hence are descriptively stated
to be alike. It is a singleness or uniformity of action that determines the
behavior, and this is dependent upon the stability of the physico-chemical com-
plex in the germinal material, and not at all upon the nature or array of poten-
tialities present.

The first problem with any natural population is to discover if there are pres-
ent minor stable groups which can exist without selection to maintain them,
but which, nevertheless, interbreed to a greater or less extent in the population.
With a species like L. midtitceniata it is, as has been shown, easy to isolate many
true-breeding strains. How many of these will remain without " selection "
when carried as group cultures ? To what extent can these be modified by cumu-
lative " selection " ?

Analysis of Heterogeneity in Complex Chaeacters 249

primary biotypes.

In the population of L. muUitcBniata I have been able to isolate from the
pronotal pattern 13 groups that interbreed, overlap, but can be isolated and
which remain constant through an indefinite number of generations. These I
have called " primary biotypes/' and they can be distinguished as follows :

Primary biotype 1 — Complete black surface both above and below, without
trace of hypodermal color or pattern. (Fig. 35; fig. 36, 1.)

Primary biotype 2 — Spots b', a' , am, pm, a, b and anterior marginal spots
forming a solid central triangular area. Spots c, e, f, and d and posterior mar-
ginal area fused to form an irregular triangular spot in each posterior outer
angle. These two larger areas meet along the posterior border in the median
line and fuse with the central area through medianward extremes of the d
spots. This type may vary from complete fusion of all areas to form a central
dark area with a lighter margin (fig. 35, 2) to the extreme shown in figure 36, 2.

Primary biotype 3 — Differs from No. 2 as follows : The central triangular
area is broken anteriorly, traces of anterior marginal spots appearing, and spot c
is free from the e-f-g posterior marginal group. As an extreme c may fuse with
the lateral group and the anterior spots may by fluctuation give the appearance of
a No. 2. Breeding, however, always reveals the real character of the individuals
in question.

Primary biotype k — Differs from No. 2 and No. 3 in that c is never united to
the lateral fused mass of d+e + f, and the anterior marginal spots are never
united to the central triangular area and may be much reduced or even not
visible, while the central area becomes more open, showing median separation
into two lateral a, h groups, which are widely divergent anteriorly. The d, e, f
posterior marginal area is also reduced, although still connected and fused
usually with the a' b' a b group by a medianward elongation of d, which is
deflected cephalward to the anterior end of the a areas. This group is variable,
as shown in figure 35, 4, and figure 36, 4.

The primary biotypes 1 to 4 overlap and form by their " variations " a graded
series, as shown in figure 41, and are distinguished by changes in the relation
of the elements of the pattern and not at all by the amount of pigment present.

Primary biotype 5 — The central spots form a broadly open V composed of

6' -fa' A 2^a + h. The anterior marginal spots are always entirely wanting

^ pm ^
on all. The posterior marginal d, e, f are fused and d prolonged medianward at
90° to median axis, and fuses with a (rarely as a variation; " fluctuation " e is
prolonged anteriorly to meet c). Figure 35, 5, and figure 41, 5, show the char-
acter of this biotype. In figure 36 it is shown as forming a diverging line from
the meeting-point of the different biotypes and tends to form in appearance a
pronotal pattern with a transverse dark band on the posterior half of the
pronotum.

Primary biotype 6 — Precisely the same as biotype 5, except that the anterior
marginal spots are always present in varying degree, as are also the posterior
marginal areas. The central group is broadly open, as in No. 5, and united to
lateral groups in the same manner. In figure 35, 6, this biotype is shown as
another divergent group. The tendency of this group is to form a pattern with
the posterior half of the pronotum darkened to a greater or less extent,
17

250 The Mechanism of Evolution in Leptinotaesa

Primary hiotype 7 — Occupies a central position in the series. The central
system of spots is always fused into a broadly open V, of which none of the ele-
ments ever becomes free. C is always free and distinct, and d, e, f group are
fused but never united to c or medianward to the middle group. The anterior
marginal spots are always absent and also the posterior marginal. This biotype
exhibits as little fluctuation as any excepting 1, and shows only increase or
decrease. (Fig, 35, 7.) It is the pivotal pattern type of the whole array, and
by adding to or taking from it almost any combination can be built up. It is
also a common one in nature.
Primary hiotype 8 — This comes from biotype 7 by the breaking up of the
am
groups a' + 6' ^ ^ a-\-h and the d + e + f group into the component centers,

^ fm ^
figure 35, 8. This is a variable biotype. The anterior marginal spots and
posterior marginal spots are never present. C is always free and the central
and lateral areas never united. Spot d may extend anteriorly towards h, but
rarely fuses therewith.

Primary hiotype 9 — Shows a sharp change in the position and character of the
a spots where they are parallel with the median line and not widely divergent
anteriorly as in all the preceding types. This is accomplished by the median-
ward movement of the anterior center in spot a, and a cephalward elongation of
the spot beyond the h spots, giving a more or less squared appearance of the
central system. The d, e, f group are united and d is often fused with h; e and c
send prolongations towards each other, which rarely meet. This results in giv-
ing an anterior-posterior striping appearance to the pattern, as shown in figure
35, 9. The anterior and posterior marginals are always absent and there is
never lateralward fusion between the central and lateral groups.

Primary hiotype 10 — Eepresents a divergent line from 9, in which the same
type of pattern persists, but is heavier, the fusion being always marked and
heavy, and the anterior marginal spots being present and fused with a, forming
a prong projecting medianward. This type is represented in figure 35, 10, as
a quite distinct but not common one in the population.

Primary hiotype 11 — Differs from biotypes 9 and 10 by the absence of all
marginal spots, the fact that a, h, and c never fuse, and the tendency to fuse
between d and e, which do not fuse with either a,h, or c. (Fig. 35, 11.)

Primary hiotype 12 — This is comparable to No. 8, in that it represents a case
in which all centers are free, the only fusion being produced by somatic vari-
ations. A tendency to restrict the pigment formation to the color centers also
accompanies this biotype. Figure 35, 12, shows the usual range of variation
of this group.

These 12 biotypes of the pronotal pattern are based, as has appeared in the
above description, upon a system of color centers shown in figure 40, between
which certain combinations are made, and to the occurrence of certain marginal
spots which enter into the combinations.

These biotypes overlap in their " variations," giving a complete intergrading
series in any population, and all interbreed freely when present, so that the
natural population is exceedingly complex with respect to this character. These
primary hiotjjpes may easily be entirely isolated and manipulated as " units," but
are to a greater or less extent associated with other characters or groups, form-

Analysis of Heterogenetty in Complex Chaeacters 251

ing a larger biotypic group. Thus No. 1 is in nature usually associated with the
melanothorax form, broad and rounded, while the biotypes 9, 10, 11, and 12 are
always present in and correlated with the appearance of the muUilineata form-
factor. Biotypes 2 to 8 are found associated in the species with the form and
body characters conmion to multitceniata. In any population there are thus
complicating factors in any character, so that a complete analysis would be an
analysis of all characters and their interrelations. The pronotal pattern, how-
ever, is sufficiently independent and so accurately a gametically determined char-
acter that it serves well for certain purposes of investigation.

To recognize these biotypes is of little or no use unless they be tested fully with
regard to their properties and capacities for existence. Can such groups exist in
freely-breeding cultures without change? Can they be selectively changed by
quantitative selection without adding something to the complex ? What is the
relation of the different groups when crossed? These are some of the deter-
minations that must be made concerning the nature of these biotypes.

THE BREEDING OF PRIMARY BIOTYPES SELECTION.

When one of these biotypes has been isolated by any process, can it stand
alone, isolated from intercrossing, without constant selection? This I have
tested as follows : Biotypes extracted as shown in this section were started from
1, 2, 3, or more pairs of like, homozygous-extracted types. These were either the
progeny from a single pair or from one or more pairs, and were allowed to inbreed
and continued to test the stability of the type.

The simplest to deal with is the type 1, and this I have reared in such cultures
many times. A pair of F^ A' primary biotype 1 were taken from the culture
shown in figure 37 and allowed to breed. They gave 97 progeny, 47 males and 50
females, all of the black type, with no variation in pattern. These are not
melanothorax, but the black thorax transferred onto the multitceniata form. Of
these, 20 males and 20 females taken at random from the population were allowed
to breed freely and gave a progeny of 224 males and 217 females, all of the par-
ent type. From this population, 20 males and 20 females taken at random were
again allowed to breed, and these gave in the third generation 196 males and 194
females, all black. This operation I continued until the sixth generation with
exactly the same result, when it was discontinued. At no time was there trace
of other type or of variation in this line.

An example of the breeding of a primary biotype as a group culture is shown
in figure 40, which represents a group culture of biotype 2. The original pair
were an extracted stock bred pure in pedigree culture for four generations and
came from parents obtained at the Chapultepec colony. 4 males and 4 females,
2 of each of the kind shown, were mated and these gave a progeny (FJ of 64
males and 80 females, distributed as shown in figure 40, on the scale of the range
in this biotype shown in figure 35, 2. From these 3 males and 3 females from
each class represented were taken at random and again inbred and gave a progeny
(Fj) of 183 males and 195 females, distributed between the two main classes. 3
males and 3 females from each of these classes were taken at random, mated, and
gave a progeny (F3) of 200 males and 197 females, distributed in four classes,
as shown. From these, 3 of each class were taken at random, as far as possible,
mated as the parents of generation 4, and gave a progeny of 369 males and 360

353

The Mechanism of Evolution in Leptinotaesa

females, distributed in the four classes common to this biotype, and one group of
6 males'and 1 female having some of the characters of primary biotype 3 (see fig.
40e). These divergent individuals were mated, the single male and a female,
and gave a progeny in Fg of 31 males and 36 females, which were, however, all of
the primary bfotype 3 character. From the normal group in F^, 3 of each sex
were taken'^at random from each of the classes, mated as the parents of F.,, and
gave a progeny of 360 males and 374 females, with the usual distribution. In
Fg each of the two groups were bred from and both gave in Fg from the normal
131 males and 93 females. In the group B, 36 males and 35 females, all in
both sets typical of the strain. The lines A and B were continued, giving in F^
in (A) 333 males, 191 females, (B) 65 males and 60 females, all true to type,
and in Fg (A) gave 248 males and 379 females, (B) 131 males and 133 females,
likewise true to type. At the end of Fg the culture was killed off.

GENERATION

Progeny:
Mated:

ZSZf zSZf

20^ I9f 47<f5/p
3^3f 3<i3f

T

d

7'irof

Progeny:
UcUeA:

11^81$ I06ill4f

3i

3<f 3t ,

X

Progeny: a b c

A'Sif 99^ 106 f ST'iBZf
Mated: .Z'^tf siSf 3'^ 3f

d
3^3^

Progeny: a
Mated: If

Progeny: a
Mated :

Progeny
Mated.:

bed

30^96$ 190^1871 77<!8lf
Zi Zf Zi Zf Z'SZf

bed

niaif i-fzo'issf 'n^sBf

Zi Z$ Z^Zf Z^Zf

abed

4-'^ 3!^4-Zf 86^39 f willf

zi zi Zf zizf z^z$

Progeny: abed

jtif 44Sigf I4e<!l38i 4Z^3tf

Mated.: i^Zf z^ Zf 2^ Zf Z^ Zf

\ — T ;

Progeny: abed

li 99^108^ 146'i!7lf Z^

' b c
wSizi 9^nf
z<Sz^ Z^Zf

d

Z'^3f

r

a

ri3f
liZf

b e

9'Sl4f zzil8$
Zi Zf zi Zf

d '

4i

zi

r

a

Z^!f

b C
rS-fzif 42^38^
Z^Zf Z^ Zf

d
zS

2^

Y

Z^4f 3l^47f 86'f74-f

Fio. 40.— Showing breeding record of a primary biotype 2 of L. mtiltit^niata
tlirough a series of generations and tlie constancy of the pattern produced in these
biotypic lines.

In this test with primary biotype 3, when reared as a group culture with
matings at random and perfectly free interbreeding of the parent groups, the
culture remained stable throughout. Only in generation 4 did the population
overstep the normal range, and these, separated and reared independently,
proved not to be a gametic change, but only a somatic ( ?) disturbance, as shown
by the giving of typical biotype 3 pattern all through the line (B), with no
reappearance of the divergent examples of generation 4. In a culture of this
character there is no intentional selection and no evidence of selection of any
sort. The conditions of life were kept as uniform as possible and care was

Analysis of Hetehogeneity in Complex Chaeactebs 253

taken not to allow anything to happen, such as sudden changes of the conditions
or violent stimuli which might upset the constitution of some gametes and thus
introduce another factor and complicate the results.

Another example will suffice to show the manner of reaction of these biotypes
to these breeding-tests in the testing of biotype 9, as shown in figure 41. Two
males and two females, each from classes a and b of this biotype, that had been
derived from extracted stocks from the Texcoco colony and had been pedigreed
for four generations, were mated and allowed to breed freely and gave in F^ a
progeny of 87 males and 101 females, distributed over the four classes common

GENERATION
P [j^iiea : b c

\ ZiZf ZiZf

[Progeny: a t> c d '

\Mited.: i^Zf ziz$ zizf zizf

V , .

VProgeny: "a t> c "d e ^

^Z\ r^'Zf r7<ll6f 77<!59f Z6<f3f^ 7^7 f

\A£iiea: zizf zizf z^ZfZ^f Z'l Zf

I
Progeny/ a bed

tS^IZf ^€^45^ SSii-Hf

MaieaC: ^ zj zf zi z^ z^Zf

"r •

A

[Progeny-: a b c d, e ^~c d 7""

r^S 6iSf !Oirz= ;8^Z4f Z^f 4-^5$ 7^8^ 3^49

[Afated: ^ zizf zizf z^ Zf z^z^ z^Zf z^zf z^z^

~T ^

I , \ I 1 ^ -^.

\J>rogeny:'^ 1 c 'd P 'i 2 2

r^i S^ilf rf^rof <;^40f jz^ssf zirf zisf 7^Sf /z^r4f

\Mated.: ^z^z^ z^Zf z^Z^- Z^Zf z^Zf, ^ Not continued

zi

T

(Progenj'-. ^"i 5 c 5' '

4^7^ Z10^'99f 7B^9ef

'aieci: ^ z^z^ z^ Zf z^Zf

Y^o^eny. abed

fji. Zl^ZZf 96il06^ -M-ifSf

\Mated.: ^ zi Zf zizf 2^2$,

^_, I C Biotype fZ

{Progeny/ a b c d ^ ^a b c '

^eS i'Hr7f /4-z^/99f re'^igf 4izf iz^rof /s^isf

\Mated.: z^ Zf Z^Zf Z^Zf, z'zf zizf Z^Z^,

[progeny: abed a b c-

'^SS 'f-J7f /7r^l9Zf /S^7f 4^SSf 23^44 f 39^Z7f

t Not continued Z^Zf Z^ Zf Z^Zs

-j-c .

[progeny: 'a b c d '

/>/,■< 3i^4Zf I06iti4f ra^/Of

Not continued

'I

Fig. 41. — Breeding record through a series of generations of a race of primary
biotype 9 in L. mxilUianiatO; again showing the constancy of the race within this
line.

to this biotype, as shown in figure 41. From these there were taken 2 males
and 3 females from each class, except class a, and mated. This group gave a
progeny of 136 males and 124 females in Fj. This population showed a group
of 7 males and 7 females, which were quite beyond the normal range of this bio-
type; they were not removed, but were bred back into the population in the
mating for Fg. From this population the usual random matings were made
and they gave in Fg 101 males and 102 females. In this population 3 males and
4 females stood quite without the range of the normal biotype and were separated

254 The Mechaijism: of Evolution in Leptinotaesa

and inbred as division B, division A being the normal culture. In F^ the normal
group (A) from a routine mating gave 36 males and 45 females, while all of the
progeny of the divergent B group were entirely of the normal type arrayed in
classes c, d, and e, there being 14 males and 17 females. From these F^ groups
matings were made as before from A and B, and in both normal progeny resulted
in A, 94 males and 94 females, and in B, 23 males and 22 females. The B group
was not carried any longer, while the A group continued from the usual mating,
giving Fe all normal, 292 males and 282 females. From these a mating of the
ordinary type gave F^ with two groups, the normal (A) and a divergent one (C)
well outside the normal range. In the first there were 164 males and 166
females, and in the second, 1 male and 2 females. A mating of the first group
gave normal progeny in Fg, and again in Fg, when the A line was terminated.
The C group gave a progeny in Fg all of primary biotype 12, 29 males and 30
females, distributed in classes a, b, and c. A mating from these gave Fg, again all
of type 12, 67 males and 76 females, and from a mating taken from these a
progeny was reared in F^o, again all of type 12, 156 males and 166 females. At
the end of F^, this line was also discontinued.

This culture of biotype 9 shows two common types of behavior that are found
in these groups, namely, individuals that appear beyond the normal range but
which, when inbred, give the normal population, and a group (c) beyond the nor-
mal range and without intergrades, which, when inbred, give individuals true to
their own type, which continued to breed true to that type. That is, in the lan-
guage of the adherents of the hypothesis of pure lines, biotype 9 had " mutated,"
giving biotype 12. The individuals of the group c, however, were the last to
emerge ; indeed, it was thought that all that were going to emerge had done so
and the cages were therefore set aside before being cleared for the next mating.
In this period of over a week they dried completely and were often heated to
nearly 100 °F. The modified group (c) emerged after this treatment. Whether
the treatment and the divergent group are cause and effect is a matter of opinion.
It seems plausible to think they are, but the test is too crude to be more than a
plausibility. This occurrence does, however, show one point of importance —
the delicacy of balance of some of these groups and the ease with which incident
forces may possibly give rise to sudden changes, " mutations," to other biotypes.

Each one of the biotypes shown in figure 35 has been reared in this same
manner many times with uniform results. They are groups which, under uni-
form conditions, are permanent and stable even in unselected group cultures.
Under uniform conditions and isolated they remain true to type, fluctuating
within a fairly normal and fixed range, but in all of them there is considerable
unstability manifested in the presence of extreme conditions or of intense
stimuli, or even of rapidly varying conditions, giving as a result sudden changes
of a few individuals to other biotype groups.

In this array there are also differences as regards their stability. Biotype 1
is highly stable and difficult of disarrangement, while biotype 7 is the least stable
of all. Biotypes 5, 6, and 10 are fairly stable, while between 9, 11 and 12, and 2,
3, and 4 sudden transmutations are common and easily produced, and the con-
viction is soon forced upon the observer that these guoups, although distinct and
stable under constant conditions, realities in every respect, would nevertheless
under natural conditions present a never-ending series of transmutations as the
series of external forces passed over the population.

I

1

Analysis of Heterogeneity in Complex Characters 255

As far as the continuity of this group is concerned and their gametic identity
of constitution, I am fully agreed with Johannsen and others that they are capa-
ble of isolation, are analytical realities, and breed true under constant condi-
tions, and further, that they frequently mutate to other biotypes within the gen-
eral population. I am not convinced that they are stable, elemental units in the
species, as many consider them. They represent more or less stable combi-
nations attained in the gametic complex, and are but momentary pauses in the
ceaseless metathetic recombinations which each organic group is constantly
undergoing. That these biotypes are not capable of modification, or are capable
of modification only by sudden change, is true for some, not true for all, and in
the next section I shall show how these biotypes can be changed by different
agencies.

THE MODIFICATION OF PWMARY BIOTYPES.

In the attempt to modify any of these minor groups, be it genotype, clone, or
biotype, an hereditary historic influence suggests selection and external condi-
tions, and when these have been adequately or inadequately tested, other possi-
bilities may then receive attention. Again and again have workers with pure
lines, clones, genotypes, or biotypes recorded their inability to produce changes
in the types they had isolated. No difficulty is encountered in discovering and
isolating races that are alike in most of their characters, but differ in one essential
character which sets it off, and when reared in " pure lines " keep it distinct from
its fellows. Nevertheless, no effort seems to be able to modify this into another
one.

Jennings, in his masterly studies in Paramoecium, where sexually produced
" clones " were used, finds that he was unable to modify or transmute any of his
" clones " into another, either new or existing. Johannsen and others in beans,
Nillson-Ehle with grains. East, Shull, Emmerson, working with genotypes and
biotypes, found difficulty where modification was attempted. Many workers
using phenotypes or elementary species, as De Vries, have found difficulty in
modifying them. Why this difficulty? Is it real, a limitation upon one's
experimental ability, or is it due to lack of skill and understanding?

The attempt to modify these groups by so-called selective methods seems an
indifferent characterization of what one has tried to do, and the word " selec-
tion " implies a choice between two or more possibilities rather than a chance
determination of what happens. This selective problem is a large one and is one
upon which I shall wish to dwell at length later on. I have used, as methods of
attempting modification, accumulation of pigment surface in the pronotum as a
whole; accentuation of directions of variation in the elements of the pattern
(particular spots) ; synthetic combination of directions of variation; external
incident climatic forces ; and food and metabolic disturbances.

EFFECTS OF QUANTITATIVE ACCUMULATION OF PIGMENT.

Almost all selection investigations degenerate into the effort to accumulate the
same quality or attribute by quantitative additions ad infinitum. The odd idea
that existing strange modifications of supposed adaptions in nature have arisen
by continuous accumulations of minute amounts of the quality lies at the base of
this experimental effort. It is notorious that this quantitative accumulation soon
reaches a limit beyond which the modification does not extend. This limit in the

256 The Mechanism of Evolution in Leptinotaesa

huge majority of cases corresponds to the upper limits of the natural " vari-
ation " of the species, does not transcend the natural variability, although a
fairly stable race is often capable of being created at this upper limit or at any
other point by this process.

Of late years it has become the custom to interpret any such result on the
assumption that the race obtained is an isolated genotype or biotype. Is this
the truth of the matter ? It is the experience of so many observers that these
selected races revert to the parental standard when selection has ceased that
there must be some foundation in truth, and it must represent some result of
natural operations. Isolated genotypes or biotypes are asserted not to change,
to be difficult of modification, and Jennings says no one has thus far been able to
modify one of these isolated groups into another. It is not likely that intercross-
ing with other biotypes is solely responsible for this regression, and why should
the " selected " group so constantly move back to the mediocre of the entire
population ? Why this submergence of an improved, " efficient " ( ?) race by the
mediocre.

In the population of L. muUitceniata I have shown that biotypes of the prono-
tal color-pattern are present, have been separated, and the distinguishing charac-
ters of these have been given. In the general population I can easily create a
race at any of the extremes of the natural variability by the use of quantitative
accumulation. The relation of this to the biotype question is not as simple as
one might suppose and will be considered later. In the isolated biotype, what
can quantitative accumulation accomplish ?

A good test of this can be made by starting with biotype 4 and trying to
accumulate pigment and produce an end-result like biotype 1. Three sources
of error must be rigorously eliminated : ( 1 ) it must be certain that the biotype
4 is pure, contains only biotype 4; (2) the culture must be kept under uniform
conditions to prevent any action of external agents upon the pattern; (3) the
choice must be made upon the amount of pigment, and no attention paid to the
fluctuations in the pattern type. The problem is, can accumulation of pig-
ment or any other character produce directly or indirectly a change of type?
I have made many tests of this kind, of which a good example is the following,
wherein the attempt was made to change biotype 4 into biotype 1.

Start was made upon a pure extracted biotype 4 stock derived from parents
which were obtained in nature at Chapultepec, and which in Fj gave only bio-
types 4, 5, and 6. In Fg pure-breeding examples (homozygous) of biotype 4
were found and these were carefully pedigreed through F3, F4, Fg, and Fg, and
from the latter generation the basis of this culture came, while the stock was
continued until F^^, when it was accidentally lost. It did not show any diver-
gent individuals or tendency to be transmuted into any other type.

Three males and three females of normal average aspect were mated, and gave
a progeny, Fj, of 143, distributed as shown in figure 42. From these, 4 males
and 4 females of the most extreme class were mated and gave a progeny of 79
individuals, with the distribution shown in figure 43, of which the upper range
was above the normal range of the biotype. In F^, F3, and F4 the same method
was followed of mating from the extreme upper class, and this rapidly caused
the movement of the population towards a condition of extreme pigmentation,
until in Fg the upper limit had reached a state just below that of biotype 1.

Analysis of Heterogeneity in Complex Chaeacteks

257

From Fg to F^o the same method of mating and selection of parents was followed,
giving a race of constantly decreasing variability, and, as far as amount of
pigment was concerned, much above that of the normal parent strain. In F^
there appeared 1 male entirely black above on the pronotum, but not on the
sides. This individual was separated and tested separately.

Fio

F9

^ m m

IP ^ ^

m ^ <^

m m m^

F8-

F7-

F6

Tf,-

F4

F3

.F2

Fi

< , . <— , ,

^m ^ ^ ^ ^ ^

1

V

B

m

m

^

^

^

^

m

m

^

m

^

m

m w

^

m

^ m m

m ^ m w^

^ m

FiQ. 42. — Diagrammatic representation of the effort to modify through accumu-
lative selection the amount of pigment in a pure extracted biotype 4 stock of
L. multitceniata. The figure shows graphically the Ineffectiveness of quantitative
selection in permanently altering the type of this race.

In Fg two matings were made; one (A) continued the intensely selected strain
through to Fjo; the other (B) was a random mating from all classes and was
allowed to inbreed freely. Figure 42 shows, without further comment, the
result. The selected race was kept up to the standard, the nonselected at once
began to move backwards and soon showed only the normal biotype 4 pattern.

258

The Mechanism of Evolution- in Leptinotarsa

In F5 there were 4 with almost totally black pronotmn, or biotype 2 form, and in
Fb and later such were constantly present in the selected strain. Superficial
examination of the culture would at once decide that biotype 4 had been changed

F4

F3

F2

Fi

m m mm

p-

Fig. 43. — Graphic representation of a normal cross between a true homozygous
acting biotype 1 and biotype 4 in L. multitwniata.

to biotype 2, and the single male in F^ would ordinarily be interpreted as evi-
dence of the modification to biotype 1. This I tested out as follows : If the
extreme male of F^ is a biotype 1, or even partly so, breeding would test it fully

^ m

m^m^^

»_ — , — >

Biotype 1

Biotype 4
9

Divergent 5
of F7

Fig. 44. — Graphic representation of a test between a selectively divergent
male of biotype 4 population, shown in figure 42, in which a divergent male was
crossed with an homozygous female of biotype 1 and biotype 4. The test shows
clearly that the divergent male was distinctly a normal biotype 4 and had not been
altered selectively in any respect.

as follows : This male was first mated with a pure female type 1, and second
with a pure female type 4 of another strain. At the same time there were mated
four cultures in reciprocally mated pairs from the two test strains of biotypes 1

Analysis of Heterogeneity in Complex Chaeactees

259

and 4 ; all gave progeny, and in all the expected results were obtained. Each
test strain behaved as a homozygous line, giving in F^ an intermediate between
biotypes 1 and 4, and these in Fo segregated into the three well-defined groups
shown in figure 43. Fg mating in 8 pairs gave exactly the expected results of
segregation and permanency and in close approximation to expected ratios.

The extreme male in this experiment in F^ was kept a day or two with the
female of biotype 1, and then an equal period with the female of type 4, and
this repeated as long as the pairs continued breeding. In this way any error
from an assumed influence of age, strength, environment, or the determination
of anything by these factors is eliminated, as both lines A and B were served
alike by the divergent male. In the cross with the biotype 1 female and Fi
population were black, or black with a narrow margin of light color, 19 of the
former to 37 of the latter. Six matings from each of these groups, all giving
progeny, gave in all identical results, a very ordinary behavior in Fo, with
segregation into blacks of biotype 1, clearly marked biotype 4, and intermediate
types which were heterozygous, as shown by the breeding of F3 and shown in
figure 44. In Fj the totals from the twelve pairs is shown in table 34, which was
a fair approximation to a normal ratio, although biotype 1 in all sets of progeny
was slightly in the lead in point of numbers.

Table 34.

Biotype 1.

Heterozygous.

Biotype 4.

Observed

553

447.25

945 451

974.5 447.25

Expected

1

The mating B with the biotype 4 female gave quite different results. In F^
a variable progeny, largely like type 4 in character, resulted, and of these 10
pairs were mated, and all gave in Fg like results, and all gave a rather invariable
biotype 4 progeny in F3 with low amounts of pigmentation ; of these another 10
pairs of F3 mated at random from the population gave in F4 another population
of the biotype 4. This result is also shown in figure 44.

The result of this test of this divergent black male is in every respect to show
in an indubitable manner that, irrespective of its superficial aspects, it was not
modified at all as far as its homozygous biotype 4 nature was concerned. It was
black over the entire surface, but its gametes were not of biotj'pe 1, but were still
unmodified biotype 4. No discoverable effect of the quantitative accumulation
of pigment was found and every chance was given this extreme male to show its
capacities.

A second series of tests was made of this selected race in F9, where matings of
extreme individuals were made in reciprocal crosses with a pure-breeding,
homozygous-acting strain of biotype 12. In this cross other characters of alter-
native behavior are involved — i. e., body-form, elytral color, etc. These are,
however, not considered here — only the array presented of the particular pair
of contrasted characters — i. e., selectively modified biotype 4 and homozygous-

260

The Mechanism of Evolution in Leptinotaksa

acting biotype 13. In figure 45 I have shown the behavior when biotypes 10
and 12, both from homozygous-acting extracted strains, are crossed. In F^,
uniformly, type 4 is completely dominant and in F^ there is segregation,
usually into three classes, on the average of 1:2:1, as far as this pair of char-
acters is concerned.

Fig. 45. — A second test made with respect to the same modified character as
shown in figure 42.

In the test (fig. 46) 6 reciprocal pairs were mated between Pg of the modified
biotype 4 and biotype 12, and gave an F^ population all alike with biotype 4
dominant, 247 in all. Of these, 10 pairs mated gave progeny, an Fg generation
in three classes, variable biotypes 4, 12, and typical biotype 4 nonvariable.
Inbreeding each class in F3 showed the type 12 were true-breeding extractives

F3

mm m

J

F2

I

m ^ ^

^-

"I

m

Fig. 46. — Graphic representation of a further test
between a selectively modified race crossed with
biotype 12, showing that it was not modified but
was the normal biotype 4 as far as its gametic com-
position was concerned.

in the 7 pairs tested, and that the biotype 4 nonvariable were pure, while vari-
able biotype 4 were heterozygous dominants. At no point in these tests have
any indications of gametic alteration in the biotype 4 as the result of the quan-
titative accumulation of pigmentation been observed.

In this entire experiment and its testing great care was exercised to keep out
extraneous factors which might vitiate the series. At no time has there been any
indication of gametic change. How, therefore, are these changes, often beyond

Analysis of Heterogeneity in Complex Characters 261

the normal range as far as operation is concerned, to be interpreted? This
intense " quantitative accumulation " of a character did not in any way change
the gametic nature of the strain. Only in a certain portion of the strain there
was an accentuated condition of the organism, such that pigment was produced
over a wider area of the surface than was usual in the race experimented with.
The result is comparable to the quantitative accumulation of any other substance
or condition in organisms which may have been recorded. In the experiment
described it is clear that increased amount is persistent through many gener-
ations, so long as only parents with high pigment-forming values are used as the
progenitors of the next population, and it is evident that some germinal constit-
uent is exaggerated, but in what respect? The pigment itself is passive, a
product of the oxidation of a chromogen, and a capacity for pigment production
exists at all times in all portions of the organism. Whatever determines pattern,
either itself has, or has associated with it, something which inhibits pigment for-
mation over certain areas and allows it in others. In the selective accumulation
that factor which limits pigmentation in the normal strain is weakened so that
it allows pigment to be formed over wider areas than is normal, but it appears
to be impossible to do more than diminish (or increase) this factor by the cumu-
lative method, and no fundamental change is made either in the factors present
or in their relations, and only a distorted action (a condition) is produced which
may be varied at will.

Another series of experiments similar in import was made by starting with
biotype 4 and attempting, by quantitative diminution, to produce biotype 8 in
its extreme form. For this material from the same culture of homozygous-act-
ing biotype 4 stock was taken and matings were made from the extreme lower
limit of the type present. The results of this attempt are shown in figure 47.
No modification, no change of pattern, and little decrease of pigment was found,
and the whole culture presented the appearance of being against a limit below
which it was not able to be forced by mere subtraction of substance.

At different times I have attempted modification of other biotypes in L.
multitceniata, hut never with any success when the method of quantitative
accumulation or diminution tvas v^ed. The continued attempts to produce
changes by this method have thus far resulted in failure by all workers and in all
kinds of organisms. The reason for the failures appears to be because there is
nothing in the method of operation which could either remove any factor or
change its relation in the germinal complex. These biotype groups provide ade-
quately refined materials for such tests, and while with complex populations
(mixed) results of supposed quantitative accumulation may be obtained, the
results are due entirely to other processes. In refined strains such as I have
used or as have been used by Jennings, Johannsen, and others, when properly
protected from errors due to improper experimental culture, results of the same
kind have uniformly been secured.

The results show that there is in the method employed no point of entrance
produced in the organic system whereby anything can be added to or taken away,
or any relation within changed, and these are the only known means of inducing
change. In the strains the continued homozygous-acting gametes give in each
reproduction and in gametogenesis the same processes over and over again,
with only increase or decrease of some factorial action and manifestation. There
is no opportunity for metathesis or for possible change following this. The

262

The Mechanism of Evolution in Leptinotaesa

^

inefiBciency of the quantitative accumulation method is because it is without a
physical means of producing real change in any refined homozygous-acting popu-
lation, regardless of whether it is large or small. Whether genotype, biotype,
phenotype, or species, so long as the homozygous action of the gametes is main-
tained, no quantitative accumulation can become effective for change of any kind,
and this is the real reason why quantitative accumulation is inefficient in organic
transmutation, and is not an agent in evolution.

F7

F6

F5

F4

F31

F2

p:

m

-^

m

m m m

m ^m m

'^ m m m m

Biotype 4

Biotype 7

Biotype 8

Fio. 47. — Graphic representation of the efifort to reduce the amount of pigment
and thus alter the character of biotype 4 In L. multitwniata. No effect was pro-
duced.

The far-reaching and fundamental truth of this only becomes apparent when
one is dealing with pure, homozygous-acting, gametic constitutions, and there
the reason for its inefficiency appears. With impure lines " the method " will no-
doubt be in the future asserted to be efficient and productive of results, which in
reality are due to other agencies acting in heterogeneous material. There is no
doubt that quantitative accumulation plays a role in many operations of change,
but not directly, as I shall show in other portions of this report. Indirectly it
may be still an active agent in evolution in mixed populations, but with pure
materials in organisms and in non-organized matter no effective means is pro-
vided whereby quantitative accumulation can add new, take away, or produce
rearrangements independent of other more active and effective agents as inci-
dent forces and the interaction of the prime factors of constitution.

Analysis of Heterogeneity in Complex Characters 363

effects of quantitative accentuation of direction of

VARIATION IN ELEMENTS OF PATTERN.

In this application of the idea of modification through quantitative accumula-
tion or subtraction I shall deal with changes involving only the directions of
variation in simplest characters, the present indivisible elements of the pronotal
pattern. It has been shown in an earlier section how the elements of the
pronotal pattern " vary " in diflFerent directions, and how this results in the pro-
duction of quite diverse combinations or patterns. It has further been shown
that the biotype groups are mainly dependent upon this difference in combina-
tion of the elements into stable, separated groups, capable of isolation by
modern methods. In this section is considered the possibility of modifying
quantitatively the fluctuations in the lines of variation present in the simplest
characters.

The problem can be attacked by attempting to change biotype 4 into biotype 5,
by the attempt to change the direction of fusions between (a) and (d) from one
in an antero-mediad direction to the anterior end of (a), to a strictly transverse
direction as in biotype 5, and at the same time obtain by selective reduction a
loss of the anterior and posterior marginal spots of biotype 4. In these experi-
ments only homozygous-acting, carefully pedigreed stocks, reared under uniform
conditions, can be used, and any deviation introduces errors that obscure or
falsify the results.

Many attempts have been made to accomplish the result proposed by quanti-
tative change. Thus far no success has rewarded these attempts. At the outset
difficulty is encountered in finding the proper fluctuations upon which to work.
In many series I have repeatedly failed because the supposed fluctuation in the
desired direction proved to be only a somatic disturbance and not at all a gametic
change, while others proved to be gametic in character, but of too low capacity
for use in accentuation. So, likewise, the attempt to remove the anterior and
posterior fusion lines did not succeed, although it was possible to maintain with
ease much reduced states of these. In no case did these prove to be wanting in
the selected strains. Crosses with these and some other biotype always showed
exactly the behavior of a biotype 4 when crossed with the test strains.

The same result has thus far attended my efforts to transmute biotype 7 into
either 9, 10, 11, or 12, by quantitative accentuation. The best efforts of which
I am capable have been put upon tests of this kind, and I have everywhere been
rewarded by complete failure to attain by quantitative change any transmutation
from one biotype to another. An example of the difficulty attending the work-
ing out of experiments of this type is found in the experiments wherein I was
trying to change biotype 7 into either 1, 10, 11, or 12, and tests were made of the
real nature of the accentuation shown by crossing back upon another strain of
type 7. In 11 instances it happened that there appeared in such tests biotypes
of the desired kind, but in all it was found that the test strain had carried in
" unseen condition " the pronotal form-factor for the "' muUilineata form," and
this crossed with type 7 always by synthetic combination gives fixed pure-
breeding stocks in F, of biotypes 9, 10, and 11, rarely of 12. The changes
found in these tests were not at all a product of quantitative action, but a known
result following the presence of a definite factor when introduced into the game-
tic complex. To those familiar by actual experience with the delicate nature of
some of the gametic factorial effects, the above will appear most obvious. It is

264 The Mechanism of Evolutiok in Leptinotarsa

only by the elimination of errors of this kind, and of many others, that an ade-
quate test of quantitative accumulation effects can be made. Upon a gross, unre-
fined population, or unsimplified strain, no such attempt can profitably be made,
and any result obtained will be only a change induced by an unknown or at best
by a series of two or more interacting forces. Any permanent change, as far as
evidence goes, is most probably due to a new gametic state brought about unin-
tentionally by synthetic combination and not to the action of a " selective
process."

