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G.J. Viljoen
A.G. Luckins
I. Naletoski

Stable Isotopes
to Trace Migratory
Birds and to
Identify Harmful
Diseases
An Introductory Guide
Stable Isotopes to Trace Migratory Birds
and to Identify Harmful Diseases
G.J. Viljoen • A.G. Luckins • I. Naletoski

Stable Isotopes to Trace

Migratory Birds and to
Identify Harmful Diseases
An Introductory Guide

Front Photo
FAO Mediabase; Credit ©FAO/Giulio Napolitano/FAO
G.J. Viljoen A.G. Luckins
Joint FAO/IAEA Division Schiehallion
Animal Production and Health Section Blairgowrie Schotland, UK
Vienna, Austria

I. Naletoski
Joint FAO/IAEA Division
Animal Production and Health Section
Vienna, Austria

ISBN 978-3-319-28297-8 ISBN 978-3-319-28298-5 (eBook)

DOI 10.1007/978-3-319-28298-5

Library of Congress Control Number: 2016933681

© IAEA 2016
Open Access This book is distributed under the terms of the Creative Commons Attribution-
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Foreword

Highly pathogenic avian influenza (HPAI) caused by the H5N1 virus is a dangerous
transboundary animal disease (TAD) that seriously impacts on poultry production,
especially on smallholder farmers and commercial enterprises in Member States
(MS) in Asia and Africa. With the added danger that this disease can become
zoonotic, i.e. it has the potential to transfer to humans, it is essential that every effort
is made to understand its epidemiology, particularly those factors that determine the
dissemination of the virus across countries and continents. One area in which there
is an urgent need for clarity is the role that migratory wild birds might play in the
spread of HPAI to domestic poultry. This is a matter of international importance
since millions of wild birds fly from areas where HPAI is endemic to all corners of
the globe, potentially spreading the disease wherever they land.
MS are acutely aware of the need to develop technologies that allow disease
detection before clinical signs of infection became apparent. Under the aegis of the
project “Reducing risk from transboundary animal diseases (TADs) and those of
zoonotic importance”, the IAEA, through the Animal Production and Health Section
of the Joint FAO/IAEA Division, has supported MS in acquiring sensitive, specific
and rapid nuclear, nuclear-related and nuclear-associated diagnostic tests for
HPAI. Now that MS laboratories have the required technologies to develop appro-
priate surveillance strategies, it will be possible to enlarge the scope of studies on
HPAI to include verification of the role of the wild bird hosts. Consequently, the
Animal Production and Health Section (APHS) has initiated a new Coordinated
Research Project (CRP) that uses innovative technologies to sample wild birds and
their habitats to detect virus and also introduce stable isotope analysis (SIA) of bird
tissues to determine their place of origin.
Migration patterns of wild birds have been studied for many years using a variety
of techniques, including recent various electronic tracking methods. However, more
effective and cheaper options will be required if specific information is needed on a
particular bird species, especially infected wild birds. The envisaged approach is to
determine migrant origins by determining the stable isotope “signature” of the bird
and match it to the most likely environmental habitat. Stable isotopes pose no health

v
vi Foreword

or environmental risks since they are not radioactive and are present throughout the
environment. However only a small number of them, namely, H, O, C, N and S, will
be useful for tracking the origin of wild birds. The technologies used to infer migra-
tory connectivity in migrant birds and for detection have become increasingly
sophisticated, as have the models for identifying habitats. It is now felt that such
technologies are mature enough to inform scientists engaged in studies of HPAI on
so far unanswered questions concerning the biology and epidemiology of HPAI in
wild birds.
The purpose of this publication is to make scientists, particularly those involved
in IAEA CRP and TC projects, aware of the potential of SIA by providing an intro-
duction of their application and technical requirements needed to facilitate studies.

Animal Production and Health Section Gerrit Viljoen

Joint FAO/IAEA Division for Nuclear Applications
in Food and Agriculture, Department of Nuclear
Sciences and Applications
International Atomic Energy Agency
Vienna International Centre
Vienna, Austria
Acknowledgements

This guide was developed with substantial support of the team involved in the
development and implementation of the IAEA CRP: “Use of Stable Isotopes to
Trace Bird Migrations and Molecular Nuclear Techniques to Investigate the
Epidemiology and Ecology of the Highly Pathogenic Avian Influenza” (Code:
D32030).

vii
Contents

1 General Introduction ................................................................................ 1

1.1 Background Analysis .......................................................................... 1
1.2 Migratory Birds and HPAI.................................................................. 4
1.3 Using SIA to Understand the Dissemination
of HPAI – The Way Ahead! ................................................................ 5
2 Animal Migration Tracking Methods ..................................................... 11
2.1 Extrinsic Markers ............................................................................... 11
2.2 Intrinsic Markers ................................................................................ 12
2.2.1 The Nitrogen Cycle ................................................................. 17
2.2.2 The Sulphur Cycle .................................................................. 17
2.2.3 The Oxygen Cycle .................................................................. 18
2.2.4 The Hydrogen Cycle ............................................................... 18
2.3 The Stable Isotopes of Water on a Spatial Scale ................................ 19
2.4 Deriving Isoscapes in the Absence of GNIP Data .............................. 21
2.5 Use of Stable Isotopes for Migration Studies ..................................... 22
2.6 Approaches for Determining Migratory Connectivity ....................... 25
2.6.1 Classification Trees to Predict Origins.................................... 28
2.6.2 Likelihood-Based Methods to Predict Origins ....................... 28
2.6.3 Migration Studies Using Stable Isotopes ................................ 29
2.6.4 Determining Migratory Connectivity
for Waterfowl in Asia .............................................................. 30
3 Practical Considerations .......................................................................... 35
3.1 Sample Collection and Tissue Preparation ......................................... 35
3.1.1 Questions of Sample Heterogeneity........................................ 36
3.1.2 Preparation of Feather Samples .............................................. 36
3.2 Other Stable Isotopes for Migration Research ................................... 38
3.2.1 Sampling Instructions for Water δ18O
and δ2H – Rivers/Lakes/Groundwater .................................... 38

ix
x Contents

3.2.2 Sampling Instructions for Bird Feathers

for Use in SIA ......................................................................... 39
3.2.3 Sampling Wild Birds in the Field............................................ 40
3.2.4 Sampling Strategy for H5N1 .................................................. 41

References ........................................................................................................ 45

Index ................................................................................................................. 49
List of Acronyms

Acronym Interpretation
APHS Animal Production and Health Section of the Joint FAO/IAEA
Division
BWB Bowhead Whale Baleen
C Carbon
CDT Canyon Diablo Troilite
CF-IRMS Continuous flow isotope ratio mass spectrometer
CFS Chicken feather standard
CHS Cow hoof standard
CRP Coordinated Research Project
D Deuterium
DNA Deoxyribonucleic acid
ELISA Enzyme-linked immunosorbent assay
EU European Union
FAO Food and Agricultural Organization of the United Nations
GIS Global information system
GLM General linear model
GNIP Global Network for Isotopes in Precipitation
GPS Global Positioning System
GS Growing season
H Hydrogen
HDPE High-density polyethylene
HPAI Highly pathogenic avian influenza
IAEA International Atomic Energy Agency
IRMS Isotope ratio mass spectrometer(spectrometry)
LPAI Low pathogenic avian influenza
MA Mean annual
MS Member states
N Nitrogen
O Oxygen
OPIC Online Isotopes in Precipitation Calculator

xi
xii List of Acronyms

PDB Pee Dee Formation

QA Quality assurance
RBC Red blood cells
Rs Ratio of the heavy and the light isotope of the sample
Rstd Ratio of the heavy and the light isotope of the standard
S Sulphur
SIA Stable isotope analysis
SLAP Standard light Antarctic precipitation
SMOW Standard Mean Ocean Water
TAD Transboundary animal disease
TC Technical cooperation
VPDB International Atomic Energy Agency – Pee Dee Formation
VSMOW International Atomic Energy Agency – Standard Mean Ocean Water
WMO World Meteorological Organization
Chapter 1
General Introduction

1.1 Background Analysis

Birds are amongst the few terrestrial vertebrates that share with humans the pecu-
liarity of travelling in a few hours across national and intercontinental borders
(Hobson 2002). The record for distance covered in a single year belongs to the
arctic tern, which travels over 50,000 km between Antarctica and northern
Scandinavia. Overall, billions of birds travel between continents twice a year in
only a few weeks. Migration is critical in the life cycle of a bird, and without this
annual journey many birds would not be able to raise their young. More than 5000
species of birds manage annual round-trip migrations of thousands of miles, often
returning to the exact same nesting and wintering locations from year to year. Birds
migrate to find the richest, most abundant food sources that will provide adequate
energy to nurture young birds. If no birds migrated, competition for adequate food
during breeding seasons would be fierce and many birds would starve. Instead,
birds have evolved different migration patterns, times and routes to give themselves
and their offspring the greatest chance of survival. Birds gauge the changing of the
seasons based on light level from the angle of the sun in the sky and the amount of
daily light. When the timing is right for their migrating needs, they will begin their
journey. Several minor factors can affect the precise day any bird species begins its
migration, including available food supplies, poor weather or storms and air tem-
peratures and wind patterns. While these factors may affect migration by a day or
two, most bird species follow precise migration calendars. Those calendars vary
widely for different species, however, and while autumn and spring are peak migra-
tion periods when many birds are on the move, migration is actually an ongoing
process and at any time of the year, there are always birds at some stage of their
journeys. The distance the birds must fly, the length of time it takes to mate and
produce a healthy brood, the amount of parental nurturing young birds receive and
the location of birds’ breeding and wintering grounds all affect when any one spe-
cies migrates to stay alive.