EFFECTS OF SYNTHETIC COMBINATION OF DIRECTIONS OF VARIATION
IN ELEMENTS OF PATTERN.

Quantitative modification has, in every instance thus far investigated, resulted
in no change of a gametically homogeneous group, clone, genotype, or biotype,
but on the other hand, the effects of intelligent synthetic combinations have
given different results. This is due to the fact that in these biotypes a system of
elements exists in a pattern system, and between these elements many combina-
tions are possible, but in every instance it requires the concerted interaction of
two or more agents to produce a change ; that is, one element alone, however
changed, can not combine with some other gametically ; but it is the experience
that both must be involved and show directions of variation towards each other
which unite and hold fast the two elements until disrupted by some agency ex-
ternal to the particular group. The existence of these bonds or directions of
combination affinities differ in their capacity for permanency in the same type
and in different types and species. It has already been shown how these bonds
change direction ; that their combinations are permanent or transient, and here
it remains to show how the biotype groups of the pronotal pattern that can be
isolated are also capable of being transmuted by proper treatment. The method
of change employed is, in its essential character, organic metathesis, producing
interchange and recombination of the characters of the elements of the pattern
of the pronotum.

An examination of the pattern of this part reveals the fact that the V — a' —
a — h group is either widely divergent anteriorly in the form of a widely open V,
or parallel with the h' h at the level of the a' a area. This results entirely from
the presence of two form-detenniners interacting with a pronotal form-factor.
I have shown that in the natural material of L. muUitceniata there are two gen-
eral body-shapes which are expressed by the phenotype group names, " multi-
lineata " and " muUitceniata/' Whenever the muUitceniata factor is present,
the spots b' — a' — a—h are a broadly open V, and when the muUilineata factor is
present they are closed and parallel. This can be shown as follows :

If a perfectly pure homozygous-acting strain of the form melanothorax be
obtained, and this combined with the proper forms, tests to prove this above
assertion can be made. The melanothorax form is another complex; the centers
are lost, its ontogeny and everything show it to be different from the rest of the
species when in pure strains, even though it is not to be distinguished in the adult
condition. If such a pure strain is crossed with a biotype 12, also homozygous in
action, there results in F^ a progeny with the melanothorax dominant, either
completely or nearly so. These inbred give in F^ three complex groups — one
with the form of muUitceniata, one intermediate, and one of the muUilineata
form. The proportions are variable, but in the long run are a close approximate

Analysis of Heterogeneity in Complex Chaeactees

265

of 1:2:1. Withiu each of these groups, however, the pronotal pattern shows
considerable diversity.

The least numerous groups are those with the narrow form of multilineata,
and here two types of pronotal pattern occur in alwut equal frequency — a com-
pletely black one and one or more of the biotypes in the 9, 10, 11, and 12 groups.

.yj

F3

'M>

F2

Fi

P-

Biotype 1 Biotvpe 12

+ +

.^multitaeniata form multilineata form

Fig. 48. — Graphic representation of the action of the form-determiner in the
alteration of the color pattern of the pronotum.

The second class shows wholly black, while the most common group shows black
and a variable array of pattern, as shown in figure 48. In the least numerous
group are found blacks that are both homozygous and heterozygous, and from
these it is a simple matter to extract a homozygous-acting strain that has the
melanothorax pronotal pattern in combination with the determiner for multi-
lineata, a narrow elongated type. Two test agents, races, are now available, each
with the same pronotal complex but associated with the two different form-deter-
miners, and each in pure homozygous-acting biotypes.
18

266 The Mechanism of EvoLUTioisr in Leptinotarsa

One test of the action of these determiners can be made as follows : A race of
biotype 4, which has normally the same form-determiner as the normal melano-
thorax type, when crossed with the melanothorax, gives an F^ population variable
from dominant type 1 to dominant type 4 (fig. 49) . In F2 always there occurs
the one form and three pronotal types — type 4, variable intermediates, and type
1. The first and last are homozygous, the second heterozygous. The only
gametic differences contrasted are the pronotal biotypes 1 and 4. If, however, a
cross is made between the biotype 4 and the black pronotum plus the multilineata
form-factor, there results an F^ progeny variable but of biotype 4 character. In
F2, however, there result again three general groups in form, and each variable
as regards pronotal pattern. There appears a group of long, narrow forms —
broad, oval, rounded, and intermediates. The ratios of these are variable, but
appear to be present as 1 : 2 : 4 ; at least the most numerous group is always the
broad, oval multitcejiiata form, the least numerous are the multilineata form. It
is in this latter that there occurs the evidence of the action of the form-deter-
miner, because here occurs in all instances numerous examples of the biotypes 9,
10, 11, and 12, where a' h' a h series are parallel and not a broadly open V,
as in the other biotypes. Between the two crosses of biotype 4, the only differ-

F2-

m w^ m

L- Biotype 1 Biotype 4

Fig. 49. — a graphic representation of the action of these determiners showing
the appearance of pionotal pattern biotypes not present in the original stocl£.

ence is the presence of the form-determiner, and clearly the presence of the form-
determiner acts to change definitely the pattern of this central system of spots
and their position, configuration, and interreiacions. Its presence has produced,
and does produce each time, this definite and exact transmutation, in individuals
possessing the factor, the pronotal pattern of either types 9, 10, 11, or 12, or of
entire black. There is in this a clear transmutation of biotypes, a transmuta-
tion due to a determiner for form, whose presence and action can be experiment-
ally proven. The process which is productive of this change is a synthesis, and
recombination following upon an organic metathesis produced by combining
unlikes in an interacting complex. This result is shown in figure 50, where bio-
types 9, 10, and 11 result, and could by extraction be obtained pure in F3, F^, or

In the same way and by the same method other changes less marked in visible
manifestation, but none the less definite and capable of demonstration, can be
produced. Thus pure homozygous biotype 3 X biotype 8 will give in Fj and Fg
homozygous biotype 6 individuals, from which a de novo strain of biotype 6 can
be obtained. Biotype 8, like biotype 7, is a neutral one with fusions not estab-
lished and lacking the marginal areas. Biotype 3 carries the marginal centers
and the interaction of 8 x 3 gives a transverse system of combinations, as shown

Analysis of Heterogeneity in Complex Chaeactees

267

in biotype 6, where the addition of the posterior marginal areas may produce a
pattern with the posterior half almost uniformly black. From the same com-
bination there not infrequently results biotype 5, which lacks entirely the mar-
ginal areas, but is otherwise like the pattern of biotype 6.

F2-

Biotype 4 + Biotype 1 +

multitaeniata form multilineata form

Pig. 50. — Graphic representation of effect of form-factors upon the pronotal
pattern type.

Biotype 8 Biotype 7

Fig. 51. — Showing effects produced In crossing of biotype 7 with biotype 12 and
results produced in the alteration of pattern.

Different results follow when the neutral biotype 7 is combined with biotype
12, which gives as an F3 extracted biotype 8 (fig. 51,) and in the same way bio-
type 8 crossed with biotype 9 will give in F2 and F3, as an extracted type, the

268

The Mechanism of Evolution in Leptinotaesa

neutral biotype 7 and biotype 12 (fig. 53). Combination of biotypes 7 and 9
give biotypes 10 and 5 in Pj and F3 as extracted forms (fig. 53). This is not
the place for an extensive analysis of the gametic constitution and of heredity
behavior, and only enough has been presented to show how exactly these " vari-
ation " phenomena are a product of gametic constitution, and to show how differ-
ing determiners are capable of rearrangement, of extensive manipulation, and
are able to transmute one biotype into another by altering, through its presence,
the configuration of the pattern complex of the pronotum. The biotypes are
realities, capable of independent stable existence; the determiners are no less
real, even though unseen, and known only through their action. Not one deter-
miner but several are involved, each more or less independent and capable of
altered relations, of being present and acting, or of being entirely removed or
made inactive in the complex.

F3-

F2'

Fl-

F3-

.^ F2

J I

I

Pi-

JL

Biotype 8 Biotype 0

A

Biotype 7 Biotype 9
53

Fio. 52. — Showing behavior of biotypes 8 and 9 when pure homozygous lines
are crossed.

Fio. 53. — Showing effect produced In the crossing of biotypes 10 and 5.

In nature there is constant and unlimited mixing of these factors and deter-
miners in all kinds of combinations ; but out of this complex of interacting agen-
cies certain definite patterns always come, so that the net result is a rather stable
population as far as the patterns presented in any given location. The hetero-
geneity presented, however, is not one of quantity nor of directions of departure,
but is at least analogous to the diversity found in many chemical operations
where nearly related compounds are easily transmuted into some other through
the presence or absence of something whose presence determines a different
configuration of the system, and whose absence permits of another and diverse
arrangement. In this pronotal pattern, which is a system as intricate as one can
wish, the presence of one or two form-determiners decides the type of the central
a' b' h a group. The determiner of the posterior and anterior marginal spots
likewise determines the resultant changes found in certain biotypes, and so on
through the series.

From this point of view the variation in the complex pattern as found in
nature presents a very different aspect. It is no longer " variation," but
metathesis, and the phenomena must be viewed as purely physical in character

Analysis of Heterogeneity in Complex Characters 269

and of chance occurrence, dependent upon chance gametic agents, combinations,
and conditions present, as far as their appearance and frequency in nature are
concerned.

The discussion and description of the foregoing relations in the pronotal pat-
tern are based upon materials held under uniform conditions of growth and
development and provided with optimum food, temperature, and other life rela-
tions. In nature the conditions of life are neither constant, uniform, nor
optimum, and probably no population ever exists that does not encounter more or
less in the way of adverse conditions at some time. It requires but little obser-
vation in nature to show that from generation to generation different aspects of
the population are presented, and these have usually been attributed to the effects
of external conditions. That this is true to some extent no one doubts, but the
exact role of external forces in any given case has yet to be determined. These
pronotal patterns are no exception to the rule, and this aspect of the problem will
be considered after a discussion of the conditions in nature have been presented.

CHAPTER IX.
ANALYSIS OF HETEROGENEITY IN THE POPULATION.

It has been shown how the elements of the pattern found upon the pronotum —
simplest characters — behave with reference to different factors that are met in
the life of the individual. How some of these are related in the complex pat-
tern that is found on this portion of the body and the different aspects of some
of the problems of heterogeneity presented by this portion of the organism.
There remains the complex character as it is found in nature in the population,
the manner in which this system acts as it meets the ever-changing conditions of
its habitat, and the metathesis of simplest characters within the population
itself. A picture and an analysis of the conditions found will help materially to
gain a complete vista of the problems of heterogeneity in a complex character,
and help in some measure to aid in the understanding of the causes of this
heterogeneity, its limitation, and control within the population.

For this survey of the conditions in the population the tropical forms have
afforded the finest material. The species L. multitceniata Stal has been particu-
larly favorable for this analysis in nature and for the experimental studies, both
in nature and in the laboratory. The fact of its complexity, the fortunate
diversity of its natural habitat, and the close proximity of habitats of widely
differing conditions have, in the aggregate, produced conditions of uncommon
excellence for the proper and successful pursuit of the problems. Naturally the
other species in the group have come in for their share of the same treatment,
but none of the other species has been given the time or attention that this one
has, and none has given the least indication of principles that were in any man-
ner different from those found in the most exploited species. In the presenta-
tion of the rather extensive data, the condition found in each colony during the
time of observation, with brief comments upon the findings, will be first pre-
sented, and at the end a comparison and discussion of the results of the examina-
tion and census-taking, after which some of the experimental results will be
presented.

IN LEPTINOTARSA MULTIT/ENIATA.
THE CHAPULTEPEC COLONY.

The character and general topography of this location have been already
described, as well as the methods of taking the observations, and the source of
the climatological data used to show the conditions that prevailed in the location
as nearly as possible during the time that the observations were being made. It
would have been far better if the records could have been made on the exact spot
where the organisms were living, but this was not possible, and the records
presented show fairly well the general prevailing difference of conditions in this
and in the other colonies where observations were made.

270

Analysis of Heterogeneity in the Population 271

The observations were begun in 1904 at this colony and ended at the close of
1909, covering 13 generations in the same location, and are lineal descendant
populations, with probably some additions of new blood from outside of the
colony. The records show the response of the population as a whole at this
location to all of the conditions it had to meet, and as registered on the pattern
of the pronotum.

The first census of the population, made in 1904, showed a total count of 3,550
in the colony, with the distribution of the different biotypic groups without
breaks, and the array when plotted gave complete gradations along all of the
lines with extremes lacking, with the exception of the series composed of bio-
types 1, 2, 5, and 10. Comparatively little " somatic " variability in the amount
of pigment is shown in the population.

The second census, made in 1904, showed somewhat different conditions, with
a population of 1,970 in the colony, and instead of the homogeneous array which
the first census presented, the distribution in the second is broken and shows
many gaps. In the males there was a clearly isolated group of biotj'pe 1,
another of 10 and 12, while the extreme conditions of biotypes 8, 6, and 5 stood
apart from the population as distinct groups of considerable strength. The
males showed much more variability than did the females where the only dis-
tinctly isolated biotype was the extremes of 12. Comparison of the two cen-
suses for the year shows quite different conditions for the population, and illus-
trates the utter uselessness of a single determination of the condition in the
population as a means of discovering the true condition therein. These census
records I have shown in figures 54 and 55.

In the third census, made in the early portion of the season of 1905, the
population was small in number, 1,897, with a distribution much like that at the
beginning of 1904, with no separated groups, and the entire range fairly well
represented, with the exception of biotype 12, and the curtailment of the
extreme conditions of biotypes 5, 6, and 8.

The fourth census, made in the latter portion of the season of 1905, showed an
increase in numbers, 3,230, and the separation of the population into isolated
groups, the probable absence of biotypes 5 and 6, the isolation of the extremes
of 8 in the males, and the isolation in both sexes of 10 and 11. On the whole, the
population in the fourth generation showed much restriction in its range, and
this came in a season and the portion thereof when the precipitation was low
and distributed in such a manner that the critical periods in the life of the
population were passed in relatively dry conditions with high rates of evapora-
tion. The condition in the population for the two censuses of the 3'ear is shown
in figures 56 and 57, and exhibit about the same condition as in the year 1904,
with the absence of extremes, excepting in the series composed of biotypes 1, 2,
3, and 4, and the isolation from the mass of the population of the same groups
that were isolated in the second generation of 1904.

In the year 1906 the first census made (the fifth) showed a new arrangement
of the distribution of the population, in that the 9, 11, and 12 series was well
developed in both sexes and strong in numbers, whereas the groups 5, 6, and 8
were practically wanting in the population. The series 1, 2, 3, and 4, while
present, were, in proportion to the whole, not as strong as in previous censuses.
The only isolated group was a small number of males of biotype 5. This condi-

272 The Mechanism of Evolution in Leptinotaesa

tion in the population occurred in the early portion of the season, when the
rainfall was low but well distributed, with uncommonly widely ranging humid-
ity percentages and consequent desiccation during the reproduction of the
population that had emerged from hibernation from the previous year.

,96-t^ ^i^?» 77-ife

85 -\
127— ^•
92—

I ! '92-1-;

( / 86— t^

-iS*

:109-^.-^
114_ — _^-;:;

75 ;_^.

10 ^r-m-'' . J^i 92 1^ V^'^'. ^- — 77

60 '-^ ■ ^ m-- n 65 --^rr-m :\ ^^— ,r— 100

92_-^_.,..::-:-|^,^^: ^i* 86—^:-^---%^ ^-,?— "219

im-m^^^ ^..: 219- ^.^.^ ^--- 155

f 7 'Im. ^- 141 / / /■■~'^^::^ 32

/ / / \ \ / / /

21 Tl -101 4 18 }9 71 92 18

1705 Males. Total 3349. 1644 Females.

Fig 54. — Census of first annual generation of 1904 at the Chapultepec colony,
Bhowing actual pattern conditions In population and number of Individuals In total
population showing the pattern observed.

■41-^\ 11 4 17 ?

^^' i ,-Mv^ 1— 4i^x ! I / ^^-^

41— ^-«»

mi

31 WB-k 59 T— «^ ^ £l 31

14 -^^:^a ,17 :-—*»- : -,j^:^- 64

87 -V#\ :im 31' 14 ----«* mr- 77

74 ^ i^ 79 74 .- — -*W» ■«*"^ 141

1^ V, -iM* «¥ii 186 -^ «¥t— --: 211

' ?^,, *^ 92 / ^^ *^--- 41

i|W|^? 8

/ / \ m

! /ms-K / \\-

8 31 3 141 *^* ^ 46 13 91

906 Males. Total 1970. 1064 Females.

Fig 55 — Census of second annual generation at the Chapultepec colany in 1004,
showing pattern conditions In the pronotum and the fact that sharp dlfterencea
in the population may arise between successive generations.

The second generation showed in the census essentially the same condition as
in the first for this year. The same groups were present, in about the same
strength, but in this generation there were no isolated portions of the popula-
tion. Biotypes 11 and 12 seem to have been lacking, and in their place 8

Analysis of Heteeogeneity in the Population

273

appeared in full strength, while 5 and 6 were still practically absent. This
generation was the product of the first, but under conditions that were unusual
for the place, in that the rains had been light in the earlier portion of the
season, the ranges in the humidity had been large, and the desiccation high,

Total 1897

/ / / \ \ \

7 12 5 4 7 58

1034 Females.

Fio. 56. — Census of first annual generation at the Chapultepec colony In lOOS,
showing conditions In the population with respect to pronot.il pattern sod rela-
tlye frequency of each type.

.^^^.

^^.-31

41-1 ^

74 ^

ao ^ ^ ^^^^ gg

214 ^^ .^^^^^ 34

«18 -i^ ^V^ 4- 211

< «¥^- 196

31--^''::> !

141 — ---<©»

192 \--^ \

.96 -^5», I

43 ^<^:i .^

88 y^^-^,,'^.

171 _A.^ ^.

•''j^\

^-9
5S#-46

41

55

77

319

41

\^-

17

1624 Males. Total 3253. 1629 Females.

irio. 57. — Census of second annual generation In the Chapultepec colony In 1905,
showing the array in the pronotal pattern, and a sudden change in aspect of the
population from that shown in preceding censuses.

while in the latter portion of the season the rains had been copious and well
distributed, with low ranges in the desiccation and moderate and uniform tem-
peratures. The comparison of the two censuses of this season with those of
previous years shows different responses in the population to the conditions of

274 The Mechanism of Evolution in Leptinotarsa

the total complex in which they were existing. Figures 58 and 59 illustrate
well the condition in the population, and the fact that the observer who saw
only the 1905 generations would have continuity, and the one that observed the
populations of 1904 would have " mutations," and the stage would be set for a
fine series of pointless " brief communications."

14 —

24 ->^^

46 %^i^ J'r. ^^r^fs^k

17 —

## 96 S^^

12 H^^^ fO!^.:^ 39 14 ^^^

31 .a:*e«? y^^- 417 42 -^|#f^'^1i^^ 246

/

C^; ;'^>f| 261 ^^-^^#^'jr 292

:;*^^,.^^ ^192 / /''^^^^^ 141

^;.=«s.. 17 i . , -.;

, , y1^ 34 / / \^- 21

/ / \'' / /

4 7 31 2 8 46

1655 Males. Total 3350. 1695 Females.

Fig. 58. — Census of first annual generation at Chapultepec colony In 1908,
Bhowing the array of population in pronotal pattern.

17 -^>-^--, 42- ^Ml^

31— ^»4 y—Wi^

42- — ^lil^ 9— -^f<e^-,

61 1^^*;, 38 1,.^ -- ^

94 "— iiit . .^sm^ .. 191 ^-

66

83 ---yiCSJ- ^mr- 31 51 -«^^(^^\ .^ 92

41 ;-'-<«•■ g.-^i-- 192 64 -ffl^^"^"^" 219

f^3*_ «¥»-•? 481 ^ ^h^ 386 '9

/'•^^^-- 314 /\^^,^. 217

/ \v;*^'':t 161 / '^'*'^^^" 211 j

/ ^^ft ^^ " / \'

/ ^"s"^^^ 45 / \

17 19 22 4

1842 Males. Total 3549. 1707 Females.

Fio. 59. — Census of second annual generation at Chapultepec colony in 1906,
showing the array in the pronotal pattern.

The season of 1907 was in many respects an optimum one, with the different
factors of the climatic complexes distributed in such fashion that they afforded
the best conditions for the growth of the population at all times and essentially
equal conditions in the early and late portions of the season, so that the condi-
tions in the population were unusually uniform throughout the season.

The first census was made in the middle of July at the maximum of the first
summer generation, and showed full development of all the lines, with the
exception of biotypes 5 and 6, which were not well represented, if present at
aU. N"o isolated groups were found, with the exception of the females where

Analysis of Heteeogeneity in the Population 275

biotj-pes 10 and 11 were separated by slight gaps, and in the females the extreme
end of biotype 8 was wanting.

The second generation in this season showed the nearly complete development
of all of the lines in the population, no biotypic groups being absent, and none
separated from the mass of the population by discontinuities of any sort. This

61^^ 4J^1^ 43^ 50 22 18

7^^^ i' M-'' ''■ m :^ i i / g-«s

m---r--i0- =,. ^s, .|i^£is^;,..^ .-.: ^■s^^

7- . afc :-- :>:a^^^^ 22 141 —

86 f-j@^ , '''<i^^ 71 -» ^

107 ^-.rrmy i^^-3 146 44 -^-4^^;

79 -W"^^^ ^feV-..^- 36 186 -^^^-^;, _^

81 -^x,>e^::*gi^^-- 219 210 jf~^ ^ ^^®

l^ ic^-V 417 ''^ -.^Z^ ■***

y0,; ^.. 209 / :^<^-^ 199

/ \ -tV" 44 / \

/ V-v*^'"- 31 / \

5 7 12 17

2289 Males. Total 4507. 2218 Females.

Fig. 60. — Census of first annu.il generation at Chapultepec colony in 1907.
(Showing great difference in the array presented by the two censuses in the pronotal
pattern and also different from those present in the preceding censuses.

12 44 31

3170 Males. Total 6782. 3612 Females.

Fio. 61. — Census of second annual generation at Chapultepec colony in 1907.
The population was numerous and showed nearly the complete array possible for
the pattern in the population. Only a few of the extreme members In each of the
general lines are absent.

condition in the population is not often attained at any location in this species,
and appears apparently only under the influence of optimum conditions in the
climatic complex. The findings in the two censuses of the season of 1907
are shown in figures 60 and 61, the conditions of the climatic complex in
climatic record 4,

276 The Mechanism of Evolution in Leptinotaksa

The season of 1908 was also a highly favorable one for this species at this
location, and therefore tlie results of the examinations of the population are
much like those of the previous year. In the first census the population shows
some curtailment of biotypes 5 and 6, the absence of 12, the reduction of 10,
about equally in both sexes, so that the distribution of the sexes is essentially the-
same.

51 -^#-f, ,, 7 4

10- t^ ' ; ;

42- ^ I I

71 \®''

£5 ' 4^

42 Mi

8

i^ -'---42
77 Tr^-,:i -^^^---si 81 -m 4s^ - — m

42 '-^'f.-^^:^ _^:^;-^_._- — 44 46 W m 58

58 ":--f.^'ift^-- 88 17 '-:— '^ iSH^ 71

*ip„^^ ,^-:! 2191 '^^'* **^ ' 246

/ ff^r^:^'. 86 / , '^ '^f*^h 79

/ Ji^-'X 0^'^ — --31 / /«« ^i'J 51

/ /^^\ \ ',^,-.-.19 / /=«*. ,^ ^ -S^; 4

// \\ X'W— 7 / / \\ X di^-10

16 41 12 19 U 12 55 1 19 86

1290 Males. Total 2621. 1331 Females.

Fig 62 — Census of first annual generation at Chapultepee colony In 1908,
showing about the same range of pattern distribution, but with fewer numbers
throughout and a tendency to restrict the extreme numbers, especially in biotypes
10, 11, and 12.

18-11pi I 40-^-,^
31— --TfiBS'v. i'jci. 42 ^^IB>M

42-— ^-^o-g% ^-36 86- — f--«^\, ^^—^ S

51 -•^__^^. , .^_.^_...42 <, — --- 4ge isft/-.,,.^.^

64 _.___^, j|^-^-- 39 31 —r — -^.] :**--- 146

^ — r'^-.-Xm W;-- 166 S2 TJ^**^; ^•■- 248

'^^Ji^ '^-J- 141 '-^^ if^'-^- 319

f-'-'i r-'M:,^^- 76 /' *^. W 12

/ / /^v \ ..^ 12 l^:. \

/ ' J \ \ \ I \ \ \

9 41 8 4 14 31 13 7 18 ^6

1083 Males. Total 2613. 1530 Females.

Fio. 63. — Census of second annual generation at Chapultepee colony In 1908,
showing the array in the pronotal pattern of the population.

The second census taken in this season showed much the same conditions that
were found in the first, with the exception that biotype 10 was present in the
males as an isolated group and wanting in the female population. Some minor
differences in the proportions of biotypes 5, 6, and 8 are shown in the array of
the two sexes, but are not of any significance. The conditions of the habitat
as shown by the meteorological records were much the same as in the previous
year — uniformity of distribution of the elements and lack of extreme conditions.
The findings for the two censuses are given in figures 62 and 63.

Analysis of Heterogeneity in the Population 277

The season of 1909 was the last in which this series was followed methodically
in this location, and completed a series of 12 censuses of 12 successive genera-
tions at one location, in a population of fairly restricted character and habitat.
The first census of this season did not show any conditions greatly different from
those found in pj-evious determinations. The season opened with rains at
unusually early dates, so that there was much reproductive activity in late May,

n-^mx oft ^'"" v^Sp ^%i i^'-'

19 ..j^ ^ , :^-- 31

41 sm.^- m^- 9

42 j^ 5^ 4j ^^ ^

51 j^ ^ 77 ^^ Xj^ 1^2. 45

44 «. ^ ---19 ^g....^.,_..:..^^^. 101

,61— -•■ ^ -^ ^21 ^mi^- m-- 1«

** ^^ "^ T^'t"/^^^ 22

, ** ■**'^^ 3^ / / ^^^*'\ -^^ -31

/ \ / / myy^^^^-- ^

/ \ / / / \ \ \"%^: — «

6 19 18 14 12 15 8"^

799 Males. Total 1523. 724 Females.

Fio. 64. — Census of first annual generation at Chapultepec colony In 1909,
showing the array in the pronotal pattern.

9- iA'

8-
9-

14- - ^ _

4- ^. '^ 31 -^ m^

8 - - ^ '^^'^ ^
19 -^ X,,,_ 1** *^ ^^ 19

^"" 31 43 ^ ^ g^

31 ,ai? -av 61

IS - - - ife!* *6. - 116

«*»«»*« «^ 145

^Isf^ 'I2 / / Jf* ^?*^ 51

41 ^ Vjft¥--^^ 45

43 ^; ^ _;:^ 41

8 ^. -- - tj^ W- — 92

''m >m ^ <^-^- 161

/ .' .' ^ ' / / ^ ^ '

9 4 15 31 7 9 14 1 4 22

632 Males. Total 1401. 769 Females.

Fio. 65. — Census of second annual generation at Chapultepec colony in 1909,
showing the array of the pronotal pattern.

with widely ranging conditions in the humidity, rainfall, and desiccation. The
population that started its development in this period showed that the males
were much reduced in all biotype lines, with the exception of 1, 2, 3, and 4, the
absence of 12, and portions of 11, 10, and 9, while the extreme of 10 and part of
11 were present as isolated groups. The females, however, showed nearly all of
the lines present to their full extent, and no isolated groups, so that the condi-
tions in the two sexes in the population were quite different.

278 The Mechanism of Evolution in Leptinotarsa

The second census was taken at the optimum of the season in the second
annual generation, when the conditions were somewhat more severe than they
had been in this portion of the season in previous years. In this census there
were no isolated groups in either sex; biotypes 10, 11 and 12 were wanting in
the population in both sexes; biotype 8 was reduced to the merest trace, and
might have been absent entirely ; while biotypes 5 and 6 were variable in their
presence in the sexes, but were certainly present in both. The general condi-
tion in the population in this year shows a uniform restriction in the range
which might in some measure be connected up with the prevailingly harder con-
ditions of the environment in the location during this season. The findings in
the distribution of the population are given in figures 64 and 65.

These 12 censuses, taken in the same location, when each of the two annual
generations were at their maximum, do not show for the location uniformity in
the population as a mass, but diversity. During the progress of the observations
the climatic conditions were given as full study and measurement as possible,
and while it would have been vastly better to have had measurements made on
the spot, by methods better suited to the purposes of the study, the government
observations are the best on the whole that are available for the entire period
of the observations, and do show in rough fashion the march of events in the
habitat.

The population is constantly shifting in this location, and has shown in the
distribution of the quality chosen for examination — conditions which if seen but
once would have been quite differently interpreted. It would in this instance
not be difficult to build up from the data of this set of observations rather plausi-
ble arguments concerning the action of the environmental conditions in the pro-
duction of the diversified arrays presented in the different censuses. I have
never been enthusiastic in this, because all that I have been able to perceive in
the situation is that the determination in nature has given me data concerning
the sequence of events in two series of phenomena that were passing at the same
place and time, and in some instances the oscillations in the one coincided in
some respects with the oscillations in the other, but in so far as I am able to dis-
cover, there exists in these determinations plausibilities and suggestive situa-
tions that may well be the motive for exact experimental analysis in nature and in
the laboratory. It is situations of this sort that have given the basis for numer-
ous arguments in faunistic and ecological literature, as well as in the field of
evolution essayism, in which the argument is from effect to cause.

THE TEXCOCO COLONY.

The records at this colony cover the period from 1903 to the middle of 1909,
13 censuses in the population being made, of which the first two were from col-
lections made in the summer of 1903. The location and its general topographic
setting have been previously described and differ from the Chapultepec location
in many respects. For a record of the climatic conditions prevailing at this
location I have been indebted to the local observers connected with the service
in the state of Mexico. These are not so complete as those from the observatory
at Tacubaya, and in the main are monthly averages, with distribution of some of
the elements. These records, while more might be desired, are far better than
none at all and are the best that one can do in work of this sort where trained
observers are not available to make the proper measurements.

Analysis of Heterogeneity in the Population 279

The first conditions in this colony were determined from collections made at
random in the year 1903. The first census for this location shows at once a
decided difference from the condition prevailing in the Chapultepec colony.
Conspicuously isolated from the mass of the population is a group composed of
biotype 1, while 2 and 3 are wanting in this line of development in both the
sexes. The biotypes 10, 11, and 12 are also wanting in both sexes, and both
show 9 fairly well developed, while 5, 6, and 8 are poorly developed, if repre-
sented at all. The distribution in the population is uniform for the sexes.

9

31 ^

f * 12

Sti 31

. * ■" - ''ml 20 42 *#■■> fm^'--' 48

24 :^-'*«:f 5J 51 ,^-m m^- «

agi_- 9G

i?^ ^ ^i^^ 42

»3g:

17

/ •"'^■■■\^^ 4

/ ^r^^ir " / \^"

9 3 12 9

342 Males. Total 857. 515 Females.
Fig. 66. — Census of first annual generation at Texcoco in 1903, showing condi-
tion of the pronotal pattern.

7 ^^

6 ,-'it''-.„ „ viS^.. ..<W-

14 .-«► \ ^^

11 -'-- iSfr /'m- 31

31 -,-\^'.; ■&»-- 55

12 -m

i^--'^ 42

7 -^-m]- I «*--- 51

14 :-"i@« , ; Wy- — 86

41 tjn^ if^-_^ 45

19 ''■-'■i^ iiff..-; 92 :l^ ^^ : 92

i«* ««*-

/ W, fV=f:^ 39

I m, ^ 41 / iiti-\ - ^,^ 14

/ "" \ \

9' 17 8 '5 n

418 Males. Total 903. 485 Females.
Fig. 67. — Census of second annual generation at Texcoco in 1903, showing con-
dition of the pronotal pattern.

The second generation from this location of which the census was made
showed essentially the same condition in the population as was found in the
first yearly generation. These determinations are shown in figures 66 and 67.

In the season of 1901: two censuses were taken ; the first showing in both sexes
considerable restriction of the range in the character, with the complete absence
of any isolated groups and no trace of the isolated biotype 1 group that was
found in the previous year. Biotypes 1, 2, and 3 were entirely wanting, as were
10, 11, and 12 in the males and 11 and 12 in the females. In both biotypes 5
and 6 were wanting or weak, but in both 8 was well developed in the population.
Compared with the corresponding generation of the Chapultepec colony this
population is strikingly different.

280 The Mechanism of Evolution in Leptinotarsa

In the second generation of this year the distribution of the population over
the range of values was much different from that of the first. The line com-
posed of biotypes 1, 2, 3, and 4 was nearly restored in the population, completely
so in the males and with only the lack of 2 and 3 in the females, while in both
sexes 10 was present and isolated, as was also 11, while 5, 6, and 8 were wanting
or weak in their manifestation. The conditions at this location seemed in this
year to be much more variable than at the Chapultepec colony, and this seemed
in some respects to be reflected in the behavior of the population. The findings
in the censuses are shown in figures 68 and 69.

5

-42

12 —f

■46-— -:^'-'«^--:-l — -77 66--'--rm\ miy^- 73

■42 --m *^:r-- 91 "— ^'*«^'>^?- 84

$^: ^ :f- 99 ^:.. '^--V-- 104

/ \ W? ■ 51

/ \ ''\^^^ 31

5 19^^ 4 8 i2^

653 Males. Total 1390. 737 Females.

Fig. 68. — Census of first annual generation at Texcoco in 1904, showing condi-
tion of the pronotal pattern.

738 Males. Total 1352. 614 Females.

Fig. 69. — Census of second annual generation at Texcoco in 1904, showing con-
dition in the pronotal pattern and increased variation thereof, but with marked
gaps between the extreme members.

In the season of 1905 the population of the colony showed even more diversity
in the range and in the number of isolated groups than in the previous season.
In the first census made this year, biotype 1 was present as a conspicuously iso-
lated group, not numerous, but vigorous in appearance, and would easily have
passed in the population as a distinct species. In the males, biotypes 12, 11, 10,
and 9 were all present and formed a continuous series, but in the females 11
was wanting, leaving 10 and 12 separated in groups. Biotype 8 was practically
wanting in both sexes and 6 was absent from the female population, and repre-
sented in the males by a small isolated group. Biotype 5 was present in both,
but in the males it was broken, the two extremes existing, with the average con-
ditions wanting, while the females presented a uniform array in this series.

Analysis of Heterogeneity in the Population

281

The second generation in this season showed still different conditions from
that of the first, in that there was an entire absence of biotypes 1, 2, and 3, in
both sexes; 10 was represented by a well-defined group that while small in num-
bers stood well apart from the population, and the female showed a weak develop-
ment of 13, while 8 was wanting in both. Biotypes 5 and 6 showed the same
general conditions that were found in the first generation of this year. Com-
parison of the findings in this generation of the population with that in the

10
12

', ^-V— 19

,'• ^^^^- 51

W^^ 86

-i^^ *lk^-- 77

#« ^^^- 98

JW^-:^^A 61

/ "■' V^^ 42

m

9 f^^^

26 ^t

43 ^

44 . .^

31—/-- ' - ^

fr

34

'^W 41

lUf'f 88

MIK ^ ^-\ 92

<ih ^ 51

Total 1478,

6 14 16

598 Females.

Fio.
tion of
group.

70.-
the

-Census of first annual generation at Texcoco in 1905, showing condl-
population with regard to the pronotal pattern and in this isolated

^n

22

40

61

54

41-_. VJ-

■* i# 4

*» tm-- — 15-
'~"V."~"'^\. ** 19

--f—'-y--_-'*t^ *^ 81

^ H# fftjt i^ - J04

r^r'7^ ^ 5^

/ / i?*l . ^,';— — 45

III \ \ \

11 4 12

I 19 6

34 ^

46 -.-^

,«^ WW «•}» ^.-v —

/ / >^x*"

i i /""\ \

6 12 8 3 9

7

51

86

44

597 Males. Total 1006. 409 Females.

Fig. 71. — Census of second annual generation at Texcoco In 1905, showing
reduction in the array presented by the population and the diminished number
in Isolated groups.

corresponding generation at Chapultepec still shows differences that are of con-
siderable magnitude, chief of which is the evident tendency in the Texcoco loca-
tion not to present so wide a range in the character in question, or the frequent
complete series in the different lines, and the greater frequency of groups iso-
lated by considerable gaps. The conditions of life on the whole were more severe
at the Texcoco location, with wider ranges in the temperature and desiccation
and more variable rainfall. Coupled with these conditions is the apparent dif-
ference in the population shown in the records. The findings for the two
censuses of 1905 are shown in figures 70 and 71.
19

383 The Mechanism of Evolution in Leptinotaksa

In the season of 1906 the first generation showed no especial change from the
conditions shown at the close of the previous year. In both sexes biotype 5 was
much reduced, 6 was also reduced, and 8 represented by a few at the upper end
in the males and by a distinctly isolated group in the females. Biotype 1 was
present in both in small numbers, and 11 was found only in the females. The
two distributions are essentially the same.

9 —

^ 62 / ^^

y^ « ^, \ P---^

18 19 12 8 '"

429 Males. Total 976. 547 Females.

Fig. 72. — Census of first annual generation at Texcoco in 1906, stiowing con-
tinuation of the same condition present in the last generation of previous year.

I

6 ,j,n

11 '^

"- * ■ .^—-33

9 ^ .^ ^^

12 .. ^ ^ ^g

^ ^ 103

-' i3^^ ■^-- 42

' w y av?- - — 81

; \ \ ■^"T" 36

/ \ \ V^:@#- 19

8 41 17 42 ^^ 12

573 Males. Total 1113. 540 Females.

k

Fig. 73. — Census of second annual generation at Texcoco in 1906, showing con-
dition with regard to pionotal pattern.

In the census of the second generation, biotype 1 was absent in both sexes and
5 was represented by a mere trace ; 6 and 8 were fairly shown in the males, but
in the females were weakly represented, while 9, 10, and 12 were best developed
in the females, where 13 was found as a distinctly isolated group, the only one
in the entire population. The records of the two generations are shown in the
figures 73 and 73. Comparison of these two generations in the season of 1906
at Texcoco and at Chapultepec shows differences that seem permanent to the
two locations in the population living there. In this year, at the Chapultepec

Analysis of Heterogeneity in the Population

283

colony, there was a wide range in the character of the population with no well-
developed isolated groups, but in the Texcoco colony the sharp restrictions of
the central mass of the population and the evident propensity to produce widely
and sharply isolated groups would seem to indicate for the two a different condi-
tion either in the population, in the medium, or both. Comparison of the condi-
tions in the two locations on the basis of the climatic data that I can give is not
satisfactory. In the latter part of the season of 1906 the conditions as regards

6 14 9

14 --"-"IM

36 ^^^

3, '1^0

29 ^^

36

19

42-— ^ )f^ 98

^iS^iW ^ 95

y j' I ^ ^ 46

'^

"X-- 12

i^ vk- — 31

m^>^ —42
W 51

«a* 56

it» 104

/

/ / /

/ / m \ \

5 18 12

I I /

I I / ^-6
' j / /<^---46

' ' :=^-51
•iC|2).. 5g

imm/- — 61

V ^ 77

- is*-- 86

_ ^^ 4^

-r^^-^-l^"mr 58

'^M m-i 77

. \

14 9

11 9

^ X

\ <^^ 5

13 '

876 Males. Total 1874. 998 Females.

Fig. 74. — Census of first annual generation at Texcoco in 1907, showing sudden
marked extension of pronotal pattern in the array in all directions.

im.

h-4
-—7

21

6

9

W^m^r 46

\ ^^^»r 61

378 Males. Total 747. 369 Females.

Fig. 75. — Census of second annual generation at Texcoco in 1907, showing
reduction of the general mass of the population as far as pronotal pattern Is
concerned and Increased number of isolated groups.

the precipitation and its distribution were more uniform and propitious to the
development of this species than common, a fact that is partly indicated on the
climatic record by the high rainfall in the latter portion of the season, which
was also well distributed. The somewhat lower temperatures in this portion
of the season acted perhaps to check or counteract the opportunity presented by
the precipitation for the population to show wider ranges than common, which it
did not do.

284 The Mechanism of Evolution in Leptinotaesa

In the season of 1907 this favorable condition existing in the population at
the close of 1906, and the still more favorable conditions in the early portion of
1907, with plentiful, well-distributed precipitation and the higher temperatures
of late May and June, were the series of conditions that preceded and ensued
during the production of the first generation in this year, and which showed the
unusual condition for this location of almost no isolated groups and widely
ranging distribution of the character over the possible range of manifestations.
In both sexes biotypes 1, 2, 3, 4, 5, 7, 9, 10, 11, and 12 were present in full inten-
sity, while 6 and 8 were weakly present in the males and in the females as the
only two isolated groups in the entire population. The condition of the popula-
tion in this generation is for once not unlike that in the Chapultepec colony in
corresponding generation.

The second generation in this season showed the rapidity with which the
population may change its aspect on the advent of an efficient cause. In this
generation the solid, widely distributed condition of the pattern was again dis-
turbed, and in its stead there was a narrowly restricted central mass of the
population, and several sharply isolated groups. In the population biotypes 2,
4, 5, 6, and 11 were entirely wanting in both sexes; 9 was present as a weak
development in both sexes; 8 was strongly marked in both males and females.
Biotypes 1, 3, 10, and 12 showed distinctly segregated groups of considerable
numbers in each instance, so that the aspect of the population at this time
presented an entirely different one from that shown in the first generation of
this season. The record of the two censuses are shown in figures 74 and 75.

The season of 1908 was favorable at this location on the whole, and from the
point of view of climatic conditions there should have been a condition not
unlike that in the Chapultepec colony at the same time. The census of the first
generation for this season showed the characteristic, much-restricted central
mass of the population and three large groups of isolated patterns. In the cen-
sus biotypes 1 and 2 as a large group, 12 and 10 in the males, and 12 in the
females were sharply separated from the rest of the population. Within the
mass of the population biotypes 6 and 8 showed strong development in both sexes.
The second generation at the time of census-taking showed essentially the same
state as in the first generation, with the entire absence of bio type 12, and some
minor changes in the rest of the population. The combined group, consisting
of biotypes 1 and 2, was still strong and prominent in the population, in marked
contrast to the condition of these groups in some other generations that had been
examined earlier in the series. The results of the censuses for this season I have
given in figures 76 and 77.

In the following year (1909) the colony was visited once in the early portion of
the season, and the last census of the population made in the series at Texcoco.
The condition in the population then found showed as in the first generation
of 1907 that the conditions or something in the complex, either within or with-
out the organisms, had produced an array of pattern conditions that was on the
whole the widest that had been found in this location during the observations,
and on the whole it presented a uniform distribution over the range of the
possible arrangements. No marked or large groups isolated from the popula-
tion were found and the three shown in the males are unimportant and few in
number. The findings in this census are shown in figure 78.

Analysis of Heterogeneity in the Population

285

Compared with the Chapultepec population for corresponding generations,
this series at the Texcoco colony show constant differences, chief of which is the
uniform restriction of the mass of the population to a rather narrow range in and
about biotype 7 ; and second, the striking presence in the population of distinctly
isolated groups, none of which have any constancy of numbers or occurrence.
With the onset of environmental conditions which at other locations are opti-

17--
4 -

11

17

22

36

11 12
I /

17— %^yg

'^/- 1

— v--^ ^ 51

m' )tlS/T 65

-1S» K©^ 76

«» J«^-? 87

/ SWt ^^ 55

/ 'JftSt \ VV5. 41

/ \ \ ^

5 9 4

11

36

29

-55

'W' *«f— v-*^-- — 74

— -^ m, 89

IS' m- 81

/-m ^-^ 49

19

\ "dsO^ 15

\

1 5 7

612 Males. Total 1186. 574 Females.

Fig. 76. — Census of first annual generation at Texcoco in 1908, showing pres-
ence of numerous isolated groups of considerable strength and an increased array
in the general population.

1 4 S

3 6

I

5

19

37 ,

m mr — 10

f^ 31'-

'^ ; fm- 40

^ . H^r 41

/..^ ^^^ ^

^■'•:W^- 61

55

31

9^

17^

22

65

%^^.:— 29

\ .«X 55

^^ ' W 61

-hW^ 'im^ 83

y--'^ m^' 77

'^' ^ 105

-92
-46
-51

/

«% ^V--

\

19

/ \

730 Males. Total 1522.

11 4

792 Females.

Fig. 77. — Census of second annual generation at Texcoco in 1908, showing con-
dition In pronotal pattern.

mum conditions in the medium, the population at Texcoco was on the whole
inclined to exhibit an increased response to the conditions in the form of
widened ranges of the pattern combinations presented, which may be cause and
effect, but not necessarily so. Commonly it would be so considered and the rela-
tion considered as established ; but it is in any such " establishment " assumed
that the material is uniform at the two locations, which may or may not be true.
If the populations were uniform, then the certainty of such determinations

286

The Mechanism of Evolution in Leptinotaesa

would be increased, but the populations are rarely uniform in two separate loca-
tions, and this is especially so in the case of organisms that are restricted to
narrow habitats, and from which they do not move far. In such restricted habi-
tats small peculiarities may and often do develop and become the potent cause of
wide differences in the reaction of two supposedly like organisms to the same set
of conditions in the environment. There is one outcome of these determina-
tions of the conditions in these populations that is of interest and of some
moment as a practical consideration in investigations of this nature, namely, the

ifljjfh:.

9

11

31
12
46
47

-0^^

m0 I

I ir^ 9

^^^- 7

* ii^" 4

i^ «SC' 11

'-.'^' -mf 46

--^ «*- 92

^ ^»-- 55

/ m:. m't 31

11

9-v|»-\

17 — ^^i

21 --ig^

7 J^

9 ^

32 -':

31 ..

11 8 14

1 /

i /

fif

7

\ \

9 11

14

/ /
///

4 12 8

'i^-12
^^-61
SSf'-— 58
f^— -64
-51
--«»' J«Sr 74

«*■ im-i 77

/'J^^-\#- 36

-^h^^

\:

\

510 Males. Total 1154. 644 Females.

Fig. 78. — Census of first annu; 1 geueration at Tc.xooco in 1909. showing con-
dition of pronotal pattern and extension of pattern in several directions, so a.
fairly continuous whole is presented with only small isolated groups In the males.

difficulty of finding twice the same condition in the state or array of the popula-
tion in successive generations or seasons. The significance of this will appear
more evident later.

THE TLALNEPANTLA COLONY.

This location, in the northern portion of the valley of Mexico, the last of the
locations in that physiographic unit, has been described, and it presents a
different pliysiographic setting and, owing to local topography and climatic
conditions, the same in plan and range, but differing in intensity and dis-
tribution in the growing-season of the year. On the whole, the conditions at
this location are more severe than at either of the other two, and this in general
finds corresponding changes in the response of the population of our subject
living there.

The first census recorded from that location is from a collection made in the
second generation of 1903 ; the remainder, to the end of 1907, have been made
without removal of any of the population from the location. This was especially
necessary in this location during most of the period of observation, owing to the
relatively small number of inhabitants of this particular kind found in the
location.

The condition found in the second generation of the season of 1903 indicated
a narrowly limited range in the population without isolated groups in the
population. This was true for both sexes. Biotypes 1, 2, 3, 10, 11, and 12
were entirely lacking in the population, and 6 and 8 were but poorly developed

Analysis of Heterogeneity in the Population 287

in the array, which might have been due to the small number in the original
collection in this generation. The findings in this generation are given in
figure 79.

In the season of 1904 two censuses were made, comparable in time with those
at the two other colonies in the valley of Mexico. The first one for the first
generation did not exhibit any considerable change from the condition presented
in the population of the preceding year, with the exception of the better develop-

9 — 'liE\,.,

12 .4S^. ^^r... 14 --iaifc ■ .is^^

21 jg^ ^:^^ 22 20—^---^^ -^:i^ 29

39-^— -^ ^ 46 44---^--^^ %^" ^ 35

-^WSt^S* v*t 59 ^^^,_M'T-r- 74

I I ^ '^ ^eV^ 39 / y — v'*^%i" ^^

III \ ^ 46 / /^ '^¥^ 33

/ / / \ ^ / / \ \

7 11 21 ft 6 14 2 9

350 Males. Total 704. 354 Females.

Fig. 79. — Census of second annual generation at Tlalnepantla In 1903, showing
conditions of pronotal pattern.

' ^ #- 16 i7-:-«^'„

46- -m W 38 3^ —m ■ m- 31

■29 ^ ^ 51 33 — - -^ m~ 77

^ -^S* 77 "^^''^L *^— . 8&

/ ^ -if* ~T 46 / /'^ AS|^----19

I \ / / ^

9 7 8 5 1

384 Males. Total 747. 363 Females.

Fig. 80. — Census of first annual generation at Tlalnepantla In 1904, showing
condition of the pronotal pattern at that time.

11 ^ 14-, ;||^

29 5^ 9 m

31 «t .1^ 4s 29 m m-—M

44—- ^ ^i 52 46 ^^ m 29

.^m m 77 ^*fe: — ^

/-/^3|* ^^^ 38. /^<^^ 46

/ / \ ^ 29

7 14 5 5 22

383 Males. Total 718. 335 Females.

Fig. 81. — Census of second annual generation at Tlalnepantla in 1904, showing
the presence of a single isolated group of females, the rest of the population
fairly uniform in its distribution.

ment of biotype 4 and a few biotype 3 individuals in the female population. On
the whole, the population was massed on or near to biotype 7, the pivotal one in
the series. The second generation did not show anything different from the
first — only a somewhat more restricted range in the extremes of the population.
The conditions as found are recorded in figures 80 and 81. In amount and dis-

288 The Mecha^jism of Evolution in Leptinotaesa

tribution of precipitation, temperature, and other factors the climatic complex
was such as to indicate optimum conditions, basing the estimate of the condi-
tions upon the conditions that had been found to be optimum in other locations.
In the Tlalnepantla habitat, however, no response to the conditions was mani-
fested in the population, such as had been observed in other locations.

In 1905 the first generation in the location showed some change, but no iso-
lated groups, the area of the distribution of the pattern being " continuous," but
showing development along the direction of the biotypes 3 and 4, also in the
direction of 9 and 11. Biotypes 5, 6, and 8 were feebly represented in both
sexes. In the second generation of this year the census showed the same con-

9 — -

31 M-'s^i

n—M§j^

t- — "^ '^> — 21 •21—-^;:^ ^^^ — 47

47 - -_^^ ^^ 5j ^_jig(jj^_^ 83

''f^if^m V^ 96 m m 77

/ / / ^^ ^ 39 7 m- ^r- 56

/ / / \ / \ m^^ 39

.8 17_?5. 9 22 n

553 Males. Total 1072. 519 Females.

Fig. 82. — Census of first annual generation at Tlalnepantla in 1905, showing
condition of the pronotal pattern.

12

-14 11.

--^ 17

m —21 ,, ,j^% ^^:^m---2i

41— jfc %»=. ^1 34 j_p ^

46--^^.^ W -45 ^^ — ^ ^ m 38

61 -:^tK|gi^ ^ ___.^g 46 -^ ^ _..3j

49 .^ ^ ^^ _g2 49- -^ m *«► 42

/ X <V7 ^ 26 / \ -V? 29

/ \ / \ ^ -36

14 31 19 z2

665 Males. Total 1314. 649 Females.

Fio. 83. — Census of second annual generation at Tlalnepantla in 1905, showing
an Increased array in pronotal pattern.

ditions as in the previous generations. None of the isolated groups that were
so characteristic of other locations was found, and the poor development of the
more divergent biotypes in the population is characteristic of the location. Bio-
types 3 and 4 were fairly well developed, as was 5 in the males, otherwise the
census showed nothing of interest. The record of the censuses of these two
generations are shown in figures 82 and 83.

The year 1906 showed in the location the first indication of any of the diver-
gent groups, there appearing in the males biotype 1, which were found in freshly
emerging condition, showing that they were a portion of the population, but

Analysis of Heterogeneity in the Population 289

whether they were the product of an introduced individual of this biotype or not
is impossible to determine. The remainder of the population showed only the
customary array at this location in both sexes. The second generation in this
year showed biotype 1 in both males and females as widely isolated groups. The
remainder of the population was fairly well massed, but showed indications of
extending the range of pattern in several directions. The recorded conditions I
have also shown in figures 8-1 and 85.

81— -V jj0t ^ 42

29 . ^^ ^ 72 45- --.S^\.i^^t 55-

46.

^ ii^^—-86 51 ---j^:.- ^1^4^— -61
i» ^ . 77 <^^jm-i 74

17 14 26 31

462 Males. Total 970. 508 Females.

Fio. 84. — Census of first annual generation at Tlalnepantla in 1906, showing
the arrav In pronotal pattern. In this generation there appears the introduction
(apparently) of biotype 1 in the pronotal pattern which was present only in the
males.

12;

34 m - ^-^^ 00 .: *L m 22

22 46.__

29 ^r^'-m ■ 'm-' 51

47 -—S^ m-' 42

61 ,-^--v~^-l6g*. H^--' 86

110 m m m--: 74

-- iflP ^ 3ft

83 ^ ^ 35

51 ]^ j^ 31

^ ^ 42

/^ 4^ 46

/ / / \ 9- -:- / \

/ / / ' .V, —42 / \

9 31 38 6 12 15

713 Males. Total 1186. 473 Females.

Fig. 85. — Census of second annual generation at Tlalnepantla in 1906, showing
the presence of biotype 1 in both sexes and an increased array present in the gen-
eral mass of population.

In the year 1907, the last at this location, the first generation showed no indi-
cations of isolated groups and only a much restricted range in the pattern,
nearly identical to that found in previous years. The census of the second
generation showed the usual well-defined and restricted mass, with little
tendency to diverge, and isolated groups of biotypes 1, 8, 10, and 12 in the males,
and of 1 and 8 in the females, all of which were fairly strong in numbers and

290 The Mechanism of Evolution in Leptinotarsa

were clearly the product of the breeding of the population that had preceded, and
not due to introductions. These records I have shown in figures 86 and 87.

The following year (1908), and again in 1909, the location was visited and a
rough census made of the conditions in the population, but with no added indi-
cation of change in the characters of the population of the place. The same iso-
lated groups occurred as in the previous years, and with frequency and in such
numbers that it was evident that their occurrence in the location was not entirely
the product of introductions, but was, in the main, the result of the response of
the population to the conditions of the habitat into which they were by chance
placed.

31 ^. ■■ 22- ^f^-] .,^.___.9

46-4^^ /B^ 29 63— ---^gg^v ^--- 51

51— —'-m m-- ''^ ■65—--^-^ im— — ''^

^m m- 106 ^Mm ■^---:- 98

f'W.''m— 83 / /'^^ .^- 77

/'^pV'-^\^-%V5*-W 41 / /^,; -, ^v ^^

/ '\ \ i§---— -42 / / \ \

12 15 8 ■" 9 4G 29 22

577 Males. Total 1204. 627 Females.

Fig. 86. — Census of first annual generation at Tlalnepantla in 1907, showing
the pattern conditions.

17 -^ 19 2H|li|

I

! -.'£53-12

9' — -m^

31 \^--

38 ^, 21-

** ** 21

■42- ^ .^ 42 -m «(^ 45

41 ^ 3^ 92 31 ^- J«^ 96

v*l^^ . ^ .^.7;.% 114

1-^<$^ 46 ^^g^=j 77

/ \ ^ 24 ■ ^ 31.

583 Males. Total 1109. 526 Females.

Fig. 87. — Census of second annual generation at Tlalnepantla in 1907, showing
the restriction of the mass of the population to a narrow range and the existence
of several distinctly separate groups in both sexes.

The series of observation at the Tlalnepantla colony were in the character of
the population, as indicated by these censuses, quite different from those in the
other locations examined. Here as elsewhere the attempt to tie the observed
conditions to any portion of or to the environmental conditions as a whole is
argument from effect to cause or possible cause. The series may and often does
make a plausible array, one that strongly indicates the relation of cause and
effect.

Comparison of the conditions presented in the population at the three loca-
tions in the valley of Mexico shows decided differences in the character of the
individual generations and in the sum total at each of the colonies. The
Chapultepec colony presents the most diversity and the greatest range. Tex-

Analysis of Heterogeneity in the Population 291

coco is next in the series, and the Tlalnepantla location least diverse in its
population. The diversity is indicated in another way by the occurrence of
biotype 1, which may with certainty be expected in the population of the
Chapultepec colony. At Texcoco it is about a 1 to 1 chance that one will find it
in the population, while at Tlalnepantla, the chances are far less, on the average
10 to 1, against finding it present. With reference to the other biotypes much
the same set of chances might be derived, differing of course in the chances and
in the locations.

That these three locations, situated on the rim of this great basin, presented
constant differences in the character of the population was at first surprising,
and when was added to this the weight of the determination from other locations
in the basin, giving still other differences in the character of the population, so
that at no two points in the whole area was I able to find like conditions in the
population of this species, it became increasingly clear that the phenomena were
well worth analysis. There has been little trace of migration of the population
from one colony to another, although this has been sought for most diligently in
the years of the study of the locations. Especially in the close of the season, in
the interim between the maturing of the second summer generation and its
entrance into hibernation for the dry season, there was opportunity for dis-
persion of the population at any colony to nearby locations, producing thereby
a mixing and keeping the population of the valley fairly uniform. Any move-
ments would, on account of the topography, be almost entirely confined to the
valley of Mexico, and the only probable chance of foreign individuals would be
from the plains of Apam entering through the pass at the northeast comer of
the valley. All other sides were well cut off by topograpliic or other barriers
that in the main prevented either entrance or exit to this basin. One exception
to this must be noted in that, in especially favorable seasons, when the country
to the north of the basin has had high temperatures and plenty of rain, there
might be produced opportunity for food and dissemination, such that there
might be entrance or exit on the north. However, I have not been able in two
seasons of good conditions in these northern districts to discover any movement
in either directions, so that I have come to regard the basin as one that is prac-
tically physiographically isolated as far as this form is concerned, as it is
known to be for other organisms, especially those of aquatic habitat (Meek).
It seems from all that I have been able to find out about the dissemination of
these forms in the valley with respect to the regions about, that there is relatively
little interchange as a regular thing, and only in exceptional years is there pas-
sage of many of the barriers to dissemination.

Within the valley there is of course more or less in the way of interchange of
materials between the nearby locations where the animals are able to grow,
although this is not as extensive as might at first seem probable. In the north-
ern species, L. decemlineata, it has long been kno^vn that the autumn is a time
of extensive dissemination by means of flying, and this is influenced by the
winds to a considerable degree as has been shown in an earlier publication. In
this species, in which the flying habit in the end of the growing season is also
developed, I was interested to find if it there too played any considerable part in
the dissemination of the animals from one location into another. Observation
in the field established the fact that the forms did fly more readily at this season
than at any other, and in captivity they do the same, so that the behavior is not a

292 The Mechanism of Evolution in Leptinotaesa

product of the external conditions, or at least not entirely so, but in nature I
have not been able to find them flying for more than a few yards at any one time.
Thus far I have never seen one of these beetles fly more than 75 yards at any
one time in nature, and after such a flight I have never seen it repeated, or
rarely do they fly again until after some time has elapsed, and on resuming
flight they are as apt to return to the original place as to start out in a forward
direction, so that the end-result of the flying would, as far as I have seen it, be
relatively little or no dissemination at all. From witnessing single exceptional
flight and assuming that all flights were in a forward direction, quite false
conclusions would be reached. I have followed a single animal for hours
at a time in the proper season, with net result of the day's movements —
10 or 12 yards' movement from the original starting-point and still within
the original colony. On one occasion I followed one female three days
at the Chapultepec location. The first day was passed in aimless walk-
ing of undetermined length, 87 meters of flight, and 5 hours of rest on the
underside of leaves, passing the night in the ground in a crack beside a stone.
This was at the height of the flying season in 1905. I returned to the location
in the early morning and found the beetle in the same location, and as the
morning was cool it did not move from the location until 10 : 23 o'clock, spent
the time until noon walking and feeding on the nearby plants, took one short
flight of less than 10 meters, and then came to rest on the top of a plant in almost
the identical spot from which it had started the day before, moved about on the
plant for a few moments, and then crawled down the plant to its foot and under
some leaves at the base, from which it did not emerge. I left the location at 5
o'clock in the evening, returning the next morning at sunrise, and found the
animal in the position in which it was left the night before. About mid-day it
crawled up on the plant, remaining about an hour, but the dry, desiccating winds
of the early afternoon soon drove it down again into the loose leaves at the foot
of the plant, where at 2 o'clock in the afternoon it began burrowing its way into
the soil, and in a half hour had passed out of sight.

This I have every reason to believe is a typical behavior of these animals in
these locations; at least nothing to the contrary has been seen to indicate dif-
ferently. It seems probable that in these restricted locations there are relatively
few instances of interchange in the population, and especially so in those chosen
for the examinations made.

In the course of this study a considerable number of locations of a highly
restricted character was investigated, and while most of these were too small to
provide the numbers that it was felt should be examined for the work, they did
indicate a remarkable degree of difference as a result of the isolation which the
topography and the climatic conditions enforced. For example, several loca-
tions in the region of the huge larva-flow in the neighborhood of Tlalpam
showed exactly this condition in the character of the population, and some of
these I saw at least yearly for 6 or more years.

This condition, prevailing in the valley of Mexico, received further confirma-
tion from the data derived in the examination of the populations at the Puebla
station and at the location near Chalcicomula, which were so isolated and sepa-
rated from the three locations already examined that any interchange of popula-
tion is highly improbable, or at least is not of common enough occurrence to be
of any moment in analyses as crude as we are, at present, able to make in nature

Analysis of Heterogeneity in the Population 293

with the methods of the census-taker. Both locations present differing climatic
complexes, different topographic conditions, and, as might be expected, pre-
sented different aspects in the character of the population. A priori it might
be expected that the populations would differ in the two locations. The problem
is, how and why do they differ ? The " how different " is of no importance,
excepting as it helps in the solution of the method of becoming different and the
analysis of this operation.

THE CHALCICOMULA COLONY.

This location on the western slope of the volcano of Citlaltepetl differs in
many respects from the location in the valley of Mexico and the colony at
Puebla. Situated in the lee of the huge volcanic pile, at an altitude of 8,500
feet, it was, as a result of the physiographic conditions, a dry, cold, grass-covered
plain, with a short growing-season and cold nights, even in the best of seasons.
In this general region the development of the colonies of this form were not at
any time on a par with those at Puebla or at Chapultepec in the valley of
Mexico. The location chosen for examination was 2 miles west and north of the
town of Chalcicomula and about 1 mile west of the Barrio de San Augustin, in
the open plain, with rainy-season pools. Observations were begun in 1904 and
continued until the middle of 1910, covering 13 generations in the location.
The censuses at this location were not as satisfactory as those at other locations,
owing to the small numbers and the frequent difficulty of being sure that a con-
siderable number of the previous population had not persisted and formed a
part of those present at the second census in the season. This was due to the
frequent spell of low temperature in the summer months, which kept the indi-
viduals alive, inactive, and present in the population longer than in other loca-
tions. This error exists in any census determinations and can hardly be com-
pensated for or eliminated unless the individual by being killed is put beyond
the possibility of again entering into the count. The determinations, defective
as they may be, show decided differences between this colony and those previously
studied.

In the first year of observation (1901), the first census showed a small popu-
lation, which was centered around biotype 7 in both sexes and with 8 well devel-
oped in both. No divergent or isolated groups were found in the popula-
tion. The records are shown in figures 88 and 89. The second census, made
in September, showed the same general condition, a small population, aggregated
closely about biotype 7, and in this generation 8 was not represented any more
than the others that center immediately about the pivotal biotype. Comparison
of these two censuses with those made in the same year at the other locations
shows a quite different condition than exists elsewhere.

In the season of 1905 the first census made showed in the males a strong
development of biotype 8 and something of 4 and 5, but in the female the condi-
tion was not changed. No isolated groups of any kind were found in the loca-
tion. The second determination for the year showed in both sexes about the
same condition that existed in the males of the previous generation, this time
without divergent or isolated groups. The records of the conditions found are
shown in figures 90 and 91. The two generations of this year compare well in
range and numbers with those of the previous years, the four showing that the
location is more constant in the appearance of the population than any of the
other locations examined.

294

The Mechanism of Evolution in Leptinotaesa

In 1906 two generations were again examined, with the results shown in
figures 92 and 93, no essential change being found in the population, the only
item of interest being the existence of the extreme of biotype 8 present in the

/

31

— 31

--109'

— 87

— 55
—51

46

27
471 Males.

Total 971.

42
500 Females

Fig gg Census of first annual generation at Chalcicomula in 1004, showing

condition in pronotal pattern, and the restriction thereof to a very narrow range.

17

524 Males. Total 1025. 501 Females.

p,Q 89. — Census of second annual generation at Chalcicomula in 1904, showing
further reduction in the range of pronotal pattern.

39 — i^; ^.-s

27— --,r^\ ^,-^; 19

35--;-,--— <ys< ii# 38

^ *4*: im • 151

/"''''^-i^...'fe 88

'\g^^— -41

' " "'^'"^— -56

19

12 19

22

3e—~$^i^$^^ 74

%fi* ^^ '-—196
/'30,.<,^^-— 55

580 Males. Total 1009. 429 Females.

Fig. 90. — Census of first annual generation at Chalcicomula in 1905, showing
a somewhat increased array of the pattern.

19--. ^ .

28— -Sgp .^,^-
21 -^^ W* *^

11

- 44
-171

—-92

:^i — 56

&'^— 39

\ •^'^^—■42

29
496 Males.

Total 1036.

42
540 Females.

Fig 91. — Census of second annual generation at Chalcicomula in 1905, showing
the stable condition of the population with reference to pronotal pattern.

males as an isolated group. The population was small, and it is probable that
twice as many would have shown intergrades in this direction.

Analysis of Heterogeneity in the Population

295

Two censuses were taken in the season of 1907, with the results shown in
figures 94 and 95, where no difference in the character of the population was
found in either generation. There were now records of 8 generations without

u —

42 —

18 —

410 Males. Total 815. 405 Females.

)M^ «*,' 83

Fio. 92. — Census of first annual generation at Chalcicomula In 1906, showing
a reduction of the array in population.

411 Males. Total 875.

11 14

464 Females.

Fio. 93. — Census of second annual generation at Chalcicomula in 1906, showing
a marked reduction in the array.

Total 1141.

Females

Fig. 94. — Census of first annual generation at Chalcicomula in 1907, showing
a somewhat increased array of conditions in the pronotal pattern.

625 Males. Total 1200. 575 Females.

Fig. 95. — Census of second annual generation at Chalcicomula in 1907, showing
a marked reduction of the conditions in all lines with the exception of blotypes 8
and 9, into which the majority of the population seem to have been thrown.

trace of any of the behaviors found in the other locations, so at the end of this
year a large sample was taken to Chicago for testing in the laboratory, and there
were introduced into the colony in September 425 individuals at random from

296

The Mechanism of Evolution" in Leptinotaesa

the Chapultepec colony, which went into hibernation with the native population
and passed the cold, dry season in the new location. The purpose of the intro-
duction was to discover, if possible, whether the condition of the population as
shown in the censuses was the result of the conditions of the environment or the
constitution of the materials, which in some unknown manner had become
limited to the narrow range of heterogeneity shown. It might be expected from
the result of the introduction that the condition of the population would change
in its nature, or that the new introduced conditions would be obliterated.

W^S 99

fc^^M 151

61

506 Males. Total 1062. 556 Females.

Fig. 96. — Census of first annual generation at Chalclcomula In 1908, showing
return of the population to the condition observed most frequently in the location.

18-

mp

401 Males. Total 896.

12 19
495 Females.

Fig. 97. — Census of second annual generation at Chalclcomula in 1908, showing
presence of isolated groups of blotypes 1, 10, 9 and 7, after the introduction of the
heterozygous mass from the outside. The population in geueral is closely massed
around biotype 7 as in previous instances.

I do not know how many survived the cold, dry winter in this location, but
when I saw the product of the native population and the introduced materials
in the first summer generation in 1908 there was little trace of the introduced
materials, with the exception of an isolated group of females of biotype 13, as is
shown in the record in figure 96. The second census of the season showed the
same imiform population, but present in it were divergent isolated groups of
biotype 1 in both sexes, 6 and 12 in the males, and extreme 8 in the females.
These groups were strong in proportion to the population (fig. 97).

Analysis of Heteeogeneitt in the Population

297

In the following season (1909), none of the introduced conditions were found
in the population in either of the censuses, the only trace being a single de-
formed weak female of biotype 13 in the first generation of the season, which
did not reappear in the second annual population. In the second generation a
small group of isolated males of biotype 6 were present, but were too few in pro-
portion to the total population to have any meaning (figs. 98, 99).

Evidently something that has happened in the location and in the population
has obliterated the introduced stock in a relatively short time and held the state
of the population at a fairly constant average, in spite of the disturbances of

541 Males. Total 1082. 541 Females.

FiQ. 98. — Census of first annual generation at Chalcicomula In 1909, showing
massing of population in or close to biotypes 7 and 8, with only a few present
in the divergent group of biotype 11 in the females.

12-

25 ^ iM 78

14

389 Males. Total 846. 457 Females.

Fig. 99. — Census of second annual generation at Chalcicomula In 1909, showing
massing of population in still more restricted zones around biotype 7, practically
eliminating all biotypes introduced from outside.

the introduced strains carrying divergent factorial conditions that might easily
disrupt the population of this restricted area. The experiment is suggestive,
but from it one can not decide whether the resulting rapid incorporation of the
introduced stock and its characters is due to the dominant character of the
native material, to the conditions of the environment, which either eliminated
the introduced forms, or has made them recessive in the population, with final
eradication from the constitution of the race at that localized area.

At the end of this season I introduced from the Puebla colony a large army of
immigrants, 1,845 females and 2,134 females, a horde outnumbering the native
population several times. These hibernated with the natives, and apparently
20

298 The Mechanism of Evolution in Leptinotaksa

there emerged in the spring a large number, and they interbred with the native
population and inter se, giving the resultant distribution of the population in
the first generation shown in figure 100. In this array the mass of the popula-
tion is still gathered about the same condition as in the other generations exam-
ined, and the development of the line composed of biotypes 1, 2, 3, and 4 is, rela-
tive to the entire population, weak and may well have been due to the inbreed-
ing of some of the Puebla immigrants, of which all of these conditions were
present in the population introduced, and not to the inbreeding of the native,
and introduced with the dominance of the introduced over the native. Census
methods could not decide this in nature.

9- '"'

31 — -'^^wiB^^.,

14

26

19

31

42

-m^'m

--^^ V

m

—m.

;*v~-'

Fig. 100. — Census of first annual generation at Chalcicomula in 1910, showing
array in population after the introduction of a large foreign population from
Puebla.

I have no further records of the fate of the colony with its immigrants, owing
to the disturbed condition of the area in the seasons of 1911 and 1912. An
attempt to gain information of the condition of the colony in the winter of 1911-
12 by digging for the hibernating beetles also gave no results, owing to the
reluctance of the labor in town to go on a strange errand into the country. To
judge by past experiences in the location, it is probable that the introduced
material was progressively incorporated into the population, leaving its presence
indicated for longer or shorter periods by changed ranges in the character most
considered.

THE PUEBLA COLONY.

The location of this colony in the Eio Atoyac Valley, on the brink of the
Mexican Plateau, in a location open to the warm winds from the low, hot valleys
to the southward, and protected from the cold winds from the northern plateau
and high volcanoes, with higher rainfall distributed over a somewhat longer
portion of the year, gave the location a more favorable set of conditions than any
of those previously examined, with the exception of Chapultepec, whose peculiar
topographic situation gave it especial advantages over other locations in the
valley of Mexico. At Puebla two collections near the location chosen, made in
1903, gave data showing the difference that existed in that year between this
location and others examined, and in 1904 observations were begun and con-
tinued to the close of 1908, covering 10 generations in the location.

Analysis of Heterogeneity in the Population

299

In the first census of 1904 the population showed a condition not unlike some
of those already encountered in the first two examined, namely, a large central
mass grouped about the central biotype 7, and isolated groups of biotypes 1, 10,
and 13 in both sexes. In the second generation in this season the distribution
of the population showed about the same condition of the central mass of the

32 44 21

31 ~r-.~m- m^-

/ / ■ '■■

46
1488 Males.

Total 3310.

19 77
1822 Females.

— 141

318

—416

109

96

41

Fig. 101. — Census of first annual generation at Puebla in 1904, showing condi-
tion of the pronotal pattern and presence of several strongly marked isolated
groups.

Sl-pJUjir^

29

X-^'^ 71

44

171 ^^ p^

124 C^ ^___

89 --^-^- jj)^ i^

• — 549
■— 541
--319

2300 Males. Total 5010. 2620 Females.

Fig. 102. — Census of second annual generation at Fuebia in 1904, showing
essentially the same conditions as found in previous censuses.

population and the same isolated groups, with the exception that in the males
the groups composed of biotypes 10 and 12 were wanting. The records of the
conditions in these two censuses are shown in figures 101 and 102.

In the season of 1905 both censuses showed the complete absence of isolated
groups and a nearly complete distribution of the population over the entire area
that the pattern covers. The season was especially favorable, with regularly

300 The Mechanism of Evolution in Leptinotaesa

distributed precipitation and other conditions optimum in their arrangement.
The findings in the population are shown in figures 103 and 104.

The year of 1906 was a favorable year as measured by the range in the char-
acter presented in the population, as shown in figure 105, wherein no separate

V -i^^^-- 408

/ €:X:M^-—''' / / \\

I I '"^"ms^—n I / \ \

31 84 46 71 77 21 29

3034 Males. Total 5501. 2467 Females.

Fio. 103. — Census of first annual generation at Puebla in 1905, showing entire
lack of Isolated groups and relatively wide range of population with respect to
conditions of pronotal pattern.

12 19 21

2463 Males. Total 5157. 2694 Females.

Fig. 104. — Census of second annual generation at Puebla in 1905, showing
further increase in array of pattern and also a sharp increase in the population ia
regard to size. No isolated groups.

groups are present in the population; but the second generation of the year
showed isolated groups again in biotypes 5 and 10 in the males and in 10 and 13
in the females, all relatively small in numbers, as shown in figure 106,

Analysis of Heterogeneity in the Population 301

In the season of 1907 the first generation showed a curious reduction of the
line composed of biotypcs 1, 2, 3, and 4; 1 and 2 were entirely absent in the

12-|^ 4 29 31 W-r'iJl^A 9 21 44

42-:*iK% I j ' 46 -V^|$, , 1 i' I

17-,-liir'- i i / .W-7 81

46 aft ^' ' ^V"--42 42 m '■■'-'^' ^^''--^

«, ^ «Wf/ 1:8;^— -63 21 -^ <«Jt;^^||i^_..77

9 ^ <^«te^^.— .74 ' ^ ; ^«»^.__..8i

45 jm^ ^, jj^ 94 -^< ^..____..88

47 ^ ^ - __i4g 109 ■-'^^■mt^-r-^..~-m

51 )eySi 4»> 219 108 '-4ISfl»' iS^-- 196

iW* i^ 314 , ^ 431

^/iB^ -«^- 198 '^.'^, ^--^-----219

/ \ 'V? '- -- 85

/ V-W- 9 \

14 19 " 41

46

1955 Males. Total 4221. 22G6 Females.

Fig. 105. — Census of first annual generation at Puebla In 1006, showing same
array of pattern conditions present in figure 104, but somewhat decreased in
amount of fluctuating variability.