© IAEA 2016 1
G.J. Viljoen et al., Stable Isotopes to Trace Migratory Birds and to Identify
Harmful Diseases, DOI 10.1007/978-3-319-28298-5_1
2 1 General Introduction

During these yearly migrations, birds have the potential of dispersing microor-
ganisms that can be dangerous for human as well as animal health. For instance,
birds are believed to be responsible for the wide geographic distribution of various
pathogens, including viruses [e.g., West Nile, Sindbis, Highly Pathogenic Avian
Influenza (HPAI), Newcastle disease], bacteria (e.g., Borrelia, Mycobacterium,
Salmonella), and protozoa (e.g., Cryptosporidium). An insight into the ecology of
bird populations is necessary to understand fully the epidemiology of bird-associated
emerging diseases. Furthermore, data about avian movements might be used to
improve disease surveillance or to adapt preventive measures. However, the links
between bird ecology and livestock and human disease have yet to be completely
understood so there is a need to increase knowledge of avian migration patterns and
infectious diseases to help predict future outbreaks of emerging diseases.
Animal influenza viruses threaten animal health, livestock productivity and food
security in poor countries, but they can also evolve into dangerous human patho-
gens. This has been seen with the emergence of HPAI. Its main impact has been on
domesticated poultry, with over 300 million birds killed or destroyed, but of consid-
erable public health concern is the transmission of the virus from birds to humans,
resulting in over 500 occurrences of disease in which over half of the infected indi-
viduals have died.
The threat from animal influenza viruses makes it essential for animal health
professionals to take the lead in detecting and monitoring the occurrence of the
viruses and sharing the information with the international community. Since avian
influenza appears to be associated with migratory bird movements (over 1000
reported AI outbreaks since 2006, involving 25 species of wild birds in EU alone)
surveillance would need to focus on detection of HPAI in both wild birds as well as
domesticated poultry and it will also be necessary to establish the migratory path-
ways of wild birds to increase the capacity to assess their risk in spreading the virus.
In March 2010 the International Scientific Task Force on Avian Influenza and
Wild Birds, led by FAO and the United Nations Environment Programme –
Convention on Migratory Species, reported that waning attention to HPAI was
reducing opportunities for surveillance and research, thereby affecting efforts to
understand the epidemiology of the disease. The disease continues to be a major
problem in Egypt and parts of Asia and outbreaks have occurred in poultry in
Romania and in wild birds in Russia, China and Mongolia (Fig. 1.1). One of the
most important issues that need addressing is the surveillance of wild bird popula-
tions to improve understanding of the role that they play in the dissemination of
infection. Although various methods have been used to track the migration of birds
there is increasing interest in utilizing methodologies that would enable tracing of
migratory movement based on the birds’ stable isotope signatures. A small number
of stable isotopes are involved in important biological and ecological processes and
there is a strong correlation between levels of these isotopes in the environment and
the concentration of the same isotopes in avian tissues. Of most interest are stable
hydrogen and oxygen ratios in tissues that accurately reflect those in lakes, rivers,
oceans and in groundwater, along the wild bird flyways. Using stable isotopes to
characterize a population involves examining the isotopic signatures of a few
1.1 Background Analysis 3

Fig. 1.1 Characteristics of the reported outbreaks of AI in domesticated poultry and wild migra-
tory birds in the period from 1 January 2011 until 13 May 2015 (Source: FAO 2015).
Map A symbols: red circle [ ] = H5 AI; blue square [ ] = H7 AI; green triangle [ ] = H9 AI;
grey star [ ];
Map B symbols: AI outbreaks reported during 2011 (grey star [ ]), 2012 (green square [ ]),
2013 (blue square [ ]), 2014 (red triangle [ ]) and 2015 (yellow circle [ ]);
Map C symbols: AI outbreaks in wild (red circle [ ]) and captive birds (blue triangle [ ])

individuals that are representative of the entire population. The hydrogen and oxy-
gen isotope composition of environmental water varies spatially across the globe
and because it is a constituent of many biosynthetic pathways, the isotopes’ pres-
4 1 General Introduction

ence is relayed to animal tissues, providing the means to link data on groundwater
isoscapes with isotope levels in biological tissues such as feathers. This isotope data
would reveal migration patterns and enable identification of the breeding areas of
birds sampled at non-breeding grounds and disease outbreak sites.

1.2 Migratory Birds and HPAI

To date, only a small number of migratory birds have been tracked by satellite trans-
mitters to establish links with disease outbreaks in domestic poultry, with little evi-
dence so far of direct correlation with HPAI. Furthermore, surveillance of 750,000
“healthy” wild birds has not revealed many infected individuals. Circumstantial
evidence suggesting that the spread of the H5N1virus to new areas can be facilitated
by migratory wild birds has come from studies on ducks in Asia marked with satel-
lite transmitters (Yamaguchi et al. 2010) that were tracked during an outbreak of
highly pathogenic H5N1 avian influenza virus.
The satellite transmitters were attached to northern pintail ducks several months
before the H5N1 virus was discovered in dead and dying whooper swans at wet-
lands in Japan. Twelve percent of marked pintails used the same wetlands as
infected swans and the pintail ducks were present at those sites on dates the virus
was discovered in swans. During the first week after they become infected with
H5N1 virus, ducks such as pintails can shed the virus orally or in their faeces, con-
tributing to the virus’ spread. Some of the marked pintails migrated 700 miles
within 4 days of leaving the outbreak sites; marked pintails ultimately migrated
more than 2000 miles to nesting areas in eastern Russia. The discovery that northern
pintails made long-distance migrations during the period when an infected duck
would likely shed the virus offers insight into how H5N1 could be spread by wild
birds across large areas (Yamaguchi et al. 2010).
In another study, the movements of bar-headed geese marked with GPS satellite
transmitters at Qinghai Lake, China were traced in relation to virus outbreaks and
disease risk factors. A previously undocumented migratory pathway between
Qinghai Lake and the Lhasa Valley of Tibet where 93 % of the 29 marked geese
overwintered was discovered. From 2003 to 2009, 16 outbreaks in poultry or wild
birds were confirmed on the Qinghai-Tibet Plateau, and the majority were located
within the migratory pathway of the geese. Spatial and temporal concordance
between goose movements and three potential H5N1 virus sources (poultry farms,
a captive bar-headed goose facility, and H5N1 outbreak locations) indicated that
ample opportunities existed for virus spill over and infection of migratory geese on
the wintering grounds. Their potential as vectors of H5N1 was supported by rapid
migration movements of some geese and genetic relatedness of H5N1 virus isolated
from geese in Tibet and Qinghai Lake. This study was the first to compare phyloge-
netics of the virus with spatial ecology of its host, and the combined results suggest
that the geese play a role in the spread of H5N1 in this region (Prosser et al. 2006;
Zhou et al. 2011).
1.3 Using SIA to Understand the Dissemination of HPAI – The Way Ahead! 5

Defining the migratory behaviour of animals is fundamental to understanding

their evolution and life history; much of the effort to study migration in birds, has
been driven by the needs for conservation in changing habitats. The advances in
understanding of migration, particularly in North America and Europe have been
aided considerably by stable isotope techniques. The methods have not yet been
applied to study the migratory birds and the dissemination of HPAI, but this is likely
to be remedied in the near future. This manual provides background information on
Stable Isotope Analysis (SIA) for scientists investigating HPAI from Member States
engaged in Collaborative Research Projects or Technical Contract Projects and wish
to understand how the disease is disseminated to provide information that would
enable better risk assessment and more effective control and prevention measures.
In order to follow the global distribution of animal diseases, as well as to enable
rapid information sharing, FAO has established an Animal Disease Information
System (EMPRES-i), (FAO 2015) which enables for filtering disease records
according to different predefined criteria. Specifically for AI, record-sets can be
generated, which enable for correlation according to the species of origin of the
outbreak (domesticated or wild birds), period of the observation/reporting of the
outbreak and the location of the outbreak. The data on the evolution of the AI glob-
ally, obtained from EMPRES-i system over the period of 5 years (January 2011–
May 2015) is shown on Fig. 1.1 and Table 1.1.

1.3 Using SIA to Understand the Dissemination

of HPAI – The Way Ahead!

Most elements consist of one or more stable isotopes – elements having the same
number of protons, but differing in the numbers of neutrons. Stable isotopes are
those isotopes of an element that do not decay through radioactive processes over
time. For instance, the element carbon (C) exists as two stable isotopes, 12C and 13C,
and the element hydrogen (H) exist as two stable isotopes, 1H and 2H.
Stable isotope contents are expressed in ‘delta’ notation as δ values in parts per
thousand (‰), where:

 R ‰  (Rs / RStd – 1)  1000

and Rs and RStd are the ratios of the heavy to light isotope (e.g. 13C/12C) in the
sample and the standard, respectively. The stable isotope ratios of hydrogen, carbon,
nitrogen, oxygen and sulphur are denoted in delta notation as δ2H, δ13C, δ15N, δ18O
and δ34S, respectively. R values have been carefully measured for internationally
recognized standards. The standard used for both H and O is Standard Mean Ocean
Water (SMOW), where (2H/1H) standard is 0.0001558 and 18O/16O is 0.0020052.
The original SMOW standard is no longer available and has been replaced by a new
International Atomic Energy Agency (IAEA) standard, VSMOW. The international
 6

Table 1.1 Quantitative information on the reported outbreaks of AI in domesticated poultry and wild migratory birds in the period from 1 January 2011 until
13 May 2015 (Source: FAO 2015)
Pathogenicity
(HP or
LP) – H
Species subtype of
category the AIV 2011 2012 2013 2014 2015 Sub total
Captive Number of outbreaks/number of cases affected during the utbreak
HP-H5 1/1 1/ 8/5 10/6
LP-H5 2/8 2/8
LP-H7 1/4 1/4
Total captive 1/1 2/8 1/4 1/ 8/5 13/18
Domestic HP-H5 1927/763,007 551/666,346 489/306,795 891/520,219 1614/2,359,345 5472/4,615,712
HP-H7 1/10 48/2,699,685 86/780,143 4/3 3/139 142/3,479,980
HP-NoInfo 1/10 1/10
1

LP-H5 2/40 24/1584 37/6071 25/8179 24/15,526 112/31,400

LP-H7 24/4369 1/ 40/7186 34/3039 6/2373 105/16,967
LP-H9 39/45,003 69/3 35/6 8/ 4/ 155/45,012
LP-NoInfo 6/26 1/1 2/ 9/27
NoInfo- 34/2519 34/2519
NoInfo
Total 2000/812,465 693/3,367,618 722/1,102,721 962/531,440 1653/2,377,383 6030/8,191,627
domestic
General Introduction
 1.3

Pathogenicity
(HP or
LP) – H
Species subtype of
category the AIV 2011 2012 2013 2014 2015 Sub total
Wild HP-H5 63/101 25/1183 4/2 37/35 70/217 199/1538
HP-H7 2/11 1/5 1/8 4/24
HP-NoInfo 1/ 1/
LP-H10 1/ 1/
LP-H5 1/ 2/ 3/
LP-H7 1/ 1/ 2/
LP-H9 1/ 1/
Total wild 64/101 27/1194 6/7 42/35 72/225 211/1562
Grand total 2126/812,567 757/3,368,820 921/1,102,732 1376/531,475 2066/2,377,613 7246/8,193,207
NoInfo HP-H5 59/ 34/ 38/ 39/ 147/ 317/
HP-H7 1/ 1/
LP-H10 1/ 2/ 3/
LP-H7 152/ 329/ 185/ 666/
LP-H9 2/ 1/ 1/ 1/ 5/
Total NoInfo 61/ 35/ 192/ 371/ 333/ 992/
Confirmed AI outbreaks with no record on the total number of cases and the species affected (Source: FAO 2015)
Using SIA to Understand the Dissemination of HPAI – The Way Ahead!
7
8 1 General Introduction

carbon standard is the PDB, where (13C/12C) is 0.0112372 and is based on a belem-
nite from the Pee Dee Formation. As with SMOW, the original PDB standard is no
longer available, but the IAEA provides Vienna-PDB with a similar R value.
Atmospheric nitrogen is the internationally recognized standard with an R value
(15N/14N) of 0.0036765. Lastly, the internationally recognized standard for sulfur is
CDT, the Canyon Diablo Troilite, with a value (34S/32S) of 0.0450045. Typically,
during most stable isotope analyses, investigators would not use IAEA standards on
a routine basis. Instead, laboratories establish secondary reference materials to use
each day that are traceable to IAEA standards and that bracket the range of isotope
ratio values anticipated for the samples.
Although ecologists refer to “isotope signatures or isotope fingerprints” the val-
ues obtained for a sample do not provide a unique fingerprint but a distinctive pro-
file; a more neutral notation is isotope value, rather than signatures.
The δ notation is derived as follows:

 Isotopic ratio sample 

 H (‰)    1   1000
0
 Isotopic ratio standard 

The right side of the equation is the measure of the light to heavy isotope (2H/1H)
and that ratio is multiplied by 1000 transform the values into whole numbers. The
isotope reference points were established many years ago and results reported can
be negative or positive (‰) relative to the accepted international standard. Thus, a
δH value of +150 (‰) means the sample has 150 parts per thousand (15 %) more
deuterium in it than the standard; while if the value were negative it would be 150
times less deuterium than the standard. Primary reference material is limited in
quantity so laboratories tend to use local standards calibrated against a reference
standard. The primary isotopic reference standard for hydrogen and oxygen is
Vienna Standard Mean Ocean Water (VSMOW).
It will be necessary to develop suitable procedures for monitoring wild birds to
detect HPAI, determine their origin and migratory pathway to estimate the duration
of their stay at the place of capture. This will require the application of detailed
standard operating procedures for feather and tissue collection, data recording, sam-
ple storage, and designation of a laboratory to carry out the SIA. Since tissue sam-
ples could come from birds suspected of carrying avian influenza virus it will be
necessary to install safeguards to prevent possible infection. Soft tissues could be
freeze-dried and both these and keratinaceous samples heated to 100 °C for 20 min
to destroy any viruses. Alternative methods would be to irradiate specimens at a
central location before making them available for study. Various innovative meth-
ods for virus detection should also be considered, including environmental sam-
pling to detect H5N1 virus in water, or faeces (Khalenkov et al. 2008; Cheung et al.
2009; Dovas et al. 2010) in locations where wild birds congregate. Other proce-
dures that will be useful could include identification of bird species using DNA
barcoding from feather or faeces (Lee et al. 2010) to provide better links to identify-
ing those birds that are truly implicated in dissemination of HPAI.
1.3 Using SIA to Understand the Dissemination of HPAI – The Way Ahead! 9

There are already available data on variation in stable isotope ratios across the
globe and how this is reflected in tissue samples from birds inhabiting different
regions. It might be necessary however to collect environmental samples from dif-
ferent locations if there is not sufficient data already available. Collection of wild
birds is a specialized procedure and it will be necessary to liaise with wildlife groups
skilled in this task, who are also able to provide basic data on the ecology of differ-
ent bird species. The FAO Wildlife Unit can provide inputs in this area through its
links with various wildlife organizations in order to carry out sampling with greater
efficiency.
Consideration needs to be given to which isotopes to analyse. While stable
hydrogen can be used to obtain information on the wider geographical location,
further details might be inferred on the local habitat by analysing carbon, nitrogen
and sulphur. Samples from keratinaceous tissues e.g. feathers and claws, will pro-
vide an isotope ratio of the place when they were grown. In blood, stable isotope
ratios could be used to determine timing of arrival on breeding or wintering grounds.
Isotope ratios in the blood relate to those in the current environment hence a differ-
ence between isotope ratios in blood and the environment would indicate newly
arrived birds compared with birds that had been staying in a particular location for
some time. If infected birds were to be found it should be possible to establish
where the disease was acquired – at the site or in a previous location. By under-
standing migratory movements it will be possible to predict risk and derive models
to show the spread of HPAI and identify areas that pose a significant potential for
being locations where the dissemination of HPAI is greater due to the particular
congregation of wild birds, H5N1 virus and domestic poultry.

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Chapter 2
Animal Migration Tracking Methods

2.1 Extrinsic Markers

The migration of birds has been studied for many years relying mostly on extrinsic
passive markers attached to individual animals at the point of capture, with the
expectation that a proportion of the marked individuals will then be identified in
another location at a different point in time. Over the past 100 years, the most wide-
spread approach has been through the application of markers such as leg bands,
neck collars, or dyes. Many millions of birds have been tagged in this way but
although this method has provided insights into migration, for the vast majority of
bird species examined the recovery rate is low. An alternative to these simple devices
is to use miniature transmitting devices – radio transmitters, radar and satellite
tracking – that serve as active markers and are small enough (<0.5 g) to be attached
to even small birds or mammals. The location of the marked animal can be inferred
by tracing the individual using a receiver, or by triangulation using several receivers.
Since the devices are miniaturized their range and battery life are restricted and they
can provide information over only a few kilometers. Radar technology has also
made useful contributions to studies on migration since it can provide information
on animal movements over considerable distances, but since radar installations are
fixed it is not possible to trace movements over the whole spectrum of migration
routes used by birds. The most significant advances in tracking migratory animals
have come from the use of satellite transmitters that allow highly accurate position-
ing of individual animals (Hiroyoshi and Pierre 2005; Whitworth et al. 2007; http://
www.fao.org/avianflu/en/wildlife/sat_telemetry.htm). Much of the globe is covered
by satellites so that animals can be monitored over thousands of kilometers. The
technique can only be used on relatively large animals as the weight of the smallest
transmitters is approximately 10 g, restricting their use to an animal weighing about
250 g, thereby excluding 80 % of the world’s birds and 70 % of the mammals. With
the exception of satellite transmitters all extrinsic markers require that individuals
be recaptured, re-sighted or move within a detector’s range at some time after initial

© IAEA 2016 11
G.J. Viljoen et al., Stable Isotopes to Trace Migratory Birds and to Identify
Harmful Diseases, DOI 10.1007/978-3-319-28298-5_2
12 2 Animal Migration Tracking Methods

marking. The probability of recapture depends on the number of observers, the

regions and habitats and the chances of success are low. In addition, extrinsic meth-
ods tend to be biased towards regions with a high likelihood of mark-recapture
(Hobson et al. 2004). A fundamental flaw in the use of an extrinsic marker is that it
provides information only on the marked individuals. Geolocators and satellite
tracking rely on small sample sizes and the devices may affect the behavior of the
marked bird (Stutchbury et al. 2009). Extrapolating the findings from one individual
to a whole population depends on how representative the marked individuals are. A
single recovery or satellite track may not reveal what the population is doing.