1787 Males. Total 3695. 1908 Females.

Fig. 106. — Census of second annual generation at Puebla in 1906, showing a
restriction in the array of pattern conditions, the appearance of isolated groups
and increased fluctuation in the line composed of biotypes 1, 2, 3, and 4.

males, while 1 was present and 2 and 3 absent in the females. The same condi-
tion prevailed in the second generation of this year, where biotype 1 was present
in both sexes, but 2, 3, and most of 4 were absent, as were also 10, 11, and 12 in

303 The Mechanism of Evolution in Leptinotaesa

both sexes. These conditions in the population are shown in figures 107 and
108. This behavior of the population in this season is difficult to account for.
The season, as far as the recorded conditions are concerned, was not below those
of the two preceding years, and on my visits to the location in the middle and
end of the season nothing was discovered that would indicate that the climatic
conditions had anything to do with the array presented by the population and
the practical obliteration of the major portion of the biotypes in lines 1, 2, 3, and
4. I saw the second generation twice in its life — once during the time when it

^,.--.-..81 ; ^-- 108

21 ^^i# igfj,:^- 219 46 rW i*f — 186

IS '—^ m— 322 31 -~.^ ^. 192

: ■ 3i^.-.^ 319 m .-m"' 414

^<;'^..^-' 191 -f-^.^ '^ 109

'"^■-^.^ \ ^:i 89 / '\^ Agn- 96

\^ "\ «Vi£^-- 49 / \ ^^: 83

\ '\-'rm^ 12 / \ ^ 4L

31 36 19 12

2106 Males. Total 3995. 1889 Females.

Fig. 107. — Census of first annual generation at Puebia in 1907, showing reduc-
tion in array in population and also absence of Isolated groups.

40) 211 ^^ W ^^

49 --^ ^ 321 54 ^ jwr— -286

21-—/ m **. 319 36 ^ ^ — -291

^ ^^ 414 ^ ^ 439

/ m. V 31* / '^^ ^" — 244

\ ;v, 141 / \-^— 186

/ \ ^"ijL IflC / \

,-Sf^ 106

•i-Wy

/

/ \ .y.^ 81 / \

38 29 58 41

2048 Males. Total 3955. 1907 Females.

Fig. 108. — Census of second annual generation at Puebia in 1907, sliowlng an
extensive reduction, as far as the array is concerned, with isolated groups com-
posed of biotypes 1 and 2 in both males and females.

was mainly in the larval and pupal stages and again when in the adult condition,
and at no time was there any indication of an epidemic of disease or of parasit-
ism which might have been responsible for the condition found. Further, I
have never seen disease or parasitic attacks select out a portion of the population
in this exact manner. The condition found is more probably the result of some
condition in the population itself than entirely the product of external condi-
tions, which may have been a contributing factor. Whatever the cause of the
condition, it is all too evident that only experimental analysis of the situation
and its experimental duplication can solve the problems involved. The census

Analysis of Heteeogeneity in the Population

303

and observation in nature can suggest possible solutions, but can solve none with-
out the aid of analytical experiment.

In the following season (1908), the population was examined with much
interest in the first generation to see if the condition prevailing in the previous
season persisted into the following generations. The census of the first gener-
ation showed that the line of biotypes 1, 2, 3, and 4 were present in full in both
sexes and strongly developed, as were 5, 6, and 8. In the 9, 10, 11, and 12 series

66-
«6-
92-
41
7.1-
92-
106-
77-
61-
93-

m
»

45

^

58

92 - m0

97 IP

106 ^tgir^

93— - 40t

m-

■m^-

-31
-96

m

:m-

"- m *si-

! , , m ^

31 92 77 4 21 19

-318
--414
--211
-96

71 —
106-
68-
92--

-m^

m m —

'^mm ^

/ / ^

—161

— 219

— 341

— 497

— 293

/ / -
12 39 44

5 21 46

2232 Males. Total 4936. 2704 Females.

Fig. 109. — Census of first annual generation at Puebla in 1908, showing a sharp
increase in the array in the population, with the presence of two added isolated
groups in both males and females.

74-1
92-
108
93--
44-
88-
71-
59--
86--
92

■m'

219

-^ m^ 338

•5ll?M^^ mi^,- -518

I I Xm ^^ 219

I

I j
71 44

\
"•A

10 14 31
2450 Males.

\ \

9 47 31 19 21 44
Total 5018. 2568 Females.

Fig. 110. — Census of second annual generation at Puebla in 1908, showing array
and increased development along the line composed of biotypes 1, 2, 3, and 4, and
isolated groups in 10, 11, and 12.

there was decided derangement, 10 being isolated in both, 11 in the males, and
12 in the females as distinct groups. In the second generation the same condi-
tion prevailed in the population, with the exception that 10 was absent in the
males and only present in the females as a small group, while 11 was present
in the males and 12 in the females. Otherwise the distribution of the popula-
tion was uncommonly well developed. These conditions are shown in figures 109
and 110. In this season the conditions of the previous one were repeated, but

304 The Mechanism of Evolution in Leptinotaesa

with a different series of the population involved in the operations. The cli-
matic conditions in the location were, on the whole, as indicated in the records,
more favorable for the entire population than in the previotis season, both
being of such a character that one would hardly look for extensive derangements
in the array of the population due to the conditions of the environment. There
is little doubt that the condition is a perfectly normal one in the population and
that the change in its character in successive seasons and generations represents
the product of operations that are far too delicate and intricate in their organ-
ization and operation to be analyzed by the crude methods that must at present
be employed in investigations of this kind in nature. The fact that the climatic
records are the product of a meteorological station of itself introduces a huge
error, and they are not at this station, nor at any other station that might be
chosen, of much service in determining the role of the external condition in the
operations that take place in the organisms living there. I have not the least
doubt that if it had been possible to have had proper observations at the location,
some element in the complex of the environment would have been found that
was playing a part in the production of the diversity observed.

COMPARISON OF THE FINDINGS IN THE COLONIES.

The conditions presented by the arrays observed in five locations, in different
generations and seasons, show phenomena that are of interest and helpful in the
further study of the problems of heterogeneity. Most striking is the manner in
which this complex character, simple in comparison to the entire organism, shows
a ceaseless movement over the field of possible form or pattern conditions in the
part. In no two locations, at no two points in time, is one sure of finding the
condition in the population the same, even in the array that is present, much
less in the proportions and distribution of the population over the area it can
occupy. The conditions found are fascinating and suggest vividly the manner
in which some distribution enigmas may well have arisen. This production in
the population of conditions, first in one direction, then in another, withdrawing
from the first but dropping behind, isolated remnants, that conceivably might
thenceforward become isolated permanently through diverse natural processes,
and so a stem-form with the differing factorial potentialities that this one has
might, without further endowment or other natural operations, be productive of
a considerable amount of " species formation " as it moved over the substratum
on which it lived. Figure 111 shows the conditions of all of the populations in
outline, so that the nature of the movements become more striking in their diver-
sity in the different locations and generations.

Another point of interest is the mass action of the population in its response
from generation to generation and in the different locations, in the manifesta-
tion of the responses in " variation," either determinate or indeterminate, in the
sense of Darwin. Both are in this series present together and manifested in the
different locations in different ways. Each of the locations chosen showed that
the populations respond differently to the complex in which they lived from the
response shown in other locations at the same time and generation by the same
" species." No question exists that in the Chalcicomula location the population
responded definitely, uniformly, in the same manner, to the conditions of the
medium, else why so certainly should the introduced strains have gone from

Analysis of Heterogfneity in the Population

305

sight so soon ? The Tlalnepantla colony also shows the same definite response
in the population as a whole to the complex, and the Chapultepec and Puebla
colonies each shows its characteristic difference in the population at the loca-
tion. Thus each of the population masses varies in its location in differing
degrees in several directions, and all may well be present at one time, producing
a well-defined condition of " indefinite variation " in the population, which in
turn may be followed by decided " definite variation " in Darwin's sense as the
result of some minor change in the direction of incidence or intensity of some of
the factors in the medium, of which instances are fairly frequent in the data
presented.

19g4

I9£5_

1906 1907

1908

1909

_iL

_an_

MM JT^

|V AVI

jva f,vm jiy^ i.'x

XT giffl gun $mf

Q o'

Fig. 111. — Composite figure representing results observed at five colonies de-
scribed in preceding pages and change in the appearance of the population In suc-
cessive generations through a period of years. Corresponding determinations are
arranged one above the other, so that conditions prevailing in the population at a
given time In the colonies are comparable. This figure shows graphically the
behavior in the population which the different colonies presented and which are
apparently constant conditions in the habitats studied.

The condition presented in a complex character of this kind, as a type of
what one might expect to find in more complicated forms or in organisms as a
whole were it possible to analyze the relations fully, is suggestive, and is one
that may well have played a role in the production of the heterogeneity that
exists in the organized world. Darwin certainly saw the conditions in nature
in this respect and tried to estimate them on the basis of their value in species
formation, on the basis of a working hypothesis that he was not able to put to an
exhaustive experimental test. With Darwin, as since, it has been entirely a
matter of opinion as to how conditions like these operated in nature to produce
heterogeneity and stable permanent groups under natural conditions. This is
still too much a matter of opinion and plausibilities, but should be a matter

306 The Mechanism of Evolution in Leptinotaesa

of direct experimental analysis, in the laboratory and in nature, in at least a
few instances. These materials do show precisely the condition that Darwin
saw and considered in the variation phenomena in organisms and the condition
upon which much of the controversy concerning variations, their direction and
selection, have been based. In this an unfortunate cul-de-sac was produced by
the biometricians following Galton, which for many years completely befogged
the true situation as Darwin had seen it, and as it is now seen more clearly with
the aid of present methods of analysis.

Throughout the series of observations there is constant delimited " variation,"
no trace of heterogeneous undelimited conditions being present, nor is such to
be expected in a complex pattern-system of this sort, which is itself a product of
the interactions of simpler elements, " simplest characters," each the product of
an exact and demonstrable group of productive agents, so that the system dealt
with, as far as it is known, is physically exact in its constitution and action, and
delimited behavior is to be expected. Any other condition would be the basis for
most searching analysis to discover hitherto unrecognized agents and to deter-
mine their role and place in the system.

This series of observations upon L. multitceniata Stal at the locations given
has been supplemented and tested by the minor test series made at different
points in the south Mexican region where this form is found. Some of these
locations have been mentioned in an earlier portion of this chapter. The results
at none of these were in any way different from those already described, and are
not given, purely on account of the space they would occupy.

All that an examination of this sort can ever establish, even under the best of
conditions in nature, with natural species, is the determination of the results
of unknown antecedent series of causes. In the organic population a series of
events is passing, certain stable conditions which are capable of convenient
expression, in one form or another, and so in the medium another series is pass-
ing, some elements of which are measured, and the two are matched up as
seems most plausible on the basis of some hypothesis which may or may not
be true and applicable to the case in hand. The net result of an examination of
materials such as is presented in the preceding pages is solely a determination of
the range, extent, and direction of the diversity of the materials in nature, no
more, and at the same time some of the conspicuous associations of the factors
in the medium, and of their range and direction of diversity, and in its totality
of both series, there is accomplished only the preliminary rough blocking-out of
the problems that are necessary in any investigation. All further progress rests
in this, as in all other instances, on the analysis of the materials and conditions,
their relations, and the recombination of separated materials and elements by
synthesis under divergent conditions, and in these laborious, complicated analyt-
ical and synthetic operations alone lies the sole basis of further knowledge.

One other point in this series of observations is of interest in this place,
namely, the effect produced by the treatment of the data by the methods of the
biometrician. If the series of data had been treated in this manner and only
the amount of the pigment present been considered, the result of the examina-
tion would have been a series of polygons of distribution, in which the phe-
nomena revealed by the analysis here employed would have been obliterated and
the resultant curves meaningless. One coujd find different polygons at different

Analysis of Heterogeneity in the Population

307

places and at the same place in different generations, which would then he inter-
preted on the hasis of some favored hypothesis, and that would be the end of the
study. The condition of the data thus gathered and analyzed left nothing upon
which to initiate further investigations into the analysis of the " variation "
phenomena that were present. The present method of analysis of the condi-
tions in the character examined, and the same would be true of any other, does
leave a broad foundation upon which to build further investigations.

Similar series of observations in other species in the group have been made
and brief statements of the findings in some of these are presented, not that they
add anything in principle to the conditions seen in L. multitcsnuita, but they
show that the state found in that species is not limited to it, nor are the prin-
ciples which it shows specific in any degree. This species is of the high-plateau
group, and for comparison I shall give briefly the conditions in a lowland
savannah species L. undecimlineata Stal and L. decemlineata of the temperate
portions of the northern United States. Data of the former at two locations
Tierra Blanca and San Marcos in the lowland savannahs of Mexico, at Chicago
for the latter form in the north.

IN LEPTINOTARSA UNDECIMLINEATA.

The condition presented in the pattern of the pronotum of this species
shows many points of likeness to the pattern in the species already examined.
The same elements are present in the sys-
tem, with some wanting, and a few are
added. As in the former species, by
proper breeding analyses it can be broken
up into a series of groups that are uniform
in character, breed true in mass cultures,
and in all respects biotypic groups of this
character and species. In figure 112 is
shown the condition of the pattern as a
whole, as far as it is known to exhibit dif-
ferences in the pattern within the known
range of the species in nature, represent-
ing the conditions over the whole known
extent of the species. The known range
shows that the different conditions can, for
convenience, be arranged around the neu-
tral or indifferent biotype 7. In L. un-
decimlineata the same elements are present
as in L. multitceniata, the combinations in
both are the same, and in both similar
directions of the modification are found.
From biotype 7 in both, 9, 8, 5, and 4 are
present in the same condition, displaying
the same elements in the identical combi-
nations thereof, and with the same general range ; biot}T)e 6 is also present, but
with an added element, the area forming a connecting element between c and e,
and designated in this species as biot)T)e 6a. Biotypes 1, 2, and 3 are uniformly
absent in this species, and 4 is not as a rule much developed in any location thus

Fig. 11 2. ^Diagram representing bio-
tvpes and their arrangement in the popu-
lation in the species as L. undecimUneata
and L. panamensis.

308 The Mechanism of Evolution in Leptinotaesa

far seen. Biotypes 10, 11, and 12 are entirely absent, but a new line of arrange-
ment is introduced in this form that is represented by biotypes 13, 14, and 15;
pattern arrangements that were not present in the population of the first species.

ADDITIONAL PRIMARY BIOTYPES.

Biotype 13 : A condition of the pattern in which the marginal elements are
all wanting; the areas much reduced with the fusions a' + a and d + e the
only ones present. There is also a reduction of the anterior a center and a
widely divergent axis of the a, the spot giving them a sharp broad V-shape that
is quite distinctive and unlike anything seen in L. multitceniata. Biotype 14
is in most respects like 13, with the exception that the a spots are parallel and
fused along their entire median sides, giving a central oval or oval spot notched
at the ends. This biotype is also distinctly separated from the others by the
absence of spot c. Biotype 15 differs from all of the rest in the complete lack of
any permanent associations between the elements of the pattern and the
tendency to reduce the areas into their component elements where compound.
The a areas are often separated, and the e areas are divided into a three-lobed
area or into three isolated centers. Considerable somatic disturbance occurs in
this pattern and often obscures the true condition in the system, as is shown in
the figure, where the member of this biotype nearest to 13 and 14 shows the a
areas fused, when in reality such an individual would not breed as such.

The two species then present 16 biotypic conditions that can be analyzed out
of the populations, that are capable of being isolated, breed true, and act when
crossed as alternatives, giving in Fj a segregation into the same extracted bio-
types. There is one conspicuous difference between the condition in L. mul-
titaniata and undecimlineata, namely, that in the first the entire range in the
character may be present in the population at one location, as it was at Chapul-
tepec on certain occasions, while in L. undecimlineata the entire known
range of the system is never exhibited by the population of any location
at one time or through any length of time ; and furthermore, the different bio-
types have a geographical distribution in the habitats occupied by this species
that is interesting and would have been of interest to have followed in a series of
locations had this been practicable. The difficulties of transportation in the
southern portions of the range inhabited by this species, from the Rio Coatza-
coalcos and southward, made any project of this sort out of the question. Had
such been possible, it is certain that the results of the examination would only
have shown the same principles found in the locations that could be followed
with ease, and have added perhaps instances of localized distribution of some
biotypes, a condition that has little interest to us, and no possibility of solution.
The fact that biotypes may be geographically limited is at least interest-
ing and suggestive of another productive cause of heterogeneity in nature.

Two locations were chosen for examination of this species in the savannahs of
lower Vera Cruz, on the Vera Cruz al Pacifico Railroad, that were comparable
but different in topography and in the environmental complex, as far as it is
possible in the savannah areas. These locations at Tierra Blanca and San
Marcos have already been described in Chapter II in the description of the
habitats and the sources of the material that had been used in the experimental
work. No records of the climate for either place are available that are of any

Analysis of Heteeogeneity in the Population 309

value in this connection, and the few measurements and the maxima and minima
that I obtained are not of any significance in this connection.

THE TIERRA BLANCA COLONY.

Eleven censuses of the population were made at this point, beginning in 1904
and ending in 1909. These probably do not represent as purely generation
determinations as do those for muUitceniata, owing to the fact that the species
breeds over a longer time than the former, and that the generations become
much mixed from the middle of the summer on, and in nature the population is
mixed with respect to the number of generations in the yearly cycle. In captiv-
ity in pure lines the cycle in my cultures has been uniformly two, with a period of
rest between that varies with the conditions of the medium and the stock. In
nature the population is immensely diversified in this character, and stock from
nature is heterozygous, giving all sorts of combinations at the start, so that the
censuses taken here represent censuses of the natural population, in which the
relations of generations to one another and to the censuses could not be deter-
mined with accuracy. This condition will not invalidate the general results
that may come from the examination of the materials, and will give data on the
species comparable with that already obtained.

27 41 39 92 21 55 77

604 Males. Total 1201. 597 Females.

Fig. 113. — First census of condition in the population at Tierra Blanca In 1904,
showing the pronotal pattern.

..^ 51 — f«<g?'^

*!y^ -M'i'Sr — 10^ ','^ *^ 'V' —95

"^ ^^ / ^> V '-83

^^§— -75 / "^\ ,^.^-59

\^*^ / \ ';••'<*?*''

29 46 \ \

522 Males. Total 1104. 582 Females.
Fig. 114. — Second census at Tierra Blanca in 1904.

The first census of 190-1 showed in both sexes a ujiiform population, centered
mainly in biotype 7, with indications of 5, 6a, 8, and 9 poorly developed, and no
isolated groups of any sort, and on the whole an extremely uniform set of indi-
viduals and homogeneous in the total mass of the population. The second census
showed essentially the same condition in range and in the biotj'pes present, the
chief difference being the increased development of 8. As far as I could observe
and determine from the data to be obtained, the conditions of the climatic com-
plex had been favorable to the colony in the year; at least no striking adverse
conditions had been present. The records of the population at the two censuses
are shown in fisrures 113 and 114.

310 The Mechanism of Evolution in Leptinotaesa

In the season of 1905 the first census showed no change in the character of the
population ; if anything it was more completely limited to the central mass and
almost entirely in biotype 7. The second determination showed increased
ranges within the general mass of the population, and also the presence of
marked, divergent groups that were well separated from the rest of the popula-
tion. In the males there was a strong group of biotype 5, and in the females
a strong one of 14 and a minor one of 15. The records of the two censuses are
shown in figures 115 and 116, The environmental conditions, as far as known,
showed no divergence of interest.

In the season of 1906, the first census showed an increase of the number and
strength of the isolated groups and changes in the distribution of the population

tV^ .V. -414 ^^^^^^-—349

V'liV^-— 192 / '^^«f^^?— -174

77 34 71 92

1099 Males. Total 2300. 1201 Females.

Fig. 115. — First census at Tierra Blanca in 1905, showing restriction of the
population to relatively narrow conditions centering around biotype 7.

9 — €ii '^M-u

.22

92

158

86--*::W-'^-,

'iiM^ -'^r-'T— 31

J^-.;;^,i:,Kr-i4i

C \<J*J> 196

91 ---^-^ ^v> ^'' :>':

-77

-192

311

-196

77 81

29 41 84

1128 Males. Total

2258.

1130 Females.

^sma

Fig. 116. — Second census at Tierra Blanca in 1905, showing somewhat increased
array and the existence in the females of two highly divergent isolated groups.

as a whole, especially in the males. In this sex, biotypes 4 and 5 were strongly
developed ; 6a was present as a marked group isolated from the population, and
14 and 15 were also isolated and of considerable strength in the population.
The females showed almost complete absence of biotypes, with the exception of 7,
and an isolated group composed of the extremes of 4 and some few individuals
of 9, both standing well apart from the mass of the population. In the second
determination of the condition in the population a sharp change had taken place
in that the mass of the population in range and in the groups that were present
had been much extended in both sexes and was about equal in the array of
biotypes presented. In both, 5, 6a, and 8 were strongly present ; 4 was strong in
the males but weak in the females; 7 strong in both ; and in the males, 14 was
present as a well-marked isolated group. The conditions found are given in
figures 117 and 118. The only item to be noted in the environmental conditions

Analysis of Heterogeneity in the Population 311

in this year is the early onset of the rainy season, the amount and uniform distri-
bution of the precipitation, and accompanying this there was an uncommon
development in growth and luxuriance of the flora and fauna noted throughout
the season.

In 1907 the season was an average one, and in both determinations the popu-
lation showed only restricted ranges in the immediate proximity of biotype 7,

8 — ^^

58-- 0^ ^& «

'^m^.mw- — 217 te^Siis^-211

/ / / -^:<M^^-^-"^ / ^\

25 34 21 ^^^ 5 49 49 45 196

922 Males. Total 1720. 798 Females.

Fig. 117. — First census at Tierra F.lanca in 1906, showing condition in the
population as a whole and ('xi.>--tence of an increased numljer of isolated groups.

31 -«^^

56 -^^:,,

92- -p^'^^'i ..-, 45 ^^^^

141

i!?pi'^'^^4^?^s-::----2i4 |*;;??*i >^ tyi: {i;v>) ->, 283

/ ' / 'i. , \ . / / / - ^' .V' -- 1.30

/ / iQ^^s \^;^.'""^^ I I it"^ , v.^-J-105

/ / /^^\\ \ / / / ^.;."-'\ \ "

8 31 33 9 24 20 47 41 42 31 41 83 91

1240 Males. Total 2475. 1235 Females.

Fig. 118. — Second census at Tierra Blanca in 1006, showing increased array
in the population as a whole and the restriction of isolated groups to a single one
present in the males only.

with no divergent groups. The conditions of the population in the two censuses
are shown in figures 119 and 120 ; distribution about like that in 1904 and 1905.
In 1908, which was a season in character like that of 1906, the population
again showed increased ranges in the array of the central mass of the population
and considerable developments of isolated groups. In the first census the two
sexes showed essentially identical conditions ; the central mass showed a heavy
development of biotype 7 and conspicuous showing of 5. Biotype 4 was feebly

312

The Mechanism of Evolution in Leptinotarsa

41 83

697 Males,

Total 1535. 838 Females.

Fig. 119. — First census at Tlerra Blanca in 1907 showing condition in the popu-
lation with regard to pronotal pattern.

34 91

438 Males.

4--^
81—

43

l#

—103
^^\Cit"219

29 87

Total 1059. 621 Females.

Fig. 120. — Second census at Tlerra Blanca in 1907, showing condition In pro-
notal pattern.

'"S::"-^^^^ 194

^%iM;:^^p 318

/ / / ^^r---i47
/ / '

i/i/

/

7m-
\

92
946 Males.

56

Total 1973.

1^
44 13 51 7

1027 Females.

Fig. 121.— First census at Tierra Blanca In 1908, showing sudden Increase In
certain directions in pronotal pattern and the sudden rise of three strongly
developed isolated blotyplc groups in both sexes.

41—

<t^%,

14 91 58

1231 Males.

Total 2141. 910 Females.

Fig. 122. — Second census at Tlerra Blanca in 1908, showing condition in the
population as a whole and the persistence of isolated groups in previous censuses.

Analysis of Heterogeneity in the Population

313

represented, and 8 was present in small numbers. In both sexes biotype 6a
stood apart from the population as a marked group, as did the extreme condi-
tions in 4 and a fairly strong group of 13. In the males some of the extreme
conditions of 9 were present as a poorly developed group. In the second census
the two sexes again showed the same conditions, in that the central mass had
changed in its shape by cutting off the extremes of biotype 5 as an isolated group
of some strength, and the increased development of 4 into a strong group.
Biotypes 6a and 13 were still represented in the population by isolated groups.
These conditions in the population are shown in figures 121 and 122.

The last census made at this location was the first in 1909 and showed little
of interest. The body of the population showed the same condition that was
seen in the last census of the previous season, but the isolated groups had
dropped out, with the single exception of a small group of biotype 13 in the
females (fig. 123).

In principle these arrays of the population through a series of generations and
seasons does not show anything different from that found in the data of L. mul-
titceniata. There are present in the population the same biotypes to some extent,
shovnng that in part at least the same complex system is being dealt with, but
in the population there appear other combinations that are not known in the

"^ *^ '-V^l 199

-181
41 77

--'^- ■.^^\

22

25

41 83

--51
-228
-161
-107

909 Males. Total 1632. 723 Females.

Fig. 123. — First census at Tierra Blanca In 1909, showing condition of the
pronotal pattern.

former series, as in biotypes 13, 14, and 15. In this series the same ranging
condition of the population over the possible pattern conditions is seen, but for
the location the range is limited to a relatively narrow series of changes, and the
isolated groups are uniformly conspicuously isolated from the mass of the popu-
lation.

THE SAN MARCOS COLONY.

I introduce the data of this location briefly, as a comparison of two locations
that are much alike in their topography, climate, and other conditions of life,
covering the same time and the same number of determinations as in the first
colony, but showing that the two are not at any time the same in population,
even though the same elements may be present (figs. 124 to 134). Com-
parison of the two series of determinations given will show the differences found
purely local in character, but of enough importance so that the differences
might well be productive of unlike results in different investigations, unless the
local differences in the constitution of the races are taken into consideration.
For purposes of some of the latter portions of this work it is necessary to have the
conditions present in these two locations for a basis of comparison, as they are
the base locations for most of the materials that have entered into some of the
more important experiments in nature and in the laboratory.
21

314

The Mechanism of Evolution in Leptinotaesa

'jJA» ,^i 196

T», ^v> .j^ 214

--319
—111

87-
■96-

■'ti,-^ «V» ^v^-

/
/

38

57 29
1034 Males.

77 92 104

— 81
"519.

— 2U

Total 2304. 1270 Females.

Fio. 124. — First census at San Marcos In 1904, showing condition in the pro-
notal pattern.

11—
51—

V

..^ V^ tV=» jV

M$ 86

; 214

• ^ 479

79. =55^>i-;.

/ / /

25 11 81

^*

\ \~Q^^— -241.
\ \'^'— 38
\ \ "
56 92

/ \ \ ^

24 31 85

1385 Males. Total 2466. 1081 Females.
Fig. 125. — Second census at San Marcos in 1904.

• — 4S
— 541
— -219
— -56

^""

"\^\

21"

86 -^«Sl^\

441 ?^

•5.?.

^5--^^ _ ^ 'i.f. .■'19

m^^.'^ *>?' .V2^> -415

/ / ,\> ^^-^ i,&(JK 109

/ / / ^jsr\ \ r^

/

'47 21 4) 9 24 51 97

1559 Males. Total 3032. 1473 Females.

Fio. 126. — First census at San Marcos in 1905, showini? increased array of
pattern conditions and existence of an isolated group in biotype 12.

3^ -mi-^

56 -m:\

p3 ■"^>^*jv,

^"— :;:-;-' *^' i>-"V -sss

/ / .' fci'ga'-i^f,..^^. 4J3

-146

■' / /^. \ Is

//

4 51 65

/ m^

C'^J

12 22 131

2105 Males.

77

92

106

99

^^'W^^

/

14

II

-219

12 17

Total 3716. 1611 Females.

W

58 92

Fig. 127. — Second census at San Marcos in 1905, showing condition in the popu-
lation as a whole and the existence of two isolated groups in both sexes.

Analysis of Heterogeneity in the Population

315

25 : C

11

61— ■

T"

■ ^

^^Sfe ' t;^^^^ 417

'^^' \-^m 214

46

/
92

46 92

1839 Males. Total 3364. 1525 Females.
FiQ. 128. — First census at San Marcos In 1906.

18 ^^^^_

61 — -vssifcV

19 X^i 6j _

''- : ^" '^>V 145' " 5S^4Vf ?^^-:-::|«

' / \ \ «3S®«^ — °"* / / \ \

! ' \ \ ' ' ^ \

31 56 29 41 33 56 21 92

727 Males. Total 1566. 839 Females.

Fig. 129. — Second census at San Marcos in 1906, showing condition of pronotal
pattern.

„ — ii,

19 ^^

31 — ^m^

46

m ^ ^ftg»— 210

\ '^Vj- 92

56 71
852 Males.

83

■77

Total 1559.

^ -i^ tV. .V- 92

/ / "^^^ x^iii^-107

/ / w

40 41 96 88

707 Females.

Fig. 130. — First census at San Marcos in 1907, showing conditions in pronotal
pattern.

40

— ;4

m.

55

— -'

m.

51

. —

w-

17

i»V

- >w^'^^ .^

-106

-511

/' ,

/ /

{0--.

-45
-51

11 17

12

4G

WW^::^

i^— — i«^

/ / / "\^-''
22 11 91 44 58

962 Males. Total 2256. 1294 Females.
Fig. 131. — Second census at San Marcos in 1907, showing the pronotal pattern.

316

The Mechanism of Evolution in Leptinotaesa

55 41

818 Males. Total 1546.
Fio. 132. — First census at San Marcos In 1908, showing the pronotal pattern.

41 1

^^f^^^M — ^^^

38 56 41

728 Females.

•55- H

47

85

90

■-29

214

19

46--^

1073 Males. Total 2263. 1190 Females.

Fio. 133. — Second census at San Marcos in 1908. showing sudden change In
the population as a whole and sudden appearance of isolated groups In biotypes
6, 8, 12, and 10.

^^1— 87-

11 — f^\

41 '-^J^

II

/ /

51 12

"mm

---17

V \ V

9 31 55

1230 Males.

61 ^Csm.

60 -^mm). ,^f— 141

\— 96

44--^---3P^^^--109

86

26 25 31 11 41 33

Total 2531. 1301 Females.

Fig. 134. — First census at San Marcos in 1909, showing an increased array
In pattern conditions with total elimination of isolated groups present in the popu-
lation in previous censuses.

Analysis of Hetekogeneity in the Population

317

Comparison of the two locations is shown in figure 135, in which the outlines
of the populations are shown as in figure 111 for L. multiiceniata for its different
locations. L. undecimlineata, while conspicuously less widely ranging in its
pattern, an " invariable " species, shows the same set of conditions present and
acting in the same system, as was seen in the first species, and could the investi-
gation of this species have been followed advantageously for a widely separated
set of places most interesting arrays quite different in their composition would
have been seen. As it is, in the consideration of the geographic aspects of this
complex pattern some indication of the diversity present in many localities is
obtained. These, while not presenting the entire series of ever-changing differ-
ences that are found in the population, reveal that over the range of the species,
as a whole, different locations in space constantly show differences in the array
of the pattern presented — instances of determinate variation.

1904 1905 1906 1907 1908 |909

Fig. 135. — Diagram representing the conditions found In the two populations
of L. undecimlineata in the colonies of San Marcos and Tierra Blanca, during
the period of observation. Although the colonies are essentially alilte, as far as
populations are concerned, there appear to be minor constant differences present
In both.

One other instance of the series, namely, L. decemlineata at Chicago, for a
series of years, will complete the presentation of this portion of the data.

IN LEPTINOTARSA DECEMLINEATA.

In the populations of this species I had examined in Massachusetts, Long
Island, and Ohio, I had found that there were present in this character associ-
ated conditions that could be grouped into assemblages that were capable of
isolation into true-breeding groups, as far as the analysis had then been carried.
Further analysis of this condition at Chicago between 1902 and 1907 showed
that in the population there were five groups that could be isolated and that
would breed true in mass cultures without further selection, once they had been
purified. This species is poor in elements of the pattern, none of the extra
areas being present, and only the main elements are represented. So, also, the
species is poor in possible combinations and is quite distinctive and specific in
this respect.

THE CHICAGO COLONY.

There are present at Chicago five biotypes, 7, 4, 8, 9, and 13. In some other
locations in the range of this species there are indications of the presence in the

318

The Mechanism of Evolution in Leptinotaesa

population of other biotype conditions, but these I have not examined or
attempted to follow, the localities being too remotely separated from each other

and the materials inferior in most respects to
those available to me in the tropical areas,
where the topography had concentrated the
distribution without decreasing the diversity.
These conditions, with good transportation in
the region, determined the location of most of
my work in this direction.

At Chicago, between 1902 and 1907, 10
generations in a restricted location to the
south of the city were examined, 2 gen-
erations being missed, one in 1905 and one
in the early part of 1906. The condition
in the population of this species is shown in
figure 136, with the arrangement of the dif-
ferent biotype groups in the pattern array.
This species is, in part, like the condition in
multitceniata and in the presence of biotype
13 is like undecimlineata, although the pres-
ence of the same biotypic conditions in the
pattern has no necessary connection phylo-
genctically, the condition representing solely
similar arrangements in the effective forces that produce the conditions observed,
and is the sole result of the interaction of the effective pattern-producing agents
and the conditions of the medium at the time of the reactions producing the
patterns.

Fig. 136. — Diagram showing bio-
types commonly present In L. decem-
lineata as observed at Chicago and
the biotypic groups which have been
isolated.

14

11

19

21---!^ \ \

56 -^ '

77-—^ ]

29 ^^

31 ^

m

10 -t^

91

42-
71-
86-

iij^--31
^ 42

^ 17

Cr~^ ,• ^ 51

^^ ^ 29

-■mi^ ^ 77

121

314

:Vyt> 211

SjMs^ 97

^ 31

^ 44

t^ 77

^-^ 86

^- 141

211

t^ 151.

^r -77

^h — -83

(^ 46

^ 51

1256 Males. Total 2695. 1439 Females.
Fig. 137. — First annual generation at Chicago in 1902.

It is not worth while describing at any length the conditions in the different
generations examined, the series of determinations showing a restricted range
in the array, with not much in the presence of isolated groups in the population,
although such occur, as, for example, in the census of 1903 and again in 1905.
These are never far separated from the general mass of the population, but are
the extreme members of some of the biotypes, between which and the general
population there are no intermediate conditions presented. The array shown
in the censuses that are given in figures 137 to 146 show in principle the same
conditions that were found in thp formpr snppipa

Analysis of Heteeogeneity in the Population 319

10 14 9 11 14

31--J^
\^ 21 —

22 ^^) (^^^ 214

^. 311

fi^ 214

4^V 171

^ loe

88 ® ^k- 166

<^) 341

^^) 207

(^ 2C0

@^ 104

Q^ 97

^232^- — 81

1898 Males. Total 3329. 1431 Females.

Fig. 138. — Second annual generation at Chicago in 1902, showing the pronotal
pattern.

I 7 4

/
9- ®^^^ j^^ / ^ 106

31-

86—--^^ ^ 71

r..^ 45 (^ ^ 217

^^ 111

73-— <ijM>j) (^ 15,

^) 411

<§M0 131

M^ 56

\B 44

(^ 192

^ 219

4Sj> 155

/^. 143

(55) 76

1090 Males. Total 2682. 1592 Females.

Fio. 139. — First annual generation at Chicago in 1903, showing the pronotal
pattern.

8 51 11
9"(^\\\ 8-^

14

r.^

^ ^ 9 11 (^ ^

^ 9

_ 56

11 -^ij}^ ^ 41 31 J^ ^ 44

31 — -^m ^^ ^ 42 -^ <^- '

^ 77 ^j j^

^ 55 ^ ^ ^^

®- 88

55

482 Males. Total 1054. 572 Females.

Fio. 140. — Second annual generation at Chicago in 1903, showing condition
in population and existence of isolated groups in biotypcs 3 and 4.

320

The Mechanism of Evolution in Leptinotaksa

5 15 8
29 -- ^'\ \ \

42 ^\\^ ^^ 51

47 (^ \^^ 1^ 74

86 (^ («^ ^^

45

74 i^w;^ li^ 91

83 (^ 1^ 183

(^>- 171

^). 146

W- 211

^- — 145

^SB> 77

1653 Males. Total 2774.

17
20--^

51 &

42 (^

26 ^

21

14 2 12 9

.' /

m
^

(^—14
7 ^ 49

^ m- 74

81

192

186

^L 144

^^ 93

^ 74

1121 Females.

FiQ. 141. — First annual generation at Chicago in 1904.

W <^— 20

.58

\ \

41- i^

77— (^ \^

92 (^ ^

56

19

-m

^ 55

^ 92

^^ 46

(^ -183

$'- 1'^

m^ 155

^ 177

-92

-36
-41
• 17
Total 2152.

27

25--^
33—-^

31-—^
17

8 5 4 1

\ I ' f

1 1 :

mi

m —

<^— --

1454 Males.

^ 9

^ 31

^ 44

^ 88

ii 166

75

^. 56

42

698 Females.

Fia. 142. — Second annual generation at Chicago in 1904, showing the pronotal
pattern.

8 17
X \31 4

5-^\\\\

^ 46

--^

^ ^

92

& ^;

-107

18 ^

41 @ (^ 100

12- -^ "^ 58

(^ 177

^ 96

^:. 109

^, 77

4

71 J^

66 &

69

41

9 5

//

/ ^— 31

' (^ 20

^ 21

74

55

_^ ^. 83

155

^. 86

®- 77

^ 42

(^ 51

^. 12

1145 Males. Total 2084. 939 Females.

Fig. 143. — First annual generation at Chicago in 1905, showing conditions In
population and existence of biotype 4 as an isolated group in both sexes.

Analysis of Heterogeneity in the Population

■--9

^ 25

^ 86

^ 104

7 4 3

26 '^ WW

28 -^ \^

44 <^ ^ i@--- -in

39 4^ ^ 92

;^:. 214

(^ 119

(^, 42

(^ 11

^ 4

^ 1

1203 Males. Total 2327.

14

26— til
51—

42 -i^

31 -®

20 -M

/

/ ^—-2
/ ^ 7

^^ 34

(^ jgj

34 ^ ^^ 217

^> 318

^- 141

^^ 12

^^5 7

1124 Femalea.

paKn'a's'fTr^a'' observed"' generation at Chicago in 1906. showing the pronotal

12 11

14--^ \\^
21—-^ Vg)

56 ® ,^ ^^._ gg

33 'W (^ 92

11 '^^^ (^^ 171

^ 314

^- 211

@#- 171

1 7 4 «
' /

!5^— 86
1271 Males.