2.2 Intrinsic Markers

The major advantage of an intrinsic marker is that it is not necessary to capture and
mark an individual in advance, and every capture provides information on the origin
of that animal. There is therefore, less bias than with extrinsic markers, where the
location of the origin is dependent on accessibility; for example, remote areas might
not be easily included in any migration study. Man-made contaminants such as
dioxins and methyl mercury or other heavy metals could provide a means of infer-
ring migratory origins as exposure to them varies throughout the world. Exposure
of migratory animals to parasites or pathogens also varies geographically and could
be used for determining movements but little research has been conducted as to how
such markers might assist in deciphering migration patterns. In a study of Sharp-
shinned Hawks (Accipiter striatus) in New Mexico, stable-hydrogen isotope ratios
of feathers were estimated and blood was collected to quantify the prevalence and
intensity of haematozoan infection in the birds (Smith et al. 2004). Twenty four
percent of the birds were infected with Leucocytozoon toddi, 37 % with
Haemoproteus elani, and 5 % with H. janovyi. This was the first documented occur-
rence of H. janovyi in North America in these hawks and the stable-hydrogen iso-
tope analyses indicated that only birds originating from south-western North
America harboured H. janovyi which could be of significance in regard to biogeog-
raphy of that parasite species. The isotope studies also showed that birds infected
with Haemoproteus spp were more widespread geographically than those with
Leucocytozoon species. Another type of intrinsic markers that could potentially
track animal movement geographically is the trace elements. Animals acquire quite
distinctive chemical profiles relative to a particular geographical location and retain
that profile when they move to another area. Trace element profiles have been used
to determine if populations of breeding birds originate from different winter loca-
tions (Sze´p et al. 2008). However, there is little information on how trace elements
vary over the globe, so it is of only limited potential. Stable isotope tools to trace
animal migration have been used extensively in ecological studies but have not yet
been utilized in work on the dissemination of transboundary animal diseases. There
is potential for greater understanding the epidemiology of HPAI in relation to the
migration of wild waterfowl by using stable isotope studies to identify the origins and
stopover points of birds suspected of carrying HPAI. A summary of the advantages
and disadvantages of extrinsic and intrinsic methods is given in Tables 1.1 and 2.1.
2.2 Intrinsic Markers 13

Table 2.1 Extrinsic markers for tracking animal migration

Technique Advantages Disadvantages
Extrinsic 1. Can apply to a 1. Requires capture/recapture
broad range of
animals
2. High spatial 2. Biased towards initial
resolution captured population
Phenotypic variation 1. Inexpensive 1. Low resolution
2. Can be used for 2. Not applicable to all
historical specimen species
Banding/Marking 1. Inexpensive 1. Low recovery rates <0.5 %
2. Gives 2. Data must be acquired
information on start over many years
and end of 3. Only few banding stations
migration across the world
4. Marking and recovery are
biased towards the banding
station location
Radio-transmitter 1. Gives precise 1. Low range
locations
2. Gives precise 2. Expensive
trajectory 3. Transmitters might affect
behaviour
Satellite transmitters 1. Precise animal 1. Expensive
trajectory 2. Only for use on large
animals
3. Transmitters might affect
behaviour
International Cooperation for Animal 1. Transmitter 1. High start-up investment
Research Using Space ICARUS (www. allows many
IcarusInitiative.org) different species to
be tracked
2. Can track 2. Technology not proven
individuals over the
globe
Satellite tracking to determine the 3. Inexpensive after 3. Transmitters might affect
spread the spread of infectious diseases start-up costs behaviour
Passive radar 1. Coverage over 1. Coverage only from
large geographical existing stations or portable
region instruments
2. Inexpensive 2. Poor ability to determine
3. Individuals need species and individual
not be captured movements
Transponders 1. Small size 1. Requires external
activation
2. Low range
3. Coverage only form
existing stations or portable
instruments
(continued)
14 2 Animal Migration Tracking Methods

Table 2.1 (continued)

Technique Advantages Disadvantages
Geolocation tags 1. Produces animal 1. Individuals must be
trajectory captured to download data
2. Low weight 2. Accuracy relative to
3. Inexpensive satellite tags low
relative to satellite
tags

Studies of avian migration and population structure take advantage of geographi-

cal variation in stable isotope ratios, in particular the ratio of the two stable isotopes
of hydrogen in precipitation. In North America, for instance, the ratio of growing
season precipitation decreases from −20‰ VSMOW (Vienna Standard Mean
Ocean Water) in Florida to −140‰ VSMOW in north-western Canada (McKechnie
2004). Because the deuterium of precipitation is incorporated into avian tissues via
food webs and feathers are metabolically inert once grown, the deuterium values of
feathers reflect the latitude at which they were grown. In species that moult prior to
migration, feather deuterium can be used to identify the latitudinal origin of indi-
viduals, and hence link breeding and wintering grounds. Isotopic data can also be
combined with information on capture dates or ring recovery. Stable isotope studies
require a detailed knowledge of a species’ moult schedule, and the assumption that
the isotope signature of feathers reflects that of food webs in the region where the
feather is grown must be verified. The technique has the best potential where there
is a good spatial variation in the environmental locations in which the particular bird
species is found.
The basic requirements in the use of stable isotopes are that:
• The characteristics of the environment through which the animal of interest
moves are known. The term used to describe the mapping of large-scale and
spatiotemporal distributions of stable isotope ratios in the environment and ani-
mals is an isoscape or isotopic landscape (Bowen and West 2008).
• Isotopic values in animal tissues can be discriminated from baseline isoscape
values,
• The time period for acquisition into a particular animal tissue is understood. For
instance, turnover of isotope will be greater in metabolically active tissues than
in tissues with a slow, or no, turnover e.g. feathers (Table 2.2).
Just a handful of these stable isotopes are of any practical interest to studies on
animal migration. These are the “light isotopes” of H, O, C, N, and S that are pres-
ent in all components of the biosphere (plants and animals), the hydrosphere (water)
and the atmosphere (gaseous O2, N2 and H2O). These five elements comprise the
bulk of all animal tissues (Table 2.3). These elements and their isotopes circulate in
the biosphere to produce characteristic isotope distributions globally. Large pools of
these elements provide stability in the overall isotope circulation e.g. in the ocean,
for hydrogen and oxygen isotopes, inorganic carbon pool in the ocean for carbon
2.2 Intrinsic Markers 15

Table 2.2 Intrinsic markers for tracking animal migration stable isotopes
Technique Advantages Disadvantages
Intrinsic 1. Not biased to initial capture 1. Biased to final capture population
population
2. Less labour intensive than 2. Lower resolution than extrinsic
most extrinsic methods methods
Contaminants 1. Potentially high spatial 1. Lack of distribution maps
resolution 2. Transport of contaminants could
give unreliable geographical signal
Parasites/Genetics 1. Several possible markers 1. Species specific
2. Low resolution
Trace elements 1. Measurement of a large 1. Lack of distribution maps
number of elements
2. Potentially high spatial 2. Expensive
resolution 3. Requires more sample tissue
4. Requires tissue that is
metabolically inactive after growth
5. Spatial resolution could be too
high
6. Some elements may be integrated
into inactive tissue after growth is
complete
Stable isotopes 1. Inexpensive 1. Low resolution
2. Not species- or 2. No base maps
taxon-specific
3. Several isotopes can be 3. Ideal tissue is metabolically
combined to increase spatial inactive after growth
resolution 4. Turnover rate of elements in
active tissue is unknown
5. Interpretation may be complicated
by animals’ physiology

isotopes, and sulphate in the sea for sulphur isotopes and the atmospheric reservoir
for nitrogen. The presence of the stable isotopes varies widely in nature. All of the
light isotopes have a common or abundant form, e.g. 1H; 99.985 % and a more rare
“heavier form”, 2H; 0.015 % (Table 2.3). The abundance ratios of these isotopes
vary because of physical and chemical processes they undergo in nature and it is
these variations that enable the use of stable isotopes in tracing migration. As it is
difficult to measure the precise concentrations of the isotopes in samples, measuring
the relative differences in isotopic ratios between a sample and a reference by means of
a mass spectrometer is the way in which data are acquired. Since gas source isotope
ratio mass spectrometers (IRMS) are used to measure light isotopes it is not possible
to measure isotope ratios directly from a tissue sample – feather, blood, muscle,
claw, hair – instead, the sample must first be combusted to form a gaseous analyte
in an elemental analyzer, a gas chromatograph or a laser and the resulting gas can
then be used to measure the isotopic ratios relative to a calibrated reference gas of
16 2 Animal Migration Tracking Methods

Table 2.3 Dry weight % abundance of light stable isotope ratios of interest in determining
migratory connectivity in tissues
Element Weight (%) Isotope ratios δ Range (0/00) Mass required (mg)
13
Carbon 30–40 C/12C 05 to −65 0.2–1.5
18
Oxygen 27–40 O/19O +10 to +30 0.2–00.5
15
Nitrogen 12–19 N/14N −2 to +25 0.5–1.5
2
Hydrogen 6–8 H/1H −250 to +90 0.1–0.4
34 32
Sulphur 5–20 S/ S −20 to +30 1–2