6--^ ^/ ^.„. ^

41-—-^ ^- 174

18 ^^ ^ 218

326

^ 211

(^ 44

Total 2568. 1297 Females.

paftern^^^""^*'^^'' *°°"'''^ generation at Chicago in 1907, showing the pronotal

11 7^
/iSf^ ^ \ ^t!ll^^— 17

5 - -'^5?) >. \ rr~

9 ---^^ .^^ ^^^

-27 <?W^ '^ £^

41 (^ ^.

8 i^s ^, ^^^

'^ 176

^fU). 144

^W0) -107

^- 193

(^ 86

112 4

41

66

21

'55

,-^

27

46

.--^^

34-___J^

1163 Males. Total 2265. 1102 Females.

56

44

(^ 92

$ 191

317

-45

-81

^ 22

^ 26

^—74

^^^^^^•^ 1*6.— Second annual generation at Chicago in 1907, showing the pronotal
22

321

322 The Mechanism of Evolution" in Leptinotaesa

SOME RESULTS OF THESE ANALYSES.

The analysis of the condition in the population covering a series of genera-
tions in the same location has not often been attempted, and when it has, not
infrequently the outcome was to arrive at incorrect results, owing to the short
time that the observations were continued, or to the wide gaps that intervened
between different determinations. One of the best examples of this is the experi-
ence of Kellogg with Diabrotica soror in California, who examined the popula-
tion of this beetle, finding certain conditions in the population as expressed in
the variations of the elytral pattern, and later, with an interval of several genera-
tions, a second set of determinations gave results that looked like movement of
the population, which was strengthened by the determination in one or two
succeeding generations without change ; and so the conclusion was reached that
the population had changed in one direction with respect to this character,
between the first and the second censuses, and the results were described as
determinate evolution. Later determinations showed that the population had
moved back to the first position with respect to the same wing characters. What
happened in this instance is that the lack of continuity in the observations gave
false appearance in the determinations taken at random times. Out of the
data that I have had at my disposal, any number of instances of this sort could be
realized by precisely these methods.

The data that I have presented in the preceding pages would, by the statisti-
cian and his measuring-stick, be massed by force into curves of error, interpreted
in terms of some hypothesis, and then put forth as " proof " of the same hy-
pothesis. So, also, the f aunistic and ecological worker and the geographic varia-
tion worker could and would utilize the arrays presented as examples of the
direct action of environment upon an extremely labile species, and derive
various agents that may or may not have been operative in the actual conditions
in nature. These plausible relations, easy to create, soon become fixed in the
minds of the worker and are soon " established facts " in support of the especial
proposition with regard to the action of geographic influences.

Some, no doubt, conceive of the population in terms of minor groups which
the census does not reveal, and can therefore be interpreted upon the basis of the
presence in the population of numerous elemental conditions "without inter-
grades," but whose " fluctuations " overlap and thereby produce the appearance
of continuity, wherein there is no real " continuity " excepting in the " fluctua-
tions of the minor groups." No census can show anything with regard to the
truth of this proposition.

With these materials I have gone carefully over the series and have applied
the census methods, but nowhere have I been able to discover that the real nature
of the materials and of the processes at work was even indicated. These
are problems for the laboratory and the experimentalist and not for the
statistician.

I, for one, do not care to discard either the method or the point of view of the
statistician, as some would do, because the method does give information con-
cerning the mass of the population that is of real value in the attempt to experi-
mentally analyze its constitution and the processes at work; but more, it gives
accurate information that could never be provided by the laboratory and the
experimental garden with respect to the sum total result produced in and the
aspect of the population after it has been subjected to the operations of nature.

Analysis of Heterogeneity in the Population 323

The methods here employed, with the information of the constitution of the
character and the experimental analysis of it have given the opportunity to
understand rather well the meaning of the conditions in the natural populations
that the censuses have revealed, and this is of value in the analysis of some of
the problems of the production and origin of species and minor groups in nature
and in experiment.

I have been especially interested in the constant presence in the population of
" definite variation," which is true not only in the single population, but in each
location there is an unmistakable place response in which the population
" varied " in the same direction as the result of the conditions generally present
in the population and in the medium. I do not recall at present anyone who
has attempted continuous analyses of these responses in any population in
nature; rather most of the instances that are in the literature are the plausi-
bilities of records from few specimens or from determinations made at one time.
Nor do I recall that the use and recognition of the principle that Darwin so
clearly saw and believed might be of so much importance in the evolution of
organisms has been often mentioned or considered. There is an abundance of
argument concerning the production of the " directed " conditions which are
supposed to exist by " selection," but almost no attempts at the analysis of the
situation. Some few, as in the crab Carcinus, investigated by Weldon, gave
information that is at once suggestive and at the same time too incomplete to be
of much service in the analysis of the problem, and the results can be interpreted
on any basis one wishes. The incomplete facts as determined show only that
changes of some sort were in progress, either permanent in character or tem-
porary, ontogenetic or germinal, due to selection, environment, or any other
cause one wishes to introduce. The unquestioned fact of change existed, but
was not analyzed.

There is no question but that the conditions as presented in the populations
represented here show this definite place " variation " in the population as a
whole in this complex character, and the census methods employed leave no
doubt of the reality of the conditions revealed. If these responses in the popu-
lation of a place as a whole to the conditions of the habitat have any meaning
with respect to the production of heterogeneity in nature, this set of materials
provides at least one opportunity to test it out. The populations as found in
the determinations and the general place tendencies in the population may be
produced by one of two general conditions ; either the place response may be due
to a real germinal difference in the race, or it may be due to the ever-present
action of the conditions, influencing the soma in a definite direction in its
" fluctuating variations," or it may be due to the presence in the location of
influences that eliminate a proportion of the population possessing certain char-
acters, so that the maturing population presents only the aspects of the portion
thereof that was able to escape the eliminating agents. A further possibility
is that the conditions seen in the population are the product of the combined
interaction of the character of the combining materials and the conditions of
the medium in the habitat.

If the condition in the population at any location is a permanent one, then
the difi'erences that are presented may well be the basis of further differentia-
tion and in the end result in the production of a distinct localized race and

324 The Mechanism of Evolution in Leptinotarsa

add another element to existing heterogeneity of race and form in nature.
These questions I have put to an experimental test as an aid in the further
analysis of the problems of heterogeneity in complex characters.

PATTERN IN DIFFERENT HABITATS.

In my 1906* paper, mider the terms "place variation'' and "geographical
variation," I have discussed the differences found in the population in several
species of beetles belonging to this genus, using the current biometric methods
of expression and interpretation. Also, the experimental data then presented
were purely of the sort indicated by the quantitative, qualitative aspects of the
problems, although I then knew well enough that the whole aspect of the prob-
lem would soon change its setting and method of analysis. From the point of
view of the presentation in the 1906 paper nothing more is to be added to the
data and the conclusions there presented, and nothing further is to be expected
by continuation of the method of examination. Analyses had shown that all the
materials examined presented the phenomena of pJace variation in different
degrees, and the same species in the same place showed different variations,
depending upon the season. These biometric results and the legitimate deduc-
tions to be derived from them, with the experimental attempts to fix mere quan-
tity as measured statistically, while showing the presence of the condition with
regard to the phenomena and their difference in the several species examined,
lead to some points of view and conclusions that are essential to tie back to as a
part of the general problem that receives further analysis in this report.

It was there recognized that on the basis of the results presented place varia-
tion was a generally present phenomenon in the organism examined, and also
in general that it affects every portion of the animal (p. 103), and that not all
animals are alike in their variability. The methods employed led to the con-
clusion that the " variations " were not germinal, but somatic fluctuations, the
product of the varying conditions of the environment (p. 102), and that some
of these conditions of the amount of pigment or other characters were in favor-
able instances to be rather accurately tied in interpretation to conditions in the
environment. The fact that in experiment the increase in the amount of
decrease was easy to produce, and had been produced in many experiments, led
to the conclusion with respect to the permanence of place variation that it was
not, as far as the evidence then went, permanent and a probable factor in the
production of evolution changes (p. 102). A possible correlation between the
production of extreme variations, simultaneous with the occurrence in the envir-
onment of divergent conditions, was noted, its possible action indicated from
actual examples, and its bearing upon the relation of these productions to
varying conditions, suggested lines of experiment (p. 104).

With respect to the condition as there measured by statistics, the conclusion
is valid as expressed, that the conditions thus determined were not permanent,
capable of fixation in experiment, and it is further true that many of the difPer-
ences in the array of the measurements are mere ontogenetic exaggerations and
nothing more, due to the action of incident conditions at appropriate periods in
the life of the individuals. At present I fully concur in the result expressed in

^ An Investigation of Evolution in Chrysomelid Beetles of the Genus Leptino-
tarsa. Carnegie Inst. Wash. Pub. 48.

Analysis of Heteeogeneity in the Population 325

1906 as to the permanence of this sort of " variation " when determined in this
manner. Attempts since 1906 have been as positive failures in this respect as
were the earlier ones.

Biometrically the assumption is made that the material is homogeneous; in
reality it is not so, and eliminating by proper methods the larger portion of the
somatic variations, the proposition is still true that any instance of statistically
determined amount is not permanent and does not become fixed by continued
selection, due to the fact that the statistical determination does not choose
identical conditions in the character. No better demonstration of this could
be asked for than that presented in the history and improvement of the sugar
beet. The area of the pigment exposed on the pronota of my material may well
be equal in area or proportion of surface exposed, but the pattern conditions
presented, the array of active agents, factors, and determiners present and that
are brought into this array gives so extensive opportunity for heterogeneous con-
ditions that the real nature of the series is constantly shifting and is hopelessly
confused, so that the result statistically is to end with the " conclusion " that
the " variations " are somatic and so not inheritable. They may be so or not ;
the method has not been able to test the point. I have seen series in which the
conditions present were known to be accurately inherited and recognizable by
the arrangements in the pattern, but the biometric method resulted in a hopeless
array of ranging areas of pigment, which could not be fixed. It is this recogni-
tion of the results of the confusion of real conditions under the blanket of bio-
metrics that formed the transition from the former situation to the present one.
The results attained in this first set are correct as far as they can be with the
method employed, and were carried as far as it was possible to go in nature and
in the experimental analysis of the problem of place variation.

As has been shown in the preceding pages, this pattern is not a haphazard
array of colored areas, but an accurately constructed system, in which the color
is the end-result of many agents, and from this point of view quite different
conditions are conceived of in the population as the cause of the heterogeneity
found, and so the phenomena of place and of geographic variation take on a new
aspect, becoming capable of analysis along new and profitable lines. This is not
the place to deal with the gametic constitution of the materials used or of
the factors that have been recognized as present and acting in the population in
this complex system.

It is from this point of view that the phenomena of place and geographical
" variation " deserve analysis, so that the result will be a determination in
terms of effective agents, internal and external, in the production of the results
observed. Further, the examination will answer accurately questions as to the
actual, permanent, germinal differences that may or may not exist in the sepa-
rate portions of a widely distributed race, and in experiment these differences
can be tied to combinations of environmental conditions that exist in nature, so
that in the aggregate a more complete understanding and picture of the condi-
tions in nature will be produced. Out of investigations of this sort must come
the rational explanation of the origin and meaning of the facts presented by
closely allied, locally placed isolated races, as for example the Achatinellidae in
the Hawaiian Islands, as described by Gulick, and many other similar instances
in nature. The explanation of the origin and the experimental duplication of

326 The Mechanism of Evolution" in Leptinotaesa

them will rest upon the interaction of the conditions of the medium and the
character of the factors or determiners, and their location, association in the
system, and capacity to react uniformly under a given set of surroundings and
associations. A most desirable result of the method of analysis and the deter-
mination of the interacting agents will be the passing of choice, selection, pur-
pose, utility, isolation, segregation, and the rest of the array of " biological
explanations " current in the literature as an " explanation " of the position in
closely placed habitats of forms, much alike but suflSciently different in minor
aspects to give true taxonomic value to them.

Two main questions arise in this connection and can be successfully attacked
through experimental analysis : (1) In a given " species " or " race," in any or
all its habitats, does the actual germinal condition of the race differ in successive
generations, or is it constant throughout for the location and form ? (3) In dif-
ferent habitats does the constitution of the race change in constant, recognizable
gametic differences ? These are tests that should be made upon species or races
that are considered by the best of taxonomists as uniformly good species, which
are real things in nature. Eecognized geographical races are to be excluded from
this analysis, which is planned to reveal, if possible, whether in nature, separa-
tion into habitudinal groups of naturally miiform race may not begin in local
areas and be the result of processes that can be determined and utilized in
experiment.

In the investigation of these problems I have used three methods of attack.
(1) The introduction of stocks from one colony into other isolated loca-
tions and the watching of the following generations and the recording of them,
to determine what they did in terms of the census-taker in the new locations.
This will give rather useful data with regard to the readiness of the stocks to
respond to different conditions, and also the direction and extent of the
response. (2) The transportation of samples of the materials taken at ran-
dom in the population from the locations under observation to some central
location, where all are reared in isolation under one set of conditions, and in
this manner can be tested the reality and permanence of the differences in the
populations from different locations. If the samples are random, and if the
breeding is in group culture, with no selection in the matings, then the result
will be an accurate demonstration of whether the differences present in the orig-
inal location are able to persist alongside of the differences found in other loca-
tions, when both are living under the same conditions. This I have tested by
taking the materials to Chicago and growing them under conditions that were
uniform and neutral to all of the complexes that were under examination.
Under experimental conditions of this sort the result will be an accurate measure
of the permanency of the character of the material in both its germinal and
somatic capacities to be reproduced in like values in successive generations.
(3) The analysis of the conditions of the population with pedigree analysis,
especially of the extreme conditions and of the most common condition. Collec-
tively the three will give answers to the problems investigated.

EFFECTS OF TRANSPORTATION INTO A NEW HABITAT.

In this method of testing the permanency of the array shown in a local popula-
tion, the results that have been obtained in transplanting materials from the
colonies at Chapultepec, Tlalnepantla, and Chalcicomula will serve to show the

Analysis of Heterogeneity in the Population 327

general type of results arrived at in L. muUitceniata, and in the same way with
materials from Tierra Blanca and Campeche the conditions in L. undecim-
lineata, which will be fairly illustrative of the method and principles. In this
work it would have been fortunate if the opportunity had been available to have
used cages in some of the habitats, to contain materials taken to that point from
some other, so that the tests would be made more accurate and much more
critical. One method employed was to find isolated locations in which the
species was not living, stock them with food, and then introduce the materials to
be tested. This was not so difiicult in southern Mexico, where the topography
lends itself admirably to the needs of problems of this sort. Most fortunate
conditions of isolation were found ready or easily created in the southwest por-
tion of the valley of Mexico. At this point in the valley a huge surface lava-
flow has broken out of the sides of the volcano of Ajusco and flowed down over
the sides of the valley, covering the whole mountainside from Contreras and San
Angel on the northwest to the Eio San Buenaventura on the south and to
Tlalpam on the northeast and east. Although recent in geologic time, there
had been sufficient erosion and denudation to provide the region with a rather
well-developed but peculiar flora, and, most important, the forming of pockets of
soil in the much-broken surface of the flow that could be utilized as locations
for growing the food for these animals for the introduced colonies. In the
area as I found it there was no food for these animals and none of them was
found living in the area, due to the fact that the agencies of dissemination in the
valley were entirely against their introduction. The pedregal had become
inhabited by plants and animals from the higher slopes of the mountainside that
had been carried down by the wash of the storms. No roads traversed the
area, and only a few footpaths crossed the edges thereof, so that introductions
by transportation were trivial factors. The added character of the area that
it was of no use as a grazing-ground made it immune to the introduction of 8.
rostralum, the food of these animals, by grazing cattle, one of the most common
and efficient means of disseminating the plant. By going into the area from 1
to 2 miles, conditions of complete isolation were obtained, and with consider-
able labor and trouble the proper locations were obtained. These had, within
certain limits, the same general set of conditions, which, located as they were,
were not greatly different from the location at Chapultepec. It was found in
practice that the dissemination from the colonies was slight, owing to the char-
acter of the bare, dry lava-flows about each of the locations chosen, so that if
any of the beetles did wander from the colony the chances were immensely
against their ever gaining the next one in the maze of pits, cracks, caverns,
bare, hot rock surfaces, and so on, that completely filled the area, so that the
random wandering of one of these animals would with large probability end in
elimination and not in reaching the next location, even when only a few hun-
dred yards away. So broken and bad was the surface, so cut by pits, huge,
deep cracks in the lava, that passage of the portion used, on either horse, mule,
or burro, was impossible, while foot travel was slow and tiresome.

The first tests were begun in 1905, when 10 good locations were found,
located in the midst of the pedregal and isolated by distances varying from 300
yards to a mile apart. The majority of these locations were depressions in the
face of the flow into which denudation from above had washed accumulations
of soil, in some instances to a depth of several feet. The areas were mostly

328 The Mechanism of Evolution- in Leptinotaesa

oval or roughly so in outline and on the average were about 60 feet in diameter.
They were denuded of the original vegetation, sown with the seeds of *S'. rostra-
turn from the valley, and in a few days the location was ready for the test.

Early in June 1905, 1 introduced into the locations prepared random samples
of L. multitceniata from the colonies at Chapultepec, Texcoco, Tlalnepantla,
and Chalcicomula, and all thrived and became self-sustaining with the excep-
tion of the introduction from Texcoco, which died off, owing to failure of the
food-supply. This was the result of too shallow soil in the spot chosen. In
each test the same method was followed as was employed in the natural colonies,
that of making a census of the population twice each year. At the time I did
not know what the condition in any of the colonies would be shown to be, but I
was certain from previous experiences with the biometric methods of study that
the problems had to be attacked, and that this was the best method in this one
direction, so that in the series the test and the study of the conditions in the
colony went on simultaneously, a combination that has obvious advantages.
The other locations were utilized for other tests of the same sort that weri
made on other species. Observations were continued at the three locations,
two determinations in each season from 1905 to the end of 1908, eight genera-
tions in all. One exception in the census determinations was made, namely,
the dropping of the rating of the amount of the pigment present in each of the
pattern types shown, only the types being recorded in the test locations, because
it was thought that the persistence of the types was the point to be determined
and not the amount of " fluctuation." In presenting the records of the tests
in this paper the sexes are not separated, although they were in the original
determinations. There are no sex limitations or dimorphisms with regard to
this pattern, and the mixing of the two into one population statement does
not, as far as is known, hide or alter any of the conditions of the test.

THE CHAPULTEPEC COLONY TEST.

The character of the introduction of the stock from the Chapultepec colony
is exhibited in figure 147 in the array of the population for the first genera-
tion of the year 1905, and were the overwintering members of the previous year's
last brood. The chance distribution of the parent test group is shown in the
figure in comparison with the progeny of the group at the normal location. At
the test location the whole population was allowed to take part in the operations
of reproduction throughout the series, so that the test series was treated, in all
respects, like an independent isolated colony. The results of the test are
shown in figures 147 and 148, and do not reveal change in any essential in the
test location. The two locations were not entirely identical, and it was
thought that there might be some difference in the new location, due to the
change, and some little was found, but it is not at all certain that it was the
product of the unlikeness in the conditions, and might as well have been due to
altered relations of agents in the gametic complex as to the environment.
There was certainly nothing at any point to indicate the environment as the
sole active agent in producing the observed departures (figs. 147-151).

Analysis of Heterogeneity in the Population

329

7-
22-

8-
31-
42-
26-
19-
31-
42-
51-

m

^ 9

m^ 31

-29

I

^•

11 12

Population 748, First Generation

-6
-51
-42
117
101
-38
-29

-m

11— <

4__

7

29

31

26

41--

22

14

11

m^-

7 4 5 9 11. 7

Population 620, Second Generation.

— 4

— 11
--42

— 77
- 92

—86
—31
•—29

Fio. 147. — Census showing condition in the test colony of Chapultepec in 1905.

9

4 ^

36 w> m-

12 ^ ^-__.

mm m —
/ /»; m—

11

'^>- 14'

-12
-51
-42
-86
-31
-7

4 19 11

Population 394, First Generation.

4^

4;—
13 i

4

12

21
17.

12-
31-

41-

— -» m^

m m-

0 ^—

<^ — 5

-17
-31
-42

-86;

-91

^ 17

(^ 4

5

Population 473, Second Generation.

Fig. 148. — Census showing conditions in the first and second generations in
1906 at Chapultepec test colony.

330

The Mechanism of Evolution in Leptinotaesa

14-1
18-

24 -^

12 ^

■31 m

33

29-

16

-m

1^ — 11

W> 46

31

Wl^ 55

m 61

42 —^ m 74

177

W- 51

42.

14

' \ ■*■
/ \ \ <^ 31

U 4 9

Population 830, First Generation.

19
15--

20

31

42

14

14

19

11 r^ ^

5 _^ ^_.

..jii

m

I

1 « 5 7

Population 901, Second Generation.

^-4
^---1
— 12

!> 38

35

40

41

83

96

211

86

,45

Fig. 149. — Census stiowing conditions in tlie first and second generations In
1907 at Chapultepec test colony.

4 5

7--

9

11

21

44

48

71

-m

m-
m-

^ m —

^S^-9

»— 5

(^ 7

- 46
-•83
-92

-•77

— 43

11

-^m

1 —

42 m^

m — 4
-^ m — 1^
-■m m — ^
^ (® 8^

51

^ 4

Population 697, First Generation. Population 370, Second Generation.

Fig. 150. — Census showing conditions in first and second generations in 1908
at Chapultepec test colony.

Analysis of Heterogeneity in the Population

331

6-

31-
42-
40-
61.

'(^ 8

14

26— i

1--

7 ^

31 --^

8 ^

6 ^

29-

19

--1^ ^ 96

m-

I

\

101

83

44

26

@*i 5

1 2

/' ' '

(^ 20

35

^m — 41

m 71

m 92

^ 3g

141

77

6

^ 9

\ ^ 2

Population 607, First Generation. Population 719, Second Generation.

Fig. 151. — Census showing conditions in first and second generations in 1908
at Chapultepec test colony.

THE CHALCICOMULA COLONY TEST.

About the middle of June of 1905 a representative culture from this location
was introduced into a test location about a mile from the Chapultepec test, and
there thrived well and gave in the following generations a well-defined group,
that was constant in appearance. In figures 152 to 156 are shown the resulte

51

22

11

9

//

13

^ m 22

61

45

^ 42

\^ ^-—11
(^--- 4

44

— 11
-40

77

_ 89

^^ ®— — 46

^ 9

\

\

11 21

Population 305, First Generation. Population 399, Second Generation.

Fig. 152. — Census showing conditions in first and second generations in Chal-
cicomula test colony in 1905. In the first generation the introduced generation are
indicated along side of their progeny.

of the tests in these locations, with the summated generations in the original
colony for the corresponding generation and time, with the result that in the new
location the test colony remained well within the range of the population in the
parent location during the same period. In the original location the population

333 The Mechanism of Evolutiox in Leptinotaesa

was characterized by the absence of the factors in the make-up of the popvilation
that produced biotypes 1, 2, 3, 10, 11, and 12, and these were not present in the
introduced group and did not appear during the test, whereas in the test of the

11-

9— » 11

31 — -^ ^ — ^^ 22 ^m mr—i2

42-

39 1^ ^ 26

-7

-86 -^ ^ip^ 8^ ^j^ 77

42 / / / m^ ®— -45

11
/ \ ^-—1

/ \ ^m 11 /

I I I ^. \
III \ \
III ^ \

1 4 4 11 7 8

Population 256, First Generation. Population 253, Second Generation.

Fig. 153. — Census showing conditions in the first and second generations in
1906 at Chalcicomula test colony.

17

14 J^

31 ^^ ^j^ 41.__

47

/

"W> ^——-46

-131 «fP» 9m %i^ 78

/ "^S^ ^^ 74

^ 12 / /

---4

^*)— 17

12 9 10 4 6

Population 294, First Generation. Population 267, Second Generation.

Fig. 154. — Census showing conditions in first and second generations in 1907
at Chalcicomula test colony.

40—

11 ^ 46 ^ ^^ 70

29 — ^ m-—^i ' — ^ ^^

— 31

36 -mt SS&- 44 22 ---1^ ^ 58

161

77

171

72

/ \ <S^— 70 / \ m> 76

/ \ »— -29

/ \ / \^--l4

Population 529, First Generation. Population 642, Second Generation.

i.^9- ,^?^- — Census showing conditions in first and second generations in 1908
at Chalcicomula test colony.

Chapultepec location under the same conditions all of the biotypes were present
and none were added or lost. The same is true of the test of the Chalcicomula
colony ; none was added or lost in the test location. The test in this instance

Analysis of Heterogeneity in the Population 333

represents considerable change in the environment to more optimum conditions,
nevertheless the change did not put into the test series the agents to produce the
conditions in the pattern that are not present in the parent location to bring it
up to the same range that is found in the species native in adjacent portions of
the valley of Mexico.

These two tests show that the condition in the population in at least two
locations is not temporar}-, to be shifted about at the will of the oscillating
environment, but is relatively stable and the product of the present and work-
able factorial composition of the population, and of no other source. The
unchanged condition shown by the tests of the Chalcicomula colony is in all
respects the result of this, and if by any means there had been introduced into
the test series the multilineata form-factor, the melano thorax pronotal pattern-
factor, an extension factor, and the factor and determiner for the anterior

11 —

14 — m> m 12 m—-i2

^--^ ^"""Z n -m ^- -

''—"Z^^ mm^ »—-- -83

/^ »"-— / / /^ \ ^.-..

jm \ \ ^—21 ; / /T; \ \

\ \ \ ^i— -11 / / / \ \ \

12 \ \ 9 31 44 12 8 4. 14

Population 434, First Generation. Population 322, Second Generation.

Fig. 156. — Census showing conditions in first and second generations in 1909
at Chalcicomula test colony.

marginal spots, there would have been produced in two or three generations all
of the missing biotypical conditions in the pattern.

OTHER TESTS.

As stated in an earlier place, a test was made with the colony at Tlalnepantla,
at a location in the pedregal about 0.75 mile from the Chapultepec test location,
but with identical results, which I have not thought it worth while to burden
this paper with. The population in the Tlalnepantla location showed some
interesting differences from the other locations in the valley at that time,
especially in the absence of biotype 3, in which the operation of an extension
factor plays a considerable role, and this seemed to be entirely wanting in that
location, and the test series showed the essential conditions that were present
in the parent colony. At diverse times tests have been made for two or more
generations with the stocks from other locations at points in the pedregal in the
manner indicated. Thus the location at Puebla was tested at Esperanza in
1906 and 1907, Ometusco in 1907 and 1908, Tlascala in 1906, and in none have
I thus far obtained any but the same kind of results as here shown.

In this portion of the study I have been careful to use only the materials of
this species that are well within its central range, and at no point in the range
employed is there anything like geographical races, so that the tests have been

334 The Mechanism of Evolution in Leptinotaesa

made upon the portions of a natural uniform species, variable to be true, and
complex in its make-up, and for that very reason probably a good one upon
which to test out some of these points, showing as do the results that in a uni-
form but variable species (in the taxonomic and faunistic sense) the materials
taken at different points over its natural range are not of necessity identical in
their germinal constitution, but that the conditions differ in neighboring placed
habitats, and that these gametic habitudinal differences are permanent.

Similar tests were made with less satisfaction, owing to practical difficulties
in the years 1905 to 1907 in some of the steep-sided and well-isolated ravines
that are found in the valley-head above Maltrata. These ravines are the loca-
tion of small streams falling precipitously over the edge of the Mexican
Plateau to the level of the Maltrata Valley. The location was much more
difficult to operate in, and the results were not so interesting, owing to the all
too frequent damage to some of the locations by heavy torrential rains that
come in the rainy season, often washing everything before them in some of
these ravines, and one can not pxedict when one of these will strike the
chosen spot.

The outcome of the tests that were made was in all respects identical with
those on the pedregal in the valley of Mexico. Of most interest were the
results of the series from Chapultepec, Chalcicomula, and Tlalnepantla, none
of which changed in any respect during the time that they were in the test loca-
tions, giving uniformity of action in all.

One point is of practical importance in this connection, namely, in placing
these colonies for testing the condition in the population, it must be as certain
as possible that the location chosen for the test shall not be such in its conditions
that they will serve as a modifying agent and so give confusion in the determina-
tions. I used all of the precautions that were possible to avoid this, and seem
to have been most fortunate in my selections, not one of the test-series locations
showing anything in the way of changes in the population that could in any
way be regarded as a direct modifying action of the environment.

TESTS WITH L. UNDECIMUNEATA.

The populations of this species at Tierra Blanca and San Marcos are appar-
ently not sufficiently different to provide much in the way of contrasting condi-
tions for testing, so that the permanency of the condition in the pattern was
tested in this species by taking some of the population from the Tierra Blanca
to the region of Campeche, and from a point near Campeche to the Tierra
Blanca in 1908. The population in the two locations apparently differs per-
manently in several respects. The chief difference between the two is the
absence in the Campeche location, near Seibacabecera of biotypes 4, 5, and 9,
with the preponderance of the groups 6a, 7, 13, 14, and 15, making a population
in which there was little breaking in the continuity of the array. The Tierra
Blanca colony, on the other hand, shows the 6a, 13, 14, and 15 series weak, and
in any given generation separated by large gaps. The stocks were inter-
changed in May 1908, and each put in a location not in contact with the original
colony, but isolated from it as well as the conditions in each location would per-
mit. The samples introduced into each location were fairly random. I did
not see the colony again until the winter of 1909-10, when a portion of the
colony at Tierra Blanca was probably in the groimd in aestivation, but enough

Analysis of Hetekogeneity in the Population 335

remained upon the plants to make a fairly satisfactory analysis of the popula-
tion, while the conditions at Seibacabecera were more satisfactory in this
respect. In each some three or more generations had passed since the intro-
duction, and at each location there was the probability that some of the native
population entered the introduced colony and bred therewith. In spite of this,
in the changed locations involving considerable climatic differences and the
most probable interbreeding with native stock, the census made in the loca-
tions showed that in the time elapsed and with the combined action of the
slightly changed conditions and the mixing with local conditions, the two
introductions had changed but little if any in the period of the test. I did not
see the Tierra Blanca location again until the winter of 1911-12, when the
adverse conditions had driven two-thirds of the population into the ground for
the dry season ; but of these that remained, or that were recovered from beneath
the debris on the surface, it was evident that a considerable mixing or environ-
mental effect had been produced. This was indicated by the abundant presence
in the population of biotypes 4, 5, and 9, that were only represented by traces
in the previous census and that were wanting in the original location. The
companion test location has not been visited since 1909, owing to the disturbed
political condition of the country.

The conditions observed in the Tierra Blanca test location are certainly not
the product of the external conditions, because conditions in the pattern-system,
represented by biotypes 4, 5, Ga, and 9 are the known product of the pres-
ence or absence in the gametic system of exact agents, and at no point in all of
my experiments or observations is there the least evidence that conditions in the
environment, especially so little different in the two locations, could or had ever
produced these changes in the pattern of any group. It is certain, therefore, that
the change in the condition of the test colony, which was not and could not be
perfectly isolated, had resulted from the incoming of native materials and the
incorporation of the missing agents into the introduced group to produce
the absent biotypes. So likewise other near locations in the region were show-
ing the effects of the introduction by the increased numbers of the biotypes
most characteristic of the Campeche stocks. These crude operations in nature
show the permanence of the condition in the pattern in the different locations,
regardless of the conditions of the new location, provided that the location does
not have conditions that are intense enough to act as agents in directly pro-
ducing germinal changes. In instances of this sort the action of the intro-
duced colony is always different from that seen in this series of tests.

At one time or another, to different degrees, all of the stocks that I have used
in these investigations have been tested in this manner, and in no instance
have I found that when the test was made from the point of view of the consti-
tution of the character did the condition change when the habitat was changed
within reasonable limits, which is a very different ending from the condition
found when the permanency was tested in terms of the statistical value of pig-
mented area present, when the conditions were not permanent in any instance
tested. This is due, of course, to the fact that the area, or the measured bio-
metrical character in general, is not the character, or a character, in the
individual or the race.

In the materials that I have used it was necessary to establish the permanent
nature of the differences that are shown in restricted locations in nature for two

336 The Mechanism of Evolution in Leptinotaesa

reasons: (1) It is all too commonly assumed that natural species and
materials in nature are " pure," by purity being meant uniformity of action
and homogeneity of constitution, which in my experience in using materials that
are wild in nature is not true in any instance thus far found. There is taxo-
nomic purity, or limitation to the range of the characters present, and this is a
real, permanent condition in these natural species, but within these limits the
population shows all degrees of diversity in its composition and in the factors
and agents present, so that for accurate investigation it is necessary that all
materials from nature be reduced to homogeneous conditions before they are
used in the conduct of refined experiments. Because of the many lines of
investigation in different places that these materials have and are being used in,
it Avas necessary that this point be driven home, so that repetition of these
experiments or the orientation of others of similar character must have always
this point in mind, to either employ the same or identical materials in the
operation, or else endless confusion will sujely result. (2) The condition
determined has considerable value in the insight that it gives into the nature
of heterogeneity in nature, of a sort that might well be productive of the initial
stages of wide separation of portions of the original stock, and the origin in
nature of localized groups that might become the basis of " varieties," " spe-
cies," or other taxonomic groupings of whose reality there is not the least doubt,
and of which the problems of their origin are well worth careful experimental
investigation.

In the time following the publication of Darwin's work, the existence of
groups in the population of this sort were frequently postulated and rather
generally believed in, but I think were not accurately demonstrated in any
instance. With the rise of the biometricians the effort to recognize and deter-
mine these groups was taken up anew, and with what hopefulness is fully indi-
cated in the writings of the most eminent of the biometricians — Galton,
Pierson, Davenport, Dunker and others — but the methods and the tests pro-
duced no real progress, save keeping alive the question. The investigations of
De Vries and his postulate of " elementary species " presented the old question
in a new light, and the pure-line conception of Johannsen added but a new
method of analytically isolating some of the possible population conditions upon
which divergence and heterogeneity in nature might be based. I believe that
the investigation of conditions in the population by the combined methods of
biometrics and genetic analysis, as is employed here, may lead directly to the
formation in terms of modern conceptions of hypothesis of the methods of species
formation that are capable of experimental proof. I shall have occasion to
consider these conditions in further experiment and analysis as a basis for the
development of conceptions of species formation in nature, without the aid of
abrupt and cataclysmic happenings or the long-time accumulation of undeter-
minate utilitarian " variations."

In this analysis of heterogeneity diverse aspects of many problems have been
presented and discussed. I have utilized mainly one set of characters, for the
reasons given, and combined in this investigation I used statistical methods, the
tests by breeding, and modern analyses of any sort that would elucidate the
problems considered.

Analysis of Heterogeneity in the Population 337

It is shown that the systems investigated and their component elements, the
simplest characters, are the product of gametic agents in the composition of the
organic system; that they are specific in position, relations, and in result. It
further is evident that the same agent may at different times and in different
systems sustain different relations; hence the diversity of the arrangements
presented between the elements of the pattern-system, an array that in many
respects reminds one of the series presented in many complex inorganic series
where the same members are present, but in differing arrangements. It is
shown that the system, although complex and the product of many interacting
agents, nevertheless acts as a unit in many reactions, passing through the opera-
tions of reproduction and crossing in its entirety, or at other times emerging
from the reactions changed in relations and arrangements of the elemental
simplest characters, indicating altered relations between the conditioning
agents. All of this complexity of arrangement and operation is completely
submerged by any attempt to treat the conditions in the pattern in the usual
quantitative biometric manner.

Thus far in the analysis of the conditions found in a complex array in nature,
I have found out some of the things that were there and some of the agents that
are productive of the heterogeneity in the system investigated. There appears
no evidence for the distinction between quantitative and qualitative that has
been drawn by De Vries and Weismann, and the position and role or even the
existence of the much discussed and abused fluctuation must be questioned and
demonstration demanded, and the causes actually determined in a more certain
manner, before it is accepted as a constantly present feature of the phenomena
of pure lines.

It is clear that it is possible to divide a population into different, homozy-
gous-acting, well-defined biotypic groups that are pragmatic divisions and as
such they are in the arrangement of a system, which remain permanent so long
as the structure of the system is not disarranged. Within some of these
further separations are possible into finer groups, but how many or how far
this could be carried is not shown.

In successive generations in the same place and in different places it is clear
that there is heterogeneity, that it is delimited and shows in the main deter-
minate response to the total set of conditions, internal and external, that the
organism has to meet. It is shown as the result of some of the tests in which
populations were transplanted from the different colonies to like conditions in
nature or in the laboratory, that the gametic composition of the original colony
is a permanent feature of the population at that point. This is important, and
one is made to reflect upon how many possible sets of these local permanent dif-
ferences there may be in a population with a geographic range possessed by
many organisms. In my experience it has been certain that in nature it was
by no means easy to obtain identical materials from different locations, hence
the necessity of the standard locations for the source of the organisms for investi-
gation. My experience with L. mulfitceniata, signaticollis, decemlineata, unde-
cimlineata, and others shows the diversity in the strains from different locations
with regard to minor factorial composition in many characters of all kinds.

Any analysis of the situation, as is made here, only gives the state of hetero-
geneity as it exists at present — some indication of the agents that are operative
in the production of the results — but no proof of how the state discovered was
23

338 The Mechanism of Evolution in Leptinotarsa

produced in nature. It might seem as if the proper arrangement of experi-
ment and investigation in nature would give the proof of how the heterogeneity
arose, but in most of the conditions investigated, even in the simpler ones, the
production of a specific end-result is attained through diverse combinations of
processes.