Table 2.4 Average terrestrial Element Isotope Abundance (%)

abundances of the stable 1
Hydrogen H 99.985
isotopes of major elements of 2
interest in ecological studies H 0.015
12
the carbon cycle Carbon C 98.89
13
C 1.11
14
Nitrogen N 99.63
15
N 0.37
16
Oxygen O 99.759
17
O 0.037
18
O 0.204
32
Sulphur S 95.00
33
S 0.76
34
S 4.22
36
S 0.014

the same type. Development of IRMS equipment has led to automatic sample pro-
cessing to obtain multiple isotope assays from a single sample, on decreasing sam-
ple size and improving throughput rates. IRMS can measure isotopic ratio differences
to the sixth decimal place or ±0.01 % (Table 2.4).
The carbon cycle involves active exchanges of CO2 among the atmosphere, ter-
restrial ecosystems and the surface ocean. The 13C value of atmospheric CO2 is
decreasing in response to inputs of 13C depleted CO2 from fossil fuel plus biomass
burning and decomposition. Over the past 100 years the decrease may have been
almost 1‰, from about −7‰ to −8‰.
Depending on how plants’ photosynthesis process is materialized, they are clas-
sified in two large groups, C3 and C4, with very different values for δ13C. In the C3
group, the first photosynthesized organic compound has 3 atoms of carbon while in
group C4, there are 4. Most plants (85 %) (E.g. trees and crops) follow the C3 pho-
tosynthesis pathway and have lower values of δ13C, between −22‰ and −30‰. The
remaining 15 % of the plants are of type C4. The majority are tropical herbs and
have high values of δ13C, between −10‰ and −14‰ (http://homepage.mac.com/
uriarte/carbon13.html).
Carbon uptake by the dominant C3 plants on land involves a net fractionation of
about 20‰ between the atmospheric CO2 and plant biomass (−28‰). Carbon
uptake by C4 plants, mainly tropical and salt grasses, involves a small net fraction-
2.2 Intrinsic Markers 17

ation of about 5‰. Soil organic matter globally contains several-fold more carbon
than either the atmosphere or living plant biomass and is similar or slightly enriched
in 13C in comparison with the dominant vegetation. The exchange of CO2 between
the atmosphere and the surface of the ocean involves an equilibrium chemical frac-
tionation between atmospheric CO2 (−8‰) and the total CO2 in surface ocean water.

2.2.1 The Nitrogen Cycle

Most nitrogen in the biosphere is present as N2 gas in the atmosphere. This massive
reservoir is well mixed with an isotope composition that is essentially constant at
0‰. Nitrogen in most other parts of the biosphere also has an isotope composition
near the 0‰ value, from −10 to +10‰, primarily because the rate of nitrogen sup-
ply often limits reactions such as plant growth and bacterial mineralization. Under
these conditions all available nitrogen can be consumed, without regard to isotope
content and with no overall isotope fractionation. Thus, slow rates of N supply and
limiting amounts of substrate N are often important for understanding nitrogen iso-
tope distributions. Some cumulative and large fractionations do occur in the nitro-
gen cycle. Lakes appear more variable in isotope composition than the large world
ocean. Large isotope contrasts might be expected between lakes in which primary
production is limited by N (little fractionation by phytoplankton) versus P (abun-
dant N → large possible fractionations during N uptake by phytoplankton). Where
phytoplankton have different 15N values than terrestrial vegetation, the nitrogen iso-
topes may function as source markers for autochthonous and allochthonous organic
matter.

2.2.2 The Sulphur Cycle

Sulphate in the ocean is a large well-mixed sulphur reservoir whose isotope compo-
sition is 21‰ heavier than primordial sulphur in the earth and solar system at large.
Fixation of sulphate by phytoplankton occurs with a small isotope effect, but sul-
phate reduction in marine sediment occurs with a large effect of 30–70‰. Over
geological time, and partially in response to global-scale fluctuations in sulphate
reduction activities, the 34S values of oceanic sulphate have varied from about +10
to +33‰. Uplift and preservation of marine sedimentary sulphides and sulphate-
containing evaporates on land have produced a patchwork of sulphur in terrestrial
environments, each with different 34S values for bedrock sulphur. Thus, large 34S
ranges must be assigned in general sulphur cycle diagrams. In spite of this, conti-
nental vegetation seems to average near +2 to +6‰ over large areas and is quite
distinct from the +∼17 to +21‰ values of marine plankton and seaweeds. The sta-
ble isotope composition of sulphur entering the atmosphere can also be quite
variable.
Another random document with
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 Nouna, who was playing at packing, having been busy for twenty
minutes with a delicate Sèvres tea-pot and some yards of tissue
paper, let the china fall from her hands at these words, in a torrent of
indignation. She scarcely glanced at the broken fragments on the
floor, as she burst forth with great haughtiness in the high-flown
language she habitually used when her passions were roused:
“Indeed! Does then the wife of this miserable little wooden soldier
think the granddaughter of a Maharajah unworthy to bear the light of
her eyes? We will see, we will see. Perhaps she is a little too
imperious; there may be powers in the earth greater than hers! I will
write to my mother, who has never yet failed to fulfil my wishes, and I
will tell her to search if she can find means to humble this proud lady
of the fens, so that she may sue to me to receive me in her house,
heathen, foreigner, though I am!”
And with a superb gesture Nouna signified her contempt for the
ironical laughter her husband could not restrain.
“Oh, little empress,” he said, good-humouredly, “you will have to
learn that all magicians have limits, and that even a mother so
devoted as yours can’t carry out all the freaks that enter into one little
feminine head. The very king of the black art could not move Lady
Florencecourt!”
“The king! Perhaps not, because he is a foolish male thing,”
retorted Nouna coldly, “but what my mother wills to do she does, and
I trust her.”
And she would not suffer any further word on the subject.
George was in the depths of his heart not without a little anxiety
about this Norfolk visit. Unconventionality is so much more
unconventional in the country, where every trifling detail in which a
man differs from his neighbours is nodded over far and wide as a
sign of mental aberration, while in the case of a woman it is held to
warrant even graver doubts. Nouna herself was in the highest spirits
at the prospect; delightful as life in London was, a change after five
weeks of her new home was more delightful still. She had had made
for the occasion a varied assortment of dainty white frocks, of the
kind that charm men by their simplicity, and women by their
costliness, and a white costume with fine lines of red and gold, for
yachting on the broads, which might have carried off the palm at
Cowes. Nouna had the instinct of dress, a regal instinct which
revelled in combinations and contrasts, in forms and folds, which
everyday English women might admire or marvel at, but copied at
their peril. She travelled down, the day being cool, in a Spanish cloak
of mouse-grey velvet, lined with ivory silk, and fastened with clasps
of smoked pearl and silver. On her head she wore a cap of the same
colours. The milliner, an artist spoiled by ministering to a long course
of puppets, was aghast at the order, and suggested that it would
make Mrs. Lauriston look, well—er—brown. Nouna replied, with a
great sweep of the eyelashes, that she was brown, and she should
be sorry to look anything else. And indeed her beauty was seen to
great advantage in this original setting, and its tints might pleasantly
have suggested to the fanciful brown woods in the haze of a grey
October day.
They reached Gorleth, the nearest railway-station to Maple Lodge,
at half-past four. Sir Henry and his daughter Cicely were on the
platform, Cicely in a short grey riding habit, looking in this practical
garment a thousand times handsomer and more captivating than she
had done in her most brilliant ball-dress, according to the wont of her
countrywomen, who, from the royal ladies downwards, never look
worse than when dressed solely with the view to charm.
Let it be acknowledged once for all: the Englishwoman, to her
credit be it said, is a riding animal, a walking animal, a boating
animal, a cooking animal, a creature fond of hard work and hard
play, full of energy and capabilities for better things than the piano-
strumming and Oxford local cramming which are now drummed into
her so diligently. But her social qualities are so poor as to be scarce
worth cultivation unless some better methods be discovered than
those now in vogue. Her dancing is more vigorous than graceful, her
conversation is inane, her deportment in full dress uneasy and
deplorable, and her manner at social gatherings where no active
muscular exertion is required of her, dull and constrained. Ten girls
are handsome and attractive in a boat or on the tennis-ground to one
who at an “at-home” or a dance is passable enough to make a man
want an introduction. The metropolis has the pick of the market, if
the term may be allowed, in marriageable maidens as in flowers and
fruit. But all alike lose their freshest, greatest charm when they are
plucked from their natural setting of country green.
George had an inkling of this truth as he helped his tiny wife out of
the railway carriage, amidst the stares of a crowd of country market-
folk, who gaped as they would have done if a regulation fairy, gauze
wings, wand and all, had suddenly descended down the wide
chimney on to their cottage hearth. He should love and admire her
whatever she did, but he wanted her to sway the sceptre of conquest
over all these friends at Willingham and Maple Lodge, and his heart
ached with fear lest a breath of disapproval should touch her, lest
she should appear to any disadvantage under such new conditions.
She herself, happily, was tormented by no such fear. She ran up to
Sir Henry, who was dressed in a vile suit of coarse mustard-coloured
stuff, a common little hat on his head, and a broad smile of
recovered bliss on his face, looking as no self-respecting farmer
among his tenants would have dared to look, and rejoicing in his
escape from town and tight clothes.
“Why, you little town-mouse,” he said, laughing good-humouredly
as he looked down on the tiny lady, “I don’t know how you will live
down here. We shall have to feed you on butterflies’ wings and dew-
drops; I should think a mouthful of plain roast beef would kill you.”
“Oh no, it wouldn’t, Sir Henry,” cried Nouna, distressed and
offended by these doubts cast on her accomplishments. “I eat a
great deal, don’t I, George?”
“Well, more than one would expect, to look at you,” admitted her
husband, remembering the fiasco of the wedding breakfast.
“Besides,” said Nouna astutely, “everything that one eats comes
from the country. The town produces nothing but soot; perhaps you
think I live upon that, and that’s what made me half a black woman.”
The genuine black woman, Sundran, was meanwhile creating a
great sensation; so that, to save her from the rustic wit, which made
up in blunt obtrusiveness what it lacked in point, she was packed
with her mistress inside the Millards’ one-horse brougham, which,
like all their surroundings in their country retreat, was almost
ostentatiously modest and even shabby. George was content
enough to share the coachman’s seat.
 “I thought the maid would sit outside; I hadn’t reckoned upon your
bringing a lady of so striking a complexion, George,” said Sir Henry
apologetically. “The old carriage is such a lumbering concern that I
thought the brougham would be quicker, and there’s a cart for the
luggage.”
George laughed. “If I had my choice I’d go on the cart,” said he. “I
am yet unspoiled by my promotion to matrimony.”
It was a pleasant drive over the flat country, too marshy to be dry
and burnt up even in summer. Sir Henry and his pretty daughter kept
pace with the carriage, and flung breezy commonplaces at their
guests with smiling, healthy faces that made their conversational
efforts more than brilliant. Nouna peeped out like a little bird at the
flat green fields and the pollard willows with an expression which
seemed to say that she had quite fathomed the hidden humour of
the whole thing.
“I like the country,” she called to Cicely with an exhaustive nod, as
if she had lived in and loved the fields for years.
And at sight of the Lodge itself she grew rapturous.
Sir Henry Millard’s modest country residence was nothing more
than a fair-sized one-storied white cottage, close to the road, from
which it was separated only by a little garden just big enough to
contain a semicircular drive, a small half-moon lawn, and two side-
beds full of roses. A stone-paved verandah ran the whole length of
the house, and a hammock swung between two of the supports of
the green roof, in what would have been glaring publicity if there had
ever been any public to speak of on the quiet road in front. It would
have been rather a pretty little place if Sir Henry, to meet the
requirements of his family, had not preferred enlarging it by adding at
the back various hideous red brick wings and outbuildings of his own
designing, to the more reasonable course of taking a larger house.
The pleasure of conceiving and superintending these original
“improvements” had indeed, while it lasted, been the most unalloyed
joy of Sir Henry’s simple life; to worry the architect, who had had to
be called in at the last to put a restraining check on Sir Henry’s
inspirations, which threatened to dispense with the vulgar adjuncts of
passages and staircases; to test the building materials, samples of
which lay about the sitting-rooms for days; above all, to do a little
amateur bricklaying during the workmen’s dinner hour—were joys
the mere memory of which thrilled him more than any recollection of
his honeymoon.
Whatever the architectural defects of the house might be, Nouna
had nothing but admiration for it. The tiny little hall; the box-like
drawing-room to the right, with high glass cupboards on each side of
the fireplace containing apostle spoons, old china bowls, fragments
of quartz and the like; the bare-looking dining-room to the left,
furnished as plainly as a school-room, and even the bake-house
which led out from it, all enchanted her by their novelty; while the
bedroom up stairs, ten feet square, into which she was shown, put
the climax to this deliciously new experience, and made her feel, as
she expressed herself to her husband, “that she wished she had
married a farmer.”
To George’s delight she ran down stairs within twenty minutes of
her arrival in the simplest of white muslin frocks, with a wonderful
scarlet and gold sash. But he had no time to congratulate her on her
good sense in dressing so appropriately before she was off, in a
huge garden-hat taken with instinctive knowledge of what was most
becoming from a collection in the hall, to see the farmyard—Sir
Henry’s pride. They made an odd pair—the broad-shouldered, solid-
looking country gentleman, in his rough suit, and the small airily-clad
person who varied her progress by occasional ecstatic bounds in the
air, which made the ends of her sash swirl in the breeze like the
wings of some gorgeous butterfly. George and the girls, with Lady
Millard, followed much more sedately. When, after due admiration of
cows and horses, pigs and poultry, they all returned to the verandah,
fresh objects of interest presented themselves in a pretty group of
riders at that moment climbing the hill upon which the lodge stood.
“Uncle Horace!” cried the girls, as Nouna recognised in the eldest
of the party Lord Florencecourt. He was accompanied by two pretty
boys of about eight and ten on ponies which they already managed
as if boy and pony had been one creature.
“How Horace worships those boys!” muttered Sir Henry enviously.
Charlotte had run down to open the gate, and there was much
clatter of lively greeting. Lord Florencecourt, though he seemed
happier down here with his children than he had been in town,
showed his old constraint with Nouna. It was therefore with great
surprise not only to the young husband and wife, but to their host
and his family that they learnt the object of his visit.
“You see I haven’t lost much time in paying my respects, Mrs.
Lauriston,” he said, speaking in a lively tone, but with an ill-
concealed reluctance to meet her eyes. “Those girls would like to
flatter themselves that my visit is for them, but they are all wrong.”
“Never mind, uncle, Regie and Bertie come to see us,” cried Ella,
giving a kiss to the youngest boy.
Lord Florencecourt continued: “The fact is, Mrs. Lauriston, we
know that you will be so run after down here, that when you have
been seen a little there will be no getting hold of you. So my wife
sent me to ask you and George to stay with us from Friday to
Monday the week after next. Mr. Birch, our member, will be there,
and we thought as he has come to the front so much lately you might
like to meet him.” Nouna stole a triumphant glance at her husband,
and the girls, who were near enough to hear, could not forbear little
unseen eyebrow-raisings of astonishment. He went on: “Lady
Florencecourt will call upon you on Monday, but she thought it best
to send her invitation at once to make sure of you.”
“It is very kind of Lady Florencecourt; I shall like to come very