At Chalcicomula there was a precisely restricted type of pattern-system
which was permanent in the strain. There and elsewhere it never produced
the arrangements of the pattern that were found in thvj population at Chapul-
tepec, especially biotypes 1, 2, and 3. Why did it not produce them? The
answer is easy as to why the production of these pattern conditions was not
found; there were not in the race the requisite agents in its germinal endow-
ment to produce the reactions that are necessary to bring them into existence.
These I could introduce most easily into the race through the medium of cross-
ing. It is probable that some of them might have been produced in other ways
without the crossing, and so the "why" of the difference between this and
other locations is easy to discover. How did the locations become different?
That is another and most difficult question and one that can not, in any instance
that I have had to do with, be answered. At Chalcicomula I can never know
the source, condition of the original population, the events that have taken
place in the colony du,ring its history, any or all of which may be of vital im-
portance in the production of the present condition. From a race taken at
some other location I might produce the same conditions by environmental
agencies, by elimination, by utilitarian selection; but it is no proof that the
conditions in the colony at Chalcicomula arose in the same way and as the
result of the same forces. So long as evolution was regarded as a progressive
series of events, of monophyletic origins, and dichotomizing schemes of phyletic
relationship, there might be some basis for this plausible array of causes and
results having necessary relationship. Even upon this basis there is only the
poorest of plausible suppositions of cause and effect. I have most earnestly,
in this investigation, in numerous instances, made effort to certainly discover
the productive agents of conditions found in nature. A successful termination
would have been of practical aid in other portions of my problem and otherwise
satisfying in being able to determine exactly the antecedent cause of present
conditions in a state of nature. In no instance have I been able to eliminate
some possible agents, and so restrict possibly the probable efficient factors to a
lesser number than at the start, but in no instance thus far have I been able to
attain to the desired end of a proof of the actual cause of the conditions
observed.

It is possible to determine methods of producing different races — the agents
that must enter into the several compositions — and in this it is possible to
arrive at an understanding of how in nature the heterogeneity may have been
produced in these localized habitudinally different races. In plants and in
animals with low migratory capacities instances of the close proximity of
nearly related " species " have been described. Classic examples are the
Achatinellidae in the Hawaiian Islands, described by Gulick, and the species of
Partula discovered by Mayer and recently investigated by Crampton in the
Polenesian Islands. These and other instances that exist of remarkably re-
stricted and localized races in nature have been the basis upon which exten-
sive discussions are based. Gulick has presented the combined results of his

Analysis of Hetekogeneity in the Population 339

investigations in the Hawaiian Islands, with the interpretation of the meaning
and causes of the conditions in the popiU^ation of the Achatinellidae. In no
instance has the cause of the difference between the forms been determined.
In these instances, isolation or separation of some sort is assumed to be an
active and efficient agent in the production of the result. As far as the data
available are concerned, the isolation may as well be the result of the causes that
have produced the heterogeneity in the organisms as that it is the cause thereof.
A further unguarded assumption is that the conditions in the environment are
uniform, and so are not of any moment in the production of the results in the
population. It would have been profitable to have measured accurately even
some of the factors in the climatic complex in different mountain valleys
inhabited by these animals, and determined the identity or diversity therein.
I have been very desirous to find some mountain valleys that are identical in
their climatic complexes, but have not been able thus far to locate them.

In the analysis of heterogeneity the problems center about the determination
of the factorial composition of the character or of the system investigated.
This lies at the foundation of the entire investigation, and without this deter-
mination the investigation of heterogeneity is meaningless and futile. The
problem is for the biologist as for the lithologist, crystallographer, or chemist,
the determination, first of the different internal and external agents that enter
into the production of a character of the simplest kind, then the relation of
these simplest characters in the diverse systems into which they enter. In
organisms, as in inorganic materials, these agents or factors are of diverse kinds,
in no instance bearers of anything, but determinative of ensuing or resultant
reactions and products by their presence or absence in the system at different
periods of its reaction.

Having determined the factorial composition of any character or system,
one is then in position to experimenta,lly determine the laws of heterogeneity
through the influence of external incident agents, by metathesis, by the advent
or loss of factors in the system, and so to discover how heterogeneity in nature
may be produced, to produce it in experiment, and analytically to determine
the factorial differences between groups in nature.

In this the categories of variation must vanish ; fluctuations will have to be
determined as to cause, whether due to states of stability, impurities, misplaced
factors with missing complements or loosely combined in the system and non-
productive of anything but erratic disturbances, or to the action of incident
forces. Continuity and discontinuity become terms that are merely descriptive
of the results of interactions of the factors, and unit-characters become obsolete.
In organisms there are unit-characters no more than in rocks or minerals.

Had we the understanding of the constitution of living substance that we
have of the nonliving, it would be easy to isolate and determine the precise
nature of any or all factors, to take the entire organism apart and reassemble
it in the laboratory. At present no one factor is determined, nor has one been
isolated, in a chemical sense.

We thus come to regard the heterogeneity of organisms in a new light, phys-
ical in expression, dynamic in action, nonvitalistic in conception. The
methods of investigation change from plausible arrangements to analytical
determinations of factorial constitution, action, and result, and in experi-
ment, as in nature, the responses in heterogeneity, whether definite or indefinite.

340 The Mechanism of Evolution in Leptinotaksa

delimited or undelimited, attain increased significance and open up possibili-
ties such that heterogeneity in nature and the origin of natural groups may
become a matter of direct experimentation. No doubt methods of their pro-
duction will be discovered that are capable of being duplicated at any time or
place, and no longer will species be the plausible product of adaptations, selec-
tions, and survivals of the fittest that have adorned the literature of evolution
in the last half century.

The geographic phases of heterogeneity must assume different aspects and
the problems become more directly open to analytical investigation, with regard
to both internal and external agents.

For me at least the phenomena of heterogeneity in nature take on a different
meaning, than when viewed from the one-time aspect of " variation," its cate-
gories, definitions, and kinds, utile and non-utile, introducing into the subject
and its conceptions elements of causation impossible of interpretation from a
physical viewpoint. The conceptions applied in the analysis of heterogeneity
in this investigation offer opportunity for unlimited analytical investigation,
and in place of biometrics and its plausibilities there are possible determina-
tions of the actual productive agents, their testing, rearrangement into diverse
combinations, and the entire removal of the essential factors in heterogeneity
and evolution from the situations into which Weismann and others would
place them, in inaccessible bearers of characters, operating in an inacessible
world of their own. Hope for progress in the future lies only in analytical
experiment, and at present the factorial conception of the constitution and
evolution of organisms provides the only purely physical, working hypothesis.

■^ a

o

■ H

APPENDIX

THE RELATION OF WATER TO THE

BEHAVIOR OF THE POTATO

BEETLE IN A DESERT

BY i

J. K. BREITENBECHER

OF THE BIOLOGICAL LABORATORY OF WESTERN RESERVE UNIVERSITY

CONTENTS.

PAGE

Introduction 343

Instrumentation and conditions of experiment 343

Materials 344

Role of water in reproductive activity 346

Experiments with soil moisture 348

Experiments with evaporation rates 349

Role of water in the preservation of life 350

Relation of water-loss in insects when exposed to changes in the relative
humidity of the surrounding medium, and its effect on the activities

of such organisms 354

Experiments upon evaporation, transpiration, and behavior 355

Role of water in hibernation 366

Entrance into hibernation 366

Activities during hibernation 371

Water relation of soils and hibernating beetles 371

Emergence from hibernation 372

Summary and discussion upon the relation of water to hibernation 373

Effect of changes in water content upon alterations in tropic activities 375

Experiments upon the role of water in geotropism 376

Relation of temperature to outgo and intake of water 377

Metabolism and water relation 378

General discussion upon the role of water in living things 379

Summary and conclusion 381

Bibliography 382

THE RELATION OF WATER TO THE BEHAVIOR OF
THE POTATO BEETLE IN A DESERT.

INTRODUCTION.

In a series of experiments maintained by Professor Tower to determine the
action of the Tucson Desert upon evolutionary processes in ehrysomelid beetles,
it was observed that soil-moisture, humidity, and the like played an important
role in modifying the activities of these organisms when introduced into the
arid region ; so, as a result of these observations, the author undertook a series
of investigations to discover any possible connection between this water-relation
and the reactions of the potato beetle, Leptinotarsa decemlineata (Say), when
transplanted into the desert from a temperate habitat.

A large stock of this species was sent to Tucson from Chicago in June 1911,
so that comparative studies under different environmental complexes could be
made. Three cultures were established in several open-air breeding-cages at the
stations already equipped for Professor Tower at Tucson and Chicago. Two of
these stations, which were arid in character, are designated as Tucson Station A
and Tucson Station B, while the third one, known as the Chicago Station, was
temperate and located at the University of Chicago. The former of the two
desert stations was situated at the base of the northern slope of Tumamoc Hill,
just within the flood-plain of the Santa Cruz River, at an altitude of 2,370 feet,
while the latter was located on the shoulder of this hill, on which the Desert
Laboratory is situated, at an elevation of 3,705 feet. The biological significance
of the conditions at these stations, as indicated, is given elsewhere, and the
problems dealt with concern the relations which exist between the activities of
the beetles when allowed to reproduce at these localities and the changes pro-
duced in the water-content of the animals through the action of the various
environmental factors.

INSTRUMENTATION AND CONDITIONS OF EXPERIMENT.

At each station the evaporation rates were obtained by the Livingston at-
mometers, and Friez self-recording thermographs were employed to measure
the temperatures, air and soil, and the same maker's hygrographs were also
used for the relative humidities ; these instruments were calibrated and stand-
ardized fortnightly. The rainfall-readings were obtained from a standard
weather bureau rain-gage at the Laboratory site. It is interesting to notice that
the environmental data as recorded from these experiments showed for the arid
complex that the greatest daily fluctuations occurred at Station A and the
highest evaporation at Station B, while the lowest evaporation-rates and air-
temperatures were at Station C. The Tucson region as a whole, when contrasted
with the Chicago conditions, has a higher rate of evaporation, a lower relative

343

344

Eelation of Water to the Behavior of

humidity, a stronger light intensity, and a wider daily fluctuation in both air
and soil temperatures ; excessive nocturnal radiations and convectional currents
were also potent factors in the desert.

Four seasons were apparent in the arid region : A winter rainy season extend-
ing from November until April ; a dry fore-summer season, from April until
July ; a midsummer rainy season, from July until the middle of September ; and
a dry after-summer season, from the middle of September until early in ISTovem-
ber. At Chicago rain occurred throughout the year. The annual rainfall from
several years' data was about 13 inches at Tucson and 30 at Chicago. The
monthly records for both stations are given in Table 1 for the three years during
which these experiments were in progress.

Table 1.

station.

Tucson .

Chicago.

Year. Jan.

191011.00
191l!l.31
1912.0.00

19103.07
1911 1.17
19120.84

Feb.

T.
0.90
0.37

0.89
2.27
1.57

Mch.

Apr. May. I June.

T. 0.05; T. jo. 22
0.25,0.27 0.00 0.07
2.120.280.320.61

0.29 3.84 4.67 0.91
1.45 3.03 3.37 2.54
2.202.55 3.97 1.78

July.

4.20
1.57
3.00

1.79
2.65
3.86

Aug.

4.65
2.06
0.98

3.08
3.72
3.59

Sept.

0.62
2.65
0.01

3.90
4.03
3,26

Oct.

0.08
1.23
1.78

1.79
3.79
3.52

Nov.

1.74

T.

0.00

1.31
3.27
1.45

Dec.

0.06
0.85
0.39

1.32
2.54
1.08

Total.

12.62

11.25

9.84

26.86
33.83
29.67

Note— The rainfall records at Tucson were obtained from a standard Weather
Bureau rain-gage at the Laboratory site. The Chicago data were taken from the
records of the Weather Bureau.

MATERIALS.

The potato beetles were collected in May 1911, near Chicago, as they emerged
from hibernation, and were allowed to breed there as a group-culture within a
large cage filled with potato plants until late in June. A part of this material
was then sent to Tucson, where it became the progenitor of the animals used
in the majority of the experiments. Organisms when collected from nature are
often hybrids, so that crossing of different generations may have taken place, but
for complete breeding-records and life-histories of stocks used see Table 2, which
shows that these materials reacted homozygously. A brief description of their
activities follows.

At Tucson Station A these stocks were received on June 26, 1911, and
immediately bred as a group-culture, so that 1,328 adults were produced in 25
days, giving generation I. After feeding upon the potato plants for a few days,
these first-generation individuals were bred as a group-culture and produced
generation II, numbering 2,312 progeny, in 26 days. Many of the beetles of this
second generation provided the materials for a large number of the hibernation
experiments Avhich were carried on in 1911, but many of the emerged animals
were allowed to hibernate during the winter of 1911-12 as stock for work during
the following year. When, in June, water was added to the soil within the cage,
the organisms emerged from hibernation as a group-culture, which in 29 days
gave generation III, of 1,743 offspring. From this material 50 females and
50 males were mated and allowed to breed at random, giving generation IV, of
4,049 progeny, in 26 days. For the above data, see Table 2.

The Potato Beetle in a Desert

345

At Tucson Station B the parent group of 104 adults for this culture was
received on July 15 and bred as a group-culture, producing 204 offspring in
25 days, thus giving generation I. As soon as the adults from the first genera-
tion appeared they were removed to another breeding-cage, but they immediately
burrowed into the ground within the experimental cages and hibernated there
until September, when they began breeding and in 21 days produced generation
II, of 293 progeny. A few days after emerging from pupation all of the beetles
went into hibernation without feeding, where they remained until the following
summer, when, on May 31, 7 males and 16 females emerged and bred immedi-
ately, giving generation III of 283 adults in 31 days. These were allowed to
reproduce as a group-culture, giving in 31 days generation IV of 127 offspring.

Table 2.

Year, station, and

Duration of

Period of

First

Second

Third

generation.

breeding.

oviposition.

stage larv.R.

stage larvx.

stage larva.

1911, Tucson A, g. I

June 26- July 17

July 7-19

July 12-21

July 13-27

July 17-31

1911, Tucson A, g. II

Aug. 1-18

Aug. 4-22

Aug. 10-26

Aug. 16-31

Aug. 22-Sept. 1

1912, Tucson A, g. Ill ..

Autumn of 1911

June 4-15

June 12-21

June 15-28

June 13-30

1912, Tucson A, g. IV...

July 8-29

July 15-29

July 17-Aug. 2

July 20-Aug. 5

July 25-Aug. 8

1911, Tucson B, g. I

July 15-23

July 17-23

July 21-29

July 23-Aug. 2

July 27-Aug. 6

1911, Tucson B, g. II....

Sept. 3-7

Sept. 3-7

Sept. 5-n

Sept. 7-19

Sept. 11-21....

1912, Tucson B. g. III...

May31-June2n

June 5-20

June 14-25

June 16-30

June20-Julyl0

1912, Tucson B. g. IV...

July 10-Aug. 12

July24-Aug.l2

July27-Aug.l4

Aug. 4-27

Aug. 12-29

Aug. 1-15

June 2-19

Aug. 1-15

Year, station, and

Pupa

Emerged as

No. of

Adults

Duration of

generation'.

state.

adults.

adults.

hibernated.

life cycle.

1911, Tucson A, g. I

1911, Tucson A, g II

July 19-Aug. 1
Aug. 26-Sept. 2

July31-Aug.l4
Sept. 2-13

1328

25-26 days.
22-29 days.

2312

Over winter. ..

1912, Tucson A, g. III...
1912, Tucson' A. g. IV...
1911, Tucson B, g. I

June21-July 8
July29-Aug. 11
July 29-Aug. 8

July 4-16

Aug. 8-26

Aug. 10-18

1743

28-30 days.

4049

Oct. 1

24-28 days.

204

Aug. IS-Sept. 3

25-26 days.

1911, Tucson B, g. II

Sept. 12-22....

Sept. 19-29....

298

Sept. 30

20-22 days.

1912. Tucson B, g. III...
1912, Tucson B, g. IV...

June25-July 15
Aug. 14-31

July 10 23 ...

283

33-35 days.

Sept. 1-7

127

Sept. 2-8

26-36 days.

July 1-20

None

25-35 days.
25-80 days
23-38 days.
34-36 days.

Se^t. 1-10

Sept. 1-10

None

July 10-12

Sept. 4-20

Sept. 20-22....

Early in September all were in hibernation and remained in the ground during
the winter. On the other hand, at the Chicago Station, the original wild parents
were allowed to breed as a group-culture, and gave generation I in 30 days.
These adults bred as a group-culture and produced generation II in 33 days,
which now hibernated during the winter from September until May, when they
reproduced and gave generation IV in 35 days : for the above data, see Table 2.
The animals for experiment were reared upon potato plants in cages of uni-
form size (6 by 6 by 6 feet), with sides of wire-netting, 16 meshes to the inch.
These cages were furnished with wooden bottoms (6 by 6 by 3 feet), which were
filled with a mixture of equal parts of adobe ^ and sand. This mixture proved to

^ At Station A, where this soil was obtained, the adobe consisted of a clay loam,
which constituted the soil-mass of the river flood-plain. This soil was about 8 to 9
meters deep and rested on sand and gravel. Livingston (1910) found it to have a
water-holding power of about 18 per cent of its dry weight. The sand used in all
experiments was obtained from an arroyo near at hand and had a water-holding
power of about 39 per cent of its dry weight.

346 Relation" of Watee to the Behavior of

furnish favorable conditions for plant growth as well as pupation and hiberna-
tion activities.

The condition of the experiment required that a certain routine be repeated
each time, and some of the most ordinary methods follow. The rates of evapora-
tion were obtained with the Livingston atmometers, which were cut down to a
cone of 50 mm. in length to avoid an error introduced by having shellacked
bases. These were standardized on the rotating machine in the Genetics Labora-
tory at Chicago, and showed after standardization a maximum range of 3 per
cent from the normal. The dry weights of the insects were obtained as follows :
first by killing them in potassium cyanide, and desiccating them at a constant
temperature in a vacuum over concentrated sulphuric acid until the dry weights
became approximately constant. The soil samples when collected were placed
in glass-stoppered weighing-bottles and carried to the laboratory, where they
were weighed and dried at a constant temperature of 100° C. The tropic reac-
tions were tested in the constant-temperature room (18° to 20° C.) and the
beetles were exposed in wire-netting tubes (30 cm. long and 5 cm. in diameter).
All geotropic reactions were tested in the dark, and if the animals crawled to
the top of the tube when held in a vertical position they were considered positive,
and if they moved to the bottom of the tube, negative. The phototropic reac-
tions were tested with an ordinary 33 c. p. electric lamp in a constant-tempera-
ture room. If the organisms crawled toward the direct rays of light when the
tube was in a horizontal plane they were recorded as positive and if they moved
away from the source of light as negative.

ROLE OF WATER IN THE REPRODUCTIVE ACTIVITY.

A striking fact that one observes in desert biology is that a remarkable degree
of coincidence is shown between the rainy season and the reproductive period
of the animals native to such a region. Corresponding periods of inactivity for
such organisms occur during the dry season. In studying this problem. Tower
(1906) finds this is true for most of the species of Leptinotarsa distributed
over the American deserts and similar observations were made by Semper
(1881) for several desert forms.

For many years Tower has introduced chrysomelid beetles into desert com-
plexes of Arizona from a wide range of habitats, and the majority of these
experiments, which were placed under my care, showed that food, enemies, and
the like were not the determining factors in the survival of such organisms, but
that in most instances the survival was successful if the proper complex for the
reproductive activity was attained.

Frequent observations at Tucson indicated that the optimum breeding
activity of the potato beetle was coincident with the highest water-content of the
medium, since periods of egg-laying were exactly concurrent with those of rain
and with the low rates of evaporation. Therefore, it was important to determine
experimentally what relations existed between reproductive behavior and changes
of water-content within the medium surrounding these animals.

The literature of the subject contains much data in regard to the effects of
temperature upon the reproductive activity, but almost none upon the relation
of water to reproduction. In reference to the genus Leptinotarsa, Tower ( 1906)
states :

The Potato Beetle iit a Deseet 347

" In both tropical and temperate latitudes, the germ-cells do not develop nor
reproduction take place until the conditions of temperature and moisture are

favorable Likewise, in the northern United States and Canada, decemli-

neata may emerge from the ground in April, but the germ-cells do not begin to
grow until the coming of the warm moist days in May or possibly June."

Kammer's (1907) experiments show that Salamandra {maculosa and atra)
can be induced in varying ways to lay their eggs, depending in part upon the
moisture relation. Jacobs (1909) states for the rotifer Philodina rosela:

" The period of maximum egg-production had been preceded by a period of
desiccation and furthermore, that each desiccation for any length of time has
been followed by an increase in the reproductive activity."

Hennings (1907), working on the bark beetle Tomicus typographus Linn.,
finds that the amount of water present acted as a regulatory factor for such
activities. The results of these investigators show that egg-production may be
modified through changed water-relations.

At Tucson Station A, comparative study of the environmental records indi-
cated that when the atmosphere had a high water-content, and when the
evaporation rates were low, then egg-laying took place most frequently. This
was self-evident, for during the breeding of four generations of the stock at this
locality egg-laying occurred during the following periods : July 7 to 19, August
4 to 22, 1911 ; June 4 to 15, and July 15 to 29, 1912, which were exactly coinci-
dent with the maximum rainy periods at this station. At Tucson Station B the
results were similar to those of Station A, since the periods of oviposition were
as follows : July 17 to 23, September 3 to 7, 1911 ; June 8 to 20 and July 24 to
August 12, 1912, which coincided with high humidities and low rates of
evaporation. At the Chicago station, however, the evaporation rates were lower
and the egg-laying was controlled by other factors. For the desert complex
these results indicated that the optimum for egg-laying was reached when the
organism was subjected to a moist medium.

Since these conclusions were only probable, it was advisable that they be sub-
stantiated by further experiment. Therefore, it was necessary to produce
artificial differences in the moisture-content of the medium, and observe its
effect upon beetles during hibernation and after emergence.

For the purpose of these experiments, 120 beetles (Tucson A, g. II) were
removed from hibernation at 8 p. m. June 19 and were placed in ground-glass
stoppered weighing-bottles, so that no moisture could enter. They were removed
immediately to the constant-temperature room of the Desert Laboratory, where
reactions were tested and all responded positively to light and negatively to
gravity. At the same time a sample of the soil surrounding the beetles was
taken and found to contain 11.3 per cent of water by weight. The animals were
now divided into two lots of 60 adults each (30 of each sex), which were found
to weigh 10.007 grams and 9.845 grams, respectively. The first batch was sub-
jected to differences in soil-moisture and its effect upon egg-production observed.
In the second case the effect of changing evaporation-rates upon oviposition was
also observed.

348

Relation of Water to the Behavior of

EXPERIMENTS WITH SOIL MOISTURE.

It was important to determine the effect of a varying soil-moisture upon
oviposition in these organisms during hibernation. Tubes used in experiments
upon this subject were 30 cm. long and 15 cm. in diameter, and made of wire
netting surrounded by several layers of tinfoil to prevent the egress and ingress
of moisture. Three of these tubes were filled with a mixture consisting of equal
parts of sand and adobe, and then sunk in a large box of sand, so that only the
tops were exposed. The sand in the box was kept damp by means of self-
watering automatic soil-cups, which were devised by Hawkins (1910), the
purpose of this wet sand being to keep the organisms within the tubes at a
uniform temperature, a result attained through rapid evaporation of water-
vapor from the surface of the soil.

The desired differences in soil-moisture were produced in the above tubes in
the following manner: In one tube were placed two porous soil-cups, which
gave the soil a high water-content ; in the second one, however, a small porous
soil-cup was placed, which kept the soil -within at a lower moisture than in the
former; and in the third, no soil-cup was employed, thus keeping the soil dry.
It was thus possible to obtain differences in soil moisture with other conditions
approximately uniform. But to make certain that the above apparatus produced
the desired results, determinations of soil-moisture within these tubes were
made every second day throughout the experiment. These data are tabulated
in Table 3, which shows that the moist soil contained 15.8 per cent moisture,
the medium moist soil 8.8 per cent moisture, and the dry soil 1.9 per cent
moisture. Thermometers were placed in these tubes at a depth of 15 cm. and
readings were made at 5 and 9 a. m. and at 1, 5, and 9 p. m. When tabulated,
these soil temperatures throughout the test indicated a close agreement for all
experimental tubes.

Table 3.

Soil samples obtained.

Tube 1. Moist soil.

Tube 2. Medium
moist soil.

Tube 3. Dry soil.

.Tiinp 21

Per cent.
15.9
15.6
16.0
15.8

Per cent.
8.9
9.2
8.4
8.7

Per cent.
1.7
2.1
1.8
2.0

Tiinp 2.^

.Tune 25

June 27

Average

15.8

8.8

1.9

The 60 beetles of batch 1 were now divided into three groups, and when
tested in the constant-temperature chamber were found to react positively to
light and negatively to gravity. Each group was now weighed, group A weigh-
ing 3.3-41 grams, group B, 3.329 grams, and group C, 3.337 grams, respectively.
Each group was next buried on June 19 at a depth of 15 cm. in each of the three
experimental tubes as indicated, members of A being buried in a moist tube,
those of B in one less moist, and those of group C in a dry tube. These animals
remained as buried until June 27, 6 p. m., when their weights were again tested
in the constant-temperature room, and group A was found to weigh 3.213 grams,
group B 2.962 grams, and group C 2.107 grams, respectively.

The Potato Beetle in a Desert 349

Thus it was discovered that the beetles of group A from the moist soil showed
a loss of only 0.128 gram, while their reactions were, as before, positive to light
and negative to gravity. The beetles of group B, however, from the medium
moist soil, showed a much greater loss in weight (0.367 gram), although their
responses were unchanged, except in the case of 3 which were positive to gravity.
The beetles of group C from the dry soil indicated the greatest decrease in
weight (1.123 grams), and showed a reversal in their behavior.

After this test the beetles were put into separate cages out-of-doors and
allowed to breed under natural conditions. A comparison of the rates of
evaporation, obtained with Livingston atmometers when placed within these
cages, indicated that the environment was uniform. When the activities of
these insects were closely observed, the following results were obtained : Those
beetles from the wet soil, whose reactions as previously tested, were still positive
to light and negative to gravity, moved immediately upward on the potato
plants, and began feeding on the uppermost leaves. This indicated that their
activities were normal, and on June 30 eggs were laid. On the other hand,
those animals from the medium-wet soil also fed on these plants, but no eggs
were laid until July 4. This showed that oviposition was postponed 4 days,
but that the dry-soil beetles, whose responses were now reversed, were negative
to light and positive to gravity. They immediately burrowed into the ground
and remained there until the arrival of the summer rains, July 13, when they
emerged and laid eggs on July 15. In this case oviposition was delayed 15 days.
An analysis of these results follows :

These experiments showed that differences in soil-moisture produced changes
in the water-content of these animals as well as modified their behavior. Since
the egg-production was changed, we must conclude that beetles emerging from
soils of high moisture-content lay their eggs sooner than those issuing from drv
soils. The soil no doubt has played an important role in the economy of desert
organisms, which are known to respond accurately to environmental changes
such as we have described; otherwise many forms would have ceased to exist
where they are now widely distributed in desert regions.

EXPERIMENTS WITH EVAPORATION RATES.

The second batch of beetles was used to determine the effect of differences
in rates of evaporation upon insects just emerged from hibernation. The
apparatus for this experiment consisted of three uniform bell-jars placed over
pots of potato plants. Each pot was sunk into adobe soil in the bottom of a
vivarium, which was an open inclosure covered with wire netting. One of the
bell-jars was provided with 8 atmometers, which, through the evaporation of
water-vapor from their surfaces, produced both a high relative humidity and a
low rate of evaporation. The second jar was furnished with 2 atmometers, and
the evaporation-rate was greater in this jar than in the first ; but the third was
kept dry, for, since no water or atmometer whatsoever was used, a high rate of
evaporation was secured. The food-plants in this test were kept in a normal
healthy condition by the use of automatic soil-watering cups placed in the
earth near the bottom of the pots. A few preliminary experiments demon-
strated that the dry bell- jar in direct sunshine would become several degrees
warmer than the more moist ; consequently a shade was so placed as to give

350 Eelation of Water to the Behavior of

closer agreement in temperature readings. A small strip of wire-netting was
then fastened around the base of each bell-jar to afford a free circulation of air.

The environmental conditions produced artificially within each bell- jar were
measured in the following manner : Thermometers were suspended in each jar
and temperature readings were taken five times daily at 5 and 9 a. m., 1, 5, and
9 p. m. ; they show a close agreement of the three jars. The evaporation-rate
was obtained by means of a single atmometer placed in each jar, and the cubic
centimeters evaporated by this instrument were recorded twice each day at 8 a. m.
and 8 p. m. In averaging the different rates, it was found that the moist jar
showed an evaporation rate of 14.5 c. c. daily, the medium moist 20.7 c. c, and
the dry 25.0 c. c, respectively. The results show that the air temperatures
within each jar were approximately uniform during progress of the experiment,
and furthermore that the anticipated differences in the rates of evaporation were
produced in this manner. Thus the environmental conditions for this test were
experimentally attained.

Sixty beetles of batch 2 were now divided into three groups of 20 each, and
on June 20 were distributed to the jars mentioned above. In the jar with a low
rate of evaporation the beetles reacted normally in feeding upon the potato
plants, and laid eggs on the third day, June 23, but in the jar with the medium
evaporation-rate, the animals, though resting upon the plant leaves, laid no
eggs for 9 days, or until June 29. In the third jar, with a high rate of evapora-
tion, which produced the greatest degree of desiccation, they stayed upon the
potato vines for 5 days or until June 25, when they entered the ground and
there remained until the summer rains began July 11 ; they emerged, however,
from the soil on July 13, and oviposition occurred within 3 days.

These results showed that egg-production was modified by differences in the
evaporating power of the air surrounding these animals, and that a low rate of
evaporation, coincident with a high water-content, encouraged oviposition, but
that a high rate of evaporation, which reduced their water-content, retarded
reproduction. So with a high rate of evaporation the beetles were desiccated ;
their tropisms were reversed and they entered the soil, where they remained
until their moisture-content was sufficiently increased; they absorbed water
until their reactions became normal, when emergence resulted.

It was shown clearly that reproductive activity occurred during a period of
high water-content in the surrounding medium, whether atmosphere or soil,
and that desiccation, by reversing the animal's tropisms, inhibited and post-
poned reproductive reactions of the L. decemlineata, which is a typical grassland
organism. This demonstrated that the introduced stock had adjusted itself to
the complex of desert conditions and reacted in the same manner as did the
indigenous organisms of the surrounding region. Accordingly, in the majority
of species, we should expect reproduction to coincide with the season of high
water-content and a dormant period to follow the dry season, regardless of any
discovered structural adaptations.

ROLE OF WATER IN THE PRESERVATION OF LIFE.

In the desert it was found that hibernating insects could continue life during
long dry seasons of one or more years ; therefore, experiments were undertaken
to answer the following questions : How was such vitality preserved ? Was this

The Potato Beetle in a Desert 351

relation due to a reduction of normal physiological activities through desicca-
tion? If so, what relation exists between such organisms and changes in the
physical composition and especially the moisture-content of the medium sur-
rounding them ? For these tests, animals of different physiological activities,
induced by differences in humidity, were buried for certain periods of time in
soils of varying degrees of moisture and texture. The criteria used in deter-
mining the power of resistance were the number of individuals surviving in
the test at the end of a given period of time, and the differences in activities of
the insects produced through desiccation, as compared to a similar set in which
the behavior was normal. The capacity of these animals to resist was tested,
and beetles were placed in wire-netting tubes (50 cm. long by 10 cm. in diam-
eter) which permitted a free circulation of air and moisture. The tubes con-
taining the insects were buried upright, so that the base of each was 60 cm. deep
in the soil. The plots were 10 meters apart, in the open, at Tucson Station A.
Earth was removed from each plot so as to leave two holes 6 meters square by
1 meter deep, and each cavity was further divided into equal parts by a partition
of red- wood boards 1 inch thick ; one side was filled in with sand, the other with
adobe. One of these plots was exposed under natural conditions in the open,
while the other was covered with a roof, which extended on each side 1 meter
beyond the limits of the plot. This roof was raised 1.5 meters above the ground
in order to give a free circulation of air and to keep the soil dry underneath.
Water from rains was collected in ditches which conveyed it beyond the plot.
The plot in the open was designated Plot A, and that under roof as Plot B.
The soils in both plots were kept moist by adding water by means of a garden
hose until October 20, but after this date they were exposed to the conditions
of the winter of 1911-12 at Tucson.

The first set of experiments concerned only beetles of the summer generation
that were emerging from the pupa state. Such insects do not normally hiber-
nate, but may be caused to do so through adverse conditions such as those which
cause desiccation. It should be noticed that in one instance the animals were
buried with all activities normal; in the other, they were first induced to
hibernate in cages in the vivarium, and they were sifted out in the soil; in
either case they were finally buried under the conditions described above.

In the former test in which the beetles were buried with all their activities
normal, 400 emerging individuals (Tucson A, g. I) were collected on August
12 ; 50 of these were then placed into each of the 8 wire-netting tubes ; 4 of these
tubes were filled with sand and 4 with adobe soil; the insects were placed at
corresponding levels in each of the 8 tubes ; 2 of these containing sand were
buried in the open plot and 2 under the shelter, while the 4 tubes with adobe soil
were sunk in the adobe sections of the plots, 2 in each. These were left unmo-
lested until May 1, when one tube under each set of conditions was examined
but no living beetles were found. On October 1, the remaining 4 tubes were
inspected with the same results ; there were no living organisms found. These
results showed that no beetles of the summer generation with their activities
normal hibernated successfully when buried under the conditions of this experi-
ment. These observations are tabulated in Table 4.

352

Eelation" of Water to the Behavior of

In the latter test, however, the insects were first induced to hibernate in
cages in the vivarium, and were then immediately sifted out of the soil, to be
finally buried in Plots A and B. The following methods were used in this test :
During the period of August 13 to 20, emerging adults to the number of 1,613
( Tucson A, g. I ) were collected and placed in pedigree cages under adverse con-
ditions within the vivarium. These conditions were produced by having their
food reduced to sliced potato tubers and by adding just enough water to keep
the soil slightly moist, so that the beetles were partially desiccated. A census
taken September 10 showed that 1,209 insects had successfully hibernated in

Table 4. — Census of counts on covered plot and open plot.

Conditions of the experiment.

Covered plot.

Open

plot.

Adobe.

Sand.

Adobe.

Sand.

>

T3

>•

■a

oJ

•a

6

>

•6

01

<

Q

<

Q

<

Q

<

o

A. Beetles of summer generation:

1. With activities normal when buried

on August 12. On May 1 the fol-

lowing count was made

0
0

50
50

0
0

50
50

0
0

50
50

0
0

50

50

Another census on Oct. 1 showed.. .

2. Others were desiccated and buried on

Sept. 10. On May 1 the following

count was made

44
12

6

38

0
0

50
50

46
0

4
50

4
0

46
50

Another census on Oct. 1 showed.. .

B. Beetles of winter generation:

1. With activities normal when buried

on Sept. 10. On May 1 the fol-

lowing count was made

0

50

0

50

0

50

0

50

Another census on Oct. 1 showed.. .

0

50

0

50

0

50

0

50

2. Others were desiccated and buried on

Oct. 2. On May 1 the following

count was made

48
39

2
11

0
0

50
50

45
0

5
50

26
0

24
50

Another census on Oct. 1 showed.. .

3. Others were hibernated and buried on

Oct. 7. On May 1 the following

count was made

49

1

4

46

47

3

39

11

Another census on Oct. 1 showed.. .

46

4

0

50

0

50

0

50

these cages, and from this result, it was discovered that their vitality was greatly
diminished, for 67 per cent of them were killed by these conditions. Of the
survivors, 400 were divided into 8 groups of 50 individuals each, which were
placed in tubes buried beside the 8 which were described in the former test. All
were left unmolested during the winter and until May 1, when 4 tubes from
each plot were examined. Tubes from the covered plot showed that those in
sand contained no life ; 2 from adobe contained 54 living beetles ; while 4 were
alive in the sand from the covered plot; the adobe portion of this harbored 46
living insects. On October 1 the remaining 4 tubes were also examined ; those
in adobe soil under shelter contained 12 living beetles, but all were dead in the
sand and there were no living animals in either part of the open plot. These
experiments proved that beetles of the summer generation, which normally

The Potato Beetle in a Deseet 353

breed and produce a hibernating winter generation, may be buried after having
been induced to enter the ground through desiccation; furthermore, that the
animals lived many months when buried under these conditions, but that the
death-rate was greater in the sand, and the majority in the open plot succumbed.
These data are also given in Table 4.

On the other hand, the second set of experiments concerned beetles of the
winter generation which had just emerged from the pupa state. This problem
was considered from three aspects : (1) Some animals were buried with all their
activities normal; (2) others were induced to hibernate through partial desicca-
tion produced through adverse conditions, and were then buried; (3) many
were allowed to hibernate normally before they were finally buried in the two
plots.

For the first test in which the animals were buried with all their activities
normal, 400 emerging adults (Tucson A, g. II) were collected on September 10 ;
they were placed within the sand and adobe portions of the two plots. During
the winter, and until May 1, they were left unmolested, when tubes from each
plot were examined and no living individuals were discovered. On October 1
the remaining tubes were exhumed, but no live animals were found. These
results showed that, when beetles of the winter generation, which normally
hibernate, were buried with all their activities normal, hibernation was unsuc-
cessful and all the animals succumbed (Table 4).

In the second test, where beetles were induced to hibernate through desicca-
tion, 1,000 emerging adults (Tucson A, g. II) were collected on September 11,
and were placed under adverse conditions in pedigree cages in the vivarium,
where their food was sliced potato, as in a previous test. On October 2, hiber-
nating adults to the number of 692 were sifted from the soil, and 308 dead ones
were gathered from its surface ; 400 of the living insects were then divided into
8 groups, and were buried within the soils of the two plots, in order to afford
the opportunity of winter hibernation. On May 1 these tubes were examined
for living beetles ; the tube from sand under the covered plot contained no living
adults, while in the one from adobe earth were found 48 living adults ; also those
in sand from the open plot contained only 26 living individuals, while 45 beetles
were removed from the tubes in adobe. In a similar manner, on October 1, the
remaining 4 tubes were removed. In the adobe soil tube, under the shelter,
were found 39 animals, but no individuals hibernated successfully in sand;
moreover, tubes from the open plot harbored no life. The results indicate that
induced hibernation was effected in the winter generation through desiccation,
which increased the resistance of these animals by decreasing their normal
activities. The only insects alive at the end of the experiment were found in
adobe under the covered plot, which proved that potato beetles, when hiber-
nating in adobe, possessed a greater resistance to desiccation than when buried
in sand (Table 4) .