much,” said Nouna, who was brimming over with delight and
triumph. “Only I don’t think I could do much to entertain a rising
member of Parliament. I can’t talk politics; but perhaps he’d like to
learn to make cocked hats out of newspaper, and then he can
amuse himself when the other members are making dull speeches.”
“I’m sure he’d like it immensely if you will teach him,” said Lord
Florencecourt, with cold civility, which would have damped frivolity
less aerial than Nouna’s.
The girls thought Lady Florencecourt must have been bewitched
thus to transgress her own well-known rule of ignoring any stranger
whose pedigree was not at her fingers’ ends. She had, besides,
gone so far as to gibe at her brother for admitting “a loose-mannered
young woman of unknown and questionable antecedents”—as she
styled young Mrs. Lauriston—into the society of his daughters. And
now she was sending a pressing invitation by the mouth of her
husband, whose prejudice against the interloper was hardly
concealed! Decidedly Nouna had a dash of Eastern magic about her.
Meanwhile the young lady herself was troubling her head very little
with the problem. She was much struck with the blue eyes and curly
dark hair of the younger of the two boys, and bending down to him
with her little head perched on one side in the coquettish manner she
used alike to man, woman, child or animal, she asked with a smile
what his name was.
“Allow me to present him with proper ceremony,” said Ella
playfully. “Permit me to introduce you,” gravely to her small cousin,
“to Mrs. George Lauriston. Mrs. Lauriston,” turning to the lady, “the
Honourable Bertram Kilmorna!”
She had scarcely uttered the last word when Nouna shot up from
her bending attitude as if at an electric shock, and fixed her great
eyes, wide with bewilderment and surprise, on Lord Florencecourt,
who was standing behind Ella and his son, near enough to hear
these words and to see their effect.
“Kilmorna!” she repeated in a whisper, still looking full at the
Colonel, whose rugged face had grown suddenly rigid and grey.
Then, without further ceremony, she ran away to her husband, who
was talking to Lady Millard at a little distance.
“George, George!” she said in a tumultuous whisper, her face
quivering with excitement, “I don’t want to go to Lady
Florencecourt’s; tell him I don’t want to go!”
“Why, what’s this? How has the Colonel offended you?” asked
George laughing.
“He hasn’t offended me at all. Only I’ve changed my mind. I know I
—I shouldn’t like Lady Florencecourt. I’d rather not go.”
 But as George insisted that it was impossible to break an
engagement just made, without any reason, she broke from him with
an impatient push, and disappeared into the house just before Lord
Florencecourt, who had abruptly discovered that he was in a hurry to
be off, took his leave. Ella prevented George from fetching his wife
out.
“It is only some little caprice of hers,” she said persuasively, not
guessing that there was any mystery in the matter, and considering
the young bride’s conduct as the result of some girlish freak. “I think
she was offended because uncle didn’t introduce the boys to her.
She will be all right if you leave her alone a few minutes.”
But George was not unnaturally annoyed at his wife’s rudeness,
and he followed her into the little drawing-room, where he found her
with her nose flattened against the window, staring at Lord
Florencecourt’s retreating figure. She had no explanation to give of
her conduct, but persisted in begging him not to take her to
Willingham. As he remained firm on this point, and continued to
press her for her reasons, she grew mutinous, and at last peace was
only made between them on the conditions that she would go to
Willingham if he would not tease her with any more questions.
And George had to be content with this arrangement, being above
all things anxious to learn the meaning of the miraculous change of
front on the part of Lady Florencecourt and her husband.
 CHAPTER XIX.
At dinner at Maple Lodge on the evening of their arrival, George
Lauriston and his wife met the gentle Dicky Wood, who had come
down the day before, and spent the afternoon riding with the son of
Sir Henry’s steward. Nouna was much pleased by this compliance
with her wishes, and showed her appreciation of it by flirting very
prettily at dinner with the young guardsman. Later in the evening she
held in the verandah a little court, and chanted them some half-wild,
half-monotonous Indian songs in a tiny thread of sweet voice, with
some plaintive low notes that lived in the memory. And George, who
was standing with Ella some yards away from the rest of the group,
felt thrilled through and through by the weird melodies, and liked to
fancy that in these native songs of hers the soul-voice, that, in the
tumultuous life of emotions and sensations in which she found her
happiness, had small opportunity to be heard, forced up its little note
and promised a richer fulness of melody by and by.
It was not by the man’s choice, but the girl’s, that he and Ella
found themselves together. At the present time there was only one
woman in Lauriston’s world, and in his absorption in his wife the
ungrateful fellow was incapable even of feeling his old friendly
pleasure in Ella’s society. Her interest in him, on the other hand, as is
the way with that splendid institution for the comfort and consolation
of man—plain women, had grown tenfold stronger since he had
lowered himself to the usual dead level of his foolish sex, by
marrying through his eyes. To Ella this downfall was quite tragic; she
had thought and hoped so much for him; he had feeling, sense,
ambition, was, in fact, not the mere beautifully turned out figure-head
of a man who, under various disguises of light or dark complexion,
slim or heavy build, was continually saying to her the same
commonplaces, betraying to her the same idea-less vacuity, at
dinner, ball, and garden-party. Yet here he was, bound for life, and by
his own choice, to a beautiful pet animal, with all the fascinating
ways of a kitten, who could gambol and scratch, and bask in warmth
and shiver in cold, and whom nevertheless he undoubtedly
worshipped. Ella, whose mind was of an intellectual cast, and in
whom the passions had as yet only developed in an ardent but hazy
adoration of dead-and-gone heroes, very naturally underrated the
strength of one side of a man’s nature, and was cast down when the
creature whose sympathetic comprehension of her highest
aspirations had made her raise him to a demi-god proved to be in
truth only a very man. She fancied, poor child, that he showed
deterioration already; when she reminded him at dinner that she had
not yet returned a book of Emerson’s he had lent her, George
laughed carelessly, and said he had forgotten all about it.
“Don’t you remember you particularly advised me to read the
articles on ‘Goethe’ and ‘Napoleon’?” she asked rather acidly.
“Oh, yes, they’re very good,” said he, with a man’s irritating
frivolity, smiling at his wife, who was shutting one eye and holding
her glass of claret up to the light, in imitation of an elderly
connoisseur, for the amusement of Dicky. Then, perceiving in a
pause that he had offended Ella, he hastened to say penitently: “I
haven’t done much reading lately; but you have, I suppose; you are
always so good.”
“I don’t read because I am good, but because I like it,” she
answered coldly.
And George, reflecting on the oddity of Ella’s trying to improve him
as he had tried to improve Nouna, had taken the snub meekly as a
bolt of retributive providence.
But when she got an opportunity of speech with him alone in the
verandah, in a rather melancholy and remorseful frame of mind, she
“had her say” after her sex’s fashion.
“One mustn’t expect you to be the same person that you were
three months ago, George,” she began, with a very humble,
deprecating manner. “Otherwise I would ask you why we don’t hear
of your coming to the front as a writer, as we heard then there was a
probability of your doing.”
George laughed with the same maddening indifference to his
deterioration, and asked if he might smoke. With a cigarette between
his lips, flourishing before her eyes the privilege of a man, he felt
more of a man’s commanding position.
 “I haven’t come to the front,” said he, “because I haven’t made any
steps at all, either forward, backward, or in any direction. I’ve been
lazy, Ella, miserably, culpably lazy, and if my great thoughts have not
yet stirred the world, it is no doubt only because they have not been
committed to paper.”
“Oh, if you are satisfied, of course that is everything. Ambition, I
see, is not the great, never pausing, never ceasing motive-power
that we poor foolish women are taught to believe; it is a pretty whim,
to be taken up alternately with a fit of smoking, or mountain-climbing,
as we girls change about between tennis and tatting.”
“Not quite, Ella,” said George, doing her the justice to grow serious
when he saw how deeply and unselfishly she was in earnest.
“Ambition does not die for lying a short time hidden by other feelings;
and surely even if it loses a little of its bitter keenness, it gains by
being no longer wholly selfish.”
“A beautiful answer, at least. And no doubt contentment is better
than ambition.”
“I don’t know what contentment is, except by seeing it in the faces
of cows and pigs. No passion could be stifled by such a tepid feeling
as that. I am not contented, I am happy. So will you be some day,
and you will let your bright wits rest a little while, and you will
understand.”
Understand? No, she felt that was impossible, as she looked down
at the big, handsome man sitting on the hammock below her, his
eyes bright, not with serene, but with ardent happiness, content to
bend all his faculties to the will of a creature whom he must know to
be his inferior in every way. She did not wish to understand such a
decadence as that.
“Then you will give up all idea of writing?”
“No. I am more anxious to distinguish myself than ever, as things
have turned out. A man who suddenly finds himself to be married to
a rich wife feels as if he had got off at a false start, and is put at a
disadvantage. But so far I own my wife has taken up all my time. You
see, she didn’t know she was going to be rich any more than I did,
and being hardly more than a child, she wants as much looking after
at first as a baby at the edge of a pond.”
 “And this is the sort of woman who gets a man’s best love!”
thought Ella half bitterly, half disdainfully.
“And of course you choose her friends for her,” suggested Ella, not
quite hiding her feeling.
“I can’t quite do that, yet at least,” said George. “Nobody but all of
you has got further than acquaintance yet.”
“But of course you are very particular about those acquaintances?”
Decidedly Ella was in her most disagreeable mood to-night.
“I do my best,” said he briefly.
“And of course it’s all nonsense about the smoking-parties, and
Captain Pascoe being there nearly every night.”
George felt a shock. Mentioned in that manner, the evening calls
of his friends, the admittance among the callers of a man whom he
cordially disliked but whom he had no grounds strong enough for
insulting, were heavy accusations.
“I see my own friends as freely as I did when I was a bachelor,
certainly,” said he, cold in his turn. “Nouna is too sensible to prevent
that. As for Captain Pascoe, he has not been in our house more than
three times at the outside.”
Ella dared not say more on this subject, even if she had had more
to say. She looked out at the swallows, flying low over the young
trees of the plantation on the other side of the road, and asked
musingly:
“Do you like being rich?”
“It’s not bad for a change,” answered George philosophically.
“I hate it. I always feel with papa, so glad to shake off the big
house and the footmen and the feeling that the great human world is
surging round without touching you, and to get back to my tiny room
where I can almost water the plants in my window without coming in
at the door, and to the farm and my pensioners that I take tracts to.
They never read them, but it is quite as much a matter of etiquette to
leave them as it is to make calls in town, and they are dreadfully
insulted if I forget.”
“But you’ve always been well off?”
“Yes, but that doesn’t make any difference. Money rolls together in
such ugly fashions. Look at mamma’s. When her father made his
millions, thousands of people were ruined. Well, you know, that’s
horrible!”
“They chose to speculate, remember. They must have known no
lottery has all prizes.”
“It’s hideous to think of, all the same. On the other hand, if your
property descends to you by a long line of greedy land-scrapers, you
know it has grown in value because other people’s has decreased,
and that your tenants have to pinch themselves to make up your
handsome rent-roll. And you haven’t even done the wretched work
for it that the speculator has done to get his!”
“It’s lucky all capitalists are not so soft-hearted, or there’d be an
end to enterprise, which by the by is brother to your god Ambition.”
“Oh, I’m not making preparations for re-organising the universe,
only lifting up a little weak mew of discontent with my corner of it.
And your wife’s money: is it the result of a robbery of recent date,
like ours, or plunder that has been rolling down for generations, like
Lord Florencecourt’s?”
“Well, really, I’ve never put it to her trustees in that way, and, now I
think of it, why I really don’t know. But as Nouna’s father was a
soldier, and there’s very little loot to be got in our days, I expect it
has rolled down.”
“And you don’t really care how it was got together?”
“Yes, I do, now I think of it. But to tell you the truth, the lawyers
have managed things so easily for us that all we’ve been called upon
to do is to spend the money, a very elementary process.”
“What a strange thing!”
“Why? By the by, so it is, when one comes to think about it. It’s
altogether contrary to one’s personal and traditional experience of
lawyers.”
“When mamma married,” said Ella, pursuing her own train of
thought, “her money was tied up and fenced round with as many
precautions as if poor dear old papa had been a brigand. He often
laughs about it, and says she couldn’t buy a pair of gloves without a
power of attorney. So that it really does seem very astonishing.”
“It does,” assented George, who, never before having had
experience of money in any but infinitesimal quantities, had been
much readier to take things for granted than was this granddaughter
of a Chicago millionaire.
“What would you do, George, if you found out it had been made by
supplying bad bayonets to the English army, or anything like that?”
she asked, half-laughing, but not without a secret wonder whether
this easy-got gold would turn out to have unimpeachable
antecedents.
The question gave George a great shock. He jumped up from the
hammock across which he had been sitting, with a white face.
“Good heavens, Ella! What makes you say that?” he asked in a
low voice, each word sounding as if it were being ground out of him.
“Don’t take it like that,” said she nervously, almost as much moved
as he, and impelled by his strong feeling to be more impressed than
she had been at first with her own surmise. “I only suggested—it
came into my head—I don’t know anything about it.”
“Oh, well, you shouldn’t say things like that, you know, Ella, even
in fun. The mere suggestion gives one such an awful shock. It’s like
cold water down one’s back,” said he, trying to laugh.
“I didn’t mean it, indeed,” said she, quite unable to take a jesting
tone. “As if one would say a thing like that in earnest! I never
guessed you would think twice about a foolish speech like that!”
But they both felt uncomfortable; and both were glad when
George, noticing that Dicky Wood was standing near anxious to get
a word with the jolly nice girl Ella, but much too diffident to come
forward at the risk of intruding unwelcomely upon a tête-à-tête, drew
him into their group by asking if he had been in Norfolk before.
And so both George and Ella were able to shuffle off the burden of
a conversation which had grown decidedly difficult to keep up, and
the memory of which made a slight constraint between them, on the
man’s side especially, for two or three days.
Nouna, to her husband’s great comfort and gladness, was
behaving beautifully, and putting new life by her gaiety into the whole
household, the younger members of which, in spite of Ella’s
intelligence and her sisters’ beauty, were a little wanting in those
electric high spirits which, in the routine of a quiet country-house, are
as sunshine to the crops. The day after their arrival was Sunday, and
the morning church-going had been a fiery ordeal for George, not
from religious indifference, but from the misgiving that if Nouna could
not keep from smiling in the course of a well-conducted service in a
West End church, she would certainly be carried out in convulsions
from the Willingham place of worship, where the school children,
summer and winter, sniffed through the service in a distressing
chorus, while the loud-voiced clerk’s eccentric English rang through
the building, drowning the old vicar’s feeble voice; and where the
vicar’s wife, a strong-minded lady, whose district-visiting was a sort
of assize, had been known to “pull up” her reverend husband publicly
from her pew immediately below the pulpit when, as not infrequently
happened, he turned over two leaves of his sermon instead of one,
and went quietly on as if nothing had happened. “Turned over two
leaves? Bless me, so I have!” he would murmur, and rectify his
mistake with a tranquil nod.
So George had put his wife through a very severe drill before
starting, and had strictly forbidden her so much as to sneeze without
his permission. She had a narrow escape at the offertory, when one
of the churchwardens, with a lively remembrance of the artifices of
his own youth, shovelled a penny into the fingers of each of his
offspring with one hand, while he presented the plate menacingly
with the other. But glancing up at her husband and perceiving a
frown of acute terror on his face, she contrived to choke in silence;
and the day was gained.
On the following Monday too, when the dreaded Lady
Florencecourt fulfilled her threat of calling and proved equal to her
reputation for unamiability, the young wife was, as she triumphantly
averred afterwards, “very good.” The county censor proved to be a
fair, florid woman of middle height, rather stout, and with features so
commonplace that, without the saving shield of her title, they would
have been called common. She had arrogant and capricious
manners, an oily self-satisfied voice, and an ill word for everybody.
Whenever her husband, who accompanied her on this occasion,
ventured to make a remark, she turned to look at him with a resigned
air, as if she were used to being made a martyr at the stake of his
imbecility. She examined Nouna from head to foot through a gold
double eyeglass, as if the young wife had been a charity-girl
convicted of misconduct, and made no remark to her except to ask
her if she was interested in the Zenana Missions, to which Nouna
replied rather haughtily that Indian ladies were no more in need of
missionaries than English ones: after which thrust and counter-thrust
it may be imagined that the conversation languished, and that later in
the day George had great difficulty in persuading his wife not to
break off their engagement to go to Willingham. She said it would
just spoil the end of their visit to the Millards, for one of whom she
had begun to feel a real affection. This was the sharp-tongued Ella,
whose intelligence she had the wit to recognise, and whose smart
sayings amused her.
It was on the evening of a day in the course of which this oddly-
assorted pair of friends had been a good deal together that George,
on going up stairs to his room after a last cigar with his host, found
his wife, not as usual fast asleep like a child, but perched upon the
bed in the attitude of a Hindoo idol, with a big book open on her
crossed knees, and her eyes fixed upon the nearest candle.
“Hallo!” said he, “what’s the matter?”
She turned her eyes upon him slowly, with an air of suspicion and
curiosity.
“Nothing is the matter,” she said gravely, and turning down a whole
half-leaf of the book before her to keep the place, she closed it
carefully, and handed it to him with an affectation of solemn
indifference. “I have been reading,” she added with decision.
George looked at the title of the ponderous volume, and observed
that it was The Complete Works of Xenophon. He opened it without
a smile at the page she had turned down, and remarking that it was
about half-way through the volume, said she had got on very well if
she had read so much in one evening.
“I skipped a little—the dry parts,” she observed modestly, but in
such a tone that it was impossible for George to tell whether she
meant to be taken seriously or not.
“Dry!” he exclaimed, raising his eyebrows, “why, he is the very
lightest of light reading. Xenophon was the most frivolous man I ever
knew; he was at school with me.”
She crawled to the foot of the bed, and stretching over the rail to
the dressing-table, on which George had placed the volume, she
recovered it with a violent muscular effort, and turned back the
leaves to the title-page.
“This book was published in 1823; so you are much older than you
told me you were, I see,” she said simply, while George, unable to
contain himself longer, burst out into a long laugh, and made a dive
at her, which she evaded like a squirrel, still staring at him with
unmoved gravity, so that his mirth died away in wonderment and in a
rush of tenderness as he perceived the pathos of this futile plunge
into the mazes of learning.
As he recovered his gravity the expression of her mobile face also
changed; after a moment’s shy silence their eyes met, and each saw
the other through a luminous mist.
“What are you crying for?” she asked tremulously; and in a
moment flung herself impulsively into his ready arms. “Why didn’t
you marry Ella?” was her next question, shot suddenly into his ear in
the midst of an incoherent outburst of the passionate tenderness that
glowed ever in his heart for her.
“Marry Ella!” said he, feeling a shock of surprise at the
remembrance that he had indeed once offered to make the good
little blue-stocking his wife. “Why, what makes you ask such a
question as that? Are you jealous?”
“Oh, no. But I see that she would have had you, and therefore you
were foolish not to have her.”
“Well, I’m afraid it’s too late now, and I shall have to put up with the
consequences of my folly,” said he, pressing her tenderly to him.
“That’s just what I thought,” she agreed quite plaintively. “Miss
Glass says a good wife must cook, Ella says a good wife must read,
but nobody says a good wife must just sing and laugh and amuse
herself as I do. And so when you’re tired of kissing me, you will feel
you had better not have married me, but only have amused yourself
like Dicky Wood—” She paused significantly.
“Dicky Wood!” echoed he sharply.
“—With Chloris White.”
George moved uneasily; he was angry and disturbed.
“You must not say such things—you must not think them. The
name of such a woman as that is not fit to pass your lips.”
 “But, George,” she argued, looking straight into his eyes with
penetrating shrewdness, “if you had not been you, say, if you had
been Rahas or Captain Pascoe, I might—”
He stopped the words upon her lips with a great gravity which
awed her and kept her very still, very attentive, while he spoke.
“When God throws an innocent girl into the arms of an honest
man, Nouna, as you came into mine, she is a sacred gift, received
with such reverent love that she must always hold herself holy and
pure, and never even let any thought of evil come into her heart, so
that she may be the blessing God intended. I was born into the world
to protect you and shield you from harm, my darling; and so my love
was ready for you at the moment when your innocence might have
put you in danger, just as it will be to the end of your life.”
“Supposing you were to die first?” suggested she, not flippantly,
but with an awestruck consideration of possibilities.
“A soldier can always last out till his duty’s done,” said George,
with quiet conviction.
After this Nouna remained silent a little while, but that her ideas
had not been working in quite the desired direction was evident
when she next spoke.
“If, as you say, your love will keep me safe and good whatever I
do, I needn’t be so particular,” she argued, “and it won’t do me any
harm to go and see this Mrs. Chloris White, and ask her to leave
poor Dicky alone, and let him meet some one who will be a blessing
to him. I want him to marry Ella.”
George was thunderstruck.
“Go and see Chloris White! I’d as soon let you go to the Morgue!”
“But I know I could persuade her to give him up; I know just what I
would say, just how I would look. I’ve thought it all over; and surely
anything’s better than that he should rush back to her as soon as he
gets to town, and undo all the good we’ve done him in the country.”
She spoke with a pretty little matronly air of perfectly sincere
benevolence.
“My dear child,” said her husband decisively, laying his hand on
her head with his gravest air of authority, “you cannot go; it is out of
the question. You must not even mention such a wild idea to any
one; they would be horribly shocked. But we’ll keep poor Dicky safe
among us by much better means than that, I promise you. So now
go to sleep, and don’t ever let such an idea come into your head
again.”
She let herself be kissed quite brightly and submissively, and
rubbed her cheek against his with affection which might have been
taken to argue docility. But her own fantastic notion of helping her
friend remained in her mind quite unmoved by her husband’s
prohibition.
 CHAPTER XX.
When the time came for them to finish their stay in Norfolk by the
dreaded three days at Willingham, neither George nor Nouna made
any secret of the fact that they felt the coming visit to be a severe
ordeal. Undoubtedly it would be a cruelly abrupt change from the
cheerful homeliness of the Lodge, to the penitential atmosphere in
which the household of Lady Florencecourt passed their days. So
notorious was the character of the gracious châtelaine that
Willingham Hall was commonly known in the neighbourhood as the
House of Correction, a title to which the severely simple style of its
architecture gave no very flat denial. Willingham Church stood in the
grounds belonging to the Hall, so that Nouna had had an opportunity
of shuddering at the sombre dreariness of the mansion even before
the return call she had made with Lady Millard and Cicely, on which
occasion she had sat almost mute on a high-backed chair, looking as
insignificant and unhappy as a starved mouse, thinking that Lady
Florencecourt’s light eyes looked like the glass marbles with which
she played at solitaire, and what a good model her face would be for
one of those indiarubber heads that children squeeze up into
grotesque grimaces.
She cried at parting with the Millards, like a little girl sent to school
for the first time. Sir Henry, with his simple good humour; Lady
Millard, with her quiet manners, and the quick black eyes whose
flashing keenness and sympathy showed the burning soul of the
New World flickering in uneasy brightness among the glowing
embers of the Old; Cicely and Charlotte, fair, kind creatures, who
filled up the pauses gracefully, the one by merely smiling, the other
by a gentle rain of chatter which she had been taught to think a
fascinating social accomplishment; and, above all, Ella, of the sallow
face, the sharp tongue, and the warm heart, were a group to live
pleasantly in the memory, and to make the approaching encounter
with the unamiable hosts of Willingham more disagreeable by
contrast. It added to poor Nouna’s forlornness in these