In the last test, insects were permitted to hibernate normally, then buried in
Plots A and B. This was a control for former tests, since it showed that no
error was introduced through handling or digging up the animals. For this
test, 1,000 emerging adults (Tucson A, g. II) were collected on September 13;
they were placed in a large out-of-door cage that was provided with potato
plants, and other environmental conditions were apparently normal. After
consuming much food, these animals were in hibernation by October 7 ; then,
24

354 Eelation- of Water to the Behavior of

400 of these were sifted from the soil and buried in the adobe and sand portions
of the two plots, after having been placed in tubes as in previous tests. On
May 1, when 4 tubes from these plots were examined, it was found that the tube
from a'dobe in the covered shelter showed 49 living beetles, while the one from
sand contained only 4 ; on the other hand, the tube from the adobe portion of
the open plot was found to have 47 living insects, and those from sand 39. On
October 1, when the remaining 4 tubes were removed, those in adobe from the
sheltered plot contained 46 live animals, but those in the sand none ; those from
the open plot contained no individuals which had hibernated successfully. This
natural type exhibited the greatest resistance because of the large number of
survivals, and it also appeared that adobe was more favorable to the main-
tenance of life than was sand.

In Table 4 the results are briefly indicated; it is shown there that insects
with all activities normal die when buried, for no beetles were found under any
of these conditions. It appears also that either the summer or winter genera-
tion may be buried and still live, providing the animals were desiccated previous
to burial. It is also shown that a covered plot with adobe soil is a most favor-
able condition for the preservation of life. It is also demonstrated that insects
can be desiccated at any time, when they will burrow into the ground, and may
remain there many months without apparent injury. These tests further show
that the large pores in sand permitted too rapid drying, so that the animals were
desiccated beyond recovery. Livingston (1910) shows that this adobe soil has
a water-holding power twice as great as sand, which agrees with the above results
and explains why these insects continued to live. Lastly, since the adobe soil in
an arid region does contain such a high percentage of moisture, it therefore
is the best medium for the sustentation of desert life.

RELATION OF WATER LOSS IN INSECTS WHEN EXPOSED TO
CHANGES IN THE RELATIVE HUMIDITY OF THE SUR-
ROUNDING MEDIUM AND ITS EFFECT ON THE ACTIVITIES
OF SUCH ORGANISMS.

The relation of water-loss, i. e., transpiration and respiration from exposed
surfaces, to the behavior of plants and animals has already received some atten-
tion. This is especially true of plants, the water-relations of which have been
studied by Livingston (1906), Lloyd (1912), MacDougal (1913), Eenner
(1910, 1911), and other plant physiologists. The results of Livingston (1906)
are of interest in this connection, since they show that there is a close relation
between the daily march of evaporation, as measured by the atmometer, and
transpiration in plants. The following experiments upon insects show that
these animals exhibit a physiological behavior not unlike that of transpiration
in plants, but the results further show that tropisms of insects are modified by
loss of water, which in turn is governed by the evaporating power of the air.
There is wide literature upon perspiration, but it does not bear directly upon
our problem ; accordingly we shall consider such researches as have been made
upon transpiration and evaporation and the efficiency of these processes in
modifying behavior.

The results which follow upon the behavior of insects and other desert animals
and upon the relation of evaporation to their behavior and life economy was

The Potato Beetle in a Desert 355

reported, by the present writer (1911, 1912), and these results have been
substantiated by Shelford (1914 a, I) and his students, Weese (1917), Hamil-
ton (1917), and Chenoweth (1917). The writer's experiments upon the potato
beetle and other desert animals (1911) showed that "the fundamental activi-
ties of this beetle, as well as those of many desert organisms, are directly con-
ditioned by their water-content or water-balance. The water-content of the
beetle is determined by the evaporating capacity of the air, the leaf-moisture
content of its food plant, soil-moisture, and temperature. Variation in any one
of these factors may influence not only hibernation, but other habits and
reactions." This work was carried on during the next year (1912), in which I
stated regarding the behavior of desert animals that " the proportion of water
held in the body, or the water-balance, is correlated with various activities, and
the lowering of this balance, or surplus, inhibits several functions or processes,
and is also followed by reversed response to various external agencies which may
exert a stimulatory action."

Shelford. (1913) records that certain spiders, ground-beetles, wasps, milli-
peds, frogs, and salamanders react in consequence of evaporation, and that a
short exposure to evaporation conditions increases sensibility to it. Aside from
this experimental data, Shelford and Deere (1913) established laboratory
methods for determining the reactions of the above animals to evaporation
gradients. My experiments differ from Shelford's in being made under natural
conditions out-of-doors, while his studies were undertaken in the laboratory.

Hamilton (1917) studied certain soil insects, in the full-grown larval and
adult state, of the family Carabidae, and his results tended to show that an
increase in the rate of air-flow did not effect the larvae as much as did an
increase in temperature or a decrease in relative humidity; the adults, more-
over, offered greater resistance to evaporation and temperature. The experi-
ments upon the horned lizard by Weese (1917) demonstrated a clear-cut reac-
tion to the substratum temperature gradient, while the evaporation gradient
was not the limiting factor. On the other hand, Chenoweth (1917) concludes
that the evaporating power of the air is the best index of environmental con-
ditions affecting the white-footed woodland mouse, as well as other land
mammals, and that the mice reacted to evaporation whether it was produced
by movement, dryness, or heat.

EXPERIMENTS UPON EVAPORATION. TRANSPIRATION. AND BEHAVIOR.

Previous experiments upon L. decemlineata show that tropic activities for
light and gravity can be reversed through desiccation, and furthermore, that
normal reactions are restored if the beetles were surrounded by a moist medium.
On the other hand, it seemed important in this connection to perform certain
experiments, in order to determine if these insects in nature react to losses of
water, which might be produced through desiccation by means of the evaporating
power of the air immediately surrounding them. Therefore, it seemed advisable
to devise certain tests which would show the daily march of evaporation and
transpiration when compared with their behavior.

The first experiment was made to determine the relation between the daily
progress of evaporation and transpiration rates of L. decemlineata when exposed
at three different strata, which were produced by an association of potato plants
that completely filled the bottom of a cage, 6 feet square by 4 feet high, and

356 Eelation' of Water to the Behavior of

covered with wire-netting. This dense growth produced horizontal zones with
atmospherical moisture, varying from high water-content at the bottom of the
cage to one of low content above the plants in the open.

All beetle exposures and environmental measurements were made every
2 hours for a period of 12 observations at 3 strata within the cage, where insects
and instruments were exposed within wire-netting tubes, 30 cm. long and
5 cm. in diameter. Stratum A was 5 cm. above the ground near the base of the
potato plants, and contained the greatest moisture, thus giving the lowest
evaporation rate ; stratum B was 60 cm. above the ground, near the center of
the cage among the plants, and was directly above stratum A, so that it con-
tained less moisture, which gave a medium rate of evaporation ; while stratum
C was 90 cm. above ground and 5 cm. above the tops of the plants, and furnished
the driest conditions, with a high evaporation-rate, which was the only exposure
to true desert conditions. Each stratum was directly above the other, and all
exposures were made near the center of the cage. The environmental measure-
ments were obtained as follows: The evaporation rates, by using Livingston
atmometers ; relative humidities from wet and dry bulb readings ; temperatures,
from uniform standard centigrade thermometers.

The environmental measurements were made every 2 hours for a period of
12 observations at the 3 strata within the experimental cage as previously
described and at the beginning of each period a new batch of beetles was
exposed to these conditions for 2 hours. The results are given in table 5.

The beetles used in this experiment (Tucson A, g. II) were collected as soon
as possible after their emergence. Since these newly emerged individuals take
no food until after 24 hours, all collections were made previous to this time, so
that no error might be introduced in consequence of feeding ; moreover, no food
was given them at any time, and no excretion of waste products by the animals
was observed throughout the test. The beetles were placed at once in bell-jars
of uniform size in the constant-temperature room, which stood at 2-1° C, and
a high but uniform relative humidity was produced by placing wet filter-paper
inside the jars; this kept the air of the jars approximately saturated and the
beetles absorbed moisture until their reactions and physiological states were
uniform, as was proved by tests made later.

The animals were retained in the jars until needed for further experiment.
Three batches of 10 beetles were removed from the constant-temperature room,
and exposed every 2 hours in wire-netting tubes, at the 3 strata within the cage.
Each batch was made up of similar stocks as follows : 4 individuals of 180 adults
which had emerged on July 5 were placed in the constant-temperature room at
8 a. m. July 6; 3 adults of 110 individuals which had emerged on July 6 were
also placed in this chamber on July 7 ; and 3 animals of 124 adults which had
emerged on July 7 were likewise placed in this room at 10 a. m. July 8 ; while a
batch of 30 beetles which emerged July 8 received the same treatment at 12 p. m.
July 8, and were used during the last 2 hours of the experiment, beginning at
4 p. m. on July 10. Thus all the organisms used were of the same culture and
of the same parents ; in general they were of the same age, and approximately
of similar states, so the conditions of the test were uniform.

Aside from the environmental records, the following was also determined as
far as the insects were concerned : the total weight in grams of 10 beetles when
exposed, their weight in grams after 2 hours' exposure, the total grams of dry

The Potato Beetle in a Desert

357

weight for each batch exposed, the grams of water in the beetles, the total per-
centage of water in the animals, the loss percentage of water in terms of entire
weight, and the loss percentage of water in terms of dry weight (Table 5) .

Table 5.

c

JO

>

o

o

<u

B

H

E

3

c
o

«

o

c

>

o

Q)

«

3

5

v
hi

•5
E

3

.a
>

as
1

u

.11

CO ^

o o

« 3

il

1"

3
•V

ic
u

li

.5 0
£ X

h3

Is

0 -g

01 .C

^ rr.

•=•§
So

0
K

01

C
Eh

01
c
0

0

ii

— >.

C-o

.^ o*

0 ■"

c. c.

' C.

p. Ct.

gms.

gms.

gms.

gms.

gms.

p. Ct.

p. Ct.

p. ct.

r^

2.0

26.8

36

1.2957

1.2700

0.0257

0.239

1.0567

81.55

2.43

10.75

8 a.

m..

Jb

2.3

26.8

33

1.2879

1.2574

0.0305

0.235

1.0529

81.75

2.89

12.98

Ic

3.6

27.3

30

1.2367

1.2028

0.0339

0.229

1.0077

81.48

3.36

14.80

r^

2.2

27.9

35

1.2435

1.2150

0.0285

0.230

1.0135

81.50

2.81

12.40

10 a.

m..

Jb

3.3

33.2

23

1.2860

1.2405

0.0455

0.275

1.0105

78.42

4.50

16.51

Ic

6.0

33.9

22

1.2400

1.1585

0.0815

0.226

1.0135

81.73

8.04

35.98

fA

2.7

30.8

35

1.1840

1.1496

0.0344

0.223

0.9610

81.16

3.58

15.42

12 noon .

.. B

4.6

36.2

19

1.1740

1.1196

0.0544

0.213

0.9610

81.85

5.66

25.53

^C

9.5

36.2

15

1.1870

1.0500

0.1370

0.218

0.9690

81.63

14.14

62.84

f^

4.3

33.3

30

1.2090

1.1320

0.0770

0.232

0.9770

80.81

7.88

33.19

2 p.

m..

Jb

8.4

40.2

15

1.2750

1.1590

0.1160

0.272

1.0030

80.86

11.56

42.64

Ic

17.6

40.0

12

1.2140

1.0405

0.1735

0.236

0.9780

80.56

17.74

73.51

r^

4.6

33.9

28

1.2415

1.1610

0.0800

0.229

1.0125

81.. 55

7.90

34.93

4 p.

m..

Jb

8.9

40.5

13

1.1005

0.9805

0.1200

0.209

0.8915

81.00

13.46

57.41

Ic

19.4

40.0

11

1.2540

0.9800

0.2740

0.232

1.0215

81.46

26.82

117.84

r^

5.3

28.3

33

1.2565

1.2015

0.0550

0.215

1.0415

82.73

5.28

25.57

6 p.

m..

Jb

8.1

37.3

15

1.2230

1.1185

0.1045

0.213

1.0100

82.58

10.34

49.06

Ic

16.1

37.1

12

1.1965

1.0550

0.1415

0.217

0.9795

81.86

14.44

65.20

r^

4.6

28.4

35

1.20.30

1.1815

0.0215

0.210

0.9930

82.54

2.16

10.24

8 p.

m..

. B

7.6

34.0

19

1.2.380

1.1885

0.0495

0.219

1.0190

82.31

4.85

22.60

Ic

14.3

33.4

14

1.2780

1.2110

0.0670

0.234

1.0440

81.69

6.41

28.63

r^

3.0

25.4

43

1.2080

1.1845

0.0235

0.213

0.9945

82.32

2.36

11.00

10 p.

m..

1^

4.6

30.6

24

1 . 1580

1 . 1295

0.0285

0.214

0.9435

81.47

3.02

13.28

Ic

9.0

30.0

17

1.2565

1.2160

0.0405

0.239

1.0175

80.97

3.98

16.95

r^

2.0

25.0

44

1.2660

1.2375

0.0285

0.228

1.0380

81.99

2.74

12.50

12 p.

m..

\^

3.1

29.7

27

1.2230

1.1900

0.0330

0.223

1.0000

81.76

3.30

14.79

Ic

6.9

29.0

23

1.2055

1.1606

0.0449

0.220

0.9S55

81.75

4.55

20.40

fA

2.1

24.1

47

1.1727

1 . 1420

0.0307

0.224

0.9487

80.89

3.23

13.70

2 a.

m..

- B

3.2

28.0

31

1 . 1693

1.1380

0.0313

0.222

0.9473

80.01

3.30

14.09

,C

6.7

27.5

27

1.1384

1.0489

0.0489

0.213

0.9254

81.29

5.28

22.91

r^

1.3

24.3

45

1.1422

1.1122

0.0.300

0.224

0.9182

80.39

3.26

13.39

4 a.

m..

1^

3.1

27.1

28

1.2815

1.2435

0.0380'

0.251

1.0305

80.40

3.68

15.13

Ic

5.1

27.2

25

1.1390

1.0990

0.0400

0.225

0.9140

80.25

4.37

17.77

r^

1.2

22.3

56

1.4.385

1.3975

0.0410

0.301

1.1375

79.07

3.60

13.62

6 a

m..

1^

3.1

25.2

33

1.4550

1.4010

0.0540

0.312

1.1430

78.56

4.72

17.30

Ic

4.9

25.2

27

1.5375

1.4795

0.0580

0.328

1.2095

78.66

4.79

17.68

358

Relation of Water to the Behavioe of

Hourly -
Units

8 10 12 2 4 6 8 10 12 2 4 ^ 8 10 12 2 4 6 8 10 12 2 4
A.M. P.M. P.M. AM. AM. P.M. P.M. AM.

Fig. 1.— Curves showing relation between daily progress of evaporation and
transpiration rates of beetles when exposed to different strata produced by an
association of potato plants. Consult table 5 for above data.

15
14
13
12
11
10
9
8

6
5
4
3
2

'"

~]

"

1 1

1 1 1

~

~

"

~

~

~

Strat

am C

15
14
13
12
U
10
9
8
7
6
.5
4
3
2
1

1 I L 1
Stratum A

Stratum B

1

Water Loss
urve 2 = Rate of

Evaporation
urve 3 = Air Tern p.

Curve 1 = Rate of

Water Loss -
Curve 2= Rate of

(

'urve 1= Transpiration Rate or

Rale of Water Loss
'urve 2= Evaporation Rate

c

1

r

Evaporation
Curve 3 = Air Temp.

1

¥

/

!S

/

\

D

f

1

\

\

,1

1

X

\

1

\

\

f

1

\

1 ^

\

\

?•

^

^

1

\

i

1

\

\

\

/

3

\,

\

L

1

n

\

\

^

€

^,.

3

-*.

\

/

f ,

■v.

\

•-■

<

?^

—^

t-

4

"•.

..s

■^

*^

' —

\

,

s

'^

'

i

1

10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12 2 4
KM. P.M. PM. A.M. A.M. P.M. P.M. A.M. A.M. P.M. P.M. A.M.

Fig. 2. — Results recorded in table 5 reduced to unity and curves plotted in
order to draw a closer comparison than is given in figure 1. The same conclusions
are self-evident.

The Potato Beetle in a Desert

359

Figure 1 shows the plotted results for the data of this experiment. The broad,
heavy lines give the results for stratum A, the broken line, the results at stratum
B, and the narrow line those for stratum C. The abscissae represent the 2-hour
time intervals, and each unit along the ordinate represents for the evaporation
curves, 2.5 c. c; for the transpiration curves in terms of entire weight, 3 per
cent; for the transpiration curves in terms of dry weight, 13.5 per cent; for the
relative humidity curves, 5 per cent; and for the temperature curves 2.5° C.
These graphs are interesting in that they show a close agreement between the
evaporation-rate and transpiration curve for each stratum. Since the humidity
and temperature curves coincide closely for the two upper strata, and the
evaporation and transpiration curves for these strata vary in a similar direction,
it appears that the rate of loss of water from the animals when exposed to the
atmosphere agrees closely with the evaporation rates; i. e., the transpiration
curves of these organisms, as Livingston (1906) found with plants, are largely
controlled by the evaporating power of the air.

Table 6. — Summary of rate of evaporation in each stratum comMned with loss of

water from the beetle.

Location.

Evaporation.

Transpiration.

Stratum.

cm.

c. c.

Ratio.

Lose per cent HjO.

Ratio.

A
B
C

5
60
90

35.3

60.3

119.1

1.0
1.7
3.4

47.2

71.3

113.9

1.0
1.5
2.4

Figure 2 shows the water-loss in percentage, the evaporation-rates, and the
air-temperatures, all reduced to unity. The broad, unbroken line represents
the rate of water-loss in each case ; the narrow, unbroken line, the evaporation
rate for each stratum; and the broken line, the air-temperatures for each
stratum. In making comparisons broadly, the air-temperatures agree in being
represented by nearly straight lines, so that they were negligible; but the
evaporation curves and curves of water-loss differ for each stratum, yet are
similar when compared. At stratum A, the evaporation curve rises more
rapidly and higher than the curve of water-loss, and the drop in the evaporation
curve is faster than in the curve of water-loss. At stratum B, the curves of
evaporation and of water-loss parallel each other until 6 p. m. when the evapora-
tion curve drops more suddenly. At stratum C the increased air-movement is
an added factor in the environmental complex, so that both the water-loss and
evaporation rates rise much higher, although the temperature curves remain
the same. The curve of water-loss at stratum C rises more rapidly and higher
than the evaporation curves. The latter drops sooner than the curve for
evaporation. This appears to be due to the fact that the beetles are more
sensitive to the environmental fluctuations than the porous-cup atmometer, so
that difference might account for the lagging effect.

These differences in the rate of evaporation in the three strata and the water-
losses are given in Table 6. This table shows that approximately 1 c. c. loss
from the cup is equal to 1 per cent loss of water from the beetles exposed.

360

Eelation of Water to the Behavioe of

The evaporation rates here shown gave greater differences within this experi-
mental cage than were obtained by Fuller (1911) for all the plant associations
studied. Shelf ord (1913) uses Fuller's data with tables and compares them
with conditions in certain animals, stating that distribution and succession of
the animals is clearly correlated with evaporating power of air. By a further
comparison with the description of stations, Shelford shows that the evaporating
power of the air may be taken in this case as an index of materials, abode,
and the like. Since the evaporation ratios existing inside of this cage
filled with potato plants are greater than those obtained by Fuller for the
stations such as Shelford used, it appeared that L. decemUneata reared in such

Table 7.

EnTironmen'tal conditions
determined.

Leptinotarsa decemlineata subjected to desert conditions.

V c

s

•6
E

D
J3

01

0

1

c
c

c
0 .

C is

to -a
0 <»
^- 0 .

0 0 .

2 1

■t 2 .

Time.

^1

0 i>

0.

>
a >-.

0
6

'^ 0

•0 J

ii

S (U S

J3 -w P

0-" S

0. posit

ercent 1
to lighl

General Remarks.

O

s

<

a

»

Ed

0

K

0

K

^i^

c. e.

c.c.

° C.

%

No.

gms.

gms.

gms.

%fl20

%n„o

2Vo. %

10 a. m.

0.0

0.030.2

52

25

3.4650

0.0000

0.0000

00.00

00.00

15

60

11 a. m.

2.1

2.1 31.4

49

25

23

92

1 p. m.

6.1

3.134.0

32

25

'.'.'.'.'.'.

23

92

2 p. m.

2.7

2.7 34.2 33

25

3.'327()

' iiaso

'.'6345

'is'.bk

"3!76

15

60

3 p. m.

3.1

3.135.01 32

25

21

84

4 p. m.

3.9! 3.937.7: 32

25

21

84

5 p. m.

3.2i 3.2 35.6 33

25

25

100

6 p. m.

4.3; 4.3 34.2 31

25

3. '2365

"logos

'!6226

"9.86

"2!47

25

100

7 p. m.

2.8' 2.832.3; 35

25

25

100

10 p. m.

5.2

1.7128.3

46

25

3. '1870

'!6495

".0124

"5.39

"l.Zb

25

100

6 a. m.

7.0

0.9!23.7

77

25

3.1490

.0380

.0050

4.14

.52

24

96

10 a. m.

4.4

1.133.2

49

25

3.0725

.0765

.0191

8.33

2.08

24

96

2 p. m.

14.8

3.7I22.5J 32

25

2.9895

.0830

.0208

9.04

2.26

23

92

4 p. m.

6.0

3.032.4! 42

25

0

0

Very cloudy.

S p. m.

0.0\ 0.022. 8\ 90

25

""(Bet

ties incr

eased in

weight.)

11

U

Absorbed B^O

from

6 p. m.

i.s\ 1. Site. 8

Gi

25

S.i2S0

.iSS5

.1168

1,7.22

11.81

21

Si

the air.

9 p. m.

3.0! 1.0125.2

69

25

3.3940

.0290

.0097

3.16

1.05

25

100

12 p. m.

4.3

1.4|23.4

73

25

3.3715

.0225

.0075

2.45

.82

25

100

3 a. m.

1.7

0.6

23.5

70

25

3.3540

.0175

.0058

1.91

.64

25

100

6 a. m.

2.0

0.7

22.7

82

25

3.3295

.0245

.0082

2.67

.89

25

100

9 a. m.

1.8

0.6

28.6

66

25

3.2880

.0415

.0138

4.52

1.51

23

92

12 a. m.

6.4

2.1

31. 4i 41

25

3.1825

.1055

.0362

11.49

3.83

22

88

3 p. m.

9.7

3.233.6 42

25

3.0840

.0985

.0328

10.73

3.58

21

84

0.0705 gm. deducted for|

6 p. m.

6.7

2.2

31.2 43

23

2.9405

.0730

.0243

8.42

2.81

14 1 61

2 died.

9 p. m.

6.2j 2.1

28.0

52

23

2.9035

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12 p. m.

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2.71

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from

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the air.

a cage has a great range of adaptability. From the base of the potato plants to
just a little above their tops, there existed zones of evaporation of wide extremes.
To illustrate, Table 5 shows that from 2 to 4 p. m. at stratum A the evaporation-
rate was 4.6 c. c. and loss in water-weight of beetles was 7.9 per cent of their
entire weight; at stratum B the evaporation rate was 8.9 c. c. and loss of weight
of animals exposed, 13.46 per cent; and at stratum C the rate was 19.4 c. c. and
loss in weight of the beetles was 26.82 per cent. This shows the evaporation
ratios to be 1.00: 1.93:4.22, and the transpiration ratios to be 1.0: 1.7:3.4.
For the whole experiment, the ratios of evaporation were 1.0 : 1.7 : 3.4, while the
transpiration ratios were 1.0 : 1.5 : 2.4. In general, these ratios are very similar,
showing that there is a direct relation between the evaporation-rates and trans-
piration percentages, and it answers in part the question already raised: Do

The Potato Beetle in a Desert

361

the evaporation-rates, the transpiration curves, and the reaction graphs of these
organisms correspond ? The following experiments give us a further affirmative
response by showing that such a correspondence does exist in the potato-beetle
and other insects.

The first experiment was performed out-of-doors at the foot of Tumamoc hill
near the experimental cages at Tucson Station A. The beetles which had
newly emerged from the pupa state were collected at night. They were kept
under saturated bell jars at a constant temperature until morning when they
were placed in wire netting tubes. At 11 a. m. on July 19 they were exposed
to the desert conditions in the open mi til 6 p. m. on July 21. The following
environmental conditions were determined hourly : the rate of evaporation, the

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2:5 24 25 26 Z(

Fig 3 — Curves 2 and 3 show the relation of evaporation to transpiration
(water-loss) of beetles when exposed out-of-doors at the foot of Tumamoc hill;
however, Curve 1 shows that the reactions of this insect to light were in general
the reciprocal of the evaporation and transpiration curves.

air temperature, and the relative humidity. During this experiment, the entire
weight of the beetles was also determined, as well as periods when these insects
gave off water to the atmosphere. It is of importance to plant and animal
physiologists to state that these beetles absorbed water directly from the
atmosphere. Aside from these determinations, others were made, which
include the hourly loss in weight of these insects, the observed loss of water in
terms of their dry weight, the daily loss of water in terms of dry weight, and
the number of the beetles which were positive to light as well as the percentage
positive to light. These results are given in Table 7 and the figures in italics in
Table 7 show periods when the beetles absorbed water directly from the air. The
evaporation rates and the transpiration and reaction rates of Leptinotarsa are
plotted in figure 3.

362

Relation of "Water to the Behavior of

By consulting this figure, one can see at a glance that the curves for trans-
piration and evaporation correspond. Moreover, the reciprocal of the reaction
or behavior curve also corresponds to these curves in a similar manner. Thus
in the reaction of the potato beetle, its percentages of positiveness was, broadly
speaking, the reciprocal of the transpiration curve. This seems to show that the
loss of water from the animal, when exposed in the open, determined the reac-
tions of the insect.

Table 8.-

-Results of subjecting other insects to the same environmental conditions
as Leptinotarsa decemlineata.

In-
sects.

Time.

So .

™ '" '3
3 * J

X

General Remarks.

10 a. m.

11 a. m.

1 p. m.

2 p. m.

3 p. m.

4 p. m.
6 p. m.

6 p. m.

7 p. m.
10 p. m.

C a. m.
10 a. m.

10 a. m.

11 a. m.

1 p. m.

2 p. m.

8 p. m.

4 p. m.

5 p. m.

6 p. m.

7 p. m.
10 p. m.

6 a. m,
10 a. m.

10 a. m.

11 a. m.

1 p. m.

2 p. m.

3 p. m.

4 p. m.

5 p. m.

6 p. m.

7 p. m.
10 p. m.

10 a. m.

11 a. m.

1 p. m.

2 p. m.

3 p. m.

4 p. m.

5 p. m.

6 p. m.

7 p. m.
10 p. m.

gms.
7.2700

6.7025

6 ! 4970
6.3620
6.1735

2.1790

1.5715

1.0545
0.8030

8 ] 2.9510

2.7845

1 .3055
1.2705

5.1725

4.6005
4!6346

gmi.
0.0000

2690

2056
1350
1885

0000

0575
0290

1666

0880

0380
0350

0000
3462

2258
1690

gm».
0.0000

0

0740
0675

osii

0170
0471

0000

0

0473

oiei

0144
0036

0000

0

0416

6226

0095
0044

0000
0866

0565
0398

%H^0
0.00

7.90
5.19
7.26

0.00

22.16

10.23
7.30

18.79
'i4!42

"^'.i'i

9.07
0.00

27'.54

17.96
4!ii

%B^0
0.00

2.77

1.98
0.65
1.81

2.56
0.91

4.70

2!46
1.13

0.00
6!89

3.53

No.

9

10

10

10

10

10

10

10

10

0

0

0

0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

p. ct.

90

100

100

100

100

100

100

100

100

0

0

0

0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

5

56

6

66

8

89

4

44

4

44

5

56

4

44

3

33

0

0

0

0

All doad.

3 dead ; deduct 0.235 gm,

5 dead = 0.4695 gm.
2 dead = 0.2225 gm.
All dead.

2 dead = 0.7525 gm.
2 dead = 0.6005 gm.
All dead.

1 dead = 0.4075 gm.
All dead.

The second experiment was performed to determine the relation of evapora-
tion to the transpiration and reactions of insects when exposed for several days
under natural conditions. To get a comparison between L. decemlineata and
other insects, a cricket {Gryllus), a beetle {Catalpa lanigera), and two species
of June-bugs (Lachnosternae) were used, since they could be collected in large
numbers. These insects, with the exception of the potato-beetles, were obtained

The Potato Beetle in a Desert

363

at night by aid of a light, and were collected as soon as possible after emergence.
All were placed at once in bell-jars in the constant-temperature room. A high
relative humidity was produced as before by placing a wet filter-paper inside
the jars, which permitted the animals, if not already saturated, to absorb water,
so that their water-contents would be as uniform as possible, and they would
attain, in this respect, a similar physiological equilibrium. The insects were
retained under these conditions until 10 a. m. the following morning, when they
were exposed in similar cylindrical tubes. The instruments for measuring
environmental conditions were also placed in these tubes, which were suspended
to a wire and placed in the open, so that each tube was inclined toward the north.
The direct rays of the sun were thus permitted to fall upon the tubes at right
angles. Unless otherwise indicated in Table 8, the observations were made
hourly from 11 a. m. July 19 to 6 p. m. July 21, and for the environmental
conditions consult Table 7.

I

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If one should plot the results given in Table 8, he will find that all of the
organisms used give a transpiration curve which corresponds with their curves
of evaporation. Catalpa lanigera and the crickets reacted to transpiration in a
manner not unlike the potato beetles, while the Lachnosternge always gave a
negative response, regardless of conditions.

Another experiment upon loss of water and insect activity was made as a
conformatory test, in which the methods and materials used were similar. The
animals consisted of 31 L. decemlineata (Tucson A, g. Ill), 10 Catalpa lani-
gera, and 20 Lachnosternas. The latter were collected around an electric light
and then placed in a refrigerator. All instruments and insects were exposed
in the open in large netting spheres and hourly observations were made from
9 p. m. August 9 to 4 p. m. August 11 ; the complete data are given in Table 9.
The evaporation-rates, the air-temperatures, and the relative humidities are

3.0?.

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The Potato Beetle in a Desert

365

shown in figure 4, while the transpiration and reaction curves of Leptinotarsa
decemlineata and Catalpa lanigera are given in figure 5. The upper diagram
contrasts for the potato beetle its transpiration rate with the percentage posi-
tive to light, while the lower half of the cut does the same thing for Catalpa
lanigera. This experiment was similar to the former ones, in that the insects
were subjected to the environmental conditions out-of-doors at the foot of
Tumamoc hill.

For other data and comparisons Table 9 should be consulted; the results
given show that the evaporation curve as measured by the porous-cup atmometer
and the transpiration curves of the insects are similar, as was previously found
to be true. Moreover, the positive reaction curve of the potato beetle was the

55.0%

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jcptinotarsa decemlineata

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Units 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Fig. 5.

reciprocal of its transpiration curve. In the same way the reaction curve of
C. lanigera was associated with its transpiration curve until 9 a. m., when all
reactions became negative. The Lachnosternae appeared here only with a
negative reaction, regardless of their transpiration curve, which agreed with
the curve of evaporation.

These results prove, in the first instance, that the evaporating power of the
air was the determining factor in the transpiration of these animals, a result
similar to that obtained by Livingston (1906) for plants; secondly, that there
existed from the base to the top of associated plants, in an arid region, an
extreme zonation, in which great differences were found in the evaporating
power of the air, and that this in turn controlled the rate of transpiration ; and
finally, that the evaporating power of the air surrounding the organisms deter-
mined their behavior through transpiration. Moreover, many animal organisms
of the desert exhibited great localization in their distribution and the ruling
feature of the environmental complex, whether it entailed a habitat of trees,
among rocks, or in soils, was that of the moisture-relation.

366 Relation' of Watee to the Behavioe of

ROLE OF WATER IN HIBERNATION.

It is an established fact (Tower, 1906) that in the second or winter genera-
tion Leptinotarsa decemlineata in its homozygous state always hibernates under
normal conditions, but that desiccation, or cold, or both, might produce hiber-
nation at any time. At Tucson it was also found that whenever the conditions
became adverse enough to produce desiccation, hibernation was produced. Tower
(1906) showed that preparation for hibernation in the winter generation con-
sists largely in the reduction of the watery contents of the body and in an
elimination of all food and other substances from the alimentary canal.

These facts indicated that the loss of water appeared to be produced by two
different mechanisms. One was controlled by an external medium, while the
other was determined by heredity. In the former, water was extracted from
the tissues through desiccation due to conditions in the medium, while in the
latter water was eliminated from the tissues through internal processes under
normal conditions. Tower states :

" Preparation for hibernation consists in a physiological change in the con-
stitution of the body for the time being and a consequent lowering of the
freezing-point of its tissues in exactly the same way that spores of many plants
and the over-wintering eggs of rotifers prepare for the coming of the unfavor-
able conditions in their environment."

The following experiments were performed to show how the above results can
be brought about through desiccation. At Tucson this type of hibernation was
quite common, but did not occur in nature at Chicago.

ENTRANCE INTO HIBERNATION.

It is evident that in the potato beetle entrance into hibernation may be
" induced hibernation," which occurred whenever the evaporating power of the
air surrounding the insect removed more water by weight in a given time than
was introduced into the organism by food and other agencies. Such desiccation
produced in the course of one or two days depended upon the adversity of the
conditions, thus effecting change in the beetle's behavior, so that its reactions
were reversed and it burrowed in the soil.

Extensive observations were made at Tucson Station A, where it was dis-
covered that this type of hibernation took place whenever sufficiently desiccating
conditions existed. The evidence of such a reaction in a large population was
determined by comparing the daily environmental readings with counts of the
non-hibernating population, which showed that during the rainy season and as
long as water was added to the soil no entrance in this type was found, but when
water was discontinued desiccation occurred and hibernation resulted. On the
other hand, at Tucson Station B, the " induced hibernation " was always
observed as the prevailing type of behavior, since in this habitat the conditions
were more adverse. Moreover, at this locality, the growth of the food plants was
retarded, since the leaves were tougher and showed less water-content ; desicca-
tion was also much greater, so that the response of the organisms to such
rigorous conditions was sharper than in any other locality under observation.
This clearly demonstrated when a comparison of the daily environmental
records were made with the daily count of beetles, which were found out of the

The Potato Beetle in a Desert 367

ground during one month of the rainy season. At Chicago, however, no induced
entrance was discovered, since the conditions were more favorable there for
normal activities, as the daily environmental readings indicate. From these
observations it is evident that a type of hibernation occurred during periods
of low water-content in the surrounding medium ; this produced a lowering of
the beetles' content and induced a set of reactions so that a type of behavior,
potentially hibernation, resulted ; to determine exactly the role of water-loss in
the observed reactions other experimental tests were performed.

For the purposes of this particular problem, the first test consisted of inducing
aestivation by desiccating adults of the summer generation, which do not
normally hibernate. The beetles for this experiment consisted of 259 adults
(Tucson A, g. Ill) which had been placed in a culture cage filled with potato
plants. They were allowed to feed until July 15, when a few bunches of eggs
were deposited, and at 4 p. m. 200 of these beetles were collected and divided
into two groups of 100 each, regardless of sex. The beetles were weighed,
group A weighing 12.16 grams and group B 11.52 grams, respectively. Group
A was now placed under a bell-jar with calcium chloride and group B under a
similar jar filled with wet filter-paper. The jars with the beetles were placed
side by side in an adobe building under identical conditions, except for differ-
ences in the desiccating capacity of the medium within the bell-jars. Through-
out the experiment the temperatures ranged from 26° to 38° C. At 8 p. m.
July 24 these insects were removed from the soil in the bell- jar and re weighed.
Group A from the desiccator weighed 8.53 grams, showing a loss of 3.63 grams,
and group B from the humidor weighed 10.92 grams. Previously a box of soil
(90 by 60 by 15 cm.) had been filled with a mixture of equal parts of sand and
adobe, which also had been already saturated with water and was kept slightly
moist throughout the experiment. Two bell-jars (a humidor and a desiccator)
were placed side by side over the slightly moist soil in the above box ; insects of
group A were placed in the latter, and those of group B in the former; after
28 hours group A was in hibernation, but group B did not hibernate, although
the beetles remained active upon the filter-paper. The calcium chloride was
now removed from the bell-jar over the hibernated group A, and the soil was
kept saturated ; at the end of 3 days 12 beetles emerged and after 6 days 57 more
beetles were discovered, but at the end of 2 days no others had emerged; the
soil was then sifted and 31 dead beetles were found. The individuals of group
B still remained active, but none, however, had hibernated. These results
show clearly that in the summer generation " induced " hibernation with a high
death-rate may be produced through desiccation and, furthermore, that when
water-balances were restored, all the living individuals emerged again and
resumed the activities normal to their generation and season.

To further substantiate the above conclusions other tests follow. In the
fall generation, which hibernates normally, 1,000 newly emerged adults (Tucson
A, g. IV) were collected on August 14, and divided equally, regardless of sex,
into the following four groups: Each group was placed immediately in a
separate wire-netting tube in the screened vivarium at Station A, and the tubes
were made of wire-netting (95 cm. deep and 35 cm. in diameter), with a similar
material covering the top. These were sunk 55 cm. into adobe soil composing
the bottom of the vivarium, and each tube was filled to a depth of 53 cm. with a
mixture of equal parts of sand and adobe.

368

Kelation of Water to the Behavioe of

The following conditions were experimentally planned in tube 1, containing
250 beetles, to give a low rate of evaporation, and the soil was kept moist by
adding water each morning and evening. This tube was kept filled with sprays
of Solanum hertivigii, which were kept fresh by having the stems in 250 c. c.
bottles filled with water, and the sprays were renewed twice daily; it was also
necessary to wrap tinfoil about the top of the bottles to prevent the beetles from
drowning. The environmental conditions were apparently normal, for the

Table

10.

Tube 1.

Tube 2.

Tube S.

Tube 4.

When observed.

Food and moJBt.

Food and dry.

No food and dry.

No food but moist.

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Tubes removed, soil sifted, and all
to be alive

hibernated £

idults found

water-condition, the food-supply, the low rate of evaporation, and the soil-
moisture were all favorable for the normal activities of the animals used (Table
10 for tube 1) in this problem.

The following set of experimental conditions was maintained in tube 2, which
contained 250 beetles; however, in this case the soil was kept only slightly
moist and the first 5 cm. was used as a dry mulch, so that less moisture was lost
through evaporation. The same food plants, Solanum hertwigii, were added

The Potato Beetle ik" a Deseet 369

thrice daily, but only a few small sprays were used each time, so that the air
within the tube was free from dampness, a condition which would have increased
the evaporation rate and assisted in making the environmental situation un-
favorable. The environmental conditions in this case were normal, in so far as
the food-supply was a factor; but the other surroundings were modified, at
least as to the high rate of evaporation (Table 10 for tube 2) . Both tubes 1 and
2 were planned to give the ascertained differences in evaporation-rates ; accord-
ingly tube 1 produced a lower and tube 2 a higher rate, but the food relations
for both were approximately normal.

No plants were used in tubes 3 and 4, but each tube was wrapped with several
thicknesses of coarse absorbent paper ; at the beginning of the test the soil within
each was saturated with water, but no water was added during the experiment.
Around the outside of tube 4 was placed a coil of lead-tubing drilled full of
holes, and this was connected to a carboy of water. This device kept the
absorbent paper surrounding the tube saturated and, furthermore, a large piece
of oil-cloth was wrapped to the height of 15 cm. around the base of the tube
and beneath the absorbent paper. This oil-cloth was extended out in all direc-
tions for about 60 cm. from the bottom of the cage, so that the dripping water
did not come in direct contact with the soil in the tube. On the other hand, no
water was added to tube 3, so that this cage was kept dry during the experiment.
These conditions, therefore, produced a high rate of evaporation in tube 4 and a
low one in tube 3 (Table 10) . The results for each of these tests follow.

The 1,000 beetles used in this experiment were grown under the same environ-
mental conditions and from the same parents, and, moreover, these animals had
emerged as adults from the pupa state synchronously; so they were then as
nearly uniform physiologically as it was possible to obtain them. The con-
clusions showed for tube 1 with plenty of food and moisture, when the indi-
viduals were counted at the end of the experiment, that there was no hiberna-
tion ; 28 were found dead upon the soil. In tube 2, with plenty of food, but in
which the air was kept dry, the census, when taken at the end of the test, showed
that 157 had successfully hibernated and that 93 had died. At the end of the
experiment tube 3, which contained dry air and no food plants, showed 129
beetles in hibernation and 121 dead ones. Tube 4, which was supplied with
moisture but with no food, at the end of the test gave no evidence of hibernation ;
73 of the 250 beetles originally present were found dead upon the soil. These
results proved that newly emerged adults of the fall generation can not be
caused to hibernate under normal conditions, but that if the surrounding
medium was dry a type of hibernation reaction did result through desiccation.
Such deductions are possible, since the evaporation rates in Table 10 show that
a low rate retarded hibernation and a high one accelerated this behavior.

It is also true that this entrance into hibernation may be of the normal type,
which always occurs under normal conditions in the winter generation. Low
temperature, however, was an important factor at Chicago, but this kind of
hibernation also took place in the pure winter-generation stock, even under high
temperatures. This behavior was further studied in the second generation of
the year under the following set of experimental conditions.

At Tucson Station A this type of hibernation reaction in beetles of the winter
generation (Tucson A, g. II) was observed to appear under a normal environ-
mental complex in the fall of 1911, but all other hibernations (Tucson A, g. IV),
25

370 Kelation of Watek to the Behaviok of

which took place under adverse circumstances in the early fall of 1912, were of
the induced type. At Tucson Station B this behavior was not discovered in
either the winter generation of 1911 or that of 1912, since the environmental
conditions always produced desiccation in the early fall at this locality, thus
causing the beetles to be in hibernation about 10 months each year ; they emerged
about the middle of July, and after feeding for a short period re-entered hiber-
nation late in August. At the Chicago Station, however, normal hibernation
always occurred, because the environment was normal, for no excessive desicca-
tion or any other climatic adversities appeared. It became necessary, therefore,
to determine if these results could be confirmed by further data, so the following
hibernation tests were carried out.

For these tests 30 emerging adults (Tucson A, g. II) on September 2 were
placed in a hibernating pedigree-cage containing potato plants for food; the
soil was a mixture of equal parts of sand and adobe, and water was added twice
daily, but the plants completely filled the cage. The experimental conditions,
therefore, were apparently normal throughout the test. It was discovered by
daily observations that these animals were in hibernation on September 18 and
when dug up on October 2, the first adults were uncovered at a depth of 20 cm.,
but the larger number of beetles were found at the bottom of the pot. The
beetles were inactive when first removed, but began to move in a few minutes at
an air-temperature of 33° C. Various tests in the field demonstrated that they
possessed no reactions to food or dry soil, but within 5 minutes they did respond,
and all burrowed into the moist earth at a temperature of 21° C. This indicated
that a cool moist soil accelerated the entrance reaction. These results were
further tested to determine if similar reactions always took place.

On September 2, 130 emerging adults (Tucson A, g. II) were put into a
hibernating cage, which had been previously filled with potato plants, and in
which the soil consisted of a mixture of equal parts of adobe and sand, to which
water was added twice daily; thus the conditions were approximately normal,
for no desiccation occurred. The beetles responded to this set of conditions,
for they were in hibernation by September 22. When dug up on October 21
the insects were found distributed through the soil from the top to the bottom
of the cage, but when tested in the field showed no reaction to food or dry soil,
and when brought into contact with cool moist soil, out-of-doors, they burrowed
into it within 5 minutes.

This activity was again tested in the following manner: 51 emerging adults
(Tucson A, g. II) were removed September 3 and were placed in a hibernating
cage filled with Solanum hertwigii for food. In this experiment a different
food plant was also used, but with no apparent result upon their behavior. The
soil was also of equal parts of sand and adobe, and water was added each morning
and evening, so that the experimental conditions were apparently normal. All
the animals were in hibernation by September 19, but when dug up on October 3
only 46 adults were alive. The first individuals, however, were discovered at
a depth of 29 cm., and the majority were at the bottom of the pot. When
tested in the field no reactions to food or dry soil were observed, but when
brought into direct contact with cool moist earth all burrowed into it imme-
diately. Thus when normally hibernating beetles of the winter generation were
removed from hibernation a cool moist soil was necessary to initiate this
behavior.

The Potato Beetle in a Deseet

371

ACTIVITIES DURING HIBERNATION.

It was observed that hibernating beetles were inactive when first dug from the
soil, and if the insects were moved to a warm room they soon began to crawl ;
then entrance into hibernation occurred if they were brought into contact with
moist earth. At Chicago, during the winter of 1911-12, it was observed that
beetles which had hibernated out-of-doors migrated more deeply than usual
during the cold winter. From the following test it appeared that beetles would
move to moist regions in the earth during hibernation, for late in April 1912,
at Tucson Station A, several hundred individuals were found to be hibernating
in the open air cage. Accordingly a corner of this cage was watered and the
soil was sifted ; thus, all the beetles were removed in that locality, but the newly
dug soil was kept moist, and each week it was examined for adults ; during each
observation a large number was always discovered.

WATER RELATION OF SOILS AND HIBERNATING BEETLES.

The beetles hibernate in small cavities or cells, which contain air of a relative
humidity, that is in proportion to the water-content of the surrounding earth.
During heavy rains the soil becomes flooded with water, so that some air is
driven from the cavities, but if the rain continues for too long a period the
beetles may die. On the other hand, if the soil is too dry, desiccation of the
insects takes place and death may result ; therefore, the part soil-moisture plays
in mortality during hibernation is of great significance. Tower (1906), in
discussing results with soils, states:

" The water-content of soil is controlled, not by an abundant rainfall, nor by
telluric water, but almost wholly by adhesion and capillarity in the soil — that
is, physical conditions alone, such as permeability, capillarity, and the power to
absorb and to retain water are the factors which influence the moisture content
of soil .... In all soils the pores which do not contain water are filled with air
in which the percentage of relative humidity is controlled by the amount of
water in neighboring pores. Likewise, the cells in which these beetles pupate
are filled with air ; the relative humidity is controlled by water in the pores of
the surrounding earth."

Thus the physical composition of the soil is important in preserving insect
life, and the adobe soil of the Tucson Desert, although it contains but little
moisture, does possess other physical potentialities which act in retaining
moisture, for through drying it becomes impervious to water and hibernating
animals are sealed up in their cells and thus preserved from desiccation. To
determine how dry the adobe soil was when containing living beetles, samples
of it were taken from the walls of the cells (Table 11) and those of June 30

Table 11.

Date.

Depth.

Wet soil.

Dried soil.

Water-loss.

Water-content.

cm.

gms.

gms.

gms. HjO.

per cent H3O.

April 1

25

60.93

56.18

4.75

8.45

April 8

25

122.22

110.00

12.22

11.11

April 30

25

123.84

115.89

7.95

6.85

5

58.94

57.64

1.30

2.25

June 30

10

76.55

73.55

3.00

4.07

20

58.09

55.45

2.54

4.45

372 Kelation of Watek to the Behavior of

showed an average of less than 4 per cent water of their dry weights. This
was during the dry season, when no beetles had emerged from hibernation up
to the above date, but on July 1, the day following, 0.98 inch of rain fell; 73
beetles emerged on July 2, and by July 3 eggs were laid. This sharp response in
behavior must be attributed to the water-content of the soil, for the beetles
emerged immediately after the first rain and oviposition took place within
48 hours. The insects of the surrounding desert showed a similar response,
inasmuch as the adobe soil held them imprisoned until the first rain, which
raised their water-content and softened the soil so that emergence of immense
numbers occurred.

The following experiment was performed to show the relation of physical
composition of soils to mortality during hibernation. Three hibernating cages
were prepared containing soils, one was composed of sand, another of adobe, and
a third of equal parts of sand and adobe. On October 1, 500 hibernating adults
were caused to hibernate artificially in each tube by placing the insects at a
depth of 40 cm. in the soil, and the soil within each tube was kept slightly moist
until late in October; they were then allowed to remain out-of-doors under
natural conditions during the winter until May 1, when water was added to each
cage. On May 3, 362 adults emerged from the soil mixture, 251 from the adobe,
but none from the sand. On May 5, the soil of each cage was sifted, when it
was found that all in the pure sand were dead but that only 27 in the adobe
and 23 in the soil mixture had succumbed. It appeared, then, that there was a
relation between soil composition and mortality during hibernation, for the
sand permitted excessive desiccation to occur, the adobe and mixed soils did
not admit of great desiccation, but most individuals were hibernated suc-
cessfully in the mixture of equal parts of adobe and sand.

EMERGENCE FROM HIBERNATION.

The physiological complex of emerging beetles was next considered in refer-
ence to two phases of the problem. In one case the reactions of hibernating
beetles caused to emerge by applying water to the soil were determined, and in
the other the reactions of similar adults, in which emergence was produced by
sifting the soil, were tested. In the first experiment hibernating beetles (Tucson
A, g. II) were encouraged to emerge by applying water on June 1, when they
were removed to the constant-temperature room ; their reactions were found to
be positive to light, but negative to gravity. The light response was further
tested by placing 5 beetles in each of 10 test-tubes and each tube was placed so
that one-half of it was in the shade of the laboratory roof and the other half
in direct sunlight. The beetles all oriented and moved out into the sunlight
at the end of each tube, where they remained, and in a few minutes were dead.
The air-temperature in the exposed ends of the tubes was 57° C. In this experi-
ment the organisms reacted to sunlight and the suggestion that possibly the red
or blue rays might have influenced this result led to the next test.

Of 50 hibernating individuals (Tucson A, g. II) emerged after adding water
on June 3, 25 were put in each of two test-tubes ; one of which was placed in
direct sunlight under a red bell-Jar containing a potassium-bichromate solution,
and, while no deaths occurred, no definite reaction was observed ; the other was
placed in the direct sunlight beside the former, under a blue bell-jar provided
with a solution of copper sulphate, when all became positive to the rays and

The Potato Beetle in a Deseet 373

none died. We may therefore conclude that death in the first experiment was
due to heat.

Many tests of various kinds have been given elsewhere and with the same
result — that when hibernating beetles were caused to emerge by applying water,
they reacted positively to light and negatively to gravity. The next experiment
consisted in testing the reactions of hibernating beetles, in which emergence
was attained by digging and no water was added to the cage.

For this test 47 hibernating beetles (Tucson A, g. II) were removed by
digging at 8 p. m. on June 12. They were negative to a 32 c. p. lamp, but 23 of
these insects were positive to candle-light of a weak intensity. These were
immediately placed under a bell-jar containing moist filter paper, and when
tested on June 13 they were found to be positive to light but negative to gravity.
This behavior was repeated in the following case.

Twelve hibernating beetles (Tucson A, g. II), when removed by digging at
7 p. m. on June 20, were found to weigh 1.2573 grams, but they gave no response
to light or gravity. (A soil-sample taken from the earth surrounding the beetles
showed that it contained water to the extent of 12.7 per cent of dry weight.)
When the animals were placed in a humidor at 10 a. m. on June 22, they
weighed 1.3057 grams, having absorbed 0.1484 gram of water from the moist
chamber; 10 of these beetles, when tested, were positive to light, but 2, which
were inactive, died in a very short time after the experiment. The 10 adults
were also negative to gravity, for when placed in the constant-temperature dark-
room, all crawled to the top of the cylindrical wire-netting tube. These experi-
ments showed that positive phototropic and negative geotropic reactions were
induced in hibernating beetles by increasing the water-content of the surround-
ing medium, because the beetles under the moist bell-jar increased in weight and
imbibed water directly from the moist air. It was also true that they absorbed
water from the air in the soil. This relation was further shown in the following
observations, which were made upon the emergence response.

The time of emergence is controlled by the environmental complex, for if
water were added to the medium surrounding hibernating beetles, when the soil
temperatures were above 14° to 16° C, emergence resulted. This was evident
at Tucson, for no emergence was discovered at either station until the rainy
season in July, and furthermore, the winter rainy season caused no emergence
because of low temperatures. At Chicago, emergence occurred whenever the
soil-temperatures reached 14° to 16° C, for enough precipitation always took
place during the winter and spring months so that emergence occurred as soon
as the proper temperature relations existed, which was from May 20 to June 25.

SUMMARY AND DISCUSSION UPON THE RELATION OF WATER
TO HIBERNATION.

The conclusions arrived at from these previous results indicated that a type
of hibernation might be produced at any time through desiccation, except with
low temperatures, when little desiccation took place. This condition produced
a loss of water from the beetles in sucli a way that they responded negatively to
light and positively to gravity, so that they burrowed into the soil and remained
there until the moisture-content of the soil was sufficiently high. They then
absorbed hydroscopic water, which raised their water-content and reversed their
reactions, so that they became positive to light and negative to gravity, hence

374 Eelation" of Water to the Behavioe of

their emergence, so that now, in case other conditions were suitable, they were
ready to enter upon their reproductive activities. Below 12° to 15° C. in soil-
temperature the water-relation was not the controlling factor, but the duration
of the hibernating period depended upon the length of the dry season in an arid
complex and upon the length of winter (low temperature) in a temperate
region.

Baumberger (1914) reviews, at length, the literature on hibernation of insects
and reaches the following conclusions, chiefly from his own researches :

" 1. That temperature is but a single factor and not necessarily the con-
trolling one in hibernation.

" 2. That hibernation is usually concomitant with overfeeding and may be
the result of that condition or the result of accumulation of inactive substances
in the cytoplasm of the cell due to feeding on innutritive food.

" 3, That the loss of water which is general in hibernation probably results
in a discharge of insoluable alveolar cytoplasmic structures which have accumu-
lated and produced premature senility with an accompanying lowering the rate
of metabolic processes.

" 4. That starvation during hibernation, together with loss of water, may
result in rejuvenation, when aided by histolysis, and an increase in permeability.

" 5. That this rejuvenated condition and increased permeability will, if
stimulated to activity by heat, permit pupation in codling-moth larvae, which
in this case is the termination of the hibernating conditions."

The results of Sanderson and Peairs (1913) add another condition for
hibernation that was also discovered by Tower (1906) for the potato-beetle;
this is the influence of heredity. The former authors reached the following
conclusions :

" That our first work was an effort to show that emergence from hibernation
was due to an accumulation of temperature, but it soon became apparent that
hibernation is very largely controlled by the influence of heredity, and that the
relation of the temperature and inheritance must be determined for each

species."

For the Mexican cotton boll weevil. Hunter and Hinds (1904) found that
dryness was more desirable for hibernation and that mortality during hiberna-
tion was greater from exposure to moisture than from cold; but, on the other
hand, high temperatures and moisture were the best conditions for the develop-
ment of such beetle larvae. In this connection, Baumberger (1914) stated:

" The effects of ether on plants is similar to hibernation and since the action
of ether is probably a drying one, this may throw light on the importance of
moisture in hibernation."

Loeb (1906) says:

" The lack of water acts similarly to a low temperature. This is the reason
why seeds can be kept alive so long. Lack of water may reduce the reaction
velocity of the hydrolytic processes in seeds at ordinary temperature so con-
siderably that it may become practically zero."

The snail, according to the results of Kiihn (1914) loses weight in winter
and reacts to drought in summer. Unless it contains a large amount of water,
no dry food is taken, and if placed under moist conditions when in hibernation
it will come out of its closed shell. Bellion (1909) finds that a low moisture-
content of the air is the determining hibernating factor in the European snail,

The Potato Beetle in a Desert 375

and Baker (1911) shows that snails during dry seasons form an epiphragm;
they usually bury themselves during hibernation and aestivation. On the other
hand, Pearl (1901) finds that the terrestrial slug Agriolimax can hibernate in
cold water.

In the vertebrates, Rulot (1901) determines for the bat that the proportion
of water increases during hibernation from Xovember to April; but there
actually was a loss of water, more in proportion at the end than at the beginning.
Polimanti (190-1) finds that an increase in humidity increases the pounds in a
marmot during hibernation.

In conclusion, the work of Sanderson (1908) agrees most closely with my
results upon hibernation. In discussing the relation of temperature to the
hibernation of insects, he states:

" In come cases, however, the time of emergence from hibernation is con-
trolled by moisture conditions as well as temperature, or independent of tem-
perature. Thus Tower kept the potato beetle in hibernation for 18 months at a
high temperature but with a dry atmosphere, and they emerged as soon as
normal moisture conditions were produced. Webster and Hopkins have shown
a similar effect of the lack of rainfall on the emergence of the Hessian fly in the
fall. In relation to hibernation in humid climates the matter of moisture is
probably not a controlling factor, but undoubtedly has the most important
influence upon the time of emergence of forms in aestivation during the summer
or in an arid region."

My results upon the potato-beetle substantiate the work of Sanderson.

EFFECT OF CHANGES IN WATER-CONTENT UPON ALTERATIONS
IN TROPIC ACTIVITIES.

The experiments and observations upon L. decemlineata proved that, when
surrounded by a moist medium, the beetles were positive to light and negative
to gravity. It is also evident from previous tests that if the moisture of the
surrounding medium was decreased, desiccation resulted, so that the insects
were reversed in their behavior and reacted negatively to light and positively to
gravity. These beetles, however, responded to any intensity of light if moved
from a lesser to a greater intensity, and accordingly when moved from darkness
into the moonlight at Tucson they always reacted ; and in many instances insects
which were negative to a strong light were also positive to a weak one.

It was shown by Burdin (1913) that heat and dryness stimulate positive
reactions in terrestrial amphipods, while cold, moisture, and quiet favor nega-
tive reactions. The results of Dice (1914) prove that light of high intensity
makes daphnias positively geotropic, but a decrease in light intensity has the
reverse effect; and furthermore, these animals tend to become positively
geotropic in high temperatures and negatively geotropic in low. Kanda
(1916a), in studying geotropism in a marine snail, found that it is negatively
geotropic, but most individuals would orient positively if placed on a dry glass
or wooden plate. Later, Kanda (1916&) demonstrates for fresh-water snails
that they are negatively geotropic when their lungs are empty and positively
geotropic when their lungs are full of air. Olmsted (1917) finds that food is a
factor in the reversal of the behavior to gravity in Planaria maculata. Adams
(1903) concludes that earthworms retreat into their burrows during the day-
time because of their negative phototropism, but they emerge at night not so

376 Eelation of Water to the Behavioe of

much because of darkness as because of their positive phototropism for faint
light. Wilson (1891) shows that Hydra is negative to bright light and positive
to dim light. According to McGinnis (1912), Branchipus serratus is positively
geotropic in light and negatively geotropic in darkness; darkness rather than
light may furnish the stimulus to this reversal. In studying the reactions of
Drosophila to gravity, Cole (1917) finds that the response to gravity is much
less marked in flying than in creeping, where it is very definite.

Many animals orient in the field in relation to the center of gravity of the
earth. Loeb (1905) found that some animals turn their heads upward and
others downward. To this latter class belongs the garden spider, which he
found may hang in this position in the center of its web for hours. He dis-
covered the same behavior in some diptera. Shelford (1917) states that such
animals as the grasshopper usually orient with the head up, while aphids and
katydids orient with the head down. In the potato beetle the majority of
larvae and a large number of adults orient with the dorsal side down.

There is a vast amount of literature dealing upon reversibility in photo-
tropism through chemical agencies. Loeb (1893 and 1904) proves that it was
possible to reverse the reactions of a large number of water forms through
chemicals such as salts, acids, and the like. According to Moore (1912a, 1912&,
and 1913), phototropism in Daphnia and Diaptomus may be influenced through
the agency of caffein, strychnin, atropin, acids, alcohol, and ether. Moore
(1913) says:

"While negative phototropism in Diaptomus can be reversed by acids, but
positive phototropism brought about by chemical means can not be reversed by
strychnin (atropin or caffein)."

Wolfgang (1912) determines that electrolites influence phototaxis, and
Kanda (1914) reversed geotropism in Arenicola larvffi by means of salts.

EXPERIMENTS UPON THE ROLE OF WATER IN GEOTROPISM.

On May 15, at 8 p. m., 21 freshly emerged beetles (Tucson A, g. I) were
moved to a constant- temperature room and tested 10 times as to their reactions
to gravity, and in each test all were negative. These geotropic reactions were
tested in the dark, and if the beetles crawled to the top of the tube, when held
in a vertical position, they were considered as positive and if they crawled to
the bottom as negative. The thermograph tracings showed a constant tempera-
ture of 21° C, with a daily variation of 1° C, throughout the test. Again at
11 a. m. on May 16, when tested as previously (10 times), they were still
negative to gravity, and at this time weighed 2.0009 grams; furthermore, on
May 17 at lO*" 30'° a. m., they weighed 1.9294 grams, and again gave the same
test to gravity, so that these results proved that the beetles under these con-
ditions were uniform for this reaction. For experimental purposes these insects
were divided into three groups. The first group of 7 adults was put into a
calcium-chloride chamber, which produced so high a rate of evaporation as to
desiccate them; the second of 7 individuals was subjected to a low rate of
evaporation by placing wet filter-paper under the bell-jar, so that little water
was removed from them under these conditions ; in the third chamber 7 adults

The Potato Beetle in a Desert

377

were used as a control. In table 12, the results are given, which shows that
group 1 at the beginning of the test was negative, but by 9*^ SO'" a. m., while
under the dry bell-jar, all became positive, but when moist conditions were
restored in the jar, by 10 a. m. on May 26, they were again negative. In group 2,
at the beginning, all were negative and remained thus as long as they were kept
under moist conditions, but at 10 a. m. on May 30, all had become positive. In

Table 12. — Reversal in the potato 'beetle to gravity.

Date and hour of observation.

3

Group 1.

Group 2.

Group 8.

•4 o

o

Ori

o
, si

o

0, >.

"S m

o

b

-*j,5J

.t>

■*^ >

S*^

>■-

'Z! >

j: ^

.t>

'Z >

■u a

M a

Ml»>

•M ca

u

i>JS

■gti

Sf&

4)J3

4> .O

S&

^u

<

^

Oi

K

^

0.

S5

^

a,

2

°C. gms.

p. ct.

p. ct.

gms.

p. ct.

p. Ct. gms.

p. Ct.

p. ct.

Mav 17, 10" 30" a.m

20 0.692

0

100

0.640

0

100 ,0.597

0

100

May 19, 12 30 p. m

May 21, 9 30 a.m

May 22, 10 00 a.m

May 24, 10 00 a.m

May 26, 10 00 a.m

May 28, 10 00 a.m

May 30, 10 00 a.m

20

.573

80

20

.783

0

100

.590

0

100

20

.496

100

0

.801

0

100

.586

0

100

20

.577

25

75

.810

0

100

.577

0

100

20

.682

15

85

.810

0

100

.571

0

100

20

.684

0

100

.703

0

100

.570

30

70

20

.685

0

100

.594

15

85

.563

100

0

20

.683 0

100

.556

100

0

.560

100

0

Note.— In group 1 the conditions in bell-jar were dry on May 17, 19, and 21, and
moist on the other days. In group 2 said conditions were dry on May 26, 28, and 30,
and moist on other days. In group 3 said conditions were xmiform throughout.

group 3, which were the control individuals, a gradual loss of weight occurred
until 10 a. m. on May 28, when all were positive. These results showed clearly
that reactions to gravity may be reversed through changes in the moisture-
content of the surrounding medium.

RELATION OF TEMPERATURE TO THE OUTGO AND
INTAKE OF WATER.

An interesting discovery was the determination that there was little absorp-
tion of water below 12° C. as was shown in a test in which beetles emerging
from pupation were collected on July 28 ; they were placed under a bell-jar
containing wet filter-paper, where they remain until 13 midnight on July 30,
when equal numbers of insects were placed in two bell- jars, in a refrigerator, at
a temperature of 10° to 13° C. One bell-jar contained wet filter-paper and the
other calcium chloride, but weighings made at frequent intervals showed that
in the humidor there was no appreciable loss during the 84 hours in the
refrigerator. In another test, desiccated beetles were placed under saturated
bell- jars in the refrigerator, but weighings made at frequent intervals gave no
evidence of water-absorption. These results showed how much organisms were
protected from absorbing water during winter rains, which would otherwise
result in their freezing, and further demonstrated that desiccation, occurring
slowly at a low temperature, was a factor in the economy of the organism.

A similar result was obtained with upper temperature limits. It is known
that the coagulation temperature of colloids varies with the amount of contained

378 Relation" of Water to the Behavior of

water, a condition with which our results on L. decemlineata agreed generally,
since the death-point in potato beetles with a high water-content was 58° to
60° C, and desiccated ones withstood 1° to °5 higher temperature. Bachmet-
jew (1902) shows that the temperature of the insect's body varied with the
conditions, namely, moisture, temperature, and the like. If the air was damp,
the body-temperature was higher than that of the surrounding medium, since no
evaporation occurred; but if the air was dry it cooled through evaporation.
He also pointed out that the smaller the percentage of fluids in a unit of the
living insect body, the lower was the normal congealing-point of the fluids.
Tower (1906) states that soil-temperatures taken on the savannas of Vera Cruz
in April 1904, in places where L. decemlineata was aestivating, were frequently
as high as 60° to 65° C, and that success in passing through these high tem-
peratures at the end of the dry season depended upon the completeness of the
physiological changes preceding entrance into hibernation. These results were
similar to those Just given for this insect, which showed that the lower and
upper temperature limits were influenced through water-relation.

In studying longevity in insects, Baumberger (1914) shows that the tem-
perature at which colloidal substances coagulate lowers with a decrease in
water-content and that long exposure to cold or high temperature may result
in this decrease in water. He explains that the result of a long exposure to cold
is the same as short exposure to heat, while intensity of cold shortens the length
of the period. He also demonstrates that the point of coagulation varies with
the water-content of the insects studied. Greely (1901) concludes that:

" A reduction of the temperature and a loss of the water have similar effects,
because the cell loses water when the temperature is lowered, as well as when
the concentration of the surrounding medium is raised."

The results of Livingston (1903) show for Spirogyra that a cell loses water
when the temperature is lowered. In discussing the reversal in animal instincts,
Loeb (1900) concludes that a decrease in temperature has the same physio-
logical effects as a loss of water.

METABOLISM AND THE WATER-RELATION.

The results of various workers show that desiccation modifies the rate of
metabolism; thus, the alterations in the behavior of the potato beetle may be
due to differences in metabolic activity, brought about through combined rela-
tions of water and temperature of the organism. Shelf ord (1913) states that
the changes in activity of the animals used in his experiments were due to the
withdrawal of water.

It is known that anything which disturbs the rate of metabolism in an animal
alters its response to a stimulus, and that reversed reactions in behavior are
caused by changes in this metabolic process. According to Jennings (1904),
Child (1910), Wodsedalek (1911), Alice (1912), Phipps (1915), and others a
stimulus may change the physiological state of an animal, which produces a
modified type of reaction. Many depressing agents are also known, such as
potassium cyanide, chloretone, and a low oxygen content. Baumberger (1914)
shows that starvation is an agent of this character, since it decreases metabolism
by removing material to be oxidized. Loeb (1906), Mast (1911), Shelford
(1914), and others further demonstrate that acids and alkalis increase irri-

The Potato Beetle in a Desert 379

tability. The results of Shelford (1914) and of Chenoweth (1917), however,
agree most closely with those which are recorded in this paper. Both of these
observers conclude that a high rate of evaporation increases sensibility or irri-
tability through loss of water, a condition which might account for the altera-
tions in the reactions of the potato beetle.

GENERAL DISCUSSION UPON THE ROLE OF WATER IN
LIVING THINGS.

In order to show why such a large number of reactions in the potato beetle
were controlled by its water-relation, it was considered necessary to review only
literature which bore directly upon these studies. "It was assuredly not
chance," to quote Henderson (1913), " that led Thales to found philosophy and
science with the assertion that water is the origin of all things." He also states
that the action of water now appears to be a momentous factor in geological
evolution, and the physiologist has found that water is invariably the principal
constituent of living organisms. Thus, according to this observer, water makes
up from 70 to 85 per cent of fishes, about 87 per cent of oysters, 85 per cent of
apples, 78 per cent of potatoes, and 95 per cent of the edible portion of lettuce.
It is interesting in this connection to add that my results upon the potato beetle
show it to contain 80 per cent water. Henderson further says that the organism
itself is essentially an aqueous solution in which are spread out colloidal sub-
stances of vast complexity, and as a result of these conditions there is hardly a
physiological process in which water is not of fundamental importance. Accord-
ing to Livingston (1903), it is absolutely essential that every living mass of
protoplasm be saturated with water, since vital phenomena occur solely in
aqueous solutions.

Physiologists have long recognized that water is of the greatest importance
for normal activity of tissues and that all exchanges of material, all supplies of
food, and metabolic processes in general are dependent upon it. Aberhalden
and Hall (1908) assert that water is absolutely necessary as a solvent for
numerous compounds, for it brings into play various chemical reactions, which
take part in building up and breaking down substances without number ; it is
also a carrier of nourishment to the body and provides the means for the removal
of its waste products. In discussing the physical importance of water, Hammar-
sten and Mendel (1911) show that water by its evaporation is an important
regulator of temperature. Davenport (1897) states that growth is due chiefly
to imbibed water, and Estabrook (1910) also demonstrates that growth in
paramoecium is due almost solely to inhibition of water. MacDougal (1912),
Lloyd (1905), and others demonstrated that many plants absorb water directly
from the air. In respect to this subject, in animals the frog has perhaps
received most attention, and according to Hill (1908) frogs take up water
through the skin ; they do not drink, for a thirsting frog with its gullet tied
increases in weight no less than one with the gullet open. He also states that a
frog can be gradually dried to less than 39 per cent of its normal weight with-
out fatal results. My own data for the potato beetle show that it can be desic-
cated to less than 50 per cent and still live, while CataJpa Janigera will die if
reduced by 25 per cent and the June bug if dried less than 15 per cent of its
normal weight.

380 Eelation- of Water to the Behavioe of

It is not true that all animals do absorb water, for my experiments upon the
scorpion and horned lizard (Phrynosoma cornutum) of the Tucson Desert indi-
cated that these animals would not imbide any aerial water, and even when
immersed in it no absorption was detected; furthermore, when desiccated no
difference in weight was observed. A large scorpion lived for more than 3
months in a desiccator without food, but it probably died of starvation. If
lizards do not absorb any appreciable amount of water or lose any through
desiccation, then such a condition might demonstrate why they are distributed
in a desert as well as in a hot humid region ; therefore, the water-relation would
not be the determining factor in a lizard's habitat, but the temperature-relation
should be of greater importance in determining its distribution. This might
also account for the results of Weese (1917), since he studied the reactions of
the horned lizard to evaporation and temperature gradients, but found that the
lizard responded definitely to temperature, while there was no marked reaction
to evaporation. In this connection the work of Matthews (1913) is important.
He says:

" There is a mechanism for rendering mammals tolerably independent of the
moisture content of their environment, a mechanism most highly developed in
the reptiles. A mechanism formed by the replacing of the wet skin of the
amphibian by a dry or scaly skin; the perfecting of the kidneys to maintain
osmotic pressure of the blood ; the control of the sweat glands and loss of water
by the intestines ; the development of membranes non-permeable to salts so that

animals may sit in fresh water and lose their salts By this improvement

reptiles have secured almost complete independence of the water-content of
their surroundings."

Water is essential to life, says Babcock (1912), for during the period of
development it is the most abundant constituent of living organisms. He
continues :

" Some of this water is imbibed directly, some of it is taken with solid food
which is rarely dry, and some of it is formed within the organisms by metabolic
changes in the organic constituents of the food and tissues, induced by respira-
tion and other vital processes. The relative amount of water derived from each
of these sources depends upon the kind of organisms, its period of growth, the
nature of its food, its environment, and its activities."

His experiments show that many varieties of insects, such as the clothes-moth,
the bee-moth, and the flour-beetle, the flour-moth, and others live during all
stages of development upon foods containing less than 10 per cent of water.
He concludes that nearly all the water used by insects feeding upon air-dried
foods is metabolic. In my own experiments upon the potato-beetle and other
animals there are no data upon metabolic water and its relation to behavior.

The results of Hegner (1916) further illustrate the water-relation in animals.
He arranged an experiment upon oviposition in the potato-beetle, so that 35
batches of eggs were in the sunlight and 15 were in the shade. Those in the sun
came to nothing, but all in the shade were hatched. It was found that develop-
ment had started in the sunlight, but that desiccation probably arrested this
process; therefore he concludes that the advantage of concealment is not so
great as that secured by shielding the eggs from the desiccating properties of the
sun. My results show that the majority of adults orient to gravity with their
dorsal side down, which might explain why eggs are usually deposited on the
under side of a potato-leaf.

The Potato Beetle in a Deseet 381

SUMMARY AND CONCLUSION.

In general the results indicate for the potato-beetle that :

(1) The optimum breeding activity of this insect coincides with the highest
water-content of the atmosphere, since periods of oviposition are exactly con-
current with those of rain and low rates of evaporation.

(2) Differences in soil-moisture produce alterations in the water-content
of these animals which modify their behavior, since beetles will lay their eggs
sooner if they emerge from a soil of high moisture-content than if they issue
from a dry soil.

(3) Egg-production is also modified by differences in the evaporating power
of the air which surrounds these insects, since a low rate of evaporation en-
courages oviposition.

(4) The beetle dies if buried when all its activities are normal, but either
the summer or winter generation may be buried without injury if previously
desiccated.

(5) The adobe soil of the arid region retains a relatively high percentage of
water and is thus an excellent medium for the sustentation of the life of this
beetle, as in other desert animals.

(6) These insects exhibit a physiological behavior not unlike that of trans-
piration in plants; but further, their tropisms are modified by loss of water,
which is governed by the evaporating power of the air.

(7) The evaporating power of the air surrounding these insects determines
their behavior through transpiration ; even their responses to light and gravity
are controlled by evaporation.

(8) Entrance into hibernation in a desert region may be produced at any
time through desiccation, except at low temperatures, when little desiccation
takes place.

(9) The hibernating period in an arid region is controlled by the duration of
the dry season, but is dependent upon the length of the winter in a temperate
region.

(10) The water-relation is the controlling factor in the emergence of this
insect from hibernation if the temperature is above 15° C.

(11) When surrounded by a moist medium (above 15° C.) , either atmosphere
or soil, these beetles are positive to light and negative to gravity, but desiccation
reverses this behavior.

(12) This insect absorbs very little water below 12° C. ; its death-point under
a high water-content was found to be 58° to 60° C, but when desiccated it can
withstand from 1° to 5° C. more of heat.

(13) Alterations in the behavior of the potato-beetle may be due to differ-
ences in metabolic activity as influenced through the water-relation.

(14) This animal also imbibed water directly, but no studies were made upon
metabolic water.

We may conclude that Leptinotarsa decemlineata, when introduced from its
grassland habitat into an arid region, is equilibrated immediately with respect
to its surroundings, especially in regard to its water and temperature medium ;
that its behavior is changed to resemble those responses still present in an
organism long accustomed to a desert complex, and that, since water is the

383 Eelation op Water to the Behavior of

limiting factor in an arid region and the prime essential for metabolism, the
behavior of the potato beetle in a desert is determined chiefly by the water-
content of its environment. Henderson (1913) states:

" Water, of its very nature, as it occurs automatically in the process of cosmic
evolution, is fit, vs'ith a fitness no less marvelous and varied than that fitness of
the organism which has been won by the process of adaptation in the course of
organic evolution. ... In truth, Darwinian fitness is a perfectly reciprocal
relationship. In the world of modern science a fit organism inhabits a fit
environment."

These results upon the potato beetle indicate that its marvelous fitness and
adaptation to water is such a " reciprocal relationship."

The experiments were performed at the Desert Laboratory of the Carnegie
Institution of Washington, and it is a pleasure to acknowledge my indebtedness
to its director, Dr. D. T. MacDougal, for his interest manifested. I must also
acknowledge my great indebtedness to Professor W. L. Tower, of the University
of Chicago, who made it possible for me to continue this problem at Tucson.
The following bibliography gives only the literature cited.

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